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Clustering patterns of human papillomavirus genotypes in multiple infections
Virus Research 142 (2009) 154–159
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Clustering patterns of human papillomavirus genotypes in multiple infections Arsenio Spinillo a , Barbara Dal Bello b,∗ , Paola Alberizzi b , Stefania Cesari b , Barbara Gardella a , Marianna Roccio a , Enrico Maria Silini c a
Department of Obstetrics and Gynaecology, IRCCS-Fondazione Policlinico San Matteo and University of Pavia, p.le Golgi 1, 27100 Pavia, Italy Department of Pathology, IRCCS-Fondazione Policlinico San Matteo, Via Forlanini 16, 27100 Pavia, Italy c Department of Pathology, Azienda Ospedaliero-Universitaria di Parma, Viale Gramsci 14, 43100 Parma, Italy b
a r t i c l e
i n f o
Article history: Received 12 December 2008 Received in revised form 5 February 2009 Accepted 6 February 2009 Available online 20 February 2009 Keywords: Human papillomavirus (HPV) Cervical intraepithelial neoplasia (CIN) Squamous intraepithelial lesion (SIL) Coinfection Cervix Cancer
a b s t r a c t Many human papillomavirus (HPV) infections are sustained by multiple viral genotypes whose effect on the risk of cervical intraepithelial neoplasia (CIN) is unknown. The study investigated whether specific HPV types or species may affect the likelihood of multiple infections and have a clustered distribution in a consecutive series of 681 women with a histological diagnosis of CIN. HPV typing was performed by the SPF10 -LIPA assay; associations were evaluated by loglinear analysis of multiple contingency tables after stratification by age and CIN grade. HPV prevalence was 99.4% with a 72.1% rate of coinfection. The risk of coinfection was higher for types 6, 11, 16, 18, 31, 33, 51, 52, 56. Significant interactions were found for species A7–A9–A10, A6–A9 and A7–A10. Coinfection by types 31–35–56, 16–51–52, 16–18 and 51–52 was more frequent than expected. Interactions between viral species and HPV 16–18 were maintained among CIN1, whereas interactions of 16–51–52 and 31–51–56 were significant only in CIN ≥ 2. Interactions between species and types were lost among women younger than 32 years. Significant clustering of HPV types and species occurs among women with CIN. This has implications for the assessment of the oncogenic potential and the prevention of HPV infections. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Coinfection with multiple human papillomavirus (HPV) types is common and can be observed in 20–50% of HPV-positive women (Chaturvedi et al., 2005a; Cuschieri et al., 2004; Rousseau et al., 2003a; Trottier et al., 2006). The virological, epidemiological and clinical significance of multiple HPV infection is still debated; in particular, its possible role in determining the risk and natural history of precancerous cervical lesions remains uncertain (Gargiulo et al., 2007; Liaw et al., 2001; Mendez et al., 2005; Trottier et al., 2006; Wheeler et al., 2006). Epidemiological investigations have contributed to identify the risk factors of multiple HPV infection (Cuschieri et al., 2004; Mendez et al., 2005; Rousseau et al., 2003a) and prospective studies have clarified the dynamics of viral acquisition and clearance (Liaw et al., 2001; Plummer et al., 2007; Rousseau et al., 2001; Thomas et al., 2000). Coinfection may increase HPV persistence, impact on viral oncogenic potential (Ho et al., 1998; Trottier et al., 2008) and affect risk and severity of cervical intraepithelial neoplasia (CIN) (Fife et
∗ Corresponding author at: U.O. Anatomia Patologica, Fond. IRCCS Policlinico San Matteo, Via Forlanini 16, 27100 Pavia, Italy. Tel.: +39 382 502517 fax: +39 382 525866. E-mail address: [email protected] (B. Dal Bello). 0168-1702/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2009.02.004
al., 2001; Trottier et al., 2006). Few studies, however, have investigated the possible interactions of viral genotypes or species in multiple HPV infection (Chaturvedi et al., 2005a; Liaw et al., 2001; Mendez et al., 2005; Trottier et al., 2006; Wheeler et al., 2006). Experimental studies and preliminary vaccination trials indicate that immunity against HPV is predominantly type-specific with limited cross-protection (Roden and Wu, 2006). Therefore, understanding the molecular correlates of multiple HPV infection has become clinically relevant both in the planning of multivalent vaccines and in post-vaccination monitoring. The present study aimed to assess whether specific viral types or species influence the likelihood of multiple HPV infection and show a clustered distribution. It takes advantage of extensive typing data of a large series of women undergoing cytology, colposcopy and targeted biopsy in a tertiary referral center. 2. Materials and methods 2.1. Patients The study base included all patients aged 18–70 years attending the colposcopy clinic of the Department of Obstetrics and Gynaecology in the period 2005–2007. Indications for colposcopy were current diagnosis of squamous intraepithelial lesion (SIL)
A. Spinillo et al. / Virus Research 142 (2009) 154–159
or atypical squamous/glandular cell of unknown significance (ASC/AGC-US) in 1665 (77.9%) women, follow-up of previous SIL in 214 cases (10%) and miscellaneous reasons (vulvar or vaginal condylomas, vulvodynia, referral from sexually transmitted disease clinic, etc.) in the remaining 257 (12%) subjects. Pregnancy, total hysterectomy, lack of a recent (1 month) Pap smear and use of vaginal medication in the previous 2 days were exclusion criteria. The baseline sample was composed of 2136 women with a mean age of 36.5 ± 10.7 years, 84% of whom were of Italian descent. Seventy-eight (3.2%) subjects were excluded from the study because of inadequacy of PAP smear or DNA extraction. The Institutional Review Board of our Hospital approved the study. Informed consent for HPV testing was obtained from all subjects. 2.2. HPV-DNA detection and typing Cervical samples for HPV typing were obtained before colposcopy. After speculum examination, scrapes were taken with a cervix brush, suspended in Thin Prep-PreservCyt Solution (Cytec Corporation, Marlborough, MA), and stored at 4 ◦ C. DNA extraction was performed by lysis and digestion with proteinase K. HPV sequences from the L1 region were PCR (polymerase chain reaction)-amplified using SPF10 primers (Kleter et al., 1999) in a final reaction volume 50 l for 40 cycles. Appropriate positive and negative controls were introduced for each set of reactions. Concurrent amplification of beta-globin sequences was used as control for DNA adequacy. HPV type-specific sequences were detected by the line probe, INNO-LiPA HPV genotyping assay (Innogenetics N.V., Ghent, Belgium) according to manufacturer’s instructions. The current version of the assay allows the simultaneous and separate detection of 15 high-risk (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 70), and 10 low-risk HPV types (6, 11, 34, 40, 42–44, 53, 54, and 74). Hybridization patterns were visually interpreted by two independent readers. 2.3. Colposcopy and targeted cervical biopsy Following cytological sampling, a detailed and standardized colposcopic examination of the lower genital tract was performed. PAP smears preceding colposcopy were available in all subjects; they were performed by referring physicians and not institutionally reviewed. Four hundred and seventy-eight women (25%) were cytologically negative, 376 (19.7%) had ASC/AGC-US, 927 (48.5%), low SIL (L-SIL) and 128 (6.7%) high SIL (H-SIL). Targeted biopsies were obtained as judged by the physician performing colposcopy in 1206 women. Biopsies were processed by standard H&E staining and examined on multiple levels; in selected cases, ancillary immunohistochemical stains for Ki67 and p16INK4a were performed. All histological diagnoses were rendered by the same experienced gynaeco-pathologists (EMS and BDB), in most cases by consensus reading, and were reported according to WHO (Tavassoli and Devilee, 2003). In the analysis of data, the histological diagnosis of punch biopsy was used or, when more severe, the diagnosis after cone biopsy obtained by loop electrosurgical or cold-knife excision (430 cases).
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2.4. Statistical analysis Statistical analysis included the Mann–Whitney U-test and chi-square test to compare continuous and discrete variables, respectively. The chi-square for trend and Spearman Rho were used to test for linear trends across ordered and continuous categories, respectively. Binary logistic regression analysis was applied to test for association between individual HPV species and multiple infections adjusting for potential confounders (Hosmer and Lemeshow, 1989). Explanatory terms of logistic equations included HPV species or type, age, previous history of SIL, and histology. Loglinear analysis of multiple contingency tables was used to test the interactions (associations) between multiple HPV types or species (Agresti and Coull, 1996). Loglinear analysis is specifically designed for analysing data when both the independent and dependent variables are categorical or nominal and permits the measurements of the strength of interactions without conceptually distinguishing between a response (dependent) variable and a set of explanatory (independent) variables. Interactions between HPV species and types were analysed separately. An automated hierarchical stepwise method was used to select the model containing the least number of significant interactions necessary to fit the observed tables of associations. In this model, non-significant interactions between variables are progressively eliminated (backward elimination) from the saturated model based on likelihood ratio chi-square. The model with the least number of interactions was subsequently corrected by the addition of age as continuous covariate. Under the assumption of independence, the expected rate of simultaneous infection resulting from two or more species was computed as the product of the rate of each individual species. Rate ratio of observed and expected frequencies and 95% confidence intervals based on Poisson distribution were calculated using Winpepi Program Vers. 7.4 (Abramson, 2004). Hierarchical and general loglinear analysis was carried out with SPSS 15.0. 3. Results Out of 1206 colposcopically targeted cervical biopsies obtained from the series under study, 27 (2.3%) were not adequate for diagnosis, 432 (35.8%) were negative for dysplasia, 716 (59.4) had CIN and 31 (2.6%) invasive cancer. The study was restricted to subjects with CIN. Overall, we identified 23 HPV genotypes belonging to 7 phylogenetic species. For statistical purposes, we excluded from further analysis 25 subjects infected by HPV-40 (species A8), and 10 subjects infected by HPV-42 (species A1) or HPV-43 (unclassified). The prevalence of HPV infection in the final 681 patients was 99.4% (677/681). The mean age was 34.8 ± 9.4 years in the 287 women with high-grade lesions (CIN ≥ 2) as compared to 33.4 ± 10 in the 390 subjects with low-grade lesions (CIN1) (p = 0.038). Women with CIN1 also had a more frequent history of SIL (40/390 vs. 15/287, p = 0.026) compared to those with CIN ≥ 2. Coinfection with at least two HPV types was identified in 491 (72.1%) subjects. There was a significant linear trend relating the number of HPV types to CIN grade (Table 1). The mean age of women with single and multiple infections was similar (34 ± 10.1 vs. 34 ± 9.6, p = 0.99); however, there was a marginally significant linear trend relating age with increasing number of HPV types (Spearman Rho = 0.075, p = 0.05).
Table 1 Relationship between the number of infecting HPV types and the severity of cervical intraepithelial neoplasia (CIN). No. of HPV types
Low-grade CIN High-grade CIN Chi-square for trend = 85.3, p < .0001.
1 (n = 186); n. (%)
2 (n = 299); n. (%)
3 (n = 125); n. (%)
>3 (n = 67); n. (%)
140 (75.3) 46 (24.7)
192 (64.2) 107 (35.8)
43 (34.4) 82 (65.6)
15 (22.4) 52 (77.6)
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A. Spinillo et al. / Virus Research 142 (2009) 154–159
Table 2 Distribution of HPV species and types in single and multiple infections in 677 subjects with CIN. Species and types
Single (n = 186); n (%)
Multiple (n = 491); n (%)
Odds ratio (95% CI)a ; n (%) 4.83 (2.6–9.05) 4.83 (2.6–9.05)
A5 51
12 (6.5) 12 (6.5)
124 (25.3) 124 (25.3)
A6 53 56 66
12 (6.5) 5 (2.7) 5 (2.7) 2 (1.1)
93(18.9) 37 (7.5) 41 (8.4) 20 (4.1)
3.15 (1.67–5.94) 2.82 (1.07–7.38) 2.87 (1.10–7.47) 4.27 (0.97–18.81)
A7 18 39 45 59 68 70
27 (14.59 10 (5.4) 5 (2.7) 10 (5.4) 1 (0.5) – 1 (0.5)
156 (31.8) 108 (22) 11 (2.2) 30 (6.1) 6 (1.29 11 (2.2) 1 (0.2)
2.34 (1.48–3.72) 4.11 (2.08–8.13) 0.75 (0.25–2.26) 0.97 (0.46–2.08) 2.71 (0.32–23.22) Undefined 0.33 (0.02–5.94)
A9 16 31 33 35 52 58
114 (61.3) 56 (30.1) 37 (19.9) 2 (1.1) 1 (0.5) 17 (9.1) 1 (0.5)
414 (84.3) 262 (53.4) 193 (39.3) 27 (5.5) 12 (2.4) 111 (22.6) 28 (5.7)
2.89 (1.96–4.29) 2.28 (1.58–3.31) 2.50 (1.66–3.77) 4.90 (1.14–21.1) 4.26 (0.54–33.72) 2.48 (1.43–4.3) 10.2 (1.36–76.14)
A10 6 11 44 74
13(7) 8 (4.3) 3 (1.6) 2 (1.1) –
168 (34.2) 118 (24) 59 (12) 14 (2.4) 5(1)
8.0 (4.37–14.6) 7.73 (3.67–16.3) 9.57 (2.94–31.14) 2.76 (0.61–12.52) Undefined
Bold indicates species and italics indicates genotypes. a As assessed by logistic regression equations including multiple infection as outcome variable and HPV species or type, severity of CIN, and history of SIL and age as explanatory variables.
The distribution of HPV types and species in single and multiple infections is reported in Table 2. HPV-16 was the commonest type and was identified in 46.7% (318/681) of the subjects. Logistic models were calculated to assess the risk of coinfection associated with individual HPV species and types. Each species was significantly associated with an increased risk of multiple HPV infection when compared to all others. The amount of risk was heterogeneous across species being lower for A7 and higher for A10 (chi-square for heterogeneity of odds ratios = 12.1, 4 d.f., p = 0.016). As to HPV types, HPV-6, 11, 16, 18, 31, 33, 51, 52, 56, and 58 were individually associated with an increased risk of multiple infection when compared to all other types. Hierarchical loglinear analysis with backward elimination was used to fit models of interaction between HPV species and types. The analysis indicated that a model containing three-way and two-way interactions best accomodated the data of coinfection by either HPV types or species. After correction for age, a significant three-way interaction was observed for the coinfection by species A7–A9–A10 (ratio of observed/expected cases = 0.68, 95% confidence intervals (CI) = 0.45–1, p = 0.045). Two-way interactions between species are detailed in Table 3. The rates of coinfection by species A6–A9 (p = 0.04) and A7–A10 (p = 0.031) were significantly lower than expected. In the analysis of interaction between types, only the six more frequent high-risk types (16, 18, 31, 51, 52, and 56) were considered to avoid excessive stratification of data. After correction for age, significant three-way interactions were found for coinfections by types 31–51–56 (rate ratio = 2.67, 95% CI = 1.15–5.25, p = 0.008) and 16–51–52 (rate ratio = 1.75, 95% CI = 1.08–2.67, p = 0.04). Twoway interactions between types are detailed in Table 3. The rates of coinfection by types 16–18 (p = 0.007) and 51–52 (p = 0.04) were higher than expected, whereas these were lower for types 31–52 (p = 0.03).
Table 3 Two-ways interactions between species and types of HPV in multiple infections calculated in 677 subjects with CINa . Coinfection
Observed/expected
Ratio (95% CI)
A5–A6 A5–A7 A5–A9 A5–A10 A6–A7 A6–A9 A6–A10 A7–A9 A7–A10 A9–A10
24/21 29/37 102/106 30/36 19/28 65/82 26/28 127/142 35/49 124/141
1.14 (0.73–1.7) 0.78 (0.52–1.12) 0.96 (0.78–1.68) 0.83 (0.56–1.89) 0.68 (0.41–1.06) 0.79 (0.61–1.0) 0.93 (0.61–1.36) 0.89 (0.75–1.07) 0.71 (0.50–0.99) 0.88 (0.73–1.05)
16–18 16–31 16–51 16–52 16–56 18–31 18–51 18–52 18–56 31–51 31–52 31–56 51–52 51–56 52–56
75/55 93/108 56/64 59/60 17/22 42/40 19/24 19/22 9/8 45/46 30/43 14/16 37/26 12/9 12/9
1.36 (1.07–1.71) 0.86 (0.69–1.05) 0.87 (0.66–1.74) 0.98 (0.75–1.27) 0.77 (0.45–1.24) 1.05 (0.77–1.42) 1.26 (0.81–1.88) 0.86 (0.52–1.35) 1.12 (0.51–2.13) 0.98 (0.71–1.31) 0.7 (0.47–0.99) 0.87 (0.48–1.47) 1.42 (1.0–1.96) 1.33 (0.69–2.32) 1.33 (0.69–2.32)
a Observed/expected cases of coinfection and rate ratio were calculated by hierarchical loglinear analysis with backward elimination.
Data of coinfection were then stratified by severity of CIN. Both three- and two-way interactions were calculated for HPV species and types; two-way interactions are detailed in Table 4. Among CIN1, the three-way interaction of species A7–A9–A10 (rate ratio = 0.5, 95% CI = 0.21–0.98, p = 0.03) was confirmed lower than expected. Coinfections by species A6–A9 (p = 0.026) and A7–A10 (p = 0.022) were also lower than expected in CIN1. Conversely, no Table 4 Two-ways interactions between species and types of HPV in multiple infections according to the grade of CINa . Coinfection
High grade CIN (n = 287)
Low grade CIN (n = 390)
O/E
Ratio (95% CI)
O/E
Ratio (95% CI)
A5–A6 A5–A7 A5–A9 A5–A10 A6–A7 A6–A9 A6–A10 A7–A9 A7–A10 A9–A10
16/12 20/23 54/56 16/16 13/19 42/47 14/13 85/90 22/26 61/62
1.33 (0.76–2.16) 0.87 (0.53–1.34) 0.96 (0.72–1.26) 1.0 (0.57–1.62) 0.68 (0.36–1.17) 0.89 (0.64–1.21) 1.07 (0.59–1.81) 0.94 (0.75–5.95) 0.84 (0.53–1.28) 0.98 (0.75–1.26)
8/9 9/14 48/50 14/20 6/9 23/35 12/15 42/32 13/23 63/79
0.89 (0.38–1.75) 0.64 (0.29–1.22) 0.96 (0.71–1.28) 0.7 (0.38–1.17) 0.66 (0.24–1.45) 0.66 (0.42–0.98) 0.8 (0.41–1.4) 1.31 (0.45–1.77) 0.56 (0.3–0.97) 0.81 (0.61–1.02)
16–18 16–31 16–51 16–52 16–56 18–31 18–51 18–52 18–56 31–51 31–52 31–56 51–52 51–56 52–56
55/44 56/65 35/38 50/44 14/16 27/29 16/17 17/19 8/7 25/25 20/29 10/10 22/17 10/6 10/7
1.25 (0.94–1.62) 0.86 (0.65–1.28) 0.92 (0.64–1.28) 1.14 (0.84–1.50) 0.87 (0.48–1.47) 0.93 (0.61–1.35) 0.94 (0.54–1.53) 0.89 (0.52–1.43) 1.14 (0.49–2.25) 1.0 (0.65–1.47) 0.69 (0.42–1.06) 1.0 (0.48–1.84) 1.29 (0.81–1.96) 1.67 (0.80–3.06) 1.43 (0.68–2.62)
20/11 43/43 21/26 9/16 3/6 15/11 3/7 2/3 1/1 20/21 10/14 4/6 15/9 2/3 2/2
1.82 (1.11–2.81) 1.0 (0.72–1.75) 0.8 (0.5–1.23) 0.56 (0.26–1.07) 0.5 (0.1–1.48) 1.36 (0.76–2.25) 0.43 (0.09–1.25) 0.67 (0.08–2.4) 1 (0.02–5.57) 0.95 (0.58–1.47) 0.71 (0.34–1.30) 0.67 (0.18–1.71) 1.67 (0.93–2.75) 1.5 (0.31–4.38) 1.0 (0.12–3.61)
a Observed/expected cases of coinfection and rate ratio were calculated by hierarchical loglinear analysis with backward elimination.
A. Spinillo et al. / Virus Research 142 (2009) 154–159 Table 5 Two-ways interactions between species and types of HPV in multiple infections according to the age at diagnosisa . Age ≤ 32 years (n = 338)
Age > 32 years (n = 339)
O/E
Ratio (95% CI)
O/E
Ratio (95% CI)
A5–A6 A5–A7 A5–A9 A5–A10 A6–A7 A6–A9 A6–A10 A7–A9 A7–A10 A9–A10
9/12 13/18 52/55 13/18 11/15 30/42 13/14 59/68 15/22 53/63
0.75 (0.34–1.42) 0.72 (0.38–1.24) 0.94 (0.71–1.24) 0.72 (0.38–1.24) 0.73 (0.37–1.31) 0.71 (0.48–1.0) 0.93 (0.49–1.59) 0.87 (0.66–1.12) 0.68 (0.38–1.12) 0.84 (0.63–1.10)
15/12 16/19 50/51 17/18 8/13 35/40 13/14 68/74 20/27 71/78
1.25 (0.70–2.06) 0.84 (0.48–1.37) 0.98 (0.73–1.99) 0.94 (0.55–1.51) 0.61 (0.26–1.21) 0.87 (0.61–1.22) 0.93 (0.49–1.59) 0.92 (0.71–1.16) 0.74 (0.45–1.14) 0.91 (0.71–1.15)
16–18 16–31 16–51 16–52 16–56 18–31 18–51 18–52 18–56 31–51 31–52 31–56 51–52 51–56 52–56
32/26 41/49 25/33 23/26 8/11 24/18 8/12 5/10 5/4 21/24 11/19 7/8 15/13 4/5 3/4
1.23 (0.84–1.74) 0.84 (0.60–1.14) 0.76 (0.49–1.11) 0.88 (0.56–1.33) 0.73 (0.31–1.43) 1.33 (0.85–1.98) 0.67 (0.29–1.31) 0.5 (0.16–1.17) 1.25 (0.41–2.92) 0.87 (0.54–1.33) 0.58 (0.29–1.04) 0.87 (0.35–1.80) 1.15 (0.65–1.90) 0.8 (0.22–2.04) 0.75 (0.15–2.2)
43/29 52/59 31/31 36/34 9/11 18/22 11/12 14/12 4/4 24/22 19/24 7/8 22/13 8/4 9/5
1.48 (1.07–2.0) 0.88 (0.66–1.16) 1.0 (0.68–1.42) 1.06 (0.74–1.46) 0.82 (0.37–1.85) 0.82 (0.48–1.29) 0.92 (0.46–1.64) 1.16 (0.64–1.96) 1 (0.27–2.56) 1.1 (0.70–1.62) 0.79 (0.47–1.24) 0.88 (0.21–1.86) 1.69 (1.05–2.56) 2 (0.86–3.94) 1.8 (0.82–3.41)
Coinfection
a Observed/expected cases of coinfection and rate ratio were calculated by hierarchical loglinear analysis with backward elimination.
significant clustering between species was found for CIN ≥ 2. As to HPV types, three-way interactions between types 16–51–52 (rate ratio = 1.8, 95% CI = 1.07–2.84, p = 0.02) and 31–51–56 (rate ratio = 3.5, 95% CI = 1.41–7.21, p = 0.006) were statistically significant in CIN ≥ 2. Among two-way interactions, only coinfection by HPV 16–18 was more frequent than expected (p = 0.012) among women with CIN1. Data were further stratified by the age of the patients considering a cut-off of 32 years, the mean age of the series. Both threeand two-way interactions were calculated, the latter are detailed in Table 5. As to HPV species, no significant three-way clustering was observed in the two groups. Younger women had lower than expected rates of coinfection by species A6–A9 (p = 0.05). Among older women, three-way interactions between types 16–51–52 (rate ratio = 3.25, 95% CI = 1.73–5.56, p = 0.0004) and 31–51–56 (rate ratio = 5, 95% CI = 1.62–11.7, p = 0.004) significantly deviated from chance. Coinfection by types 16–18 (p = 0.014) and 51–52 (p = 0.02) also occurred more frequently in women older than 32 years. 4. Discussion Viral interactions in multiple HPV infection may have relevant implications in the assessment of oncogenic risk and, consequently, in the clinical management of women with CIN. Establishing whether there is cross-protection, facilitation or competition between different HPVs may also help to predict the impact of vaccines and in post-vaccination monitoring (Franco and Cuzick, 2008). Issues concerning the equilibrium and clustering patterns of HPV types or species in concurrent and sequential infections have been limitedly addressed in previous studies and with controversial results (Chaturvedi et al., 2005a; Liaw et al., 2001; Mendez et al., 2005; Trottier et al., 2006; Wheeler et al., 2006). Studies of sequential HPV acquisition have shown that coinfection is common and most women experience multiple infections over time (Chaturvedi et al., 2005a; Liaw et al., 2001; Mendez et al., 2005; Trottier et al., 2006; Wheeler et al., 2006). Coinfection occurs
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more likely than expected by chance (Liaw et al., 2001; Mendez et al., 2005; Thomas et al., 2000) and is established more frequently in previously infected women (Liaw et al., 2001; Rousseau et al., 2001). In agreement with these results, the present study showed that among patients referred to colposcopy the rate of multiple HPV infection was high and strongly related to the severity of CIN (Fife et al., 2001; Liaw et al., 2001; Trottier et al., 2006), whereas, at variance with other authors, no effect of age was observed (Cuschieri et al., 2004). HPV coinfection was found in 72.1% of women, a higher prevalence than reported in smaller studies from Italy (Capra et al., 2008; Gargiulo et al., 2007; Tornesello et al., 2006). Multiple infections have been reported in a proportion variable from 10% to 80% of HPV-positive women (Chaturvedi et al., 2005a; Cuschieri et al., 2004; Fife et al., 2001; Herrero et al., 2000; Mendez et al., 2005; Rousseau et al., 2003b; Trottier et al., 2006; Wheeler et al., 2006). Considering the selection criteria of the present series and the sensitivity of the typing assay, the observed rate of coinfection seems justifiable. In agreement with other authors, the A9 species and the HPV-16 type were most frequently represented. All HPV species had a risk of multiple infection higher than expected, although the amount of risk was lower for species A7 and A10. As to individual types, the risk of coinfection was higher for types 6, 11, 16, 18, 31, 33, 51, 52, 56. In a comparable series of prevalent SIL cases (Chaturvedi et al., 2005b), the species A9 was associated with a 32% reduction of the risk of coinfection. Differences in study design and statistical power may account for this discrepancy. In fact, that study used HPV types as units of observation, predominantly enrolled young AfroAmericans, had one-third the number of coinfections—70% of which occurred in HIV+ subjects −, and exclusively considered cytological outcomes. Most previous studies on multiple HPV infection have reported that individual types or species associate at random based on their relative frequency (Chaturvedi et al., 2005b; Trottier et al., 2006; Wheeler et al., 2006). Conversely, the present results showed significant clustering of HPV types and species some of which were maintained after stratification by age and CIN severity. Overall, the combinatorial patterns between species did not deviate from chance except for the interactions of A7–A9–A10, A6–A9 and A7–A10 that were more frequent than expected. Among types, the rates of coinfection by HPV 31–35–56, 16–51–52, 16–18 and 51–52 were higher than expected whereas they were lower for HPV 31–52. After stratification by severity of cervical lesions, the interactions between species and between types 16–18 were maintained in CIN1, whereas types 16–51–52 and 31–51–56 clustered significantly in CIN ≥ 2. As to the effect of age, it abolished all interactions between species. Associations of types 31–35–56, 16–51–52, 16–18 and 51–52 were significant only among women aged more than 32 years. Clustering of HPV types was observed by Mendez et al. (2005) who calculated that HPV-16/18 infected subjects had a 5–6 times higher odds ratio of acquiring a subsequent HPV-58 infection. Conversely, in their smaller series of persistent infections, Chaturvedi et al. (Chaturvedi et al., 2005b) could not detect any difference in 21 combinatorial patterns across HPV, age and PAP smear strata. Given the complex network of variables that influence the dynamics of HPV infection, assessing viral interactions is methodologically challenging. Previous studies have considered different designs and variable clinical settings and outcomes, each approach having its inherent limitations (Chaturvedi et al., 2005b). The main interest of exploring viral interactions is to make inferences about potential effects on viral life cycle and disease progression. Therefore, the study was restricted to a uniform series of women with a well-defined outcome (CIN) of known clinical relevance. This implies that the results cannot be applied to a general population of HPV-infected women. Other major methodological points of the study are as follows. First, a large number of histologi-
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cally confirmed lesions were considered. They were derived from a consecutive series of women with cytological abnormalities undergoing systematic colposcopy, accrued in a single institution over a short period, ethnically homogenous, and examined by punch biopsy in 56.5% of cases and by cone biopsy in 20%. Second, the SPF10 -Lipa typing assay (Kleter et al., 1999) was used. This is a highly sensitive test with a large coverage of HPV types that has been extensively validated in clinical and epidemiological studies (Castle et al., 2008; Perrons et al., 2005; Safaeian et al., 2007). Third, infections were classified according to both HPV type and species, an approach that accounts for the higher number of phylogenetically related types in larger species (e.g. A7, A9, and A10). Fourth, interactions were tested by multiway contingency tables using loglinear modelling, a multivariate statistical technique that avoids assumptions about the distribution of events and that has been used to test associations between independent variables in several medical settings (Agresti and Coull, 1996). Fifth, viral interactions were verified across strata of age and CIN severity. Finally, logistic regression was used to control for confounding by age, history of CIN and lesion severity. Clustering of HPV types/species does not necessarily imply a direct biological interaction, alternative explanations must be considered. First, it should be acknowledged that broad-spectrum typing assays do not have the same specificity and sensitivity for each viral type. HPV types present at low relative concentrations can be under-represented by PCR because of primer competition (van Doorn et al., 2002). This is unlikely as the study base spans a wide spectrum of disease expressions and, presumably, of viral titres (Moberg et al., 2004). Furthermore, the viral types showing the more significant interactions are not differentially detected by SPF10 -Lipa compared to other typing assays (Castle et al., 2008; Klug et al., 2008; van Hamont et al., 2006). Second, although the sample size is large, a type I error cannot be fully excluded due to the frequency of individual type/species or to residual geographical, demographic or behavioural covariables. Third, clustering might derive from differential or shared transmission of HPV types/clades. However, risk factors of viral acquisition do not differ between single and multiple infections and new infections are predominantly established at random (Plummer et al., 2007; Rousseau et al., 2003a). Fourth, the host’s immunity might affect rates of coinfection and/or clearance of viral types (Chaturvedi and Goedert, 2006). Available evidence does not suggest that immune status may affect associations between types (Chaturvedi et al., 2005b) and immunological studies and vaccination trials do not support a main role for cross-protection between HPV types (Stanley, 2006). HPV clustering might ultimately derive from specific biological features of individual types or species (Hiller et al., 2006; Vinokurova et al., 2008). Differences in tissue preference, replicative ability or carcinogenic potential (Castle et al., 2006, 2005) might favour the persistence of specific HPVs during disease progression and result in a biased distribution of types or species. Coexistence of two oncogenic HPV types within the same cell has been observed ‘in vivo’ by ‘in situ’ hybridization (Birner et al., 2001; Vermeulen et al., 2007) and it has been experimentally obtained by electroporation of viral genomes into primary keratinocytes (McLaughlin-Drubin and Meyers, 2004). Viral interference in the maintenance of genome copy number was observed by both approaches. Antagonism between low- and high-risk HPV types has also been suggested by prospective studies of viral seropositivity (Luostarinen et al., 1999). 4.1. Conclusions The study has some methodological limitations and the results cannot be extrapolated to the viral dynamics in a population setting. Nonetheless, this is the major effort so far exerted to explore
the issue of HPV interactions in women with abnormal cytology and histologically confirmed CIN. The demonstration of significant HPV clustering underscores the need of further studies on the mechanisms of viral interference and on type distribution in the general population. Monitoring is also warranted to assess the impact of multiple HPV infection on cervical disease progression and vaccine efficacy. Acknowledgements This research was partially supported by grants from to Italian Health Ministry to IRCCS Fondazione Policlinico San Matteo, Pavia. References Abramson, J.H., 2004. WINPEPI (PEPI-for-Windows): computer programs for epidemiologists. Epidemiol. Perspect. Innov. 1 (1), 6. Agresti, A., Coull, B.A., 1996. Order-restricted tests for stratified comparisons of binomial proportions. Biometrics 52 (3), 1103–1111. Birner, P., Bachtiary, B., Dreier, B., Schindl, M., Joura, E.A., Breitenecker, G., Oberhuber, G., 2001. 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Virus Research journal homepage: www.elsevier.com/locate/virusres
Clustering patterns of human papillomavirus genotypes in multiple infections Arsenio Spinillo a , Barbara Dal Bello b,∗ , Paola Alberizzi b , Stefania Cesari b , Barbara Gardella a , Marianna Roccio a , Enrico Maria Silini c a
Department of Obstetrics and Gynaecology, IRCCS-Fondazione Policlinico San Matteo and University of Pavia, p.le Golgi 1, 27100 Pavia, Italy Department of Pathology, IRCCS-Fondazione Policlinico San Matteo, Via Forlanini 16, 27100 Pavia, Italy c Department of Pathology, Azienda Ospedaliero-Universitaria di Parma, Viale Gramsci 14, 43100 Parma, Italy b
a r t i c l e
i n f o
Article history: Received 12 December 2008 Received in revised form 5 February 2009 Accepted 6 February 2009 Available online 20 February 2009 Keywords: Human papillomavirus (HPV) Cervical intraepithelial neoplasia (CIN) Squamous intraepithelial lesion (SIL) Coinfection Cervix Cancer
a b s t r a c t Many human papillomavirus (HPV) infections are sustained by multiple viral genotypes whose effect on the risk of cervical intraepithelial neoplasia (CIN) is unknown. The study investigated whether specific HPV types or species may affect the likelihood of multiple infections and have a clustered distribution in a consecutive series of 681 women with a histological diagnosis of CIN. HPV typing was performed by the SPF10 -LIPA assay; associations were evaluated by loglinear analysis of multiple contingency tables after stratification by age and CIN grade. HPV prevalence was 99.4% with a 72.1% rate of coinfection. The risk of coinfection was higher for types 6, 11, 16, 18, 31, 33, 51, 52, 56. Significant interactions were found for species A7–A9–A10, A6–A9 and A7–A10. Coinfection by types 31–35–56, 16–51–52, 16–18 and 51–52 was more frequent than expected. Interactions between viral species and HPV 16–18 were maintained among CIN1, whereas interactions of 16–51–52 and 31–51–56 were significant only in CIN ≥ 2. Interactions between species and types were lost among women younger than 32 years. Significant clustering of HPV types and species occurs among women with CIN. This has implications for the assessment of the oncogenic potential and the prevention of HPV infections. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Coinfection with multiple human papillomavirus (HPV) types is common and can be observed in 20–50% of HPV-positive women (Chaturvedi et al., 2005a; Cuschieri et al., 2004; Rousseau et al., 2003a; Trottier et al., 2006). The virological, epidemiological and clinical significance of multiple HPV infection is still debated; in particular, its possible role in determining the risk and natural history of precancerous cervical lesions remains uncertain (Gargiulo et al., 2007; Liaw et al., 2001; Mendez et al., 2005; Trottier et al., 2006; Wheeler et al., 2006). Epidemiological investigations have contributed to identify the risk factors of multiple HPV infection (Cuschieri et al., 2004; Mendez et al., 2005; Rousseau et al., 2003a) and prospective studies have clarified the dynamics of viral acquisition and clearance (Liaw et al., 2001; Plummer et al., 2007; Rousseau et al., 2001; Thomas et al., 2000). Coinfection may increase HPV persistence, impact on viral oncogenic potential (Ho et al., 1998; Trottier et al., 2008) and affect risk and severity of cervical intraepithelial neoplasia (CIN) (Fife et
∗ Corresponding author at: U.O. Anatomia Patologica, Fond. IRCCS Policlinico San Matteo, Via Forlanini 16, 27100 Pavia, Italy. Tel.: +39 382 502517 fax: +39 382 525866. E-mail address: [email protected] (B. Dal Bello). 0168-1702/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2009.02.004
al., 2001; Trottier et al., 2006). Few studies, however, have investigated the possible interactions of viral genotypes or species in multiple HPV infection (Chaturvedi et al., 2005a; Liaw et al., 2001; Mendez et al., 2005; Trottier et al., 2006; Wheeler et al., 2006). Experimental studies and preliminary vaccination trials indicate that immunity against HPV is predominantly type-specific with limited cross-protection (Roden and Wu, 2006). Therefore, understanding the molecular correlates of multiple HPV infection has become clinically relevant both in the planning of multivalent vaccines and in post-vaccination monitoring. The present study aimed to assess whether specific viral types or species influence the likelihood of multiple HPV infection and show a clustered distribution. It takes advantage of extensive typing data of a large series of women undergoing cytology, colposcopy and targeted biopsy in a tertiary referral center. 2. Materials and methods 2.1. Patients The study base included all patients aged 18–70 years attending the colposcopy clinic of the Department of Obstetrics and Gynaecology in the period 2005–2007. Indications for colposcopy were current diagnosis of squamous intraepithelial lesion (SIL)
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or atypical squamous/glandular cell of unknown significance (ASC/AGC-US) in 1665 (77.9%) women, follow-up of previous SIL in 214 cases (10%) and miscellaneous reasons (vulvar or vaginal condylomas, vulvodynia, referral from sexually transmitted disease clinic, etc.) in the remaining 257 (12%) subjects. Pregnancy, total hysterectomy, lack of a recent (1 month) Pap smear and use of vaginal medication in the previous 2 days were exclusion criteria. The baseline sample was composed of 2136 women with a mean age of 36.5 ± 10.7 years, 84% of whom were of Italian descent. Seventy-eight (3.2%) subjects were excluded from the study because of inadequacy of PAP smear or DNA extraction. The Institutional Review Board of our Hospital approved the study. Informed consent for HPV testing was obtained from all subjects. 2.2. HPV-DNA detection and typing Cervical samples for HPV typing were obtained before colposcopy. After speculum examination, scrapes were taken with a cervix brush, suspended in Thin Prep-PreservCyt Solution (Cytec Corporation, Marlborough, MA), and stored at 4 ◦ C. DNA extraction was performed by lysis and digestion with proteinase K. HPV sequences from the L1 region were PCR (polymerase chain reaction)-amplified using SPF10 primers (Kleter et al., 1999) in a final reaction volume 50 l for 40 cycles. Appropriate positive and negative controls were introduced for each set of reactions. Concurrent amplification of beta-globin sequences was used as control for DNA adequacy. HPV type-specific sequences were detected by the line probe, INNO-LiPA HPV genotyping assay (Innogenetics N.V., Ghent, Belgium) according to manufacturer’s instructions. The current version of the assay allows the simultaneous and separate detection of 15 high-risk (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 70), and 10 low-risk HPV types (6, 11, 34, 40, 42–44, 53, 54, and 74). Hybridization patterns were visually interpreted by two independent readers. 2.3. Colposcopy and targeted cervical biopsy Following cytological sampling, a detailed and standardized colposcopic examination of the lower genital tract was performed. PAP smears preceding colposcopy were available in all subjects; they were performed by referring physicians and not institutionally reviewed. Four hundred and seventy-eight women (25%) were cytologically negative, 376 (19.7%) had ASC/AGC-US, 927 (48.5%), low SIL (L-SIL) and 128 (6.7%) high SIL (H-SIL). Targeted biopsies were obtained as judged by the physician performing colposcopy in 1206 women. Biopsies were processed by standard H&E staining and examined on multiple levels; in selected cases, ancillary immunohistochemical stains for Ki67 and p16INK4a were performed. All histological diagnoses were rendered by the same experienced gynaeco-pathologists (EMS and BDB), in most cases by consensus reading, and were reported according to WHO (Tavassoli and Devilee, 2003). In the analysis of data, the histological diagnosis of punch biopsy was used or, when more severe, the diagnosis after cone biopsy obtained by loop electrosurgical or cold-knife excision (430 cases).
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2.4. Statistical analysis Statistical analysis included the Mann–Whitney U-test and chi-square test to compare continuous and discrete variables, respectively. The chi-square for trend and Spearman Rho were used to test for linear trends across ordered and continuous categories, respectively. Binary logistic regression analysis was applied to test for association between individual HPV species and multiple infections adjusting for potential confounders (Hosmer and Lemeshow, 1989). Explanatory terms of logistic equations included HPV species or type, age, previous history of SIL, and histology. Loglinear analysis of multiple contingency tables was used to test the interactions (associations) between multiple HPV types or species (Agresti and Coull, 1996). Loglinear analysis is specifically designed for analysing data when both the independent and dependent variables are categorical or nominal and permits the measurements of the strength of interactions without conceptually distinguishing between a response (dependent) variable and a set of explanatory (independent) variables. Interactions between HPV species and types were analysed separately. An automated hierarchical stepwise method was used to select the model containing the least number of significant interactions necessary to fit the observed tables of associations. In this model, non-significant interactions between variables are progressively eliminated (backward elimination) from the saturated model based on likelihood ratio chi-square. The model with the least number of interactions was subsequently corrected by the addition of age as continuous covariate. Under the assumption of independence, the expected rate of simultaneous infection resulting from two or more species was computed as the product of the rate of each individual species. Rate ratio of observed and expected frequencies and 95% confidence intervals based on Poisson distribution were calculated using Winpepi Program Vers. 7.4 (Abramson, 2004). Hierarchical and general loglinear analysis was carried out with SPSS 15.0. 3. Results Out of 1206 colposcopically targeted cervical biopsies obtained from the series under study, 27 (2.3%) were not adequate for diagnosis, 432 (35.8%) were negative for dysplasia, 716 (59.4) had CIN and 31 (2.6%) invasive cancer. The study was restricted to subjects with CIN. Overall, we identified 23 HPV genotypes belonging to 7 phylogenetic species. For statistical purposes, we excluded from further analysis 25 subjects infected by HPV-40 (species A8), and 10 subjects infected by HPV-42 (species A1) or HPV-43 (unclassified). The prevalence of HPV infection in the final 681 patients was 99.4% (677/681). The mean age was 34.8 ± 9.4 years in the 287 women with high-grade lesions (CIN ≥ 2) as compared to 33.4 ± 10 in the 390 subjects with low-grade lesions (CIN1) (p = 0.038). Women with CIN1 also had a more frequent history of SIL (40/390 vs. 15/287, p = 0.026) compared to those with CIN ≥ 2. Coinfection with at least two HPV types was identified in 491 (72.1%) subjects. There was a significant linear trend relating the number of HPV types to CIN grade (Table 1). The mean age of women with single and multiple infections was similar (34 ± 10.1 vs. 34 ± 9.6, p = 0.99); however, there was a marginally significant linear trend relating age with increasing number of HPV types (Spearman Rho = 0.075, p = 0.05).
Table 1 Relationship between the number of infecting HPV types and the severity of cervical intraepithelial neoplasia (CIN). No. of HPV types
Low-grade CIN High-grade CIN Chi-square for trend = 85.3, p < .0001.
1 (n = 186); n. (%)
2 (n = 299); n. (%)
3 (n = 125); n. (%)
>3 (n = 67); n. (%)
140 (75.3) 46 (24.7)
192 (64.2) 107 (35.8)
43 (34.4) 82 (65.6)
15 (22.4) 52 (77.6)
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Table 2 Distribution of HPV species and types in single and multiple infections in 677 subjects with CIN. Species and types
Single (n = 186); n (%)
Multiple (n = 491); n (%)
Odds ratio (95% CI)a ; n (%) 4.83 (2.6–9.05) 4.83 (2.6–9.05)
A5 51
12 (6.5) 12 (6.5)
124 (25.3) 124 (25.3)
A6 53 56 66
12 (6.5) 5 (2.7) 5 (2.7) 2 (1.1)
93(18.9) 37 (7.5) 41 (8.4) 20 (4.1)
3.15 (1.67–5.94) 2.82 (1.07–7.38) 2.87 (1.10–7.47) 4.27 (0.97–18.81)
A7 18 39 45 59 68 70
27 (14.59 10 (5.4) 5 (2.7) 10 (5.4) 1 (0.5) – 1 (0.5)
156 (31.8) 108 (22) 11 (2.2) 30 (6.1) 6 (1.29 11 (2.2) 1 (0.2)
2.34 (1.48–3.72) 4.11 (2.08–8.13) 0.75 (0.25–2.26) 0.97 (0.46–2.08) 2.71 (0.32–23.22) Undefined 0.33 (0.02–5.94)
A9 16 31 33 35 52 58
114 (61.3) 56 (30.1) 37 (19.9) 2 (1.1) 1 (0.5) 17 (9.1) 1 (0.5)
414 (84.3) 262 (53.4) 193 (39.3) 27 (5.5) 12 (2.4) 111 (22.6) 28 (5.7)
2.89 (1.96–4.29) 2.28 (1.58–3.31) 2.50 (1.66–3.77) 4.90 (1.14–21.1) 4.26 (0.54–33.72) 2.48 (1.43–4.3) 10.2 (1.36–76.14)
A10 6 11 44 74
13(7) 8 (4.3) 3 (1.6) 2 (1.1) –
168 (34.2) 118 (24) 59 (12) 14 (2.4) 5(1)
8.0 (4.37–14.6) 7.73 (3.67–16.3) 9.57 (2.94–31.14) 2.76 (0.61–12.52) Undefined
Bold indicates species and italics indicates genotypes. a As assessed by logistic regression equations including multiple infection as outcome variable and HPV species or type, severity of CIN, and history of SIL and age as explanatory variables.
The distribution of HPV types and species in single and multiple infections is reported in Table 2. HPV-16 was the commonest type and was identified in 46.7% (318/681) of the subjects. Logistic models were calculated to assess the risk of coinfection associated with individual HPV species and types. Each species was significantly associated with an increased risk of multiple HPV infection when compared to all others. The amount of risk was heterogeneous across species being lower for A7 and higher for A10 (chi-square for heterogeneity of odds ratios = 12.1, 4 d.f., p = 0.016). As to HPV types, HPV-6, 11, 16, 18, 31, 33, 51, 52, 56, and 58 were individually associated with an increased risk of multiple infection when compared to all other types. Hierarchical loglinear analysis with backward elimination was used to fit models of interaction between HPV species and types. The analysis indicated that a model containing three-way and two-way interactions best accomodated the data of coinfection by either HPV types or species. After correction for age, a significant three-way interaction was observed for the coinfection by species A7–A9–A10 (ratio of observed/expected cases = 0.68, 95% confidence intervals (CI) = 0.45–1, p = 0.045). Two-way interactions between species are detailed in Table 3. The rates of coinfection by species A6–A9 (p = 0.04) and A7–A10 (p = 0.031) were significantly lower than expected. In the analysis of interaction between types, only the six more frequent high-risk types (16, 18, 31, 51, 52, and 56) were considered to avoid excessive stratification of data. After correction for age, significant three-way interactions were found for coinfections by types 31–51–56 (rate ratio = 2.67, 95% CI = 1.15–5.25, p = 0.008) and 16–51–52 (rate ratio = 1.75, 95% CI = 1.08–2.67, p = 0.04). Twoway interactions between types are detailed in Table 3. The rates of coinfection by types 16–18 (p = 0.007) and 51–52 (p = 0.04) were higher than expected, whereas these were lower for types 31–52 (p = 0.03).
Table 3 Two-ways interactions between species and types of HPV in multiple infections calculated in 677 subjects with CINa . Coinfection
Observed/expected
Ratio (95% CI)
A5–A6 A5–A7 A5–A9 A5–A10 A6–A7 A6–A9 A6–A10 A7–A9 A7–A10 A9–A10
24/21 29/37 102/106 30/36 19/28 65/82 26/28 127/142 35/49 124/141
1.14 (0.73–1.7) 0.78 (0.52–1.12) 0.96 (0.78–1.68) 0.83 (0.56–1.89) 0.68 (0.41–1.06) 0.79 (0.61–1.0) 0.93 (0.61–1.36) 0.89 (0.75–1.07) 0.71 (0.50–0.99) 0.88 (0.73–1.05)
16–18 16–31 16–51 16–52 16–56 18–31 18–51 18–52 18–56 31–51 31–52 31–56 51–52 51–56 52–56
75/55 93/108 56/64 59/60 17/22 42/40 19/24 19/22 9/8 45/46 30/43 14/16 37/26 12/9 12/9
1.36 (1.07–1.71) 0.86 (0.69–1.05) 0.87 (0.66–1.74) 0.98 (0.75–1.27) 0.77 (0.45–1.24) 1.05 (0.77–1.42) 1.26 (0.81–1.88) 0.86 (0.52–1.35) 1.12 (0.51–2.13) 0.98 (0.71–1.31) 0.7 (0.47–0.99) 0.87 (0.48–1.47) 1.42 (1.0–1.96) 1.33 (0.69–2.32) 1.33 (0.69–2.32)
a Observed/expected cases of coinfection and rate ratio were calculated by hierarchical loglinear analysis with backward elimination.
Data of coinfection were then stratified by severity of CIN. Both three- and two-way interactions were calculated for HPV species and types; two-way interactions are detailed in Table 4. Among CIN1, the three-way interaction of species A7–A9–A10 (rate ratio = 0.5, 95% CI = 0.21–0.98, p = 0.03) was confirmed lower than expected. Coinfections by species A6–A9 (p = 0.026) and A7–A10 (p = 0.022) were also lower than expected in CIN1. Conversely, no Table 4 Two-ways interactions between species and types of HPV in multiple infections according to the grade of CINa . Coinfection
High grade CIN (n = 287)
Low grade CIN (n = 390)
O/E
Ratio (95% CI)
O/E
Ratio (95% CI)
A5–A6 A5–A7 A5–A9 A5–A10 A6–A7 A6–A9 A6–A10 A7–A9 A7–A10 A9–A10
16/12 20/23 54/56 16/16 13/19 42/47 14/13 85/90 22/26 61/62
1.33 (0.76–2.16) 0.87 (0.53–1.34) 0.96 (0.72–1.26) 1.0 (0.57–1.62) 0.68 (0.36–1.17) 0.89 (0.64–1.21) 1.07 (0.59–1.81) 0.94 (0.75–5.95) 0.84 (0.53–1.28) 0.98 (0.75–1.26)
8/9 9/14 48/50 14/20 6/9 23/35 12/15 42/32 13/23 63/79
0.89 (0.38–1.75) 0.64 (0.29–1.22) 0.96 (0.71–1.28) 0.7 (0.38–1.17) 0.66 (0.24–1.45) 0.66 (0.42–0.98) 0.8 (0.41–1.4) 1.31 (0.45–1.77) 0.56 (0.3–0.97) 0.81 (0.61–1.02)
16–18 16–31 16–51 16–52 16–56 18–31 18–51 18–52 18–56 31–51 31–52 31–56 51–52 51–56 52–56
55/44 56/65 35/38 50/44 14/16 27/29 16/17 17/19 8/7 25/25 20/29 10/10 22/17 10/6 10/7
1.25 (0.94–1.62) 0.86 (0.65–1.28) 0.92 (0.64–1.28) 1.14 (0.84–1.50) 0.87 (0.48–1.47) 0.93 (0.61–1.35) 0.94 (0.54–1.53) 0.89 (0.52–1.43) 1.14 (0.49–2.25) 1.0 (0.65–1.47) 0.69 (0.42–1.06) 1.0 (0.48–1.84) 1.29 (0.81–1.96) 1.67 (0.80–3.06) 1.43 (0.68–2.62)
20/11 43/43 21/26 9/16 3/6 15/11 3/7 2/3 1/1 20/21 10/14 4/6 15/9 2/3 2/2
1.82 (1.11–2.81) 1.0 (0.72–1.75) 0.8 (0.5–1.23) 0.56 (0.26–1.07) 0.5 (0.1–1.48) 1.36 (0.76–2.25) 0.43 (0.09–1.25) 0.67 (0.08–2.4) 1 (0.02–5.57) 0.95 (0.58–1.47) 0.71 (0.34–1.30) 0.67 (0.18–1.71) 1.67 (0.93–2.75) 1.5 (0.31–4.38) 1.0 (0.12–3.61)
a Observed/expected cases of coinfection and rate ratio were calculated by hierarchical loglinear analysis with backward elimination.
A. Spinillo et al. / Virus Research 142 (2009) 154–159 Table 5 Two-ways interactions between species and types of HPV in multiple infections according to the age at diagnosisa . Age ≤ 32 years (n = 338)
Age > 32 years (n = 339)
O/E
Ratio (95% CI)
O/E
Ratio (95% CI)
A5–A6 A5–A7 A5–A9 A5–A10 A6–A7 A6–A9 A6–A10 A7–A9 A7–A10 A9–A10
9/12 13/18 52/55 13/18 11/15 30/42 13/14 59/68 15/22 53/63
0.75 (0.34–1.42) 0.72 (0.38–1.24) 0.94 (0.71–1.24) 0.72 (0.38–1.24) 0.73 (0.37–1.31) 0.71 (0.48–1.0) 0.93 (0.49–1.59) 0.87 (0.66–1.12) 0.68 (0.38–1.12) 0.84 (0.63–1.10)
15/12 16/19 50/51 17/18 8/13 35/40 13/14 68/74 20/27 71/78
1.25 (0.70–2.06) 0.84 (0.48–1.37) 0.98 (0.73–1.99) 0.94 (0.55–1.51) 0.61 (0.26–1.21) 0.87 (0.61–1.22) 0.93 (0.49–1.59) 0.92 (0.71–1.16) 0.74 (0.45–1.14) 0.91 (0.71–1.15)
16–18 16–31 16–51 16–52 16–56 18–31 18–51 18–52 18–56 31–51 31–52 31–56 51–52 51–56 52–56
32/26 41/49 25/33 23/26 8/11 24/18 8/12 5/10 5/4 21/24 11/19 7/8 15/13 4/5 3/4
1.23 (0.84–1.74) 0.84 (0.60–1.14) 0.76 (0.49–1.11) 0.88 (0.56–1.33) 0.73 (0.31–1.43) 1.33 (0.85–1.98) 0.67 (0.29–1.31) 0.5 (0.16–1.17) 1.25 (0.41–2.92) 0.87 (0.54–1.33) 0.58 (0.29–1.04) 0.87 (0.35–1.80) 1.15 (0.65–1.90) 0.8 (0.22–2.04) 0.75 (0.15–2.2)
43/29 52/59 31/31 36/34 9/11 18/22 11/12 14/12 4/4 24/22 19/24 7/8 22/13 8/4 9/5
1.48 (1.07–2.0) 0.88 (0.66–1.16) 1.0 (0.68–1.42) 1.06 (0.74–1.46) 0.82 (0.37–1.85) 0.82 (0.48–1.29) 0.92 (0.46–1.64) 1.16 (0.64–1.96) 1 (0.27–2.56) 1.1 (0.70–1.62) 0.79 (0.47–1.24) 0.88 (0.21–1.86) 1.69 (1.05–2.56) 2 (0.86–3.94) 1.8 (0.82–3.41)
Coinfection
a Observed/expected cases of coinfection and rate ratio were calculated by hierarchical loglinear analysis with backward elimination.
significant clustering between species was found for CIN ≥ 2. As to HPV types, three-way interactions between types 16–51–52 (rate ratio = 1.8, 95% CI = 1.07–2.84, p = 0.02) and 31–51–56 (rate ratio = 3.5, 95% CI = 1.41–7.21, p = 0.006) were statistically significant in CIN ≥ 2. Among two-way interactions, only coinfection by HPV 16–18 was more frequent than expected (p = 0.012) among women with CIN1. Data were further stratified by the age of the patients considering a cut-off of 32 years, the mean age of the series. Both threeand two-way interactions were calculated, the latter are detailed in Table 5. As to HPV species, no significant three-way clustering was observed in the two groups. Younger women had lower than expected rates of coinfection by species A6–A9 (p = 0.05). Among older women, three-way interactions between types 16–51–52 (rate ratio = 3.25, 95% CI = 1.73–5.56, p = 0.0004) and 31–51–56 (rate ratio = 5, 95% CI = 1.62–11.7, p = 0.004) significantly deviated from chance. Coinfection by types 16–18 (p = 0.014) and 51–52 (p = 0.02) also occurred more frequently in women older than 32 years. 4. Discussion Viral interactions in multiple HPV infection may have relevant implications in the assessment of oncogenic risk and, consequently, in the clinical management of women with CIN. Establishing whether there is cross-protection, facilitation or competition between different HPVs may also help to predict the impact of vaccines and in post-vaccination monitoring (Franco and Cuzick, 2008). Issues concerning the equilibrium and clustering patterns of HPV types or species in concurrent and sequential infections have been limitedly addressed in previous studies and with controversial results (Chaturvedi et al., 2005a; Liaw et al., 2001; Mendez et al., 2005; Trottier et al., 2006; Wheeler et al., 2006). Studies of sequential HPV acquisition have shown that coinfection is common and most women experience multiple infections over time (Chaturvedi et al., 2005a; Liaw et al., 2001; Mendez et al., 2005; Trottier et al., 2006; Wheeler et al., 2006). Coinfection occurs
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more likely than expected by chance (Liaw et al., 2001; Mendez et al., 2005; Thomas et al., 2000) and is established more frequently in previously infected women (Liaw et al., 2001; Rousseau et al., 2001). In agreement with these results, the present study showed that among patients referred to colposcopy the rate of multiple HPV infection was high and strongly related to the severity of CIN (Fife et al., 2001; Liaw et al., 2001; Trottier et al., 2006), whereas, at variance with other authors, no effect of age was observed (Cuschieri et al., 2004). HPV coinfection was found in 72.1% of women, a higher prevalence than reported in smaller studies from Italy (Capra et al., 2008; Gargiulo et al., 2007; Tornesello et al., 2006). Multiple infections have been reported in a proportion variable from 10% to 80% of HPV-positive women (Chaturvedi et al., 2005a; Cuschieri et al., 2004; Fife et al., 2001; Herrero et al., 2000; Mendez et al., 2005; Rousseau et al., 2003b; Trottier et al., 2006; Wheeler et al., 2006). Considering the selection criteria of the present series and the sensitivity of the typing assay, the observed rate of coinfection seems justifiable. In agreement with other authors, the A9 species and the HPV-16 type were most frequently represented. All HPV species had a risk of multiple infection higher than expected, although the amount of risk was lower for species A7 and A10. As to individual types, the risk of coinfection was higher for types 6, 11, 16, 18, 31, 33, 51, 52, 56. In a comparable series of prevalent SIL cases (Chaturvedi et al., 2005b), the species A9 was associated with a 32% reduction of the risk of coinfection. Differences in study design and statistical power may account for this discrepancy. In fact, that study used HPV types as units of observation, predominantly enrolled young AfroAmericans, had one-third the number of coinfections—70% of which occurred in HIV+ subjects −, and exclusively considered cytological outcomes. Most previous studies on multiple HPV infection have reported that individual types or species associate at random based on their relative frequency (Chaturvedi et al., 2005b; Trottier et al., 2006; Wheeler et al., 2006). Conversely, the present results showed significant clustering of HPV types and species some of which were maintained after stratification by age and CIN severity. Overall, the combinatorial patterns between species did not deviate from chance except for the interactions of A7–A9–A10, A6–A9 and A7–A10 that were more frequent than expected. Among types, the rates of coinfection by HPV 31–35–56, 16–51–52, 16–18 and 51–52 were higher than expected whereas they were lower for HPV 31–52. After stratification by severity of cervical lesions, the interactions between species and between types 16–18 were maintained in CIN1, whereas types 16–51–52 and 31–51–56 clustered significantly in CIN ≥ 2. As to the effect of age, it abolished all interactions between species. Associations of types 31–35–56, 16–51–52, 16–18 and 51–52 were significant only among women aged more than 32 years. Clustering of HPV types was observed by Mendez et al. (2005) who calculated that HPV-16/18 infected subjects had a 5–6 times higher odds ratio of acquiring a subsequent HPV-58 infection. Conversely, in their smaller series of persistent infections, Chaturvedi et al. (Chaturvedi et al., 2005b) could not detect any difference in 21 combinatorial patterns across HPV, age and PAP smear strata. Given the complex network of variables that influence the dynamics of HPV infection, assessing viral interactions is methodologically challenging. Previous studies have considered different designs and variable clinical settings and outcomes, each approach having its inherent limitations (Chaturvedi et al., 2005b). The main interest of exploring viral interactions is to make inferences about potential effects on viral life cycle and disease progression. Therefore, the study was restricted to a uniform series of women with a well-defined outcome (CIN) of known clinical relevance. This implies that the results cannot be applied to a general population of HPV-infected women. Other major methodological points of the study are as follows. First, a large number of histologi-
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cally confirmed lesions were considered. They were derived from a consecutive series of women with cytological abnormalities undergoing systematic colposcopy, accrued in a single institution over a short period, ethnically homogenous, and examined by punch biopsy in 56.5% of cases and by cone biopsy in 20%. Second, the SPF10 -Lipa typing assay (Kleter et al., 1999) was used. This is a highly sensitive test with a large coverage of HPV types that has been extensively validated in clinical and epidemiological studies (Castle et al., 2008; Perrons et al., 2005; Safaeian et al., 2007). Third, infections were classified according to both HPV type and species, an approach that accounts for the higher number of phylogenetically related types in larger species (e.g. A7, A9, and A10). Fourth, interactions were tested by multiway contingency tables using loglinear modelling, a multivariate statistical technique that avoids assumptions about the distribution of events and that has been used to test associations between independent variables in several medical settings (Agresti and Coull, 1996). Fifth, viral interactions were verified across strata of age and CIN severity. Finally, logistic regression was used to control for confounding by age, history of CIN and lesion severity. Clustering of HPV types/species does not necessarily imply a direct biological interaction, alternative explanations must be considered. First, it should be acknowledged that broad-spectrum typing assays do not have the same specificity and sensitivity for each viral type. HPV types present at low relative concentrations can be under-represented by PCR because of primer competition (van Doorn et al., 2002). This is unlikely as the study base spans a wide spectrum of disease expressions and, presumably, of viral titres (Moberg et al., 2004). Furthermore, the viral types showing the more significant interactions are not differentially detected by SPF10 -Lipa compared to other typing assays (Castle et al., 2008; Klug et al., 2008; van Hamont et al., 2006). Second, although the sample size is large, a type I error cannot be fully excluded due to the frequency of individual type/species or to residual geographical, demographic or behavioural covariables. Third, clustering might derive from differential or shared transmission of HPV types/clades. However, risk factors of viral acquisition do not differ between single and multiple infections and new infections are predominantly established at random (Plummer et al., 2007; Rousseau et al., 2003a). Fourth, the host’s immunity might affect rates of coinfection and/or clearance of viral types (Chaturvedi and Goedert, 2006). Available evidence does not suggest that immune status may affect associations between types (Chaturvedi et al., 2005b) and immunological studies and vaccination trials do not support a main role for cross-protection between HPV types (Stanley, 2006). HPV clustering might ultimately derive from specific biological features of individual types or species (Hiller et al., 2006; Vinokurova et al., 2008). Differences in tissue preference, replicative ability or carcinogenic potential (Castle et al., 2006, 2005) might favour the persistence of specific HPVs during disease progression and result in a biased distribution of types or species. Coexistence of two oncogenic HPV types within the same cell has been observed ‘in vivo’ by ‘in situ’ hybridization (Birner et al., 2001; Vermeulen et al., 2007) and it has been experimentally obtained by electroporation of viral genomes into primary keratinocytes (McLaughlin-Drubin and Meyers, 2004). Viral interference in the maintenance of genome copy number was observed by both approaches. Antagonism between low- and high-risk HPV types has also been suggested by prospective studies of viral seropositivity (Luostarinen et al., 1999). 4.1. Conclusions The study has some methodological limitations and the results cannot be extrapolated to the viral dynamics in a population setting. Nonetheless, this is the major effort so far exerted to explore
the issue of HPV interactions in women with abnormal cytology and histologically confirmed CIN. The demonstration of significant HPV clustering underscores the need of further studies on the mechanisms of viral interference and on type distribution in the general population. Monitoring is also warranted to assess the impact of multiple HPV infection on cervical disease progression and vaccine efficacy. Acknowledgements This research was partially supported by grants from to Italian Health Ministry to IRCCS Fondazione Policlinico San Matteo, Pavia. References Abramson, J.H., 2004. WINPEPI (PEPI-for-Windows): computer programs for epidemiologists. Epidemiol. Perspect. Innov. 1 (1), 6. Agresti, A., Coull, B.A., 1996. Order-restricted tests for stratified comparisons of binomial proportions. Biometrics 52 (3), 1103–1111. Birner, P., Bachtiary, B., Dreier, B., Schindl, M., Joura, E.A., Breitenecker, G., Oberhuber, G., 2001. 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