Clinical Focus

  • Clinical Pathology
  • Virology
  • Molecular Pathology
  • Clinical Laboratory Techniques

Academic Appointments

Administrative Appointments

  • Medical Co-Director, Point of Care Testing, SHC (2018 - Present)
  • Medical Director, Esoteric (Send-out) Testing (2019 - Present)
  • Medical Director, Clinical Virology Laboratory (2010 - Present)
  • Medical Director, Point of Care Testing, Surgery Center at Byers Eye Institute (2012 - Present)
  • Medical Director, Point of Care Testing, Stanford Primary Care Multisite (2016 - Present)
  • Senior Fellow, Center for Innovation in Global Health (2015 - Present)
  • Faculty Affiliate, Stanford Bio-X (2016 - Present)

Boards, Advisory Committees, Professional Organizations

  • Microbiology Resource Committee, College of American Pathologists (2012 - 2017)
  • Councilor, Pan American Society for Clinical Virology (2014 - 2018)
  • Training and Education Committee, Association for Molecular Pathology (2013 - 2015)
  • Clinical Practice Committee, Association for Molecular Pathology (2015 - Present)

Professional Education

  • Board Certification: Clinical Pathology, American Board of Pathology (2012)
  • Residency:Stanford Hospital and Clinics (2010) CA
  • Medical Education:University of Washington School of Medicine (2007) WA
  • Board Certification, American Board of Pathology, Clinical Pathology (2012)
  • Fellowship, Stanford Hospital & Clinics, Molecular Pathology (2010)
  • Ph.D., University of Washington Medical Scientist Training Program-Fred Hutchinson Cancer Research Center, Molecular and Cellular Biology (2005)


  • Benjamin Pinsky. "United States Patent 9725774 B2 Methods and reagents for detection, quantitation, and serotyping of dengue viruses.", Leland Stanford Junior University, Aug 8, 2017

Research & Scholarship

Current Research and Scholarly Interests

Development and application of molecular assays for the diagnosis and management of infectious diseases.


  • Dengue Virus Detection among Febrile Children Clinically Diagnosed with a non-Dengue Illness


    Managua, Nicaragua

  • Multiplex Molecular Diagnostics for Undifferentiated Febrile Illness in Brazil


    Rio de Janeiro, Brazil

  • Multiplex Molecular Diagnostics for Undifferentiated Febrile Illness in Sri Lanka


    Colombo, Sri Lanka

  • Multiplex Molecular Diagnostics for Undifferentiated Febrile Illness in Zimbabwe


    Mutare, Zimbabwe


2018-19 Courses


All Publications

  • 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
  • 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


    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)


    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


    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


    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


    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


     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


    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

  • 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


    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

  • 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., Doan, T. 2019


    PURPOSE: The etiology of conjunctivitis is often misdiagnosed. An ideal diagnostic test would identify all possible infectious causes. In this study, we apply unbiased metagenomic RNA deep sequencing (MDS) to identify pathogens causing conjunctivitis.DESIGN: Molecular study of prospectively collected conjunctival swabs from patients with presumed infectious conjunctivitis.PARTICIPANTS: Patients with presumed acute infectious conjunctivitis.METHODS: Conjunctival swabs were collected from patients presenting with acute conjunctivitis. Swabs were processed for MDS. Pathogens were identified using a rapid computational pipeline to analyze the non-host sequences obtained from MDS. Differential gene expression analysis was performed to evaluate for host transcriptome signatures for infectious types. Clinical samples were de-identified and laboratory personnel handling the samples and interpreting the data were masked.MAIN OUTCOME MEASURES: Pathogens and differential transcripts identified by MDS.RESULTS: MDS detected pathogens in 86% (12/14) of the patients tested. Swabs from 10 of 14 patients were positive for human adenovirus (HAdV) while swabs from 2 of 14 patients were positive for Vittaforma corneae (a parasitic fungal species of the microsporidia group). Samples positive for HAdV by RNA-seq were independently verified in a CLIA-certified laboratory. Directed pathogen-PCR confirmed the presence of V. corneae genome in the samples positive by RNA-seq. Local host transcriptome analysis identified 12 differentially expressed genes that provided distinct expression signatures for patients infected with HAdV compared to V. corneae.CONCLUSIONS: MDS can reliably detect and quantify common and rare pathogens causing conjunctivitis, and identify strains. The unbiased nature of metagenomic RNA deep sequencing allowed an expanded scope of pathogen detection, including fungal species not commonly associated with acute conjunctivitis. In addition, the identification of infection type-specific local host transcriptome signatures may allow for pathogen detection even when the pathogen load is too low for direct identification.

    View details for PubMedID 30953744

  • 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
  • Impact of Pre-Transplant Donor BK Viruria in Kidney Transplant Recipients. The 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


    BACKGROUND: BK virus (BKV) is a significant cause of nephropathy in kidney transplantation. The goal of this study was to characterize the course and source of BKV in kidney transplant recipients.METHODS: We prospectively collected pre-transplant plasma and urine samples from living and deceased kidney donors and performed BKV PCR and IgG testing on pre-transplant and serially collected post-transplant samples in kidney transplant recipients.RESULTS: Among deceased donors, 8.1%(17/208) had detectable BKV DNA in urine prior to organ procurement. BK viruria was observed in 15.4%(6/39) of living donors and 8.5%(4/47) of deceased donors of recipients at our institution (p=0.50). BKV VP1 sequencing revealed identical virus between donor-recipient pairs to suggest donor transmission of virus. Recipients of BK viruric donors were more likely to develop BK viruria (66.6%vs.7.8%, p<0.001) and viremia (66.6%vs.8.9%, p<0.001) with a shorter time to onset (log-rank, p<0.001). Though donor BKV IgG titers were higher in recipients who developed BK viremia, pre-transplant donor, recipient, and combined donor/recipient serology status was not associated with BK viremia (p=0.31,0.75,0.51,respectively).DISCUSSION: Donor BK viruria is associated with early BK viruria and viremia in kidney transplant recipients. BKV PCR testing of donor urine may be useful in identifying recipients at-risk for BKV complications.

    View details for PubMedID 30869132

  • 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


    HCV RNA quantitation is the primary method by which active HCV infections are identified and the response to direct acting antiviral therapy is monitored. This study describes evaluation of the Aptima HCV Quant Dx assay (Aptima HCV) performed on the Panther system. The clinical performance of Aptima HCV was compared to the COBAS AmpliPrep/COBAS TaqMan HCV Test v2.0 (CAP/CTM). Overall agreement was 84.9% (186/219) with a kappa statistic of 0.755 [Standard Error 0.037; 95% confidence interval (CI) 0.682 to 0.828]. Passing-Bablok regression of log10 IU/mL values revealed a regression line of Y = 1.163*X - 0.991 [95% CI of the slope (1.103 to 1.221) and intercept (-1.341to -0.642)]. The 95% LLOD for Aptima HCV on DBS samples was calculated to be 2.43 log10 IU/mL (267 IU/mL) [95% confidence interval, 2.31 to 2.73 log10 IU/mL (204 to 540 IU/mL)]. Comparison of Aptima HCV testing on paired dried blood spot (DBS) and serum specimens collected from patients at the time of routine blood collection for CAP/CTM demonstrated an overall agreement of 90.1% (82/91) with a kappa statistic of 0.657 (Standard Error 0.101; 95% confidence interval 0.458 to 0.855) In conclusion, Aptima HCV provides a suitable alternative for HCV RNA testing on serum and DBS.

    View details for PubMedID 30760534

  • 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


    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

  • Dual-target, real-time PCR for the diagnosis of intraocular Toxoplasma gondii infections. The British journal of ophthalmology Gomez, C. A., Sahoo, M. K., Kahn, G. Y., Zhong, L., Montoya, J. G., Pinsky, B. A., Doan, T. 2019


    BACKGROUND: Toxoplasma gondii is the most common infectious cause of posterior uveitis worldwide. Two multicopy targets (B1 and Rep529) are commonly used in T. gondii PCR assays, but studies evaluating these targets in ocular fluid samples are limited. Herein, we determine the analytical characteristics of a single-reaction, internally controlled, dual-target, real-time T. gondii PCR and evaluate the clinical performance of this assay in intraocular fluid samples obtained at a reference ophthalmologic centre in the USA.METHODS: Lower limits of detection for the B1 and Rep529 components of the dual-target assay were determined using serial dilutions of cultured T. gondii strain Z185. The dual-target assay was then used to test 148 archived intraocular samples (132 vitreous,16 aqueous humour) collected at the Francis I. Proctor Foundation between January 2010 and December 2015 for testing by a nested, conventional PCR targeting the B1 gene.RESULTS: The 95% lower limits of detection for the dual-target assay was determined to be 1.05 tachyzoites/mL for B1 and 0.83 tachyzoites/mL for Rep529. Using archived clinical intraocular specimens, the dual-target assay demonstrated 97.2% positive per cent agreement (n=35/36; 95% CI 83.7% to 99.9%) and 99.1% negative per cent agreement (n=111/112; 95% CI 94.4% to 100%) compared with the nested, conventional B1 PCR.CONCLUSION: This single-reaction, internally controlled, dual-target (B1, Rep529) real-time PCR for the detection of T. gondii DNA in intraocular specimens demonstrated excellent agreement with nested, conventional, B1 PCR. The dual-target design may ensure T. gondii detection when variation is present in one of two target regions.

    View details for PubMedID 30636207

  • Multiplex Solid-Phase Melt Curve Analysis for the Point-of-Care Detection of HIV-1 Drug Resistance. The Journal of molecular diagnostics : JMD 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


    A point-of-care HIV-1 genotypic resistance assay that could be performed during a clinic visit would enable care providers to make informed treatment decisions for patients starting therapy or experiencing virological failure on therapy. The main challenge for such an assay is the genetic variability at and surrounding each drug-resistance mutation (DRM). We analyzed a database of diverse global HIV sequences and used thermodynamic simulations to design an array of surface-bound oligonucleotide probe sets with each set sharing distinct 5' and 3' flanking sequences but having different centrally located nucleotides complementary to six codons at HIV-1 DRM reverse transcriptase position 103: AAA, AAC, AAG, AAT, AGA, and AGC. We then performed in vitro experiments using 80-mer oligonucleotides and PCR-amplified DNA from clinical plasma HIV-1 samples and culture supernatants containing subtype A, B, C, D, CRF01_AE, and CRF02_AG viruses. Multiplexed solid-phase melt-curve analysis discriminated perfectly among each of the six reported reverse transcriptase position 103 codons in both 80-mers and clinical samples. The sensitivity and specificity for detecting targets containing AAC mixed with targets containing AAA were above 98% when AAC was present at a proportion of at or above 10%. Multiplexed solid-phase melt-curve analysis is a promising approach for developing point-of-care assays to distinguish between different codons in genetically variable regions such as those surrounding HIV-1 DRMs.

    View details for PubMedID 31026601

  • 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


    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


    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

  • Evaluating for human herpesvirus 6 in the liver explants of children with liver failure of unknown etiology. The Journal of infectious diseases Yang, C. H., Sahoo, M. K., Fitzpatrick, M., Lau, A. H., Pinsky, B. A., Martinez, O. M. 2018


    Background: Liver failure of unknown etiology (LFUE) has a transplant-free survival rate of less than 25%. Human herpesvirus 6 (HHV-6) may be associated with LFUE, but existing studies are limited by small sample size.Methods: We identified all children who underwent liver transplant for LFUE at a single quaternary children's hospital. 51/65 cases were able to be age-matched with controls (children who underwent liver transplant for metabolic liver disease). Quantitative polymerase chain reaction (PCR) for HHV-6 was performed on DNA from formalin-fixed paraffin-embedded liver explant tissue. Results: HHV-6 was detected in 34/51 cases (66.7%), and 19/51 controls (37.3%) (p=0.005). Average HHV-6 viral load was 213,207 copies/10 6 cells in positive cases (range: 7,293-1,102,030), and 38,115 copies/10 6 cells in positive controls (range: 1,382-122,375) (p=0.0008). HHV-6 was present significantly more often in cases compared to controls in patients less than 6 years in age; in particular, in patients less than 3 years in age, HHV-6 was present in 13/27 cases (48.1%), and 2/27 controls (7.4%) (p=0.0009).Conclusions: HHV-6 was detected in liver explants significantly more often and in higher quantities in children transplanted for LFUE compared to controls. This suggests HHV-6 should be evaluated for in young children who present with LFUE.

    View details for PubMedID 30418598

  • 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
  • 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


    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

  • 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)


    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

  • Trends in the Molecular Epidemiology and Genetic Mechanisms of Transmitted HIV-1 Drug Resistance in a Large U.S. Clinic Population. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America Rhee, S., Clutter, D., Fessel, W. J., Klein, D., Slome, S., Pinsky, B. A., Marcus, J. L., Hurley, L., Silverberg, M. J., Kosakovsky Pond, S. L., Shafer, R. W. 2018


    Background: There are few large studies of transmitted drug resistance (TDR) prevalence and the drug resistance mutations (DRMs) responsible for TDR in the U.S.Methods: HIV-1 RT and protease sequences were obtained from 4,253 antiretroviral therapy (ART)-naive individuals in a California clinic population from 2003-2016. Phylogenetic analyses were performed to study linkages between TDR strains and selection pressure on TDR-associated DRMs.Results: From 2003-2016, there was a significant increase in overall (odds ratio [OR]=1.05 per year; 95% CI: 1.03 - 1.08; p<0.001) and nonnucleoside RT inhibitor (NNRTI)-associated TDR (OR=1.11 per year; 95% CI: 1.08 - 1.15; p<0.001). Between 2012 and 2016, TDR rates to any drug class ranged from 15.7%-19.2%, and class-specific rates ranged from 10.0%-12.8% for NNRTIs, 4.1%-8.1% for nucleoside RT inhibitors (NRTIs), and 3.6%-5.2% for protease inhibitors. K103N/S, Y181C, Y188L, and G190A mutations accounted for 88.5% of NNRTI-associated TDR. The thymidine analog mutations, M184V/I, and the tenofovir-associated DRMs K65R and K70E/Q/G/N/T accounted for 82.9%, 7.3%, and 1.4% of NRTI-associated TDR, respectively. The proportions of individuals with low-level resistance or higher to boosted atazanavir and darunavir were 2.2% and 0.3%, respectively. 37% of TDR strains clustered with other TDR strains sharing the same DRMs.Conclusions: Although TDR has increased significantly in this large cohort, many TDR strains are unlikely to influence the activity of currently preferred first-line ART regimens. The high proportion of DRMs associated with infrequently used regimens combined with the clustering of TDR strains suggest that some TDR strains are being transmitted between ART-naive individuals.

    View details for PubMedID 29846534

  • 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)


    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


    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/μL 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

  • 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


    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
  • 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


    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/μL, and the lower limit of 95% detection was 8.2copies/μL. 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


    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

  • Evaluating for human herpesvirus-6 in children with liver failure of unknown etiology Yang, C., Sahoo, M., Lau, A. H., Pinsky, B., Martinez, O. M. WILEY. 2017: 658A–659A
  • 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


    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


    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

  • 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


    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)


    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

  • 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


    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

  • 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

  • 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


    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

  • IgG antibodies to dengue enhanced for FcγRIIIA 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


    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 FcγRIIIA 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

  • IgG antibodies to dengue enhanced for FcgRIIIA binding determine disease severity Science Wang, T. T., et al 2017; 355 (6323): 395-398


    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 FcγRIIIA 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 DOI 10.1126/science.aai8128

  • 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


    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/μL, and the lower limit of 95% detection was 22.4 copies/μL. 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

  • 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. 2017; 4 (1): ofw245-?


    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 PubMedID 28540320

  • 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


    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


    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

  • 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


    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

  • 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

  • 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


    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

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


    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

  • 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


     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

  • 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


    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


    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


    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/μL for each DENV serotype and CHIKV, and the lower limits of 95% detection were 7.9-37.4copies/μL. 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

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


    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

  • 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


    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

  • 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)

    View details for DOI 10.1371/journal.pone.0142216

    View details for PubMedID 26562786

  • 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


    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

    View details for DOI 10.1128/JCM.01876-15

    View details for PubMedID 26354810

  • 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


    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


    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

  • 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


    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

  • 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


    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

  • Case Report: 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
  • 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)
  • 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


    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


    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
  • 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 : the official publication of the Pan American Society for Clinical Virology Tan, S. K., Waggoner, J. J., Pinsky, B. A. 2015; 69: 179–83


    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 PubMedID 26209403

  • 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


    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)


    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


    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)
  • 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., Le, Q., Pinsky, B. A. 2014; 52 (10): 3802-3804


    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 PubMedID 25078918

  • 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


    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


    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


    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


    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

  • 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


    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

  • 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


    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

  • 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

    View details for DOI 10.7717/peerj.334

    View details for PubMedID 24765569

  • 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)


    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


    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


    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


    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


    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


    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


    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 β-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


    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/μL and a lower limit of 95% detection ranging from 1.7 to 7.6 cDNA equivalents/μL 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


    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


    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


    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


    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

  • 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


    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


    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 β-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)


    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 log₁₀ 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


    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

  • Development of an Amplicon Enrichment and Barcoding Strategy for Cytomegalovirus (CMV) Genotypic Drug Resistance Testing by Next-Generation Sequencing Annual Meeting of the Association-for-Molecular-Pathology Lefterova, M., Sahoo, M. K., Yamamoto, F., Farina, H., ANDERSON, M. W., Pinsky, B. A. ELSEVIER SCIENCE INC. 2012: 679–79
  • 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


    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


    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

  • 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


    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
  • Rare Variant Sensitive High Resolution Melting Demonstrates that the His275Tyr Substitution in Pandemic H1N1 Influenza A Virus Appears after Oseltamivir Treatment in Infected Patients 16th Annual Meeting of the Association-for-Molecular-Pathology Chen, N., Pinsky, B. A., Lee, B., Lin, M., Schrijver, I. ELSEVIER SCIENCE INC. 2010: 875–75
  • 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


    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


    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


    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

  • 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


    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

  • 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


    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

  • 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


    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)


    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


    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


    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


    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


    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


    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


    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


    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


    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