Sexually Transmitted Diseases:
Evaluation of the Polyclonal ELISA HPV Serology Assay as a Biomarker for Human Papillomavirus Exposure
Coseo, Sarah E. MPH*; Porras, Carolina MS†; Dodd, Lori E. PhD‡; Hildesheim, Allan PhD*; Rodriguez, Ana Cecilia MD†; Schiffman, Mark MD, MPH*; Herrero, Rolando MD, PhD†; Wacholder, Sholom PhD*; Gonzalez, Paula MD†; Sherman, Mark E. MD*; Jimenez, Silvia MBA†; Solomon, Diane MD§; Bougelet, Catherine PhD¶; van Doorn, Leen-Jan PhD∥; Quint, Wim PhD∥; Safaeian, Mahboobeh PhD, MPH*; for the Costa Rica HPV Vaccine Trial (CVT) Group
From the *Infections and Immunoepidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD; †Proyecto Epidemiológico Guanacaste, Fundación INCIENSA, Liberia, Costa Rica; ‡Biostatistics Research Branch, National Institute of Allergy and Infectious Diseases, Bethesda, MD; §Division of Cancer Prevention, National Cancer Institute, Bethesda, MD; ¶GlaxoSmithKline Biologicals, Rixensart, Belgium; and ∥DDL Diagnostic Laboratory, Voorburg, The Netherlands
The authors thanks to the women of Guanacaste and Puntarenas, Costa Rica, who consented for participating in this effort. They also acknowledge the tremendous effort and dedication of the staff in Costa Rica involved in this project, including Bernardo Blanco and his team (census), Ricardo Cerdas and Ana Hernández (blood processing), José Miguel González, Osman López, Johnny Matamoros, Manuel Sánchez, Rafael Thompson, and Jorge Umaña (field activity coordinators), Su Yen Araya, Hazel Alvarez, Hayleen Campos, Muriel Grijalba, Ana Cristina Monge, Ana Peraza, Diana Robles, María Fernanda Sáenz, Dorita Vargas, and Jessica Vindas (clinic coordinators), Paola Alvarez, Dinia Angulo, Ana Live Arias, Betzaida Barrantes, Andrea Bolaños, Diego Bonilla, Marianela Bonilla, Mary José Calvo, Loretto Carvajal, Jessenia Chinchilla, Blanca Cruz, Marianela Herrera, Andrea Interiano, Fabiola Jiménez, Erick Lagos, Viviana Loría, Andrea Messeguer, Rebeca Ocampo, Ana Cristina Ocampo, Silvia Padilla, Angie Ramírez, Daniela Romero, Byron Romero, Yessenia Ruiz, Daniela Ruiz, Genie Saborío, Sofía Soto, Malena Salas, Adriana Torrez, Natalia Ugalde, Adriana Vallejos, Yesenia Vásquez, Maricela Villegas (clinicians), Marta Alvarado, Ana Cristina Arroyo, Gloriana Barrientos, Diana Díaz, Marlen Jara, Maureen Matarrita, María Ester Molina, Elida Ordóñez, Adriana Ramírez, Gina Sánchez, and Sihara Villegas (nurses), Arianne Castrillo and Vivian López (education and outreach effort coordinators), Karla Coronado (appointment coordinator), Ricardo Alfaro (quality control coordinator), Yenory Estrada (pharmacist) Charles Sánchez and Livia Romero (document center coordinators), Cristian Montero (quality assurance, regulatory) and Carlos Avila and Eric Alpízar (IT coordinators). Special recognition is also extended to Sofía Elizondo, Executive Director of Fundación INCIENSA and her staff for their administrative support. In the United States, they thank the team from Information Management Services (IMS) responsible for the development and maintenance of the data system used in the trial and who served as the data management center for this effort. They thank for the invaluable contributions made by Julie Buckland and Laurie Rich. They also thank for the contributions made by individuals at Westat, Inc., who provided project development and/or monitoring support, including Maribel Gomez, Kirk Midkiff, Isabel Trejos and Susan Truitt, and the assistance provided by Carla Chorley, Troy Moore, Kathi Shea, Mindy Collins, and Heather Siefers in the establishment of a specimen and vaccine repository for the trial and in their continued assistance with the handling and shipment of specimens. From GSK Biologicals,they thank Gary Dubin, Anne Schuind, Kelechi Lawrence, Darrick Fu, and Bruce Innis for their contribution to discussions regarding trial conduct and Francis Dessy and Catherine Bougelet for HPV-16/18 antibody testing. They thank members of the Data and Safety Monitoring Board charged with protecting the safety and interest of participants in their trial (Steve Self, Chair, Adriana Benavides, Luis Diego Calzada, Ruth Karron, Ritu Nayar, and Nancy Roach) and members of the external Scientific HPV Working Group who have contributed to the success of their efforts over the years (Joanna Cain, Chair, Diane Davey, Francisco Fuster, Ann Gershon, Elizabeth Holly, Silvia Lara, Wasima Rida, Henriette Raventós, Luis Rosero-Bixby, and Sarah Thomas).
Supported by NCI (N01-CP-11005) with support from the NIH Office of Research on Women's Health and conducted in agreement with the Ministry of Health of Costa Rica. S.E.C. is supported in part by training grant NIH R25 CA098566.
W.Q. and L.-J.v.D. are employees of DDL Diagnostic Laboratory; C.B. is an employee of GSK Biologicals. None of the authors have any potential conflicts of interest to report.
The Costa Rican Vaccine Trial is a longstanding collaboration between investigators in Costa Rica and NCI. The NCI and Costa Rica investigators are responsible for the design and conduct of the study; collection, management, analysis, and interpretation of the data; and preparation of the manuscript. Vaccine was provided for our trial by GSK Biologicals, under a Clinical Trials Agreement with NCI. GSK also provided support for aspects of the trial associated with regulatory submission needs of the company under FDA BB-IND 7920. Douglas Lowy and John Schiller are named inventors on US government owned HPV vaccine patents that are licensed to GSK and Merck, and so are entitled to limited royalties as specified by federal law. None of the other coauthors have any potential conflicts of interest to report.
Cervarix is a registered trade mark of the Glaxo Smith Kline Biologicals group of companies.
Gardasil is a registered trade mark of Merck and Co. Inc.
Names and Affiliations of investigators in the Costa Rica Vaccine Trial (CVT) group are as follows: Proyecto Epidemiológico Guanacaste, Fundación INCIENSA, San José, Costa Rica: Mario Alfaro (Cytopathologist); Manuel Barrantes (Field Supervisor); M. Concepción Bratti (co-Investigator); Fernando Cárdenas (General Field Supervisor); Bernal Cortés (Specimen and Repository Manager); Albert Espinoza (Head, Coding and Data Entry); Paula González (co-Investigator); Diego Guillén (Pathologist); Rolando Herrero (co-Principal Investigator); Silvia E. Jiménez (Trial Coordinator); Jorge Morales (Colposcopist); Lidia Ana Morera (Head Study Nurse); Elmer Pérez (Field Supervisor); Carolina Porras (co-Investigator); Ana Cecilia Rodríguez (co-Investigator); Libia Rivas (Clinician′s coordinator); Luis Villegas (Colposcopist).
University of Costa Rica, San José, Costa Rica: Ivannia Atmella (Microbiologist, Immunology Laboratory); José Bonilla (Head, HPV Immunology Laboratory); Enrique Freer (Director, HPV Diagnostics Laboratory); Alfonso García-Piñeres (Immunologist); Margarita Ramírez (Microbiologist, Immunology Laboratory); Sandra Silva (Head Microbiologist, HPV Diagnostics Laboratory).
United States National Cancer Institute, Bethesda, MD: Allan Hildesheim (co-Principal Investigator and NCI co-Project Officer); Aimee Kreimer (Investigator); Douglas R. Lowy (HPV Virologist); Nora Macklin (Trial Coordinator); Mark Schiffman (Medical Monitor & NCI co-Project Officer); John T. Schiller (HPV Virologist); Mark Sherman (QC Pathologist); Diane Solomon (Medical Monitor and QC Pathologist); Sholom Wacholder (Statistician).
SAIC, NCI-Frederick, Frederick, MD: Ligia Pinto (Head, HPV Immunology Laboratory); Troy Kemp (Scientist, HPV Immunology Laboratory).
Womens and Infants' Hospital, Providence, RI: Claire Eklund (QC Cytology); Martha Hutchinson (QC Cytology).
Georgetown University, Washington, DC: Mary Sidawy (Histopathologist)
DDL Diagnostic Laboratory, The Netherlands: Wim Quint (Virologist, HPV DNA Testing); Leen-Jan van Doorn (HPV DNA Testing).
Correspondence: Mahboobeh Safaeian, PhD, MPH, Infections and Immunoepidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, DHHS, 6120 Executive Blvd, EPS, Room 7086, Rockville, MD 20852. E-mail: firstname.lastname@example.org.
Received for publication December 17, 2010, and accepted May 19, 2011.
Background: Seropositivity to human papillomavirus (HPV)16 and 18 antibodies is used as a measure of cumulative HPV exposure and as a stratifier of HPV exposure for vaccine efficacy analyses. Overall performance of these assays, as a measure of HPV exposure, has not been evaluated.
Methods: Using data from the enrollment phase of the HPV16/18 vaccine trial in Costa Rica, we evaluated the performance of the polyclonal enzyme-linked immunosorbent assay (ELISA) HPV16 and 18 serological assays as a measure of HPV exposure. Biologic (e.g., HPV infection at the cervix) and behavioral characteristics (e.g., lifetime number of sexual partners) with known associations with current and past HPV infection were used to define cases and controls (HPV exposed vs. not exposed). Prevaccination serum was measured for antibodies against HPV16 and 18 by ELISA; cervical samples were tested for HPV DNA using PCR SPF10/LiPA25. ELISA results were analyzed using receiver–operator characteristic curves; performance was evaluated at the manufacturer set cut point (HPV16 = 8, HPV18 = 7) and at cut points chosen to optimize sensitivity and specificity (HPV16 = 34, HPV18 = 60).
Results: Defining cases as type-specific HPV DNA positive with high-grade abnormal cytology (i.e., combined molecular and microscopic markers of infection), HPV16-ELISA gave sensitivity that was lower at the optimal cut point than the manufacturer cut point (62.2 compared with 75.7, respectively; P = 0.44). However, specificity was higher (85.3 compared with 70.4, respectively; P < 0.0001). Similarly, HPV18-ELISA gave sensitivity that was lower at the optimal cut point than the manufacturer cut point (34.5 compared with 51.7, respectively; P = 0.40), with higher specificities (94.9 compared with 72.6, respectively; P < 0.0001).
Conclusions: Modifying cut points did not improve the low sensitivity. The low sensitivity of this assay does not support its use for risk stratification or clinical settings.
Human papillomavirus (HPV) is a common sexually transmitted infection, often acquired shortly after initiation of sexual life.1,2 In epidemiologic research, current HPV DNA infections are detected using sensitive polymerase chain reaction (PCR) assays on cervical exfoliated samples.3 HPV DNA-based assays are highly sensitive and specific for measuring current infections. However, they are limited as they provide information on current infection only and HPV clearance (i.e., loss of DNA detectability) occurs within months to a few years in the majority of infections.4 Thus, DNA-based assays do not provide information on lifetime, cumulative HPV exposure.
Several serological assays with different properties are currently available for research purposes; they measure a wide range of anti-HPV16 and 18 antibodies. Seropositivity to HPV16 and 18 antibodies is being used as an epidemiologic measure of cumulative HPV exposure, a marker of immunity or protection from subsequent infections,5 and in conjunction with DNA-based measures, to define or stratify subgroups of potentially HPV-naïve women for vaccine efficacy analyses. Despite the use of HPV serological assays in many studies, the overall performance of the assays as a measure of HPV exposure have not been evaluated.
The aim of this analysis was to evaluate the performance of the polyclonal enzyme-linked immunosorbent assay (ELISA) HPV assay as a measure of HPV exposure and to determine whether alternative cut points would improve the sensitivity/specificity of the ELISA HPV serology assay as a biomarker of HPV exposure. We also compared the correlates of HPV16 and18 seropositivity (separately) at the manufacturer cutoff, and the cutpoint determined to maximize sensitivity and specificity.
MATERIALS AND METHODS
Data are collected from the enrollment/prevaccination phase of the ongoing publicly funded, community-based randomized phase III HPV16/18 vaccine trial in Costa Rica. The study has been described elsewhere.6 Briefly, the main objectives of the trial are to evaluate the efficacy of a prophylactic HPV16/18 vaccine manufactured by GlaxoSmithKline for prevention of HPV16/18 infection and related precancerous lesions compared to women receiving hepatitis A vaccine as controls. A total of 7466 women provided written, informed consent at the time of enrollment.
At enrollment participants were asked detailed questions regarding demographics, sexual practices, contraceptive use, reproductive and menstrual history, and smoking. Sexually experienced women underwent a pelvic examination and exfoliated cervical cells were collected for cytology, HPV DNA, Chlamydia trachomatis DNA, and Neisseria gonorrhoeae DNA testing. ThinPrep slides were prepared to obtain a Pap stain for cervical cytology interpretation.
All testing were masked to the results of randomization arm and other test results. Protocols were approved by the US National Cancer Institute and a Costa Rican institutional review board.
HPV Serological Measurements
Serum collected at enrollment was used to determine HPV16 and 18 IgG serostatus at GSK Biologicals (Rixensart, Belgium), using a virus-like particle (VLP)-based direct ELISA developed by GSK that measures polyclonal antibodies as described previously.7,8 All research and development of the assay and testing of the samples were conducted at GSK. Briefly, ELISA microtiter plates were separately coated with 2.7 μg/mL of either HPV16 or HPV18 VLPs that were produced in a baculovirus expression system. The plates were blocked with phosphate buffered saline containing 4% skim milk with 0.2% Tween 20. Serum samples from participants were serially diluted in the blocking solution starting at 1:100 in two-fold increments. Serial dilutions of samples, standard and quality control specimens were added to the microtiter plates. After incubation and washing steps, a peroxidase- conjugated antihuman polyclonal antibody was added. After incubation and washing, enzyme substrate and chromogen were added to allow color development. Reactions were stopped, and optical density (OD) read at 450 and 620 nm, with background measured at 620 nm and subtracted from the OD reading at 450 nm. Antibody levels, expressed as ELISA units (EU)/mL, were calculated by interpolation of OD values from the standard curve by averaging the calculated concentrations from all dilutions that fell within the working range of the reference curve. The seropositivity cut points were determined by GSK and calculated from antibody titer values of 3 standard deviations above the geometric mean titers taken from 2 groups of known HPV-negative individuals. These groups included: (1) human serum samples previously incubated with corresponding VLP to remove specific antibodies and (2) human serum taken at day 0 before vaccination from women who did not show an increased immune response after 7 days after the first vaccine.8 Cut points were set at OD ≥8 EU/mL for anti-HPV16 and OD ≥7 EU/mL for anti-HPV18.8
HPV DNA detection and genotyping was performed at DDL Diagnostic Laboratory (Voorburg, Netherlands), as described previously.9,10 Extracted DNA was used for PCR amplification with the SPF10 primer sets.9,10 The samples were run through an HPV DNA enzyme immunoassay (DEIA) to obtain an OD reading, and categorized as HPV DNA negative, positive, or borderline. The same SPF10 amplimers were used on SPF10-DEIA-positive samples to identify HPV genotype by reverse hybridization on a line probe assay (LiPA) (SPF10-DEIA/HPV LiPA25, version 1; Labo Bio-Medical Products, Rijswijk, Netherlands), which detects 25 HPV genotypes.
Since Costa Rica HPV Vaccine Trial uses the bivalent HPV16/18 vaccine, to ensure detection for these types, HPV16 and 18 type-specific PCR primer sets were used to selectively amplify HPV16 and 18 from specimens tested SPF10-DEIApositive, but LiPA25 HPV16 and/or HPV18 negative.9 Amplimers from the type-specific PCRs were detected by DEIA similar to the method used for SPF10 amplimer detection.9–11
All analyses were conducted separately for HPV16 and 18. We noted that the results from the HPV16 and 18 models cannot be directly compared to one another because the HPV16 and 18 ELISAs are not identical. We excluded virgin women (n = 1592) from this analysis, because they did not have a pelvic examination and thus we could not assess their HPV DNA or cytology status. However, for completeness, we also performed the analyses including the virgin women, assuming that they were HPV DNA and cytology negative. As expected, specificity increased; however, the main conclusions of the analysis were not altered by restricting to sexually active women (data not shown). We also excluded 3 women who were outside the age range required for the trial.
We used nonparametric empirical receiver operating characteristic (ROC) analysis to calculate the area under the curve (AUC).12,13 We sought to evaluate HPV serology as a biomarker of exposure to HPV. Because HPV DNA is not sensitive for detecting prior infections, we used both biologic and behavioral characteristics known to be associated with current and past HPV infection to define cases and controls. Specifically, in separate analyses, cases were defined as either: (1) current type-specific HPV DNA positive at cervix (HPV16 DNA positive for the HPV16 analysis; HPV18 DNA positive for HPV18 analysis); (2) abnormal cytology/histology, defined as ≥HSIL/CIN2+ or ASC-H; (3) lifetime number of sexual partners >4 (that is the 90th percentile among nonvirgin women); (4) years since sexual debut >4 (median among nonvirgin women); and (5) combined current DNA infection with abnormal cytology/histology (≥HSIL/CIN2+/ASC-H). Given that we cannot attribute specific HPV type to a cytologic lesion, for definition number 5, we conditioned first on HPV16 DNA positivity (or 18 DNA positivity for the HPV18 analysis) to increase the likelihood of type specificity. We chose the 90th percentile of number of lifetime sexual partners and median years since sexual debut to increase the probability of accurately classifying women as HPV exposed. The main conclusions were not altered by dichotomizing at different points (data not shown). A priori, we considered the last definition to provide the strongest evidence of past exposure because it incorporated both molecular and microscopic evidence of infection. For each of the aforementioned 5 case definitions, controls were accordingly defined as women not having the specific case definition.
We estimated that how well continuous levels of HPV16 serology (and HPV18 serology for the HPV18 analysis) collected at enrollment discriminates between cases and controls. The y-axis of the ROC graph represents sensitivity, or the true-positive rate, that is, the proportion correctly discriminated or predicted as such by serology among cases. The x-axis represents the complement of specificity, or the false-positive rate, which is the proportion incorrectly discriminated by serology among noncases. An area under the ROC curve is a commonly used summary measure. An AUC of 1.0 represents a perfect test, whereas an AUC of 0.5 represents random classification.12 Using the continuous measures of HPV16 and 18 serology, we determined the cut point that maximized the sum of sensitivity and specificity. This method implicitly weighs sensitivity and specificity equally.
In addition, we compared the sensitivity and specificity at the current standard cut points (8 EU/mL and 7 EU/mL, for HPV16 and 18, respectively) to 3 a priori specified cut points (the 25th, 50th, and 75th percentiles among nonvirgin seropositive women) and the cut point that maximizes the sum of sensitivity and specificity to show the trade-offs in loss and gain of sensitivity and specificity at different cut points.
Finally, to investigate whether different serology cut points would alter our previous estimates of enrollment correlates of HPV16/18 seropositivity,14 we compared multivariate logistic regression models using the current cut points for HPV16 and 18 (8 EU/mL and 7 EU/mL, respectively), to the cut point that maximized the sum of sensitivity and specificity.
Of the 7466 women enrolled in the trial, 5871 (78.7%) sexually experienced subjects had a pelvic examination, constituting the population for this analysis. The median age of the women included in the analysis was 21 years (interquartile range [IQR], 19–23 years); the median time since sexual debut was 4 years (IQR, 3–7 years); and the median number of lifetime sexual partners was 2 (IQR, 1–3). HPV16 DNA prevalence was 8.3% (n = 488/5868) and HPV18 DNA prevalence was 3.2% (n = 188/5868).
Figure 1 shows the ROC graphs and corresponding AUCs comparing how this polyclonal ELISA discriminated cases and controls based on the 5 different case definitions. For each analysis, the biomarker discriminated significantly better than a completely uninformative model (P < 0.001 for each test of the null hypothesis of AUC = 0.5). The AUC for the HPV16 DNA-only model was 0.70; adding cytology increased AUC to 0.77. For HPV18, the AUC for the HPV18 DNA-only model was 0.69, whereas for combined DNA and cytology was 0.65.
Focusing on the most stringent case definition “current DNA infection and abnormal cytology,” using the continuous serology measure, and acknowledging that sensitivity and specificity are weighted equally, for HPV16 a cut point of 34 EU/mL yielded the best combination of sensitivity and specificity (sensitivity: 62.2%; specificity: 85.3%); for HPV18, a cut point of 60 EU/ mL yielded the best sum of sensitivity and specificity (sensitivity: 34.5%; specificity: 94.9%).
Using the same case definition of current DNA infection and abnormal cytology, from the lowest to highest cut point, sensitivity ranged from 75.7% to 37.8%; specificity ranged from 70.4% to 92.8% (Table 1). Comparing the cut point of 34 EU/mL to 8 EU/mL showed that there was not a statistically significant difference in sensitivity (P = 0.44), but a statistically significant difference in specificity (P < 0.0001). Similarly for HPV18 (Table 2), sensitivity ranged from 51.7% to 34.5%; specificity ranged from 72.6% to 94.9%. Comparison of 60 to 7 EU/mL showed that there was a statistically significant difference in specificity (P < 0.0001), but a nonstatistically significant difference in sensitivity (P = 0.40).
Finally, to investigate whether these new cut points would alter our previous estimates of correlates of HPV16/18 seropositivity,14 we compared models using the current cut points of 8 and 7 EU/mL to the cut points that maximized combined sensitivity and specificity for HPV16 and 18, respectively (Table 3). Although the adjusted odds ratios of the correlates of HPV16 seropositivity, we reported previously,14 did not change substantially using the higher cut point, some correlates lost statistical significance, possibly due to loss of power. Similar results were observed for HPV18 seropositivity (Table 3). As expected, HPV16 and 18 seroprevalence among virgins decreased using the higher cut points; however, virginal seroprevalence did not reach 0% (data not shown).
HPV serology (mainly against HPV16 and 18, the 2 most carcinogenic HPV types and the main targets of the prophylactic vaccines) is being used in research settings to identify current and past HPV-exposed individuals. However, the utility and performance of the different serological assays to discriminate HPV exposure is currently unknown. Using data from a large study of unvaccinated, sexually active, young adult women at peak exposure to HPV, we evaluated the sensitivity and specificity of the polyclonal HPV16 and HPV18 ELISA assay developed by GSK over a wide range of antibody levels and at various prespecified case definitions. Our results showed that the ELISA assays provided moderate discriminative ability to detect cases with a current HPV DNA infection (AUC = 0.7 for both HPV16 and 18), and for HPV16, it improved slightly with the addition of abnormal cytology (AUC = 0.77). Interestingly, addition of cytology to HPV18 DNA decreased the AUC relative to HPV18 DNA alone. Although reasons for these discrepancies are unclear, it could be as a result of the differences in the HPV18 serology assay and also detection of HPV18 DNA or cytology. It is not possible to directly compare the 2 assays, however, it is important to note that the HPV18 VLPs in general are more difficult to make and reproduce. It is also noteworthy that HPV18-related lesions are difficult to detect with cytology15–18 compared with HPV16-related lesions. Finally, using a case definition of HPV DNA-positive and abnormal cytology, serology at cut points identified by the ROC analysis (34 and 60 EU/mL, for HPV16 and 18, respectively) had lower sensitivity and statistically significantly higher specificity than manufacturer set cut points (8 and 7 EU/mL, for HPV16 and 18, respectively).
Accurate classification of both HPV DNA and serostatus is important consideration for epidemiologic studies of HPV infection and vaccine efficacy. For HPV16, using a case definition of HPV16 DNA-positive and abnormal cytology, a cut point of 8 EU/mL set by the manufacturer had the highest sensitivity (75.7%), resulting in a 24.3% false-negative rate. Similarly for HPV18, the set cut point of 7 EU/mL (51.7% sensitivity), results in a 48.3% false-negative rate. Therefore, even at the highest sensitivity, there is misclassification if HPV serology is used as a biomarker of HPV exposure. This could have implications for studies investigating cofactors influencing progression of HPV-infected cells to precancer and cancer, whereby associations found could be due to residual confounding by HPV positivity. As an example, some studies that have found an association between presence of Chlamydia trachomatis and cervical cancer, in the absence of HPV DNA exposure, have used HPV serology to account or adjust for HPV status.19–21 Based on our results, given that they used an assay with similar misclassification of HPV exposure as the one we evaluated in this report, 25% of HPV16 and 48% of HPV18 exposed women would be classified as HPV negative. Thus, an observed positive association could be due to HPV-positive women who were misclassified as negative by the serology assay. In addition, it is premature to use this assay clinically to distinguish HPV-exposed from unexposed women.
Our correlates of HPV16 and 18 seropositivity found in a previous analysis did not change substantially at the higher cut points, which indicates that the cut point set by the manufacturer is specific for epidemiologic studies of correlates of seropositivity. Although some correlates lost statistical significance at the higher cut points possibly due to decreased power, risk estimates remained similar. Additionally, we found virginal seroprevalences never reached 0%, which is not unexpected as virginal seroprevalence can reflect the inherent biases in self-report of virginity, a technical artifact of nonspecific binding, possible transmission through other nonpenetrative sexual contact, or perinatal transmission, which we are unable to assess in this analysis.
There are some limitations to be considered. Currently there is no gold standard to classify HPV exposure, and we know from natural history studies that the majority of HPV infections clear.4 Additionally, natural history studies of HPV prevalence and incidence consistently show that the strongest correlates/predictors of HPV infection are those associated with increased exposure and persistence of the virus, such as higher number of lifetime sexual partners, increased years since sexual debut, abnormal cytology, and a current infection. Taken together, because DNA positivity does not reflect cumulative infection, we used the aforementioned predictors of past exposure when defining our cases. In addition, not all HPV-infected women develop antibody response to natural infection; some seroconvert at very low antibody levels, often below the assays' limit of detection; and, it may take time and repeated exposures to develop such a detectable response.22 Therefore, it is unknown whether the moderate AUCs we obtained are due to poor assay, poor “gold standard,” a combination of the 2, or due to biology of HPV infection. Thus, restricting HPV-exposed women to biologic and behavioral characteristics could have resulted in misclassification of our outcome.
Our analysis is also limited by the cross-sectional nature of our data. Only longitudinal studies can evaluate the serological response to HPV and its relation to HPV exposure, infection, persistence, and immunity. It is also important to note that the results from this study are not applicable to studies using other HPV serology assays. Finally, this study was performed in a young, healthy population; whether the performance of assay is different in older women, is unknown.
In summary, this polyclonal ELISA assay provided moderate discriminative ability to detect HPV exposure. In addition, for HPV16 and 18, respectively, serology at cut points of 34 and 60 EU/mL yielded the best combination of sensitivity and specificity, and had lower sensitivity and statistically higher specificity than the current cut points (8 and 7 EU/mL) to discriminate HPV-exposed women. It is also important to note that even at the highest sensitivity (current cut points) a substantial proportion of women are still misclassified. The application of the polyclonal ELISA HPV assays should be considered in the context of the outcome of interest and balance between sensitivity and specificity. If the intention is measuring exposure, the goal may be to maximize sensitivity; thus, the current cut points are appropriate as the higher cut points yielded higher specificity, but at a detriment to sensitivity. However, if the objective is to measure immunity, it may be more appropriate to maximize specificity and positive predictive value; thus, a higher cut point may be more appropriate.
Present evidence does not support use of the ELISA serological assays in risk stratification or clinical setting. Future research on this topic should consider application of the ELISA assays and the important balance between sensitivity and specificity in the specific research setting.
1. Cogliano V, Baan R, Straif K, et al. Carcinogenicity of human papillomaviruses. Lancet Oncol 2005; 6:204.
2. Dunne EF, Unger ER, Sternberg M, et al. Prevalence of HPV infection among females in the United States. JAMA 2007; 297:813–819.
3. Schiffman M, Herrero R, Hildesheim A, et al. HPV DNA testing in cervical cancer screening: Results from women in a high-risk province in Costa Rica. JAMA 2000; 283:87–93.
4. Rodriguez AC, Schiffman M, Herrero R, et al. Longitudinal study of human papillomavirus persistence and cervical intraepithelial neoplasia grade 2/3: Critical role of duration of infection. J Natl Cancer Inst 2010; 102:315–324.
5. Schiffman M, Safaeian M, Wentzensen N. The Use of human papillomavirus seroepidemiology to inform vaccine policy. Sex Transm Dis 2009; 36:675–679.
6. Herrero R, Hildesheim A, Rodriguez AC, et al. Rationale and design of a community-based double-blind randomized clinical trial of an HPV 16 and 18 vaccine in Guanacaste, Costa Rica. Vaccine 2008; 26:4795–4808.
7. Dessy FJ, Giannini SL, Bougelet CA, et al. Correlation between direct ELISA, single epitope-based inhibition ELISA and pseudovirion-based neutralization assay for measuring anti-HPV-16 and anti-HPV-18 antibody response after vaccination with the AS04-adjuvanted HPV-16/18 cervical cancer vaccine. Hum Vaccin 2008; 4:425–434.
8. Harper DM, Franco EL, Wheeler C, et al. Efficacy of a bivalent L1 virus-like particle vaccine in prevention of infection with human Papillomavirus types 16 and 18 in young women: A randomised controlled trial. Lancet 2004; 364:1757–1765.
9. van Doorn LJ, Molijn A, Kleter B, et al. Highly effective detection of human papillomavirus 16 and 18 DNA by a testing algorithm combining broad-spectrum and type-specific PCR. J Clin Microbiol 2006; 44:3292–3298.
10. Safaeian M, Herrero R, Hildesheim A, et al. Comparison of the SPF10-LiPA system to the Hybrid Capture 2 Assay for detection of carcinogenic human papillomavirus genotypes among 5683 young women in Guanacaste, Costa Rica. J Clin Microbiol 2007; 45:1447–1454.
11. Baay MF, Quint WG, Koudstall J, et al. Comprehensive study of several general and type-specific primer pairs for detection of human papillomavirus DNA by PCR in paraffin-embedded cervical carcinomas. J Clin Microbiol 1996; 34:745–747.
12. Hanley JA, McNeil BJ. The meaning and use of the area under a receiver operating characteristic (ROC) curve. Radiology 1982; 143:29–36.
13. Gonen M. Receiver operating characteristic (ROC) curves. SUGI 2006; 31:210–231. Statistics and Data Analysis Paper.
14. Coseo S, Porras C, Hildesheim A, et al. Seroprevalence and correlates of human papillomavirus 16/18 seropositivity among young women in Costa Rica. Sex Transm Dis 2010; 37:706–714.
15. Clifford GM, Smith JS, Aquado T, et al. Comparison of HPV type distribution in high-grade cervical lesions and cervical cancer: A meta-analysis. Br J Cancer 2003; 89:101–105.
16. Smith JS, Lindsay L, Hoots B, et al. Human papillomavirus type distribution in invasive cervical cancer and high-grade cervical lesions: A meta-analysis update. Int J Cancer 2007; 121:621–632.
17. Kovacic MB, Castle PE, Herrero R, et al. Relationships of human papillomavirus type, qualitative viral load, and age with cytologic abnormality. Cancer Res 2006; 66:10112–10119.
18. Safaeian M, Schiffman M, Gage J, et al. Detection of precancerous cervical lesions is differential by human papillomavirus type. Cancer Res 2009; 69:3262–3266.
19. Anttila T, Saikku P, Koskela P, et al. Serotypes of Chlamydia trachomatis
and risk for development of cervical squamous cell carcinoma. JAMA 2001; 285:47–51.
20. Koskela P, Anttila T, Bjorge T, et al. Chlamydia trachomatis
infection as a risk factor for invasive cervical cancer. Int J Cancer 2000; 85:35–39.
21. Matsumoto K, Yasugi T, Oki A, et al. Area smoking and chlamydial infection risk factors for CIN? Different results after adjustment for HPV DNA and antibodies. Br J Cancer 2003; 89:831–833.
22. Stanley M. Immunobiology of HPV and HPV vaccines. Gynecol Oncol 2008; 109:S15–S21.
This article has been cited 1 time(s).
Plos OneHPV16 Seropositivity and Subsequent HPV16 Infection Risk in a Naturally Infected Population: Comparison of Serological AssaysPlos One
© Copyright 2011 American Sexually Transmitted Diseases Association