Human papillomavirus genotypes in genital warts in Latin America: a cross-sectional study in Bogota,Colombia

Abstract

Summary

Epidemiological studies on benign lesions related to human papillomavirus (HPV) infection are scarce in Latin America. We enrolled 342 consecutive patients with lesions suspected of being genital warts (GW). All patients underwent confirmatory biopsy and GP5+/GP6+/− Reverse Line Blot HPV testing on frozen tissue. In 261 (81%) cases, the diagnosis was confirmed by histopathology and HPV was detected in 90.6% of men and 87.7% of women. HPV 6 was by far the most common type in both women (62%) and men (56%), followed by HPV 11 (∼20%). Co-infection with these two types occurred in 7% and 12% of women and men, respectively. HPV16 ranked third in prevalence, with 16% of patients testing positive. Twenty-five percent of cases tested positive for multiple HPV genotypes. Although HPV 6 and HPV 11 were the main types detected and no differences between men and women were observed, we found HPV 11 contributed more to GW aetiology compared with previous reports, showing a variability of HPV type distribution in GW across populations. This information is valuable baseline data in Latin America for future estimations of the burden of GW in men and women and shows the potential benefit obtainable by prophylactic vaccination against HPV types 6 and 11.

Keywords

sexually transmitted infections human papillomavirus HPV genital warts genotype HPV vaccination Latin America

BACKGROUND

Human papillomavirus (HPV) infection has been recognized as the most prevalent sexually transmitted infection worldwide. Some 600 million persons globally are estimated to be currently infected (both men and women) and at least 10% of this infection corresponds to genital warts (GW) (∼60,000,000)^1,2

There is much less information available on GW compared with other HPV-related diseases. This includes lack of information on type-specific HPV frequency differences across populations, the costs of medical treatments and the knowledge and perceptions towards the disease and its prevention.³

Although it is well established that HPV genotypes 6 and 11 are the major types associated with GW cases worldwide,⁴ the proportion of lesions attributed to each type may vary across populations and gender. Recent information derived from the FUTURE trials⁵ (The Females United to Unilaterally Reduce Endo/Ectocervical Disease) showed that 85% of GWs in women were associated with these two HPV types. In contrast a recent prospective multinational cohort study of men⁶ showed that only 55% of GW cases tested positive for HPV 6 and HPV 11. Compared with large amount of information on HPV distribution of cervical cancer in Latin America⁷ there is lack of information on the distribution of HPV types in GW in the region. The aim of this paper is to describe the prevalence of HPV genotypes in biopsy-confirmed GW from men and women attending a major reproductive and sexual health clinic in Bogotá, Colombia (Clinica Piloto-PROFAMILIA), to contribute information on the burden of benign HPV-related lesions in the region.

METHODS

Study design and population

Patients were enrolled between December 2009 and September 2010, with prior approval by the Ethics committee at PROFAMILIA. Clinica Piloto is one of the 34 clinics that PROFAMILIA foundation runs in 25 cities around the country. PROFAMILIA foundation (www.profamilia.org.co) is the most important and respected organization providing services on sexual and reproductive health in Colombia for the last 45 years.⁸ Consecutive men and women age 18–45 attending an outpatient consultation with a lesion suspected of being HPV related were eligible to enter in the study. Patients with known immunosuppressive disease, or those previously vaccinated with commercially available HPV vaccines (Gardasil or Cervarix) were not invited to participate. After written informed consent was given, patients were asked to answer a short epidemiological survey including risk factors for HPV-related diseases and clinical history of GW.

Study sample size

Results from women enrolled in placebo arm of FUTURE I and II trials⁵ reported a relative frequency of 85% for HPV genotypes 6 and 11. To asses this result in our population, under the assumption that HPV prevalence in GW does not vary with gender, we estimated a sample size of 300 cases would give a confidence of 95% to estimate a proportion of 85% with an absolute error of 4%.

Clinical procedures and histopathological assessment

Patients were examined by experienced gynecologists or urologists. Examinations included a naked eye inspection of the genital area. Cauliflower-like condylomata acuminata lesions, keratotic and smooth papular or flat warts were considered for the study. Biopsies were taken through an excisional procedure under local anaesthesia. Haemostasis was achieved using topical formulations of Monsel's solution. Samples were divided for histological diagnosis (kept in 10% formalin solution) and HPV genotyping (kept in liquid nitrogen). Biopsy diagnosis of GW was made evaluating the following histological criteria:⁹ koilocytosis, basal cell hyperplasia (four or more layers of basal cells), acanthosis, hyperkeratosis and parakeratosis. The remaining sample was preserved in liquid nitrogen for HPV DNA analysis.

HPV DNA detection

HPV DNA was extracted from frozen samples and genotyping was made by reverse line blot (RLB) of PCR GP5+/GP6+ oligoprobes using a direct typing approach.¹⁰ This included direct genotyping of 37 HPV genotypes (23 considered low-risk [LR] HPV types: 6, 11, 26, 34, 40, 42, 43, 44, 53, 54, 55, 57, 61, 70, 71, 72, 73, 81, CP6108, 82/IS39, 82/MM4, 83 and 84 and 14 high-risk [HR] types:16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68).¹¹

Statistical analysis

All comparisons of frequencies and proportions were assessed using chi squared tests. We estimated the proportions for the epidemiologically-defined HPV groups (i.e. high- and low-risk types) and for the most common HPV type observed in the samples for inclusion in the analysis. To estimate the precision of the proportions observed in the study we calculated Wald 95% confidence intervals for proportions.¹²

RESULTS

Biopsy assessment

A total of 342 (138 male and 204 female) patients were included in the study. Individuals who did not assent to the biopsy procedure (n = 21) were excluded from the analysis. Overall, 261 (81%) patients received a histopathological diagnosis of GW; other diagnoses include of papillomatous hyperplasia (8%), chronic inflammation (4%) and verruca vulgaris (1%). Two male cases were diagnosed with low-grade penile intraepithelial neoplasia (PIN I); four patients were diagnosed with lesions not related to HPV (2 molluscum contagiosum, 2 intradermal naevus) and eight showed no histological changes. All patients were informed of the results and were clinically evaluated afterwards. No differences in diagnoses were observed between genders. Hereafter all results are based on the 261 histopathologically-confirmed cases of GW.

Sociodemographic, sexual behaviours and clinical features

The study population was predominantly female (male:female ratio=0.7), >35 years old, employed, literate and with adequate access to health-care services (Table 1).

Table 1

Demographic characteristics of the study population by gender

Characteristic	Women	Men	P
Characteristic	(n = 155)	(n = 106)	P
Age (years)
18–24	55.5	17.9
25–34	34.8	59.4
35–44	9.7	22.6	<0.01
Education (highest level)
Primary	7.1	5.7
High school	66.5	48.1
University/College	23.2	23.6
Postgraduate	3.2	22.6	<0.01
Civil status
Single	68.4	64.2
Married	17.4	24.5
Widow	0.7	0.0
Divorced	3.2	3.8
Union	10.3	7.6	0.5
Social security
HMO – workers	64.5	82.1
Subsidiary	5.8	1.9
No affiliation	29.7	16.0	<0.01
Main economical activity
Work	58.1	82.1
Study	36.1	14.2
Unemployed	5.8	3.8	<0.01

HMO, health maintenance organization

There were clear differences in age distribution between men and women, which also played a role in the distribution in education level, main activity and affiliation to the health system between genders. On average, men reported an earlier age of sexual debut (∼75% before age 18 for men versus 56% for women), larger number of lifetime sexual partners (∼92% of men had four or more sexual partners versus ∼24% for women) and having sex with persons other than their ‘regular partner’ in the last year. Fewer men reported using any contraceptive method compared with women (Table 2).

Table 2

Sexual behaviours related to HPV infection and genital warts by gender

	Women	Men	P
	(n = 155)	(n = 106)
Sexual debut age (years)
<15	7.1	29.3
15–17	49.7	46.2
18+	43.2	24.5	0.001
Lifetime sexual partners
1–4	75.5	7.6
5–9	21.9	40.6
10+	2.6	51.9	0.001
Sexual partner concurrency
Yes	21.3	60.4
No	78.7	39.6	0.001
Use of any contraception methods
Yes	27.0	41.3
No	73.0	58.7	0.001
Regular condom use during intercourse
Yes	83.9	46.2
No	16.1	53.8	0.001

HPV, human papillomavirus

Clinical features showed that GW diagnosed in men and women were commonly acuminate lesions, compromising a single anatomic location (Supplementary Table S1). Labia (87/187) and penile shaft (62/129) were the most affected areas in women and men, respectively. The time reported since having noticed the GW by patients was similar in men and women. At least half of the patients had the lesion for no more than three months and no more than 11% reported to have first noticed the lesions more than one year prior.

HPV genotypes

Overall, less than a third of LR (7/23) and more than half (8/14) of HR-HPV types tested were detected in the study population. HPV was detected in 90.6% (CI: 85–96%) of the GW cases in men and 87.7% (CI: 83–93%) of the cases in women. Approximately 71% of cases featured HPV type 6, 11 or 16 as a single infection, being the most common HPV types detected (Table 3). Multiple infections were detected in 25% of GW cases. In women, no more than three HPV types were simultaneously detected, whereas in men two cases were detected with five HPV types in the same lesion. Aside from HPV 6 and HPV 11 no other LR virus was detected more than once in the single infection cases.

Table 3

Distribution of human papillomavirus (HPV) genotypes (percent and 95% CI) in biopsy samples from lesions diagnosed as genital warts by gender

Women				Men
(n = 155)				(n = 106)
Genotypes	n	%	Binomial Wald	Genotypes	n	%	Binomial Wald
			[95% CI]				[95% CI]
Negative	19	12.3	(7.0–17.5)	Negative	10	9.4	(3.8–15.1)
One single genotype detected							–
6	63	40.6	(32.8–48.5)	6	40	37.7	(28.4–47.1)
11	24	15.5	(9.7–21.2)	11	20	18.9	(11.3–26.4)
16	5	3.2	(0.4–6.0)	16	3	2.8	(0.0–6.0)
18	1	0.6	(0.0–1.9)	58	2	1.9	(0.0–4.5)
56	1	0.6	(0.0–1.9)	56	1	0.9	(0.0–2.8)
43	1	0.6	(0.0–1.9)	40	1	0.9	(0.0–2.8)
				42	1	0.9	(0.0–2.8)
				55	1	0.9	(0.0–2.8)
				66	1	0.9	(0.0–2.8)
Only two genotypes detected
6–16	13	8.4	(4.0–12.8)	6–11	5	4.7	(0.6–7.5)
6–11	6	3.9	(0.8–6.9)	6–16	4	3.8	(0.1–4.5)
11–16	4	2.6	(0.1–5.1)	11–16	2	1.9	(0.0–2.8)
11–33	2	1.3	(0.0–3.1)	11–33	1	0.9	(0.0–2.8)
6–45	2	1.3	(0.0–3.1)	16–42	1	0.9	(0.0–2.8)
6–56	2	1.3	(0.0–3.1)	6–56	1	0.9	(0.0–2.8)
6–81	2	1.3	(0.0–3.1)	6–35	1	0.9	(0.0–2.8)
6–42	1	0.6	(0.0–1.9)	6–42	1	0.9	(0.0–2.8)
11–45	1	0.6	(0.0–1.9)	42–56	1	0.9	(0.0–8.8)
35–58	1	0.6	(0.0–1.9)		16
Three or more genotypes detected
6–11–16	4	2.6	(0.1–5.1)	6–11–16	5	4.7	(0.6–2.8)
6–11–33	1	0.6	(0.0–1.9)	6–11–18	1	0.9	(0.0–2.8)
6–16–56	1	0.6	(0.0–1.9)	42–56–58	1	0.9	(0.0–2.8)
6–45–56	1	0.6	(0.0–1.9)	6–11–16–33–40	0.9	(0.0–2.8)
				6–11–42–58–66	0.9	(0.0–2.8)

HPV 6 was by far the most detected type in both women (62%) and men (56%), followed by HPV 11 (21%). Co-infection of HPV 6 and 11 (with or without other HPV types) was detected in 7% of women and 12% of men (Figure 1). HPV 16 ranked third, being detected in ∼16% of patients. Interestingly, HPV 16 was detected in GW more frequently than the sum of all LR types other than type 6 and 11 (Figure 2). At least one of these three HPV types was detected in 85% of cases. Although more HPV types were detected among men, no significant difference was observed in the distribution of HPV types between men and women.

Figure 1

Distribution of HPV types 6, 11 and 16 in genital warts by gender. HPV, human papillomavirus

Figure 2

Frequency of HPV types 6, 11, 16 and other types in genital warts according to gender. HPV, human papillomavirus; LR, low risk; HR, high risk

DISCUSSION

To our knowledge this is the largest study in Latin America reporting the prevalence of HPV genotypes in GW in both women and men drawn from the same population, and is also the first study in the region reporting results for 37 of the most common HPV types known to infect the anogenital tract.

Clinical presentation of GW was similar to other reports.^{12
–14} In men, the penile shaft has been described as the most affected location in both the circumcised and non-circumcised, followed by the foreskin and the glans. In women, the labia (minora and majora) and vulval introitus were the most affected locations, as observed in other studies.^12,15

Our results support the large contribution of HPV 6 and HPV 11 to the burden of GW, depicting no major differences between men and women in our study population but showing certain differences from other studies.^{4
–6,15
–17} Although the overall HPV detection was similar to that observed in the placebo arm of the FUTURE trials,⁵ we found a larger relative contribution of HPV 11 to GW aetiology in women. The prevalence of HPV 6 and HPV 11 in the placebo arm of the FUTURE I and II trials (age range 16–24) was 74% and 14%, respectively, differing notably from those observed in the present study (HPV 6: 55% [95% CI: 47–63]; HPV 11 26% [95% CI: 19–33]). Moreover, this difference stands out when comparing the detection of HPV 11 as a single infection. While Future I and II trials reported 6.7% detection of HPV11 as a single infection we found it in 15.5% of women cases. These contrasting results may be related to age range differences between studies, however we also observed a higher detection of HPV11 (18%) compared with that observed in the multinational male cohort⁶ (11%), which included individuals in broader age range (18–70 years). Overall our results add evidence to the observed differences of HPV genotype distribution in the anogenital area across populations.^18,19

We found a lower proportion of multiple infections compared with previous reports.^6,20 This might be explained partially due to the lower sensitivity to detect multiple infections shown by the PCR GP5+/GP6+ reverse line technique used in our study.²¹ HPV 6 or HPV 11 were present in virtually all cases with multiple types detected. Previous reports suggest that replicative activity of HPV 6 or 11 types is predominant when present in multiple infections, indicating that a single HPV drives the natural history of the proliferative lesion.²⁰

28% of GW cases here reported were infected or co-infected with HR-HPV. Here we tested for most of HPV types involved in anogenital tract infection, including 23 LR types; nevertheless we found that 5% of cases had only HR-HPV, suggesting a causative roll of HR-HPV in GW, mainly for HPV type 16. A similar result was reported in a sample of 130 Chinese men diagnosed with GW and enrolled in a STD clinic,¹⁶ in the multiethnic male cohort study and in the placebo arm of the FUTURE trials.⁵

We observed that male cases reported a higher prevalence of related risk factors and were diagnosed later than women. These findings are consistent with previous studies reporting age differences at diagnosis in men and women.¹⁵ These differences could be partially explained by a more permissive attitude towards sex in men,²² and increased health-seeking behaviour in women diagnosed with GW.²³ However, we did not observe a difference in the time since noticing a GW lesion between men and women.

There was a high correlation between clinical and histological diagnosis in this study. Only four clinically diagnosed cases resulted in diseases not related to HPV infection. Interestingly, 75% (36/48) of those cases with clinical diagnosis of GW but not confirmed by biopsy also tested HPV-positive, most often for HPV 6 (23/48). These lesions featured papillomatous hyperplasia (26), non-specific epithelial inflammation (12) or no visible histological alteration (10). The spectrum of microscopic patterns observed in clinical compatible GW might represent the various stages of HPV infection dynamics: epithelium proliferation, intense viral replication with cytopathic effects and lastly, a phase characterized by regression of HPV activity due to the host immune response.²⁴

Our study had several strengths. First, HPV testing was done on frozen fresh preserved samples and not from skin surface scrapes. HPV detection from the lesion itself rather than from a scrape, minimizes the detection of HPV types not related to the lesion but could be found in normal skin in the same body area.^20,25 Also, we tested for 37 of the most common HPV types infecting the genital area using a highly reliable technique.¹⁰ We included patients of both genders not known to be related, allowing comparison of the HPV type profile of a sexually transmitted infection. Lastly, we confirmed the diagnosis of GW with histology. Eighty-one percent of clinically-diagnosed lesions were confirmed by microscopy and only four lesions had a histological diagnosis not related to HPV infection. The main limitation of this study could be the generalization of our findings to the entire Colombian population, as PROFAMILA is a private institution where patients pay for medical attention. Access to medical care in Colombia has improved in the last few decades so that most patients with genital warts could seek medical care that would be covered by their insurance. It is possible that patients who decided to pay for medical care for GW may represent only a small fraction of the spectrum.

This information is valuable as regional reference data for future estimations of the burden of HPV-related benign disease in men and women in Latin America and confirms the potential benefit achievable by prophylactic vaccination against HPV types 6 and 11.

Footnotes

ACKNOWLEDGEMENTS

We are most grateful to the participants in the study. We thank all gynecologists and urologists at PROFAMILIA clinic in Bogota (Clinica Piloto) for their collaboration and support enrolling patients for the study. We also are grateful to Dr Gloria Sanchez and her team for the HPV DNA analysis at the Universidad de Antioquia. Special thanks to Johana Dechamps, Luisa Medina and Arlene Cano for administrative support and monitoring of this study. Thanks to Scott Vuocolo for editing with respect to content and language.

Competing interests: GH and AE have received grants and support for travel to meetings for the study or other purposes from Merck, Sharp and Dohme Laboratories. MP has received support for travel to meetings for the study or other purposes. GP is employee of Merck, Sharp and Dohme and may have options or shares from the company. JV, LO, FH, CP and DT do not declared any conflict of interest.

Contributions statement: GH participated in the conception and study design, statistical analysis of data, draft of manuscript and revision and approval of final version of manuscript. MP participated in the conception and study design, interpretation of results, draft of manuscript and revision and approval of final version of manuscript. LO participated in the collection of samples, draft of manuscript and revision and approval of final version of manuscript. JV participated in the collection of samples and clinical data, draft of manuscript and revision and approval of final version of manuscript. DT, FH and CP, participated in the collection of clinical data, draft of manuscript and revision and approval of final version of manuscript. GP participated in the conception and study design, interpretation of results, draft of manuscript and revision and approval of final version of manuscript.

Funding: IISP Program (Independent Investigator Studies Program) of Merck, Sharp and Dohme. MSD was not involved in the study design, analysis of results or drafting of the manuscript.

Appendix

Supplementary Table S1

Clinical features of men and women diagnosed with biopsy-confirmed genital warts

	Women	Men	P
	(n = 155)	(n = 106)
Clinical features	%	%
Time since GW were first noticed
<1 month	29.0	31.1
1–2 months	34.2	22.6
3–5 months	21.3	20.8
6–11 months	7.7	15.1
1 year +	7.7	10.4	0.15
Diagnosed with GW for first time
No	95.5	91.5
Yes	4.5	8.5	0.23
Anatomic locations affected by GW
Single	83.9	76.4	0.13
Multiple	16.1	23.6
GW presentation
Acuminate	97.4	98.1
Flat	2.6	1.9	0.63
Specific location of GW*
Labia	87
Vaginal introitus	80
Perineum	20	15
Glans/sulcus		19
Shaft		62
Foreskin		33

GW, genital warts

*Individuals may have lesions in more than one site

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