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1.  Efficient Methodologies for Sensitive HIV-1 RNA Quantitation from Plasma and Vaginal Secretions 
Background
Quantifying HIV levels in mucosal secretions is essential to study compartmentalized expression of HIV and facilitate development of intervention strategies to prevent disease progression and transmission.
Objectives
To develop a sensitive, reliable, and cost-effective technique to quantify HIV from blood and vaginal secretions that is compatible with efficient implementation in clinical research environments.
Study Design
A sensitive, reliable, internally-controlled real-time reverse transcriptase (RT) PCR assay, which uses the HIV-1 pol gene as a target (Hpol assay) was developed to quantify HIV levels in plasma and genital secretions, and compared to the widely-used Roche Amplicor HIV-1 Monitor assay. In addition, a simplified method of sample collection and processing of genital secretions (self-collection and use of RNAlater with batch processing) was compared to provider collection of samples and immediate processing.
Results
The sensitivity and reliability of HIV levels detected by the assay described herein correlate well with measurements from Roche Amplicor™ HIV-1 Monitor assay for both plasma and vaginal secretions (R2= 0.9179 and R2=0.942, respectively). The Hpol assay reproducibly quantifies a lower limit of 5 HIV-1 RNA copies per reaction, with low-levels of inter-assay and intra-assay variation. Additionally, vaginal viral levels and detection frequency did not differ significantly between the two the collection and processing methods.
Conclusions
The methodologies developed here provide sensitive, reliable, and cost-effective quantification of HIV levels in plasma and mucosal secretions, and are compatible with efficient use in clinical research studies.
doi:10.1016/j.jcv.2009.08.015
PMCID: PMC3969843  PMID: 19783472
Human immunodeficiency virus; real-time PCR; genital; plasma; RNAlater
2.  A comparison of methylation levels in HPV18, HPV31 and HPV33 genomes reveals similar associations with cervical precancers☆ 
Journal of Clinical Virology  2014;59(3):161-166.
Background
High risk human papillomavirus (HR-HPV) infection is common and only a small minority of infections become persistent and lead to cervical cancers. Women positive for HR-HPV usually require a second test to avoid unnecessary colposcopies and over treatment. Elevated DNA methylation of HR-HPV L1 and L2 genes in high grade disease has emerged as a promising molecular triage tool.
Objectives
Our aim was to accurately measure methylation levels at selected CpG positions in the HPV18, HPV31 and HPV33 genomes. We focused on the L2, L1, URR and E6 regions because these were previously shown to be interesting areas for study.
Study design
Pyrosequencing was used to measure methylation in 208 HPV18, 207 HPV31, and 126 HPV33 positive women selected from a London colposcopy referral population.
Results
After adjustment for multiple testing, at FDR 5%, elevated methylation was significantly associated with cervical intraepithelial neoplasia grades 2 or worse (CIN2+) in all investigated CpGs in HPV18 L2 and L1. Two of 6 L2 and 12 of 15 L1 sites in HPV31 and 6 of 8 L2 and 3 of 13 L1 sites in HPV33 showed significantly elevated methylation in CIN2+. Methylation of CpG sites in the URR and E6 region of the HPV types was low and most differences were not significant.
Conclusion
Elevated methylation of CpG sites in the L1 and L2 regions of HPV18, HPV31 and HPV33 is associated with CIN2+ and a panel test may be useful for triage of women with HR-HPV infections.
doi:10.1016/j.jcv.2013.12.014
PMCID: PMC3969303  PMID: 24468012
HR-HPV, high risk human papilloma virus; CIN, cervical intraepithelial neoplasia; CI, confidence interval; FDR, false discovery rate; P1, predictors 1; P2, predictors 2; ROC, receiver operator characteristics; AUC, area under the curve; SCC, squamous cell carcinoma; High risk human papillomavirus; Methylation; L1; L2; Pyrosequencing
3.  Recovery of Live Virus after Storage at Ambient Temperature Using ViveST™ 
Background
A major impediment to performing virological field studies in developing nations is the lack of ultra-low freezers as well as the expense and difficulty of shipping frozen samples. A commercially available product, ViveST™, was developed to preserve nucleic acids at ambient temperature for use in specimen storage and transportation. However, its applications as a viral storage, transport and recovery device have not been evaluated.
Objective
To examine the ability of ViveST to preserve live virus following storage at ambient temperature.
Study Design
A panel of six viruses was stored at ambient temperature (~22°C) in ViveST with fetal bovine serum (FBS), or ViveST with minimal essential media (MEM) and compared with virus stored in universal transport media (M4RT), MEM, and FBS alone. Stored viruses included: human adenovirus (14p), dengue virus 2 (16608), echovirus 3 (Morrisey), human rhinovirus 15 (1734), Coxsackie virus B5 (Faulkner), and herpes simplex virus 1 (HF). After 7 days storage at ambient temperature, virus recovery was measured via titration using viral plaque assays or focus-forming unit assays.
Results
Viral titer studies indicate that ViveST with either FBS or M4RT preserved/recovered 5 different viruses for 1 week at ambient temperature. MEM preserved 4 viruses while FBS and ViveST with MEM preserved 3 viruses each. Statistical analyses indicate that M4RT and ViveST with FBS preserved significantly more virus than the other treatments.
Conclusions
These data suggest that ViveST with either FBS or M4RT may be useful in field specimen collection scenarios where ultra-cold storage is not available.
doi:10.1016/j.jcv.2012.09.005
PMCID: PMC3529791  PMID: 23046621
virus; preservation; recovery; storage; transport
4.  Rhino/Enteroviruses in Hospitalized Children: A Comparison to Influenza Viruses 
Background
The relative impact of human rhino/enteroviruses (HRV/EV) compared to influenza viruses on hospitalized children is unknown.
Objectives
This retrospective study compared the epidemiology and clinical characteristics of hospitalized patients with HRV/EV to patients hospitalized with influenza virus.
Study Design
Respiratory specimens from hospitalized children submitted between January 1, 2009 and December 31, 2009 to Children’s Hospital Colorado Virology Laboratory in Aurora, CO were tested by a commercial multiplex PCR for 16 respiratory viruses and subtypes. Patients with specimens positive for HRV/EV or influenza virus without bacterial or viral co-infection were selected for retrospective chart review.
Results
Of the 2299 patients with specimens tested during the study period, 427 (18.6%) were singly positive for HRV/EV and 202 (8.8%) for influenza virus (p<0.01). Children with HRV/EV were more likely to present with increased work of breathing (67.9% vs. 52.5%, p<0.01) with crackles (36.3% vs. 23.3%, p<0.01) and wheezing (41.7% vs. 22.8%, p<0.01) noted on exam. Children hospitalized with HRV/EV had a shorter median length of stay (2 vs. 3 days, p<0.01), duration of fever (1 vs. 3 days, p<0.01), and duration of hypoxemia (2 vs. 3 days, p <0.01) than children with influenza virus. Similar percentages of children with HRV/EV and influenza virus were admitted to the PICU and required positive pressure ventilation. There were no deaths in children hospitalized with HRV/EV, whereas 6 children with influenza virus expired.
Conclusions
HRV/EVs are common pathogens in hospitalized children associated with serious lower respiratory tract disease and significant morbidity, similar to influenza viruses.
doi:10.1016/j.jcv.2012.09.012
PMCID: PMC3529827  PMID: 23078869
Rhinovirus; Enterovirus; Influenza virus; Respiratory virus
5.  Analysis of residual perinatal transmission of hepatitis B virus (HBV) and of genetic variants in human immunodeficiency virus and HBV co-infected women and their offspring 
Background
Despite implementation of universal infant hepatitis B (HB) vaccination, mother-to-child transmission (MTCT) of hepatitis B virus (HBV) still occurs. Limited data are available on the residual MTCT of HBV in human immunodeficiency virus (HIV)-HBV co-infected women.
Objectives
We assessed the prevalence of HBV infection among HIV-infected pregnant women and the rate of residual MTCT of HBV from HIV-HBV co-infected women and analyzed the viral determinants in mothers and their HBV-infected children.
Study design
HIV-1 infected pregnant women enrolled in two nationwide perinatal HIV prevention trials in Thailand were screened for HB surface antigen (HBsAg) and tested for HBeAg and HBV DNA load. Infants born to HBsAg-positive women had HBsAg and HBV DNA tested at 4–6 months. HBV diversity within each HBV-infected mother-infant pair was analyzed by direct sequencing of amplified HBsAg-encoding gene and cloning of amplified products.
Results
Among 3,312 HIV-1 infected pregnant women, 245 (7.4%) were HBsAg-positive, of whom 125 were HBeAg-positive. Of 230 evaluable infants born to HBsAg-positive women, 11 (4.8%) were found HBsAg and HBV DNA positive at 4–6 months; 8 were born to HBeAg-positive mothers. HBV genetic analysis was performed in 9 mother-infant pairs and showed that 5 infants were infected with maternal HBV variants harboring mutations within the HBsAg “a” determinant, and four were infected with wild-type HBV present in highly viremic mothers.
Conclusions
HBV-MTCT still occurs when women have high HBV DNA load and/or are infected with HBV variants. Additional interventions targeting highly viremic women are thus needed to reduce further HBV-MTCT.
doi:10.1016/j.jcv.2013.06.025
PMCID: PMC3872003  PMID: 23916828
HBs antigen variants; Hepatitis B vaccine failure; HIV pregnant women; mother-to-child transmission; Thailand
6.  Kinetics of DNA load predict HPV 16 viral clearance 
Introduction
While high HPV 16 viral load measured at a single time point is associated with cervical disease outcomes, few studies have assessed changes in HPV 16 viral load on viral clearance.
Objective
To measure the association between changes in HPV 16 viral load and viral clearance in a cohort of Thai women infected with HPV 16.
Study design
Fifty women (n = 50) between the ages of 18–35 years enrolled in a prospective cohort study were followed up every three months for two years. Women positive for HPV 16 DNA by multiplex TaqMan© assay at two or more study visits were selected for viral load quantitation using a type-specific TaqMan© based real-time PCR assay. The strength of the association of change in viral load between two visits and viral clearance at the subsequent visit was assessed using a GEE model for binary outcomes.
Results
At study entry, HPV 16 viral load did not vary by infection outcome. A >2 log decline in viral load across two study visits was found to be strongly associated with viral clearance (AOR: 5.5, 95% CI: 1.4–21.3). HPV 16 viral load measured at a single time point was not associated with viral clearance.
Conclusions
These results demonstrate that repeated measurement of HPV 16 viral load may be a useful predictor in determining the outcome of early endpoints of viral infection.
doi:10.1016/j.jcv.2011.01.011
PMCID: PMC3837526  PMID: 21388867
HPV; DNA; Viral load; Epidemiology; Thailand
7.  WU and KI polyomavirus infections in pediatric hematology/oncology patients with acute respiratory tract illness 
Background
WU and KI polyomaviruses (PyV) were discovered in 2007 in respiratory tract samples in adults and children. Other polyomaviruses (BKPyV and JCPyV) have been associated with illness in immunocompromised patients, and some studies suggest a higher prevalence of WUPyV and KIPyV in this population.
Objective
To determine whether a higher prevalence or viral load for WUPyV and KIPyV exists in immuno-compromised children compared with immunocompetent children.
Study design
We measured the prevalence and viral load of WU and KI PyV by quantitative real-time PCR of viral DNA in respiratory tract specimens from pediatric hematology/oncology patients and immuno-competent controls with acute respiratory illnesses.
Results
The prevalence of WUPyV in the immunocompromised population was 5/161 (3%) versus 14/295 (5%) in the control population (P = 0.5), and 9/161 (5.6%) versus 7/295 (2.3%) respectively for KIPyV (P = 0.13). The mean viral load (in copies per cell or mL of sample) for KIPyV, was higher in the immuno-compromised group compared to the control group (P = 0.019), but was not statistically different for WUPyV. A higher prevalence was seen in the hematopoietic stem cell transplant recipients compared with other immunocompromised patients (6/26 versus 3/43, P = 0.054). Viral persistence was demonstrated only in 1/25 (4%) of sequential samples for KIPyV, and no persistence was seen for WUPyV.
Conclusions
A higher prevalence of WUPyV or KIPyV in the immunocompromised population compared with the immunocompetent group was not demonstrated. Higher viral loads for KIPyV in the immunocompromised group may suggest an increased pathogenic potential in this population.
doi:10.1016/j.jcv.2011.05.024
PMCID: PMC3816538  PMID: 21705268
WU polyomavirus; KI polyomavirus; Respiratory viruses; Immunocompromised patients; Acute respiratory illness; Viral load
8.  Toscana meningoencephalitis: a comparison to other viral central nervous system infections 
Background
Toscana virus (TOSV) is an emerging pathogen causing central nervous system (CNS) infection in Mediterranean countries, mostly during summer season.
Objectives
To compare the clinical and laboratory characteristics of Toscana CNS infections to the most common viral pathogens seen in the United States.
Study Design
We performed a case series of patients with 41 TOSV infection and compared the clinical characteristics, laboratory findings, imaging results and clinical outcomes to the most commonly recognized viral causes of meningoencephalitis in the US (enterovirus (n=60), herpes simplex virus (n=48), and west nile virus (n=30) from our multi-center study of patients with aseptic meningoencephalitis syndromes in the Greater Houston area.
Results
TOSV infection occurs in different age groups compared to enterovirus, HSV, and WNV. All infections most frequently occur during summer-fall except HSV which distributes throughout the year. All patients with TOSV had history of travel to endemic areas. There are differences in clinical presentation and CSF findings comparing TOSV and enterovirus, HSV, and WNV infection. There are no significant differences in outcomes of each infection except WNV meningoencephalitis which had a poorer outcome compared to TOSV infection.
Conclusions
TOSV is an emerging pathogen that should be considered in the differential diagnosis of patients with CNS infections and a recent travel history to endemic areas.
doi:10.1016/j.jcv.2012.07.007
PMCID: PMC3445752  PMID: 22867730
Toscana virus; enterovirus; west nile virus; herpes simplex virus; meningoencephalitis
9.  Characterization of a novel gyrovirus in human stool and chicken meat 
Background
Sequence-independent amplification of clinical specimens can lead to the identification of novel pathogens.
Objectives
To identify novel viruses in human stool specimens from patients with diarrhea and to investigate the ecology and clinical significance of such viruses.
Study Design
Nucleic acid extracted from stool specimens from patients with diarrhea with no known aetiology were subjected to random PCR amplification and Roche/454 pyrosequencing. Novel viruses identified were genetically and epidemiologically characterized.
Results
Four gyroviruses, chicken anemia virus (CAV), human gyrovirus (HGV) / avian gyrovirus 2 (AGV2), gyrovirus 3 (GyV3) and a novel gyrovirus (tentatively designated as gyrovirus 4 (GyV4)) were identified in human stool specimens. GyV4, as well as CAV and AGV2/HGV were also detected in chicken skin and meat used for human consumption.
Conclusions
A novel gyrovirus (GyV4) was identified in human stool and in chicken meat sold for human consumption. This virus was phylogenetically distinct from previously reported gyroviruses in chicken and humans (chicken anemia virus, human gyrovirus, avian gyrovirus 2 and recently reported gyrovirus 3). The epidemiology and pathogenesis of this virus in humans and in chicken needs to be further investigated.
doi:10.1016/j.jcv.2012.07.001
PMCID: PMC3449218  PMID: 22824231
diarrhea; virus; gyrovirus; virus discovery; human; chicken; chicken anaemia virus
10.  Utility of Rapid Antibody Tests to Exclude HIV-1 Infection among Infants and Children aged < 18 Months in a Low-Resource Setting 
Background
Excluding HIV infection among infants and young children in resource-poor settings where nucleic acid amplification tests (NAAT) are not routinely available remains a considerable challenge.
Objectives
To assess the performance of two rapid HIV antibody tests (RT) used alone and in parallel for excluding HIV infection among acutely ill infants and children < 18 months in comparison to NAAT in a region where maternal HIV prevalence was approximately 7%.
Study Design
Infants and children ≥2 <18 months admitted to hospital with an acute febrile illness had two rapid antibody tests in parallel, with single and parallel results subsequently compared against NAAT.
Results
HIV prevalence among 1,602 enrolled infants was 3.4%. All 1,526 infants with 2 negative RT were HIV negative by NAAT. All 46 infants with 2 positive RT were HIV positive by NAAT. The overall specificity of two rapid tests for excluding HIV infection was 99.5%. Sensitivity and specificity were ≥ 99% and >98%, respectively, across all age brackets ≥2 <18 months. Overall sensitivity and specificity for a single RT was 98.2% and 99%, respectively, for Determine, and 85.5% and 99.6%, respectively, for Capillus.
Conclusions
In a setting with a maternal HIV prevalence rate of <10%, a single negative RT had excellent specificity and two negative RT performed in parallel had a perfect negative predictive value for HIV infection among acutely ill patients < 18 months of age. In this and similar settings, RT could assist with excluding HIV infection with much lower complexity and cost than NAAT.
doi:10.1016/j.jcv.2012.08.001
PMCID: PMC3449279  PMID: 22925720
Human immunodeficiency virus; rapid antibody test; infants; children; early diagnosis
11.  Validation for Clinical Use of a Novel HIV-2 Plasma RNA Viral Load Assay Using the Abbott m2000 Platform 
Background
Optimal care of persons infected with human immunodeficiency virus type 2 (HIV-2) requires an accurate assessment of HIV-2 plasma viral load (VL), but no clinically-approved quantitative HIV-2 RNA VL assay exists.
Objectives
To validate a novel quantitative HIV-2 RNA assay for clinical and research use.
Study Design
The Abbott m2000sp/rt platform was adapted for quantification of HIV-2 RNA in plasma. Amplification targeted a region of the long terminal repeat conserved in Group A and B HIV-2. Electron microscopy-counted-HIV-2 standards, the WHO/NIBSC HIV-2 International Standard and clinical specimens (N=162) were used to determine the precision, sensitivity, specificity, linear range, accuracy, and clinical performance of the assay.
Results
The quantitative linear range of the HIV-2 RNA assay was 10–1,000,000 copies/mL (R2 >0.99), with a limit of detection of 8 copies/mL (95% CI, 5–18 copies/ml). The assay did not cross-react with HIV-1, and quantification of HIV-2 RNA was not affected by the presence of >5 log10 HIV-1 RNA copies/mL. The total standard deviation (SD) and intra- and inter- run SD were 0.095, 0.093 and 0.162, respectively, at nominal inputs of 3.7, 1.7 and 1.0 log10 HIV-2 RNA copies/mL. The HIV-2 WHO/NIBSC International Standard (1000 IU) was shown to contain 152 RNA copies/mL (95% CI 141–163). Overall, HIV-2 RNA was quantified at ≥ 10 copies/mL from 86 (53%) clinical specimens (median, 2.24 log10copies/mL; range 10–16870), and nine specimens (6%) had HIV-2 RNA detected at <10 copies/mL.
Conclusions
We developed and validated a highly-sensitive HIV-2 VL assay that is suitable for clinical and research use.
doi:10.1016/j.jcv.2012.06.024
PMCID: PMC3444162  PMID: 22832059
HIV-2; PCR; Real-time; diagnostic; plasma viral loads
12.  Ecological and taxonomic variation among human RNA viruses 
Journal of Clinical Virology  2013;58(2):344-345.
Only a minority of RNA viruses that can infect humans are capable of spreading in human populations independently of a zoonotic reservoir. This is especially true of vector-borne RNA viruses; the majority of these are not transmissible (via the vector) between humans at all. Understanding the biology underlying this observation will help us evaluate the public health risk associated with novel vector-borne RNA viruses.
doi:10.1016/j.jcv.2013.02.019
PMCID: PMC3793856  PMID: 23518441
Discovery; Emerging; One health
13.  Human Polyoma Viruses and Disease with Emphasis on Clinical BK and JC 
Polyoma viruses are ubiquitous infecting many different mammalian species including humans. There are five known human polyoma viruses. JC virus and BK virus are two polyoma viruses identified nearly three decades ago. Recently WU, KI and Merkel cell polyoma viruses have been isolated from humans. The exact role of these three newly discovered viruses in human disease is not known. Most human polyoma disease is caused by BK and JC viruses which are usually acquired in childhood. Approximately 50–80% of humans have seropositivity to these viruses. Clinically apparent diseases in immunocompetent hosts are extremely rare. These viruses remain latent possibly in the lymphoid organs and kidney and under the circumstances of severe immunosuppression both these viruses reactivate. Neurotropic JC virus reaches the brain and causes progressive multifocal leukencephalopathy, a demyelinating disease of the central nervous system with a high mortality rate. BK virus is urotheliotropic and its reactivation causes a form of interstitial nephritis, known as BK or polyoma virus associated nephropathy which is associated with high graft loss if not recognized early. There are no known effective antiviral agents for any of the polyoma viruses.
doi:10.1016/j.jcv.2009.12.006
PMCID: PMC3774018  PMID: 20060360
Polyomavirus; BK virus; JC virus
14.  Enhanced Detection of Rift Valley Fever Virus using Molecular Assays on Whole Blood Samples 
Journal of Clinical Virology  2012;54(4):313-317.
Background
Rift Valley fever (RVF) is an emerging arthropod-borne zoonoses of global agricultural and public health importance. In December 2006, an RVF outbreak was recognized in Kenya which led to the deployment of international response laboratory teams to the area.
Objectives
A field laboratory was operated in Malindi, Kenya to provide safe sample handling and molecular testing for RVF virus (RVFV) as well as selected other pathogens for differential diagnosis.
Study Design
Safe sample handling was carried out using a negative pressure flexible film isolator (glovebox) and commercial reagents to inactivate clinical specimens and purify nucleic acid. Whole blood was routinely used for diagnostic testing although paired plasma samples were also tested in select cases. Subsequently, human macrophages were tested in vitro for their susceptibility to RVFV.
Results
The field laboratory received samples from 33 individuals and a definite laboratory diagnosis was provided in 16 of these cases. Using molecular diagnostic techniques, RVFV was more consistently detected in whole blood than in plasma samples most likely due to association of RVFV with blood cells. Subsequent in vitro studies identified macrophages as a target cell for RVFV replication.
Conclusions
RVFV appears to replicate in blood cells such as macrophages. Thus, the sensitivity of molecular diagnostic testing is improved if whole blood is used as the clinical specimen rather than plasma or serum.
doi:10.1016/j.jcv.2012.04.022
PMCID: PMC3398164  PMID: 22632901
15.  A national study of individuals who handle migratory birds for evidence of avian and swine-origin influenza virus infections 
Journal of Clinical Virology  2012;54(4):364-367.
Background
Persons with occupational or recreational exposure to migratory birds may be at risk for infection with highly pathogenic avian influenza and other avian influenza viruses since wild birds are the natural reservoir of influenza A. Additionally, bird handlers may host avian and swine-origin influenza (pH1N1) virus co-infections, which generate reassortant viruses with high pathogenicity in mammals.
Objectives
We assessed the prevalence of avian and swine influenza viruses in US-based bird handlers and estimated their exposure to different orders of wild birds including waterfowl (Anseriformes), songbirds (Passeriformes), and shorebirds (Charadriiformes).
Study Design
Cross-sectional serologic survey accompanied by a questionnaire to estimate behavioral risk factors. This is first survey of US-based bird handlers who also work at international sites.
Results
401 participants were recruited and tested over the course of three years. One participant with occupational exposure to migratory birds had evidence of past infections with a H5N2 virus antigenically related to A/Nopi/MN/07/462960-02, which is the first case of this influenza subtype in a human host associated with exposure to wild rather than domestic birds. We detected no avian and swine-origin influenza virus co-infections. The exposure of bird handlers to songbirds was four times greater than to shorebirds or waterfowl.
Conclusions
Though rare, the transmission of avian influenza viruses from migratory birds to US-based bird handlers has potentially significant public health and economic consequences.
doi:10.1016/j.jcv.2012.05.001
PMCID: PMC3398209  PMID: 22632900
16.  Comparative clinical evaluation of the IsoAmp® HSV assay with ELVIS® HSV culture/ID/typing test system for the detection of herpes simplex virus in genital and oral lesions 
Journal of Clinical Virology  2012;54(4):355-358.
Background
The novel IsoAmp® HSV Assay employs isothermal helicase-dependent nucleic acid amplification and a user-friendly disposable test device to achieve rapid (<1.5 hours), on-demand qualitative detection of herpes simplex virus (HSV) types 1 and 2 in oral and genital lesions.
Objectives
To compare performance of the IsoAmp® HSV Assay with the ELVIS® HSV ID/typing (shell-vial culture and DFA) test system for clinical specimens collected from oral and genital lesions in symptomatic patients.
Study design
A total of 994 specimens from male and female genital and oral lesions were obtained and evaluated at five study sites in the United States. Results from the IsoAmp® HSV Assay were compared to those from the ELVIS® system. Separate reproducibility studies were performed at 3 sites using a blinded and randomized study panel. Discrepant specimens were resolved by bidirectional sequencing analysis.
Results
After discrepant analysis, overall agreement of IsoAmp® with ELVIS® was 98.8% with 37.0% overall prevalence (all study sites). Reproducibility rates were well within expectations.
Conclusion
The IsoAmp® HSV assay showed excellent performance for clinical use for detection of HSV in genital and oral specimens. In contrast to ELVIS®, IsoAmp® HSV offers excellent sensitivity plus rapid on-demand testing and simpler specimen preparation.
doi:10.1016/j.jcv.2012.04.004
PMCID: PMC3402216  PMID: 22613012
Herpes simplex virus; Isothermal helicase-dependent amplification; Disposable detection device
17.  Real-Time Reverse Transcriptase PCR Assay for Improved Detection of Human Metapneumovirus 
Background
Human metapneumovirus (HMPV) is a paramyxovirus with multiple genetic lineages that is a leading cause of acute respiratory disease. Several RT-PCR assays have been described based on limited available sequence data.
Objectives
To develop a broadly reactive real-time RT-PCR assay for HMPV that allows for a rapid, sensitive, and specific detection in a clinical or research setting.
Study Design
Three published assays for HMPV were modified based on analysis of multiple HMPV sequences obtained from GenBank. Original and modified assays were tested against prototype HMPV strains from each genetic sublineage, multiple isolates of HMPV from different years, a collection of clinical specimens, and commercial validation panels.
Results
A number of potential sequence mismatches with diverse HMPV strains were identified. Modifications were made to oligonucleotides to improve annealing efficiency. Primers and probes based on newer sequence data offered enhanced detection of all subgroups, especially for low titer specimens. The new primers and probe detected multiple clinical isolates of HMPV collected over a twenty-year period. The modified assay improved detection of HMPV in a panel of clinical specimens, and correctly identified HMPV samples in two commercial validation sets.
Conclusions
We report a modified real-time RT-PCR assay for HMPV that detects all genetic lineages with high sensitivity.
doi:10.1016/j.jcv.2012.05.005
PMCID: PMC3412361  PMID: 22677006
Human metapneumovirus; diagnostics; real-time RT-PCR
18.  Monitoring Seasonal Influenza A Evolution: Rapid 2009 Pandemic H1N1 Surveillance with an Reverse Transcription-Polymerase Chain Reaction/Electro-Spray Ionization Mass Spectrometry Assay 
Background
The emergence of the pandemic H1N1 influenza strain in 2009 reinforced the need for improved influenza surveillance efforts. A previously described influenza typing assay that utilizes RT-PCR coupled to Electro-Spray Ionization Mass Spectrometry (ESI-MS) played an early role in the discovery of the pandemic H1N1 influenza strain, and has potential application for monitoring viral genetic diversity in ongoing influenza surveillance efforts.
Objectives
To determine the analytical sensitivity of RT-PCR/ESI-MS influenza typing assay for identifying the pandemic H1N1 strain and describe its ability to assess viral genetic diversity.
Study design
Two sets of pandemic H1N1 samples, 190 collected between April and June of 2009, and 69 collected between October 2009 and January 2010, were processed by the RT-PCR/ESI-MS influenza typing assay, and the spectral results were compared to reference laboratory results and historical sequencing data from the Nucleotide Database of the National Center for Biotechnology Information (NCBI).
Results
Strain typing concordance with reference standard testing was 100% in both sample sets, and the assay demonstrated a significant increase in influenza genetic diversity, from 10.5% non-wildtype genotypes in early samples to 69.9% in late samples (P<0.001). An NCBI search demonstrated a similar increase, from 13.4% to 45.2% (P<0.001).
Conclusions
This comparison of early versus late influenza samples analyzed by RT-PCR/ESI-MS demonstrates the influenza typing assay’s ability as a universal influenza detection platform to provide high-fidelity pH1N1 strain identification over time, despite increasing genetic diversity in the circulating virus. The genotyping data can also be leveraged for high-throughput influenza surveillance.
doi:10.1016/j.jcv.2012.05.002
PMCID: PMC3495324  PMID: 22673129
Influenza; PCR; Mass Spectrometry
19.  A Comparison of Viral Fitness and Virulence between Emergent Adenovirus 14p1 and Prototype Adenovirus 14p Strains 
Journal of Clinical Virology  2012;54(3):265-268.
Background
Epidemiological studies from the last decade have suggested that the morbidity and mortality associated with a newly emergent strain of human adenovirus (HAdV-14p1) is greater than other, more prevalent, adenovirus strains. Recent molecular analysis identified very minor genetic differences in HAdV-14p1 compared to prototype HAdV-14p. No studies have evaluated how these differences may affect virulence.
Objective
To compare HAdV-14p1 and HAdV-14p strains for competitive fitness and virulence.
Study Design
We performed in vitro and molecular assays to evaluate growth kinetics, cellular infectivity, cytotoxicity, and plaque morphology of the two strains.
Results
Growth kinetic data showed no viral replication at 30°C and minimal differences at 37°C for both strains. Cellular infectivity data showed propagation capabilities for both strains in a diverse array of cell lines, with human lung and kidney cells having the highest propagation potential. Cytotoxicity data indicated cellular distress differences induced by both strains of virus in the first 12 hours, but similar distress levels between 12 and 48 hours. Plaque morphology assays showed some differences in average plaque diameter.
Conclusions
These data suggest that the increase in morbidity and mortality observed in recent HAdV-14p1 infections is not due to viral growth or cellular infectivity differences from the prototypic HAdV-14 strain. While there were some statistically important differences detected between strains in cytotoxicity and plaque morphology assays, it seems more likely that other factors, such as environmental stressors, co-infections, or individual host response are likely contributing to the increase in morbidity.
doi:10.1016/j.jcv.2012.03.006
PMCID: PMC3367116  PMID: 22484030
adenoviruses; virology; viral diseases; virulence
20.  Successful use of Plasma Preparation Tubes™(PPTs) in the COBAS® AmpliPrep/COBAS® TaqMan® HIV-1 Test 
Background
Since switching to the COBAS® AmpliPrep/COBAS® TaqMan® HIV-1 Test, v. 1.0 from the Amplicor HIV-1 Monitor Test, v. 1.5, an increase in detectable viral load results was noted. We were concerned that this was due to the use of Plasma Preparation Tubes (PPT) in this test.
Objective
To assess the impact of different pre-analytical processing conditions on HIV-1 viral load results on the COBAS® AmpliPrep/COBAS® TaqMan® HIV-1 Test.
Study design
Sixty-three HIV-infected patients were consented and had 3 PPTs and 1 K2EDTA drawn for HIV-1 viral load testing. Three methods of PPT processing were compared against the referent K2EDTA tube which was spun at 1100 × g for 20 min, poured off and frozen; PPT1 was refrigerated with an additional centrifugation prior to testing, PPT2 was processed similarly to EDTA, and PPT3 was centrifuged, frozen and centrifuged again prior to testing.
Results
PPT1 and PPT3 yielded results that were most similar to the referent EDTA processing, with a concordance correlation coefficient (CCC) of 0.80 and 0.85, compared to PPT2 with CCC of 0.37. Both PPT1 and PPT3 involved additional centrifugation prior to testing. In 26 patients with residual samples from the PPT2 processing, 9 (34.6%) were found to have the presence of proviral DNA, which likely contributed to the elevated HIV-1 RNA viral loads in these individuals.
Conclusion
PPTs can be used in the COBAS® AmpliPrep/COBAS® TaqMan® HIV-1 Test with an additional centrifugation in order to avoid misleading elevated HIV-1 RNA viral loads that may change patient management.
doi:10.1016/j.jcv.2012.12.015
PMCID: PMC3684267  PMID: 23332979
HIV-1; Viral load; COBAS Amplicor/COBAS Ampliprep; Plasma Preparation Tubes
21.  Prevalence of hepatitis C virus infection among human immunodeficiency virus-1-infected pregnant women in Malawi: The BAN study☆ 
Background
In Sub-Saharan Africa, prevalence estimates of hepatitis C virus (HCV) vary widely.
Objectives
To assess the prevalence of HCV infection among HIV-infected, pregnant women screened for a large clinical trial in Lilongwe, Malawi.
Study design
Plasma from 2041 HIV-infected, pregnant women was screened for anti-HCV IgG using a chemiluminiscent immunometric assay (CIA). Specimens with a signal-cut-off ratio ≥ 1.00 were considered reactive and those with S/Co ratio < 1.00 non-reactive. All CIA-reactive specimens were tested by a recombinant immunoblot assay (RIBA) for anti-HCV and by PCR for HCV RNA.
Results
Of 2041 specimens, 110 (5.3%, 95% CI: 4.5–6.5%) were CIA reactive. Of the 109 CIA reactive specimens available for RIBA testing, 2 (1.8%) were positive, 28 (25.7%) were indeterminate, and 79 (72.5%) were negative. All CIA-reactive specimens were HCV RNA negative (n = 110). The estimated HCV prevalence based on the screening assay alone was 5.3%; based on supplemental RIBA testing, the status of HCV infection remained indeterminate in 1.4% (28/2040, 95% CI: 0.1–2.0) and the prevalence of confirmed HCV infections was 0.1% (2/2040, 95% CI: 0–0.4%).
Conclusions
HCV seroprevalence among HIV-infected, pregnant women in Malawi confirmed by supplemental RIBA HCV 3.0 is low (0.1%); CIA showed a high false-reactivity rate in this population.
doi:10.1016/j.jcv.2012.05.003
PMCID: PMC3652577  PMID: 22658797
HIV; HCV; Pregnant women; Malawi
22.  Diagnostic performance of two highly multiplexed respiratory virus assays in a pediatric cohort 
Background
Rapid detection of respiratory viruses is important for management and infection control in hospitalized patients. Multiplex nucleic acid tests (NATs) have begun to replace conventional methods as gold standards for respiratory virus detection.
Objective
To compare the performance of two large multiplex NATS, ResPlex II (RPII) and Respiratory Virus Surveillance kit with electrospray ionization mass spectrometry (RVS/MS) using nasopharyngeal aspirates (NPAs) from hospitalized children who had been tested previously with conventional methods.
Study design
Stored residual NPAs (N = 306) were tested concomitantly by RPII and RVS/MS. Alternate NATs were used to adjudicate discordant results.
Results
More viruses were detected with multiplex NATs (RPII, 110; RVS/MS, 109) than conventional assays (86); diagnostic gain was primarily for fastidious viruses (coronaviruses and enteroviruses [EVs]/human rhinoviruses [HRVs]). Total positive and negative agreement between the multiplex NATs for all viruses detected was quite high (86% positive agreement, 99% negative agreement). Most individual viruses were detected with fairly equivalent accuracy by the multiplex NATs, except for adenoviruses (RPII sensitivity 40%) and human metapneumovirus (RVS/MS sensitivity 42%). RPII had the advantage of detecting EVs and HRVs, however, it demonstrated considerable EV/HRV cross-reactivity (29 HRV-positive specimens by real-time PCR were positive for EV by RPII and 21 specimens positive for HRV only by RT-PCR were dual positive for EV/HRV by RPII). RPII also had reduced sensitivity for HRV detection (in 36 specimens, HRV was detected by RT-PCR but not by RPII).
Conclusions
Both multiplex NATs were promising, but had notable limitations.
doi:10.1016/j.jcv.2012.06.019
PMCID: PMC3586269  PMID: 22832060
Respiratory virus diagnostics; Respiratory virus nucleic acid tests; Respiratory virus multiplex tests
23.  Evidence of immune memory 8.5 years following administration of a prophylactic human papillomavirus type 16 vaccine 
Journal of Clinical Virology  2011;53(3):239-243.
Background
The duration of protection conferred by prophylactic human papillomavirus (HPV) L1 virus-like particle vaccines is a critical determinant of their public health impact. A feature of vaccines that confer long-term immunity is their ability to induce immune memory.
Objectives
We evaluated antibody responses against HPV types 6, 11, 16 and 18 following administration of the quadrivalent HPV-6/11/16/18 vaccine to women who had previously received a monovalent HPV-16 vaccine.
Study design
As part of an extended follow-up study conducted between 2006 and 2009 in Seattle, Washington, we administered the quadrivalent HPV-6/11/16/18 vaccine to 52 women (19 vaccine and 33 placebo recipients) who had participated in a monovalent HPV-16 vaccine trial 8.5 years earlier. Serum samples were tested for anti-HPV antibodies using competitive Luminex immunoassay.
Results
Following administration of the first dose of the quadrivalent HPV-6/11/16/18 vaccine, the anti-HPV-16 geometric mean titer among monovalent HPV-16 vaccine recipients (GMT = 5024.0 milli-Merck units per milliliter [mMU/mL]; 95% confidence interval [CI]: 2710.1, 9313.6 mMU/mL) substantially exceeded that among the placebo recipients (GMT = 136.1; 95% CI: 78.5, 235.8 mMU/mL; p < 0.01) and their own highest anti-HPV-16 response observed during the original trial (GMT at month 7 of the original trial = 1552.7 mMU/mL; 95% CI: 1072.6, 2247.7 mMU/mL; p < 0.01).
Conclusions
The findings suggest that the administration of the three-dose regimen of the monovalent HPV-16 vaccine had produced memory lymphocytes, characterized by a heightened immune response following administration of the quadrivalent HPV-6/11/16/18 vaccine that effectively served as an antigen challenge.
doi:10.1016/j.jcv.2011.12.009
PMCID: PMC3279625  PMID: 22209292
Human papillomavirus type 16; Vaccines; Immune memory
24.  Clinical Variables Identify Seronegative HCV Co-infection in HIV-Infected Individuals 
Background
A substantial number of people living with HIV (PLWH) are co-infected with Hepatitis C Virus (HCV) but have a negative screening HCV antibody test (seronegative HCV infection, or SN-HCV).
Objective
To identify a concise set of clinical variables that could be used to improve case finding for SN-HCV co-infection among PLWH.
Study design
Two hundred HIV-infected participants of the CHARTER study were selected based on 7 clinical variables associated with HCV infection but were HCV seronegative. Data were analyzed using Fisher's exact tests, receiver-operating characteristic (ROC) curves, and logistic regression.
Results
Twenty-six (13%) participants had detectable HCV RNA. SN-HCV was associated with a history of IDU, elevated ALT and AST, low platelets, black ethnicity, and undetectable HIV RNA in plasma. Each of these clinical variables, except for abnormal AST, remained independently associated with SN-HCV in a multivariate logistic regression analysis. A composite risk score correctly identified SN-HCV with sensitivity up to 85% and specificity up to 88%.
Conclusions
In a substantial minority of PLWH, seronegative HCV viremia can be predicted by a small number of clinical variables. These findings, after validation in an unselected cohort, could help focus screening in those at highest risk.
doi:10.1016/j.jcv.2011.08.021
PMCID: PMC3217127  PMID: 21924674
Seronegative HCV; HIV; co-infection; case finding
25.  The Core/E1 Domain of Hepatitis C Virus Genotype 4a in Egypt does not Contain Viral Mutations or Strains Specific for Hepatocellular Carcinoma 
Bachground
Hepatitis C virus (HCV) infection is a well-documented etiological factor for hepatocellular carcinoma (HCC). As HCV shows remarkable genetic diversity, an interesting and important issue is whether such a high viral genetic diversity plays a role in the incidence of HCC. Prior data on this subject are conflicting.
Objectives
Potential association between HCV genetic mutations or strain variability and HCC incidence has been examined through a comparative genetic analysis merely focused on a single HCV subtype (genotype 4a) in a single country (Egypt).
Study design
The study focused on three HCV sequence datasets with explicit sampling dates and disease patterns. An overlapping HCV Core/E1 domain from three datasets was used as the target for comparative analysis through genetic and phylogenetic approaches.
Results
Based on partial Core/E1 domain (387 bp), genetic and phylogenetic analysis did not identify any HCC-specific viral mutations and strains, respectively.
Conclusions
The Core/E1 domain of HCV genotype 4a in Egypt does not contain HCC-specific mutations or strains. Additionally, sequence errors resulting from the polymerase chain reaction, together with a strong evolutionary pressure on HCV in patients with end-stage liver disease, have significant potential to bias data generation and interpretation.
doi:10.1016/j.jcv.2011.08.022
PMCID: PMC3217131  PMID: 21925935
hepatitis C virus; hepatocellular carcinoma; cirrhosis; RT-PCR; Egypt

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