Prevalence and Clustering Patterns of Human Papillomavirus Genotypes in Multiple Infections Anil K. Chaturvedi, 1 Leann Myers, 2 Ansley F. Hammons, 3 Rebecca A. Clark, 3 Kathleen Dunlap, 4 Patricia J. Kissinger, 1 and Michael E. Hagensee 1,3 Departments of 1 Epidemiology and 2 Biostatistics, Tulane University School of Public Health and Tropical Medicine; and Departments of 3 Medicine, 4 Obstetrics and Gynecology, Louisiana State University Health Sciences Center, New Orleans, Louisiana Abstract Prevalence of multiple human papillomavirus (HPV) infec- tions, involvement of specific HPV phylogenetic clades in multiple infections, and clustering patterns of multiple infections at the clade level were assessed in 854 HIV () and 275 HIV (+) women cross-sectionally. Reverse line blot assay was used to detect 27 HPV genotypes. Involvement of specific clades in coinfections and clustering patterns were assessed using HPV clade/genotype as the unit of analyses. Expected frequencies assuming independence for all possible clade combinations in two-genotype infections were derived using a multinomial expansion and comparisons of observed and expected frequencies were done using a composite goodness-of-fit test. In all, 100 two-genotype infections were detected; 61 in HIV () and 39 in HIV (+) women. Clade A9 (HPV types 16, 31, 33, 35, 52, and 58) was significantly less likely to be involved in multiple infections compared with all other clades (55.2% versus 64.6%; adjusted odds ratios, 0.68; 95% confidence interval, 0.48-0.95). Observed patterns for all possible clade combinations (among HPV clades A3, A5, A6, A7, A9, and A10) in two-genotype infections did not significantly differ from those expected in the entire sample, across HIV, Pap smear, and age strata (all goodness-of-fit exact P > 0.20). These results indicate that clade A9 is less likely to be involved in multiple infections and that HPV genotypes predominantly establish multiple infections at random, with little positive/negative clustering for either phylogenetically related or unrelated types. The current method of analysis affords the opportunity to test clustering of a large number of HPV genotype/clade combinations at nominal alpha levels. (Cancer Epidemiol Biomarkers Prev 2005;14(10):2439 – 45) Introduction Among women infected with human papillomaviruses (HPV), concurrent infection with more than one HPV type is observed in 20% to 50% of HIV () (refs. 1, 2) and 50% to 80% of HIV (+) populations (3, 4). Recently, multiple HPV infections have gained increasing attention owing to reports of successful prophylactic vaccination trials (5, 6). The molecular and epidemiologic significance of infection with multiple HPV genotypes is as yet unclear. Although immunity towards HPV is predominantly type-specific (7), theoretical concerns exist regarding the impact of the removal of certain genotypes from human populations through vaccination (8). The removal of certain genotypes by type-specific vaccination could result in positive selective pressures on untargeted genotypes, thus increasing their prevalence; or type-specific vaccination could confer protective immunity against phylogenetically related genotypes (9). These concerns mandate a solid understanding of the equilibrium and clustering patterns among various HPV clades/genotypes in concurrent and sequential coinfections. Studying the clustering patterns in naturally occurring multiple infections may aid in better characterizing this equilibrium and in assessing the impact that type-specific vaccination may have on untargeted genotypes. Furthermore, studying clustering patterns may reflect on the existence of separate transmission patterns at the HPV clade/genotype level (2). Few studies have addressed the issue of clustering patterns of HPV clades or genotypes involved in concurrent or sequential multiple infections. DNA-based follow-up studies indicate that preexisting HPV infection at baseline increases the risk of acquisition of both phylogenetically related and unrelated genotypes (1, 8, 10). These studies also indicate the lack of the existence of any significant patterns over and beyond chance expectations in the clustering of genotypes involved in coinfections (1, 8-10). Previous studies addressing clustering patterns have focused on either HPV 16 or a select few genotypes. Owing to the vast heterogeneity of HPVs, these analyses focusing on HPV clades or specific genotype combinations are affected by problems arising from restrictive sample sizes and multiple statistical testing. These problems preclude a comprehensive evaluation of all possible clustering patterns involving a vast range of genotypes. In the current study, we assessed the clustering patterns at the phylogenetic clade level of 27 HPV genotypes that are involved in concurrent multiple infections. In order to protect the results from inflated type I error levels arising from multiple testing, a parallel was drawn with the Hardy- Weinberg equilibrium test (11), and observed patterns of coinfections were compared with those expected assuming independence of HPV clades using a composite goodness-of-fit test. The goals of the current study were to (a ) describe the prevalence of multiple HPV infections; (b ) assess if the distribution of number of coinfecting HPV types follows a random Poisson distribution; (c ) assess if particular HPV clades were more or less likely to be involved in multiple infections; and (d ) assess the clustering patterns of coinfections at the phylogenetic clade level, in a cross-sectional study of HIV () and HIV (+) women. Materials and Methods Study Subjects. The sample (n = 1,379) for this cross- sectional study consisted of a convenience sample of women seeking gynecologic care at the Medical Center of Louisiana in 2439 Cancer Epidemiol Biomarkers Prev 2005;14(10). October 2005 Received 6/21/05; revised 7/29/05; accepted 8/12/05. Grant support: Doris Duke clinical research grant, National Cancer Institute (grant NCI-1 R03 CA86378), and Health Excellency Fund of Louisiana. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Anil K. Chaturvedi, 6120 Executive Boulevard, EPS 8015, Rockville, MD-20852. Phone: 301-451-2495; Fax: 301-402-0817. E-mail: chaturva@mail.nih.gov Copyright D 2005 American Association for Cancer Research. doi:10.1158/1055-9965.EPI-05-0465 Downloaded from http://aacrjournals.org/cebp/article-pdf/14/10/2439/2263413/2439-2445.pdf by guest on 01 July 2022