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1.  [No title available] 
PMCID: PMC4289600  PMID: 18790666
2.  Characterization of three novel human papillomavirus types isolated from oral rinse samples of healthy individuals 
Background
Despite the strong evidence of HPV infection as the etiological agent in a subset of oral cancer, oral α-HPV detection is rare in healthy individuals, and little is known of the existing of novel HPV types in oral cavity.
Objective
We determined whether novel HPV types can be isolated from oral rinse samples collected from healthy individuals.
Study design
We performed rolling circle amplification (RCA) coupled with degenerated PCR assay on 48 oral rinse samples to amplify novel HPV types. Full length HPV DNA was cloned using long range PCR. Quantitative type specific Taqman assays were used to determine the prevalence of novel HPV types in 158 archived oral tissue samples.
Results
We were able to isolate four novel human papillomavirus types. Full length HPV DNA was cloned for three of the four novel HPV types. All four HPV types belong to the genus Gammapapillomavirus (γ-PV), where HPV 171 is most closely related to HPV 169, showing 88% similarity; HPV 172 is most closely related to HPV 156, showing 70% similarity; HPV 173 is most closely related to HPV 4, showing 73% similarity; oral sample lavage (OSL) 37 is most closely related to HPV 144, showing 69% similarity. Finally, we showed that HPV 173 was rarely present in oral tissues (2/158), HPV 172 was only detected in normal oral tissues (25/76), and HPV 171 was more prevalent in malignant oral tissues (17/82 vs 10/76, p=0.21).
Conclusions
Novel γ-HPV types are present in oral cavity of healthy individuals.
doi:10.1016/j.jcv.2013.10.028
PMCID: PMC3898882  PMID: 24268765
HPV; oral lavage; oral squamous cell carcinoma
3.  Finding those at risk: Acute HIV infection in Newark, NJ 
Background
A screening strategy combining rapid HIV-1/2 (HIV) antibody testing with pooled HIV-1 RNA testing increases identification of HIV infections, but may have other limitations that restrict its usefulness to all but the highest incidence populations.
Objective
By combining rapid antibody detection and pooled nucleic acid amplification testing (NAAT) testing, we sought to improve detection of early HIV-1 infections in an urban Newark, NJ hospital setting.
Study design
Pooled NAAT HIV-1 RNA testing was offered to emergency department patients and out-patients being screened for HIV antibodies by fingerstick-rapid HIV testing. For those negative by rapid HIV and agreeing to NAAT testing, pooled plasma samples were prepared and sent to the University of Washington where real-time reverse transcription-polymerase chain reaction (RT-PCR) amplification was performed.
Results
Of 13,226 individuals screened, 6381 had rapid antibody testing alone, and 6845 agreed to add NAAT HIV screening. Rapid testing identified 115 antibody positive individuals. Pooled NAAT increased HIV-1 case detection by 7.0% identifying 8 additional cases. Overall, acute HIV infection yield was 0.12%. While males represent only 48.1% of those tested by NAAT, all samples that screened positive for HIV-1 RNA were obtained from men.
Conclusion
HIV-1 RNA testing of pooled, HIV antibody-negative specimens permits identification of recent infections. In Newark, pooled NAAT increased HIV-1 case detection and provided an opportunity to focus on treatment and prevention messages for those most at risk of transmitting infection. Although constrained by client willingness to participate in testing associated with a need to return to receive further results, use of pooled NAAT improved early infection sensitivity.
doi:10.1016/j.jcv.2013.07.016
PMCID: PMC4266376  PMID: 23953941
HIV testing algorithms; NAAT; Rapid HIV tests; HIV screen; HIV seronegativity; Nucleic acid amplification tests
4.  Cutaneous human papillomavirus types detected on the surface of male external genital lesions: A case series within the HPV Infection in Men Study 
Background
Cutaneous human papillomaviruses (HPVs) may be associated with cutaneous epithelial lesions and non-melanoma skin cancers. No study has systematically evaluated the presence of genus beta [β]-HPV in male genital skin or external genital lesions (EGLs).
Objectives
To examine cutaneous β-HPV types detected on the surface of EGLs in men and describe their presence prior to EGL development.
Study design
A retrospective case series was conducted among 69 men with pathologically confirmed EGLs (n=72) who participated in the HPV Infection in Men Study. Archived exfoliated cells collected from the surface of each EGL and normal genital skin specimens 6–12 months preceding EGL development were tested for β-HPV DNA using a type-specific multiplex genotyping assay.
Results
β-HPV DNA was detected on 61.1% of all EGLs, with types 38 (16.7%), 5 (15.3%), and 12 (12.5%) most commonly identified. HPV prevalence differed across pathological diagnoses, with the largest number of β-HPV types detected on condylomas. Most β-HPV types were detected on normal genital skin prior to EGL development, though the prevalence was lower on EGLs compared to preceding normal genital skin.
Conclusions
EGLs and the normal genital skin of men harbor a large number of β-HPV types; however, it appears that β-HPVs are unrelated to EGL development in men. Despite evidence to support a causal role in skin carcinogenesis at UVR-exposed sites, cutaneous HPV appears unlikely to cause disease at the UVR-unexposed genitals.
doi:10.1016/j.jcv.2013.10.011
PMCID: PMC3866698  PMID: 24210970
5.  Relative Accuracy of Serum, Whole Blood, and Oral Fluid HIV Tests among Seattle Men Who Have Sex with Men 
Background
Point-of-care (POC) rapid HIV tests have sensitivity during the “window period” comparable only to earliest generation EIAs. To date, it is unclear whether any POC test performs significantly better than others.
Objective
Compare abilities of POC tests to detect early infection in real time.
Study Design
Men who have sex with men (MSM) were recruited into a prospective, cross-sectional study at two HIV testing sites and a research clinic. Procedures compared four POC tests: one performed on oral fluids and three on fingerstick whole blood specimens. Specimens from participants with negative POC results were tested by EIA and pooled nucleic acid amplification testing (NAAT). McNemar’s exact tests compared numbers of HIV-infected participants detected.
Results
Between February 2010 and May 2013, 104 men tested HIV-positive during 2479 visits. Eighty-two participants had concordant reactive POC results, 3 participants had concordant non-reactive POC tests but reactive EIAs, and 8 participants had acute infection. Of 12 participants with discordant POC results, OraQuick ADVANCE Rapid HIV-1/2 Antibody Test performed on oral fluids identified fewer infections than OraQuick performed on fingerstick (p=.005), Uni-Gold Recombigen HIV Test (p=.01), and Determine HIV-1/2 Ag/Ab Combo (p=.005).
Conclusions
These data confirm that oral fluid POC testing detects fewer infections than other methods and is best reserved for circumstances precluding fingerstick or venipuncture. Regardless of specimen type, POC tests failed to identify many HIV-infected MSM in Seattle. In populations with high HIV incidence, the currently approved POC antibody tests are inadequate unless supplemented with p24 antigen tests or NAAT.
doi:10.1016/j.jcv.2013.09.018
PMCID: PMC3867744  PMID: 24342471
HIV testing; rapid HIV test; oral fluids
6.  Galectin-9 Plasma Levels Reflect Adverse Hamatological and Immunological Features in Acute Dengue Virus Infection 
Background
Dengue virus (DENV) infection remains a major public health burden worldwide. Soluble mediators may play a critical role in the pathogenesis of acute DENV infection. Galectin-9 (Gal-9) is a soluble β-galactoside-binding lectin, with multiple immunoregulatory and inflammatory properties.
Objective
To investigate plasma Gal-9 levels as a biomarker for DENV infection.
Study design
We enrolled 65 DENV infected patients during the 2010 epidemic in the Philippines and measured their plasma Gal-9 and cytokine/chemokine levels, DENV genotypes, and copy number during the critical and recovery phases of illness.
Results
During the critical phase, Gal-9 levels were significantly higher in DENV infected patients compared to healthy or those with non-dengue febrile illness. The highest Gal-9 levels were observed in dengue hemorrhagic fever (DHF) patients (DHF: 2464 pg/ml; dengue fever patients (DF): 1407 pg/ml; non-dengue febrile illness: 616 pg/ml; healthy: 196 pg/ml). In the recovery phase, Gal-9 levels significantly declined from peak levels in DF and DHF patients. Gal-9 levels tracked viral load, and were associated with multiple cytokines and chemokines (IL-1α, IL-8, IP-10, and VEGF), including monocyte frequencies and hematologic variables of coagulation. Further discriminant analyses showed that eotaxin, Gal-9, IFN-α2, and MCP-1 could detect 92% of DHF and 79.3% of DF, specifically (P<0.01).
Conclusion
Gal-9 appears to track DENV inflammatory responses, and therefore, it could serve as an important novel biomarker of acute DENV infection and disease severity.
doi:10.1016/j.jcv.2013.10.022
PMCID: PMC3880569  PMID: 24239423
Galectin-9; dengue virus; biomarker; dengue fever; dengue hemorrhagic fever
7.  Influence of HIV-1 and/or HIV-2 infection and CD4 count on cervical HPV DNA detection in women from Senegal, West Africa 
Background
HIV infection is associated with greater risk of precancerous lesions and cervical cancer in women. However, several factors remain unclarified regarding the association between HIV infection and HPV detection, especially among those with HIV type 2 versus type 1 infection and severely immunocompromised persons.
Objectives
To evaluate HPV overall and type-specific detection among HIV-infected and uninfected women in Senegal.
Study Design
Detection of HPV DNA for 38 genotypes in cervical swabs using PCR-based methods was evaluated in HIV-positive (n=467) and HIV-negative (n=2139) women participating in studies in Senegal. Among HIV-1 and/or HIV-2 positive women, CD4 counts were assessed. Adjusted multivariable prevalence ratios (PR) were calculated.
Results
The prevalence of any HPV DNA and multiple HPV types was greater among HIV-infected individuals (78.2% and 62.3%, respectively) compared with HIV-negative women (27.1% and 11.6%). This trend was also seen for HPV types 16 and 18 (13.1% and 10.9%) compared to HIV-negative women (2.2% and 1.7%). HIV-infected women with CD4 cell counts less than 200 cells/µl had a higher likelihood of any HPV detection (PRa 1.30; 95% CI 1.07–1.59), multiple HPV types (PRa 1.52; 95% CI 1.14–2.01), and HPV-16 (PRa 9.00; 95% CI 1.66–48.67), but not HPV-18 (PRa 1.20, 95% CI 0.45–3.24) compared to those with CD4 counts 500 cells/µl or above.
Conclusion
HIV-infected women, especially those most severely immunocompromised, are more likely to harbor HPV. Measures to prevent initial HPV infection and subsequent development of cervical cancer through focused screening efforts should be implemented in these high risk populations.
doi:10.1016/j.jcv.2013.10.012
PMCID: PMC4059498  PMID: 24210330
8.  Performance of an alternative HIV diagnostic algorithm using the ARCHITECT HIV Ag/Ab Combo assay and potential utility of sample-to-cutoff ratio to discriminate primary from established infection☆ 
Background
The ARCHITECT HIV Ag/Ab Combo assay has a wide dynamic range for determining the sample-to-cutoff ratio (S/CO) values compared to other diagnostic HIV antibody assays.
Objectives
Determine the performance of an HIV testing algorithm that uses the ARCHITECT combo assay in the clinical setting and explore the utility of the signal-to-cutoff (S/CO) ratio to predict acute HIV-1 infection status.
Study design
A retrospective analysis of clinical samples from a hospital and referral population screened for HIV-1 infection between May 2011 and March 2013. Repeatedly reactive samples were tested using the Multispot HIV-1/HIV-2 rapid test and depending on that result, confirmatory orthogonal testing used the Western blot (WB) for HIV-1, Immunoblot for HIV-2 and nucleic acid amplification testing (NAAT) for HIV RNA.
Results
A total of 21,317 test results were evaluated of which 509 were ARCHITECT repeatedly reactive; of these, 422 were Multispot-reactive only for HIV-1 (413 WB-positive; 9 indeterminate), 4 were Multispot-reactive for both HIV-1 and HIV-2 (one HIV-2 immunoblot-positive with 17 HIV-2 RNA copies/mL) and 83 were Multispot-non-reactive of which 15 were HIV-1 RNA positive and represented acute HIV-1 infection. There was an association among the ARCHITECT S/CO (median; IQR) values for antibody-negative (0.14; 0.11–0.16), acute infection (33; 2.1–76) and established HIV-1 infection (794; 494–1,029) (Kruskal–Wallis, p < 0.0001).
Conclusions
The ARCHITECT combo assay with Multispot confirmation and reserved use of HIV-1 WB, HIV-2 Immunoblot and HIV NAAT for Multispot dual HIV-1/2 infection, and NAAT alone for Multispot-negative specimens, had a suitable test performance for detecting acute and established HIV infection.
doi:10.1016/j.jcv.2013.08.014
PMCID: PMC4209943  PMID: 24029686
ARCHITECT HIV Ag/Ab Combo assay Sample-to-cutoff ratio Alternative HIV diagnostic algorithm
9.  Clinical performance of the Multispot HIV-1/HIV-2 rapid test to correctly differentiate HIV-2 from HIV-1 infection in screening algorithms using third and fourth generation assays and to identify cross reactivity with the HIV-1 Western Blot 
Background
An accurate and rapid serologic method to differentiate HIV-2 from HIV-1 infection is required since the confirmatory HIV-1 Western Blot (WB) may demonstrate cross-reactivity with HIV-2 antibodies.
Objectives
To evaluate the performance of the Bio-Rad Multispot HIV-1/HIV-2 rapid assay as a supplemental test to correctly identify HIV-2 infection and identify HIV-1 WB cross-reactivity with HIV-2 in clinical samples tested at an academic medical center.
Study design
Between August 2008 and July 2012, clinical samples were screened for HIV using either 3rd-or 4th-generation HIV-1/2 antibody or combination antibody and HIV-1 p24 antigen assays, respectively. All repeatedly reactive samples were reflexed for Multispot rapid testing. Multispot HIV-2 and HIV-1 and HIV-2-reactive samples were further tested using an HIV-2 immunoblot assay and HIV-1 or HIV-2 RNA assays when possible. The HIV-1 WB was performed routinely for additional confirmation and to assess for HIV-2 antibody cross-reactivity.
Results
Of 46,061 samples screened, 890 (89.6%) of 993 repeatedly reactive samples were also Multispot-reactive: 882 for HIV-1; three for only HIV-2; and five for both HIV-1 and HIV-2. All three HIV-2-only Multispot-positives along with a single dually reactive HIV-1/2 Multispot-positive were also HIV-2 immunoblot-positive; the latter was HIV-1 RNA negative and HIV-2 RNA positive.
Conclusions
The Multispot rapid test performed well as a supplemental test for HIV-1/2 diagnostic testing. Four new HIV-2 infections (0.45%) were identified from among 890 Multispot-reactive tests. The use of HIV-1 WB alone to confirm HIV-1/2 screening assays may underestimate the true prevalence of HIV-2 infection in the United States.
doi:10.1016/j.jcv.2013.08.018
PMCID: PMC4210374  PMID: 24342468
Multispot; HIV-2; HIV testing algorithms; Nucleic acid amplification tests; Orthogonal supplemental testing
10.  Correlation of immune activation with HIV-1 RNA levels assayed by real-time RT-PCR in HIV-1 Subtype C infected patients in Northern India 
Background
Assays with specificity and cost effectiveness are needed for the measurement of HIV-1 burden to monitor disease progression or response to anti-retroviral therapy (ART) in HIV-1 subtype C infected patients.
Objectives
The objective of this study was to develop and validate an affordable; one step Real-Time RT-PCR assay with high specificity and sensitivity to measure plasma HIV-1 loads in HIV-1 subtype C infected patients.
Results
We developed an RT-PCR assay to detect and quantitate plasma HIV-1 levels in HIV-1 subtype C infected patients. An inverse correlation between plasma viral loads (PVL) and CD4+ T-cell numbers was detected at all CDC stages. Significant correlations were found between CD8+ T-cell activation and PVL, as well as with the clinical and immunological status of the patients.
Conclusions
The RT-PCR assay provides a sensitive method to measure PVL in HIV-1 subtype C infected patients. Viral loads correlated with immune activation and can be used to monitor HIV care in India.
doi:10.1016/j.jcv.2007.08.020
PMCID: PMC4230990  PMID: 17962068
real-time RT-PCR; HIV-1 viral load; HIV-1 subtype C
11.  Clinical and Molecular Epidemiological Features of Hemorrhagic Fever with Renal Syndrome in Korea over a 10-year Period 
Background
Laboratory diagnosis of hemorrhagic fever with renal syndrome (HFRS), an infectious disease caused by rodent-borne hantaviruses in Asia and Europe, depends primarily on serological methods. Since the advent of such serodiagnostic tests, few reports are available about the clinical and molecular epidemiological features of HFRS.
Objectives
To investigate the epidemioclinical features of HFRS treated at a tertiary-care teaching hospital in Seoul over a 10-year period.
Study design
Medical records of HFRS patients, visited to a tertiary-care teaching hospital during February 2002 to February 2012, were reviewed. Sera from patients were tested for Hantaan virus (HTNV) and Seoul virus (SEOV) RNA using RT-PCR.
Results
Among 35 HFRS patients (mean age was 44.2 ± 14.7 years), 29 were male (82.9%). Acute renal failure developed in 27 patients (77.1%), and 12 patients (34.3%) were admitted to the intensive care unit (ICU). Conjunctival injection (OR 10.32, 95% CI 1.09–97.77, P = .04) and initial serum albumin less than 3 g/dL (OR 22.83, 95% CI 1.45–359.93, P = .03) were risk factors for ICU admission. Of 35 acute-phase sera, 11 (31.4%) were positive for HTNV RNA. None were positive for SEOV RNA.
Conclusions
HFRS was characterized by the clinical triad of fever, renal insufficiency and gastrointestinal symptoms. Conjunctival injection and serum albumin level were related to severity. Large scaled multi-center study is needed to enhance an insight to epidemioclinical characteristics of HFRS in Korea.
doi:10.1016/j.jcv.2013.06.027
PMCID: PMC4153883  PMID: 23871164
Hantaan virus; Hantavirus; Hemorrhagic fever with renal syndrome; Korea
12.  Automated, simple, and efficient influenza RNA extraction from clinical respiratory swabs using TruTip and epMotion 
Background
Rapid, simple and efficient influenza RNA purification from clinical samples is essential for sensitive molecular detection of influenza infection. Automation of the TruTip extraction method can increase sample throughput while maintaining performance.
Objectives
To automate TruTip influenza RNA extraction using an Eppendorf epMotion robotic liquid handler, and to compare its performance to the bioMerieux easyMAG and Qiagen QIAcube instruments.
Study Design
Extraction efficacy and reproducibility of the automated TruTip/epMotion protocol was assessed from influenza-negative respiratory samples spiked with influenza A and B viruses. Clinical extraction performance from 170 influenza A and B-positive respiratory swabs was also evaluated and compared using influenza A and B real-time RT-PCR assays.
Results
TruTip/epMotion extraction efficacy was 100% in influenza virus-spiked samples with at least 745 influenza A and 370 influenza B input gene copies per extraction, and exhibited high reproducibility over four log10 concentrations of virus (<1% CV). RNA yields between the three automated methods differed by less than 0.5 log10 gene copies. 99% of clinical specimens that were PCR-positive after easyMAG or QIAcube extraction were also positive following TruTip extraction. Overall Ct value differences obtained between TruTip/epMotion and easyMAG/QIAcube clinical extracts ranged from 1.24 to 1.91. Pairwise comparisons of Ct values showed a high correlation of the TruTip/epMotion protocol to the other methods (R2 >0.90).
Conclusion
The automated TruTip/epMotion protocol is a simple and rapid extraction method that reproducibly purifies influenza RNA from respiratory swabs, with comparable efficacy and efficiency to both the easyMAG and QIAcube instruments.
doi:10.1016/j.jcv.2013.06.033
PMCID: PMC3810421  PMID: 23880159
RNA; influenza; automated extraction; epMotion; TruTip; respiratory swabs
13.  An Artesunate-Containing Antimalarial Treatment Regimen Did Not Suppress Cytomegalovirus Viremia 
Background
Additional drugs are needed for the treatment of cytomegalovirus (CMV) infection. Artesunate is an antimalarial drug that has activity against CMV in vitro and in a rodent model. Only a small number of case reports are available describing the clinical effects of artesunate on CMV infection, and these yielded inconsistent results.
Objective
To evaluate the effect of artesunate on CMV infection, using blood samples collected from children who participated in malaria treatment trials.
Study design
Quantitative CMV DNA PCR was performed on dried blood spots collected from 494 Ugandan children, who were randomized either to artesunate plus amodiaquine or sulfadoxine-pyrimethamine plus amodiaquine for acute malaria infection. Poisson regression was used to compare treatment regimens with respect to the change in the frequency and quantity of CMV detected that occurred before and after treatment.
Results
CMV was detected in 11.4% of children immediately prior to treatment and 10.7% 3 days later (p=0.70). The average quantity of CMV was 0.30 log10 copies per million cells higher on day 3 than at treatment initiation (95% CI 0.01 to 0.58, p=0.041). There was no measurable difference in either the frequency or quantity of CMV detected in blood between children randomized to the two treatment arms.
Conclusions
A standard 3-day artesunate-containing antimalarial regimen had no detectable effect on CMV viremia in children with malaria. Longer treatment courses and/or higher doses of artesunate than those routinely used for malaria may be required for effective treatment of CMV infection.
doi:10.1016/j.jcv.2013.06.008
PMCID: PMC4036801  PMID: 23827788
14.  Analysis of primary resistance mutations to HIV-1 entry inhibitors in therapy naive subtype C HIV-1 infected mother–infant pairs from Zambia 
Background
Small molecular CCR5 inhibitors represent a new class of drugs for treating HIV-1 infection. The evaluation of the primary resistance mutations associated with entry inhibitors during HIV-1 perinatal transmission is required because they may have a profound impact on the clinical management in MTCT.
Objectives
To evaluate the primary resistance mutations to maraviroc and vicriviroc during perinatal transmission and analyze the sensitivity of Env derived from mother–infant pairs to maraviroc.
Study design
Nine MIPs infected by subtype C HIV-1 were recruited to analyze the prevalence and transmission of primary resistance mutations to maraviroc and vicriviroc. Moreover, Env derived from six MIPs were employed to construct provirus clones and to analyze the sensitivity to maraviroc.
Results
Mutations A316T, conferring partial resistance to maraviroc, T307I and R315Q, both conferring partial resistance to vicriviroc are prevalent in mother and infant cohorts, indicating the transmission of primary resistance mutations during HIV-1 perinatal transmission. However, the mutations of acutely infected mothers seem to directly transmit to their corresponding infants, while some mutations at low frequency of chronically infected mothers would be lost during transmission. Moreover, provirus clones derived from acutely infected MIPs are less susceptible to maraviroc than those from chronically infected MIPs.
Conclusions
Our study suggests that the transmission mode of primary resistance mutations and the sensitivity to maraviroc are dependent on infection status of MIPs either acutely or chronically infected. These results may indicate that higher dose of maraviroc could be needed for treatment of acutely infected MIPs compared to chronically infected MIPs.
doi:10.1016/j.jcv.2013.05.022
PMCID: PMC4017664  PMID: 23809473
MTCT; HIV-1; Subtype C; Primary resistance; Maraviroc; Vicriviroc
15.  Molecular epidemiology of respiratory syncytial virus transmission in childcare 
Background
Respiratory syncytial virus (RSV) is the most important cause of serious respiratory infections in young children. No prior studies using molecular techniques to examine RSV transmission in the community childcare setting have been performed.
Objectives
We seek to characterize the molecular epidemiology of RSV transmission in childcare to evaluate the impact of RSV disease in a community-based population.
Methods
We sequenced RSV-positive nasopharyngeal samples from a prospective longitudinal study of respiratory illnesses among children enrolled in childcare during three winter seasons. Phylogenetic analysis was performed to identify unique viral strains.
Results
RSV was detected in 59 (11%) illnesses. Compared to RSV-negative illnesses, RSV-positive illnesses were associated with longer symptom duration and increased frequency of health care visits. Another respiratory virus was detected in 42 (71%) RSV-positive illnesses. RSV viral load did not differ between RSV-positive illnesses with and without another respiratory virus identified (P = 0.38). In two childcare rooms, 50% of the children had RSV detected within six days of the first case. Five (38%) of 13 illness episodes from one childcare room were sequenced and shown to be the same viral strain, suggesting rapid child-to-child transmission within the room over a 16 day period.
Conclusions
RSV is rapidly transmitted within childcare. Childcare facilities may serve as ideal sites for evaluation of new prevention strategies given the high burden of RSV disease in this population and the rapidity of RSV spread between children.
doi:10.1016/j.jcv.2013.04.011
PMCID: PMC3800193  PMID: 23684816
Respiratory syncytial virus; Childcare; Molecular epidemiology; Infection control
16.  An increased diversity of HCV isolates were characterized among 393 patients with liver disease in China representing six genotypes, 12 subtypes, and two novel genotype 6 variants 
Background
We have recently determined HCV isolates among volunteer blood donors and IDUs in southern China and revealed the genotype distribution patterns not only different between the two studied cohorts but also from what we have sampled in 2002. A changed pattern could have also occurred among patients with liver disease.
Materials/Methods
Both E1 and NS5B sequences of HCV were characterized among 393 patients with liver disease followed by phylogenetic analysis.
Results
Six HCV genotypes, 12 subtypes (1b: 65.9%, 6a: 17.1%, 2a: 7.4%, 3a: 3.6%, 3b: in 3.3%, 6e: 0.76%, and 1a, 1c, 2b, 2f, 4d, and 5a each 0.25%), and two novel genotype 6 variants were classified, showing the greatest complexity of HCV hitherto found in China. Although the predominance of 1b followed by 6a is largely consistent with what we have sampled in 2002, the identification of single isolates of 1c, 2f, 4d, 5a, and two novel HCV-6 variants were first reported. Excluding 4d from a European visitor, all the others were from Chinese patients. Since the 6a proportion (17.1%, 67/393) was unexpectedly lower than what we have recently detected among blood donors (34.8%, 82/236) and IDUs (51.5%, 70/136), further statistical analyses were conducted. Comparison of the mean ages showed that among the 393 patients, those infected with 1b were significantly (6.7 years) older than those with 6a, while the 393 patients as a whole were significantly older than the 236 blood donors (8.4 years) and 136 IDUs (12.6 years) we have recently reported. Explanations are that younger individuals had higher proportions of 6a infections while patients with liver disease could have acquired their infections earlier than volunteer blood donors and IDUs.
Conclusion
Among 393 patients with liver disease, a great diversity in HCV was detected, which reflects a constantly changing pattern of HCV genotypes in China over time.
doi:10.1016/j.jcv.2013.04.013
PMCID: PMC3743673  PMID: 23706765
Hepatitis C virus; genetic sequence; phylogenetic tree; genotype
17.  Emergence of Antiviral Resistance During Oral Valganciclovir Treatment of an Infant with Congenital Cytomegalovirus (CMV) Infection 
Congenital infection with human cytomegalovirus (CMV) is a major cause of morbidity, including sensorineural hearing loss (SNHL), in newborns. Antiviral therapy with ganciclovir (GCV) and its oral prodrug, valganciclovir (VAL-GCV) are increasingly being administered to infected infants, toward the goal of improving neurodevelopmental and auditory outcomes. In this case report, we describe a symptomatic congenitally infected infant treated with VAL-GCV in whom GCV resistance was suspected, based on a 50-fold increase in viral load after 6 weeks of oral therapy. Analyses of CMV sequences from both blood and urine demonstrated populations of viruses with M460V and L595F mutations in the UL97 phosphotransferase gene. In contrast, analysis of viral DNA retrieved from the newborn dried blood spot demonstrated wild-type UL97 sequences. DNAemia resolved after the discontinuation of VAL-GCV. Long-term VAL-GCV therapy in congenitally infected infants can select for resistant viral variants, and anticipatory virological monitoring may be warranted.
doi:10.1016/j.jcv.2013.04.004
PMCID: PMC3807863  PMID: 23688863
congenital cytomegalovirus infection; antiviral therapy; ganciclovir resistance; UL97 mutation
18.  Multiplex qPCR assay for ultra sensitive detection of JCV DNA with simultaneous identification of genotypes that discriminates non-virulent from virulent variants 
Background
JC Virus (JCV) is the etiologic agent for progressive multifocal leukoencephalopathy (PML), a demyelinating disease occurring in the brain of patients with underlying immune compromised states. All viable JCV genomes contain a conserved region in the T protein coding nucleotide sequence that when detected by PCR in CSF is a confirmatory diagnostic marker for PML along with clinical and neuroradiological evidence. The non-coding regulatory region (NCRR) is hypervariable, as evidenced by nucleotide sequence of the non-virulent variant, which is predominantly excreted in urine, versus that of virulent variants found in brain and CSF of PML patients. All variants can be found in blood.
Objective
A single assay that quantifies and identifies JCV DNA in clinical samples and discriminates between variants has significant value to physicians and patients at risk for PML.
Study Design
Separate primer pairs were tested together to quantitatively detect conserved viral DNA nucleotide sequence in patient samples, while simultaneously detecting the NCRR specific for the non-virulent variant.
Results
In testing using control plasmids and patients’ CSF, blood, and urine, PML patients predictably demonstrated the non-virulent, archetype NCRR in urine, but virulent NCRR variants in CSF and blood.
Conclusion
The JCV qPCR Multiplex assay targets two regions in JCV genomes to simultaneously identify and measure viral DNA, as well as distinguish between variants associated with PML and those that are not. The multiplex results could signal risk for PML if patients are viremic with JCV variants closely associated with PML pathogenesis.
doi:10.1016/j.jcv.2013.03.009
PMCID: PMC3698945  PMID: 23619054
19.  REACTIVATION OF LATENT VIRUSES IN INDIVIDUALS RECEIVING RITUXIMAB FOR NEW ONSET TYPE 1 DIABETES 
Background
Rituximab has been successfully used as an experimental therapy in different autoimmune diseases. Recently, a double-blind placebo-controlled phase-2 study in early onset type 1 diabetes showed that rituximab delayed progression of the disease. However, like with any immunosuppressive therapy, there is a concern of opportunistic viral reactivations with the use of rituximab, including herpes and polyomaviruses.
Objectives
To study the incidence of new infections and reactivations with BK, JC, Epstein-Barr and cytomegalovirus (BKV, JCV, EBV and CMV) in T1D participants in the phase-2 rituximab study.
Study Design
Subjects received 4 weekly doses of rituximab (N=57) or placebo (N=30) during the first month of study. Blood samples obtained at weeks 0, 12, 26, 56 and 78 were assayed for CMV, EBV, BKV and JCV by real-time DNA PCR and serology.
Results
EBV reactivations were diagnosed by PCR in 25% of placebo, but none of rituximab recipients (p<0.01). There were no episodes of CMV viremia in either treatment group. BKV viremias were significantly more common in the rituximab recipients (9%) compared with placebo controls (0, p<0.01). No JCV reactivations were detected in this study, but among 6 rituximab and 2 placebo recipients who seroconverted for JCV during the study, only one rituximab recipient had detectable viremia. All infections were asymptomatic.
Conclusions
Four doses of rituximab administered to individuals with early onset T1D decreased the incidence of asymptomatic EBV reactivations, as predicted by the rituximab-mediated elimination of memory B-cells, but increased the frequency of asymptomatic viremias caused by polyomaviruses.
doi:10.1016/j.jcv.2013.01.016
PMCID: PMC3640764  PMID: 23422292
Rituximab; Type 1 diabetes; Epstein Barr virus; JC virus; BK virus; Cytomegalovirus
20.  Acyclovir induced hypokalemia 
doi:10.1016/j.jcv.2012.10.002
PMCID: PMC4018808  PMID: 23122658
Acyclovir; Hypokalemia
21.  Individual detection of 14 high risk human papilloma virus genotypes by the PapType test for the prediction of high grade cervical lesions 
Journal of Clinical Virology  2014;60(1):44-49.
Background
HR HPV genotypes when assayed collectively, achieve high sensitivity but low specificity for the prediction of CIN2+. Knowledge of the specific genotypes in an infection may facilitate the use of HR HPV detection in routine clinical practice.
Objectives
To compare the rate of HR HPV detection and the accuracy of CIN2+ prediction between PapType test (Genera Biosystems) and other commercially available HR HPV assays, and to examine the value of full HPV genotyping.
Study design
PreservCyt samples from 1099 women referred for abnormal cervical cytology were used. CIN2+ was chosen as the primary end-point but CIN3+ was also evaluated. A hierarchy of HR HPV genotypes was created using PPV and this was used to create 3 groups of genotypes with potentially different management.
Results
The PapType assay has a specificity of 22.4% and a sensitivity of 94.6% for CIN2+ prediction. Classification into Groups A (HPV33 and HPV16, very highly predictive), B (HPV31, 18, 52, 35, 58, 51 highly predictive) and C (HPV68, 45, 39, 66, 56, 59, intermediate predictive) could double the specificity (44.5%) but only slightly reduce the sensitivity for CIN2+ (91.5%) and CIN3+ (94.0%).
Conclusions
The PapType assay is a simple, reproducible and effective test for HR HPV detection and genotyping. HPV 33 was found to have a very high PPV and should therefore be managed as for HPV16.
doi:10.1016/j.jcv.2014.02.002
PMCID: PMC4012136  PMID: 24630483
HR HPV, high risk human papillomavirus; CIN2+ and/or CIN3+, high grade cervical intraepithelial neoplasia; PPV, positive predictive value; High risk HPV; Genotypes; Cervix; CIN; Positive predictive value
22.  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
23.  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
24.  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
25.  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

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