Stability of Zika Virus in Urine: Specimen Processing Considerations and Implications for the Detection of RNA Targets in Urine.
Journal of virological methods
Calibration of BK Virus Nucleic Acid Amplification Testing to the 1st WHO International Standard for BK Virus.
Journal of clinical microbiology
2017; 55 (3): 923-930
Detection of Zika virus (ZIKV) RNA in urine is of increasing interest for the diagnosis of ZIKV infection. Pre-analytical variables can significantly impact the stability of RNA in urine.To determine optimal specimen processing protocols that would maximize detection of ZIKV RNA in urine by real-time, reverse transcriptase PCR, we investigated the effect of temperature, initial ZIKV concentration, use of nucleic acid stabilizers, and time on ZIKV RNA levels. Urine samples from healthy donors were spiked with ZIKV using the Exact Diagnostics(®) ZIKV Verification Panel, a commercially available panel composed of heat-inactivated ZIKV, at concentrations of 5.0 log10 copies/mL (ZIKV-high) and 4.0 log10 copies/mL (ZIKV-low). Samples were stored at room temperature, 4°C, or -80°C and frozen aliquots were exposed to no stabilizer (urine), Buffer ATL (Qiagen, Germantown, MD), or DNA/RNA Shield (Zymo Research, Irvine, CA).ZIKV RNA levels in urine declined steadily at room temperature, though was not significant by 48hours (ZIKV-high, p=0.09; ZIKV-low, p=0.20). ZIKV RNA titers were consistently higher when stored at 4°C, suggesting that storage at 4°C can slow the progression of RNA degradation. Freezing urine samples at -80°C resulted in a significant loss of detectable ZIKV RNA in the ZIKV-low group. ZIKV RNA was detected in 5/6 replicates at 3 days, 1/6 replicates at 10 days, and 1/3 replicates at 30 days, with findings reproducible on repeat testing. Presence of either nucleic acid stabilizer in urine corrected this effect, and resulted in recovery of ZIKV RNA in all replicates. Use of a nucleic acid stabilizer in the ZIKV-high group did not add incremental benefit for the detection or quantitation of ZIKV RNA.ZIKV RNA is prone to degradation in urine with loss of detectable virus even when specimens are frozen at -80°C for 10 days. Detection of ZIKV-positive urine samples, particularly those containing low ZIKV titers may be aided with the addition of a nucleic acid stabilizer during urine specimen processing.
View details for DOI 10.1016/j.jviromet.2017.04.018
View details for PubMedID 28472623
The Human Virome - Implications for Clinical Practice in Transplantation Medicine.
Journal of clinical microbiology
Significant interassay variability in the quantification of BK virus (BKV) DNA precludes establishing broadly applicable thresholds for the management of BKV infection in transplantation. The 1st WHO International Standard for BKV (primary standard) was introduced in 2016 as a common calibrator for improving the harmonization of BKV nucleic acid amplification testing (NAAT) and enabling comparisons of biological measurements worldwide. Here, we evaluated the Altona RealStar BKV assay (Altona) and calibrated the results to the international unit (IU) using the Exact Diagnostics BKV verification panel, a secondary standard traceable to the primary standard. The primary and secondary standards on Altona had nearly identical linear regression equations (primary standard, Y = 1.05X - 0.28, R(2) = 0.99; secondary standard, Y = 1.04X - 0.26, R(2) = 0.99) and conversion factors (primary standard, 1.11 IU/copy; secondary standard, 1.09 IU/copy). A comparison of Altona with a laboratory-developed BKV NAAT assay in IU/ml versus copies/ml using Passing-Bablok regression revealed similar regression lines, no proportional bias, and improvement in the systematic bias (95% confidence interval of intercepts: copies/ml, -0.52 to -1.01; IU/ml, 0.07 to -0.36). Additionally, Bland-Altman analyses revealed a clinically significant reduction of bias when results were reported in IU/ml (IU/ml, -0.10 log10; copies/ml, -0.70 log10). These results indicate that the use of a common calibrator improved the agreement between the two assays. As clinical laboratories worldwide use calibrators traceable to the primary standard to harmonize BKV NAAT results, we anticipate improved interassay comparisons with a potential for establishing broadly applicable quantitative BKV DNA load cutoffs for clinical practice.
View details for DOI 10.1128/JCM.02315-16
View details for PubMedID 28053213
View details for PubMedCentralID PMC5328461
Molecular and Culture-Based Bronchoalveolar Lavage Fluid Testing for the Diagnosis of Cytomegalovirus Pneumonitis.
Open forum infectious diseases
2016; 3 (1): ofv212-?
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 DOI 10.1128/JCM.00489-17
View details for PubMedID 28724557
Limited Variation in BK Virus T-Cell Epitopes Revealed by Next-Generation Sequencing.
Journal of clinical microbiology
2015; 53 (10): 3226-3233
Background. ?Cytomegalovirus (CMV) is a major cause of morbidity and mortality in immunocompromised patients, with CMV pneumonitis among the most severe manifestations of infection. Although bronchoalveolar lavage (BAL) samples are frequently tested for CMV, the clinical utility of such testing remains uncertain. Methods. ?Retrospective analysis of adult patients undergoing BAL testing via CMV polymerase chain reaction (PCR), shell vial culture, and conventional viral culture between August 2008 and May 2011 was performed. Cytomegalovirus diagnostic methods were compared with a comprehensive definition of CMV pneumonitis that takes into account signs and symptoms, underlying host immunodeficiency, radiographic findings, and laboratory results. Results. ?Seven hundred five patients underwent 1077 bronchoscopy episodes with 1090 BAL specimens sent for CMV testing. Cytomegalovirus-positive patients were more likely to be hematopoietic cell transplant recipients (26% vs 8%, P < .0001) and less likely to have an underlying condition not typically associated with lung disease (3% vs 20%, P < .0001). Histopathology was performed in only 17.3% of CMV-positive bronchoscopy episodes. When CMV diagnostic methods were evaluated against the comprehensive definition, the sensitivity and specificity of PCR, shell vial culture, and conventional culture were 91.3% and 94.6%, 54.4% and 97.4%, and 28.3% and 96.5%, respectively. Compared with culture, PCR provided significantly higher sensitivity and negative predictive value (P ? .001), without significantly lower positive predictive value. Cytomegalovirus quantitation did not improve test performance, resulting in a receiver operating characteristic curve with an area under the curve of 0.53. Conclusions. ?Cytomegalovirus PCR combined with a comprehensive clinical definition provides a pragmatic approach for the diagnosis of CMV pneumonitis.
View details for DOI 10.1093/ofid/ofv212
View details for PubMedID 26885542
View details for PubMedCentralID PMC4752011
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
2015; 69: 179-183
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
Fatal acanthamoeba encephalitis in a patient with a total artificial heart (syncardia) device.
Open forum infectious diseases
2014; 1 (2): ofu057-?
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
Hematocrit Effect in Bilateral Subdural Hematomas
JOURNAL OF GENERAL INTERNAL MEDICINE
2013; 28 (2): 321-321
A Common Fungus, an Unusual (and Deadly) Infection
AMERICAN JOURNAL OF MEDICINE
2011; 124 (11): 1023-1024
Acute Myocardial Infarction After Treatment of Thrombocytopenia in a Young Woman With Systemic Lupus Erythematosus
JCR-JOURNAL OF CLINICAL RHEUMATOLOGY
2008; 14 (6): 350-352
Acanthamoeba encephalitis is an uncommon but often fatal infection complication. Here we report the first case of Acanthamoeba encephalitis in a patient with a Total Artificial Heart device.
View details for DOI 10.1093/ofid/ofu057
View details for PubMedID 25734127
Disparities in mammographic screening for Asian women in California: a cross-sectional analysis to identify meaningful groups for targeted intervention
We describe a case of an acute myocardial infarction (MI) coincident with correction of severe thrombocytopenia in a 23-year old African American woman with systemic lupus erythematosus (SLE) in the absence of coronary artery disease on angiography. Despite a history of anticardiolipin and beta(2)-glycoprotein I antibodies, she had no prior thromboembolic events. The occurrence of an acute MI after rapid normalization in the platelet count suggests the need for close monitoring of possible cardiovascular events during and after treatment of severe thrombocytopenia in the presence of antiphospholipid antibodies.
View details for DOI 10.1097/RHU.0b013e31817de0fb
View details for Web of Science ID 000261654400008
View details for PubMedID 19086148
Breast cancer is the most commonly diagnosed cancer among the rapidly growing population of Asian Americans; it is also the most common cause of cancer mortality among Filipinas. Asian women continue to have lower rates of mammographic screening than women of most other racial/ethnic groups. While prior studies have described the effects of sociodemographic and other characteristics of women on non-adherence to screening guidelines, they have not identified the distinct segments of the population who remain at highest risk of not being screened.To better describe characteristics of Asian women associated with not having a mammogram in the last two years, we applied recursive partitioning to population-based data (N = 1521) from the 2001 California Health Interview Survey (CHIS), for seven racial/ethnic groups of interest: Chinese, Japanese, Filipino, Korean, South Asian, Vietnamese, and all Asians combined.We identified two major subgroups of Asian women who reported not having a mammogram in the past two years and therefore, did not follow mammography screening recommendations: 1) women who have never had a pap exam to screen for cervical cancer (68% had no mammogram), and 2) women who have had a pap exam, but have no women's health issues (osteoporosis, using menopausal hormone therapies, and/or hysterectomy) nor a usual source of care (62% had no mammogram). Only 19% of Asian women who have had pap screening and have women's health issues did not have a mammogram in the past two years. In virtually all ethnic subgroups, having had pap or colorectal screening were the strongest delineators of mammography usage. Other characteristics of women least likely to have had a mammogram included: Chinese non-U.S. citizens or citizens without usual source of health care, Filipinas with no health insurance, Koreans without women's health issues and public or no health insurance, South Asians less than age 50 who were unemployed or non-citizens, and Vietnamese women who were never married.We identified distinct subgroups of Asian women at highest risk of not adhering to mammography screening guidelines; these data can inform outreach efforts aimed at reducing the disparity in mammography screening among Asian women.
View details for DOI 10.1186/1471-2407-7-201
View details for Web of Science ID 000252447400001
View details for PubMedID 17961259