Prevalence and factors associated with anal lesions mediated by human papillomavirus in men with HIV/AIDS

Abstract

Summary

Prevalence and risk factors for human papillomavirus-related anal lesions were evaluated in 60 men with human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS). Patients underwent anal cytology, anuscopy under colposcopic vision and anal biopsy for detection of the lesions. The mean age was 41.9 years and the mean time of HIV infection was 6.8 years, 88.3% of them having been on highly active antiretroviral therapy for an average of 6.5 years. Homosexuals represented 43.3% and bisexuals 15.0% whereas heterosexuals comprised 41.7%. The prevalence of anal lesions detected by anuscopy under colposcopic vision, cytology and biopsy were, respectively, 35.0, 16.7 and 23.3%. Homosexuals or bisexuals accounted for 85.7% of the patients with an abnormal biopsy, the remaining 14.3% being heterosexuals (P = 0.02). The T-CD4+ lymphocyte count, HIV viral load and use of antiretrovirals did not reveal any association with anal lesions. The occurrence of anal lesions was high in the individuals with HIV/AIDS, especially in the homosexuals and bisexuals, but it also occurred in heterosexuals, justifying the screening of anal lesions of all men with the infection.

Keywords

human papillomavirus anal lesion HIV/AIDS anuscopy anal biopsy

Introduction

The acquisition of a human immunodeficiency virus and human papillomavirus (HIV–HPV) co-infection has been shown to be fairly widespread, possibly because these viroses are sexually transmitted.¹ As a result, certain epidemiological and clinical peculiarities are observed in anogenital diseases mediated by HPV in individuals infected by HIV: a higher frequency of infection by HPV in HIV-positive individuals compared with HIV-negative ones, as well as a higher prevalence of premalignant and malignant anogenital lesions.² A number of studies suggest that these changes are due to deficiencies in the immune system induced by HIV, as well as the interaction between HIV and HPV.^1,3,4 Although the potentialization of the effect of HPV by HIV is not fully understood⁴ at the cellular level a direct interaction occurs between the two viruses with the HIV favouring the activation of the E6 and E7 genes of the HPV, increasing the expression of their oncoproteins. An abnormal secretion of cytokines that may facilitate progression to neoplasm is also noted in lymphocytes infected with HIV.³

Since 1996, with the introduction of highly active antiretroviral therapy (HAART), a significant decrease has been seen in morbidity and mortality from opportunistic infections and several malignant diseases related to HIV, such as Kaposi's sarcoma.^1,2 However, little effect has been observed on the anogenital intraepithelial neoplasms and the results of the studies have been controversial, ranging from the lack of any effect to only modest benefits.^2,5

The minimal effect of HAART on the course of anal intraepithelial neoplasia (AIN) might be accounted for by the fact that the rise in the levels of T-CD₄⁺ lymphocytes induced by these drugs does not reflect the reconstitution of specific immunity for HPV, but rather an increase in the systemic immune response to other pathogens with limited action on local immunity.⁴

The interest in conducting this research was due to the nonexistence of data on anal lesions in men with HIV/AIDS in the Northeast Region of Brazil. The purpose of this study was to determine the prevalence of anal lesions mediated by HPV in the patients with HIV/AIDS seen in the Infectious and Parasitological Diseases Outpatient Clinic of the Federal University of Pernambuco Teaching Hospital, as well as the possible related factors.

Methods

This is a descriptive study carried out in the Infectious and Parasitic Diseases Outpatient Clinic of the University Hospital of the Federal University of Pernambuco and in the Colposcopy and Lower Genital Tract Sector (SCTGI) of the same hospital from July to November 2006. Out of a total of 118 male patients invited, a group of 60 agreed to take part in the study and signed the informed consent document. The patients were interviewed by means of a questionnaire on which their demographic data, personal habits, clinical history in relation to HIV infection and sexual behaviour were recorded.

After the questionnaire had been applied, the patients’ clinical records were consulted to verify their last T-CD₄⁺ lymphocyte count and quantification of the viral load. The T CD₄⁺ lymphocytes were measured by the flow cytometry method. The viral load represented by the ribonucleic acid particle of the HIV (RNA-HIV) was quantified by the nucleic acid sequence-based amplification method.

The subjects were submitted to diagnostic tests comprising collection of endoanal material for oncotic cytology, anuscopy under colposcopic vision of the perianal and endoanal regions and biopsy guided by anuscopy under colposcopic vision of any suspected lesion.

Anal cytology was performed with a cytological brush, moistened with a saline solution, in the anal canal to between 3 and 5 cm, with a rotation of 360° and removed in a spiral movement. The material adhering to the brush was rolled on a dry transparent glass slide with identification of the patient and conditioned in a cylindrical recipient containing 95% ethyl alcohol as fixing solution. The slides were stained by the Pananicolaou method (with haematoxylin–eosin dyes) and examined under light miscroscopy by a single cytopathologist. The cytological findings were classified in accordance with the Bethesda system (2001) for the classification of preinvasive cervical and anal lesions.⁶ The intraepithelial anal lesions were thus classified as low-degree squamous intraepithelial lesion, high-degree squamous intraepithelial lesion and atypical squamous cells of undetermined significance.

After the cytology collection, anuscopy was performed under colposcopic vision with the patient in the right lateral decubitus position and knees bent over the thighs. The anuscope, duly lubricated with aqueous gel, was then inserted and after removal of the embolus, the application of 3% acetic acid was made with visualization magnified by the colposcope. This procedure allowed to identify the changes in the epithelium characterized as a flat or a dense acetowhite epithelium, whether or not associated with fine or thick punctuation/mosaicism. Next, Schiler's test was performed (application of a lugol's solution). The anuscopic features were assessed using the Barcelona Colposcopic Terminology, 2002.⁷

The findings suggestive of an anal lesion on anuscopy were biopsied. The procedure was guided by the colposcope with the aid of Gaylor-Medina clamp under local anaesthesia using 2% lidocaine gel without a vasoconstrictor. When an extensive macroscopically visible lesion on the anal margin was found, the biopsy was conducted in the operating room under rachidian anaesthesia. The histological evaluation was carried out by a single pathologist and classified as one of the following: normal, atypical related to infection by HPV, condyloma acuminatum and intraepithelial anal neoplasia grade I, II and III.⁸

The outcome of interest was the presence of a perianal and an anal lesion characterized by the condyloma acuminata and intraepithelial neoplasia according to anuscopy under colposcopic vision summed to anal oncotic cytology and anal histology. The anal biopsy was used as a measure of the results for the anal and perianal lesions. The description and quantitative representation of the data were conducted from the contingency tables, as was the measurement of the prevalence of anal lesions mediated by HPV in the population studied. In order to conduct an exploratory approach, Fisher's Exact test was applied in an attempt to identify possible explanations or factors associated with anal lesion. This method is used to test the hypotheses in studies in which the population sample is a small one. All the analyses were performed with the SPSS 12.0 for Windows software.

This study was approved by the Ethics in Research Committe of the Federal University of Pernambuco's Center of Health Sciences, protocol number 109/2006–CEP/CCS.

Results

Characteristics of the population

The present study evaluated a population of 60 male patients diagnosed as having HIV/AIDS. The demographic characteristics and personal habits of the subjects are shown in Table 1. Their mean age was 41.9 years. Only 20.0% reported smoking and none reported using intravenous drugs.

Table 1

Distribution of the 60 individuals according to demographic characteristics and personal habits

Characteristics	No.	(%)	Mean
Age (years)
28–39	22	36.7	41.9
40–49	27	45.0
50–59	9	15.0
60–69	2	3.3
Education (years of formal schooling)
0–7	24	40.0
8–11	24	40.0
>12	12	20.0
Income
≤ 1 MW	30	50.0
2 × MW	11	18.3
3 × MW	8	13.4
>4 × MW	11	18.3
Smoker
Yes	12	20.0
No	48	80.0
Former smoker
Yes	21	35.0
No	39	65.0
Use of injectable drugs
Yes	0	0
No	60	100.0

MW = current national minimum wage

The average time since HIV infection diagnosis was 6.8 years. The majority (88.3%) reported being on HAART for an average of 6.5 years. A total of 43.3% of the patients presented T-CD₄⁺ lymphocytes >500 cells/mm³ and 75% had a viral load <5.000 copies of RNA/mL. Sexual behaviour was distributed as follows: 43.3% homosexuals, 41.7% heterosexuals and 15.0% bisexuals. Most of the patients (63.3%) reported having had receptive anal intercourse at some time. The mean age of the first anal intercourse was 16.6 years and 35.0% reported having had a total of more than 10 partners (Table 2).

Table 2

Distribution of the 60 individuals according to clinical status of the HIV infection and sexual behaviour

Characteristics	No.	Percentage (%)	Mean
Time since diagnosis of HIV (years)
0–5	23	38.3	6.8
6–10	26	43.4
11–18	11	18.3
Use of antiretroviral
Yes	53	88.3
No	7	11.7
Time of use of antiretroviral (years]
0–4	14	35.0	6.5
5–9	31	51.7
≥10	8	13.3
CD₄⁺ (cells/mm³)
<200	5	8.3
200–500	29	48.3
>500	26	43.4
Viral load (copies of RNA/mL)
<5.000	45	75.0
5.000–30.000	6	10.0
>30.000	9	15.0
Sexual preference
Homosexual	26	43.3
Bisexual	9	15.0
Heterosexual	25	41.7
Anal intercourse
Yes	38	63.3
No	22	36.7
Age at first anal intercourse
7–10	5	13.2
11–19	24	63.1
20–41	9	23.7
Number of partners of same sex
None	22	36.7	16.6
1–5	11	18.3
6–10	6	10.0
>10	21	35.0

HIV = human immunodeficiency virus; RNA = ribonucleicacid

Result of the anal examination

The results of the investigations carried out for the diagnosis of the anal lesions are shown in Figure 1. The frequency of the anal lesions according to the anal cytology was 16.7%, according to anuscopy under colposcopic vision 35.0% and according to anal biopsy was 23.3%.

Figure 1

Frequency of anal lesions according to anuscopy under colposcopic vision, anal oncotic cytology and anal biopsy

Evaluation of the age, clinical status of the HIV infection, sexual behaviour and the result of the anal biopsy

The patients with abnormal histology were aged between 28 and 49 years (P = 0.05), with a mean age of 38.3 years. The majority (85.7%) were homosexuals or bisexuals (P = 0.02). A similar percentage (85.7%) was found among the individuals who reported receptive anal intercourse (P = 0.06). Around 78.6% of the patients with an anal lesion on biopsy reported having had 10 or more partners of the same sex during their lifetime (P = 0.001) (Table 3). The average of T-CD₄⁺ lymphocytes, the viral load and the use of HAART showed no significant differences between the individuals who presented with and those without abnormalities on the anal biopsy (Table 4).

Table 3

Distribution of anal lesions diagnosed by biopsy according to age, sexual preference, practice of anal intercourse and number of sexual partners

	Anal lesion
	Present		Absent		Total		P
Age (years)	No.	%	No.	%	No.	%
28–49	14	100.0	35	76.1	49	81.7	0.05^∗
50–69	0	0.0	11	23.9	11	18.3
Total	14	100.0	46	100.0	60	100.0
Sexual preference
Homo/bisexual	12	85.7	23	50.0	35	58.3	0.02^∗
Heterosexual	2	14.3	23	50.0	25	41.7
Total	14	100.0	46	100.0	60	100.0
Anal intercourse
Yes	12	85.7	26	56.5	38	63.3	0.06^∗
No	2	14.3	20	43.5	22	36.7
Total	14	100.0	46	100.0	60	100.0
Number of partners
<10	3	21.4	34	73.9	37	61.7	0.001^∗
≥10	11	78.6	12	26.1	23	38.3
Total	14	100.0	46	100.0	60	100.0

∗

Fisher's Exact test

Table 4

Distribution of anal lesions diagnosed by biopsy according to use of HAART, CD₄⁺ and viral load

	Anal lesion
	Present		Absent		Total		P
HAART	No.	%	No.	%	No.	%
Yes	13	92.9	40	87.0	53	88.3	1^∗
No	1	7.1	6	13.0	7	11.7
Total	14	100.0	46	100.0	60	100.0
CD₄⁺ (cells/mm³)
<500	6	42.9	28	60.9	34	56.7	0.3^∗
>500	8	57.1	18	39.1	26	43.3
Total	14	100.0	46	100.0	60	100.0
Viral load (copies/mL)
<5.000	10	71.4	35	76.1	45	75.0	0.7^∗
>5.000	4	28.6	11	23.9	15	25.0
Total	14	100.0	46	100.0	60	100.0

HAART, highly active antiretroviral therapy

∗

Fisher's Exact test

Discussion

For some years, studies have been showing the clinical and epidemiological similarity between the cervical and anal squamous carcinomas mediated by HPV.^2,9–11 Although there is no consensus on screening programmes for anal cancer,¹² the studies recommend such an investigation in high-risk groups such as men with HIV/AIDS.^5,13 Using the diagnostic methods employed in the prevention of cervical cancer as a model, the authors suggest screening programmes using anal cytology, followed by biopsy, guided by anuscopy under colposcopic vision, as the principal components in the prevention and diagnosis of anal lesions mediated by HPV in men with HIV/AIDS.¹³

Anal cytology, regarded as a screening test,¹³ presents the lowest percentage of abnormal results when compared with the other two examinations. In a systematic review, it was reported that the effectiveness of anal cytology is not fully understood, hence the need for further investigation.² In the present study, the evaluation of the anuscopy under colposcopic vision was of fundamental importance, identifying the extension and location of the anal lesion, thereby facilitating the anal biopsy. As in the literature, the anal cytology showed the lowest percentage of abnormal results and highest frequency of non-specific findings.

The patients in this study exhibited a substantial prevalence of anal lesions, which was as high as 23% on anal biopsy. This high frequency confirms and justifies routine screening of anal lesions for men with HIV/AIDS. The evaluation of the possible association between risk factors and the presence of anal lesions was made through the histological findings, and it was noted that anal lesions occurred in the younger patients. This finding was similar to that shown by Varnai, who found a mean age of 42.2 years,¹⁴ a little higher than that of the present study. Wilking et al.¹⁵ also observed that an age of 40 years or under represented a risk for the diagnosis of an anal lesion by biopsy.¹⁵

In the present study, the sexual behaviour proved to be expressive in determining the presence of an anal lesion by biopsy, this being more prevalent among the individuals who identified themselves as homosexuals or bisexuals, among those who practised receptive anal intercourse and those who reported more than 10 partners during their lifetime. In agreement with this data, a number of studies have conclusively demonstrated the role of sexual behaviour in determining the risk of acquiring infection by HPV, AIN and anal cancer.^5,16,17 In one of these studies, a history of anal intercourse was present in 52.0% of the patients with AIN on histology.¹⁵ As far as the number of sexual partners is concerned, it is also observed in the literature that infection by HPV is more prevalent among those with multiple sexual partners (over 10 in their lifetime).^{14,16,18–21}

It is important to underline that HIV-positive heterosexuals with no history of receptive anal intercourse also constitute an at-risk population for infection by HPV, AIN and anal cancer.^11,15,16 It is emphasized that the chief risk factors in HIV-positive heterosexuals are low values of T-CD₄⁺ lymphocytes, a high viral load, a previous AIDS-defining event and the detection of anal HPV, while in homosexuals and bisexuals these factors were represented by more than 10 partners of the same sex in their lifetime and the detection of anal HPV.¹⁶ In another study, it was shown that 23.0% of the HIV-positive men with no history of anal intercourse presented a histological diagnosis of AIN, suggesting that anal intercourse is not a necessary factor for anal infection by HPV and AIN.¹⁵ In the present study, 14.3% of the patients with anal lesions were heterosexuals. One can argue that this could reflect incorrect information about sexual practices, but it may in fact be real and suggest perhaps that heterosexuals should also be routinely screened for the disease.

Another feature observed in this study is that none of the patients used intravenous drugs, the infection by HIV being ascribed to sexual transmission. This illustrates the importance of sexual transmission, favouring the high prevalence of HIV/HPV co-infection in Brazil. In this country, the number of cases of AIDS among IDU fell from 26.7% in 1994 to 9.8% in 2006 in men.²² In contrast, around 70.0% of men with AIDS acquired the infection through sexual contact. A nationwide survey conducted in 2004 revealed that almost 91% of Brazilian patients from 15 to 54 years of age mentioned sexual contact as the way of contracting HIV.²³

The assessment of sexual behaviour identified 43.3% of homosexuals, 41.7% of heterosexuals and 15.0% of bisexuals. In 1994, the cases of AIDS acquired by sexual exposure in Brazil were represented by 27.0% of homosexuals, 12.8% of bisexuals and 14.4% of heterosexuals. Little more than a decade later, in 2006, the figures changed to 16.9% of homosexuals, 10.6% of bisexuals and 42.9% of heterosexuals, which shows the changing patterns of the AIDS epidemic in Brazil.²²

In the present study, no possible protective or facilitating effect of the HAART was observed with regard to the appearance of the anal lesion, although the small size of the sample may have influenced the results. As a rule, the studies state that, when HAART is used, an increase in the incidence of anal cancer, associated with HPV, is evidenced, accounted for by the increased survival of the patients, enabling AIN to progress to anal cancer.^5,15,20,24 It is believed that the use of HAART does not result in a fall in the incidence of AIN, nor does it lead to its regression, even if it promotes the restoration of systemic immunity through the increase in T-CD₄⁺ lymphocytes. Its importance lies in the fact that it lengthens the survival of AIDS patients, although not modifying the natural history of the HIV/HPV co-infection.^1,5,25

In the present study, no possible relation was evidenced between the T-CD₄⁺ lymphocyte levels or viral load and the diagnosis of an anal lesion. Low-CD₄⁺ levels (<500 cells/mm³ and especially <200 cells/mm³) are cited as a risk factor for infection by anal HPV and AIN, but the progression from high-level AIN to anal cancer does not appear to be influenced by the T-CD₄⁺ lymphocyte count, even when this is due to the use of HAART.^1,2,4,15 A number of studies suggest that immunological suppression plays a more pre-eminent role in the initial stages of diseases associated with HPV.^1,4,5,20 Nevertheless, the persistence of high-level AIN and the development of cancer may be related to the cumulative effect of the genetic instability associated with HPV.^{1,3–5,20,24,26}

In view of the importance of HIV–HPV co-infection, it is essential that longitudinal studies be carried out in order to clarify the natural history of anal lesions. No less important and recommended by several authors are programmes for tracking anal cancer, particularly in HIV-positive individuals, with well-defined and uniform criteria of evaluation.

Conclusions

The high prevalence of anal lesions mediated by HPV in the population studied highlights the need for early diagnosis in this group by means of anuscopy under colposcopic vision, anal cytology and anal biopsy. The high-risk sexual behaviour, particularly homosexual and bisexual practices, pointed to possible associations with the appearance of anal lesions, although they also appeared in heterosexuals. The current clinical status of the patients with HIV/AIDS, however, was not shown to be important in determining the presence or absence of anal lesions, as a significant number of patients with a good immunological status, most of them on HAART, presented anal lesions.

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