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1.  Global Genomic Diversity of Human Papillomavirus 11 Based on 433 Isolates and 78 Complete Genome Sequences 
Journal of Virology  2016;90(11):5503-5513.
Human papillomavirus 11 (HPV11) is an etiological agent of anogenital warts and laryngeal papillomas and is included in the 4-valent and 9-valent prophylactic HPV vaccines. We established the largest collection of globally circulating HPV11 isolates to date and examined the genomic diversity of 433 isolates and 78 complete genomes (CGs) from six continents. The genomic variation within the 2,800-bp E5a-E5b-L1-upstream regulatory region was initially studied in 181/207 (87.4%) HPV11 isolates collected for this study. Of these, the CGs of 30 HPV11 variants containing unique single nucleotide polymorphisms (SNPs), indels (insertions or deletions), or amino acid changes were fully sequenced. A maximum likelihood tree based on the global alignment of 78 HPV11 CGs (30 CGs from our study and 48 CGs from GenBank) revealed two HPV11 lineages (lineages A and B) and four sublineages (sublineages A1, A2, A3, and A4). HPV11 (sub)lineage-specific SNPs within the CG were identified, as well as the 208-bp representative region for CG-based phylogenetic clustering within the partial E2 open reading frame and noncoding region 2. Globally, sublineage A2 was the most prevalent, followed by sublineages A1, A3, and A4 and lineage B.
IMPORTANCE This collaborative international study defined the global heterogeneity of HPV11 and established the largest collection of globally circulating HPV11 genomic variants to date. Thirty novel complete HPV11 genomes were determined and submitted to the available sequence repositories. Global phylogenetic analysis revealed two HPV11 variant lineages and four sublineages. The HPV11 (sub)lineage-specific SNPs and the representative region identified within the partial genomic region E2/noncoding region 2 (NCR2) will enable the simpler identification and comparison of HPV11 variants worldwide. This study provides an important knowledge base for HPV11 for future studies in HPV epidemiology, evolution, pathogenicity, prevention, and molecular assay development.
PMCID: PMC4934746  PMID: 27030261
2.  Distinct Ecological Niche of Anal, Oral, and Cervical Mucosal Microbiomes in Adolescent Women 
Human body sites represent ecological niches for microorganisms, each providing variations in microbial exposure, nutrient availability, microbial competition, and host immunological responses. In this study, we investigated the oral, anal, and cervical microbiomes from the same 20 sexually active adolescent females, using culture-independent, next-generation sequencing. DNA from each sample was amplified for the bacterial 16S rRNA gene and sequenced on an Illumina platform using paired-end reads. Across the three anatomical niches, we found significant differences in bacterial community composition and diversity. Overall anal samples were dominated with Prevotella and Bacteriodes, oral samples with Streptococcus and Prevotella, and cervical samples with Lactobacillus. The microbiomes of a few cervical samples clustered with anal samples in weighted principal coordinate analyses, due in part to a higher proportion of Prevotella in those samples. Additionally, cervical samples had the lowest alpha diversity. Our results demonstrate the occurrence of distinct microbial communities across body sites within the same individual.
PMCID: PMC5045137  PMID: 27698612
human microbiome; cervical microbiome; anal microbiome; oral microbiome; metacommunity theory; massively-parallel sequencing; next-generation sequencing
3.  Deep sequencing of HPV16 genomes: A new high-throughput tool for exploring the carcinogenicity and natural history of HPV16 infection 
Papillomavirus research  2015;1:3-11.
For unknown reasons, there is huge variability in risk conferred by different HPV types and, remarkably, strong differences even between closely related variant lineages within each type. HPV16 is a uniquely powerful carcinogenic type, causing approximately half of cervical cancer and most other HPV-related cancers. To permit the large-scale study of HPV genome variability and precancer/cancer, starting with HPV16 and cervical cancer, we developed a high-throughput next-generation sequencing (NGS) whole-genome method. We designed a custom HPV16 AmpliSeq™ panel that generated 47 overlapping amplicons covering 99% of the genome sequenced on the Ion Torrent Proton platform. After validating with Sanger, the current “gold standard” of sequencing, in 89 specimens with concordance of 99.9%, we used our NGS method and custom annotation pipeline to sequence 796 HPV16-positive exfoliated cervical cell specimens. The median completion rate per sample was 98.0%.
Our method enabled us to discover novel SNPs, large contiguous deletions suggestive of viral integration (OR of 27.3, 95% CI 3.3–222, P=0.002), and the sensitive detection of variant lineage coinfections. This method represents an innovative high-throughput, ultra-deep coverage technique for HPV genomic sequencing, which, in turn, enables the investigation of the role of genetic variation in HPV epidemiology and carcinogenesis.
PMCID: PMC4669577  PMID: 26645052
HPV16; HPV epidemiology; HPV genomics
4.  Cervical Precancer Risk in HIV-Infected Women Who Test Positive for Oncogenic Human Papillomavirus Despite a Normal Pap Test 
Human immunodeficiency virus-infected women with a normal Pap result who nonetheless test positive for human papillomavirus (HPV) type 16 have a high risk of cervical precancer that may warrant immediate colposcopy, whereas those positive for other oncogenic HPV types are at moderate risk.
Background. Determining cervical precancer risk among human immunodeficiency virus (HIV)–infected women who despite a normal Pap test are positive for oncogenic human papillomavirus (oncHPV) types is important for setting screening practices.
Methods. A total of 2791 HIV-infected and 975 HIV-uninfected women in the Women's Interagency HIV Study were followed semiannually with Pap tests and colposcopy. Cumulative risks of cervical intraepithelial neoplasia grade 2 or greater (CIN-2+; threshold used for CIN treatment) and grade 3 or greater (CIN-3+; threshold to set screening practices) were measured in HIV-infected and HIV-uninfected women with normal Pap tests, stratified by baseline HPV results, and also in HIV-infected women with a low-grade squamous intraepithelial lesion (LSIL; benchmark indication for colposcopy).
Results. At baseline, 1021 HIV-infected and 518 HIV-uninfected women had normal Pap tests, of whom 154 (15%) and 27 (5%), respectively, tested oncHPV positive. The 5-year CIN-2+ cumulative risk in the HIV-infected oncHPV-positive women was 22% (95% confidence interval [CI], 9%–34%), 12% (95% CI, 0%–22%), and 14% (95% CI, 2%–25%) among those with CD4 counts <350, 350–499, and ≥500 cells/µL, respectively, whereas it was 10% (95% CI, 0%–21%) in those without HIV. For CIN-3+, the cumulative risk averaged 4% (95% CI, 1%–8%) in HIV-infected oncHPV-positive women, and 10% (95% CI, 0%–23%) among those positive for HPV type 16. In HIV-infected women with LSIL, CIN-3+ risk was 7% (95% CI, 3%–11%). In multivariate analysis, HIV-infected HPV16-positive women had 13-fold (P = .001) greater CIN-3+ risk than oncHPV-negative women (referent), and HIV-infected women with LSIL had 9-fold (P < .0001) greater risk.
Conclusions. HIV-infected women with a normal Pap result who test HPV16 positive have high precancer risk (similar to those with LSIL), possibly warranting immediate colposcopy. Repeat screening in 1 year may be appropriate if non-16 oncHPV is detected.
PMCID: PMC4614409  PMID: 26187020
HIV; human papillomavirus; cervical cancer screening; Pap test
5.  High Oral Human Papillomavirus Type 16 Load Predicts Long-term Persistence in Individuals With or at Risk for HIV Infection 
The Journal of Infectious Diseases  2015;212(10):1588-1591.
The association between oral human papillomavirus 16 (HPV16) DNA load and infection clearance was evaluated among 88 individuals with oral HPV16 infection who were identified within a prospective cohort of 1470 HIV-infected and uninfected individuals. Oral rinse specimens were collected semiannually for up to 5 years. The oral HPV16 load at the time of the first positive test result was significantly associated with the time to clearance of infection (continuous P trends <.01). Notably, clearance rates by 24 months were 41% and 94% in the highest and lowest HPV16 load tertiles (P = .03), respectively. High oral HPV16 load warrants consideration as a biomarker for infection persistence, the presumed precursor of HPV16-associated oropharyngeal cancer.
PMCID: PMC4621250  PMID: 25954049
oral HPV; viral load; persistence; oropharyngeal cancer; HIV
6.  Development of the Diabetes Technology Society Blood Glucose Monitor System Surveillance Protocol 
Inaccurate blood glucsoe monitoring systems (BGMSs) can lead to adverse health effects. The Diabetes Technology Society (DTS) Surveillance Program for cleared BGMSs is intended to protect people with diabetes from inaccurate, unreliable BGMS products that are currently on the market in the United States. The Surveillance Program will provide an independent assessment of the analytical performance of cleared BGMSs.
The DTS BGMS Surveillance Program Steering Committee included experts in glucose monitoring, surveillance testing, and regulatory science. Over one year, the committee engaged in meetings and teleconferences aiming to describe how to conduct BGMS surveillance studies in a scientifically sound manner that is in compliance with good clinical practice and all relevant regulations.
A clinical surveillance protocol was created that contains performance targets and analytical accuracy-testing studies with marketed BGMS products conducted by qualified clinical and laboratory sites. This protocol entitled “Protocol for the Diabetes Technology Society Blood Glucose Monitor System Surveillance Program” is attached as supplementary material.
This program is needed because currently once a BGMS product has been cleared for use by the FDA, no systematic postmarket Surveillance Program exists that can monitor analytical performance and detect potential problems. This protocol will allow identification of inaccurate and unreliable BGMSs currently available on the US market. The DTS Surveillance Program will provide BGMS manufacturers a benchmark to understand the postmarket analytical performance of their products. Furthermore, patients, health care professionals, payers, and regulatory agencies will be able to use the results of the study to make informed decisions to, respectively, select, prescribe, finance, and regulate BGMSs on the market.
PMCID: PMC5038526  PMID: 26481642
accuracy; blood glucose monitor; FDA; meter; protocol; surveillance
8.  The Association between Cumulative Psychosocial Risk and Cervical HPV Infection Among Female Adolescents in a Free Vaccination Program 
This study investigated the association of cervical Human Papillomavirus (HPV) infection with cumulative psychosocial risk reflecting family disadvantage, psychological distress, and unhealthy life style.
The sample (N=745) was comprised of sexually-active female adolescent patients (12-19 years), primarily ethnic minorities, enrolled in a free HPV vaccination program. Subjects completed questionnaires and provided cervical swabs for HPV DNA testing. Unweighted and weighted Principal Component Analyses (PCA) for categorical data were used to derive multi-systemic psychosocial risk indices using nine indicators: low socioeconomic status, lack of adult involvement, not attending high-school/college, history of treatment for depression/anxiety, antisocial/delinquent behavior, number of recent sexual partners, use of alcohol, use of drugs, and dependency risk for alcohol/drugs. The association between cervical HPV (any-type, high risk-types, vaccine-types) assayed by polymerase chain reaction and self-reported number of psychosocial risk indicators was estimated using multivariable logistic regression.
Subjects had a median of three psychosocial risk indicators. Multiple logistic regression analyses showed associations with unweighted and weighted number of psychosocial indicators for HPV any-type (adjusted odds ratio [aOR]=1.1; 95% confidence interval [CI]: 1.0-1.2 ); with the strongest associations between weighted drug/alcohol use, drug/alcohol dependency risk, and antisocial/delinquent behavior and detection of HPV vaccine-types (aOR=1.5; 95%CI: 1.1-2.0) independent of number of recent sexual partners and vaccine dose (0-3).
Increased HPV infections including HPV vaccine-types were associated with greater number of psychosocial risk indicators even after controlling for demographics, sexual behavior, history of chlamydia, and vaccine dose.
PMCID: PMC4868125  PMID: 25985216
human papillomavirus; HPV; psychosocial risk; alcohol; drugs; sexual activity; adolescent health
9.  Oral Human Papillomavirus Infection and Oral Lesions in HIV-Positive and HIV-Negative Dental Patients 
The Journal of Infectious Diseases  2015;212(5):760-768.
We evaluated the risk factors associated with oral human papillomavirus (HPV) infection and oral lesions in 161 human immunodeficiency virus (HIV)–positive patients and 128 HIV-negative patients presenting for oral examination at 2 urban healthcare centers. Patients were interviewed on risk factors and provided oral-rinse samples for HPV DNA typing by polymerase chain reaction. Statistical associations were assessed by logistic regression. Oral HPV was prevalent in 32% and 16% of HIV-positive patients and HIV-negative patients, respectively, including high-risk HPV type 16 (8% and 2%, respectively; P = .049) and uncommon HPV types 32/42 (6% and 5%, respectively; P = .715). Among HIV-negative patients, significant risk factors for oral HPV included multiple sex partners (≥21 vs ≤5; odds ratio [OR], 9.1; 95% confidence interval [CI], 1.7–49.3), heavy tobacco smoking (>20 pack-years vs none; OR, 9.2; 95% CI, 1.4–59.4), and marijuana use (OR, 4.0; 95% CI, 1.3–12.4). Among HIV-positive patients, lower CD4+ T-cell count only was associated with oral HPV detection (≤200 vs ≥500 cells/mm3; OR, 4.5; 95% CI, 1.3–15.5). Detection of high-risk HPV was also associated with concurrent detection of potentially cancerous oral lesions among HIV-negative patients but not among HIV-positive patients. The observed risk factor associations with oral HPV in HIV-negative patients are consistent with sexual transmission and local immunity, whereas in HIV-positive patients, oral HPV detection is strongly associated with low CD4+ T-cell counts.
PMCID: PMC4539901  PMID: 25681375
human papillomavirus (HPV); oral lesions; HIV; immunity; sexual activity
10.  The role of human papillomavirus (HPV) genotyping in cervical cancer screening: A large-scale evaluation of the cobas HPV test 
The cobas® HPV Test (“cobas”, Roche Molecular Systems) detects HPV16 and HPV18 individually, and a pool of 12 other high-risk (HR) HPV types. The test is approved for 1) ASC-US triage to determine need for colposcopy, 2) combined screening with cytology (“co-testing”), and 3) primary HPV screening.
To assess the possible value of HPV16/18 typing, >17,000 specimens from a longitudinal cohort study of initially HPV-positive women (HC2, Qiagen) were retested with cobas. To study accuracy, cobas genotyping results were compared to those of an established method, the LINEAR ARRAY HPV Genotyping Test (LA, Roche Molecular Systems). Clinical value of the typing strategy was evaluated by linking the cobas results (supplemented by other available typing results) to 3-year cumulative risks of CIN3+.
Grouped hierarchically (HPV16, else HPV18, else other HR types, else negative), the kappa statistic for agreement between cobas and LA was 0.86 (95%CI=0.86-0.87). In all 3 scenarios, HPV16-positive women were at much higher 3-year risk of CIN3+ than HPV16-negative women: women aged 21 and older with ASC-US (14.5%, 95%CI=13.5%-15.5% versus 3.5%, 95%CI=3.3%-3.6%); women aged 30 and older that were HPV-positive cytology-negative (10.3%, 95%CI=9.6%-11.1% versus 2.3%, 95%CI=2.2%-2.4%); and all women 25 and older that were HPV-positive (18.5%, 95%CI=17.8%-19.2% versus 4.3%, 95%CI=4.2%-4.4%).
The cobas and LA results show excellent agreement. The data support HPV16 typing.
HPV16 typing is useful in the management of HPV- positive/cytology-negative women in co-testing, of all HPV-positive women in primary HPV testing, and perhaps in the management of HPV-positive women with ASC-US.
PMCID: PMC4560647  PMID: 26088703
11.  HPV16 CpG methyl-haplotypes are associated with cervix precancer and cancer in the Guanacaste natural history study 
Gynecologic oncology  2015;138(1):94-100.
To evaluate HPV16 CpG methylation and methyl-haplotypes and their association with cervix precancer and cancer utilizing massively parallel single molecule next-generation sequencing (NGS).
A nested case-control study of HPV16 positive women was performed in a prospective cohort from Guanacaste, Costa Rica designed to study the natural history of HPV and cervical neoplasia. Controls encompassed 31 women with transient infections; there were 44 cases, including 31 women with CIN3 and 13 with cervical cancer. DNA samples from exfoliated cervical cells were treated with bisulfite and four regions (E6, E2, L2 and L1) were amplified with barcoded primers and tested by NGS. CpG methylation was quantified using a bioinformatics pipeline.
Median methylation levels were significantly different between the CIN3+ cases versus controls in the E2, L2, and L1 regions. Methyl-haplotypes, specifically in 5 CpG sites included in the targeted L2 region, with the pattern “−−+−+” had the highest Area Under the Curve value (AUC = 88.40%) observed for CIN3 vs. controls. The most significant CpG site, L2 4277, determined by bisulfite NGS had an AUC = 78.62%.
This study demonstrates that NGS of bisulfite treated HPV DNA is a useful and efficient technique to survey methylation patterns in HPV16. This procedure provides quantitative information on both individual CpG sites and methyl-haplotypes that identify women with elevated present or subsequent risk for HPV16 CIN3 and cancer.
PMCID: PMC4867226  PMID: 26001326
12.  Neutrophil ageing is regulated by the microbiome 
Nature  2015;525(7570):528-532.
Blood polymorphonuclear neutrophils provide immune protection against pathogens but also may promote tissue injury in inflammatory diseases1,2. Although neutrophils are generally considered as a relatively homogeneous population, evidence for heterogeneity is emerging3,4. Under steady-state conditions, neutrophil heterogeneity may arise from ageing and the replenishment by newly released neutrophils from the bone marrow5. Aged neutrophils up-regulate CXCR4, a receptor allowing their clearance in the bone marrow6,7, with feedback inhibition of neutrophil production via the IL17/G-CSF axis8, and rhythmic modulation of the haematopoietic stem cell niche5. The aged subset also expresses low levels of L-selectin (CD62L)5,9. Previous studies have suggested that in vitro-aged neutrophils exhibit impaired migration and reduced pro-inflammatory properties6,10. Here, we show using in vivo ageing analyses that the neutrophil pro-inflammatory activity correlates positively with their ageing in the circulation. Aged neutrophils represent an overly active subset exhibiting enhanced αMβ2 integrin (Mac-1) activation and neutrophil extracellular trap (NET) formation under inflammatory conditions. Neutrophil ageing is driven by the microbiota via Toll-like receptors (TLRs)- and myeloid differentiation factor 88 (Myd88)-mediated signalling pathways. Depletion of the microbiota significantly reduces the number of circulating aged neutrophils and dramatically improves the pathogenesis and inflammation-related organ damage in models of sickle cell disease or endotoxin-induced septic shock. These results thus identify an unprecedented role for the microbiota in regulating a disease-promoting neutrophil subset.
PMCID: PMC4712631  PMID: 26374999
13.  HPV16 methyl-haplotypes determined by a novel next-generation sequencing method are associated with cervical precancer 
We have developed and evaluated a next-generation bisulfite sequencing (NGS) assay to distinguish HPV16 cervical precancer (CIN2-3; N=59) from HPV16-positive transient infections (N=40). Cervical DNA was isolated and treated with bisulfite and HPV16 methylation was quantified by (1) amplification with barcoded primers and massively parallel single molecule sequencing and (2) site-specific pyrosequencing. Assays were evaluated for agreement using intraclass correlation coefficients (ICC). Odds ratios (OR) for high methylation vs. low methylation were calculated. Single site pyrosequencing and NGS data were correlated (ICC=0.61) and both indicated hypermethylation was associated with precancer (ORs of 2-37). Concordant NGS and pyrosequencing results yieled ORs that were stronger when compared to using either assay separately. Within the L1 region, the ORs for CIN2-3 were 14.3 and 22.4 using pyrosequencing and NGS assays, respectively; when both methods agreed the OR was 153. NGS assays provide methylation haplotypes, termed methyl-haplotypes from single molecule reads: cases had increased methyl-haplotypes with ≥ 1 methylated CpG site(s) per fragment compared to controls, particularly in L1 (P=3.0×10−8). The maximum discrimination of cases from controls for a L1 methyl-haplotype had an AUC of 0.89 corresponding to a sensitivity of 92.5% and a specificity of 73.1%. The strengthening of the OR when the two assays were concordant suggests the true association of CpG methylation with precancer is stronger than with either assay. As cervical cancer prevention moves to DNA testing methods, DNA based biomarkers, such as HPV methylation could serve as a reflex strategy to identify women at high risk for cervix cancer.
PMCID: PMC4262737  PMID: 25081507
HPV16; methylation; next-generation sequencing; pyrosequencing; cervical precancer
14.  Risk Factors for Acquisition and Clearance of Oral Human Papillomavirus Infection Among HIV-Infected and HIV-Uninfected Adults 
American Journal of Epidemiology  2014;181(1):40-53.
Human papillomavirus (HPV) causes the majority of oropharyngeal cancers in the United States, yet the risk factors for and natural history of oral HPV infection are largely unknown. In 2010–2011, a US-based longitudinal cohort study of 761 human immunodeficiency virus (HIV)-infected and 469 at-risk HIV-uninfected participants from the Multicenter AIDS Cohort Study and the Women's Interagency HIV Study was initiated. Semiannually collected oral rinses were evaluated for 37 HPV genotypes using the Roche LINEAR ARRAY HPV Genotyping Test (Roche Molecular Systems, Pleasanton, California), and factors associated with oral HPV incidence and clearance were explored using adjusted Wei-Lin-Weissfeld modeling. Through 2013, the 2-year cumulative incidence of any type of oral HPV infection was 34% in HIV-infected persons and 19% in HIV-uninfected persons. However, many of these infections cleared. Seven percent of incident infections and 35% of prevalent infections persisted for at least 2 years. After adjustment for other risk factors, HIV infection (adjusted hazard ratio = 2.3, 95% confidence interval: 1.7, 3.2), reduced current CD4 cell count, and increased numbers of oral sex and “rimming” partners increased the risk of incident oral HPV infection, whereas male sex, older age, and current smoking increased the risk of oral HPV persistence (each P < 0.05). This helps explain the consistent associations observed between these factors and prevalent oral HPV infection in previous cross-sectional studies.
PMCID: PMC4288119  PMID: 25480823
head and neck cancer; HIV; human papillomavirus; natural history; oral HPV; risk factors
15.  Evolution and classification of oncogenic human papillomavirus types and variants associated with cervical cancer 
The nomenclature of human papillomavirus (HPV) is established by the International Committee on Taxonomy of Virus (ICTV). However, the ICTV does not set standards for HPV below species levels. This chapter describes detailed genotyping methods for determining and classifying HPV variants.
PMCID: PMC4435683  PMID: 25348294
Papillomavirus; Evolution; Taxonomy; Genus; Species; Type; Variant
16.  Geographical Distribution and Risk Association of Human Papillomavirus Genotype 52–Variant Lineages 
The Journal of Infectious Diseases  2014;210(10):1600-1604.
Human papillomavirus (HPV) genotype 52 is commonly found in Asian cases of cervical cancer but is rare elsewhere. Analysis of 611 isolates collected worldwide revealed a remarkable geographical distribution, with lineage B predominating in Asia (89.0% vs 0%–5.5%; Pcorrected < .001), whereas lineage A predominated in Africa, the Americas, and Europe. We propose that the name “Asian lineage” be used to denote lineage B, to signify this feature. Preliminary analysis suggested a higher disease risk for lineage B, although ethnogeographical confounders could not be excluded. Further studies are warranted to verify whether the reported high attribution of disease to HPV52 in Asia is due to the high prevalence of lineage B.
PMCID: PMC4539913  PMID: 24879800
HPV; cervical cancer; phylogeny; oncogenic risk; Asia
17.  Characterization of HPV DNA methylation of contiguous CpG sites by bisulfite treatment and massively parallel sequencing—the FRAGMENT approach 
Frontiers in Genetics  2014;5:150.
Invasive cervix cancer (ICC) is the third most common malignant tumor in women and human papillomavirus 16 (HPV16) causes more than 50% of ICC. DNA methylation is a covalent modification predominantly occurring at CpG dinucleotides and increased methylation across the HPV16 genome is strongly associated with ICC development. Next generation (Next Gen) sequencing has been proposed as a novel approach to determine DNA methylation. However, utilization of this method to survey CpG methylation in the HPV16 genome is not well described. Moreover, it provides additional information on methylation “haplotypes.” In the current study, we chose 12 random samples, amplified multiple segments in the HPV16 bisulfite treated genome with specific barcodes, inspected the methylation ratio at 31 CpG sites for all samples using Illumina sequencing, and compared the results with quantitative pyrosequencing. Most of the CpG sites were highly consistent between the two approaches (overall correlation, r = 0.92), thus verifying that Next Gen sequencing is an accurate and convenient method to survey HPV16 methylation and thus can be used in clinical samples for risk assessment. Moreover, the CpG methylation patterns (methylation haplotypes) in single molecules identified an excess of complete-and non-methylated molecules and a substantial amount of partial-methylated ones, thus indicating a complex dynamic for the mechanisms of HPV16 CpG methylation. In summary, the advantages of Next Gen sequencing compared to pyrosequencing for HPV genome methylation analyses include higher throughput, increased resolution, and improved efficiency of time and resources.
PMCID: PMC4042685  PMID: 24917876
human papillomavirus; methylation; next generation sequencing; CpG methylation; methylation haplotypes
18.  Strategies for Conducting Adolescent Health Research in the Clinical Setting: The Mount Sinai Adolescent Health Center HPV Experience 
Clinical research with adolescents can be challenging due to issues of informed consent, parental involvement, institutional review board requirements, and adolescent psychosocial development. This presents a dilemma, particularly in the area of sexual health research, as adolescents are disproportionately affected by sexually transmitted infections such as human papillomavirus (HPV). To successfully conduct adolescent research in the clinical setting, one requires an awareness of state statutes regarding adolescent confidentiality and consent for medical care, and a close partnership with the IRB.
Case Study
In 2007, the Mount Sinai Adolescent Health Center (MSAHC) in collaboration with the Albert Einstein College of Medicine developed a longitudinal research study to examine the natural history of oral, cervical, and anal HPV in an adolescent female population engaged in high-risk sexual behaviors. We use this research project as a case study to explore the ethical, methodological, and clinical issues related to conducting adolescent health research.
Summary and Conclusions
Several strategies were identified to promote adolescent study participation, including: (1) building a research team that is motivated to work with adolescents; (2) combining research and patient care visits to avoid duplication of services; and (3) establishing a personalized communication network with participants. Using these methods, adolescent sexual health research can successfully be integrated into the clinical setting. While retaining a prospective cohort of adolescents has its challenges, a persistent and multi-disciplinary approach can help improve recruitment, sustain participation, and acquire critical data that will lead to improved healthcare knowledge applicable to understudied populations of adolescents.
PMCID: PMC4053481  PMID: 24332677
Adolescent Research; Human papillomavirus; Sexual & reproductive health; Parental consent; Institutional Review Boards
19.  Genital Tract HIV RNA Levels and Their Associations with Human Papillomavirus Infection and Risk of Cervical Pre-Cancer 
Plasma HIV RNA levels have been associated with risk of human papillomavirus (HPV) and cervical neoplasia in HIV-seropositive women. However, little is known regarding local genital tract HIV RNA levels and their relation with cervical HPV and neoplasia.
In an HIV-seropositive women’s cohort with semi-annual follow-up, we conducted a nested case-control study of genital tract HIV RNA levels and their relation with incident high-grade squamous intraepithelial lesions sub-classified as severe (severe HSIL), as provided for under the Bethesda 2001 classification system. Specifically, 66 incident severe HSIL were matched to 130 controls by age, CD4+ count, HAART use, and other factors. We also studied HPV prevalence, incident detection, and persistence in a random sample of 250 subjects.
Risk of severe HSIL was associated with genital tract HIV RNA levels (odds ratio comparing HIV RNA ≥ the median among women with detectable levels versus undetectable [ORVL] 2.96; 95% CI: 0.99–8.84; Ptrend=0.03). However, this association became non-significant (Ptrend=0.51) following adjustment for plasma HIV RNA levels. There was also no association between genital tract HIV RNA levels and the prevalence of any HPV or oncogenic HPV. However, the incident detection of any HPV (Ptrend=0.02) and persistence of oncogenic HPV (Ptrend=0.04) were associated with genital tract HIV RNA levels, after controlling plasma HIV RNA levels.
These prospective data suggest that genital tract HIV RNA levels are not a significant independent risk factor for cervical pre-cancer in HIV-seropositive women, but leave open the possibility that they may modestly influence HPV infection, an early stage of cervical tumoriogenesis.
PMCID: PMC4267467  PMID: 24694931
Genital tract HIV viral load; cervical neoplasia; HPV natural history
20.  Degradation of Human PDZ-Proteins by Human Alphapapillomaviruses Represents an Evolutionary Adaptation to a Novel Cellular Niche 
PLoS Pathogens  2015;11(6):e1004980.
In order to complete their life cycle, papillomaviruses have evolved to manipulate a plethora of cellular pathways. The products of the human Alphapapillomavirus E6 proteins specifically interact with and target PDZ containing proteins for degradation. This viral phenotype has been suggested to play a role in viral oncogenesis. To analyze the association of HPV E6 mediated PDZ-protein degradation with cervical oncogenesis, a high-throughput cell culture assay was developed. Degradation of an epitope tagged human MAGI1 isoform was visualized by immunoblot. The correlation between HPV E6-induced degradation of hMAGI1 and epidemiologically determined HPV oncogenicity was evaluated using a Bayesian approach within a phylogenetic context. All tested oncogenic types degraded the PDZ-containing protein hMAGI1d; however, E6 proteins isolated from several related albeit non-oncogenic viral types were equally efficient at degrading hMAGI1. The relationship between both traits (oncogenicity and PDZ degradation potential) is best explained by a model in which the potential to degrade PDZ proteins was acquired prior to the oncogenic phenotype. This analysis provides evidence that the ancestor of both oncogenic and non-oncogenic HPVs acquired the potential to degrade human PDZ-containing proteins. This suggests that HPV E6 directed degradation of PDZ-proteins represents an ancient ecological niche adaptation. Phylogenetic modeling indicates that this phenotype is not specifically correlated with oncogenic risk, but may act as an enabling phenotype. The role of PDZ protein degradation in HPV fitness and oncogenesis needs to be interpreted in the context of Alphapapillomavirus evolution.
Author Summary
It is thought that the ability to degrade PDZ domain containing proteins is a hallmark of oncogenic papillomaviruses. However, since papillomaviruses did not evolve to be oncogenic, this hypothesis does not address the evolutionary importance of this phenotype. The present manuscript attempts to address whether HPV induced degradation of PDZ containing proteins is associated with oncogenic potential as determined by the clinical/epidemiological empirical cancer risk. Using Bayesian approaches to model trait evolution we show that it is highly unlikely for a virus to become oncogenic without first acquiring the ability to degrade PDZ proteins. Furthermore, the ability to degrade PDZ proteins allowed ancestral viruses to colonize a new cellular niche. However, in order to thrive in this new environment, these ancestral viruses had to acquire additional functions. We hypothesize that some of these additional phenotypes lead to oncogenicity. Importantly, our study illustrates the power of combining epidemiological, biochemical and evolutionary data with phylogenetic analysis in attempting to understand the relative role of specific pathogen phenotypes with host pathogenesis.
PMCID: PMC4472669  PMID: 26086730
21.  Human Papillomavirus 16 Non-European Variants Are Preferentially Associated with High-Grade Cervical Lesions 
PLoS ONE  2014;9(7):e100746.
HPV16 accounts for 50–70% of cervical cancer cases worldwide. Characterization of HPV16 variants previously indicated that they differ in risks for viral persistence, progression to cervical precancer and malignant cancer. The aim of this study was to examine the association of severity of disease with HPV16 variants identified in specimens (n = 281) obtained from a Cervical Pathology and Colposcopy outpatient clinic in the University Hospital of Espírito Santo State, Southeastern Brazil, from April 2010 to November 2011. All cytologic and histologic diagnoses were determined prior to definitive treatment. The DNA was isolated using QIAamp DNA Mini Kit and HPV was detected by amplification with PGMY09/11 primers and positive samples were genotyped by RFLP analyses and reverse line blot. The genomes of the HPV16 positive samples were sequenced, from which variant lineages were determined. Chi2 statistics was performed to test the association of HPV16 variants between case and control groups. The prevalence of HR-HPV types in
PMCID: PMC4077691  PMID: 24983739
PMCID: PMC4430864  PMID: 24643174
Data mining and data reduction methods to detect interactions in epidemiologic data are being developed and tested. In these analyses, multifactor dimensionality reduction, focused interaction testing framework, and traditional logistic regression models were used to identify potential interactions with up to three factors. These techniques were used in a population-based case-control study of pancreatic cancer from the San Francisco Bay Area (308 cases, 964 controls). From 7 biochemical pathways, along with tobacco smoking, 26 polymorphisms in 20 genes were included in these analyses. Combinations of genetic markers and cigarette smoking were identified as potential risk factors for pancreatic cancer, including genes in base excision repair (OGG1), nucleotide excision repair (XPD, XPA, XPC), and double-strand break repair (XRCC3). XPD.751, XPD.312, and cigarette smoking were the best single-factor predictors of pancreatic cancer risk, whereas XRCC3.241*smoking and OGG1.326*XPC.PAT were the best two-factor predictors. There was some evidence for a three-factor combination of OGG1.326*XPD.751*smoking, but the covariate-adjusted relative-risk estimates lacked precision. Multifactor dimensionality reduction and focused interaction testing framework showed little concordance, whereas logistic regression allowed for covariate adjustment and model confirmation. Our data suggest that multiple common alleles from DNA repair pathways in combination with cigarette smoking may increase the risk for pancreatic cancer, and that multiple approaches to data screening and analysis are necessary to identify potentially new risk factor combinations.
PMCID: PMC4410856  PMID: 18559563
Clinical pediatrics  2014;53(9):890-895.
Sexually transmitted infections (STIs) are common among adolescents, and multiple STIs over one’s lifetime can increase health risks. Few studies have assessed lifetime STI prevalence. This study evaluates minority, underserved adolescents’ self-reported lifetime STI history and objective STI rates.
Lifetime STI rates of female patients at an urban adolescent health center were obtained from self-administered questionnaires. Additionally, STI test results were retrieved from electronic medical records.
Patients reported a high lifetime prevalence of STIs. By comparing self-report and objective data, underreporting was identified for chlamydia, gonorrhea, and herpes.
STI rates in at-risk adolescent females are higher than in the general population and remain elevated over time. Lifetime STI reports could expand our understanding of sexual health and should be further studied. Underreporting, which may increase health risks and hinder health care delivery, requires further investigation. Improvements in STI screening and prevention targeting at-risk populations are warranted.
PMCID: PMC4239196  PMID: 24807980
sexual health; self-report; underreporting; electronic medical records; EMR; HPV vaccine
Toxicologic pathology  2012;41(6):893-901.
Genital condyloma-like lesions were observed on male and female cynomolgus macaque monkeys (Macaca fascicularis) originating from the island of Mauritius. Cytobrush and/or biopsy samples were obtained from lesions of 57 affected macaques. Primary histologic features included eosinophilic, neutrophilic, and lymphoplasmacytic penile and vulvar inflammation, epidermal hyperplasia with acanthosis, and increased collagenous stroma. Polymerase chain reaction–based assays to amplify viral DNA revealed the presence of macaque lymphocryptovirus (LCV) DNA but not papillomavirus or poxvirus DNA. Subsequent DNA analyses of 3 genomic regions of LCV identified isolates associated with lesions in 19/25 (76%) biopsies and 19/57 (33%) cytology samples. Variable immunolabeling for proteins related to the human LCV Epstein Barr Virus was observed within intralesional plasma cells, stromal cells, and epithelial cells. Further work is needed to characterize the epidemiologic features of these lesions and their association with LCV infection in Mauritian-origin macaques.
PMCID: PMC3980466  PMID: 23262641
genital; condyloma; macaque; primate; gammaherpesvirus; lymphocryptovirus; papillomavirus; Mauritius

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