Human Papillomavirus 16 Lineage A Variants Associated With Persistent Genital Infections in Men: The HPV Infection in Men (HIM) Study.

BACKGROUND: Human papillomavirus (HPV) 16 non-A lineage variants have higher carcinogenic potential for cervical cancer. HPV-16 variants natural history among males is not established. We evaluated HPV-16 variants prevalence and persistence in the external genitalia of men enrolled in the prospective HPV Infection in Men (HIM) Study. METHODS: The HIM Study included men from the United States, Brazil, and Mexico. HPV-16 variants were distinguished using polymerase chain reaction sequencing. The prevalence of HPV-16 variants was assessed, and associations with infection persistence were estimated. RESULTS: We characterized the HPV-16 variants for 1700 genital swab samples from 753 men and 22 external genital lesions in 17 men. The prevalence of HPV-16 lineages differed by country and marital status (P < .001). Overall, 90.9% of participants harbored lineage A variants. The prevalence of non-A lineages was heterogenous among countries. HPV-16 lineage A variants were associated with a 2.69-fold increased risk of long-term persistent infections compared with non-A lineages. All high-grade penile intraepithelial neoplasia harbored lineage A variants and occurred in the context of long-term persistent infections with the same variants. CONCLUSIONS: The prevalence and persistence of HPV-16 variants observed at the male external genitalia suggest differences in the natural history of these variants between men and women, which may be associated with intrinsic differences in the infected genital epithelia.

Prevalence and persistence of HPV-16 molecular variants in the anal canal of men: The HIM study.

BACKGROUND: HPV-16 causes approximately 90% of anal canal (AC) cancers worldwide. This study aimed to evaluate the prevalence and persistence of HPV-16 genetic variants in the AC of men from three different countries (Brazil, Mexico and United States) and to further identify sociodemographic and behavioral factors associated with these infections. METHODS: Participants from the multinational prospective HPV Infection in Men (HIM) Study who had at least one HPV-16 positive AC swab were included. Characterization into HPV-16 genetic variants was successfully performed by PCR-sequencing in 95.6% (217/227) samples and these were classified into HPV-16 lineages and sublineages. RESULTS: We observed higher prevalence of lineage A variants, mainly from A1 sublineage, in all countries. Non-A lineage variants were mostly detected in men from Brazil, where higher diversity of sublineage variants was detected during follow-up. Compare to men detected with Non-A HPV-16 lineage variants, men infected with lineage A reported a higher lifetime number of female sexual partners. Finally, a significantly higher prevalence of Non-A lineage variants was observed among men who have sex with men (MSM) with a transient HPV-16 AC infection (p = 0.033), but no significant differences regarding variants lineages and persistence status were observed when stratified by country, self-reported ethnicity or age. CONCLUSIONS: Our data extend previous reports which indicate that globally HPV-16 variants are unevenly distributed, and contribute further to studies of the natural history of AC HPV infections in men.

Biomarkers of human papillomavirus (HPV)-driven head and neck cancer in Latin America and Europe study: Study design and HPV DNA/p16INK4a status.

BACKGROUND: Human papillomavirus (HPV)-driven head/neck squamous cell carcinomas (HNSCC) prevalence varies globally. We evaluated HPV DNA and p16INK4a in formalin fixed paraffin embedded (FFPE) HNSCC from Argentina, Brazil, Colombia, and Peru. METHODS: HPV was genotyped by PCR-hybridization. All HPV DNA positive and some HPV DNA negative cases underwent p16INK4a immunohistochemistry. RESULTS: HPV DNA was detected in 32.8%, 11.1%, and 17.8% of oropharyngeal (OPC), oral cavity (OCC) and laryngeal (LC) cancers, respectively. OPC HPV prevalence was higher in Colombia (94.7%), and Argentina (42.6%) compared to Brazil (10.6%) and Peru (0.0%). HPV-16 was the most detected. Other HPVs were found in LC. Higher rates of p16INK4a positivity were observed among HPV positive OPC/OCC cases compared to LC cases. CONCLUSIONS: Our results support a role for HPV-16 in a subset of HNSCC, corroborate the heterogeneity observed in samples from different countries, and contribute additional etiological and biomarkers information in tumors of significant impact worldwide.

E6/E7 Functional Differences among Two Natural Human Papillomavirus 18 Variants in Human Keratinocytes.

It is suggested that HPV-18 variants from the A lineage have higher oncogenic potential compared to B variants. Some studies show uneven distribution of HPV-18 variants in cervical adenocarcinomas and squamous cell carcinomas. Regarding HPV-18 variants' functions, the few studies reported focus on E6, and none were performed using natural host cells. Here, we immortalized primary human keratinocytes (PHKs) with E6/E7 of HPV-18 A1 and B1 sublineages and functionally characterized these cells. PHK18A1 reached immortalization significantly faster than PHK18B1 and formed a higher number of colonies in monolayer and 3D cultures. Moreover, PHK18A1 showed greater invasion ability and higher resistance to apoptosis induced by actinomycin-D. Nevertheless, no differences were observed regarding morphology, proliferation after immortalization, migration, or epithelial development in raft cultures. Noteworthy, our study highlights qualitative differences among HPV-18 A1 and B1 immortalized PHKs: in contrast to PHK18A1, which formed more compact colonies and spheroids of firmly grouped cells and tended to invade and migrate as clustered cells, morphologically, PHK18B1 colonies and spheroids were looser, and migration and invasion of single cells were observed. Although these observations may be relevant for the association of these variants with cervical cancer of different histological subtypes, further studies are warranted to elucidate the mechanisms behind these findings.

Prevalence of human papillomavirus 6 and 11 variants in recurrent respiratory papillomatosis.

Human papillomavirus (HPV) types 6 and 11 are the etiological agents of recurrent respiratory papillomatosis (RRP). We examined the prevalence and distribution of HPVs 6 and 11 genetic variants in juvenile onset (JORRP) and adult onset (AORRP) laryngeal papillomas. Cases of JORRP and AORRP were collected, retrospectively. HPV detection and genotyping were accessed by polymerase chain reaction-sequencing in 67 RRP samples. Overall, the most prevalent HPV-6 variants were from B1 (55.8%) and B3 (27.9%) sublineages, whereas among HPV-11 positive samples A2 (62.5%) variants were predominant. A higher prevalence of HPV-6 B1 was observed in JORRP (83.3% B1 and 16.7% B3), compared with AORRP cases (58.3% B1 and 41.7% B3). HPV-11 A2 variants were more prevalent both in JORRP (57.2%) and in AORRP cases (70.0%). Nevertheless, with the exception that HPV-6 B1 were significantly less likely to recur, there was a lack of association between any particular HPVs 6 or 11 variant and clinicopathological features. Our data do not support an association between HPVs 6 and 11 variability and RRP.

Role of epstein-barr virus in the severity of recurrent respiratory papillomatosis.

OBJECTIVE: The objective was to investigate the prevalence of the Epstein-Barr virus (EBV) and its association with human papilloma virus (HPV) detection, clinicopathological features, and the severity of recurrent respiratory papillomatosis (RRP). METHODS: Cases of juvenile recurrent respiratory papillomatosis (JRRP) (n = 36) and adult recurrent respiratory papillomatosis (ARRP) (n = 44) were collected retrospectively and subdivided into low- and high-risk severity groups based on the Derkay score. We performed HPV detection and genotyping using a reverse hybridization protocol and investigated the presence of EBV by polymerase chain reaction (PCR) and in situ hybridization. CD21 levels were accessed by immunohistochemistry. RESULTS: All samples were HPV-positive, including 49 cases of HPV 6, 26 cases of HPV 11, four cases of HPV 6 and 11 coinfections, and one case of HPV 16. EBV-DNA was detected in nine samples by PCR, although none of the cases were positive by means of in situ hybridization. CD21 immunoexpression was not statistically associated with any of the variables analyzed. HPV 6 detection was significantly higher in ARRP cases (P = 0.03), whereas HPV 11 was more prevalent in JRRP cases (P = 0.02) and was even more prevalent in JRRP cases of greater severity (Derkay laryngoscopic scale ≥20) (P = 0.04). CONCLUSION: The presence of EBV does not seem to play an important role in the progression/severity of RRP. LEVEL OF EVIDENCE: 4 Laryngoscope, 130:E611-E618, 2020.

Lack of Association between Human Papillomavirus Types 6 and 11 Genetic Variants and Cervical Abnormalities: The Ludwig-McGill Cohort Study.

BACKGROUND: Human papillomavirus (HPV) types 6 and 11 are mainly associated with the development of genital warts and recurrent respiratory papillomatosis. We examined intratypic genetic variability of both viral types with the development of cervical cytologic abnormalities in Brazilian women. METHODS: We used PCR sequencing to characterize variants of HPVs 6 and/or 11 in cervical swabs from women in the Ludwig-McGill Cohort Study. We used a binomial generalized estimating equations (GEE) model with logit link to estimate odds ratios (OR) and 95% confidence intervals (CI) for the associations between HPV 6 and 11 variants and cytologic abnormalities. RESULTS: B1 and B3 HPV6 and A2 HPV11 variants were the most common isolates identified. Compared with HPV6-negative women, the ORs among women harboring HPV6 B1 or B3 variants were 6.3 (95% CI, 2.3-17.0) and 2.3 (95% CI, 0.6-9.7) for atypical cells of undetermined significance (ASCUS)/low squamous intraepithelial lesions (LSIL), respectively, and 1.7 (95% CI, 0.6-5.1) and 1.2 (95% CI, 0.3-4.7) for ASCUS/LSIL/high squamous intraepithelial lesions (HSIL). Respective ORs were 5.0 (95% CI, 1.7-14.6) and 2.8 (95% CI, 1.0-8.1) upon comparing women with HPV11 A2 variants to HPV11-negative women. All associations disappeared when adjusting for coinfections with high-risk HPV types. CONCLUSIONS: Our data do not support an association between low-risk HPVs 6 and 11 genetic variability and cervical abnormalities. IMPACT: Risk of cervical cytologic abnormalities is not affected by intratypic polymorphism in HPVs 6 and 11.

Prevalence of high risk HPV DNA in esophagus is high in Brazil but not related to esophageal squamous cell carcinoma.

BACKGROUNDS: The first publication that associated Human Papillomavirus (HPV) infection and esophageal cancer was published in 1982. However, data are still contradictory and require further investigation. The aim of this study was to identify high risk HPV DNA in esophageal tissue of patients with and without esophageal squamous cell carcinoma (ESCC) and correlate HPV presence with classical risk factors. METHODS: Invited patients signed the informed consent form, and interviews were conducted in order to obtain information about sociodemographic and lifestyle behavior. During endoscopy, esophageal biopsies were collected from case and controls. Multiplex polymerase chain reaction genotyping was conducted on endoscopic biopsies to identify HPV types and HPV-16 was further evaluated by specific PCR real time. RESULTS: Among 87 cases, 12 (13.8%) had tumors harboring high risk HPV DNA and among 87 controls, 12 (13.8%) had high risk HPV DNA (OR:1.025 [CI:0.405:2.592]). Variables regarding consumption of alcohol and use of tobacco continued to characterize risk factors even after adjustments by presence or absence of high risk HPV. CONCLUSION: HPV was demonstrated to be frequently and similarly associated to normal and malignant esophageal tissues, but not as an independent risk factor to esophageal cancer. IMPACT: To contribute to the Brazilian population data on this subject, which is still contradictory.

HPV-11 variability, persistence and progression to genital warts in men: the HIM study.

HPV-11 and HPV-6 are the etiological agents of about 90 % of genital warts (GWs). The intra-typic variability of HPV-11 and its association with infection persistence and GW development remains undetermined. Here, HPV infection in men (HIM) participants who had an HPV-11 genital swab and/or GW, preceded or not by a normal skin genital swab were analysed. Genomic variants were characterized by PCR-sequencing and classified within lineages (A, B) and sublineages (A1, A2, A3, A4). HPV-11 A2 variants were the most frequently detected in the genital swab samples from controls and in both genital swabs and GW samples from cases. The same HPV-11 variant was detected in the GW sample and its preceding genital swab. There was a lack of association between any particular HPV-11 variant and the increased risk for GW development.

Concordance of Beta-papillomavirus across anogenital and oral anatomic sites of men: The HIM Study.

We evaluated the concordance between ß-HPVs detected in external genital skin, anal canal, and oral cavity specimens collected simultaneously from 717 men that were participating in the multinational HIM Study. Viral genotyping was performed using the Luminex technology. Species- and type-specific concordance was measured using kappa statistics for agreement. Overall, concordance of ß-HPVs across sites was low and mainly observed among paired genital/anal canal samples. When grouped by species, solely ß-4 HPVs showed moderate concordance in genital/anal pairs (κ = 0.457), which could be attributed to the substantial concordance of HPV-92 in men from Brazil and Mexico (κ > 0.610). ß-HPV type concordance was higher in Mexico, where HPV-19 was consistently concordant in all anatomic site combinations. Our analysis indicates that type-specific concordance across sites is limited to few viral types; however, these infections seem to occur more often than would be expected by chance, suggesting that although rare, there is agreement among sites.

Cervical Infection with Cutaneous Beta and Mucosal Alpha Papillomaviruses.

Background: Alpha-human papillomavirus (α-HPV) plays a causal role in cervical cancer, but little is known about the epidemiology of genital Beta-human papillomavirus (ß-HPV) infection.Methods: We used Luminex and PCR hybridization to detect ß- and α-HPVs prevalence at enrollment and 12-month follow-up in cervical samples from 505 women enrolled in the Ludwig-McGill cohort study. We compared epidemiologic correlates of both ß- and α-HPVs and compared genotypes between these genera with respect to co-occurrence and association with cervical cytologic abnormalities.Results: Infection with ß-HPV types was more prevalent than that with α-HPV types at both visits (cumulative prevalences: 27.3% vs. 21.6%, respectively, P = 0.034). ß-HPVs were mostly transient; however, only 1.98% women retained their original positivity at 12 months, whereas persistence was higher for α-HPVs (5.15%; P = 0.007). Age, parity, and sexual activity variables were predictors of α-HPV but not of ß-HPV. α- and ß-HPV types occurred independently. Increased risk of cervical abnormalities was restricted to women infected with α-9 or α-6 HPV types. We found no epidemiologic correlates for ß-HPV infections.Conclusions: Detection of ß-HPV types in the cervix tends to occur as random and transient episodes not explained via the sexual-transmission correlates that characterize infections by α-HPVs.Impact: Although it is plausible that ß-HPVs may play a direct or indirect carcinogenic role, the lack of epidemiologic correlates for detection episodes of these viruses and lack of association with cervical lesions speak against their ancillary role as sexually transmitted agents in cervical carcinogenesis. Cancer Epidemiol Biomarkers Prev; 26(8); 1312-20. ©2017 AACR.

HPV-6 Molecular Variants Association With the Development of Genital Warts in Men: The HIM Study.

Background: Human papillomavirus type 6 (HPV-6) and HPV-11 are the etiological agents of approximately 90% of genital warts (GWs). The impact of HPV-6 genetic heterogeneity on persistence and progression to GWs remains undetermined. Methods: HPV Infection in Men (HIM) Study participants who had HPV-6 genital swabs and/or GWs preceded by a viable normal genital swab were analyzed. Variants characterization was performed by polymerase chain reaction sequencing and samples classified within lineages (A, B) and sublineages (B1, B2, B3, B4, B5). Country- and age-specific analyses were conducted for individual variants; odds ratios and 95% confidence intervals for the risk of GWs according to HPV-6 variants were calculated. Results: B3 variants were most prevalent. HPV-6 variants distribution differed between countries and case status. HPV-6 B1 variants prevalence was increased in GWs and genital swabs of cases compared to controls. There was difference in B1 and B3 variants detection in GW and the preceding genital swab. We observed significant association of HPV-6 B1 variants detection with GW development. Conclusions: HPV-6 B1 variants are more prevalent in genital swabs that precede GW development, and confer an increased risk for GW. Further research is warranted to understand the possible involvement of B1 variants in the progression to clinically relevant lesions.

Diversity of beta-papillomavirus at anogenital and oral anatomic sites of men: The HIM Study.

Our goal was to describe prevalence of ß-HPVs at three anatomic sites among 717 men from Brazil, Mexico and US enrolled in the HPV Infection in Men (HIM) Study. ß-HPVs were genotyped using Luminex technology. Overall, 77.7%, 54.3% and 29.3% men were positive for any ß-HPV at the genitals, anal canal, and oral cavity, respectively. Men from US and Brazil were significantly less likely to have ß-HPV at the anal canal than men from Mexico. Older men were more likely to have ß-HPV at the anal canal compared to younger men. Prevalence of ß-HPV at the oral cavity was significantly associated with country of origin and age. Current smokers were significantly less likely to have ß-HPV in the oral cavity than men who never smoked. Lack of associations between ß-HPV and sexual behaviors may suggest other routes of contact such as autoinoculation which need to be explored further.

Prevalence of human papillomavirus types and variants and p16(INK4a) expression in head and neck squamous cells carcinomas in São Paulo, Brazil.

BACKGROUND: Human papillomavirus (HPV) prevalence in head and neck squamous cell carcinomas (HNSCC) diverges geographically. The reliability of using p16(INK4a) expression as a marker of viral infection is controversial in HNSCC. We evaluated HPV types and HPV-16 variants prevalence, and p16(INK4a) expression in HNSCC specimens provided by two different Institutions in São Paulo. METHODS: HPV DNA from formalin-fixed specimens was accessed by Inno-LiPA, HPV-16 variants by PCR-sequencing, and p16(INK4a) protein levels by immunohistochemistry. RESULTS: Overall, HPV DNA was detected among 19.4 % of the specimens (36/186). Viral prevalence was higher in the oral cavity (25.0 %, 23/92) then in other anatomical sites (oropharynx 14,3 %, larynx 13.7 %) when samples from both Institutions were analyzed together. HPV prevalence was also higher in the oral cavity when samples from both Institutions were analyzed separately. HPV-16 was the most prevalent type identified in 69.5 % of the HPV positive smaples and specimens were assigned into Asian-American (57.2 %) or European (42.8 %) phylogenetic branches. High expression of p16(INK4a) was more common among HPV positive tumors. CONCLUSION: Our results support a role for HPV-16 in a subset of HNSCC.

High genital prevalence of cutaneous human papillomavirus DNA on male genital skin: the HPV Infection in Men Study.

BACKGROUND: The genital skin of males hosts a diversity of HPV genotypes and uncharacterized HPV genotypes. Previously we demonstrated that a specific viral genotype was not identified in 14% of all genital specimens (i.e., HPV unclassified specimens) using the Roche Linear Array method. Our goal was to identify and assess the prevalence of individual HPV types among genital HPV unclassified specimens collected in the HIM Study population, at enrollment, and examine associations with socio-demographic and behavioral characteristics. METHODS: Genital skin specimens of men that were considered unclassified (HPV PCR positive, no genotype specified) at enrollment were typed by sequencing amplified PGMY09/11 products or cloning of PGMY/GP+ nested amplicons followed by sequencing. PGMY/GP+ negative specimens were further analyzed using FAP primers. HPV type classification was conducted through comparisons with sequences in the GenBank database. RESULTS: Readable nucleotide sequences were generated for the majority of previously unclassified specimens (66%), including both characterized (77%) and yet uncharacterized (23%) HPV types. Of the characterized HPV types, most (73%) were Beta [ß]-HPVs, primarily from ß-1 and ß-2 species, followed by Alpha [α]-HPVs (20%). Smokers (current and former) were significantly more likely to have an α-HPV infection, compared with any other genus; no other factors were associated with specific HPV genera or specific ß-HPV species. CONCLUSIONS: Male genital skin harbor a large number of ß-HPV types. Knowledge concerning the prevalence of the diverse HPV types in the men genital is important to better understand the transmission of these viruses.

Broad HPV distribution in the genital region of men from the HPV infection in men (HIM) study.

The HPV infection in men (HIM) study examines the natural history of genital HPV infection in men. Genotyping methods used in this study identify 37 α-HPV types; however, the viral type could not be identified in approximately 22% of male genital specimens that were HPV PCR positive. Our aim was to genotype HPV-unclassified specimens by sequencing PGMY09/11, GP5+/6+ or FAP59/64 PCR products. Using this approach we were able to detect 86 unique HPV types among 508 of 931 specimens analyzed. We report for the first time the presence of a broad range of α-, ß- and γ-HPV at the male genitals.

Identificação do papilomavírus humano em doentes com carcinoma de células escamosas do canal anal e sua relação com o grau de diferenciação celular e estadiamento / Identification of human papillomavirus in patients with anal squamous cell carcinoma and its relation with the grade of cellular differentiation and staging

OBJETIVO: Identificar os tipos de papilomavírus humano (HPV) nos portadores de carcinoma do canal anal (CCA), relacionando-os ao grau de diferenciação celular e estadiamento da lesão, em pacientes do Belém, Pará, entre 1998 e 2000. MÉTODOS: Foi realizado um estudo de caso-controle com 75 pacientes, divididos em: Grupo Teste, com 33 portadores de carcinoma do canal anal, e o Grupo Controle, com 42 portadores de doenças não-neoplásicas do canal anal. Os tipos virais foram identificados por PCR e dot blot. O teste exato de Fischer foi utilizado para avaliar a ocorrência de HPV. Adotou-se a tabela de contingência 3x2 para representar a distribuição dos tipos de HPV. Nos testes de hipóteses, foi prefixado o nível de significância alfa=0,05 para a rejeição da hipótese de nulidade. RESULTADOS: A prevalência do HPV foi significante entre os Grupos Teste (60,6 por cento) e Controle (26,2 por cento) (p=0,0027). Os tipos virais mais comuns foram 16 (42,4 por cento) e 18 (15,2 por cento). Observaram-se diferenças entre grupos na prevalência do HPV 16 (p=0,027) e 18 (p=0,043) no Grupo Teste, e o tipos 16 (19,0 por cento,) e 18 em (2,4 por cento) no Grupo Controle. No Grupo Teste, avaliou-se a distribuição dos tipos de HPV em relação ao estadiamento e ao grau de diferenciação celular, não apresentando diferenças estatisticamente significativas. CONCLUSÃO: O carcinoma de células escamosas do canal anal está associado à presença de HPV, e os tipos 16 e 18 são os mais frequentes.

OBJECTIVE: To identify the most predominant types of human papillomavirus (HPV) in carriers of the anal canal carcinoma (ACC), relating them to the cell differentiation and lesion staging degree, in patients from Belém, Pará, Brazil, between 1998 and 2000. METHODS: A case-control study was conducted with 75 patients, divided into Test Group, with 33 carriers of the anal canal carcinoma, and Control Group, with 42 carriers of non-neoplastic diseases of the anal canal. The methods employed to identify the viral types were the polymerase chain reaction and the dot blot. The Fisher's exact test was used to assess the HPV occurrence. The distribution of HPV types was analyzed by 3x2 contingency tables, representing the distribution of HPV types. For hypothesis testing, the significance level alfa=0.05 was previously established for rejection of the null hypothesis. RESULTS: HPV was significantly prevalent (p=0.0027) in the Test (60.6 percent) and Control Groups (26.2 percent) (p=0.0027). The most prevalent viral types were HPV 16 (42.4 percent) and 18 (15.2 percent). Significant differences related to the prevalence of HPV 16 and 18 were verified between both groups (p=0.027 and p=0.043, respectively). In the Control Group, HPV 16 was found in 19.0 percent, whereas HPV 18 was observed in 2.4 percent. In the Test Group, we evaluated the distribution of HPV types according to the staging and degree of cell differentiation, and found no significant differences between the results of the different groups. CONCLUSION: Squamous cell carcinoma of the anal canal is associated with the HPV presence, and the 16 and 18 types are the most frequently found.

Human papillomavirus infection and reinfection in adult women: the role of sexual activity and natural immunity.

There is a paucity of data on whether or not women can be reinfected with human papillomavirus (HPV) types to which they were exposed to earlier in life and on the role of natural immunity. The observation of HPV infection at older ages may be explained by the reactivation of a latent infection or new exposure from sexual activity. Our objective was to analyze the association between reinfection and sexual activity. We analyzed data from 2,462 women enrolled in the Ludwig-McGill cohort and followed every 4 to 6 months for up to 10 years. We performed HPV typing and viral load measurements via PCR and determined HPV-16 seroreactivity at enrollment. Incidence of infection and reinfection were estimated for individual types. Adjusted relative risks (RR) for the association between infection/reinfection and new sexual partners were calculated using Cox regression. Rates of initial infection and reinfection postclearance were statistically comparable. RRs of initial infection or reinfection were consistently associated with new sexual partners [2.4 (95% confidence intervals; 95% CI, 2.0-3.1) for first infection, 3.7 (1.1-13.8) for reinfection with the same type, and 2.3 (1.5-3.7) for reinfection with a different type]. Reinfection in older women was also associated with new sexual partners (RR, 2.8; 95% CI, 1.4-5.3) as were new infections with HPV-16 among women with serologic evidence of prior HPV-16 exposure (RR, 3.0; 95% CI, 1.6-5.3). Viral loads at initial infection and at reinfection were comparable. HPV infection and reinfection were strongly associated with sexual activity. This study suggests that natural immunity does not play a role in controlling the extent of reinfections.

Use of the normalized absorbance ratio as an internal standardization approach to minimize measurement error in enzyme-linked immunosorbent assays for diagnosis of human papillomavirus infection.

The serological detection of antibodies against human papillomavirus (HPV) antigens is a useful tool to determine exposure to genital HPV infection and in predicting the risk of infection persistence and associated lesions. Enzyme-linked immunosorbent assays (ELISAs) are commonly used for seroepidemiological studies of HPV infection but are not standardized. Intra- and interassay performance variation is difficult to control, especially in cohort studies that require the testing of specimens over extended periods. We propose the use of normalized absorbance ratios (NARs) as a standardization procedure to control for such variations and minimize measurement error. We compared NAR and ELISA optical density (OD) values for the strength of the correlation between serological results for paired visits 4 months apart and HPV-16 DNA positivity in cervical specimens from a cohort investigation of 2,048 women tested with an ELISA using HPV-16 virus-like particles. NARs were calculated by dividing the mean blank-subtracted (net) ODs by the equivalent values of a control serum pool included in the same plate in triplicate, using different dilutions. Stronger correlations were observed with NAR values than with net ODs at every dilution, with an overall reduction in nonexplained regression variability of 39%. Using logistic regression, the ranges of odds ratios of HPV-16 DNA positivity contrasting upper and lower quintiles at different dilutions and their averages were 4.73 to 5.47 for NARs and 2.78 to 3.28 for net ODs, with corresponding significant improvements in seroreactivity-risk trends across quintiles when NARs were used. The NAR standardization is a simple procedure to reduce measurement error in seroepidemiological studies of HPV infection.