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Comparison of the SPF₁₀-LiPA System to the Hybrid Capture 2 Assay for Detection of Carcinogenic Human Papillomavirus Genotypes among 5,683 Young Women in Guanacaste, Costa Rica

Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, Maryland,¹ Proyecto Epidemiológico Guanacaste, Fundación INCIENSA, San José, Costa Rica,² Universidad de Costa Rica, San José, Costa Rica,³ Delft Diagnostic Laboratory, Delft, The Netherlands⁴

Received 22 December 2006/ Returned for modification 9 February 2007/ Accepted 26 February 2007

	ABSTRACT

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The objective of this analysis was to compare the performance characteristics of two human papillomavirus (HPV) DNA detections assays, the Hybrid Capture 2 assay (HC2) and the SPF₁₀ assay, for the detection of carcinogenic HPV. Data are from the enrollment visits of women who participated in the randomized, double-blind, placebo-controlled phase III HPV16/18 Vaccine Trial in Guanacaste, Costa Rica. We compared the results of HC2 and SPF₁₀ testing of cervical specimens. Since the line probe assay (LiPA) detection system does not distinguish between HPV type 68 (HPV68; which is targeted by HC2) and HPV73 (which is not targeted by HC2), for SPF₁₀-LiPA, we defined the carcinogenic HPV types as the 12 HC2-targeted types (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59), HPV68/73, and the HC2-cross-reactive, carcinogenic type HPV66. The kappa values and the performance characteristics for the detection of cervical abnormalities were ascertained. Paired observations were available for 5,683 sexually active, young women (median age, 21 years). The prevalence of carcinogenic HPV types was 35% (n = 1,962) by HC2 and 35% (n = 2,003) by SPF₁₀-LiPA. There were no differences in the prevalence of carcinogenic HPV types by HC2 and SPF₁₀-LiPA among women with normal, atypical squamous cells of undetermined significance and high-grade squamous intraepithelial lesion cytology. Among women with low-grade squamous intraepithelial lesion cytology, HC2 was more likely to test positive than SPF₁₀-LiPA for the carcinogenic HPV types (87% and 79%, respectively; P = 0.001) as a result of HC2 cross-reactivity with HPV types 40, 43, 44, 53, 54, 60, 70, and 74. The crude agreement between the two assays was 88%, with a kappa value of 0.75 (95% confidence limits, 0.73 to 0.76). We observed very good agreement between HC2 and SPF₁₀-LiPA for carcinogenic HPV type detection.

	INTRODUCTION

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In the 1990s, data from multiple, international epidemiologic studies established infection with a group of 15 human papillomavirus (HPV) types as a necessary cause of cervical cancer (2, 27). The steady increase in the fraction of cervical cancers attributable to HPV infection from the low estimates reported in epidemiologic studies from a decade earlier was achieved by a reduction in misclassification due to the measurement errors caused by suboptimal HPV DNA tests (3, 8, 9, 23).

Now that the causal relationship between HPV infection and cervical cancer is certain, HPV-based prevention strategies are becoming increasingly important. In some countries HPV DNA assays are used as an adjunct to cytology to identify women at risk of cervical cancer who require preventive treatment. Recently, trials of prophylactic HPV vaccines have shown that these vaccines have high degrees of efficacy in preventing new and persistent infections with the HPV types in the vaccine formulation (most notably, HPV type 16 [HPV16] and HPV18, which together cause 70% cases of cervical cancer worldwide) (11, 26). Thus, the study of cervical cancer prevention within the context of vaccines requires accurate detection of type-specific incidence and persistent HPV infections associated with cancer and precancerous lesions. Beyond the detection of HPV16 and HPV18, other types must be identified accurately as well. Important secondary aims of ongoing clinical trials of vaccines are whether they confer protection against HPV types besides those in the formulations and whether reducing the frequencies of the two most carcinogenic genotypes as a result of vaccination could lead to increased frequencies of other HPV types.

At present there is no "gold standard" for HPV detection; however, there is one FDA-approved molecular assay, the Hybrid Capture 2 assay (HC2; Digene Corporation, Gaithersburg, MD), which collectively targets 13 carcinogenic HPV types, with the limitation that it does not provide information about type-specific HPV genotypes. On the other hand, PCR-based assays are able to amplify most genital HPV genotypes in a single PCR and have the added benefit of providing HPV type discrimination following amplification. Commonly used PCR primers are consensus primers GP5+/GP6+, MY9/MY11, and PGMY9/PGMY11, which amplify 150-bp (primer GP5+/GP6+) and 450-bp (primers PGMY and MY9/MY11) regions within the conserved L1 open reading frame, which encodes the major capsid protein.

A newer primer set (SPF₁₀) that amplifies a 65-bp region in the same L1 open reading frame region (16) as the other primers mentioned above has been developed. Because of its shorter amplification product, it is thought to be more analytically sensitive but possibly less specific for HPV detection than DNA-based assays, with a potential to amplify at least 54 HPV types. Genotype identification is achieved by using a reverse line probe assay (LiPA).

We are using the SPF₁₀-LiPA system as part of the HPV Vaccine Trial in Costa Rica (CVT). CVT is a phase III randomized efficacy trial of an HPV16 and HPV18 vaccine, with the primary end point being the reduction in HPV16- and HPV18-related cervical intraepithelial neoplasia (CIN) grade 2 (CIN2) and CIN3. The secondary aims of the trial (e.g., the effect of vaccination on the persistence of the targeted and nontargeted carcinogenic HPV types) are also dependent on the results of SPF₁₀-LiPA. However, large formal evaluations of the SPF₁₀-LiPA system are lacking, with only one report comparing the performance of SPF₁₀-LiPA with that of HC2 using samples from 138 women attending a colposcopy clinic (18). Thus, we sought to compare the detection of HPV DNA by the SPF₁₀-LiPA system to that by HC2 using paired samples collected during the enrollment phase of the community-based, randomized, double-blind, placebo-controlled phase III HPV16/18 Vaccine Trial in Guanacaste, Costa Rica.

	MATERIALS AND METHODS

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Study population. Data are from the enrollment visit of women who participated in CVT. As mentioned in the introduction, the primary aim of CVT is to independently assess the efficacy of an HPV16/18 vaccine manufactured by GlaxoSmithKline for the prevention of precancerous lesions (defined for the trial as CIN2, CIN3, or adenocarcinoma in situ [AIS]) and invasive cervical cancer. Enrollment began in June 2004 and ended in December 2005. Study participants were women between 18 and 25 years of age, in good general health, and without a history of chronic conditions that required treatment; were willing to use birth control during the vaccination period; and lived in the study area and had no plans of imminent departure from the study area. Approximately one-third of the females identified in a previous census fulfilled the inclusion criteria and participated in the study.

At enrollment, the women provided written, informed consent and underwent a urine pregnancy test. Prior to randomization, the women were also administered a questionnaire that inquired about demographics, sexual activity, contraceptive use, reproductive history, cigarette use, and the family history of cancers. A detailed medical questionnaire was also administered, and medical and pelvic examinations were conducted for all consenting, sexually experienced women. During the pelvic examination, cervical cells were collected and placed in 20 ml of liquid cytology medium (PreservCyt; Cytyc Corporation, Marlborough, MA) for liquid-based cytology (ThinPrep; Cytyc Corporation) and for HPV detection by using SPF₁₀ and HC2. To minimize the chance of carryover, cytologic slides were prepared after the withdrawal of two 0.5-ml aliquots, one for SPF₁₀ PCR testing and one for confirmatory HPV testing in the future. Aliquots destined for PCR were stored in a liquid nitrogen tank, while the remaining PreservCyt samples were kept at room temperature (20°C) until they were used to make liquid cytology slides to test for carcinogenic HPV, Chlamydia trachomatis, and Neisseria gonorrhoeae. All testing was done with the investigator masked to the results of other tests or cytology results.

This analysis was based on the enrollment, prevaccination specimens from women entering the vaccine trial. All study protocols were reviewed and approved by the NCI and Costa Rican Institutional Review Boards.

HPV detection and genotyping. (i) HC2. HC2 is an FDA-approved, commercially available HPV test which collectively targets 13 carcinogenic HPV types (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) without distinguishing the HPV type present. HC2 is a signal amplification assay that uses a technique which combines antibody capture of HPV DNA and RNA probe hybrids and chemiluminescent signal detection. Additionally, because of their genetic relatedness (19, 21, 24), other cancer-associated types, such as HPV66, are also detected by HC2. HC2 was performed according to the manufacturer's instructions in a laboratory at the University of Costa Rica in San Jose with residual PreservCyt samples. HC2 results were missing for 185 (3.2%) samples, mainly due to insufficient specimen volume.

(ii) SPF₁₀-LiPA system. Total DNA was isolated from 200 µl of a PreservCyt aliquot drawn prior to ThinPrep preparation by using a MagNA Pure LC instrument (Roche Diagnostics, Almere, The Netherlands) and a Total DNA isolation kit (Roche Diagnostics). DNA was eluted in 100 µl of water. Each DNA extraction run contained positive and negative controls to monitor the DNA isolation procedure.

A 10-µl aliquot of extracted DNA was used for each SPF₁₀ PCR. The SPF₁₀ PCR primer set was used to amplify a broad spectrum of HPV genotypes, as described earlier (15, 16). Briefly, this primer set amplifies a small fragment of 65 bp from the L1 region of HPV. Reverse primers contain a biotin label at the 5' end, enabling capture of the reverse strand onto streptavidin-coated microtiter plates. The captured amplimers are denatured by alkaline treatment, and the captured strand is detected by a defined cocktail of digoxigenin-labeled probes that detect a broad spectrum of HPV genotypes. This method is designated the HPV DNA enzyme immunoassay (DEIA), which provides an optical density value. If the SPF₁₀-DEIA yielded a borderline value (75 to 100% of the cutoff value), the SFP₁₀ PCR was repeated and the sample was retested by DEIA. Each DEIA run contained separate positive, borderline, and negative controls. The broad-spectrum SFP₁₀ primers can recognize at least 54 HPV types.

The same SPF₁₀ amplimers (from SPF₁₀-DEIA-positive samples) were used to identify the HPV genotype by reverse hybridization on a LiPA containing probes for 25 different HPV genotypes (SPF₁₀ HPV LiPA, version 1; manufactured by Labo Bio-Medical Products, Rijswijk, The Netherlands). The LiPA detects HPV types 6, 11, 16, 18, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68/73, 70, and 74. Each LiPA run contained negative and positive controls. Since the interprimer regions of HPV68 and HPV73 are identical, the LiPA system cannot distinguish between HPV68 and HPV73; hence, they are designated HPV68/73. SPF₁₀-LiPA results were available for all samples.

Because the CVT is focused on HPV16 and HPV18, type-specific (TS) PCR (TS-PCR) primer sets were also used to selectively amplify HPV16 (TS16) and HPV18 (TS18) from 2,513 specimens that tested positive by the SPF₁₀ PCR but that did not contain HPV16 or HPV18, as determined by LiPA (25). The type-specific primers were based on those described by Baay et al. (1); they generate amplimers of 92 and 126 bp for HPV16 and HPV18, respectively. Amplimers from the TS-PCRs were detected by DEIA, similar to the method used for SPF₁₀ amplimer detection.

Statistical analysis. The primary outcome was the HPV prevalence determined by HC2 and SPF₁₀-LiPA. It was necessary to adjust for the differences in HPV genotypes targeted by the two assays. HC2 collectively targets 13 carcinogenic HPVs (HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68), although additional types, like HPV66, have also been shown to be detected sensitively in practice (4, 22). The SPF₁₀ PCR can detect more than 50 HPV types, whereas the genotyping system (LiPA) can identify only 25 different HPV types: 11 noncarcinogenic types (types 6, 11, 34, 40, 42, 43, 44, 53, 54, 70, and 74) and 14 carcinogenic types (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68/73). Hence, to compare the SPF₁₀ assays to HC2, we defined HPV detection by the SPF₁₀ assays at three levels: (i) PCR positive, detection of all amplified HPV genotypes by DEIA without distinguishing which genotype(s) is present, (ii) LiPA positive, detection of at least 1 of the 25 low- and high-risk HPV types; and (iii) carcinogenic positive, detection of 1 of the 14 carcinogenic HPV types.

The agreement between the two assays was determined using unweighted kappa statistics and 95% confidence intervals (CIs), which calculate the percent agreement beyond that expected by chance alone. The nonparametric test for matched data (McNemar's ² test) was used to determine whether the proportion of samples classified as positive by HC2 and negative by SPF₁₀-LiPA was equal to the proportion of samples classified as negative by HC2 and positive by SPF₁₀-LiPA.

In an effort to investigate the reasons for discordant assay findings, we compared the 307 HC2-positive, SPF₁₀-LiPA carcinogenic HPV-negative samples to the 348 HC2-negative, SPF₁₀-LiPA carcinogenic HPV-positive samples. Using two-way tabulations and Pearson's ² test, we compared selected demographic and behavioral factors for women with discordant test results. Additionally, we compared selected medical findings from the pelvic examinations.

Furthermore, because discordance between HC2 and the SPF₁₀ assays could be due to the differential viral quantity required by the two assays, we used the ratio of relative light unit (RLU) values to positive control (pc) values (RLU/pc) from the HC2 assay as a proxy measure for HPV viral burden to investigate whether a lower viral RLU/pc quantity explained why some HC2-positive samples were classified as negative by the SPF₁₀ PCR system. RLU/pc values were categorized on the basis of quartiles among HC2-positive samples: 1 to 4, 5 to 29, 30 to 267, and 268 RLU/pc. Among the HC2-negative samples, we investigated the frequency of HPV types missed.

	RESULTS

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Of the 7,466 women enrolled in the trial, 1,598 were excluded from this analysis because a pelvic examination was not performed, mainly because they were virgins (n = 1,592). The median age was 21 years (interquartile range [IQR], 19 to 23 years), and the median age at the time of their first sexual intercourse was 17 years (IQR, 15 to 18 years). Forty-four percent (n = 2,574) were single; 52% (n = 3,050) were married; and 3.4% (n = 198) reported being separated, divorced, or widowed.

Carcinogenic HPV detection. Among the 5,683 paired observations, 2,856 women (50%) were positive by PCR, 2,398 (42%) were positive by LiPA, and 2,003 (35%) were carcinogenic HPV positive. By comparison, 1,962 women (35%) were carcinogenic HPV positive by HC2. The crude agreement between the two assays for carcinogenic HPV type detection was 88%, and the kappa value was 0.75 (95% CI, 0.73 to 0.76), indicating very good agreement. Three hundred seven women (5%) were HC2 positive but carcinogenic HPV type negative by use of the SPF₁₀-LiPA system. Similarly, 348 (6%) were carcinogenic HPV type positive by use of the SPF₁₀-LiPA system but HC2 negative (McNemar's P value = 0.1) (Table 1).

View larger version (34K): [in this window] [in a new window]   FIG. 1. Comparison of clinical performance for cytologic interpretation by HC2 and SPF10-PCR system. *, comprised of 25 types detected by LiPA (types 6, 11, 16, 18, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68/73, 70, and 74); **, comprised of 13 types targeted by HC2 (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) and HPV66.

Type-specific testing for HPV16 and HPV18. Among the 5,868 women for whom SPF₁₀-LiPA results were available, 403 (6.9%) were positive for HPV16 and 145 (2.5%) were positive for HPV18, with 16 (0.3%) infected by both of those HPV types, based on SPF₁₀-LiPA. Thus, 564 (9.6%) women were infected with HPV16 and/or HPV18.

Of the 2,937 SPF₁₀-DEIA-positive women, 471 (16%) were negative for the HPV types detected by LiPA. An additional 2,450 were SPF₁₀-DEIA positive but either HPV16 or HPV18 negative by LiPA. This resulted in 2,921 women for whom results were available by additional type-specific tests with TS16 and/or TS18 primers, of whom 95 (3.2%) were found to be either HPV16 positive (n = 69) or HPV18 positive (n = 27) and of whom 1 was positive for both HPV16 and HPV18. By combining the results of both SPF₁₀-LiPA and TS testing, 488 women (8.3%) were positive for HPV16 and 188 (3.2%) were positive for HPV18, with 30 (0.5%) infected with both of those types. Thus, 646 women (11%) were infected with HPV16 and/or HPV18. SPF₁₀-LiPA detected 86% of HPV16 infections and 86% of HPV18 infections detected by both SPF₁₀-LiPA and TS testing. Eighty-one of the 95 (85%) samples positive by the TS-PCR were HC2 positive (compared with 88% of any specimens that tested positive for HPV16 and/or HPV18 and 80% of specimens that tested positive only for HPV16 or HPV18 by SPF₁₀-LiPA); among the HC2-positive specimens, 33 (35%) of the TS PCR-positive specimens had high RLU/pc values, i.e., above 268.

	DISCUSSION

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In this large, population-based cohort of young women, there was good agreement for the detection of carcinogenic HPV types by HC2 and the SPF₁₀-LiPA system. Further stratification by cytology, age, and marital status revealed similar kappa values among the different strata. From a research perspective, both the SPF₁₀ system and HC2 were comparable in detecting carcinogenic HPV, and from a clinical perspective, the HPV prevalences determined by both tests were comparable among those with normal cytologies, atypical squamous cells (ASCs) of undetermined significance (ASCUS), and high-grade squamous intraepithelial lesions (HSILs).

Unlike our results, a recent study from The Netherlands found poor agreement between the results of HC2 and the SPF₁₀-LiPA system (crude agreement, 0.70; kappa value, 0.40) (18), with a carcinogenic HPV type prevalence of 36% by HC2 compared with a carcinogenic HPV type prevalence of 50% with the SPF₁₀-LiPA system. The sample size for that study was relatively small (n = 138 at two 6-month intervals, resulting in 276 datum points), and the study consisted of women with low-grade cervical changes or women attending a clinic pre- and posttreatment for CIN, whereas our analysis consisted of younger, healthy women from the general population. It is unclear why there are such marked differences between the two studies and what methodological differences, if any, could explain such differences. Importantly, both studies showed similarly high sensitivities for the detection of HSIL by either HC2 or SPF₁₀-LiPA.

Our findings confirmed the previously reported cross-reactivity of HC2 with other HPV types not targeted in its probe set (4, 19, 20) by the use of HPV amplification and typing systems different from those used in the previous reports. We confirmed that HC2 detects HPV66 (a carcinogenic type not targeted by HC2) more frequently than HPV68/73, at least one of which (HPV68) is targeted by the HC2 probe. This is similar to the findings of other studies, which also observed that, among the targeted types, HC2 detection is weakest for HPV68 (10, 22). This might account for the equivocal data regarding the carcinogenicity of HPV68 (5, 13). However, if 50% of the specimens positive for HPV68/73 are HPV68 positive and 50% are HPV73 positive and HC2 detected HPV in 50% of the specimens, this is consistent with 100% detection of HPV68 and 0% detection of HPV73. We were also able to show that there was a modest degree of HC2 cross-reactivity with HPV types 70 and 53 and, to a lesser extent, with types 6, 40, 11, 74, 44, 54, and 43.

When we compared the misclassification of disease outcomes among those negative by both HC2 and SPF₁₀-DEIA, we observed that both assays missed similar numbers of cytologic abnormalities; however, there was a tendency for more women with LSIL to be HC2 positive than positive for carcinogenic HPV types by SPF₁₀-LiPA. Further evaluation revealed that this was mainly attributed to the cross-reactivity of HC2 to other, noncarcinogenic HPV types (types 40, 43, 44, 53, 54, 60, 70, and 74) detected in the LSIL-positive samples; these types can also cause LSILs (17).

Discordance between the two assays based on the 14 carcinogenic HPV types could be explained by a couple of factors. In general, HC2-positive, SPF₁₀-LiPA-negative specimens had lower viral quantities than those positive by both assays, suggesting that sampling error due to a low HPV viral load may be one reason for HC2-positive, SPF₁₀-system-negative findings at the PCR level. Furthermore, comparing the samples with discordant results by HC2 and with the SPF₁₀-LiPA system, we found that there were no differences between the discordant groups by comparison of factors that are critical for HPV detection. Women whose samples were carcinogenic HPV type positive (by SPF₁₀-LiPA) but HC2 negative were older and were more likely to have had a concurrent Chlamydia infection than carcinogenic HPV type-negative (by SPF₁₀-LiPA) but HC2-positive women. However, this did not provide further clarification regarding the reasons for or the directionality of the discordance, suggesting that there is some degree of misclassification of HPV infection status by either assay.

HC2 has been shown to be highly sensitive and specific for clinical outcomes (6), and its cross-reactivity with other HPV types, rather than compromising, probably contributes to its high sensitivity but probably results in false-positive findings. However, use of HC2 is limited in some applications because it does not provide information on the type-specific HPV infection. The type-specific information is valuable in studies of the natural history of HPV and specifically in the context of vaccine efficacy trials, in which the most relevant end points recommended by a FDA vaccine advisory panel for determining vaccine efficacy are a reduction in the incidence of vaccine type-specific persistent infections and associated moderate to severe CIN, approximated in this study by HSIL, or worse by cytology (7). Such desired type-specific associations are not facilitated by HC2; however, PCR-based assays and detections systems such as the SPF₁₀-LiPA system allow type-specific HPV determination.

Interestingly, the SPF₁₀ consensus primers can potentially coamplify 54 different HPV types in one PCR. However, its analytic sensitivity, especially in the presence of multiple HPV types with various viral loads, can possibly be reduced, as the different types can compete for limited PCR primers. This possibility might have been demonstrated by the additional HPV16- and HPV18-positive results when type-specific HPV16 and HPV18 primers were used to selectively amplify SPF₁₀-DEIA-positive, LiPA-negative, and all HPV16- and HPV18-negative samples. An additional 69 samples were found to be HPV16 positive, and an additional 27 were found to be HPV18 positive. From a clinical perspective, missing these additional HPV16- and HPV18-positive samples was not plainly detrimental in our population sample because all six women with HSILs that were HPV16 positive only by the TS test and the two women with HSILs that were HPV18 positive only by the TS test were also positive for at least one other carcinogenic HPV type and, hence, were identified as HPV positive because of the pooling of the data. However, more investigation is warranted to evaluate the effect of competition between types within consensus PCR assays, especially if the competition is not random, and if, rather, competition is dependent on factors such as HPV type and viral quantity.

One limitation of SPF₁₀-LiPA system, especially in studies in which it is compared to HC2, was that the LiPA detection system does not differentiate between HPV68 (a type targeted by the HC2 probe) and HPV73 (a type not targeted by the HC2 probe). By including HPV68/73 in our comparison category, we may have artificially made a more robust category, especially if HPV73 is more prevalent than HPV68 in this cohort. However, data from a previous natural history study in the same region (Guanacaste, Costa Rica) (12) showed small but equal prevalences of both HPV68 and HPV73 in that population (0.3% and 0.5%, respectively). Hence, it is unlikely that inclusion of HPV73 as a carcinogenic type would have strongly influenced our results and led to conclusions of the equal performance of SPF₁₀-LiPA and HC2.

A strength of this study is that it is unlikely to have suffered from selection bias. Both assays were performed with samples derived from one specimen from the same participant at the same time and were collected in the same media, thus reducing the chance that differences could be attributed to procedural differences or other host factors. Another strength of this study was its large, population-based sample of women.

In conclusion, we observed good agreement between HC2 and the SPF₁₀-LiPA system for the detection of carcinogenic HPV types. We suggest that the SPF₁₀-LiPA is a robust assay for the study of the natural history of type-specific HPV and vaccine-related outcomes.

	ACKNOWLEDGMENTS

 
CVT is a long-standing collaboration between investigators in Costa Rica and NCI. The trial is funded by intramural NCI and the NIH Office of Research on Women's Health and is conducted in agreement with the Ministry of Health of Costa Rica. 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 the regulatory submission needs of the company. NCI and Costa Rican investigators make final editorial decisions on this presentation and subsequent publications; GSK has the right to review/comment.

The affiliations of the members of the CVT group are as follows. At the Proyecto Epidemiológico Guanacaste, Fundación INCIENSA, San José, Costa Rica, Mario Alfaro (cytologist), Manuel Barrantes (field supervisor), M. Concepcion Bratti (coinvestigator), Fernando Cárdenas (general field supervisor), Bernal Cortés (specimen and repository manager), Albert Espinoza (head, coding and data entry), Yenory Estrada (pharmacist), Paula Gonzalez (coinvestigator), Diego Guillén (pathologist), Rolando Herrero (co-principal investigator), Silvia E. Jimenez (trial coordinator), Jorge Morales (colposcopist), Lidia Ana Morera (head study nurse), Elmer Pérez (field supervisor), Carolina Porras (coinvestigator), Ana Cecilia Rodriguez (coinvestigator), and Maricela Villegas (clinic physician); at the University of Costa Rica, San José, Costa Rica, Enrique Freer (director, HPV Diagnostics Laboratory), Jose Bonilla (head, HPV Immunology Laboratory), Sandra Silva (head technician, HPV Diagnostics Laboratory), Ivannia Atmella (immunology technician), and Margarita Ramírez (immunology technician); at the National Cancer Institute, Bethesda, MD, Pamala Gahr (trial coordinator), Allan Hildesheim (co-principal investigator and NCI co-project officer), Douglas R. Lowy (HPV virologist), Mark Schiffman (medical monitor and NCI co-project officer), John T. Schiller (HPV virologist), Mark Sherman (quality control pathologist), Diane Solomon (medical monitor and quality control pathologist), and Sholom Wacholder (statistician); at SAIC, NCI—Frederick, Frederick, MD, Ligia Pinto (head, HPV Immunology Laboratory) and Alfonso Garcia-Pineres (scientist, HPV Immunology Laboratory); at Womens and Infants’ Hospital, Providence, RI, Claire Eklund (quality control, cytology) and Martha Hutchinson (quality control, cytology); and Delft Diagnostics Laboratory, The Netherlands, Wim Quint (HPV DNA testing) and Leen-Jan van Doorn (HPV DNA testing).

	FOOTNOTES

 
* Corresponding author. Mailing address: Division of Cancer Epidemiology and Genetics, Hormonal and Reproductive Epidemiology Branch, National Cancer Institute, 6120 Executive Boulevard, Suite 550, Rockville, MD 20852. Phone: (301) 594-2934. Fax: (301) 402-0916. E-mail: safaeianm{at}mail.nih.gov

Published ahead of print on 7 March 2007.

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