Benjamin Pinsky

All Publications

• Sample Pooling as a Strategy to Detect Community Transmission of SARS-CoV-2. JAMA Hogan, C. A., Sahoo, M. K., Pinsky, B. A. 2020

  View details for DOI 10.1001/jama.2020.5445

  View details for PubMedID 32250394

• B cell clonal expansion and convergent antibody responses to SARS-CoV-2. Research square Nielsen, S. C., Yang, F., Hoh, R. A., Jackson, K. J., Roeltgen, K., Lee, J. Y., Rustagi, A., Rogers, A. J., Powell, A. E., Kim, P. S., Wang, T. T., Pinsky, B., Blish, C. A., Boyd, S. D. 2020

  Abstract

  During virus infection B cells are critical for the production of antibodies and protective immunity. Establishment of a diverse antibody repertoire occurs by rearrangement of germline DNA at the immunoglobulin heavy and light chain loci to encode the membrane-bound form of antibodies, the B cell antigen receptor. Little is known about the B cells and antigen receptors stimulated by the novel human coronavirus SARS-CoV-2. Here we show that the human B cell compartment in patients with diagnostically confirmed SARS-CoV-2 and clinical COVID-19 is rapidly altered with the early recruitment of B cells expressing a limited subset of V genes, and extensive activation of IgG and IgA subclasses without significant somatic mutation. We detect expansion of B cell clones as well as convergent antibodies with highly similar sequences across SARS-CoV-2 patients, highlighting stereotyped naïve responses to this virus. A shared convergent B cell clonotype in SARS-CoV-2 infected patients was previously seen in patients with SARS. These findings offer molecular insights into shared features of human B cell responses to SARS-CoV-2 and other zoonotic spillover coronaviruses.

  View details for DOI 10.21203/rs.3.rs-27220/v1

  View details for PubMedID 32702737

  View details for PubMedCentralID PMC7336706

• SARS-CoV-2 RNAemia in a Healthy Blood Donor 40 Days After Respiratory Illness Resolution. Annals of internal medicine Pham, T. D., Huang, C., Wirz, O. F., Röltgen, K., Sahoo, M. K., Layon, A., Pandey, S., Foung, S. K., Boyd, S. D., Pinsky, B. A. 2020

  View details for DOI 10.7326/L20-0725

  View details for PubMedID 32678685

• Occurrence and Timing of Subsequent SARS-CoV-2 RT-PCR Positivity Among Initially Negative Patients. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America Long, D. R., Gombar, S., Hogan, C. A., Greninger, A. L., Shah, V. O., Bryson-Cahn, C., Stevens, B., Rustagi, A., Jerome, K. R., Kong, C. S., Zehnder, J., Shah, N. H., Weiss, N. S., Pinsky, B. A., Sunshine, J. 2020

  Abstract

  Using data for 20,912 patients from two large academic health systems, we analyzed the frequency of SARS-CoV-2 RT-PCR test-discordance among individuals initially testing negative by nasopharyngeal swab who were retested on clinical grounds within 7 days. The frequency of subsequent positivity within this window was 3.5% and similar across institutions.

  View details for DOI 10.1093/cid/ciaa722

  View details for PubMedID 32506118

• Report from the American Society for Microbiology COVID-19 International Summit, 23 March 2020: Value of Diagnostic Testing for SARS-CoV-2/COVID-19. mBio Patel, R., Babady, E., Theel, E. S., Storch, G. A., Pinsky, B. A., St George, K., Smith, T. C., Bertuzzi, S. 2020; 11 (2)

  View details for DOI 10.1128/mBio.00722-20

  View details for PubMedID 32217609

• hrHPV prevalence and type distribution in rural Zimbabwe: A community-based self-collection study using near-point-of-care GeneXpert HPV testing INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES Fitzpatrick, M. B., Mandishora, R., Katzenstein, D. A., McCarty, K., Weber, J., Sahoo, M. K., Manasa, J., Chirenje, Z., Pinsky, B. A. 2019; 82: 21?29

  View details for DOI 10.1016/j.ijid.2019.02.022

  View details for Web of Science ID 000466424400007

• Persistence of Human Immunodeficiency Virus-1 Drug Resistance Mutations in Proviral Deoxyribonucleic Acid After Virologic Failure of Efavirenz-Containing Antiretroviral Regimens. Open forum infectious diseases De La Cruz, J., Vardhanbhuti, S., Sahoo, M. K., Rovner, R., Bosch, R. J., Manasa, J., Katzenstein, D. A., Pinsky, B. A. 2019; 6 (3): ofz034

  Abstract

  Background: Efavirenz (EFV)-based regimens select broad drug resistance to nonnucleoside reverse-transcriptase inhibitors (NNRTIs), limiting the effectiveness of EFV and other NNRTIs. The duration, persistence, and decay of drug resistance mutations (DRMs) in the proviral reservoir is not well defined.Methods: Participants with virologic failure of EFV-based regimens and drug-resistant viremia with the K103N mutation in plasma ribonucleic acid (RNA) were identified from AIDS Clinical Trials Group (ACTG) studies A364 and A5095. These individuals received a second-line, boosted protease inhibitor-based regimen with suppression of viremia for up to10 years during long-term follow-up (median = 3.6 years; interquartile range, 2.1-6.9 years). Proviral deoxyribonucleic acid (DNA) from cryopreserved peripheral blood mononuclear cells was sequenced to identify the persistence of DRM.Results: Twenty-eight participants from ACTG 364 and ACTG 5095 were evaluated. Sanger sequencing of proviral DNA detected K103N as well as additional reverse-transcriptase inhibitor (RTI) mutations. Ultradeep sequencing confirmed persistence of K103N in 71% of participants with minimal decay over time. In an adjusted model including years since suppression, persistent proviral K103N was 2.6 times more likely (95% confidence interval, 1.0-6.4) per log10 higher human immunodeficiency virus RNA at EFV failure.Conclusions: Persistence of RTI mutations in proviral DNA after virologic failure has implications for the effectiveness of future drug regimens and the recycling of RTI drugs.

  View details for PubMedID 30863788

• Yellow Fever Virus: Diagnostics for a Persistent Arboviral Threat JOURNAL OF CLINICAL MICROBIOLOGY Waggoner, J. J., Rojas, A., Pinsky, B. A. 2018; 56 (10)

  Abstract

  Yellow fever (YF) is the prototypical hemorrhagic fever and results from infection with yellow fever virus (YFV), which is endemic to regions of Africa and South America. Despite the availability of an effective vaccine, YFV continues to cause disease throughout regions where it is endemic, including intermittent large outbreaks among undervaccinated populations. A number of diagnostic methods and assays have been described for the detection of YFV infection, including viral culture, molecular testing, serology, and antigen detection. Commercial diagnostics are not widely available, and testing is generally performed at a small number of reference laboratories. The goal of this article, therefore, is to review available clinical diagnostics for YFV, which may not be familiar to many practitioners outside areas where it is endemic. Additionally, we identify gaps in our current knowledge about YF that pertain to diagnosis and describe interventions that may improve YFV detection.

  View details for PubMedID 30021822

  View details for PubMedCentralID PMC6156298

• Multiplexed identification, quantification and genotyping of infectious agents using a semiconductor biochip NATURE BIOTECHNOLOGY Hassibi, A., Manickam, A., Singh, R., Bolouki, S., Sinha, R., Jirage, K. B., McDermott, M. W., Hassibi, B., Vikalo, H., Mazarei, G., Pei, L., Bousse, L., Miller, M., Heshami, M., Savage, M. P., Taylor, M. T., Gamini, N., Wood, N., Mantina, P., Grogan, P., Kuimelis, P., Savalia, P., Conradson, S., Li, Y., Meyer, R. B., Ku, E., Ebert, J., Pinsky, B. A., Dolganov, G., Van, T., Johnson, K. A., Naraghi-Arani, P., Kuimelis, R. G., Schoolnik, G. 2018; 36 (8): 738?45

  Abstract

  The emergence of pathogens resistant to existing antimicrobial drugs is a growing worldwide health crisis that threatens a return to the pre-antibiotic era. To decrease the overuse of antibiotics, molecular diagnostics systems are needed that can rapidly identify pathogens in a clinical sample and determine the presence of mutations that confer drug resistance at the point of care. We developed a fully integrated, miniaturized semiconductor biochip and closed-tube detection chemistry that performs multiplex nucleic acid amplification and sequence analysis. The approach had a high dynamic range of quantification of microbial load and was able to perform comprehensive mutation analysis on up to 1,000 sequences or strands simultaneously in <2 h. We detected and quantified multiple DNA and RNA respiratory viruses in clinical samples with complete concordance to a commercially available test. We also identified 54 drug-resistance-associated mutations that were present in six genes of Mycobacterium tuberculosis, all of which were confirmed by next-generation sequencing.

  View details for PubMedID 30010676

• Characterization of Dengue Virus Infections Among Febrile Children Clinically Diagnosed With a Non-Dengue Illness, Managua, Nicaragua JOURNAL OF INFECTIOUS DISEASES Waggoner, J. J., Gresh, L., Mohamed-Hadley, A., Balmaseda, A., Soda, K., Abeynayake, J., Sahoo, M. K., Liu, Y., Kuan, G., Harris, E., Pinsky, B. A. 2017; 215 (12): 1816?23

  Abstract

  We sought to characterize dengue virus (DENV) infections among febrile children enrolled in a pediatric cohort study who were clinically diagnosed with a non-dengue illness ("C cases").DENV infections were detected and viral load quantitated by real-time reverse transcription-polymerase chain reaction in C cases presenting between January 2007 and January 2013.One hundred forty-one of 2892 C cases (4.88%) tested positive for DENV. Of all febrile cases in the study, DENV-positive C cases accounted for an estimated 52.0% of patients with DENV viremia at presentation. Compared with previously detected, symptomatic dengue cases, DENV-positive C cases were significantly less likely to develop long-lasting humoral immune responses to DENV, as measured in healthy annual serum samples (79.7% vs 47.8%; P < .001). Humoral immunity was associated with viral load at presentation: 40 of 43 patients (93.0%) with a viral load ?7.0 log10 copies/mL serum developed the expected rise in anti-DENV antibodies in annual samples versus 13 of 68 (19.1%) patients with a viral load below this level (P < .001).Antibody responses to DENV-positive C cases differ from responses to classic symptomatic dengue. These findings have important implications for DENV transmission modeling, immunology, and epidemiologic surveillance.

  View details for PubMedID 28863466

  View details for PubMedCentralID PMC5853235

• Diagnosis of Zika virus infection on a nanotechnology platform. Nature medicine Zhang, B., Pinsky, B. A., Ananta, J. S., Zhao, S., Arulkumar, S., Wan, H., Sahoo, M. K., Abeynayake, J., Waggoner, J. J., Hopes, C., Tang, M., Dai, H. 2017

  Abstract

  We developed a multiplexed assay on a plasmonic-gold platform for measuring IgG and IgA antibodies and IgG avidity against both Zika virus (ZIKV) and dengue virus (DENV) infections. In contrast to IgM cross-reactivity, IgG and IgA antibodies against ZIKV nonstructural protein 1 (NS1) antigen were specific to ZIKV infection, and IgG avidity revealed recent ZIKV infection and past DENV-2 infection in patients in dengue-endemic regions. This assay could enable specific diagnosis of ZIKV infection over other flaviviral infections.

  View details for DOI 10.1038/nm.4302

  View details for PubMedID 28263312

• Viremia and Clinical Presentation in Nicaraguan Patients Infected with Zika Virus, Chikungunya Virus, and Dengue Virus. Clinical infectious diseases Waggoner, J. J., Gresh, L., Vargas, M. J., Ballesteros, G., Tellez, Y., Soda, K. J., Sahoo, M. K., Nuñez, A., Balmaseda, A., Harris, E., Pinsky, B. A. 2016

  Abstract

  ?Zika virus (ZIKV), chikungunya virus (CHIKV), and dengue virus (DENV) cocirculate in Nicaragua. In this study, we sought to compare the quantified viremia and clinical presentation of patients infected with 1 or more of these viruses.?Acute-phase serum samples from 346 patients with a suspected arboviral illness were tested using a multiplex real-time reverse-transcription polymerase chain reaction for ZIKV, CHIKV, and DENV. Viremia was quantitated for each detected virus, and clinical information from request forms submitted with each sample was recorded.?A total of 263 patients tested positive for 1 or more viruses: 192 patients tested positive for a single virus (monoinfections) and 71 patients tested positive for 2 or all 3 viruses (coinfections). Quantifiable viremia was lower in ZIKV infections compared with CHIKV or DENV (mean 4.70 vs 6.42 and 5.84 log10 copies/mL serum, respectively; P < .001 for both comparisons), and for each virus, mean viremia was significantly lower in coinfections than in monoinfections. Compared with patients with CHIKV or DENV, ZIKV patients were more likely to have a rash (P < .001) and less likely to be febrile (P < .05) or require hospitalization (P < .001). Among all patients, hospitalized cases had higher viremia than those who did not require hospitalization (7.1 vs 4.1 log10 copies/mL serum, respectively; P < .001).?ZIKV, CHIKV, and DENV result in similar clinical presentations, and coinfections may be relatively common. Our findings illustrate the need for accurate, multiplex diagnostics for patient care and epidemiologic surveillance.

  View details for PubMedID 27578819

• Zika Virus: Diagnostics for an Emerging Pandemic Threat. Journal of clinical microbiology Waggoner, J. J., Pinsky, B. A. 2016; 54 (4): 860-867

  Abstract

  Zika virus (ZIKV) is anAedesmosquito-borne flavivirus that emerged in Brazil in 2015 and then rapidly spread throughout the tropical and subtropical Americas. Based on clinical criteria alone, ZIKV cannot be reliably distinguished from infections with other pathogens that cause an undifferentiated systemic febrile illness, including infections with two common arboviruses, dengue virus and chikungunya virus. This minireview details the methods that are available to diagnose ZIKV infection.

  View details for DOI 10.1128/JCM.00279-16

  View details for PubMedID 26888897

  View details for PubMedCentralID PMC4809954

• Mechanisms of Fano-resonant biosensing: Mechanical loading of plasmonic oscillators OPTICS COMMUNICATIONS Zhu, X., Cao, N., Thibeault, B. J., Pinsky, B., Yanik, A. 2020; 469

  View details for DOI 10.1016/j.optcom.2020.125780

  View details for Web of Science ID 000532673400010

• Knowledge, attitudes, and practices of cervical Cancer screening among HIV-positive and HIV-negative women participating in human papillomavirus screening in rural Zimbabwe. BMC women's health Fitzpatrick, M., Pathipati, M. P., McCarty, K., Rosenthal, A., Katzenstein, D., Chirenje, Z. M., Pinsky, B. 2020; 20 (1): 153

  Abstract

  BACKGROUND: Women in low- and middle-income countries are at the highest risk of cervical cancer yet have limited access to and participation in cervical cancer screening programs. Integrating self-collected, community-based screening offers a potential primary screening method in areas of limited resources. In this paper, we present a study evaluating knowledge, attitudes, and practices of cervical cancer and Human Papilloma Virus (HPV) in rural Zimbabwe.METHODS: We performed a community-based cross-sectional knowledge, attitudes and practices of HPV and cervical cancer study in rural Zimbabwe from January 2017-May 2017. Women were selected for the study via random number generation from complete lists of inhabitants in the study area if they satisfied the inclusion criteria (?30-years-old, ?65-years-old, not pregnant, intact uterus). If selected, they participated in a 19-question structured knowledge, attitudes and practices survey. The questionnaire included questions on demographics, education, knowledge of HPV, cervical cancer, and risk factors. Chi-squared tests were evaluated comparing knowledge, attitudes and practices relating to HPV and cervical cancer screening with actual infection with HPV. Women were also offered a voluntary HIV and self-collected HPV screening.RESULTS: Six hundred seventy-nine women were included in the knowledge, attitudes and practices survey. Most women (81%) had heard of cervical cancer while the majority had not heard of HPV (12%). The number of women that had been screened previously for cervical cancer was low (5%). There were no significant differences between and within groups regarding knowledge of cervical cancer and actual overall infection with HR-HPV, HPV 16, and HPV 18/45 test results.CONCLUSIONS: Most women in rural Zimbabwe have heard of cervical cancer, but the number that had been screened was low. Extending existing outreach services to include cervical cancer screening, potentially including HPV screening, should include cervical cancer/HPV education and screening triage. This approach would serve to bridge the gap between knowledge and screening availability to address some of the barriers to cervical cancer care still affecting women in many regions of the world.

  View details for DOI 10.1186/s12905-020-01017-2

  View details for PubMedID 32711530

• Triplex Real-Time RT-PCR for Severe Acute Respiratory Syndrome Coronavirus 2 EMERGING INFECTIOUS DISEASES Waggoner, J. J., Stittleburg, V., Pond, R., Saklawi, Y., Sahoo, M. K., Babiker, A., Hussaini, L., Kraft, C. S., Pinsky, B. A., Anderson, E. J., Rouphael, N. 2020; 26 (7): 1633?35

  Abstract

  Most reverse transcription PCR protocols for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) include 2-3 targets for detection. We developed a triplex, real-time reverse transcription PCR for SARS-CoV-2 that maintained clinical performance compared with singleplex assays. This protocol could streamline detection and decrease reagent use during current high SARS-CoV-2 testing demands.

  View details for DOI 10.3201/eid2607.201285

  View details for Web of Science ID 000551231700052

  View details for PubMedID 32294051

  View details for PubMedCentralID PMC7323516

• Measure what matters: Counts of hospitalized patients are a better metric for health system capacity planning for a reopening. Journal of the American Medical Informatics Association : JAMIA Kashyap, S., Gombar, S., Yadlowsky, S., Callahan, A., Fries, J., Pinsky, B. A., Shah, N. H. 2020

  Abstract

  OBJECTIVE: Responding to the COVID-19 pandemic requires accurate forecasting of health system capacity requirements using readily available inputs. We examined whether testing and hospitalization data could help quantify the anticipated burden on the health system given shelter-in-place (SIP) order.MATERIALS AND METHODS: 16,103 SARS-CoV-2 RT-PCR tests were performed on 15,807 patients at Stanford facilities between March 2 and April 11, 2020. We analyzed the fraction of tested patients that were confirmed positive for COVID-19, the fraction of those needing hospitalization, and the fraction requiring ICU admission over the 40 days between March 2nd and April 11th 2020.RESULTS: We find a marked slowdown in the hospitalization rate within ten days of SIP even as cases continued to rise. We also find a shift towards younger patients in the age distribution of those testing positive for COVID-19 over the four weeks of SIP. The impact of this shift is a divergence between increasing positive case confirmations and slowing new hospitalizations, both of which affects the demand on health systems.CONCLUSION: Without using local hospitalization rates and the age distribution of positive patients, current models are likely to overestimate the resource burden of COVID-19. It is imperative that health systems start using these data to quantify effects of SIP and aid reopening planning.

  View details for DOI 10.1093/jamia/ocaa076

  View details for PubMedID 32548636

• Persistent detection of SARS-CoV-2 RNA in patients and healthcare workers with COVID-19. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology Gombar, S., Chang, M., Hogan, C. A., Zehnder, J., Boyd, S., Pinsky, B. A., Shah, N. H. 2020; 129: 104477

  Abstract

  BACKGROUND: Current guidelines for returning health care workers (HCW) to service after a positive SARS-CoV-2 RT-PCR test and ceasing of transmission precautions for patients is based on two general strategies. A test-based strategy that requires negative respiratory RT-PCR tests obtained after the resolution of symptoms. Alternatively, due to the limited availability of testing, many sites employ a symptom-based strategy that recommends excluding HCW from the workforce and keeping patients on contact precautions until a fixed period of time has elapsed from symptom recovery. The underlying assumption of the symptom-based strategy is that waiting for a fixed period of time is a surrogate for negative RT-PCR testing, which itself is a surrogate for the absence of shedding infectious virus.OBJECTIVES: To better understand the appropriate length of symptom based return to work and contact precaution strategies.STUDY DESIGN: We performed an observational analysis of 150 patients and HCW that transitioned from RT-PCR SARS-CoV-2 positive to negative over the course of 2 months at a US academic medical center.RESULTS: We found that the average time to transition from RT-PCR positive to negative was 24 days after symptom onset and 10 % remained positive even 33 days after symptom onset. No difference was seen in HCW and patients.CONCLUSIONS: These findings suggest until definitive evidence of the length of infective viral shedding is obtained that the fixed length of time before returning to work or ceasing contract precautions be revised to over one-month.

  View details for DOI 10.1016/j.jcv.2020.104477

  View details for PubMedID 32505778

• A non-optical multiplexed PCR diagnostic platform for serotype-specific detection of dengue virus SENSORS AND ACTUATORS B-CHEMICAL Yin, Z., Ramshani, Z., Waggoner, J. J., Pinsky, B. A., Senapati, S., Chang, H. 2020; 310

  View details for DOI 10.1016/j.snb.2020.127854

  View details for Web of Science ID 000519306300005

• Five-minute point-of-care testing for SARS-CoV-2: Not there yet. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology Hogan, C. A., Sahoo, M. K., Huang, C., Garamani, N., Stevens, B., Zehnder, J., Pinsky, B. A. 2020; 128: 104410

  View details for DOI 10.1016/j.jcv.2020.104410

  View details for PubMedID 32403009

• Whole-Genome Analysis of Cervical Human Papillomavirus Type 35 from rural Zimbabwean Women. Scientific reports Fitzpatrick, M. B., Hahn, Z., Mandishora, R. S., Dao, J., Weber, J., Huang, C., Sahoo, M. K., Katzenstein, D. A., Pinsky, B. A. 2020; 10 (1): 7001

  Abstract

  Human papillomavirus (HPV) types differ by geographic location and the ethnicity of the human host, which may have implications for carcinogenicity. HPV35 is one of the least frequently identified high-risk types in North America and Europe but was the most common high-risk HPV (hrHPV) infection in a cohort in rural Zimbabwe. Whole genome analysis is limited for HPV35; no such studies have been performed in Zimbabwe. Of 648 women in the initial cohort in Zimbabwe, 19 (19/648, 2.9%) tested positive for HPV35, and eight samples were successfully sequenced for HPV35. The maximum number of sequence variants for the whole genome was 58 nucleotides (0.7%) compared to the prototype (58/7879). The maximum number of sequence variants in E6 and E7 was 3 (3/450, 0.7%) 2 (2/300, 0.7%), respectively. These are the first HPV35 whole genome sequences from Zimbabwe, and these data further lend support to the carcinogenicity of HPV35 despite limited sequence heterogeneity. Further studies to determine carcinogenic effects and impact of HPV vaccinations are warranted, especially in sub-Saharan Africa.

  View details for DOI 10.1038/s41598-020-63882-z

  View details for PubMedID 32332798

• Rates of Co-infection Between SARS-CoV-2 and Other Respiratory Pathogens. JAMA Kim, D., Quinn, J., Pinsky, B., Shah, N. H., Brown, I. 2020

  View details for DOI 10.1001/jama.2020.6266

  View details for PubMedID 32293646

• Antibody-Dependent Enhancement of Severe Disease Is Mediated by Serum Viral Load in Pediatric Dengue Virus Infections. The Journal of infectious diseases Waggoner, J. J., Katzelnick, L. C., Burger-Calderon, R., Gallini, J., Moore, R. H., Kuan, G., Balmaseda, A., Pinsky, B. A., Harris, E. 2020

  Abstract

  BACKGROUND: Low preexisting anti-dengue virus (DENV) antibody levels are associated with elevated disease severity. While antibody-dependent enhancement of dengue is thought to be driven by viral load, this has not been conclusively shown. We evaluated the association between preinfection anti-DENV antibody titers, viral load, and disease severity among 133 dengue cases in a Nicaraguan pediatric cohort study.METHODS: Viral load was quantified in acute-phase serum by real-time reverse transcription polymerase chain reaction and analyzed in relation to preinfection antibody titer (measured by inhibition enzyme-linked immunosorbent assay) and dengue severity, categorized using 3 definitions.RESULTS: Higher viral load was significantly associated with dengue severity; for each increase of 1.0 log10 copies/mL, the odds of severe dengue increased approximately 50%, regardless of severity definition. Viral load at presentation and the odds of severe disease were highest among patients with low to intermediate preinfection antibody titers and lowest among those with the highest antibody titers. We showed the effect of preinfection antibody titer on disease severity was mediated by viral load for each of 3 dengue severity outcomes.CONCLUSIONS: This study demonstrates the association between preinfection anti-DENV antibody titer, serum viral load, and disease severity, and provides evidence for the mechanism of antibody-dependent enhancement in dengue cases.

  View details for DOI 10.1093/infdis/jiz618

  View details for PubMedID 32236481

• Merkel Cell Carcinoma of Lymph Node is Metastatic Cutaneous Merkel Cell Carcinoma Lawrence, L., Kunder, C., Stehr, H., Saleem, A., Natkunam, Y., Zehnder, J., Pinsky, B., Sahoo, M., Tan, S. NATURE PUBLISHING GROUP. 2020: 824?26

  View details for Web of Science ID 000518328801386

• Merkel Cell Carcinoma of Lymph Node is Metastatic Cutaneous Merkel Cell Carcinoma Lawrence, L., Kunder, C., Stehr, H., Saleem, A., Natkunam, Y., Zehnder, J., Pinsky, B., Sahoo, M., Tan, S. NATURE PUBLISHING GROUP. 2020: 824?26

  View details for Web of Science ID 000518328901386

• Human papillomavirus cytopathic effect in the urine of a 76-year-old man. Diagnostic cytopathology Gottlieb, C., Pinsky, B. A., Mills, A. M., Stelow, E. B. 2020

  View details for DOI 10.1002/dc.24384

  View details for PubMedID 32043839

• Implementation of a Multiplex rRT-PCR for Zika, Chikungunya, and Dengue Viruses: Improving Arboviral Detection in an Endemic Region. The American journal of tropical medicine and hygiene Cantero, C., Cardozo, F., Waggoner, J. J., Pinsky, B. A., Espinola, A., Infanzon, B., Acosta, M. E., Aria, L., Arevalo de Guillen, Y., Cuevas, T., Rojas, V., Segovia, C., Centurion, A., Rojas, A. 2020

  Abstract

  Arboviral diagnosis has been complicated throughout the tropical and subtropical Americas by the recent co-circulation of Zika virus (ZIKV), chikungunya virus (CHIKV), and dengue virus (DENV). The aim of this study was to implement a multiplex real-time RT-PCR (rRT-PCR) for ZIKV, CHIKV, and DENV in Paraguay to test patients who were clinically suspected of having dengue. We tested 110 sera from patients who presented to the Hospital de Clinicas in 2016 and had testing for DENV nonstructural protein 1 (NS1; 40 positive and 70 negative). Using a composite reference standard, we confirmed 51 dengue cases (46.4%): 38/40 NS1 positive and 13/70 NS1 negative. Chikungunya virus and ZIKV were detected in one sample each, both were DENV NS1 negative. The NS1 test demonstrated good agreement with rRT-PCR for DENV. However, multiplex rRT-PCR identified a subset of dengue cases and additional arboviral infections that would not be detected if NS1 assays are relied upon for diagnosis.

  View details for DOI 10.4269/ajtmh.19-0707

  View details for PubMedID 31933462

• Comparison of the Accula SARS-CoV-2 Test with a Laboratory-Developed Assay for Detection of SARS-CoV-2 RNA in Clinical Nasopharyngeal Specimens. Journal of clinical microbiology Hogan, C. A., Garamani, N., Lee, A. S., Tung, J. K., Sahoo, M. K., Huang, C., Stevens, B., Zehnder, J., Pinsky, B. A. 2020

  Abstract

  Background: Several point-of-care (POC) molecular tests have received emergency use authorization (EUA) from the Food and Drug Administration (FDA) for diagnosis of SARS-CoV-2. The test performance characteristics of the Accula (Mesa Biotech) SARS-CoV-2 POC test need to be evaluated to inform its optimal use.Objectives: The aim of this study was to assess test performance of the Accula SARS-CoV-2 test.Study design: The performance of the Accula test was assessed by comparing results of 100 nasopharyngeal swab samples previously characterized by the Stanford Health Care EUA laboratory-developed test (SHC-LDT) targeting the envelope (E) gene. Assay concordance was assessed by overall percent agreement, positive percent agreement (PPA), negative percent agreement (NPA), and Cohen's kappa coefficient.Results: Overall percent agreement between the assays was 84.0% (95% confidence interval [CI] 75.3 to 90.6%), PPA was 68.0% (95% CI 53.3 to 80.5%) and the kappa coefficient was 0.68 (95% CI 0.54 to 0.82). Sixteen specimens detected by the SHC-LDT were not detected by the Accula test, and showed low viral load burden with a median cycle threshold value of 37.7. NPA was 100% (95% CI 94.2 to 100%).Conclusion: Compared to the SHC-LDT, the Accula SARS-CoV-2 test showed excellent negative agreement. However, positive agreement was low for samples with low viral load. The false negative rate of the Accula POC test calls for a more thorough evaluation of POC test performance characteristics in clinical settings, and for confirmatory testing in individuals with moderate to high pre-test probability of SARS-CoV-2 who test negative on Accula.

  View details for DOI 10.1128/JCM.01072-20

  View details for PubMedID 32461285

• Reply to Muller and Chaudhury. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America Hogan, C. A., Pinsky, B. A., Banaei, N. 2020

  View details for DOI 10.1093/cid/ciaa220

  View details for PubMedID 32140706

• A predictive tool for identification of SARS-CoV-2 PCR-negative emergency department patients using routine test results. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology Joshi, R. P., Pejaver, V., Hammarlund, N. E., Sung, H., Lee, S. K., Furmanchuk, A., Lee, H. Y., Scott, G., Gombar, S., Shah, N., Shen, S., Nassiri, A., Schneider, D., Ahmad, F. S., Liebovitz, D., Kho, A., Mooney, S., Pinsky, B. A., Banaei, N. 2020; 129: 104502

  Abstract

  Testing for COVID-19 remains limited in the United States and across the world. Poor allocation of limited testing resources leads to misutilization of health system resources, which complementary rapid testing tools could ameliorate.To predict SARS-CoV-2 PCR positivity based on complete blood count components and patient sex.A retrospective case-control design for collection of data and a logistic regression prediction model was used. Participants were emergency department patients > 18 years old who had concurrent complete blood counts and SARS-CoV-2 PCR testing. 33 confirmed SARS-CoV-2 PCR positive and 357 negative patients at Stanford Health Care were used for model training. Validation cohorts consisted of emergency department patients > 18 years old who had concurrent complete blood counts and SARS-CoV-2 PCR testing in Northern California (41 PCR positive, 495 PCR negative), Seattle, Washington (40 PCR positive, 306 PCR negative), Chicago, Illinois (245 PCR positive, 1015 PCR negative), and South Korea (9 PCR positive, 236 PCR negative).A decision support tool that utilizes components of complete blood count and patient sex for prediction of SARS-CoV-2 PCR positivity demonstrated a C-statistic of 78 %, an optimized sensitivity of 93 %, and generalizability to other emergency department populations. By restricting PCR testing to predicted positive patients in a hypothetical scenario of 1000 patients requiring testing but testing resources limited to 60 % of patients, this tool would allow a 33 % increase in properly allocated resources.A prediction tool based on complete blood count results can better allocate SARS-CoV-2 testing and other health care resources such as personal protective equipment during a pandemic surge.

  View details for DOI 10.1016/j.jcv.2020.104502

  View details for PubMedID 32544861

• Is Merkel Cell Carcinoma of Lymph Node Actually Metastatic Cutaneous Merkel Cell Carcinoma? American journal of clinical pathology Lawrence, L. E., Saleem, A., Sahoo, M. K., Tan, S. K., Pinsky, B. A., Natkunam, Y., Kunder, C. A., Stehr, H., Zehnder, J. L. 2020

  Abstract

  The possibility of a so-called primary lymph node neuroendocrine carcinoma has been described in the literature. Here we evaluate cases fitting such a diagnosis and find that the cases demonstrate a convincing and pervasive pattern consistent with metastatic Merkel cell carcinoma.Six cases of primary lymph node Merkel cell carcinoma and one case of metastatic neuroendocrine carcinoma at a bony site, all with unknown primary, were sequenced using a combination of whole-exome and targeted panel methods. Sequencing results were analyzed for the presence of an ultraviolet (UV) mutational signature or off-target detection of Merkel cell polyomavirus (MCPyV).Four of six primary lymph node cases were positive for a UV mutational signature, with the remaining two cases positive for off-target alignment of MCPyV. One case of neuroendocrine carcinoma occurring at a bony site was also positive for a UV mutational signature.We find no evidence to corroborate the existence of so-called primary Merkel cell carcinoma of lymph node.

  View details for DOI 10.1093/ajcp/aqaa051

  View details for PubMedID 32445471

• Comparison of a laboratory-developed test targeting the envelope gene with three nucleic acid amplification tests for detection of SARS-CoV-2. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology Bulterys, P. L., Garamani, N., Stevens, B., Sahoo, M. K., Huang, C., Hogan, C. A., Zehnder, J., Pinsky, B. A. 2020; 129: 104427

  Abstract

  Numerous nucleic acid amplification tests, including real-time, reverse transcription PCR (rRT-PCR) and isothermal amplification methods, have been developed to detect SARS-CoV-2 RNA, including many that have received emergency use authorization (EUA). There is a need to assess their test performance relative to one another.The aim of this study was to compare the test performance of a high complexity laboratory-developed rRT-PCR EUA from Stanford Health Care (SHC) targeting the SARS-CoV-2 envelope (E) gene with other tests: the Atila isothermal amplification assay targeting the nucleocapsid (N) gene and open reading frame 1ab (ORF1ab), the Altona E and spike (S) multiplex, real-time RT-PCR, and the US Centers for Disease Control and Prevention (CDC) N1 and N2 rRT-PCRs.A diagnostic comparison study was performed by testing nasopharyngeal samples from persons under investigation for coronavirus disease 2019 (COVID-19). Assay performance was assessed by percent agreement and Cohen's kappa coefficient.Positive percent agreement with the SHC EUA reference assay was 82.8 % (95 % confidence interval (CI) 65.0 to 92.9) for Atila, 86.7 % (95 % CI 69.7 to 95.3) for the Altona E and S targets, and 86.7 % (95 % CI 69.7 to 95.3) and 90.0 % (95 % CI 73.6 to 97.3), for the CDC N1 and N2 targets, respectively. All assays demonstrated 100 % negative percent agreement. Kappa coefficients ranged from 0.86 to 0.92, indicating excellent agreement.Performance was comparable among the SARS-CoV-2 nucleic acid amplification methods tested, with a limited number of discrepancies observed in specimens with low viral loads.

  View details for DOI 10.1016/j.jcv.2020.104427

  View details for PubMedID 32535398

  View details for PubMedCentralID PMC7207111

• Comparison of a Point-of-Care Assay and a High-Complexity Assay for Detection of SARS-CoV-2 RNA. The journal of applied laboratory medicine Stevens, B., Hogan, C. A., Sahoo, M. K., Huang, C., Garamani, N., Zehnder, J., Kurzer, J., Pinsky, B. A. 2020

  Abstract

  Numerous nucleic acid amplification assays utilizing different target genes of the SARS-CoV-2 genome have received emergency use authorization (EUA) by the United States Food and Drug Administration (FDA). Limited data are available comparing the test performance characteristics of these assays.A diagnostic comparison study was performed to evaluate the performance of the Cepheid Xpert Xpress SARS-CoV-2 assay compared to the Hologic Panther Fusion SARS-CoV-2 assay using clinical nasopharyngeal specimens. Agreement between the two assays was assessed by overall, positive, and negative percent agreement and Cohen's kappa coefficient.A total of 104 (54 positive and 50 negative) clinical nasopharyngeal samples were tested by both assays. Using the Panther Fusion as a reference standard, the Xpert demonstrated an overall agreement of 99.0% (95% confidence interval (CI): 94.8 - 100), positive percent agreement of 98.1% (95% CI: 90.1 - 100), and a negative percent agreement of 100% (95% CI: 94.2 - 100). The kappa coefficient was 0.98 (95% CI: 0.94 - 1.0). One sample positive by the Panther Fusion with a cycle threshold (Ct) of 38.6 was found to be reproducibly negative by the Xpert assay.The Cepheid Xpert Xpress SARS-CoV-2 assay provides test performance comparable to the Hologic Panther Fusion SARS-CoV-2 assay while offering laboratories rapid, on-demand testing capacity.

  View details for DOI 10.1093/jalm/jfaa135

  View details for PubMedID 32761092

• Comparison of the Panther Fusion and a laboratory-developed test targeting the envelope gene for detection of SARS-CoV-2. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology Hogan, C. A., Sahoo, M. K., Huang, C., Garamani, N., Stevens, B., Zehnder, J., Pinsky, B. A. 2020; 127: 104383

  Abstract

  Numerous nucleic acid amplification assays have recently received emergency use authorization (EUA) for the diagnosis of SARS-CoV-2 infection, and there is a need to assess their test performance relative to one another.The aim of this study was to compare the test performance of the Hologic Panther Fusion SARS-CoV-2 assay targeting two regions of open reading frame 1ab (ORF1ab) to a high complexity molecular-based, laboratory-developed EUA from Stanford Health Care (SHC) targeting the SARS-CoV-2 envelope (E) gene.We performed a diagnostic comparison study by testing nasopharyngeal samples on the two assays. Assay agreement was assessed by overall percent agreement and Cohen's kappa coefficient.A total of 184 nasopharyngeal samples were tested using the two assays, of which 180 showed valid results and were included for the comparative analysis. Overall percent agreement between the assays was 98.3 % (95 % confidence interval (CI) 95.2-99.7) and kappa coefficient was 0.97 (95 % CI 0.93-1.0). One sample was detected on the SHC laboratory developed test (LDT) and not on the Panther Fusion, and had a Ct of 35.9. Conversely, 2 samples were detected on the Panther Fusion and not on the LDT, and had Ct values of 37.2 and 36.6.The Panther Fusion SARS-CoV-2 assay and the SHC LDT perform similarly on clinical nasopharyngeal swab specimens. Other considerations, including reagent availability, turnaround time, labor requirements, cost and instrument throughput should guide the decision of which assay to perform.

  View details for DOI 10.1016/j.jcv.2020.104383

  View details for PubMedID 32353760

• Assessment of Sensitivity and Specificity of Patient-Collected Lower Nasal Specimens for Sudden Acute Respiratory Syndrome Coronavirus 2 Testing. JAMA network open Altamirano, J., Govindarajan, P., Blomkalns, A. L., Kushner, L. E., Stevens, B. A., Pinsky, B. A., Maldonado, Y. 2020; 3 (6): e2012005

  View details for DOI 10.1001/jamanetworkopen.2020.12005

  View details for PubMedID 32530469

• Utilization, yield, and accuracy of the FilmArray Meningitis/Encephalitis panel with diagnostic stewardship and testing algorithm. Journal of clinical microbiology Broadhurst, M. J., Dujari, S., Budvytiene, I., Pinsky, B. A., Gold, C. A., Banaei, N. 2020

  Abstract

  Background: The impact of diagnostic stewardship and testing algorithms on utilization and performance of the FilmArray® Meningitis/Encephalitis (ME) Panel has received limited investigation.Methods: We performed a retrospective single-center cohort study assessing all individuals with suspected ME between February 2017 and April 2019 for whom the ME Panel was ordered. Testing was restricted to patients with cerebrospinal fluid (CSF) pleocytosis. Positive ME Panel results were confirmed before reporting through correlation with direct stain (Gram and Calcofluor white) and CSF Cryptococcal antigen or by repeat ME Panel testing. Outcomes included ME Panel test utilization rate, negative predictive value of non-pleocytic CSF samples, test yield and false-positivity rate, and time to appropriate de-escalation of acyclovir.Results: Restricting testing to pleocytic CSF samples reduced ME Panel utilization by 42.7% (263 vs 459 tests performed) and increased test yield by 61.8% (18.6% vs 11.5% positivity rate; P < 0.01) with application of criteria. The negative predictive value of normal CSF WBC for ME Panel targets was 100% (195/195) for non-viral targets and 98.0% (192/196) overall. All pathogens detected in non-pleocytic CSF samples were herpesviruses. Application of a selective testing algorithm based on repeat testing of non-viral targets avoided 75% (3/4) of false-positive results without generating false-negative results. Introduction of the ME panel reduced the duration of acyclovir treatment from an average of 66 hours (SD, 43) to 46 hours (SD, 36) (P = 0.03).Conclusions: Implementation of the ME Panel with restriction criteria and a selective testing algorithm for non-viral targets optimizes its utilization, yield and accuracy.

  View details for DOI 10.1128/JCM.00311-20

  View details for PubMedID 32493787

• Evidence of transovarial transmission of Chikungunya and Dengue viruses in field-caught mosquitoes in Kenya. PLoS neglected tropical diseases Heath, C. J., Grossi-Soyster, E. N., Ndenga, B. A., Mutuku, F. M., Sahoo, M. K., Ngugi, H. N., Nbakaya, J. O., Siema, P., Kitron, U., Zahiri, N., Hortion, J., Waggoner, J. J., King, C. H., Pinsky, B. A., LaBeaud, A. D. 2020; 14 (6): e0008362

  Abstract

  Arboviruses are among the most important emerging pathogens due to their increasing public health impact. In Kenya, continued population growth and associated urbanization are conducive to vector spread in both urban and rural environments, yet mechanisms of viral amplification in vector populations is often overlooked when assessing risks for outbreaks. Thus, the characterization of local arbovirus circulation in mosquito populations is imperative to better inform risk assessments and vector control practices. Aedes species mosquitoes were captured at varying stages of their life cycle during different seasons between January 2014 and May 2016 at four distinct sites in Kenya, and tested for chikungunya (CHIKV), dengue (DENV) and Zika (ZIKV) viruses by RT-PCR. CHIKV was detected in 45 (5.9%) and DENV in 3 (0.4%) mosquito pools. No ZIKV was detected. Significant regional variation in prevalence was observed, with greater frequency of CHIKV on the coast. DENV was detected exclusively on the coast. Both viruses were detected in immature mosquitoes of both sexes, providing evidence of transovarial transmission of these arboviruses in local mosquitoes. This phenomenon may be driving underlying viral maintenance that may largely contribute to periodic re-emergence among humans in Kenya.

  View details for DOI 10.1371/journal.pntd.0008362

  View details for PubMedID 32559197

• Clinical Impact of Metagenomic Next-Generation Sequencing of Plasma Cell-Free DNA for the Diagnosis of Infectious Diseases: A Multicenter Retrospective Cohort Study. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America Hogan, C. A., Yang, S., Garner, O. B., Green, D. A., Gomez, C. A., Dien Bard, J., Pinsky, B. A., Banaei, N. 2020

  Abstract

  Metagenomic next-generation sequencing (mNGS) of plasma cell-free DNA has emerged as an attractive diagnostic modality allowing broad-range pathogen detection, noninvasive sampling, and earlier diagnosis. However, little is known about its real-world clinical impact as used in routine practice.We performed a retrospective cohort study of all patients for whom plasma mNGS (Karius test) was performed for all indications at 5 U.S. institutions over 1.5 years. Comprehensive chart review was performed, and standardized assessment of clinical impact of the mNGS based on the treating team's interpretation of Karius results and patient management was established.A total of 82 Karius tests were evaluated, from 39 (47.6%) adults and 43 (52.4%) children and a total of 53 (64.6%) immunocompromised patients. Karius positivity rate was 50/82 (61.0%), with 25 (50.0%) showing two or more organisms (range, 2-8). The Karius test results led to positive impact in 6 (7.3%), negative impact in 3 (3.7%), no impact in 71 (86.6%), and was indeterminate in 2 (2.4%). Cases with positive Karius result and clinical impact involved bacteria and/or fungi but not DNA viruses or parasites. In 10 patients who underwent 16 additional repeated tests, only one was associated with clinical impact.The real-world impact of the Karius test as currently used in routine clinical practice is limited. Further studies are needed to identify high-yield patient populations, define the complementary role of mNGS to conventional microbiological methods and how best to integrate mNGS into current testing algorithms.

  View details for DOI 10.1093/cid/ciaa035

  View details for PubMedID 31942944

• Metagenomic Next-Generation Sequencing for the Identification and Quantitation of Transplant-Related DNA Viruses. Journal of clinical microbiology Carpenter, M. L., Tan, S. K., Watson, T., Bacher, R., Nagesh, V., Watts, A., Bentley, G., Weber, J., Huang, C., Sahoo, M. K., Hinterwirth, A., Doan, T., Carter, T., Dong, Q., Gourguechon, S., Harness, E., Kermes, S., Radhakrishnan, S., Wang, G., Quiroz-Zarate, A., Ching, J., Pinsky, B. A. 2019

  Abstract

  Infections with DNA viruses are frequent causes of morbidity and mortality in transplant recipients. This study describes the analytical and clinical performance characteristics of the Arc Bio Galileo Pathogen Solution, an all-inclusive metagenomic next-generation sequencing (mNGS) reagent and bioinformatics pipeline that allows the simultaneous quantitation of 10 transplant-related dsDNA viruses (ADV, BKV, CMV, EBV, HHV-6A, HHV-6B, HSV-1, HSV-2, JCV, and VZV). The mNGS 95% limit of detection ranged from 14 international units (IU)/mL (HHV-6) to 191 copies/mL (BKV), and the lower limit of quantitation ranged from 442 IU/mL (EBV) to 661 copies/mL (VZV). Evaluation of 50 residual plasma samples with at least one DNA virus detected in prior clinical testing showed a total percent agreement of mNGS and qPCR of 89.2% (306/343), with a kappa statistic of 0.725. The positive percent agreement was 84.9% (73/86) and negative percent agreement was 90.7% (233/257). Furthermore, mNGS detected seven subsequently confirmed co-infections that were not initially requested by qPCR. Passing-Bablok regression revealed a regression line of Y = 0.953*X + 0.075 [95% CI of the slope (0.883 to 1.011) and intercept (-0.100 to 0.299)], and Bland-Altman analysis (mNGS - qPCR) showed a slight positive bias (0.28 log10 concentration, 95% limits of agreement of -0.62 to 1.18). In conclusion, the mNGS-based Galileo pipeline demonstrates comparable analytical and clinical performance to qPCR for transplant-related DNA viruses.

  View details for DOI 10.1128/JCM.01113-19

  View details for PubMedID 31554674

• Prospective Evaluation of the Vela Diagnostics Next-Generation Sequencing Platform for HIV-1 Genotypic Resistance Testing. The Journal of molecular diagnostics : JMD Weber, J., Volkova, I., Sahoo, M. K., Tzou, P. L., Shafer, R. W., Pinsky, B. A. 2019

  Abstract

  Genotypic antiretroviral drug resistance testing is a critical component of the global efforts to control the HIV-1 epidemic. This study investigates the semi-automated, next-generation sequencing (NGS)-based Vela Diagnostics Sentosa SQ HIV-1 Genotyping Assay in a prospective cohort of HIV-1 infected patients. Two-hundred and sixty-nine samples were successfully sequenced by both NGS and Sanger sequencing. Among the 261 protease/reverse transcriptase (PR/RT) sequences, a mean of 0.37 drug resistance mutations were identified by both Sanger and NGS, 0.08 by NGS alone, and 0.03 by Sanger alone. Among the 50 integrase sequences, a mean of 0.3 drug resistance mutations were detected by both Sanger and NGS, and 0.08 by NGS alone. NGS estimated higher levels of drug resistance to one or more antiretroviral drugs for 6.5% of PR/RT sequences and 4.0% of integrase sequences, whereas Sanger estimated higher levels of drug resistance for 3.8% of PR/RT sequences. Although the samples successfully sequenced by the Sentosa SQ HIV Genotyping Assay demonstrated similar predicted resistance compared with Sanger, 44% of Sentosa runs failed QC requiring 17 additional runs. This semi-automated NGS-based assay may aid in HIV-1 genotypic drug resistance testing, though numerous QC issues were observed when this platform was used in a clinical laboratory setting. With additional refinement, the Sentosa SQ HIV-1 Genotyping Assay may contribute to the global efforts to control HIV-1.

  View details for DOI 10.1016/j.jmoldx.2019.06.003

  View details for PubMedID 31382033

• Unbiased Pathogen Detection and Host Gene Profiling for Conjunctivitis OPHTHALMOLOGY Lalitha, P., Seitzman, G. D., Kotecha, R., Hinterwirth, A., Chen, C., Zhong, L., Cummings, S., Lebas, E., Sahoo, M. K., Pinsky, B. A., Lietman, T. M., Thuy Doan 2019; 126 (8): 1090?94

  View details for DOI 10.1016/j.ophtha.2019.03.039

  View details for Web of Science ID 000475845500014

• Evaluating for Human Herpesvirus 6 in the Liver Explants of Children With Liver Failure of Unknown Etiology JOURNAL OF INFECTIOUS DISEASES Yang, C. H., Sahoo, M. K., Fitzpatrick, M., Lau, A. H., Pinsky, B. A., Martinez, O. M. 2019; 220 (3): 361?69

  View details for DOI 10.1093/infdis/jiy644

  View details for Web of Science ID 000477595700004

• Impact of Pretransplant Donor BK Viruria in Kidney Transplant Recipients JOURNAL OF INFECTIOUS DISEASES Tan, S. K., Huang, C., Sahoo, M. K., Weber, J., Kurzer, J., Stedman, M. R., Concepcion, W., Gallo, A. E., Alonso, D., Srinivas, T., Storch, G. A., Subramanian, A. K., Tan, J. C., Pinsky, B. A. 2019; 220 (3): 370?76

  View details for DOI 10.1093/infdis/jiz114

  View details for Web of Science ID 000477595700005

• Multiplex Solid-Phase Melt Curve Analysis for the Point-of-Care Detection of HIV-1 Drug Resistance JOURNAL OF MOLECULAR DIAGNOSTICS Clutter, D. S., Mazarei, G., Sinha, R., Manasa, J., Nouhin, J., LaPrade, E., Bolouki, S., Tzou, P. L., Hannita-Hui, J., Sahoo, M. K., Kuimelis, P., Kuimelis, R. G., Pinsky, B. A., Schoolnik, G. K., Hassibi, A., Shafer, R. W. 2019; 21 (4): 580?92

  View details for DOI 10.1016/j.jmoldx.2019.02.005

  View details for Web of Science ID 000475412100005

• Community-based self-collected human papillomavirus screening in rural Zimbabwe. BMC public health Fitzpatrick, M. B., El-Khatib, Z., Katzenstein, D., Pinsky, B. A., Chirenje, Z. M., McCarty, K. 2019; 19 (Suppl 1): 603

  Abstract

  BACKGROUND: In low- and middle-income countries (LMIC), women have limited access to and uptake of cervical cancer screening. Delayed diagnosis leads to poorer outcomes and early mortality, and continues to impede cancer control disproportionately in LMIC. Integrating self-collected, community-based screening for High Risk-Human Papilloma Virus (HR-HPV) into existent HIV programs is a potential screening method to identify women at high risk for developing high-risk cervical lesions.METHODS: We implemented community-based cross-sectional study on self-collection HR-HPV screening in conjunction with existing community outreach models for the distribution of antiretroviral therapy (ART) and the World Health Organization Expanded Program on Immunization (EPI) outreach in villages in rural Zimbabwe from January 2017 through May 2017.RESULTS: Overall, there was an 82% response rate: 70% of respondents participated in self-collection and 12% were ineligible for the study (inclusion criteria: age 30-65, not pregnant, with an intact uterus). Women recruited in the first 2-3months of the study had more opportunities to participate and therefore significantly higher participation: 81% participation (additional 11% ineligible), while those with fewer opportunities also had lower participation: 63% (additional 13% ineligible) (p<0.001). Some village outreach centers (N=5/12) had greater than 89% participation.CONCLUSIONS: Integration of HR-HPV screening into existing community outreach models for HIV and immunizations could facilitate population-based screening to scale cancer control and prevention programs in sub-Saharan Africa. Community/village health workers (CHW/VHW) and village outreach programs offer a potential option for cervical cancer screening programs to move towards improving access of sexual and reproductive health resources for women at highest risk.

  View details for DOI 10.1186/s12889-019-6810-5

  View details for PubMedID 31138174

• Evaluation of the Aptima HCV Quant Dx Assay Using Serum and Dried Blood Spots JOURNAL OF CLINICAL MICROBIOLOGY Weber, J., Sahoo, M. K., Taylor, N., Shi, R., Pinsky, B. A. 2019; 57 (4)

  View details for DOI 10.1128/JCM.00030-19

  View details for Web of Science ID 000462714100002

• Dual-target, real-time PCR for the diagnosis of intraocular Toxoplasma gondii infections BRITISH JOURNAL OF OPHTHALMOLOGY Gomez, C. A., Sahoo, M. K., Kahn, G., Zhong, L., Montoya, J. G., Pinsky, B. A., Thuy Doan 2019; 103 (4): 569?72

  View details for DOI 10.1136/bjophthalmol-2018-313064

  View details for Web of Science ID 000471871400024

• A 20-Gene Set Predictive of Progression to Severe Dengue. Cell reports Robinson, M., Sweeney, T. E., Barouch-Bentov, R., Sahoo, M. K., Kalesinskas, L., Vallania, F., Sanz, A. M., Ortiz-Lasso, E., Albornoz, L. L., Rosso, F., Montoya, J. G., Pinsky, B. A., Khatri, P., Einav, S. 2019; 26 (5): 1104

  Abstract

  There is a need to identify biomarkers predictive of severe dengue. Single-cohort transcriptomics has not yielded generalizable results or parsimonious, predictive gene sets. We analyzed blood samples of dengue patients from seven gene expression datasets (446 samples, five countries) using an integrated multi-cohort analysis framework and identified a 20-gene set that predicts progression to severe dengue. We validated the predictive power of this 20-gene set in three retrospective dengue datasets (84 samples, three countries) and a prospective Colombia cohort (34 patients), with an area under the receiver operating characteristic curve of 0.89, 100% sensitivity, and 76% specificity. The 20-gene dengue severity scores declined during the diseasecourse, suggesting an infection-triggered host response. This 20-gene set is strongly associated with the progression to severe dengue and represents a predictive signature, generalizable across ages, host genetic factors, and virus strains, with potential implications for the development of a host response-based dengue prognostic assay.

  View details for PubMedID 30699342

• Trends in the Molecular Epidemiology and Genetic Mechanisms of Transmitted Human Immunodeficiency Virus Type 1 Drug Resistance in a Large US Clinic Population CLINICAL INFECTIOUS DISEASES Rhee, S., Clutter, D., Fessel, W., Klein, D., Slome, S., Pinsky, B. A., Marcus, J. L., Hurley, L., Silverberg, M. J., Pond, S., Shafer, R. W. 2019; 68 (2): 213?21

  View details for DOI 10.1093/cid/ciy453

  View details for Web of Science ID 000459636700010

• Molecular profiling of clear cell adenocarcinoma of the urinary tract. Virchows Archiv : an international journal of pathology Lin, C. Y., Saleem, A., Stehr, H., Zehnder, J. L., Pinsky, B. A., Kunder, C. A. 2019

  Abstract

  Clear cell adenocarcinoma (CCA) of the urinary tract is a rare type of malignancy whose molecular profiles remain undefined. Here we reported an integrated clinicopathologic and molecular profiling analysis of four cases of clear cell adenocarcinoma arising in the urethra or the bladder. Utilizing a clinically validated 130-gene exon-sequencing assay, we identified recurrent pathogenic PIK3CA (p. E545K) and KRAS (p.G12D) variants in three of four (75%) of the cases. In addition, an APC variant (P.S2310X), a TP53 variant (p.R273C), and a MYC amplification event were identified. The only CCA case without either PIK3CA or KRAS variants has a distinct pathogenesis through BK virus, demonstrated by positive BK virus PCR and SV40 immunohistochemistry. The novel finding of recurrent variants in the PI3K/AKT/mTOR pathway provides not only insights into oncogenesis but also potential clinical therapeutic targets for patients with clear cell adenocarcinoma of the urinary tract.

  View details for DOI 10.1007/s00428-019-02634-5

  View details for PubMedID 31372739

• Investigation of preanalytical variables impacting pathogen cell-free DNA in blood and urine. Journal of clinical microbiology Murugesan, K., Hogan, C. A., Palmer, Z., Reeve, B., Theron, G., Andama, A., Somoskovi, A., Steadman, A., Madan, D., Andrews, J., Croda, J., Sahoo, M. K., Cattamanchi, A., Pinsky, B. A., Banaei, N. 2019

  Abstract

  Pathogen cell-free DNA (pcfDNA) in blood and urine is an attractive biomarker; however, the impact of preanalytical factors is not well understood.Blood and urine samples from healthy donors spiked with cfDNA from Mycobacterium tuberculosis, Salmonella enterica, Aspergillus fumigatus and EBV, and samples from tuberculosis patients were used to evaluate the impact of blood collection tube, urine preservative, processing delay, processing method, freezing and thawing, and sample volume on pcfDNA. PCR cycle threshold (CT) was used to measure amplifiable cfDNA.In spiked samples, median CT for M. tuberculosis, S. enterica, and EBV cfDNA was significantly lower in blood collected in K2EDTA than Streck and PAXgene blood collection tubes, and significantly lower in EDTA-urine than Streck-urine. Blood and urine samples from TB patients preserved with K2EDTA and Tris-EDTA, respectively, showed significantly lower median M. tuberculosis CT compared with Streck blood collection tube and urine preservative. Processing delay increased median pathogen CT for Streck and PAXgene but not K2EDTA blood samples, and for urine preserved with Streck reagent but not EDTA. Double spin compared with single spin plasma separation increased median pathogen CT regardless of blood collection tube. No differences were observed between whole urine and supernatant, and between fresh and thawed plasma and urine after 24 weeks at -80 °C. Larger plasma and urine volume in contrived and patient samples showed a significantly lower median M. tuberculosis CT. These findings suggest large volume single spin K2EDTA-plasma and EDTA-whole urine with up to 24-hour processing delay may optimize pcfDNA detection.

  View details for DOI 10.1128/JCM.00782-19

  View details for PubMedID 31511335

• Comparison of Transcription-Mediated Amplification and Real-Time PCR Assays for Hepatitis B Virus DNA Quantitation in Serum. The journal of applied laboratory medicine Weber, J., Sahoo, M. K., Taylor, N., Uy, E., Shi, R. Z., Pinsky, B. A. 2019; 4 (3): 383?90

  Abstract

  The quantification of hepatitis B (HBV) DNA in serum is critical to identify patients requiring antiviral therapy and to monitor the response to treatment.This study describes the evaluation of the Aptima HBV Quant Dx assay (Aptima HBV) performed on the automated Panther system.Aptima HBV was linear from 1.70 to 7.70 log10 IU/mL with a commercial reference panel, as well as clinical specimens representing genotypes B and C, and total imprecision, as measured by the percentage coefficient of variation (%CV) at 2.0 log10 IU/mL was <10%. The specificity of Aptima HBV was 94.7% (126/133) and 96.6% (84/87) for serum specimens from individuals without HBV exposure and individuals with resolved HBV infection, respectively. The qualitative agreement and quantitative accuracy of Aptima HBV was compared to the COBAS AmpliPrep/COBAS TaqMan HBV Test v2.0 (CAP/CTM). Overall agreement was 90.8% (187/206) with a ? statistic of 0.708 (standard error, 0.063; 95% CI, 0.585-0.831). Passing-Bablok regression revealed a regression line of y = 0.953x + 0.075 (95% CI of the slope, 0.883-1.011; intercept, -0.100 to 0.299), and Bland-Altman analysis (Aptima - CAP/CTM) showed a slight negative bias (-0.054 log10 IU/mL, and 95% limits of agreement of -1.093 to 0.984).The Aptima HBV test affords a suitable alternative to CAP/CTM for serum virus load testing and provides a key component of the diagnostic algorithm for the global eradication of viral hepatitis.

  View details for DOI 10.1373/jalm.2019.029058

  View details for PubMedID 31659075

• Real-time RT-PCR for the detection and quantitation of Oropouche virus. Diagnostic microbiology and infectious disease Rojas, A., Stittleburg, V., Cardozo, F., Bopp, N., Cantero, C., López, S., Bernal, C., Mendoza, L., Aguilar, P., Pinsky, B. A., Guillén, Y., Páez, M., Waggoner, J. J. 2019: 114894

  Abstract

  Oropouche virus (OROV) causes an acute, systemic febrile illness, and in certain regions of South America, this represents the second most common human arboviral infection after dengue virus. A new real-time RT-PCR was developed for OROV and reassortant species. The new OROV rRT-PCR proved linear across 6-7 orders of magnitude with a lower limit of 95% detection of 5.6-10.8 copies/?L. Upon testing dilutions of OROV and Iquitos virus reference genomic RNA, all dilutions with >10 copies/?L were detected in both the OROV rRT-PCR and a comparator molecular assay, but the OROV rRT-PCR detected more samples with ?10 copies/?L (8/14 vs 0/13, respectively, P?=?0.002). In a set of 100 acute-phase clinical samples from Paraguay patients with a suspected arboviral illness, no patients tested positive for OROV RNA using either assay. The OROV rRT-PCR provides a sensitive molecular assay for the study of this important yet neglected tropical arboviral infection.

  View details for DOI 10.1016/j.diagmicrobio.2019.114894

  View details for PubMedID 31727377

• Characterization of dengue cases among patients with an acute illness, Central Department, Paraguay. PeerJ Rojas, A., Cardozo, F., Cantero, C., Stittleburg, V., López, S., Bernal, C., Gimenez Acosta, F. E., Mendoza, L., Pinsky, B. A., Arévalo de Guillén, I., Páez, M., Waggoner, J. 2019; 7: e7852

  Abstract

  In 2018, Paraguay experienced a large dengue virus (DENV) outbreak. The primary objective of this study was to characterize dengue cases in the Central Department, where the majority of cases occur, and identify factors associated with DENV infection.Patients were enrolled from January-May 2018 if they presented with a suspected arboviral illness. Acute-phase specimens (?8 days after symptom onset) were tested using rRT-PCR, a rapid diagnostic test for DENV nonstructural protein 1 (NS1) and anti-DENV IgM and IgG, and ELISA for IgG against NS1 from Zika virus (ZIKV).A total of 231 patients were enrolled (95.2% adults) at two sites: emergency care and an outpatient clinical site. Patients included 119 (51.5%) dengue cases confirmed by rRT-PCR (n = 115, 96.6%) and/or the detection of NS1 and anti-DENV IgM (n = 4, 3.4%). DENV-1 was the predominant serotype (109/115, 94.8%). Epidemiologically, dengue cases and non-dengue cases were similar, though dengue cases were less likely to reside in a house/apartment or report a previous dengue case. Clinical and laboratory findings associated with dengue included red eyes, absence of sore throat, leucopenia and thrombocytopenia. At an emergency care site, 26% of dengue cases (26/100) required hospitalization. In univariate analysis, hospitalization was associated with increased viral load, anti-DENV IgG, and thrombocytopenia. Among dengue cases that tested positive for IgG against ZIKV NS1, the odds of DENV NS1 detection in the acute phase were decreased 10-fold (OR 0.1, 0.0-0.3).Findings from a predominantly adult population demonstrate clinical and laboratory factors associated with DENV infections and the potential severity of dengue in this group. The combination of viral load and specific IgG antibodies warrant further study as a prognostic to identify patients at risk for severe disease.

  View details for DOI 10.7717/peerj.7852

  View details for PubMedID 31616598

  View details for PubMedCentralID PMC6790102

• Risk prediction for severe disease and better diagnostic accuracy in early dengue infection; the Colombo dengue study. BMC infectious diseases Sigera, P. C., Amarasekara, R., Rodrigo, C., Rajapakse, S., Weeratunga, P., De Silva, N. L., Huang, C. H., Sahoo, M. K., Pinsky, B. A., Pillai, D. R., Tissera, H. A., Jayasinghe, S., Handunnetti, S., Fernando, S. D. 2019; 19 (1): 680

  Abstract

  A major challenge in dengue management in resource limited settings is the confirmation of diagnosis. Clinical features of dengue often overlap with other infections and molecular diagnostic tools are not readily accessible to clinicians at hospitals. In addition, the prediction of plasma leakage in dengue is also difficult. Hematocrit level and ultrasound scans (combined with clinical parameters) are helpful to detect plasma leakage once it has happened, not before.Colombo Dengue Study (CDS) is a prospective cohort study of clinically suspected adult dengue patients recruited from the National hospital of Sri Lanka (within the first 3?days of fever) that aimed to a) identify clinical and basic laboratory test parameters to differentiate dengue from non-dengue fever, b) evaluate the comparative efficacy of loop-mediated isothermal amplification (LAMP) for dengue diagnosis (vs. NS1 antigen test and RT-qPCR) and c) identify early associations that are predictive of plasma leakage or severe dengue. The basic laboratory tests considered here included hematological parameters, serum biochemistry and inflammatory markers.Only 70% of clinically suspected patients were confirmed as having dengue by either the NS1 antigen test or RT-qPCR. On a Bayesian latent class model which assumes no "gold standard", LAMP performed equally or better than RT-qPCR and NS1 antigen test respectively. When confirmed dengue patients were compared with others, the earlier group had significantly lower lymphocyte counts and higher aspartate aminotransferase levels (AST) within the first 3?days of fever. Confirmed dengue patients with plasma leakage had a lower mean age and a higher median baseline AST level compared to those without plasma leakage (p?

  View details for DOI 10.1186/s12879-019-4304-9

  View details for PubMedID 31370795

• Virus-inclusive single-cell RNA sequencing reveals the molecular signature of progression to severe dengue. Proceedings of the National Academy of Sciences of the United States of America Zanini, F., Robinson, M. L., Croote, D., Sahoo, M. K., Sanz, A. M., Ortiz-Lasso, E., Albornoz, L. L., Rosso, F., Montoya, J. G., Goo, L., Pinsky, B. A., Quake, S. R., Einav, S. 2018

  Abstract

  Dengue virus (DENV) infection can result in severe complications. However, the understanding of the molecular correlates of severity is limited, partly due to difficulties in defining the peripheral blood mononuclear cells (PBMCs) that contain DENV RNA in vivo. Accordingly, there are currently no biomarkers predictive of progression to severe dengue (SD). Bulk transcriptomics data are difficult to interpret because blood consists of multiple cell types that may react differently to infection. Here, we applied virus-inclusive single-cell RNA-seq approach (viscRNA-Seq) to profile transcriptomes of thousands of single PBMCs derived early in the course of disease from six dengue patients and four healthy controls and to characterize distinct leukocyte subtypes that harbor viral RNA (vRNA). Multiple IFN response genes, particularly MX2 in naive B cells and CD163 in CD14+ CD16+ monocytes, were up-regulated in a cell-specific manner before progression to SD. The majority of vRNA-containing cells in the blood of two patients who progressed to SD were naive IgM B cells expressing the CD69 and CXCR4 receptors and various antiviral genes, followed by monocytes. Bystander, non-vRNA-containing B cells also demonstrated immune activation, and IgG1 plasmablasts from two patients exhibited clonal expansions. Lastly, assembly of the DENV genome sequence revealed diversity at unexpected sites. This study presents a multifaceted molecular elucidation of natural dengue infection in humans with implications for any tissue and viral infection and proposes candidate biomarkers for prediction of SD.

  View details for PubMedID 30530648

• Deep Sequencing Prompts the Modification of a Real-time RT-PCR for the Serotype-Specific Detection of Polioviruses. Journal of virological methods Holubar, M., Sahoo, M. K., Huang, C., Mohamed-Hadley, A., Liu, Y., Waggoner, J. J., Troy, S. B., Garcia-Garcia, L., Ferreyra-Reyes, L., Maldonado, Y., Pinsky, B. A. 2018

  Abstract

  Polioviruses are members of the Enterovirus C species and asymptomatic fecal shedding allows for their transmission and persistence in a community, as well as the emergence of vaccine-derived polioviruses. Using three serotype-specific real-time RT-PCR (rRT-PCR) assays, the shedding and circulation of oral poliovirus vaccine (OPV) strains was previously investigated in a prospective cohort of MFexican children, their contacts, and nearby sewage. Subsequently, a deep sequencing approach targeting the P1 genomic region was applied to characterize OPV strains previously detected by rRT-PCR. Amplifiable RNA was obtained for sequencing from 40.3% (58/144) of stool samples and 71.4% (15/21) of sewage using nucleic acids extracted directly from primary rRT-PCR-positive specimens. Sequencing detected one or more OPV serotypes in 62.1% (36/58) of stool and 53.3% (8/15) of sewage samples. All stool and sewage samples in which poliovirus was not detected by deep sequencing contained at least one non-polio enterovirus C (NPEV-C) strain. To improve screening specificity, a modified, two-step, OPV serotype-specific multiplex rRT-PCR was evaluated. In stool specimens, the overall agreement between the original assays and the multiplex was 70.3%. By serotype, the overall agreement was 95.7% for OPV serotype-1 (S1), 65.6% for S2, and 96.1% for S3. Furthermore, most original rRT-PCR positive/multiplex rRT-PCR negative results were collected in the summer and fall months, consistent with NPEV-C circulation patterns. In conclusion, this deep sequencing approach allowed for the characterization of OPV sequences directly from clinical samples and facilitated the implementation of a more specific multiplex rRT-PCR for OPV detection and serotyping.

  View details for PubMedID 30447245

• A Case Report of Pediatric Clear Cell Carcinoma of the Urinary Bladder Associated With Polyomavirus AJSP-REVIEWS AND REPORTS Saleem, A., Brown, R. A., Higgins, J. T., Troxell, M. L., Kunder, C. A., Pinsky, B. A., Zambrano, E., Kao, C. 2018; 23 (6): 291?95

  View details for DOI 10.1097/PCR.0000000000000284

  View details for Web of Science ID 000457629000012

• Native Kidney Cytomegalovirus Nephritis and Cytomegalovirus Prostatitis in a Kidney Transplant Recipient. Transplant infectious disease : an official journal of the Transplantation Society Tan, S. K., Cheng, X. S., Kao, C., Weber, J., Pinsky, B. A., Gill, H. S., Busque, S., Subramanian, A. K., Tan, J. C. 2018: e12998

  Abstract

  We present a case of cytomegalovirus (CMV) native kidney nephritis and prostatitis in a CMV D+/R- kidney transplant recipient who had completed six months of CMV prophylaxis four weeks prior to the diagnosis of genitourinary CMV disease. The patient had a history of benign prostatic hypertrophy and urinary retention that required self-catheterization to relieve high post-voiding residual volumes. At 7 months post-transplant, he was found to have a urinary tract infection, moderate hydronephrosis of the transplanted kidney, and severe hydroureteronephrosis of the native left kidney and ureter, and underwent native left nephrectomy and transurethral resection of the prostate. Histopathologic examination of kidney and prostate tissue revealed CMV inclusions consistent with invasive CMV disease. This case highlights that CMV may extend beyond the kidney allograft to involve other parts of the genitourinary tract, including the native kidneys and prostate. Furthermore, we highlight the tissue-specific risk factors that preceded CMV tissue invasion. In addition to concurrent diagnoses, health care providers should have a low threshold for considering late-onset CMV disease in high-risk solid organ transplant recipients presenting with signs and symptoms of genitourinary tract pathology. This article is protected by copyright. All rights reserved.

  View details for PubMedID 30203504

• Poor Immunogenicity, Not Vaccine Strain Egg Adaptation, May Explain the Low H3N2 Influenza Vaccine Effectiveness in 2012-2013 CLINICAL INFECTIOUS DISEASES Cobey, S., Gouma, S., Parkhouse, K., Chambers, B. S., Ertl, H. C., Schmader, K. E., Halpin, R. A., Lin, X., Stockwell, T. B., Das, S. R., Landon, E., Tesic, V., Youngster, I., Pinsky, B. A., Wentworth, D. E., Hensley, S. E., Grad, Y. H. 2018; 67 (3): 327?33

  Abstract

  Influenza vaccination aims to prevent infection by influenza virus and reduce associated morbidity and mortality; however, vaccine effectiveness (VE) can be modest, especially for subtype A(H3N2). Low VE has been attributed to mismatches between the vaccine and circulating influenza strains and to the vaccine's elicitation of protective immunity in only a subset of the population. The low H3N2 VE in the 2012-2013 season was attributed to egg-adaptive mutations that created antigenic mismatch between the actual vaccine strain (IVR-165) and both the intended vaccine strain (A/Victoria/361/2011) and the predominant circulating strains (clades 3C.2 and 3C.3).We investigated the basis of low VE in 2012-2013 by determining whether vaccinated and unvaccinated individuals were infected by different viral strains and by assessing the serologic responses to IVR-165, A/Victoria/361/2011, and 3C.2 and 3C.3 strains in an adult cohort before and after vaccination.We found no significant genetic differences between the strains that infected vaccinated and unvaccinated individuals. Vaccination increased titers to A/Victoria/361/2011 and 3C.2 and 3C.3 representative strains as much as to IVR-165. These results are consistent with the hypothesis that vaccination boosted cross-reactive immune responses instead of specific responses against unique vaccine epitopes. Only approximately one-third of the cohort achieved a ?4-fold increase in titer.In contrast to analyses based on ferret studies, low H3N2 VE in 2012-2013 in adults does not appear to be due to egg adaptation of the vaccine strain. Instead, low VE might have been caused by low vaccine immunogenicity in a subset of the population.

  View details for PubMedID 29471464

  View details for PubMedCentralID PMC6051447

• FOXP3-positive T-cell lymphomas in non-HTLV1 carriers include ALK-negative anaplastic large cell lymphoma: expanding the Spectrum of T-cell lymphomas with regulatory phenotype. Human pathology Ferreira, C. R., Zhao, S., Sahoo, M. K., Pinsky, B., Weber, J., Lage, L. A., Pereira, J., Zerbini, M. C., Natkunam, Y. 2018

  Abstract

  Forkhead box P3 (FOXP3) is a specific marker for regulatory T-cells (Tregs). We report 6 cases of T-cell lymphomas with Treg phenotype based on diffuse positivity for FOXP3 in tumor cells. The patients showed a median age of 56years with a male predominance. Sites of disease included lymph nodes (4), skin (2), subcutaneous tissue (1) and bone marrow (1). All cases showed monomorphic large cells, some with Hodgkin-like or anaplastic cells. All cases expressed pan T cell markers and lacked cytotoxic markers; one case showed diffuse PD1 staining. Only one case harbored human T-lymphotrophic virus (HTLV)-1 DNA within tumor cells and was classified as adult T-cell leukemia/lymphoma (ATLL). Among 5 HTLV1-negative cases, 3 were classified as peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS) and 2 fulfilled criteria for ALK-negative anaplastic large cell lymphoma (ALCL) with diffuse and strong CD30 positivity. We concluded that Treg phenotype may be rarely seen in HTLV1-negative cases, such as PTCL, NOS and ALK-negative ALCL. Our findings expand the spectrum of T-cell lymphomas with regulatory phenotype and suggest that consideration should be given to HTLV1 DNA testing in the appropriate clinical setting to rule out ATLL.

  View details for PubMedID 29898383

• Comparison of an In Vitro Diagnostic Next-Generation Sequencing Assay with Sanger Sequencing for HIV-1 Genotypic Resistance Testing JOURNAL OF CLINICAL MICROBIOLOGY Tzou, P. L., Ariyaratne, P., Varghese, V., Lee, C., Rakhmanaliev, E., Villy, C., Yee, M., Tan, K., Michel, G., Pinsky, B. A., Shafer, R. W. 2018; 56 (6)

  Abstract

  The ability of next-generation sequencing (NGS) technologies to detect low frequency HIV-1 drug resistance mutations (DRMs) not detected by dideoxynucleotide Sanger sequencing has potential advantages for improved patient outcomes. We compared the performance of an in vitro diagnostic (IVD) NGS assay, the Sentosa SQ HIV genotyping assay for HIV-1 genotypic resistance testing, with Sanger sequencing on 138 protease/reverse transcriptase (RT) and 39 integrase sequences. The NGS assay used a 5% threshold for reporting low-frequency variants. The level of complete plus partial nucleotide sequence concordance between Sanger sequencing and NGS was 99.9%. Among the 138 protease/RT sequences, a mean of 6.4 DRMs was identified by both Sanger and NGS, a mean of 0.5 DRM was detected by NGS alone, and a mean of 0.1 DRM was detected by Sanger sequencing alone. Among the 39 integrase sequences, a mean of 1.6 DRMs was detected by both Sanger sequencing and NGS and a mean of 0.15 DRM was detected by NGS alone. Compared with Sanger sequencing, NGS estimated higher levels of resistance to one or more antiretroviral drugs for 18.2% of protease/RT sequences and 5.1% of integrase sequences. There was little evidence for technical artifacts in the NGS sequences, but the G-to-A hypermutation was detected in three samples. In conclusion, the IVD NGS assay evaluated in this study was highly concordant with Sanger sequencing. At the 5% threshold for reporting minority variants, NGS appeared to attain a modestly increased sensitivity for detecting low-frequency DRMs without compromising sequence accuracy.

  View details for PubMedID 29618499

  View details for PubMedCentralID PMC5971553

• High human herpesvirus 6 viral load in pediatric allogeneic hematopoietic stem cell transplant patients is associated with detection in end organs and high mortality PEDIATRIC TRANSPLANTATION Winestone, L. E., Punn, R., Tamaresis, J. S., Buckingham, J., Pinsky, B. A., Waggoner, J. J., Kharbanda, S. 2018; 22 (2)

  Abstract

  Human Herpes Virus 6 (HHV-6) reactivation occurs in approximately half of patients following allogeneic hematopoietic stem cell transplant (HSCT). While encephalitis and delayed engraftment are well-documented complications of HHV-6 following HSCT, the extent to which HHV-6 viremia causes disease in children is controversial. We performed a retrospective review of HHV-6 reactivation and possible manifestations in pediatric allogeneic HSCT patients at a single institution. Of 89 children and young adults who underwent allogeneic HSCT over a three-and-a-half-year period, 34 patients reactivated HHV-6 early post-transplant. Unrelated donor stem cell source and lack of antiviral prophylaxis were risk factors for the development of HHV-6 viremia. Viremia correlated with the presence of acute graft-versus-host disease, but not chronic graft-versus-host disease. We identified two subgroups within the viremic patients-a high-risk viremic and tissue-positive group that reactivated HHV-6 and had suspected end-organ disease and a low-risk viremic but asymptomatic group that reactivated HHV-6 but did not exhibit symptoms or signs of end-organ disease. Peak viral load was found to be strongly associated with mortality. Prospective studies in larger numbers of patients are needed to further investigate the role of HHV-6 in causing symptomatic end-organ disease as well as the association of viral load with mortality.

  View details for PubMedID 29181879

  View details for PubMedCentralID PMC5820136

• Internally Controlled, Multiplex Real-Time Reverse Transcription PCR for Dengue Virus and Yellow Fever Virus Detection AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE Rojas, A., Diagne, C. T., Stittleburg, V. D., Mohamed-Hadley, A., Arevalo de Guillen, Y., Balmaseda, A., Faye, O., Faye, O., Sall, A. A., Harris, E., Pinsky, B. A., Waggoner, J. J. 2018; 98 (6): 1833?36

  Abstract

  The differential diagnosis of dengue virus (DENV) and yellow fever virus (YFV) infections in endemic areas is complicated by nonspecific early clinical manifestations. In this study, we describe an internally controlled, multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) for the detection of DENV and YFV. The DENV-YFV assay demonstrated specific detection and had a dynamic range of 2.0-8.0 log10 copies/?L of eluate for each DENV serotype and YFV. Clinical performance was similar to a published pan-DENV assay: 48/48 acute-phase samples from dengue cases were detected in both assays. For YFV detection, mock samples were prepared with nine geographically diverse YFV isolates over a range of concentrations. The DENV-YFV assay detected 62/65 replicates, whereas 54/65 were detected using a reference YFV rRT-PCR. Given the reemergence of DENV and YFV in areas around the world, the DENV-YFV assay should be a useful tool to narrow the differential diagnosis and provide early case detection.

  View details for PubMedID 29611509

• ARBOVIRUS AND MALARIA CO-INFECTIONS AMONG FEBRILE KENYAN CHILDREN Shah, M., Sahoo, M., Krystosik, A., Mutuku, F., Ndenga, B., Otuka, V., Ronga, C., Chebii, P., Maina, P., Jembe, Z., Pinsky, B., LaBeaud, A. AMER SOC TROP MED & HYGIENE. 2018: 162?63

  View details for Web of Science ID 000461386602521

• Transplant Virus Detection Using Multiplex Targeted Sequencing. The journal of applied laboratory medicine Tan, S. K., Shen, P., Lefterova, M. I., Sahoo, M. K., Fung, E., Odegaard, J. I., Davis, R. W., Pinsky, B. A., Scharfe, C. 2018; 2 (5): 757?69

  Abstract

  Viral infections are a major cause of complications and death in solid organ and hematopoietic cell transplantation.We developed a multiplex viral sequencing assay (mVseq) to simultaneously detect 20 transplant-relevant DNA viruses from small clinical samples. The assay uses a single-tube multiplex PCR to amplify highly conserved virus genomic regions without the need for previous virus enrichment or host nucleic acid subtraction. Multiplex sample sequencing was performed using Illumina MiSeq, and reads were aligned to a database of target sequences. Analytical and clinical performance was evaluated using reference viruses spiked into human plasma, as well as patient plasma and nonplasma samples, including bronchoalveolar lavage fluid, cerebrospinal fluid, urine, and tissue from immunocompromised transplant recipients.For the virus spike-in samples, mVseq's analytical sensitivity and dynamic range were similar to quantitative PCR (qPCR). In clinical specimens, mVseq showed substantial agreement with single-target qPCR (92%; k statistic, 0.77; 259 of 282 viral tests); however, clinical sensitivity was reduced (81%), ranging from 62% to 100% for specific viruses. In 12 of the 47 patients tested, mVseq identified previously unknown BK virus, human herpesvirus-7, and Epstein-Barr virus infections that were confirmed by qPCR.Our results reveal factors that can influence clinical sensitivity, such as high levels of host DNA background and loss of detection in coinfections when 1 virus was at much higher concentration than the others. The mVseq assay is flexible and scalable to incorporate RNA viruses, emerging viruses of interest, and other pathogens important in transplant recipients.

  View details for DOI 10.1373/jalm.2017.024521

  View details for PubMedID 31245786

  View details for PubMedCentralID PMC6594177

• Molecular diagnosis of Zika virus infections REVIEWS IN MEDICAL MICROBIOLOGY St George, K., Pinsky, B. A. 2018; 29 (1): 8?16

  View details for DOI 10.1097/MRM.0000000000000125

  View details for Web of Science ID 000419432200002

• Real-time RT-PCR for Mayaro virus detection in plasma and urine JOURNAL OF CLINICAL VIROLOGY Waggoner, J. J., Rojas, A., Mohamed-Hadley, A., Arevalo de Guillen, Y., Pinsky, B. A. 2018; 98: 1?4

  Abstract

  Mayaro virus (MAYV) causes an acute febrile illness which can be difficult to differentiate from dengue or chikungunya. MAYV RNA can be detected in plasma during the first 3-5days of illness, but only a single rRT-PCR has been fully evaluated in the literature.To develop an rRT-PCR for MAYV and evaluate assay performance using human plasma and urine samples spiked with different MAYV strains.A MAYV rRT-PCR targeting a region of the 5'UTR and nsp1 gene was designed from the alignment of all complete-genome MAYV sequences to be compatible with existing laboratory protocols. The assay was evaluated using human samples spiked with six MAYV strains, including strains from each of the three genotypes.The linear range of the MAYV rRT-PCR extended from 1.0 to 8.0 log10copies/?L, and the lower limit of 95% detection was 8.2copies/?L. No detection was observed when the MAYV rRT-PCR was tested with genomic RNA from related arboviruses. The assay demonstrated linear amplification of all 6 MAYV strains when spiked into human plasma samples as well as 2 strains spiked into urine.We report the design and evaluation of an rRT-PCR for MAYV. Given the concern for MAYV emergence in the Americas and the few molecular tests that have been evaluated in the literature, this assay should provide a useful diagnostic for patients with an acute febrile illness.

  View details for PubMedID 29172075

  View details for PubMedCentralID PMC5742299

• Zika and Chikungunya virus detection in naturally infected Aedes aegypti in Ecuador ACTA TROPICA Cevallos, V., Ponce, P., Waggoner, J. J., Pinsky, B. A., Coloma, J., Quiroga, C., Morales, D., Cardenas, M. 2018; 177: 74?80

  Abstract

  The wide and rapid spread of Chikungunya (CHIKV) and Zika (ZIKV) viruses represent a global public health problem, especially for tropical and subtropical environments. The early detection of CHIKV and ZIKV in mosquitoes may help to understand the dynamics of the diseases in high-risk areas, and to design data based epidemiological surveillance to activate the preparedness and response of the public health system and vector control programs. This study was done to detect ZIKV and CHIKV viruses in naturally infected fed female Aedes aegypti (L.) mosquitoes from active epidemic urban areas in Ecuador. Pools (n=193; 22 pools) and individuals (n=22) of field collected Ae. aegypti mosquitoes from high-risk arboviruses infection sites in Ecuador were analyzed for the presence of CHIKV and ZIKV using RT-PCR. Phylogenetic analysis demonstrated that both ZIKV and CHIKV viruses circulating in Ecuador correspond to the Asian lineages. Minimum infection rate (MIR) of CHIKV for Esmeraldas city was 2.3% and the maximum likelihood estimation (MLE) was 3.3%. The minimum infection rate (MIR) of ZIKV for Portoviejo city was 5.3% and for Manta city was 2.1%. Maximum likelihood estimation (MLE) for Portoviejo city was 6.9% and 2.6% for Manta city. Detection of arboviruses and infection rates in the arthropod vectors may help to predict an outbreak and serve as a warning tool in surveillance programs.

  View details for PubMedID 28982578

• Malaria and Chikungunya Detected Using Molecular Diagnostics Among Febrile Kenyan Children OPEN FORUM INFECTIOUS DISEASES Waggoner, J., Brichard, J., Mutuku, F., Ndenga, B., Heath, C., Mohamed-Hadley, A., Sahoo, M. K., Vulule, J., Lefterova, M., BanaeiA, N., Mukoko, D., Pinsky, B. A., LaBeaud, A. 2017; 4 (3): ofx110

  Abstract

  In sub-Saharan Africa, malaria is frequently overdiagnosed as the cause of an undifferentiated febrile illness, whereas arboviral illnesses are presumed to be underdiagnosed.Sera from 385 febrile Kenyan children, who presented to 1 of 4 clinical sites, were tested using microscopy and real-time molecular assays for dengue virus (DENV), chikungunya virus (CHIKV), malaria, and Leptospira.Malaria was the primary clinical diagnosis for 254 patients, and an arboviral infection (DENV or CHIKV) was the primary diagnosis for 93 patients. In total, 158 patients (41.0%) had malaria and 32 patients (8.3%) had CHIKV infections. Compared with real-time polymerase chain reaction, microscopy demonstrated a percent positive agreement of 49.7%. The percentage of malaria cases detected by microscopy varied significantly between clinical sites. Arboviral infections were the clinical diagnosis for patients on the Indian Ocean coast (91 of 238, 38.2%) significantly more often than patients in the Lake Victoria region (2 of 145, 1.4%; P < .001). However, detection of CHIKV infections was significantly higher in the Lake Victoria region (19 of 145 [13.1%] vs 13 of 239 [5.4%]; P = .012).The clinical diagnosis of patients with an acute febrile illness, even when aided by microscopy, remains inaccurate in malaria-endemic areas, contributing to inappropriate management decisions.

  View details for PubMedID 28702473

• Metagenomic DNA Sequencing for the Diagnosis of Intraocular Infections. Ophthalmology Doan, T., Acharya, N. R., Pinsky, B. A., Sahoo, M. K., Chow, E. D., Banaei, N., Budvytiene, I., Cevallos, V., Zhong, L., Zhou, Z., Lietman, T. M., DeRisi, J. L. 2017

  View details for DOI 10.1016/j.ophtha.2017.03.045

  View details for PubMedID 28526549

• Detection of Emerging Vaccine-Related Polioviruses by Deep Sequencing. Journal of clinical microbiology Sahoo, M. K., Holubar, M., Huang, C., Mohamed-Hadley, A., Liu, Y., Waggoner, J. J., Troy, S. B., Garcia-Garcia, L., Ferreyra-Reyes, L., Maldonado, Y., Pinsky, B. A. 2017

  Abstract

  Oral poliovirus vaccine can mutate to regain neurovirulence. To date, evaluation of these mutations has been performed primarily on culture-enriched isolates by using conventional Sanger sequencing. We therefore developed a culture-independent, deep-sequencing method targeting the 5' untranslated region (UTR) and P1 genomic region to characterize vaccine-related poliovirus variants. Error analysis of the deep-sequencing method demonstrated reliable detection of poliovirus mutations at levels of <1%, depending on read depth. Sequencing of viral nucleic acids from the stool of vaccinated, asymptomatic children and their close contacts collected during a prospective cohort study in Veracruz, Mexico, revealed no vaccine-derived polioviruses. This was expected given that the longest duration between sequenced sample collection and the end of the most recent national immunization week was 66 days. However, we identified many low-level variants (<5%) distributed across the 5' UTR and P1 genomic region in all three Sabin serotypes, as well as vaccine-related viruses with multiple canonical mutations associated with phenotypic reversion present at high levels (>90%). These results suggest that monitoring emerging vaccine-related poliovirus variants by deep sequencing may aid in the poliovirus endgame and efforts to ensure global polio eradication.

  View details for DOI 10.1128/JCM.00144-17

  View details for PubMedID 28468861

• Stability of Zika Virus in Urine: Specimen Processing Considerations and Implications for the Detection of RNA Targets in Urine. Journal of virological methods Tan, S. K., Sahoo, M. K., Milligan, S., Taylor, N., Pinsky, B. A. 2017

  Abstract

  Detection of Zika virus (ZIKV) RNA in urine is of increasing interest for the diagnosis of ZIKV infection. Pre-analytical variables can significantly impact the stability of RNA in urine.To determine optimal specimen processing protocols that would maximize detection of ZIKV RNA in urine by real-time, reverse transcriptase PCR, we investigated the effect of temperature, initial ZIKV concentration, use of nucleic acid stabilizers, and time on ZIKV RNA levels. Urine samples from healthy donors were spiked with ZIKV using the Exact Diagnostics(®) ZIKV Verification Panel, a commercially available panel composed of heat-inactivated ZIKV, at concentrations of 5.0 log10 copies/mL (ZIKV-high) and 4.0 log10 copies/mL (ZIKV-low). Samples were stored at room temperature, 4°C, or -80°C and frozen aliquots were exposed to no stabilizer (urine), Buffer ATL (Qiagen, Germantown, MD), or DNA/RNA Shield (Zymo Research, Irvine, CA).ZIKV RNA levels in urine declined steadily at room temperature, though was not significant by 48hours (ZIKV-high, p=0.09; ZIKV-low, p=0.20). ZIKV RNA titers were consistently higher when stored at 4°C, suggesting that storage at 4°C can slow the progression of RNA degradation. Freezing urine samples at -80°C resulted in a significant loss of detectable ZIKV RNA in the ZIKV-low group. ZIKV RNA was detected in 5/6 replicates at 3 days, 1/6 replicates at 10 days, and 1/3 replicates at 30 days, with findings reproducible on repeat testing. Presence of either nucleic acid stabilizer in urine corrected this effect, and resulted in recovery of ZIKV RNA in all replicates. Use of a nucleic acid stabilizer in the ZIKV-high group did not add incremental benefit for the detection or quantitation of ZIKV RNA.ZIKV RNA is prone to degradation in urine with loss of detectable virus even when specimens are frozen at -80°C for 10 days. Detection of ZIKV-positive urine samples, particularly those containing low ZIKV titers may be aided with the addition of a nucleic acid stabilizer during urine specimen processing.

  View details for DOI 10.1016/j.jviromet.2017.04.018

  View details for PubMedID 28472623

• Pericardial Effusion Following Hematopoietic Cell Transplantation in Children and Young Adults Is Associated with Increased Risk of Mortality. Biology of blood and marrow transplantation Cox, K., Punn, R., Weiskopf, E., Pinsky, B. A., Kharbanda, S. 2017

  Abstract

  Hematopoietic cell transplantation (HCT) is curative for many pediatric malignant and nonmalignant disorders but is associated with significant morbidity and mortality, including the development of pericardial effusion (PEF). We report the results of a retrospective chart review performed to assess the incidence, risk factors, and prognostic significance of PEF in pediatric HCT recipient at Lucile Packard Children's Hospital of Stanford University. A total of 119 patients undergoing HCT between January 2010 and December 2013 were selected through the hospital's Pediatric Stem Cell Transplant Program database. A retrospective chart review, including review of documentation, correspondence, imaging reports, laboratory values, and death records, was performed to collect data. The overall incidence of PEF in our population was 21%. Risk factors for the development of PEF included unrelated donor transplants and cord blood as the stem cell source (P?=?.005), whereas HLA mismatch approached significance (P?=?.05). The risk for development of PEF was found to not be significantly associated with acute or chronic graft-versus-host disease (GVHD), age at transplantation, sex, conditioning regimen, or viral reactivation status. Of interest, 6 of the 119 patients were found to have transplant-associated thrombotic microangiopathy (TA-TMA). Four of those 6 patients developed PEF, suggesting TA-TMA as a risk factor for PEF. Eight of the 25 patients who developed PEF (32%) required pericardiocentesis. Five out of the 8 patients requiring pericardiocentesis died owing to causes unrelated to the procedure or to PEF itself. Pericardial fluid testing in 4 of these patients (50%) was positive for human herpesvirus 6, Epstein-Barr virus, cytomegalovirus, and/or adenovirus. Finally, of significant interest, patients with PEF had a statistically significant higher likelihood of mortality compared with those without PEF (44% versus 17%; P?=?.007).

  View details for DOI 10.1016/j.bbmt.2017.03.028

  View details for PubMedID 28390986

• Current State of PCR-Based Epstein-Barr Virus DNA Testing for Nasopharyngeal Cancer JNCI-JOURNAL OF THE NATIONAL CANCER INSTITUTE Kim, K. Y., Le, Q., Yom, S. S., Pinsky, B. A., Bratman, S. V., Ng, R. W., El Mubarak, H. S., Chan, K., Sander, M., Conley, B. A. 2017; 109 (4)

  Abstract

  Clinical studies have shown plasma Epstein-Barr virus (EBV) DNA level to be an independent prognostic biomarker for nasopharyngeal carcinoma (NPC). However, the proportion of NPC patients whose tumors are associated with EBV vary with geographic location, and there are a variety of assays for plasma EBV. To develop the level of evidence needed to demonstrate the clinical utility of plasma EBV DNA detection for NPC patients and encourage widespread adoption of this biomarker test in clinical laboratories, validated harmonized assays are needed. In 2015, the National Cancer Institute (NCI) convened a Workshop on Harmonization of EBV Testing for Nasopharyngeal Cancer, where experts in head and neck oncology and laboratory medicine addressed the limitations of currently available polymerase chain reaction-based EBV DNA quantitation assays and discussed strategies for advancing the development of harmonized EBV DNA assays and their appropriate clinical use. This article presents the key recommendations to direct future efforts in assay harmonization and validation.

  View details for PubMedID 28376165

• Calibration of BK Virus Nucleic Acid Amplification Testing to the 1st WHO International Standard for BK Virus. Journal of clinical microbiology Tan, S. K., Milligan, S., Sahoo, M. K., Taylor, N., Pinsky, B. A. 2017; 55 (3): 923-930

  Abstract

  Significant interassay variability in the quantification of BK virus (BKV) DNA precludes establishing broadly applicable thresholds for the management of BKV infection in transplantation. The 1st WHO International Standard for BKV (primary standard) was introduced in 2016 as a common calibrator for improving the harmonization of BKV nucleic acid amplification testing (NAAT) and enabling comparisons of biological measurements worldwide. Here, we evaluated the Altona RealStar BKV assay (Altona) and calibrated the results to the international unit (IU) using the Exact Diagnostics BKV verification panel, a secondary standard traceable to the primary standard. The primary and secondary standards on Altona had nearly identical linear regression equations (primary standard, Y = 1.05X - 0.28, R(2) = 0.99; secondary standard, Y = 1.04X - 0.26, R(2) = 0.99) and conversion factors (primary standard, 1.11 IU/copy; secondary standard, 1.09 IU/copy). A comparison of Altona with a laboratory-developed BKV NAAT assay in IU/ml versus copies/ml using Passing-Bablok regression revealed similar regression lines, no proportional bias, and improvement in the systematic bias (95% confidence interval of intercepts: copies/ml, -0.52 to -1.01; IU/ml, 0.07 to -0.36). Additionally, Bland-Altman analyses revealed a clinically significant reduction of bias when results were reported in IU/ml (IU/ml, -0.10 log10; copies/ml, -0.70 log10). These results indicate that the use of a common calibrator improved the agreement between the two assays. As clinical laboratories worldwide use calibrators traceable to the primary standard to harmonize BKV NAAT results, we anticipate improved interassay comparisons with a potential for establishing broadly applicable quantitative BKV DNA load cutoffs for clinical practice.

  View details for DOI 10.1128/JCM.02315-16

  View details for PubMedID 28053213

  View details for PubMedCentralID PMC5328461

• T cells expand after solid organ transplantation in the absence of CMV disease. American journal of transplantation Higdon, L. E., Trofe-Clark, J., Liu, S., Margulies, K. B., Sahoo, M. K., Blumberg, E., Pinsky, B. A., Maltzman, J. S. 2017

  Abstract

  Cytomegalovirus (CMV) is a major cause of morbidity and mortality in solid-organ transplant recipients. Approximately 60% of adults are CMV seropositive indicating previous exposure. Following resolution of primary infection, CMV remains in a latent state. Reactivation is controlled by memory T cells in healthy individuals; transplant recipients have reduced memory T cell function due to chronic immunosuppressive therapies. In this study, CD8(+) T cell responses to CMV polypeptides IE-1 and pp65 were analyzed in sixteen CMV seropositive renal and cardiac transplant recipients longitudinally pre- and post-transplant. All patients received standard of care maintenance immunosuppression, antiviral prophylaxis and CMV viral load monitoring, with approximately half receiving T cell depleting induction therapy. The frequency of CMV-responsive CD8(+) T cells, defined by production of effector molecules in response to CMV peptides, increased during the course of a year post-transplant. The increase commenced after the completion of antiviral prophylaxis, and these T cells tended to be terminally differentiated effector cells. Based on this small cohort, these data suggest that even in the absence of disease, antigenic exposure may continually shape the CMV-responsive T cell population post-transplant. This article is protected by copyright. All rights reserved.

  View details for DOI 10.1111/ajt.14227

  View details for PubMedID 28199780

• High incidence of Zika virus infection detected in plasma and cervical cytology specimens from pregnant women in Guayaquil, Ecuador. American journal of reproductive immunology Zambrano, H., Waggoner, J., León, K., Pinsky, B., Vera, K., Schettino, M., Rivera, L., Landivar, J., Granda, M., Lee, A., Mor, G. 2017; 77 (2)

  Abstract

  Zika virus (ZIKV) infection during pregnancy has been linked to severe birth defects, and the epidemiologic situation of the ZIKV epidemic in Ecuador is poorly understood. Guayaquil, Ecuador, has a tropical climate and experiences frequent outbreaks of dengue and chikungunya virus, and in December 2015, ZIKV was identified. Given the well-documented effects of ZIKV in pregnancy, including microcephaly, we tested for the presence of ZIKV in both plasma and cervical cytology of pregnant women. We report the identification of a population of pregnant women with a high incidence of ZIKV infection detected in the plasma and lower reproductive tract. A case-control study was performed to determine the incidence of ZIKV infection among low-income, pregnant women at risk for preterm delivery compared to matched controls. Plasma and cervical cytology specimens were tested for ZIKV by rRT-PCR. Fifty-nine pregnant women were enrolled. The incidence of ZIKV was 54% (32/59) overall: 18/31 (58.1%) in cases and 14/28 (50.0%) in controls. ZIKV detection in plasma and cervical cytology specimens demonstrated good agreement. Overall, outcomes for neonates born to ZIKV-positive and ZIKV-negative mothers were similar. However, two neonates were born with microcephaly to case mothers who were ZIKV positive. We report a high incidence of ZIKV infection (54%) in a distinctive population in Guayaquil, Ecuador. We identify ZIKV in cervical samples that correlates with ZIKV in the plasma. These data raise concerns regarding the breadth of the ZIKV epidemic in Ecuador and demonstrate the utility of cervical cytology specimens for ZIKV testing.

  View details for DOI 10.1111/aji.12630

  View details for PubMedID 28177195

• Clinical characteristics and outcomes of pediatric patients with CMV DNA detection in bronchoalveolar lavage fluid. Pediatric pulmonology Burgener, E. B., Waggoner, J., Pinsky, B. A., Chen, S. F. 2017; 52 (1): 112-118

  Abstract

  Cytomegalovirus (CMV) infection can cause severe pulmonary disease in immunocompromised patients. There are no standard diagnostic criteria for CMV pulmonary disease beyond histopathology findings on lung tissue, which is challenging to obtain in pediatric patients. Bronchoalveolar lavage (BAL) fluid is easier to obtain. Since CMV remains latent after primary infection and can potentially reactivate due to any inflammatory response, CMV detection in BAL specimen may not indicate acute CMV pulmonary disease. Thus, we describe the clinical manifestations and outcomes of pediatric patients with CMV detection in BAL fluid.We reviewed the clinical, radiologic, and laboratory data of patients <19 years old with a BAL specimen positive for CMV during a 5-year period.Thirty-four encounters in 29 patients were found with CMV detected in their BAL specimen. Half (17/34) of the encounters were in immunocompromised patients. CMV, polymerase chain reaction (PCR) was the most common positive test. Forty-seven percent of the patients had other infections detected in BAL specimens. The majority of patients were never treated for CMV and resolved their acute respiratory illness. Only one patient had probable CMV pulmonary disease.CMV is frequently recovered from BAL specimens but does not usually indicate acute CMV pulmonary disease. We would suggest that other diagnoses be considered first, even if CMV is recovered. Pediatr Pulmonol. 2016; 9999:XX-XX. © 2016 Wiley Periodicals, Inc.

  View details for DOI 10.1002/ppul.23494

  View details for PubMedID 27280337

• The Human Virome - Implications for Clinical Practice in Transplantation Medicine. Journal of clinical microbiology Tan, S. K., Relman, D. A., Pinsky, B. A. 2017

  Abstract

  Advances in DNA sequencing technology have provided an unprecedented opportunity to study the human virome. Transplant recipients and other immunocompromised hosts are at particular risk for developing virus-related pathology; thus, the impact of the virome on health and disease may be even more relevant in this population. Here we discuss technical considerations in studying the human virome, the current literature on the virome in transplant recipients, and near future applications of sequence-based findings that can further our understanding of viruses in transplantation medicine.

  View details for PubMedID 28724557

• Global epidemiology of non-influenza RNA respiratory viruses: data gaps and a growing need for surveillance. The Lancet. Infectious diseases Tang, J. W., Lam, T. T., Zaraket, H., Lipkin, W. I., Drews, S. J., Hatchette, T. F., Heraud, J. M., Koopmans, M. P. 2017; 17 (10): e320?e326

  Abstract

  Together with influenza, the non-influenza RNA respiratory viruses (NIRVs), which include respiratory syncytial virus, parainfluenza viruses, coronavirus, rhinovirus, and human metapneumovirus, represent a considerable global health burden, as recognised by WHO's Battle against Respiratory Viruses initiative. By contrast with influenza viruses, little is known about the contemporaneous global diversity of these viruses, and the relevance of such for development of pharmaceutical interventions. Although far less advanced than for influenza, antiviral drugs and vaccines are in different stages of development for several of these viruses, but no interventions have been licensed. This scarcity of global genetic data represents a substantial knowledge gap and impediment to the eventual licensing of new antiviral drugs and vaccines for NIRVs. Enhanced genetic surveillance will assist and boost research and development into new antiviral drugs and vaccines for these viruses. Additionally, understanding the global diversity of respiratory viruses is also part of emerging disease preparedness, because non-human coronaviruses and paramyxoviruses have been listed as priority concerns in a recent WHO research and development blueprint initiative for emerging infectious diseases. In this Personal View, we explain further the rationale for expanding the genetic database of NIRVs and emphasise the need for greater investment in this area of research.

  View details for DOI 10.1016/S1473-3099(17)30238-4

  View details for PubMedID 28457597

• Prevalence of Drug-Resistant Minority Variants in Untreated HIV-1-Infected Individuals With and Those Without Transmitted Drug Resistance Detected by Sanger Sequencing. The Journal of infectious diseases Clutter, D. S., Zhou, S., Varghese, V., Rhee, S. Y., Pinsky, B. A., Jeffrey Fessel, W., Klein, D. B., Spielvogel, E., Holmes, S. P., Hurley, L. B., Silverberg, M. J., Swanstrom, R., Shafer, R. W. 2017; 216 (3): 387?91

  Abstract

  Minority variant human immunodeficiency virus type 1 (HIV-1) nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance mutations are associated with an increased risk of virological failure during treatment with NNRTI-containing regimens. To determine whether individuals to whom variants with isolated NNRTI-associated drug resistance were transmitted are at increased risk of virological failure during treatment with a non-NNRTI-containing regimen, we identified minority variant resistance mutations in 33 individuals with isolated NNRTI-associated transmitted drug resistance and 49 matched controls. We found similar proportions of overall and nucleoside reverse transcriptase inhibitor-associated minority variant resistance mutations in both groups, suggesting that isolated NNRTI-associated transmitted drug resistance may not be a risk factor for virological failure during treatment with a non-NNRTI-containing regimen.

  View details for PubMedID 28859436

• IgG antibodies to dengue enhanced for Fc?RIIIA binding determine disease severity. Science (New York, N.Y.) Wang, T. T., Sewatanon, J., Memoli, M. J., Wrammert, J., Bournazos, S., Bhaumik, S. K., Pinsky, B. A., Chokephaibulkit, K., Onlamoon, N., Pattanapanyasat, K., Taubenberger, J. K., Ahmed, R., Ravetch, J. V. 2017; 355 (6323): 395?98

  Abstract

  Dengue virus (DENV) infection in the presence of reactive, non-neutralizing immunoglobulin G (IgG) (RNNIg) is the greatest risk factor for dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). Progression to DHF/DSS is attributed to antibody-dependent enhancement (ADE); however, because only a fraction of infections occurring in the presence of RNNIg advance to DHF/DSS, the presence of RNNIg alone cannot account for disease severity. We discovered that DHF/DSS patients respond to infection by producing IgGs with enhanced affinity for the activating Fc receptor Fc?RIIIA due to afucosylated Fc glycans and IgG1 subclass. RNNIg enriched for afucosylated IgG1 triggered platelet reduction in vivo and was a significant risk factor for thrombocytopenia. Thus, therapeutics and vaccines restricting production of afucosylated, IgG1 RNNIg during infection may prevent ADE of DENV disease.

  View details for PubMedID 28126818

• Development of a Real-Time Reverse Transcription Polymerase Chain Reaction for O'nyong-nyong Virus and Evaluation with Clinical and Mosquito Specimens from Kenya AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE Waggoner, J., Heath, C., Ndenga, B., Mutuku, F., Sahoo, M. K., Mohamed-Hadley, A., Vulule, J., Mukoko, D., LaBeaud, A., Pinsky, B. A. 2017; 97 (1): 121?24

  Abstract

  O'nyong-nyong virus (ONNV), an alphavirus closely related to chikungunya virus (CHIKV), has been the documented cause of two large outbreaks in east Africa; however, little is known about the contribution of ONNV to cases of acute febrile illness during interepidemic periods. An ONNV real-time reverse transcription polymerase chain reaction (rRT-PCR) was developed and evaluated using clinical and mosquito pool samples. The ONNV rRT-PCR linear range extended from 8.0 to 2.0 log10 copies/?L, and the lower limit of 95% detection was 22.4 copies/?L. No cases of ONNV infection were identified in serum from 385 Kenyan children who presented with an acute febrile illness. Additionally, ONNV was not detected in 120 mosquito pools collected in coastal and western Kenya. The ONNV rRT-PCR demonstrated good analytical sensitivity when performed in monoplex or as a component of an ONNV-CHIKV duplex assay. This assay should provide a useful diagnostic for the detection of ONNV in surveillance studies.

  View details for PubMedID 28719301

• Closing the Brief Case: Confirmed Positive HIV-1 Serological Screening but Undetectable RNA Virus Load in a Pregnant Woman. Journal of clinical microbiology Joshi, R. P., Gomez, C. A., Steiner, D., Aziz, N., Pinsky, B. A. 2017; 55 (12): 3566?67

  View details for PubMedID 29180505

• The Brief Case: Confirmed Positive HIV-1 Serologic Screening but Undetectable RNA Virus Load in a Pregnant Woman. Journal of clinical microbiology Joshi, R. P., Gomez, C. A., Steiner, D., Aziz, N., Pinsky, B. A. 2017; 55 (12): 3316?20

  View details for PubMedID 29180504

• Zika Virus, Chikungunya Virus, and Dengue Virus in Cerebrospinal Fluid from Adults with Neurological Manifestations, Guayaquil, Ecuador. Frontiers in microbiology Acevedo, N., Waggoner, J., Rodriguez, M., Rivera, L., Landivar, J., Pinsky, B., Zambrano, H. 2017; 8: 42-?

  Abstract

  Zika virus (ZIKV), chikungunya virus (CHIKV), and dengue virus (DENV) have been associated with clinical presentations that involve acute neurological complaints. In the current study, we identified ZIKV, CHIKV, and DENV in cerebrospinal fluid (CSF) samples from patients admitted to the Hospital Luis Vernaza (Guayaquil, Ecuador) to the Emergency Room or the Intensive Care Unit, with neurological symptoms and/or concern for acute arboviral infections. Viral RNA from one or more virus was detected in 12/16 patients. Six patients were diagnosed with meningitis or encephalitis, three with Guillain-Barré Syndrome, and one with CNS vasculitis. Two additional patients had a systemic febrile illness including headache that prompted testing of CSF. Two patients, who were diagnosed with encephalitis and meningoencephalitis, died during their hospitalizations. These cases demonstrate the breadth and significance of neurological manifestations associated with ZIKV, CHIKV, and DENV infections.

  View details for DOI 10.3389/fmicb.2017.00042

  View details for PubMedID 28174559

  View details for PubMedCentralID PMC5258761

• Zika Virus and Chikungunya Virus CoInfections: A Series of Three Cases from a Single Center in Ecuador. American journal of tropical medicine and hygiene Zambrano, H., Waggoner, J. J., Almeida, C., Rivera, L., Benjamin, J. Q., Pinsky, B. A. 2016; 95 (4): 894-896

  Abstract

  Zika virus (ZIKV) and chikungunya virus (CHIKV) cocirculate throughout much of the tropical Western Hemisphere; however, few cases of coinfection with these two pathogens have been reported. Herein, we describe three cases of ZIKV-CHIKV coinfection detected at a single center in Ecuador: a patient who developed symptoms on postoperative day 5 from an orthopedic procedure, a woman who had traveled to Ecuador for fertility treatment, and a woman who was admitted for Guillain-Barré syndrome and had ZIKV and CHIKV detected in serum and cerebrospinal fluid. All cases were diagnosed using a multiplex real-time reverse transcription polymerase chain reaction, and ZIKV viremia was detected as late as 16 days after symptom onset. These cases demonstrate the varied clinical presentation of ZIKV-CHIKV coinfections as well as the importance of multiplexed arboviral testing for these pathogens.

  View details for PubMedID 27402518

• Homotypic Dengue Virus Reinfections in Nicaraguan Children. journal of infectious diseases Waggoner, J. J., Balmaseda, A., Gresh, L., Sahoo, M. K., Montoya, M., Wang, C., Abeynayake, J., Kuan, G., Pinsky, B. A., Harris, E. 2016; 214 (7): 986-993

  Abstract

  ?Infection with one of four related dengue virus serotypes (DENV-1-4) is thought to result in life-long immunity to re-infection with the same serotype (homotypic DENV re-infection).?Archived serum samples, collected as part of an ongoing pediatric dengue cohort study in Nicaragua, were tested for DENV by real-time RT-PCR. Samples were collected from 2,892 children who presented with an acute febrile illness clinically attributed to a non-dengue cause ("C" cases). Test results were added to a database of previously-identified symptomatic dengue cases in the cohort to identify repeat infections.?Four patients with homotypic DENV re-infections were identified and confirmed among 29 repeat DENV infections with serotype confirmation (13.8% of repeat symptomatic infections). Homotypic re-infections with DENV-1, -2, and -3 occurred 325-621 days after the initial infection. Each patient experienced one symptomatic dengue case and one DENV-positive C case, and two patients presented with symptomatic dengue during their second infection. These DENV-positive C cases did not elicit long-lived humoral immune responses, despite viremia up to 6.44 log10 copies/mL of serum.?We describe the first set of virologically confirmed homotypic DENV re-infections. Such cases challenge the current understanding of DENV immunity and have important implications for modeling DENV transmission.

  View details for DOI 10.1093/infdis/jiw099

  View details for PubMedID 26984144

• Molecular diagnostics for human leptospirosis. Current opinion in infectious diseases Waggoner, J. J., Pinsky, B. A. 2016; 29 (5): 440-445

  Abstract

  The definitive diagnosis of leptospirosis, which results from infection with spirochetes of the genus Leptospira, currently relies on the use of culture, serological testing (microscopic agglutination testing), and molecular detection. The purpose of this review is to describe new molecular diagnostics for Leptospira and discuss advancements in the use of available methods.Efforts have been focused on improving the clinical sensitivity of Leptospira detection using molecular methods. In this review, we describe a reoptimized pathogenic species-specific real-time PCR (targeting lipL32) that has demonstrated improved sensitivity, findings by two groups that real-time reverse-transcription PCR assays targeting the 16S rrs gene can improve detection, and two new loop-mediated amplification techniques. Quantitation of leptospiremia, detection in different specimen types, and the complementary roles played by molecular detection and microscopic agglutination testing will be discussed. Finally, a protocol for Leptospira strain subtyping using variable number tandem repeat targets and high-resolution melting will be described.Molecular diagnostics have an established role for the diagnosis of leptospirosis and provide an actionable diagnosis in the acute setting. The use of real-time reverse-transcription PCR for testing serum/plasma and cerebrospinal fluid, when available, may improve the detection of Leptospira without decreasing clinical specificity.

  View details for DOI 10.1097/QCO.0000000000000295

  View details for PubMedID 27537829

• Evaluation of the Aptima HIV-1 Quant Dx Assay Using Plasma and Dried Blood Spots. Journal of clinical microbiology Sahoo, M. K., Varghese, V., White, E., Winslow, M., Katzenstein, D. A., Shafer, R. W., Pinsky, B. A. 2016; 54 (10): 2597-2601

  Abstract

  HIV-1 RNA quantitation in plasma, or virus load testing, is the primary method by which the response to antiretroviral therapy is monitored. Here we describe evaluation of the Aptima HIV-1 Quant Dx assay (Aptima) performed on the automated Panther system. The clinical performance of Aptima was compared to that of the Cobas AmpliPrep/Cobas TaqMan HIV-1 Test v2.0 (CAP/CTM) using 162 EDTA plasma samples collected from patients undergoing HIV-1 monitoring. Overall agreement was 84.0% (136/162), with a kappa statistic of 0.723 (standard error, 0.047; 95% confidence interval [CI], 0.630 to 0.815), indicating substantial agreement. Using the 86 clinical samples quantifiable by both methods, Passing-Bablok regression revealed a regression line of Y = (1.069 × X) - 0.346 (95% CI of the slope [1.003 to 1.139] and intercept [-0.666 to -0.074]), and Bland-Altman analysis demonstrated a mean difference (Aptima-CAP/CTM) of -0.075 log10 copies/ml (95% limits of agreement of -0.624 to 0.475), consistent with negative bias. Comparison of Aptima results for paired dried blood spot (DBS) and plasma specimens archived from participants in the Peninsula AIDS Research Cohort Study (PARC) demonstrated an overall agreement of 94.7% (90/95) when 1,000 copies/ml was used as the threshold. In conclusion, the Aptima HIV-1 Quant Dx assay provides a suitable alternative for HIV-1 monitoring in plasma and DBS.

  View details for DOI 10.1128/JCM.01569-16

  View details for PubMedID 27535684

  View details for PubMedCentralID PMC5035416

• Q148N, a Novel Integrase Inhibitor Resistance Mutation Associated with Low-Level Reduction in Elvitegravir Susceptibility AIDS RESEARCH AND HUMAN RETROVIRUSES Varghese, V., Pinsky, B. A., Smith, D. S., Klein, D., Shafer, R. W. 2016; 32 (7): 702-704

  Abstract

  The integrase strand transfer inhibitor (INSTI)-resistance mutations Q148H/K/R are arguably the most important INSTI-resistance mutations as they represented the first step to high-level dolutegravir cross-resistance. We describe an individual with transmitted four-class drug resistance whose virus sequence had the previously uncharacterized mutation Q148N. Infectious molecular HIV-1 clones containing Q148N alone and in combination with G140S demonstrated ?2.4-4.5 reduced elvitegravir susceptibility depending on the virus's genetic context but retained susceptibility to raltegravir and dolutegravir. This level of reduced elvitegravir susceptibility is lower than that observed with Q148H/K/R and in fact the infected individual responded to an initial treatment regimen containing tenofovir/emtricitabine/elvitegravir/cobicistat. Q148N was associated with a higher replication capacity than Q148H, suggesting that this mutation may be more fit in the absence of selective INSTI therapy.

  View details for DOI 10.1089/aid.2016.0038

  View details for Web of Science ID 000379609100012

  View details for PubMedID 27009474

• Single-Reaction Multiplex Reverse Transcription PCR for Detection of Zika, Chikungunya, and Dengue Viruses EMERGING INFECTIOUS DISEASES Waggoner, J. J., Gresh, L., Mohamed-Hadley, A., Ballesteros, G., Vargas Davila, M. J., Tellez, Y., Sahoo, M. K., Balmaseda, A., Harris, E., Pinsky, B. A. 2016; 22 (7): 1295-1297

  Abstract

  Clinical manifestations of Zika virus, chikungunya virus, and dengue virus infections can be similar. To improve virus detection, streamline molecular workflow, and decrease test costs, we developed and evaluated a multiplex real-time reverse transcription PCR for these viruses.

  View details for DOI 10.3201/eid2207.160326

  View details for Web of Science ID 000378563900030

  View details for PubMedID 27184629

  View details for PubMedCentralID PMC4918162

• Clinical evaluation of a single-reaction real-time RT-PCR for pan-dengue and chikungunya virus detection JOURNAL OF CLINICAL VIROLOGY Waggoner, J. J., Ballesteros, G., Gresh, L., Mohamed-Hadley, A., Tellez, Y., Sahoo, M. K., Abeynayake, J., Balmaseda, A., Harris, E., Pinsky, B. A. 2016; 78: 57-61

  Abstract

  Dengue virus (DENV) and chikungunya virus (CHIKV) now co-circulate throughout tropical regions of the world, with billions of people living at risk of infection. The differentiation of these infections is important for epidemiologic surveillance as well as clinical care, though widely-used molecular diagnostics for DENV and CHIKV require the performance of two to four separate PCR reactions for detection.In the current study, we sought to develop and evaluate a single-reaction, multiplex real-time RT-PCR (rRT-PCR) for the detection and differentiation of DENV and CHIKV (the pan-DENV-CHIKV rRT-PCR).From an alignment of all available CHIKV complete genome sequences in GenBank, a new CHIKV rRT-PCR was designed for use in multiplex with a previously described assay for pan-DENV detection. Analytical evaluation was performed in accordance with published recommendations, and the pan-DENV-CHIKV rRT-PCR was clinically compared to reference molecular diagnostics for DENV and CHIKV using 182 serum samples from suspected cases in Managua, Nicaragua.The pan-DENV-CHIKV rRT-PCR had a dynamic range extending from 7.0 to 2.0 log10copies/?L for each DENV serotype and CHIKV, and the lower limits of 95% detection were 7.9-37.4copies/?L. The pan-DENV-CHIKV rRT-PCR detected DENV in 81 patients compared to 75 using a reference, hemi-nested DENV RT-PCR, and it demonstrated perfect agreement with a reference CHIKV rRT-PCR (54 positive samples).The single-reaction, multiplex format of the pan-DENV-CHIKV rRT-PCR, combined with sensitive detection of both viruses, has the potential to improve detection while decreasing testing costs and streamlining molecular workflow.

  View details for DOI 10.1016/j.jcv.2016.01.007

  View details for Web of Science ID 000374480000013

  View details for PubMedID 26991052

• Molecular and Culture-Based Bronchoalveolar Lavage Fluid Testing for the Diagnosis of Cytomegalovirus Pneumonitis. Open forum infectious diseases Tan, S. K., Burgener, E. B., Waggoner, J. J., Gajurel, K., Gonzalez, S., Chen, S. F., Pinsky, B. A. 2016; 3 (1): ofv212-?

  Abstract

  Background. ?Cytomegalovirus (CMV) is a major cause of morbidity and mortality in immunocompromised patients, with CMV pneumonitis among the most severe manifestations of infection. Although bronchoalveolar lavage (BAL) samples are frequently tested for CMV, the clinical utility of such testing remains uncertain. Methods. ?Retrospective analysis of adult patients undergoing BAL testing via CMV polymerase chain reaction (PCR), shell vial culture, and conventional viral culture between August 2008 and May 2011 was performed. Cytomegalovirus diagnostic methods were compared with a comprehensive definition of CMV pneumonitis that takes into account signs and symptoms, underlying host immunodeficiency, radiographic findings, and laboratory results. Results. ?Seven hundred five patients underwent 1077 bronchoscopy episodes with 1090 BAL specimens sent for CMV testing. Cytomegalovirus-positive patients were more likely to be hematopoietic cell transplant recipients (26% vs 8%, P < .0001) and less likely to have an underlying condition not typically associated with lung disease (3% vs 20%, P < .0001). Histopathology was performed in only 17.3% of CMV-positive bronchoscopy episodes. When CMV diagnostic methods were evaluated against the comprehensive definition, the sensitivity and specificity of PCR, shell vial culture, and conventional culture were 91.3% and 94.6%, 54.4% and 97.4%, and 28.3% and 96.5%, respectively. Compared with culture, PCR provided significantly higher sensitivity and negative predictive value (P ? .001), without significantly lower positive predictive value. Cytomegalovirus quantitation did not improve test performance, resulting in a receiver operating characteristic curve with an area under the curve of 0.53. Conclusions. ?Cytomegalovirus PCR combined with a comprehensive clinical definition provides a pragmatic approach for the diagnosis of CMV pneumonitis.

  View details for DOI 10.1093/ofid/ofv212

  View details for PubMedID 26885542

• Delayed Diagnosis of Tuberculous Meningitis Misdiagnosed as Herpes Simplex Virus-1 Encephalitis With the FilmArray Syndromic Polymerase Chain Reaction Panel Open Forum Infectious Diseases Gomez, C. A., Pinsky, B. A., Liu, A., Banaei, N. 2016: ofw245

  Abstract

  The FilmArray meningitis/encephalitis (ME) panel is a novel syndromic, nucleic acid amplification test for diagnosis of acute meningitis and encephalitis. Emerging data on its performance are concerning for false-positive results. We present a case of tuberculous meningitis misdiagnosed as herpes simplex virus-1 encephalitis with the FilmArray ME panel. Strategies to mitigate erroneous results are discussed.

  View details for DOI 10.1093/ofid/ofw245

  View details for PubMedCentralID PMC5437853

• Current and future molecular diagnostics for ocular infectious diseases. Current opinion in ophthalmology Doan, T., Pinsky, B. A. 2016; 27 (6): 561?67

  Abstract

  Confirmation of ocular infections can pose great challenges to the clinician. A fundamental limitation is the small amounts of specimen that can be obtained from the eye. Molecular diagnostics can circumvent this limitation and have been shown to be more sensitive than conventional culture. The purpose of this review is to describe new molecular methods and to discuss the applications of next-generation sequencing-based approaches in the diagnosis of ocular infections.Efforts have focused on improving the sensitivity of pathogen detection using molecular methods. This review describes a new molecular target for Toxoplasma gondii-directed polymerase chain reaction assays. Molecular diagnostics for Chlamydia trachomatis and Acanthamoeba species are also discussed. Finally, we describe a hypothesis-free approach, metagenomic deep sequencing, which can detect DNA and RNA pathogens from a single specimen in one test. In some cases, this method can provide the geographic location and timing of the infection.Pathogen-directed PCRs have been powerful tools in the diagnosis of ocular infections for over 20 years. The use of next-generation sequencing-based approaches, when available, will further improve sensitivity of detection with the potential to improve patient care.

  View details for PubMedID 27585214

• Analytical Performance Characteristics of the Cepheid GeneXpert Ebola Assay for the Detection of Ebola Virus PLOS ONE Pinsky, B. A., Sahoo, M. K., Sandlund, J., Kleman, M., Kulkarni, M., Grufman, P., Nygren, M., Kwiatkowski, R., Baron, E. J., Tenover, F., Denison, B., Higuchi, R., van Atta, R., Beer, N. R., Carrillo, A. C., Naraghi-Arani, P., Mire, C. E., Ranadheera, C., Grolla, A., Lagerqvist, N., Persing, D. H. 2015; 10 (11)

  Abstract

  The recently developed Xpert® Ebola Assay is a novel nucleic acid amplification test for simplified detection of Ebola virus (EBOV) in whole blood and buccal swab samples. The assay targets sequences in two EBOV genes, lowering the risk for new variants to escape detection in the test. The objective of this report is to present analytical characteristics of the Xpert® Ebola Assay on whole blood samples.This study evaluated the assay's analytical sensitivity, analytical specificity, inclusivity and exclusivity performance in whole blood specimens. EBOV RNA, inactivated EBOV, and infectious EBOV were used as targets. The dynamic range of the assay, the inactivation of virus, and specimen stability were also evaluated. The lower limit of detection (LoD) for the assay using inactivated virus was estimated to be 73 copies/mL (95% CI: 51-97 copies/mL). The LoD for infectious virus was estimated to be 1 plaque-forming unit/mL, and for RNA to be 232 copies/mL (95% CI 163-302 copies/mL). The assay correctly identified five different Ebola viruses, Yambuku-Mayinga, Makona-C07, Yambuku-Ecran, Gabon-Ilembe, and Kikwit-956210, and correctly excluded all non-EBOV isolates tested. The conditions used by Xpert® Ebola for inactivation of infectious virus reduced EBOV titer by ?6 logs.In summary, we found the Xpert® Ebola Assay to have high analytical sensitivity and specificity for the detection of EBOV in whole blood. It offers ease of use, fast turnaround time, and remote monitoring. The test has an efficient viral inactivation protocol, fulfills inclusivity and exclusivity criteria, and has specimen stability characteristics consistent with the need for decentralized testing. The simplicity of the assay should enable testing in a wide variety of laboratory settings, including remote laboratories that are not capable of performing highly complex nucleic acid amplification tests, and during outbreaks where time to detection is critical.

  View details for DOI 10.1371/journal.pone.0142216

  View details for Web of Science ID 000364480900020

  View details for PubMedID 26562786

  View details for PubMedCentralID PMC4643052

• Next-Generation Sequencing for Infectious Disease Diagnosis and Management A Report of the Association for Molecular Pathology JOURNAL OF MOLECULAR DIAGNOSTICS Lefterova, M. I., Suarez, C. J., Banaei, N., Pinsky, B. A. 2015; 17 (6): 623-634

  Abstract

  Next-generation sequencing (NGS) technologies are increasingly being used for diagnosis and monitoring of infectious diseases. Herein, we review the application of NGS in clinical microbiology, focusing on genotypic resistance testing, direct detection of unknown disease-associated pathogens in clinical specimens, investigation of microbial population diversity in the human host, and strain typing. We have organized the review into three main sections: i) applications in clinical virology, ii) applications in clinical bacteriology, mycobacteriology, and mycology, and iii) validation, quality control, and maintenance of proficiency. Although NGS holds enormous promise for clinical infectious disease testing, many challenges remain, including automation, standardizing technical protocols and bioinformatics pipelines, improving reference databases, establishing proficiency testing and quality control measures, and reducing cost and turnaround time, all of which would be necessary for widespread adoption of NGS in clinical microbiology laboratories.

  View details for DOI 10.1016/j.jmoldx.2015.07.004

  View details for Web of Science ID 000363830000001

  View details for PubMedID 26433313

• Molecular Testing for Plasmodium falciparum by Use of Serum or Plasma and Comparison with Microscopy and Rapid Diagnostic Testing in Febrile Nigerian Patients. Journal of clinical microbiology Waggoner, J. J., Okangba, C., Mohamed-Hadley, A., Lefterova, M. I., Banaei, N., Oyibo, W., Pinsky, B. A. 2015; 53 (11): 3596-3600

  Abstract

  Plasmodium nucleic acids have been detected in serum and plasma, but there is little published data describing the diagnostic performance of malaria nucleic acid amplification tests (NAATs) using these specimen types. Previously, our group described a multiplex NAAT for the detection of dengue virus, Leptospira, and Plasmodium species with a callout for P. falciparum (the DLM assay) that demonstrated sensitive detection of P. falciparum from plasma samples during initial evaluation. In this study, we evaluated the sensitivity and specificity of P. falciparum detection in febrile Nigerian patients using the DLM assay, microscopy, and a rapid diagnostic test (BinaxNOW Malaria). Assay performances were compared using a composite reference, which was considered positive if malaria was detected by two or more methods. Serum (n = 182) or plasma (n = 148) from 317 patients was tested; the average sample volume was 70 ?l (range, 5 to 300 ?l). The sensitivity and specificity of the DLM assay were 97.1% and 93.5%, respectively. The sensitivity of the malaria rapid diagnostic test (98.1%) was similar to that of the DLM assay, and both proved significantly more sensitive than microscopy (79%; P < 0.0001). When analysis was limited to samples with ?75 ?l of serum or plasma, the sensitivity of the DLM assay improved to 99% and specificity was 97.5%. For P. falciparum cases, cycle threshold values in the DLM assay correlated with the parasite density detected by microscopy (Spearman's rank correlation coefficient, P < 0.0001). In conclusion, malaria detection using the DLM assay on serum or plasma is more sensitive than and equal in specificity to microscopy in patients with P. falciparum malaria.

  View details for DOI 10.1128/JCM.01876-15

  View details for PubMedID 26354810

  View details for PubMedCentralID PMC4609704

• Limited Variation in BK Virus T-Cell Epitopes Revealed by Next-Generation Sequencing. Journal of clinical microbiology Sahoo, M. K., Tan, S. K., Chen, S. F., Kapusinszky, B., Concepcion, K. R., Kjelson, L., Mallempati, K., Farina, H. M., Fernández-Viña, M., Tyan, D., Grimm, P. C., Anderson, M. W., Concepcion, W., Pinsky, B. A. 2015; 53 (10): 3226-3233

  Abstract

  BK virus (BKV) infection and end-organ disease remains a formidable challenge to the hematopoietic cell transplant (HCT) and kidney transplant fields. As BKV-specific treatments are limited, immunologic-based therapies may be a promising and novel therapeutic option for transplant recipients with persistent BKV infection. Here, we describe a whole-genome, deep sequencing methodology and bioinformatics pipeline that identifies BKV variants across the genome and at BKV-specific HLA-A2, HLA-B0702, and HLA-B08 restricted CD8 T-cell epitopes. BKV whole genomes were amplified using long-range PCR with four inverse primer sets and fragmentation libraries were sequenced on the Ion Torrent PGM. An error model and variant calling algorithm were developed to accurately identify rare variants. 65 samples from 18 pediatric HCT and kidney recipients with quantifiable BKV DNAemia underwent whole-genome sequencing. Limited genetic variation was observed. The median number of amino acid variants identified per sample was 8 (range 2-37, interquartile range 10), with the majority of variants (77%) detected at a frequency of less than 5%. When normalized for length, there was no statistical difference in the median number of variants across all genes. Similarly, the predominant virus population within samples harbored T-cell epitopes similar to the reference BKV strain that was matched for BKV genotype. Despite the conservation of epitopes, low-level variants in T-cell epitopes were detected in 77.7% (14/18) of patients. Understanding epitope variation across the whole genome provides insight into the virus-immune interface and may help guide the development of protocols for novel immunologic-based therapies.

  View details for DOI 10.1128/JCM.01385-15

  View details for PubMedID 26202116

• Molecular Detection of Leptospira in Two Returned Travelers: Higher Bacterial Load in Cerebrospinal Fluid Versus Serum or Plasma. American journal of tropical medicine and hygiene Waggoner, J. J., Soda, E. A., Seibert, R., Grant, P., Pinsky, B. A. 2015; 93 (2): 238-240

  Abstract

  Leptospirosis is a potentially severe illness in returned travelers. Patients often present with fever, headache, and neck pain, which may lead to a workup for meningitis including the acquisition of cerebrospinal fluid (CSF). Although Leptospira DNA has been detected in CSF by polymerase chain reaction (PCR), little data exist regarding the utility of testing CSF in addition to serum or plasma obtained on presentation. In this report, we present two cases of leptospirosis in returned travelers presenting with fever and headache. Our first patient had neutrophilic meningitis, and Leptospira was detectable only in CSF obtained on admission. The second patient had a normal CSF profile, but Leptospira was detected in CSF at a bacterial load 5- to 10-fold higher than that in plasma. CSF is an important specimen for the diagnosis of Leptospira by molecular methods and may yield an actionable diagnosis in the absence of leptospiremia.

  View details for DOI 10.4269/ajtmh.15-0174

  View details for PubMedID 26033024

  View details for PubMedCentralID PMC4530740

• Cytomegalovirus load at treatment initiation is predictive of time to resolution of viremia and duration of therapy in hematopoietic cell transplant recipients JOURNAL OF CLINICAL VIROLOGY Tan, S. K., Waggoner, J. J., Pinsky, B. A. 2015; 69: 179-183

  Abstract

  Preemptive antiviral therapy relies on viral load measurements and is the mainstay of cytomegalovirus (CMV) prevention in hematopoietic cell transplant (HCT) recipients. However, optimal CMV levels for the initiation of preemptive therapy have not been defined.The objectives of our work were to evaluate the relationship between plasma CMV DNA levels at initiation of preemptive therapy with time to resolution of viremia and duration of treatment.Retrospective analysis of HCT recipients undergoing serial CMV PCR testing between June 2011 and June 2014 was performed.221 HCT recipients underwent preemptive therapy for 305 episodes of CMV viremia. Median time to resolution was shorter when treatment was initiated at lower CMV levels (15 days at 135-440 international units (IU)/mL, 18 days at 441-1000IU/mL, and 21 days at >1000IU/mL, P<.001). Prolonged viremia lasting >30 days occurred less frequently when treatment was initiated at 135-440IU/mL compared to 441-1000IU/mL and >1000IU/mL (1%, 15%, 24%, P<.001). Median treatment duration was also shorter in the lower viral load groups (28, 34, 37 days, P<.001).Initiation of preemptive therapy at low CMV levels was associated with shorter episodes of viremia and courses of antiviral therapy. These data support the utility of initiating preemptive CMV therapy at viral loads as low as 135IU/mL in HCT recipients.

  View details for DOI 10.1016/j.jcv.2015.06.006

  View details for Web of Science ID 000358318400037

• Fatal West Nile Virus Encephalitis in a Heart Transplant Recipient JOURNAL OF CLINICAL MICROBIOLOGY Gomez, A. J., Waggoner, J. J., Itoh, M., Hollander, S. A., Gutierrez, K. M., Budvytiene, I., Banaei, N., Pinsky, B. A. 2015; 53 (8): 2749-2752

  Abstract

  The diagnosis of encephalitis is particularly challenging in immunocompromised patients. We report here a case of fatal West Nile Virus encephalitis confounded by the presence of budding yeast in the CSF in a patient who had undergone heart transplantation for dilated cardiomyopathy 11 months prior to presentation of neurologic symptoms.

  View details for DOI 10.1128/JCM.00834-15

  View details for Web of Science ID 000358290200055

• Reverse-Transcriptase PCR Detection of Leptospira: Absence of Agreement with Single-Specimen Microscopic Agglutination Testing PLOS ONE Waggoner, J. J., Balassiano, I., Mohamed-Hadley, A., Vital-Brazil, J. M., Sahoo, M. K., Pinsky, B. A. 2015; 10 (7)

  Abstract

  Reference diagnostic tests for leptospirosis include nucleic acid amplification tests, bacterial culture, and microscopic agglutination testing (MAT) of acute and convalescent serum. However, clinical laboratories often do not receive paired specimens. In the current study, we tested serum samples using a highly sensitive real-time nucleic acid amplification test for Leptospira and compared results to MAT performed on the same specimens.478 serum samples from suspected leptospirosis cases in Rio de Janeiro were tested using a real-time RT-PCR for the diagnosis of leptospirosis, malaria and dengue (the Lepto-MD assay). The Lepto-MD assay detects all species of Leptospira (saprophytic, intermediate, and pathogenic), and in the current study, we demonstrate that this assay amplifies both Leptospira RNA and DNA. Dengue virus RNA was identified in 10 patients, and no cases of malaria were detected. A total of 65 samples (13.6%) were positive for Leptospira: 35 samples (7.3%) in the Lepto-MD assay, 33 samples (6.9%) by MAT, and 3 samples tested positive by both (kappa statistic 0.02). Poor agreement between methods was consistent regardless of the titer used to define positive MAT results or the day of disease at sample collection. Leptospira nucleic acids were detected in the Lepto-MD assay as late as day 22, and cycle threshold values did not differ based on the day of disease. When Lepto-MD assay results were added to the MAT results for all patients in 2008 (n=818), the number of detected leptospirosis cases increased by 30.4%, from 102 (12.5%) to 133 (16.3%).This study demonstrates a lack of agreement between nucleic acid detection of Leptospira and single-specimen MAT, which may result from the clearance of bacteremia coinciding with the appearance of agglutinating antibodies. A combined testing strategy for acute leptospirosis, including molecular and serologic testing, appears necessary to maximize case detection.

  View details for DOI 10.1371/journal.pone.0132988

  View details for Web of Science ID 000358197600195

  View details for PubMedCentralID PMC4503744

• How great is the threat of chikungunya virus? Expert review of anti-infective therapy Waggoner, J. J., Pinsky, B. A. 2015; 13 (3): 291-293

  Abstract

  In the last decade, chikungunya virus has emerged from an obscure arbovirus that caused limited outbreaks of disease in Africa and Asia to the cause of a pandemic affecting millions of people and spanning five continents. Two separate chikungunya virus genotypes have been responsible for outbreaks during this period, including strains adapted to transmission in Aedes albopictus mosquitoes. Further spread of this virus into new regions of the Western Hemisphere is predicted during the present rainy season in the tropics, and recurrent viral introductions and disease outbreaks, as occurred in Réunion in 2010, should be expected. Chikungunya virus no longer simply threatens; it has arrived as a significant, global pathogen.

  View details for DOI 10.1586/14787210.2015.995634

  View details for PubMedID 25537007

• Improved serotype-specific dengue virus detection in Trinidad and Tobago using a multiplex, real-time RT-PCR. Diagnostic microbiology and infectious disease Waggoner, J. J., Sahadeo, N. S., Brown, A., Mohamed-Hadley, A., Hadley, D., Carrington, L., Carrington, C. V., Pinsky, B. A. 2015; 81 (2): 105-106

  Abstract

  Dengue virus (DENV) transmission occurs throughout the Caribbean, though laboratory confirmation and epidemiologic surveillance are limited by the availability of serotype-specific molecular diagnostics. In this study, we show that a serotype-specific DENV multiplex, real-time reverse transcriptase-PCR (RT-PCR) detected DENV RNA in significantly more samples (82/182) than a reference hemi-nested RT-PCR (57/182; P=0.01).

  View details for DOI 10.1016/j.diagmicrobio.2014.10.001

  View details for PubMedID 25533614

  View details for PubMedCentralID PMC4297500

• Norovirus CLINICAL MICROBIOLOGY REVIEWS Robilotti, E., Deresinski, S., Pinsky, B. A. 2015; 28 (1): 134-164

  Abstract

  Norovirus, an RNA virus of the family Caliciviridae, is a human enteric pathogen that causes substantial morbidity across both health care and community settings. Several factors enhance the transmissibility of norovirus, including the small inoculum required to produce infection (<100 viral particles), prolonged viral shedding, and its ability to survive in the environment. In this review, we describe the basic virology and immunology of noroviruses, the clinical disease resulting from infection and its diagnosis and management, as well as host and pathogen factors that complicate vaccine development. Additionally, we discuss overall epidemiology, infection control strategies, and global reporting efforts aimed at controlling this worldwide cause of acute gastroenteritis. Prompt implementation of infection control measures remains the mainstay of norovirus outbreak management.

  View details for DOI 10.1128/CMR.00075-14

  View details for Web of Science ID 000347460400006

  View details for PubMedCentralID PMC4284304

• Fatal West Nile Virus Encephalitis in a Heart Transplant Recipient. Journal of clinical microbiology Gomez, A. J., Waggoner, J. J., Itoh, M., Hollander, S. A., Gutierrez, K. M., Budvytiene, I., Banaei, N., Pinsky, B. A. 2015

  Abstract

  The diagnosis of encephalitis is particularly challenging in immunocompromised patients. We report here a case of fatal West Nile Virus encephalitis confounded by the presence of budding yeast in the CSF in a patient who had undergone heart transplantation for dilated cardiomyopathy 11 months prior to presentation of neurologic symptoms.

  View details for PubMedID 25994169

• Reverse-Transcriptase PCR Detection of Leptospira: Absence of Agreement with Single-Specimen Microscopic Agglutination Testing. PloS one Waggoner, J. J., Balassiano, I., Mohamed-Hadley, A., Vital-Brazil, J. M., Sahoo, M. K., Pinsky, B. A. 2015; 10 (7)

  Abstract

  Reference diagnostic tests for leptospirosis include nucleic acid amplification tests, bacterial culture, and microscopic agglutination testing (MAT) of acute and convalescent serum. However, clinical laboratories often do not receive paired specimens. In the current study, we tested serum samples using a highly sensitive real-time nucleic acid amplification test for Leptospira and compared results to MAT performed on the same specimens.478 serum samples from suspected leptospirosis cases in Rio de Janeiro were tested using a real-time RT-PCR for the diagnosis of leptospirosis, malaria and dengue (the Lepto-MD assay). The Lepto-MD assay detects all species of Leptospira (saprophytic, intermediate, and pathogenic), and in the current study, we demonstrate that this assay amplifies both Leptospira RNA and DNA. Dengue virus RNA was identified in 10 patients, and no cases of malaria were detected. A total of 65 samples (13.6%) were positive for Leptospira: 35 samples (7.3%) in the Lepto-MD assay, 33 samples (6.9%) by MAT, and 3 samples tested positive by both (kappa statistic 0.02). Poor agreement between methods was consistent regardless of the titer used to define positive MAT results or the day of disease at sample collection. Leptospira nucleic acids were detected in the Lepto-MD assay as late as day 22, and cycle threshold values did not differ based on the day of disease. When Lepto-MD assay results were added to the MAT results for all patients in 2008 (n=818), the number of detected leptospirosis cases increased by 30.4%, from 102 (12.5%) to 133 (16.3%).This study demonstrates a lack of agreement between nucleic acid detection of Leptospira and single-specimen MAT, which may result from the clearance of bacteremia coinciding with the appearance of agglutinating antibodies. A combined testing strategy for acute leptospirosis, including molecular and serologic testing, appears necessary to maximize case detection.

  View details for DOI 10.1371/journal.pone.0132988

  View details for PubMedID 26177295

  View details for PubMedCentralID PMC4503744

• Norovirus. Clinical microbiology reviews Robilotti, E., Deresinski, S., Pinsky, B. A. 2015; 28 (1): 134-164

  Abstract

  Norovirus, an RNA virus of the family Caliciviridae, is a human enteric pathogen that causes substantial morbidity across both health care and community settings. Several factors enhance the transmissibility of norovirus, including the small inoculum required to produce infection (<100 viral particles), prolonged viral shedding, and its ability to survive in the environment. In this review, we describe the basic virology and immunology of noroviruses, the clinical disease resulting from infection and its diagnosis and management, as well as host and pathogen factors that complicate vaccine development. Additionally, we discuss overall epidemiology, infection control strategies, and global reporting efforts aimed at controlling this worldwide cause of acute gastroenteritis. Prompt implementation of infection control measures remains the mainstay of norovirus outbreak management.

  View details for DOI 10.1128/CMR.00075-14

  View details for PubMedID 25567225

  View details for PubMedCentralID PMC4284304

• Sensitive Real-Time PCR Detection of Pathogenic Leptospira spp. and a Comparison of Nucleic Acid Amplification Methods for the Diagnosis of Leptospirosis PLOS ONE Waggoner, J. J., Balassiano, I., Abeynayake, J., Sahoo, M. K., Mohamed-Hadley, A., Liu, Y., Vital-Brazil, J. M., Pinsky, B. A. 2014; 9 (11)

  Abstract

  Bacteria of the genus Leptospira, the causative agents of leptospirosis, are categorized into pathogenic and non-pathogenic species. However, the benefit of using a clinical diagnostic that is specific for pathogenic species remains unclear. In this study, we present the development of a real-time PCR (rtPCR) for the detection of pathogenic Leptospira (the pathogenic rtPCR), and we perform a comparison of the pathogenic rtPCR with a published assay that detects all Leptospira species [the undifferentiated febrile illness (UFI) assay] and a reference 16S Leptospira rtPCR, which was originally designed to detect pathogenic species.For the pathogenic rtPCR, a new hydrolysis probe was designed for use with primers from the UFI assay, which targets the 16S gene. The pathogenic rtPCR detected Leptospira DNA in 37/37 cultured isolates from 5 pathogenic and one intermediate species. Two strains of the non-pathogenic L. biflexa produced no signal. Clinical samples from 65 patients with suspected leptospirosis were then tested using the pathogenic rtPCR and a reference Leptospira 16S rtPCR. All 65 samples had tested positive for Leptospira using the UFI assay; 62 (95.4%) samples tested positive using the pathogenic rtPCR (p?=?0.24). Only 24 (36.9%) samples tested positive in the reference 16S rtPCR (p<0.0001 for comparison with the pathogenic rtPCR and UFI assays). Amplicon sequencing confirmed the detection of pathogenic Leptospira species in 49/50 cases, including 3 cases that were only detected using the UFI assay.The pathogenic rtPCR displayed similar sensitivity to the UFI assay when testing clinical specimens with no difference in specificity. Both assays proved significantly more sensitive than a real-time molecular test used for comparison. Future studies are needed to investigate the clinical and epidemiologic significance of more sensitive Leptospira detection using these tests.

  View details for DOI 10.1371/journal.pone.0112356

  View details for Web of Science ID 000344863100077

  View details for PubMedCentralID PMC4224423

• Commutability of the Epstein-Barr Virus WHO International Standard across Two Quantitative PCR Methods JOURNAL OF CLINICAL MICROBIOLOGY Abeynayake, J., Johnson, R., Libiran, P., Sahoo, M. K., Cao, H., Bowen, R., Chan, K. C., Quynh-Thu Le, Q. T., Pinsky, B. A. 2014; 52 (10): 3802-3804

  Abstract

  The commutability of international reference standards is critical for ensuring quantitative agreement across different viral load assays. Here, we demonstrate the commutability of the Epstein-Barr virus (EBV) WHO international standard for the BamHI-W and artus EBV assays.

  View details for DOI 10.1128/JCM.01676-14

  View details for Web of Science ID 000342371700042

  View details for PubMedCentralID PMC4187779

• Encephalitis caused by chikungunya virus in a traveler from the kingdom of tonga. Journal of clinical microbiology Nelson, J., Waggoner, J. J., Sahoo, M. K., Grant, P. M., Pinsky, B. A. 2014; 52 (9): 3459-3461

  Abstract

  Febrile travelers from countries with unique endemic pathogens pose a significant diagnostic challenge. In this report, we describe the case of a Tongan man presenting with fever, rash, and altered mental status. The diagnosis of Chikungunya encephalitis was made using a laboratory-developed real-time RT-PCR and serologic testing.

  View details for DOI 10.1128/JCM.01288-14

  View details for PubMedID 24958800

• Improved detection of emerging drug-resistant mutant cytomegalovirus subpopulations by deep sequencing. Antimicrobial agents and chemotherapy Chou, S., Ercolani, R. J., Sahoo, M. K., Lefterova, M. I., Strasfeld, L. M., Pinsky, B. A. 2014; 58 (8): 4697-4702

  Abstract

  In immunosuppressed hosts, the development of multidrug resistance complicates the treatment of cytomegalovirus (CMV) infection. Improved genotypic detection of impending drug resistance may follow from recent technical advances. A severely T-cell-depleted patient with chronic lymphocytic leukemia developed CMV pneumonia and high plasma viral loads that were poorly responsive to antiviral therapy. Serial plasma specimens were analyzed for mutant viral populations by conventional and high-throughput deep-sequencing methods. Uncharacterized mutations were phenotyped for drug resistance using recombinant viruses. Conventional genotyping detected viruses with the UL97 kinase substitution C607Y after ganciclovir treatment, a transient subpopulation of UL54 polymerase L773V mutants first detected 8 weeks after foscarnet was started, and a subpopulation of a mutant with deletion of UL54 codons 981 and 982 2 months after the addition of cidofovir. Deep sequencing of the same serial specimens revealed the same UL54 mutants sooner, along with a more complex evolution of known and newly recognized mutant subpopulations missed by conventional sequencing. The UL54 exonuclease substitutions D413N, K513R, and C539G were newly shown to confer ganciclovir-cidofovir resistance, while L773V was shown to confer foscarnet resistance and add to the ganciclovir resistance conferred by UL97 C607Y. Increased sequencing depth provided a more timely and detailed diagnosis of mutant viral subpopulations that evolved with changing anti-CMV therapy.

  View details for DOI 10.1128/AAC.03214-14

  View details for PubMedID 24890586

• Multiplex nucleic Acid amplification test for diagnosis of dengue Fever, malaria, and leptospirosis. Journal of clinical microbiology Waggoner, J. J., Abeynayake, J., Balassiano, I., Lefterova, M., Sahoo, M. K., Liu, Y., Vital-Brazil, J. M., Gresh, L., Balmaseda, A., Harris, E., Banaei, N., Pinsky, B. A. 2014; 52 (6): 2011-2018

  Abstract

  Dengue, leptospirosis, and malaria are among the most common etiologies of systemic undifferentiated febrile illness (UFI) among travelers to the developing world, and these pathogens all have the potential to cause life-threatening illness in returned travelers. The current study describes the development of an internally controlled multiplex nucleic acid amplification test for the detection of dengue virus (DENV) and Leptospira and Plasmodium species, with a specific callout for Plasmodium falciparum (referred to as the UFI assay). During analytical evaluation, the UFI assay displayed a wide dynamic range and a sensitive limit of detection for each target, including all four DENV serotypes. In a clinical evaluation including 210 previously tested samples, the sensitivities of the UFI assay were 98% for DENV (58/59 samples detected) and 100% for Leptospira and malaria (65/65 and 20/20 samples, respectively). Malaria samples included all five Plasmodium species known to cause human disease. The specificity of the UFI assay was 100% when evaluated with a panel of 66 negative clinical samples. Furthermore, no amplification was observed when extracted nucleic acids from related pathogens were tested. Compared with whole-blood samples, the UFI assay remained positive for Plasmodium in 11 plasma samples from patients with malaria (parasitemia levels of 0.0037 to 3.4%). The syndrome-based design of the UFI assay, combined with the sensitivities of the component tests, represents a significant improvement over the individual diagnostic tests available for these pathogens.

  View details for DOI 10.1128/JCM.00341-14

  View details for PubMedID 24671788

• Reply to "Inconclusive reverse transcription-PCR assay comparison for dengue virus detection and serotyping". Journal of clinical microbiology Waggoner, J. J., Pinsky, B. A. 2014; 52 (5): 1801-1802

  View details for DOI 10.1128/JCM.00013-14

  View details for PubMedID 24744403

• Evaluation of serial urine viral cultures for the diagnosis of cytomegalovirus infection in neonates and infants. Pediatric and developmental pathology Chisholm, K. M., Aziz, N., McDowell, M., Guo, F. P., Srinivas, N., Benitz, W. E., Norton, M. E., Gutierrez, K., Folkins, A. K., Pinsky, B. A. 2014; 17 (3): 176-180

  Abstract

  Cytomegalovirus (CMV) is the most common cause of congenital infection worldwide. Urine viral culture is the standard for CMV diagnosis in neonates and infants. The objectives of this study were to compare the performance of serial paired rapid shell vial cultures (SVC) and routine viral cultures (RVC), and to determine the optimal number of cultures needed to detect positive cases. From 2001 to 2011, all paired CMV SVC and RVC performed on neonates and infants less than 100 days of age were recorded. Testing episodes were defined as sets of cultures performed within 7 days of one another. A total of 1264 neonates and infants underwent 1478 testing episodes; 68 (5.4%) had at least one episode with a positive CMV culture. In episodes where CMV was detected before day 21 of life, the first specimen was positive in 100% (16/16) of cases. When testing occurred after 21 days of life, the first specimen was positive in 82.7% (43/52) of cases, requiring three cultures to reach 100% detection. The SVC was more prone to assay failure than RVC. Overall, when RVC was compared to SVC, there was 86.0% positive agreement and 99.9% negative agreement. In conclusion, three serial urine samples are necessary for detection of CMV in specimens collected between day of life 22 and 99, while one sample may be sufficient on or before day of life 21. Though SVC was more sensitive than RVC, the risk of SVC failure supports the use of multimodality testing to optimize detection.

  View details for DOI 10.2350/14-01-1432-OA.1

  View details for PubMedID 24617645

• Human Papilloma Virus is not Prevalent in Nevus Sebaceus PEDIATRIC DERMATOLOGY Kim, D., Benjamin, L. T., Sahoo, M. K., Kim, J., Pinsky, B. A. 2014; 31 (3): 326-330

  Abstract

  Nevus sebaceus (NS) is a common congenital cutaneous hamartoma that typically presents on the scalp and face at birth or in early childhood. Occasionally NS can be associated with the Schimmelpenning-Feuerstein-Mims syndrome, which presents with concomitant severe neurologic, skeletal, cardiovascular, ophthalmic, and genitourologic disorders. In a previous study, maternal transmission of the human papillomavirus (HPV) and infection of ectodermal stem cells by HPV was postulated to result in the development of NS. In this study we aimed to determine the incidence of HPV infection in pediatric NS samples to further clarify the potential link between HPV and the pathogenesis of NS. NS tissue samples (N = 16) were analyzed for HPV DNA using type-specific, real-time polymerase chain reaction (PCR) targeting HPV 6, 11, 16, and 18 and conventional PCR with modified general primers designed for broad-range HPV detection. The tissues were also histologically evaluated for evidence of HPV infection. HPV DNA was not detected in any of the NS tissue samples using PCR and HPV-associated histopathologic changes were absent in all 16 NS tissues. HPV infection is an unlikely etiologic cause of NS.

  View details for DOI 10.1111/pde.12249

  View details for PubMedID 24224641

• Comparison of automated nucleic acid extraction methods for the detection of cytomegalovirus DNA in fluids and tissues. PeerJ Waggoner, J. J., Pinsky, B. A. 2014; 2

  Abstract

  Testing for cytomegalovirus (CMV) DNA is increasingly being used for specimen types other than plasma or whole blood. However, few studies have investigated the performance of different nucleic acid extraction protocols in such specimens. In this study, CMV extraction using the Cell-free 1000 and Pathogen Complex 400 protocols on the QIAsymphony Sample Processing (SP) system were compared using bronchoalveolar lavage fluid (BAL), tissue samples, and urine. The QIAsymphonyAssay Set-up (AS) system was used to assemble reactions using artus CMV PCR reagents and amplification was carried out on the Rotor-Gene Q. Samples from 93 patients previously tested for CMV DNA and negative samples spiked with CMV AD-169 were used to evaluate assay performance. The Pathogen Complex 400 protocol yielded the following results: BAL, sensitivity 100% (33/33), specificity 87% (20/23); tissue, sensitivity 100% (25/25), specificity 100% (20/20); urine, sensitivity 100% (21/21), specificity 100% (20/20). Cell-free 1000 extraction gave comparable results for BAL and tissue, however, for urine, the sensitivity was 86% (18/21) and specimen quantitation was inaccurate. Comparative studies of different extraction protocols and DNA detection methods in body fluids and tissues are needed, as assays optimized for blood or plasma will not necessarily perform well on other specimen types.

  View details for DOI 10.7717/peerj.334

  View details for PubMedID 24765569

  View details for PubMedCentralID PMC3994632

• Sensitive real-time PCR detection of pathogenic Leptospira spp. and a comparison of nucleic acid amplification methods for the diagnosis of leptospirosis. PloS one Waggoner, J. J., Balassiano, I., Abeynayake, J., Sahoo, M. K., Mohamed-Hadley, A., Liu, Y., Vital-Brazil, J. M., Pinsky, B. A. 2014; 9 (11)

  Abstract

  Bacteria of the genus Leptospira, the causative agents of leptospirosis, are categorized into pathogenic and non-pathogenic species. However, the benefit of using a clinical diagnostic that is specific for pathogenic species remains unclear. In this study, we present the development of a real-time PCR (rtPCR) for the detection of pathogenic Leptospira (the pathogenic rtPCR), and we perform a comparison of the pathogenic rtPCR with a published assay that detects all Leptospira species [the undifferentiated febrile illness (UFI) assay] and a reference 16S Leptospira rtPCR, which was originally designed to detect pathogenic species.For the pathogenic rtPCR, a new hydrolysis probe was designed for use with primers from the UFI assay, which targets the 16S gene. The pathogenic rtPCR detected Leptospira DNA in 37/37 cultured isolates from 5 pathogenic and one intermediate species. Two strains of the non-pathogenic L. biflexa produced no signal. Clinical samples from 65 patients with suspected leptospirosis were then tested using the pathogenic rtPCR and a reference Leptospira 16S rtPCR. All 65 samples had tested positive for Leptospira using the UFI assay; 62 (95.4%) samples tested positive using the pathogenic rtPCR (p?=?0.24). Only 24 (36.9%) samples tested positive in the reference 16S rtPCR (p<0.0001 for comparison with the pathogenic rtPCR and UFI assays). Amplicon sequencing confirmed the detection of pathogenic Leptospira species in 49/50 cases, including 3 cases that were only detected using the UFI assay.The pathogenic rtPCR displayed similar sensitivity to the UFI assay when testing clinical specimens with no difference in specificity. Both assays proved significantly more sensitive than a real-time molecular test used for comparison. Future studies are needed to investigate the clinical and epidemiologic significance of more sensitive Leptospira detection using these tests.

  View details for DOI 10.1371/journal.pone.0112356

  View details for PubMedID 25379890

  View details for PubMedCentralID PMC4224423

• Comparison of automated nucleic acid extraction methods for the detection of cytomegalovirus DNA in fluids and tissues. PeerJ Waggoner, J. J., Pinsky, B. A. 2014; 2: e334

  Abstract

  Testing for cytomegalovirus (CMV) DNA is increasingly being used for specimen types other than plasma or whole blood. However, few studies have investigated the performance of different nucleic acid extraction protocols in such specimens. In this study, CMV extraction using the Cell-free 1000 and Pathogen Complex 400 protocols on the QIAsymphony Sample Processing (SP) system were compared using bronchoalveolar lavage fluid (BAL), tissue samples, and urine. The QIAsymphonyAssay Set-up (AS) system was used to assemble reactions using artus CMV PCR reagents and amplification was carried out on the Rotor-Gene Q. Samples from 93 patients previously tested for CMV DNA and negative samples spiked with CMV AD-169 were used to evaluate assay performance. The Pathogen Complex 400 protocol yielded the following results: BAL, sensitivity 100% (33/33), specificity 87% (20/23); tissue, sensitivity 100% (25/25), specificity 100% (20/20); urine, sensitivity 100% (21/21), specificity 100% (20/20). Cell-free 1000 extraction gave comparable results for BAL and tissue, however, for urine, the sensitivity was 86% (18/21) and specimen quantitation was inaccurate. Comparative studies of different extraction protocols and DNA detection methods in body fluids and tissues are needed, as assays optimized for blood or plasma will not necessarily perform well on other specimen types.

  View details for DOI 10.7717/peerj.334

  View details for PubMedID 24765569

  View details for PubMedCentralID PMC3994632

• BK Polyomavirus Subtype III in a Pediatric Renal Transplant Patient with Nephropathy. Journal of clinical microbiology Kapusinszky, B., Chen, S. F., Sahoo, M. K., Lefterova, M. I., Kjelson, L., Grimm, P. C., Kambham, N., Concepcion, W., Pinsky, B. A. 2013; 51 (12): 4255-4258

  Abstract

  BK polyomavirus (BKV) is an emerging pathogen in immunocompromised individuals. BKV subtype III is rarely identified and has not previously been associated with disease. Here we provide the whole-genome sequence of a subtype III BKV from a pediatric kidney transplant patient with polyomavirus-associated nephropathy.

  View details for DOI 10.1128/JCM.01801-13

  View details for PubMedID 24048534

  View details for PubMedCentralID PMC3838085

• Detection of cytomegalovirus drug resistance mutations by next-generation sequencing. Journal of clinical microbiology Sahoo, M. K., Lefterova, M. I., Yamamoto, F., Waggoner, J. J., Chou, S., Holmes, S. P., Anderson, M. W., Pinsky, B. A. 2013; 51 (11): 3700-3710

  Abstract

  Antiviral therapy for cytomegalovirus (CMV) plays an important role in the clinical management of solid organ and hematopoietic stem cell transplant recipients. However, CMV antiviral therapy can be complicated by drug resistance associated with mutations in the phosphotransferase UL97 and the DNA polymerase UL54. We have developed an amplicon-based high-throughput sequencing strategy for detecting CMV drug resistance mutations in clinical plasma specimens using a microfluidics PCR platform for multiplexed library preparation and a benchtop next-generation sequencing instrument. Plasmid clones of the UL97 and UL54 genes were used to demonstrate the low overall empirical error rate of the assay (0.189%) and to develop a statistical algorithm for identifying authentic low-abundance variants. The ability of the assay to detect resistance mutations was tested with mixes of wild-type and mutant plasmids, as well as clinical CMV isolates and plasma samples that were known to contain mutations that confer resistance. Finally, 48 clinical plasma specimens with a range of viral loads (394 to 2,191,011 copies/ml plasma) were sequenced using multiplexing of up to 24 specimens per run. This led to the identification of seven resistance mutations, three of which were present in <20% of the sequenced population. Thus, this assay offers more sensitive detection of minor variants and a higher multiplexing capacity than current methods for the genotypic detection of CMV drug resistance mutations.

  View details for DOI 10.1128/JCM.01605-13

  View details for PubMedID 23985916

• Profound plasmacytosis in a patient with dengue. International journal of hematology Ewalt, M. D., Abeynayake, J., Waggoner, J. J., Pinsky, B. A., Ohgami, R. S. 2013; 98 (5): 518-519

  View details for DOI 10.1007/s12185-013-1452-3

  View details for PubMedID 24114365

• Comparison of the FDA-Approved CDC DENV-1-4 Real-Time Reverse Transcription-PCR with a Laboratory-Developed Assay for Dengue Virus Detection and Serotyping. Journal of clinical microbiology Waggoner, J. J., Abeynayake, J., Sahoo, M. K., Gresh, L., Tellez, Y., Gonzalez, K., Ballesteros, G., Guo, F. P., Balmaseda, A., Karunaratne, K., Harris, E., Pinsky, B. A. 2013; 51 (10): 3418-3420

  Abstract

  Dengue virus (DENV) is the agent of the most common vector-borne disease worldwide. Using 199 clinical samples collected from Nicaragua and Sri Lanka, a laboratory-developed DENV multiplex real-time reverse transcription-PCR (rRT-PCR) proved more clinically sensitive than the FDA-approved CDC assay for DENV serotypes 1 to 4 when measured against a composite reference standard, with sensitivities of 97.4% versus 87.1%, respectively.

  View details for DOI 10.1128/JCM.01359-13

  View details for PubMedID 23903549

• Diagnosis of Congenital CMV Using PCR Performed on Formalin-fixed, Paraffin-embedded Placental Tissue. American journal of surgical pathology Folkins, A. K., Chisholm, K. M., Guo, F. P., McDowell, M., Aziz, N., Pinsky, B. A. 2013; 37 (9): 1413-1420

  Abstract

  Congenital cytomegalovirus (CMV) infection may be asymptomatic until hearing loss manifests in childhood. Because diagnosis of congenital CMV requires viral detection within an infant's first 21 days of life, CMV polymerase chain reaction (PCR) on formalin-fixed, paraffin-embedded (FFPE) placental tissue provides a unique opportunity to identify congenital exposure in cases in which CMV is not initially suspected. To assess the utility of this approach, a database of all CMV cultures performed from July 2001 to March 2012 was used to identify infants in whom urine CMV cultures were obtained within 100 days of life. Corresponding placentas were then identified through the pathology database. The database was also queried to identify placentas in which CMV immunohistochemical analysis had been performed. CMV PCR was positive in FFPE placental tissue from 100% (5/5) of cases in which the first urine culture collected before the first 21 days of life was positive. Placentas from 20 infants with negative CMV urine cultures were CMV PCR negative. Interestingly, CMV was detected in 12.5% (1/8) of placentas in which the first CMV-positive urine culture was collected after the first 21 days of life. Furthermore, 4% (1/26) of placentas with chronic villitis by histology (no urine cultures available) were CMV PCR positive. In the 10 CMV PCR-positive placentas, including 3 cases of fetal demise, CMV immunohistochemistry was positive in just 6 cases. These results suggest that the confirmation of CMV exposure in utero by PCR of FFPE placental tissue provides a useful adjunct to histologic evaluation and may identify infants requiring close clinical follow-up.

  View details for DOI 10.1097/PAS.0b013e318290f171

  View details for PubMedID 23797721

• A comparison of CMV detection in gastrointestinal mucosal biopsies using immunohistochemistry and PCR performed on formalin-fixed, paraffin-embedded tissue. American journal of surgical pathology Mills, A. M., Guo, F. P., Copland, A. P., Pai, R. K., Pinsky, B. A. 2013; 37 (7): 995-1000

  Abstract

  Cytomegalovirus (CMV) can precipitate and exacerbate gastrointestinal (GI) mucosal injury. The gold standard for CMV detection in formalin-fixed, paraffin-embedded (FFPE) tissue is immunohistochemistry (IHC). Although CMV polymerase chain reaction (PCR) on fresh tissue may be a valuable adjunct to IHC, its utility is unknown for FFPE tissues. We therefore evaluated quantitative, real-time CMV PCR in a total of 102 FFPE GI biopsy specimens from 74 patients with a history of hematopoietic stem cell or solid organ transplant, inflammatory bowel disease, human immunodeficiency virus infection, or unspecified colitis. CMV DNA was detected by PCR in 90.9% (30/33) of IHC-positive, 14.5% (8/55) of IHC-negative, and 20.0% (1/5) of IHC-equivocal FFPE tissues. Quantitation of CMV DNA copies normalized to ?-globin demonstrated a wide range of values (median 0.276; range, 0.0004 to 144.50). Importantly, 93.3% (14/15) of patients with IHC-positive, active colitis showed no evidence of CMV in matched concurrent, histologically normal biopsies tested by PCR. These results suggest that CMV PCR on FFPE GI biopsies complements IHC and has the potential to identify additional patients who may benefit from anti-CMV therapy.

  View details for DOI 10.1097/PAS.0b013e31827fcc33

  View details for PubMedID 23648457

• Development of an internally controlled real-time reverse transcriptase PCR assay for pan-dengue virus detection and comparison of four molecular dengue virus detection assays. Journal of clinical microbiology Waggoner, J. J., Abeynayake, J., Sahoo, M. K., Gresh, L., Tellez, Y., Gonzalez, K., Ballesteros, G., Balmaseda, A., Karunaratne, K., Harris, E., Pinsky, B. A. 2013; 51 (7): 2172-2181

  Abstract

  A number of diagnostic tests are available for dengue virus (DENV) detection, including a variety of nucleic-acid amplification tests (NAATs). However, reports describing the direct comparison of different NAATs are limited. In this study, we report the design of an internally-controlled, real-time reverse-transcriptase PCR (rRT-PCR) that detects all four DENV serotypes but does not distinguish between them (the pan-DENV assay). Two-hundred clinical samples were then tested using four different DENV RT-PCR assays: the pan-DENV assay; a commercially-produced, internally-controlled DENV rRT-PCR (the Altona assay); a widely-used hemi-nested RT-PCR; and a serotype-specific, multiplex rRT-PCR assay. The pan-DENV assay had a linear range extending from 7.0 to 1.0 log10 complimentary DNA (cDNA) equivalents/?L and a lower limit of 95% detection ranging from 1.7 to 7.6 cDNA equivalents/?L depending on the serotype. When measured against a composite reference standard, the pan-DENV assay proved more clinically sensitive than either the Altona or hemi-nested assays, with a sensitivity of 98.0% compared to 72.3% and 78.8%, respectively (p?0.0001 for both comparisons). The pan-DENV assay detected DENV in significantly more samples collected on or after day five of illness and in a subgroup of patients with detectable anti-DENV IgM at presentation. No significant difference in sensitivity was observed between the pan-DENV assay and the multiplex rRT-PCR, despite the presence of an internal control in the former. The detection of DENV RNA late in the course of clinical illness should serve to lengthen the period during which a confirmed, molecular diagnosis of DENV infection can be provided.

  View details for DOI 10.1128/JCM.00548-13

  View details for PubMedID 23637298

• p16 Is Superior to ProEx C in Identifying High-grade Squamous Intraepithelial Lesions (HSIL) of the Anal Canal AMERICAN JOURNAL OF SURGICAL PATHOLOGY Bala, R., Pinsky, B. A., Beck, A. H., Kong, C. S., Welton, M. L., Longacre, T. A. 2013; 37 (5): 659-668

  Abstract

  Although the incidence of human papillomavirus (HPV)-associated anal neoplasia is increasing, interobserver and intraobserver reproducibility in the grading of biopsy specimens from this area remains unacceptably low. Attempts to produce a more reproducible grading scheme have led to the use of biomarkers for the detection of high-risk HPV (HR-HPV). We evaluated the performance of standard morphology and biomarkers p16, ProEx C, and Ki-67 in a set of 75 lesions [17 nondysplastic lesions, 23 low-grade squamous intraepithelial lesions (LSIL)/condyloma, 20 high-grade squamous intraepithelial lesions (HSIL), 15 invasive squamous cell carcinomas] from the anal and perianal region in 65 patients and correlated these findings with HPV subtype on the basis of a type-specific multiplex real-time polymerase chain reaction assay designed to detect HR-HPV. A subset of cases with amplifiable HPV DNA was also sequenced. HSIL was typically flat (15/20), and only a minority (4/20) had koilocytes. In contrast, only 1 LSIL was flat (1/23), and the remainder were exophytic. The majority of LSIL had areas of koilocytic change (20/23). HR-HPV DNA was detected in the majority (89%) of invasive carcinomas and HSIL biopsies, 86% and 97% of which were accurately labeled by strong and diffuse block-positive p16 and ProEx C, respectively. LSIL cases, however, only infrequently harbored HR-HPV (13%); most harbored low-risk HPV (LR-HPV) types 6 and 11. Within the LSIL group, p16 outperformed ProEx C, resulting in fewer false-positive cases (5% vs. 75%). Ki-67 was also increased in HR-HPV-positive lesions, although biopsies with increased inflammation and reactive changes also showed higher Ki-67 indices. These data suggest that strong and diffuse block-positive nuclear and cytoplasmic labeling with p16 is a highly specific biomarker for the presence of HR-HPV in anal biopsies and that this finding correlates with high-grade lesions.

  View details for DOI 10.1097/PAS.0b013e31828706c0

  View details for PubMedID 23552383

• An international collaboration to harmonize the quantitative plasma Epstein-Barr virus DNA assay for future biomarker-guided trials in nasopharyngeal carcinoma. Clinical cancer research Le, Q., Zhang, Q., Cao, H., Cheng, A., Pinsky, B. A., Hong, R., Chang, J. T., Wang, C., Tsao, K., Lo, Y. D., Lee, N., Ang, K. K., Chan, A. T., Chan, K. C. 2013; 19 (8): 2208-2215

  Abstract

  Persistently elevated posttreatment plasma EBV DNA is a robust predictor of relapse in nasopharyngeal carcinoma (NPC). However, assay standardization is necessary for use in biomarker-driven trials. We conducted a study to harmonize the method between four centers with expertise in EBV DNA quantitation.Plasma samples of 40 patients with NPC were distributed to four centers. DNA was extracted and EBV DNA copy number was determined by real-time quantitative PCR (BamHI-W primer/probe). Centers used the same protocol but generated their own calibrators. A harmonization study was then conducted using the same calibrators and PCR master mix and validated with ten pooled samples.The initial intraclass correlations (ICC) for the first 40 samples between each center and the index center were 0.62 [95% confidence interval (CI): 0.39-0.78], 0.70 (0.50-0.83), and 0.59 (0.35-0.76). The largest variability was the use of different PCR master mixes and calibrators. Standardization improved ICC to 0.83 (0.5-0.95), 0.95 (0.83-0.99) and 0.96 (0.86-0.99), respectively, for ten archival frozen samples. For fresh plasma with spiked-in EBV DNA, correlations were more than 0.99 between the centers. At 5 EBV DNA copies per reaction or above, the coefficient of variance (CV) was less than 10% for the cycle threshold (Ct) among all centers, suggesting this concentration can be reliably used as a cutoff for defining the presence of detectable EBV DNA.Quantitative PCR assays, even when conducted in experienced clinical labs, can yield large variability in plasma EBV DNA copy numbers without harmonization. The use of common calibrators and PCR master mix can help to reduce variability.

  View details for DOI 10.1158/1078-0432.CCR-12-3702

  View details for PubMedID 23459720

  View details for PubMedCentralID PMC3630245

• CMV antigenemia and quantitative viral load assessments in hematopoietic stem cell transplant recipients JOURNAL OF CLINICAL VIROLOGY Cardenoso, L., Pinsky, B. A., Lautenschlager, I., Aslam, S., Cobb, B., Vilchez, R. A., Hirsch, H. H. 2013; 56 (2): 108-112

  Abstract

  Sensitive and reliable diagnostic tests are essential for the prevention of cytomegalovirus (CMV) disease after hematopoietic stem cell transplantation (HSCT). pp65 antigenemia and polymerase chain reaction (PCR) assays are commonly used to monitor CMV in HSCT recipients. However, there is considerable intra- and inter-laboratory variability in the results, which impact comparability and clinical practice. OBJECTIVES/STUDY DESIGN: Using 380 samples from 135 HSCT recipients, we compared the new FDA approved quantitative PCR assay, COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) CMV test (CAP/CTM CMV test) developed and standardized using the 1st WHO International Standard for CMV with pp65 antigenemia and COBAS(®) AMPLICOR MONITOR CMV tests.The median time between transplantation and testing samples was 57 days (range, 0-207 days). The median CMV load (log(10)) was 3.17 IU/mL (3.21 copies/mL). Among samples with detectable CMV load, 52% were negative by pp65 antigenemia. CMV loads were higher in pp65 antigenemia-positive than in negative samples. One pp65-antigenemia-positive cell per 100,000 leukocytes corresponded to a median CMV load of 1200 IU/mL. CMV loads determined by the CAP/CTM CMV test were slightly lower than the ones by the AMPLICOR MONITOR CMV test (-0.15 [95% CI, -0.18 to -0.13] copies/mL), but slope differences indicated only limited co-linearity.The CAP/CTM CMV test is more sensitive than pp65 antigenemia and the AMPLICOR MONITOR CMV test in HSCT recipients. The lower limit of quantification and co-linearity with the international WHO standard renders the CAP/CTM CMV test suitable for future clinical trials defining viral load thresholds of CMV therapy.

  View details for DOI 10.1016/j.jcv.2012.10.001

  View details for Web of Science ID 000313565100004

  View details for PubMedID 23146665

• An International Multicenter Performance Analysis of Cytomegalovirus Load Tests CLINICAL INFECTIOUS DISEASES Hirsch, H. H., Lautenschlager, I., Pinsky, B. A., Cardenoso, L., Aslam, S., Cobb, B., Vilchez, R. A., Valsamakis, A. 2013; 56 (3): 367-373

  Abstract

  Quantification of cytomegalovirus (CMV) load is central to the management of CMV infections in immunocompromised patients, but quantitative results currently differ significantly across methods and laboratories.The COBAS AmpliPrep/COBAS TaqMan CMV Test (CAP/CTM CMV test), developed using the first World Health Organization CMV standard in the calibration process, was compared to local assays used by 5 laboratories at transplant centers in the United States and Europe. Blinded plasma panels (n = 90) spiked with 2.18-6.7 log(10) copies/mL and clinical plasma samples from immunocompromised patients (n = 660) were tested.Observed mean panel member concentrations by site and 95% confidence intervals (CIs) of the data combined across sites were narrower for CAP/CTM CMV test compared with local assays. The 95% CI in log(10) copies/mL of the combined data per panel member for CAP/CTM CMV test vs comparator assays was .17 vs 1.5 at 2.18 log(10) copies/mL; .14 vs .52 at 2.74 log(10) copies/mL; .16 vs .6 at 3.3 log(10) copies/mL; .2 vs 1.11 at 4.3 log(10) copies/mL; .21 vs 1.13 at 4.7 log(10) copies/mL; and .18 vs 1.4 at 6.7 log(10) copies/mL. In clinical specimens, constant and variable quantification differences between the CAP/CTM CMV test and comparator assays were observed.High interlaboratory agreement and precision of CAP/CTM CMV test results across 5 different laboratories over 4 orders of magnitude suggest that this assay could be valuable in prospective studies identifying clinical viral load thresholds for CMV treatment.

  View details for DOI 10.1093/cid/cis900

  View details for Web of Science ID 000313617400014

  View details for PubMedID 23097587

  View details for PubMedCentralID PMC3540041

• Severe Hepatitis Associated with an Echovirus 18 Infection in an Immune-Compromised Adult JOURNAL OF CLINICAL MICROBIOLOGY Lefterova, M. I., Rivetta, C., George, T. I., Pinsky, B. A. 2013; 51 (2): 684-687

  Abstract

  Enteroviruses are recognized as important pathogens in pediatric patients; however, they are often overlooked as etiologic agents of disease in adults. Here, we report a case of echovirus 18-associated severe systemic infection and acute liver failure in an adult hematopoietic stem cell transplant recipient. Additionally, we illustrate the utility of molecular methods for the detection and typing of enteroviral infections.

  View details for DOI 10.1128/JCM.02405-12

  View details for PubMedID 23175267

• Laboratory-Developed L1 Sequencing and Type-Specific, Real-Time Polymerase Chain Reaction for the Detection and Typing of Human Papillomaviruses in Formalin-Fixed, Paraffin-Embedded Tissues ARCHIVES OF PATHOLOGY & LABORATORY MEDICINE Mills, A., Balasubramaniam, R., Longacre, T. A., Kong, C. S., Pinsky, B. A. 2013; 137 (1): 50-54

  Abstract

  The detection and typing of high-risk and low-risk human papillomavirus (HPV) in archival formalin-fixed, paraffin-embedded tissues by nucleic acid amplification testing is an important adjunct to immunohistochemical staining in evaluation of squamous cell proliferations of the oropharynx, larynx, and anal canal.To evaluate semiautomated, xylene-free extraction from formalin-fixed, paraffin-embedded tissues combined with laboratory-developed HPV L1 sequencing and type-specific HPV 6, 11, 16, and 18 real-time polymerase chain reaction for identification and typing of HPV in the clinical laboratory.We evaluated the adequacy of extraction using ?-globin amplification and compared L1 sequencing and real-time polymerase chain reaction methods for typing accuracy using 68 formalin-fixed, paraffin-embedded tissues, including 56 anorectal biopsy or surgical resection specimens and 12 laryngeal papilloma specimens from patients with recurrent respiratory papillomatosis.Adequate DNA was obtained from 68 of 68 specimens analyzed and all were HPV positive. In 47 cases where L1 sequencing demonstrated that the predominant HPV type was 6, 11, 16, or 18, type-specific, real-time polymerase chain reaction provided concordant results. Sequencing revealed additional low-risk (HPV 40) and high-risk HPV types (HPV 31, 33, 56, and 58) in anorectal specimens, whereas HPV 6 or 11 were the types found in laryngeal papillomas.Both L1 sequencing and type-specific, real-time polymerase chain reaction are suitable methods for routine HPV testing of formalin-fixed, paraffin-embedded tissues in a clinical laboratory setting.

  View details for DOI 10.5858/arpa.2011-0392-OA

  View details for PubMedID 23276174

• Profound plasmacytosis in a patient with dengue International Journal of Hematology Ewalt, M. D., Abeynayake, J., Waggoner, J. ., Pinsky, B. ., Ohgami, R. S. 2013: 518?19
• Single-reaction, multiplex, real-time rt-PCR for the detection, quantitation, and serotyping of dengue viruses. PLoS neglected tropical diseases Waggoner, J. J., Abeynayake, J., Sahoo, M. K., Gresh, L., Tellez, Y., Gonzalez, K., Ballesteros, G., Pierro, A. M., Gaibani, P., Guo, F. P., Sambri, V., Balmaseda, A., Karunaratne, K., Harris, E., Pinsky, B. A. 2013; 7 (4)

  Abstract

  Dengue fever results from infection with one or more of four different serotypes of dengue virus (DENV). Despite the widespread nature of this infection, available molecular diagnostics have significant limitations. The aim of this study was to develop a multiplex, real-time, reverse transcriptase-PCR (rRT-PCR) for the detection, quantitation, and serotyping of dengue viruses in a single reaction.An rRT-PCR assay targeting the 5' untranslated region and capsid gene of the DENV genome was designed using molecular beacons to provide serotype specificity. Using reference DENV strains, the assay was linear from 7.0 to 1.0 log?? cDNA equivalents/µL for each serotype. The lower limit of detection using genomic RNA was 0.3, 13.8, 0.8, and 12.4 cDNA equivalents/µL for serotypes 1-4, respectively, which was 6- to 275-fold more analytically sensitive than a widely used hemi-nested RT-PCR. Using samples from Nicaragua collected within the first five days of illness, the multiplex rRT-PCR was positive in 100% (69/69) of specimens that were positive by the hemi-nested assay, with full serotype agreement. Furthermore, the multiplex rRT-PCR detected DENV RNA in 97.2% (35/36) of specimens from Sri Lanka positive for anti-DENV IgM antibodies compared to just 44.4% (16/36) by the hemi-nested RT-PCR. No amplification was observed in 80 clinical samples sent for routine quantitative hepatitis C virus testing or when genomic RNA from other flaviviruses was tested.This single-reaction, quantitative, multiplex rRT-PCR for DENV serotyping demonstrates superior analytical and clinical performance, as well as simpler workflow compared to the hemi-nested RT-PCR reference. In particular, this multiplex rRT-PCR detects viral RNA and provides serotype information in specimens collected more than five days after fever onset and from patients who had already developed anti-DENV IgM antibodies. The implementation of this assay in dengue-endemic areas has the potential to improve both dengue diagnosis and epidemiologic surveillance.

  View details for DOI 10.1371/journal.pntd.0002116

  View details for PubMedID 23638191

  View details for PubMedCentralID PMC3630127

• Comparison of Xpert Flu rapid nucleic acid testing with rapid antigen testing for the diagnosis of influenza A and B JOURNAL OF VIROLOGICAL METHODS DiMaio, M. A., Sahoo, M. K., Waggoner, J., Pinsky, B. A. 2012; 186 (1-2): 137-140

  Abstract

  Influenza infections are associated with thousands of hospital admissions and deaths each year. Rapid detection of influenza is important for prompt initiation of antiviral therapy and appropriate patient triage. In this study the Cepheid Xpert Flu assay was compared with two rapid antigen tests, BinaxNOW Influenza A & B and BD Directigen EZ Flu A+B, as well as direct fluorescent antibody testing for the rapid detection of influenza A and B. Using real-time, hydrolysis probe-based, reverse transcriptase PCR as the reference method, influenza A sensitivity was 97.3% for Xpert Flu, 95.9% for direct fluorescent antibody testing, 62.2% for BinaxNOW, and 71.6% for BD Directigen. Influenza B sensitivity was 100% for Xpert Flu and direct fluorescent antibody testing, 54.5% for BinaxNOW, and 48.5% for BD Directigen. Specificity for influenza A was 100% for Xpert Flu, BinaxNOW, and BD Directigen, and 99.2% for direct fluorescent antibody testing. All methods demonstrated 100% specificity for influenza B. These findings support the use of the Xpert Flu assay in settings requiring urgent diagnosis of influenza A and B.

  View details for DOI 10.1016/j.jviromet.2012.07.023

  View details for Web of Science ID 000312763600024

  View details for PubMedID 22841669

• Clinical Significance of Low Cytomegalovirus DNA Levels in Human Plasma JOURNAL OF CLINICAL MICROBIOLOGY Waggoner, J., Ho, D. Y., Libiran, P., Pinsky, B. A. 2012; 50 (7): 2378-2383

  Abstract

  The clinical significance of the detection of low copy numbers of cytomegalovirus (CMV) DNA in immune-suppressed patients remains unclear. In this study, we compared the artus CMV Rotor-Gene PCR, utilizing an automated nucleic acid extraction and assay setup (the artus CMV protocol), with the COBAS Amplicor CMV Monitor test (our reference protocol). We then analyzed the results of all CMV PCR tests ordered following the implementation of the artus CMV protocol at our institution and followed 91 adult patients with positive test results. The artus CMV protocol had a linear range extending from 2.0 to 7.0 log(10) copies/ml and had a lower limit of 95% detection of 57 copies/ml. With archived plasma samples, this protocol demonstrated 100% sensitivity and 94% specificity for the detection of CMV DNA. Following implementation of the artus CMV protocol, 320 of 1,403 (22.8%) plasma samples tested positive (compared with 323/3,579 [9.0%] samples in the preceding 6 months), and 227 (16.2%) samples had copy numbers of <400/ml. Ninety-one adult patients had at least one positive test. The data were analyzed using a threshold of 200 copies/ml, and in 22 episodes, the viral load increased from <200 copies/ml to ? 200 copies/ml on sequential tests. In 21 of these 22 episodes, either the viral load continued to increase or antiviral treatment was initiated in response to the repeat value. In summary, we evaluate the performance characteristics of a protocol utilizing the artus CMV PCR and identify clinically meaningful changes in CMV DNA copy numbers even when they are initially detected at a low level.

  View details for DOI 10.1128/JCM.06800-11

  View details for Web of Science ID 000307360800033

  View details for PubMedID 22518866

  View details for PubMedCentralID PMC3405616

• Quantitation of Human Papillomavirus DNA in Plasma of Oropharyngeal Carcinoma Patients INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS Cao, H., Banh, A., Kwok, S., Shi, X., Wu, S., Krakow, T., Khong, B., Bavan, B., Bala, R., Pinsky, B. A., Colevas, D., Pourmand, N., Koong, A. C., Kong, C. S., Quynh-Thu Le, Q. T. 2012; 82 (3): E351-E358

  Abstract

  To determine whether human papillomavirus (HPV) DNA can be detected in the plasma of patients with HPV-positive oropharyngeal carcinoma (OPC) and to monitor its temporal change during radiotherapy.We used polymerase chain reaction to detect HPV DNA in the culture media of HPV-positive SCC90 and VU147T cells and the plasma of SCC90 and HeLa tumor-bearing mice, non-tumor-bearing controls, and those with HPV-negative tumors. We used real-time quantitative polymerase chain reaction to quantify the plasma HPV DNA in 40 HPV-positive OPC, 24 HPV-negative head-and-neck cancer patients and 10 non-cancer volunteers. The tumor HPV status was confirmed by p16(INK4a) staining and HPV16/18 polymerase chain reaction or HPV in situ hybridization. A total of 14 patients had serial plasma samples for HPV DNA quantification during radiotherapy.HPV DNA was detectable in the plasma samples of SCC90- and HeLa-bearing mice but not in the controls. It was detected in 65% of the pretreatment plasma samples from HPV-positive OPC patients using E6/7 quantitative polymerase chain reaction. None of the HPV-negative head-and-neck cancer patients or non-cancer controls had detectable HPV DNA. The pretreatment plasma HPV DNA copy number correlated significantly with the nodal metabolic tumor volume (assessed using (18)F-deoxyglucose positron emission tomography). The serial measurements in 14 patients showed a rapid decline in HPV DNA that had become undetectable at radiotherapy completion. In 3 patients, the HPV DNA level had increased to a discernable level at metastasis.Xenograft studies indicated that plasma HPV DNA is released from HPV-positive tumors. Circulating HPV DNA was detectable in most HPV-positive OPC patients. Thus, plasma HPV DNA might be a valuable tool for identifying relapse.

  View details for DOI 10.1016/j.ijrobp.2011.05.061

  View details for PubMedID 21985946

• A Synonymous Change in the Influenza A Virus Neuraminidase Gene Interferes with PCR-Based Subtyping and Oseltamivir Resistance Mutation Detection JOURNAL OF CLINICAL MICROBIOLOGY Trevino, C., Bihon, S., Pinsky, B. A. 2011; 49 (8): 3101-3102

  View details for DOI 10.1128/JCM.00642-11

  View details for Web of Science ID 000293221900067

  View details for PubMedID 21697334

  View details for PubMedCentralID PMC3147745

• Ultrasensitive Detection of Drug-Resistant Pandemic 2009 (H1N1) Influenza A Virus by Rare-Variant-Sensitive High-Resolution Melting-Curve Analysis JOURNAL OF CLINICAL MICROBIOLOGY Chen, N., Pinsky, B. A., Lee, B. P., Lin, M., Schrijver, I. 2011; 49 (7): 2602-2609

  Abstract

  Oseltamivir (Tamiflu), an oral neuraminidase inhibitor, has been widely used to treat pandemic 2009 (H1N1) influenza A. Although a majority of 2009 (H1N1) influenza A virus remains oseltamivir susceptible, the threat of resistance due to the His275Tyr mutation is highlighted by the limitations of alternative therapies and the potential for rapid, global fixation of this mutation in the circulating influenza A virus population. In order to better understand the emergence of resistance, we developed a rare-variant-sensitive high-resolution melting-curve analysis method (RVS-HRM) that is able to detect the His275Tyr oseltamivir resistance mutation to 0.5% in a background of susceptible virus. We applied RVS-HRM to clinical specimens from patients who developed oseltamivir resistance and demonstrated the ultrasensitive detection of influenza A virus N1 neuraminidase quasispecies. Interestingly, we were unable to detect the oseltamivir resistance mutation in pretreatment samples, suggesting that resistant virus does not reach even this very low detection threshold until exposed to selective drug pressure. Thus, patients naive to oseltamivir are most likely to be susceptible when this drug is used as a first-line treatment modality.

  View details for DOI 10.1128/JCM.00277-11

  View details for Web of Science ID 000292276200035

  View details for PubMedID 21543559

  View details for PubMedCentralID PMC3147852

• The Persistence of Influenza Infection Response EMERGING INFECTIOUS DISEASES Pinsky, B., Baron, E. J. 2010; 16 (11): 1818-1819

  View details for DOI 10.3201/eid1611.101431

  View details for Web of Science ID 000283699700043

• Long-term Shedding of Influenza A Virus in Stool of Immunocompromised Child EMERGING INFECTIOUS DISEASES Pinsky, B. A., Mix, S., Rowe, J., Ikemoto, S., Baron, E. J. 2010; 16 (7): 1165-1167

  Abstract

  In immunocompromised patients, influenza infection may progress to prolonged viral shedding from the respiratory tract despite antiviral therapy. We describe chronic influenza A virus infection in an immunocompromised child who had prolonged shedding of culturable influenza virus in stool.

  View details for DOI 10.3201/eid1607.091248

  View details for Web of Science ID 000279522200024

  View details for PubMedID 20587197

  View details for PubMedCentralID PMC3321893

• Real-Time PCR Testing for mecA Reduces Vancomycin Usage and Length of Hospitalization for Patients Infected with Methicillin-Sensitive Staphylococci JOURNAL OF CLINICAL MICROBIOLOGY Nguyen, D. T., Yeh, E., Perry, S., Luo, R. F., Pinsky, B. A., Lee, B. P., Sisodiya, D., Baron, E. J., Banaei, N. 2010; 48 (3): 785-790

  Abstract

  Nucleic acid amplification tests (NAATs) have revolutionized infectious disease diagnosis, allowing for the rapid and sensitive identification of pathogens in clinical specimens. Real-time PCR testing for the mecA gene (mecA PCR), which confers methicillin resistance in staphylococci, has the added potential to reduce antibiotic usage, improve clinical outcomes, lower health care costs, and avoid emergence of drug resistance. A retrospective study was performed to identify patients infected with methicillin-sensitive staphylococcal isolates who were receiving vancomycin treatment when susceptibility results became available. Vancomycin treatment and length of hospitalization were compared in these patients for a 6-month period before and after implementation of mecA PCR. Among 65 and 94 patients identified before and after mecA PCR, respectively, vancomycin usage (measured in days on therapy) declined from a median of 3 days (range, 1 to 44 days) in the pre-PCR period to 1 day (range, 0 to 18 days) in the post-PCR period (P < 0.0001). In total, 38.5% (25/65) of patients were switched to beta-lactam therapy in the pre-PCR period, compared to 61.7% (58/94) in the post-PCR period (P = 0.004). Patient hospitalization days also declined from a median of 8 days (range, 1 to 47 days) in the pre-PCR period to 5 days (range, 0 to 42 days) in the post-PCR period (P = 0.03). Real-time PCR testing for mecA is an effective tool for reducing vancomycin usage and length of stay of hospitalized patients infected with methicillin-sensitive staphylococci. In the face of ever-rising health care expenditures in the United States, these findings have important implications for improving outcomes and decreasing costs.

  View details for DOI 10.1128/JCM.02150-09

  View details for Web of Science ID 000274996200016

  View details for PubMedID 20071556

  View details for PubMedCentralID PMC2832423

• Preferential Lower Respiratory Tract Infection in Swine-Origin 2009 A(H1N1) Influenza CLINICAL INFECTIOUS DISEASES Yeh, E., Luo, R. F., Dyner, L., Hong, D. K., Banaei, N., Baron, E. J., Pinsky, B. A. 2010; 50 (3): 391-394

  Abstract

  We report a case of 2009 influenza A(H1N1) virus infection in which virus was detected predominantly in specimens from the lower respiratory tract but was absent or at very low levels in nasopharyngeal swab samples. This presentation suggests that, in certain hosts or for particular variants of 2009 A(H1N1) virus, the lower respiratory tract may be the preferred site of infection.

  View details for DOI 10.1086/649875

  View details for Web of Science ID 000273500300014

  View details for PubMedID 20047483

• Comparison of Real-Time PCR and Conventional Biochemical Methods for Identification of Staphylococcus lugdunensis JOURNAL OF CLINICAL MICROBIOLOGY Pinsky, B. A., Samson, D., Ghafghaichi, L., Baron, E. J., Banaei, N. 2009; 47 (11): 3472-3477

  Abstract

  Staphylococcus lugdunensis is an aggressive, virulent member of the coagulase-negative staphylococci (CoNS) that is responsible for severe, rapidly progressive skin and soft tissue infections and native valve endocarditis. To facilitate prompt identification and appropriate therapy, we describe here a rapid and robust multiplex real-time PCR assay that is able to definitively distinguish S. lugdunensis from other staphylococci. Using melting curve analysis, the assay also identifies Staphylococcus aureus and CoNS other than S. lugdunensis and determines MecA-dependent resistance to methicillin (meticillin). When applied to a panel of well-characterized staphylococcal reference strains, as well as 165 clinical isolates previously identified by conventional methods, the assay was both sensitive and specific for S. lugdunensis, correctly identifying the reference strain and all 47 S. lugdunensis isolates without inappropriate amplification of other staphylococci. Furthermore, rapid biochemical identification using the WEE-TAB system to detect ornithine decarboxylase activity was found to be unsuitable as an alternative to PCR identification, displaying just 31% sensitivity and 77% specificity when tested on a subset (90 isolates) of the clinical strains. We therefore propose that this simple, accurate PCR approach will allow for the routine and timely identification of S. lugdunensis in the clinical microbiology laboratory.

  View details for DOI 10.1128/JCM.00342-09

  View details for Web of Science ID 000271373000013

  View details for PubMedID 19741081

  View details for PubMedCentralID PMC2772579

• First documentation of isoniazid reversion in Mycobacterium tuberculosis INTERNATIONAL JOURNAL OF TUBERCULOSIS AND LUNG DISEASE Richardson, E. T., Lin, S. G., Pinsky, B. A., Desmond, E., Banaei, N. 2009; 13 (11): 1347-1354

  Abstract

  Drug-resistant strains of Mycobacterium tuberculosis are increasing worldwide and pose a major threat to global health. However, it remains unsettled whether drug-resistant mutants are fixed in the bacterial population or if they would revert in the absence of drug pressure.To document the occurrence of isoniazid (INH) reversion in a patient with multidrug-resistant tuberculosis (TB) and investigate its association with fitness cost.Genotypic and phenotypic assays were used to characterize the reversion of INH resistance in isolates from a patient with pulmonary TB. The pre-reversion katG mutation was reconstructed in a pan-susceptible laboratory strain (H37Rv DeltakatG::katG W300G) and tested for susceptibility to INH and oxidative stress.Genotyping and drug susceptibility testing showed that an isogenic strain of M. tuberculosis reverted from an INH-resistant to a susceptible phenotype in the absence of INH therapy. The genotypic basis of this reversion was mapped to the katG codon 300 which reverted from GGG (glycine, G) to a wild-type codon, TGG (tryptophan, W). The H37Rv DeltakatG::katG W300G mutant was resistant to INH, but also showed a deficiency in coping with oxidative stress.This study confirms that, in the absence of INH pressure, some INH-resistant mutants will revert to a drug-susceptible phenotype. This finding may have broader implications for INH-resistant strains and for the clinically useful lifespan of INH.

  View details for Web of Science ID 000271883400007

  View details for PubMedID 19861005

• Protein Phosphatase 1 Regulates Exit from the Spindle Checkpoint in Budding Yeast CURRENT BIOLOGY Pinsky, B. A., Nelson, C. R., Biggins, S. 2009; 19 (14): 1182-1187

  Abstract

  Accurate chromosome segregation depends on sister kinetochores coming under tension when they make bioriented attachments to microtubules from opposite poles. The spindle checkpoint halts the cell cycle in response to defects in generating proper attachments or tension on kinetochores, although the precise signal that triggers the checkpoint is unclear because tension and attachment are coupled. The target of the checkpoint is the Cdc20 protein, which initiates the anaphase-promoting complex (APC)-dependent degradation of the anaphase inhibitor Pds1/securin. Although the molecular details of spindle checkpoint activation are still being elucidated, phosphorylation by at least four kinases is a crucial requirement. However, less is known about the mechanisms that silence the checkpoint after kinetochores biorient. Here, we show that the catalytic subunit of the budding yeast protein phosphatase 1 (PP1) homolog, Glc7, regulates exit from the checkpoint. Glc7 overexpression prevents spindle checkpoint activation in response to both tension and attachment defects. Although glc7 mutant cells are able to efficiently release from a non-checkpoint-mediated metaphase arrest, they are uniquely sensitive to transient spindle checkpoint activation as a result of a failure in spindle checkpoint exit. We therefore propose that PP1 activity silences the checkpoint by reversing key phosphorylation events.

  View details for DOI 10.1016/j.cub.2009.06.043

  View details for Web of Science ID 000268530200024

  View details for PubMedID 19592248

• Hair Sheep Blood, Citrated or Defibrinated, Fulfills All Requirements of Blood Agar for Diagnostic Microbiology Laboratory Tests PLOS ONE Yeh, E., Pinsky, B. A., Banaei, N., Baron, E. J. 2009; 4 (7)

  Abstract

  Blood agar is used for the identification and antibiotic susceptibility testing of many bacterial pathogens. In the developing world, microbiologists use human blood agar because of the high cost and inhospitable conditions for raising wool sheep or horses to supply blood. Many pathogens either fail to grow entirely or exhibit morphologies and hemolytic patterns on human blood agar that confound colony recognition. Furthermore, human blood can be hazardous to handle due to HIV and hepatitis. This study investigated whether blood from hair sheep, a hardy, low-maintenance variety of sheep adapted for hot climates, was suitable for routine clinical microbiology studies.Hair sheep blood obtained by jugular venipuncture was anticoagulated by either manual defibrination or collection in human blood bank bags containing citrate-phosphate-dextrose. Trypticase soy 5% blood agar was made from both forms of hair sheep blood and commercial defibrinated wool sheep blood. Growth characteristics, colony morphologies, and hemolytic patterns of selected human pathogens, including several streptococcal species, were evaluated. Specialized identification tests, including CAMP test, reverse CAMP test, and satellite colony formation with Haemophilus influenzae and Abiotrophia defectiva were also performed. Mueller-Hinton blood agar plates prepared from the three blood types were compared in antibiotic susceptibility tests by disk diffusion and E-test.The results of all studies showed that blood agar prepared from citrated hair sheep blood is suitable for microbiological tests used in routine identification and susceptibility profiling of human pathogens. The validation of citrated hair sheep blood eliminates the labor-intensive and equipment-requiring process of manual defibrination. Use of hair sheep blood, in lieu of human blood currently used by many developing world laboratories and as an alternative to cost-prohibitive commercial sheep blood, offers the opportunity to dramatically improve the safety and accuracy of laboratory diagnosis of pathogenic bacteria in resource-poor countries.

  View details for DOI 10.1371/journal.pone.0006141

  View details for Web of Science ID 000267806300010

  View details for PubMedID 19578541

  View details for PubMedCentralID PMC2700971

• Bartholin's abscess caused by hypermucoviscous Klebsiella pneumoniae JOURNAL OF MEDICAL MICROBIOLOGY Pinsky, B. A., Baron, E. J., Janda, J. M., Banaei, N. 2009; 58 (5): 671-673

  Abstract

  Klebsiella pneumoniae serogroups displaying the hypermucoviscosity phenotype are associated with a distinct clinical syndrome characterized by liver abscesses, bacteraemia and metastatic lesions. We describe here what we believe to be the first reported case of hypermucoviscous K. pneumoniae causing a superficial Bartholin's abscess in the absence of systemic involvement.

  View details for DOI 10.1099/jmm.0.006734-0

  View details for Web of Science ID 000266018900019

  View details for PubMedID 19369531

• Multiplex real-time PCR assay for rapid identification of Mycobacterium tuberculosis complex members to the species level JOURNAL OF CLINICAL MICROBIOLOGY Pinsky, B. A., Banaei, N. 2008; 46 (7): 2241-2246

  Abstract

  The species identification of members of the Mycobacterium tuberculosis complex is critical to the timely initiation of both appropriate antibiotic therapy and proper public health control measures. However, the current commercially available molecular assays identify mycobacteria only to the complex level and are unable to differentiate M. tuberculosis from the closely related M. bovis and M. bovis BCG. We describe here a rapid and robust two-step, multiplex, real-time PCR assay based on genomic deletions to definitively identify M. tuberculosis, M. bovis, M. bovis BCG, and other members of the complex. When tested against a panel of well-characterized mycobacterial reference strains, the assay was both sensitive and specific, correctly identifying all strains. We applied this assay to 60 clinical isolates previously identified as M. tuberculosis complex and found 57 M. tuberculosis isolates and 3 M. bovis BCG isolates from patients who had received intravesical BCG. Furthermore, analysis of 15 clinical specimens previously identified as M. bovis by spoligotyping revealed an isolate of M. tuberculosis that had been misidentified. We propose that this assay will allow the routine identification of M. tuberculosis complex members in the clinical laboratory.

  View details for DOI 10.1128/JCM.00347-08

  View details for Web of Science ID 000258906800016

  View details for PubMedID 18508937

  View details for PubMedCentralID PMC2446918

• Glc7/protein phosphatase 1 regulatory subunits can oppose the Ipl1/aurora protein kinase by redistributing Glc7 MOLECULAR AND CELLULAR BIOLOGY Pinsky, B. A., Kotwaliwale, C. V., Tatsutani, S. Y., Breed, C. A., Biggins, S. 2006; 26 (7): 2648-2660

  Abstract

  Faithful chromosome segregation depends on the opposing activities of the budding yeast Glc7/PP1 protein phosphatase and Ipl1/Aurora protein kinase. We explored the relationship between Glc7 and Ipl1 and found that the phosphorylation of the Ipl1 substrate, Dam1, was altered by decreased Glc7 activity, whereas Ipl1 levels, localization, and kinase activity were not. These data strongly suggest that Glc7 ensures accurate chromosome segregation by dephosphorylating Ipl1 targets rather than regulating the Ipl1 kinase. To identify potential Glc7 and Ipl1 substrates, we isolated ipl1-321 dosage suppressors. Seven genes (SDS22, BUD14, GIP3, GIP4, SOL1, SOL2, and PEX31) encode newly identified ipl1 dosage suppressors, and all 10 suppressors encode proteins that physically interact with Glc7. The overexpression of the Gip3 and Gip4 suppressors altered Glc7 localization, indicating they are previously unidentified Glc7 regulatory subunits. In addition, the overexpression of Gip3 and Gip4 from the galactose promoter restored Dam1 phosphorylation in ipl1-321 mutant cells and caused wild-type cells to arrest in metaphase with unsegregated chromosomes, suggesting that Gip3 and Gip4 overexpression impairs Glc7's mitotic functions. We therefore propose that the overexpression of Glc7 regulatory subunits can titrate Glc7 away from relevant Ipl1 targets and thereby suppress ipl1-321 cells by restoring the balance of phosphatase/kinase activity.

  View details for DOI 10.1128/MCB.26.7.2648-2660.2006

  View details for Web of Science ID 000236312200017

  View details for PubMedID 16537909

• The Ipl1-Aurora protein kinase activates the spindle checkpoint by creating unattached kinetochores NATURE CELL BIOLOGY Pinsky, B. A., Kung, C., Shokat, K. M., Biggins, S. 2006; 8 (1): 78-U28

  Abstract

  The spindle checkpoint ensures accurate chromosome segregation by delaying cell-cycle progression until all sister kinetochores capture microtubules from opposite poles and come under tension (for reviews, see refs 1, 2). Although the checkpoint is activated by either the lack of kinetochore-microtubule attachments or defects in the tension exerted by microtubule-generated forces, it is not clear whether these signals are linked. We investigated the connection between tension and attachment by studying the conserved budding yeast Ipl1Aurora protein kinase that is required for checkpoint activation in the absence of tension but not attachment. Here, we show that spindle-checkpoint activation in kinetochore mutants that seem to have unattached kinetochores depends on Ipl1 activity. When Ipl1 function was impaired in these kinetochore mutants, the attachments were restored and the checkpoint was turned off. These data indicate that Ipl1 activates the checkpoint in response to tension defects by creating unattached kinetochores. Moreover, although the Dam1 kinetochore complex has been implicated as a key downstream target, we found the existence of unidentified Ipl1 sites on Dam1 or additional important substrates that regulate both microtuble detachment and the checkpoint.

  View details for DOI 10.1038/ncb1341

  View details for Web of Science ID 000234651500015

  View details for PubMedID 16327780

• The spindle checkpoint: tension versus attachment TRENDS IN CELL BIOLOGY Pinsky, B. A., Biggins, S. 2005; 15 (9): 486-493

  Abstract

  The spindle checkpoint ensures the fidelity of chromosome segregation by preventing cell-cycle progression until all the chromosomes make proper bipolar attachments to the mitotic spindle and come under tension. Despite significant advances in our understanding of spindle checkpoint function, the primary signal that activates the spindle checkpoint remains unclear. Whereas some experiments indicate that the checkpoint recognizes the lack of microtubule attachment to the kinetochore, others indicate that the checkpoint senses the absence of tension generated on the kinetochore by microtubules. The interdependence between tension and microtubule attachment make it difficult to determine whether these signals are separable. In this article (which is part of the Chromosome Segregation and Aneuploidy series), we consider recent evidence that supports and opposes the hypothesis that defects in tension act as the primary checkpoint signal.

  View details for DOI 10.1016/j.tcb.2005.07.005

  View details for Web of Science ID 000232333700006

  View details for PubMedID 16084093

• An Mtw1 complex promotes kinetochore biorientation that is monitored by the lpl1/Aurora protein kinase DEVELOPMENTAL CELL Pinsky, B. A., Tatsutani, S. Y., Collins, K. A., Biggins, S. 2003; 5 (5): 735-745

  Abstract

  Chromosome segregation depends on kinetochore biorientation so that sister kinetochores attach to microtubules from opposite poles and come under tension. The budding yeast Ipl1/Aurora protein kinase allows the absence of tension to activate the spindle checkpoint. We found that checkpoint activation in the mtw1-1 kinetochore mutant requires Ipl1p, suggesting that Mtw1p promotes tension. We isolated mtw1-1 dosage suppressors and identified Dsn1, a kinetochore protein that immunoprecipitates with the Mif2/CENP-C and Cse4/CENP-A proteins, as well as the Mtw1, Nnf1, and Nsl1 kinetochore proteins. mtw1 and dsn1 mutant strains exhibit similar phenotypes, suggesting that Mtw1p and Dsn1p act together. Although mtw1 mutant cells contained unattached chromosomes, attachment was restored by impairing Ipl1p function. These results suggest that mtw1 mutant kinetochores are competent to bind microtubules but Ipl1p generates unattached chromosomes. We therefore propose that an Mtw1 complex is required for kinetochore biorientation that is monitored by the Ipl1p kinase.

  View details for Web of Science ID 000186544400011

  View details for PubMedID 14602074

• Top-SUMO wrestles centromeric cohesion DEVELOPMENTAL CELL Pinsky, B. A., Biggins, S. 2002; 3 (1): 4-6

  Abstract

  Sister chromatid cohesion at the centromere is distinct from cohesion at the chromosome arms. In the June issue of Molecular Cell, Bachant et al. have shown that centromeric cohesion in budding yeast is specifically regulated by SUMO-1 modification of Topoisomerase II.

  View details for Web of Science ID 000176769500003

  View details for PubMedID 12110161

• Nlk is a murine protein kinase related to Erk/MAP kinases and localized in the nucleus PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Brott, B. K., Pinsky, B. A., Erikson, R. L. 1998; 95 (3): 963-968

  Abstract

  Extracellular-signal regulated kinases/microtubule-associated protein kinases (Erk/MAPKs) and cyclin-directed kinases (Cdks) are key regulators of many aspects of cell growth and division, as well as apoptosis. We have cloned a kinase, Nlk, that is a murine homolog of the Drosophila nemo (nmo) gene. The Nlk amino acid sequence is 54. 5% similar and 41.7% identical to murine Erk-2, and 49.6% similar and 38.4% identical to human Cdc2. It possesses an extended amino-terminal domain that is very rich in glutamine, alanine, proline, and histidine. This region bears similarity to repetitive regions found in many transcription factors. Nlk is expressed as a 4. 0-kb transcript at high levels in adult mouse brain tissue, with low levels in other tissues examined, including lung, where two smaller transcripts of 1.0 and 1.5 kb are expressed as well. A 4.0-kb Nlk message is also present during embryogenesis, detectable at day E10. 5, reaching maximal steady state levels at day E12.5, and then decreasing. Nlk transiently expressed in COS7 cells is a 60-kDa kinase detectable by its ability to autophosphorylate. Mutation of the ATP-binding Lys-155 to methionine abolishes its ability to autophosphorylate, as does mutation of a putative activating threonine in kinase domain VIII, to valine, aspartic, or glutamic acid. Subcellular fractionation indicates that 60-70% of Nlk is localized to the nucleus, whereas 30-40% of Nlk is cytoplasmic. Immunofluorescence microscopy confirms that Nlk resides predominantly in the nucleus. Nlk and Nmo may be the first members of a family of kinases with homology to both Erk/MAPKs and Cdks.

  View details for Web of Science ID 000071878500031

  View details for PubMedID 9448268