Cervical intraepithelial chagnes & HIV infection in women attending sexually transmitted disease clinics in Pune, India
Background & objectives: Cervical cancer is the most important cause of malignancy associated deaths among women in India. Western studies have reported higher risk of abnormal Pap smears in HIV infected women. A large burden of HIV infection and increasing HIV epidemic in India threatens to exacerbate incidence of cervical cancer. The objective of this study was to assess the frequency of Pap smear abnormalities and its association with HIV infection in women attending sexually transmitted disease (STD) clinics and to identify associated risk factors.
Methods: Between June 1996 and September 1999, women attending two STD clinics in Pune were screened for HIV infection, offered STD laboratory diagnosis and treatment and their Pap smears were evaluated.
Results: Squamous cell abnormality was detected in 10 per cent of HIV sero negative women attending STD clinics. This proportion was nearly double (19.2%) (Odds ratio=2.14, 95% C.1. 1.034.48, P=0.04) in HIV seropositive women. Having more than one life time partners and presence of STDs were also significantly associated with Pap smear abnormality in univariate analysis. In multivariate analysis, women presenting with STD and HIV infection both, were 2.8 times more likely to have inflammatory Pap smear and 3.5 times more likely to have abnormal Pap smear compared to HIV seronegative women presenting without STDs.
Interpretation & conclusion: Pap smear abnormalities were common in women attending STD clinics in Pune. Presence of HIV infection further increased the risk two-folds. Therefore, women suffering from STDs should undergo periodic Pap smear screening for early detection of cervical abnormalities and should receive appropriate management to reduce morbidity and mortality.
Key words Cervical cancer – HIV infection – intraepithelial changes – Pap smears
Globally 500,000 new cases of cancer of uterine cervix are recorded annually, nearly 26 per cent of these are from China and 20 per cent from India. Cancer of the cervix is the most common malignancy in Indian women and accounts for 80 per cent of all malignancies of the female genital tract2. Invasive cervical cancer is the end result of a long pathological process that begins with.precursor lesions called cervical dysplasia or SIL (squamous intraepithelial lesion)3. Human papillomavirus (HPV) plays a central etiologic role in cervical neoplasia and associated deaths in women 4-6. A high recurrence rate of cervical intraepithelial neoplasia (CIN) after standard treatment has been noted in HIV-infected women and the severity of these lesions seems to be inversely related to immune function and taking into account these data, Centers for Disease Control (CDC), USA have included invasive cervical carcinoma among the AIDS-defining conditions since 1993(7).
The HIV epidemic in India is increasing and heterosexual transmission is the most common route of transmissions. India faces the second largest burden of HIV infection in the world and over three million people were estimated to be living with HIV/ AIDS at the beginning of the millennium9. Sentinel surveillance data have indicated that HIV epidemic is well established in the general population in our country10. Considering the high prevalence of HIV infection among women in sex work as well as those not in sex worki 1, a rise in cervical abnormalities and cervical cancer can be expected in the near future in India. The extent of additional burden due to spreading HIV epidemic, on the pre-existing high burden of cervical cancer and cervical abnormalities has not been studied in India. To prevent thousands of deaths due to cervical cancer, it is necessary to determine the usefulness of various appropriate preventive strategies including Pap smears for early detection and further management of cervical abnormalities in women with high risk behaviour.
The present study was carried out to study the frequency of Pap smear abnormalities and its association with HIV infection in women attending STD clinics in Pune city in western part of Maharashtra State of India.
Material & Methods
Subjects & clinical protocol: National AIDS Research Institute (NARI) has established two clinics that provide STD diagnosis and treatment facilities for women in Pune city and enroll women in a cohort study of HIV transmission. Patients attending these clinics were screened for HIV infection after pre-test counselling and informed consent. Base line information related to demographic characteristics, sexual behaviour and medical history was collected from these patients on a pre-tested structured questionnaire. Women who were found to be HIV seronegative were invited to participate in the long-term follow up study of HIV transmission and those who were HIV seropositive at screening were called for follow up at regular intervals to study clinical progression of HIV disease. The patients were given appropriate medication for their STDs as per the CDC treatment guidelines 12 and were called back for clinical follow up and to collect report of HIV test and Pap smear after seven days. Women with squamous cell abnormality in Pap smears were referred to a tertiary care referral hospital for further evaluation and management.
A sample size of 300 was derived to yield 80 per cent power to the study with a presumed prevalence of 25 per cent Pap smear abnormality in female STD patients. Between June 1996 and September 1999, a total of 750 women made their first visit to the STD clinics and Pap smears were obtained from 225 (30%) of these patients. In addition, 76 Pap smears were collected from 183 women who came for their follow up visits. A total of 301 Pap smears were collected.
Laboratory tests: Following clinical evaluation, blood and relevant specimens for STD diagnosis including Gram stain, wet mount and dark-ground microscopy were collected. Serum samples were screened with a commercially available enzyme-linked immunosorbent assay (ELISA) (Detect-HIV TM, Biochem Immunosystems Inc, Canada) for identification of HIV-1 and HIV-2 antibodies. Samples testing positive with ELISA were confirmed by a Rapid Test (Immunocomb II, HIV I and II, BiSpot, Organics, Israel).
After exposing the cervix with a non-lubricated Cusco’s speculum, the Medscand disposable cytobrush was rotated through 360 degrees in order to get material from the full circumference of the squamo-columnar junction at the external os for Pap smear evaluation. The material was then spread on pre-labelled glass slides and the slides were fixed in methanol while still wet without any drying. After fixing for 30 min the slides were stained with Papanicolaou stains and were evaluated for any abnormality by a pathologist blinded to the HIV status of patients, using Bethesda system13. The smears were classified as normal, inflammatory (showing infection, inflammation, repair) and those showing squamous cell abnormality which includes ASCUS (abnormal squamous cells of undetermined significance), LGSIL (low grade squamous intraepithelial lesion) and HGSIL (high grade squamous intraepithelial lesion).
Statistical analysis: Initial statistical analysis involved univariate analysis with Pap smear results as an outcome variable. Factors found significant in the univariate analysis were used in the multinomial logistic regression model. In addition, best case scenario analysis was done by considering all “inflammatory” Pap smear results as “normal” and worst case scenario analysis was done by considering all “inflammatory” Pap smear results as “abnormal”. Factors found significant in the best case and worst case scenario analysis were used in the multivariate logistic regression analysis to judge if any factors had independent association with Pap smear abnormality.
A total of 301 Pap smears were collected from 933 women. The main reasons for not collecting Pap smears included refusal for physical and internal examination, presence of painful genital ulcer disease and menstruation at the time of the clinic visit.
The comparison of base line characteristics of women whose Pap smears were collected at screening visit (n=225) with those of the remaining whose Pap smears were not collected (n=525), indicated that though similar with respect to their marital status or occupation, those whose smears were not collected were younger. Comparison of base line characteristics of 76 women whose Pap smears were collected at follow up with those of 107 whose smears were not collected, showed no significant difference. Similarly, comparison of demographic characters between those whose smears could be evaluated and were included in the analysis and those whose smears were unreadable and were not included in the analysis did not show any statistically significant difference.
Out of the total 301 Pap smears collected, 57(18.9%) were unsatisfactory and not acceptable for diagnostic evaluation. The results from remaining 244 women (183 reporting for screening visit and 61 for follow up visits) with satisfactory Pap smears have been included in the analysis. The mean age of the study patients was 30.1 yr.
Table I summarizes the results of univariate analysis with Pap smear results as an outcome variable in the women studied. Of the 244 women, 125(51.2%) had normal Pap smears, 85(34.8%) had inflammatory pathology and 34(13.9%) had squamous cell abnormality. Out of the 34 squamous cell abnormalities, 14 (41.2%) had ASCUS, 15 (44.1%) had LGSIL and 5 (14.7%) had HGSIL. None of the women presented with carcinoma in situ or cancer cervix.
Of the 244 women, 140 (57.4%) were HIV seronegative and 104 (42.6%) were HIV seropositive. Among the 140 HIV seronegative women, 81 (57.9%), 45 (32.1%) and 14 (10%) women had normal, inflammatory and abnormal cytology on Pap smear respectively. Of 104 Pap smears obtained from HIV seropositive women, 44 (42.3%) were normal, 40 (38.5%) were inflammatory and 20 (19.2%) were abnormal. The increased frequency of abnormal Pap smears in HIV seropositive women was statistically significant (P = 0.03).
Majority of the women (185/244, 75.8%) were female sex workers. HIV prevalence was 49.2 per cent (91/185, 95% C.I. 42-56.4) among them as compared to 22.03 per cent (13/59; 95 per cent C.I. 11.5-32.6) among the others. This difference was statistically significant (P
The results of univariate analysis (Table I) showed that in addition to HIV serostatus, having more than one sex partner (P=0.048) and presence of STD (P=0.03) were also significantly associated with Pap smear results. Many women in the study (150/ 244, 61.5%) presented with STDs, including 119 (79.3%) women in the first clinic visit and 31 (20.7%) women returning for a follow up visit. Inflammatory cytology in Pap smears was observed in 58(38.7%) women presenting with STDs and 27(28.7%) without STDs. Abnormal Pap smears were present in 25(16.7%) women presented with STDs and 9(9.6%) without STDs.
Of the 183 smears collected at the screening visit, 93(50.8%), 63(34.4%) and 27(14.8%) showed normal, inflammatory and abnormal cytology respectively. Among the 61 smears collected from women reported for follow up visit, 32(52.5%), 22(36.1%) and 7(11.5%) had normal, inflammatory and abnormal cytology respectively. The difference was not statistically significant.
Sixteen (18.2%) of the 88 HIV seropositive women seen at their first visit, and four (25%) of 16 returning for follow up visits had abnormal Pap smear. Among the 140 HIV seronegative women, 11/95 (11.6%) and 3/45 (6.7%) women, reporting for the screening and follow up visits respectively had abnormal Pap smear. The differences were not statistically significant.
Factors found significant in the univariate analysis were used in the multinomial logistic regression model (Table II). It was observed that women presenting with STD and who were also HIV positive had 2.81 times more risk of having an inflammatory Pap smear (Adjusted odds ratio, AOR=2.81, 95% CI 1.24-6.36, P = 0.01) and 3.51 times more risk of having an abnormal Pap smear (AOR=3.51, 95% CI 1.27-9.75, P = 0.02) compared to HIV seronegative women without STDs. Women with age 30 and above were found to have 0.55 times less risk of having inflammatory cytology (AOR = 0.55, 95% CI 0.31-0.99, P = 0.047) compared to those below 30 yr.
Results of univariate and multivariate analysis of best case scenario and worst case scenario are summarized in Table III. Univariate analysis with best case scenario showed statistically significant association between Pap smear results and HIV seropositive status (P = 0.04), occupation (P = 0.02) and more than one life time partner (P = 0.01). However, multivariate logistic regression analysis showed only life time partner more than I to be independently associated with Pap smear abnormality (AOR=4.53, 95% C.I. 1.03-19.8, P = 0.045).
The univariate analysis of worst case scenario showed statistically significant association between Pap smear abnormality and HIV positive serostatus (P = 0.01), age 30 yr and above (P = 0.04) and presence of STD (P = 0.01). In multivariate logistic regression analysis, HIV positive serostatus (AOR = 1.75, 95% CI 1.04-2.96, P = 0.03) and presence of STD (AOR = 1.9, 95% CI 1.1-3.3, P = 0.02) were found to be independently associated with Pap smear abnormality.
With increasing spread of the HIV epidemic in the general population including women, an increase in the incidence and burden of cervical cancers and related morbidity can be anticipated in a country like India. Therefore studies to determine the prevalence of early pre-cancerous lesions of cervix manifesting as Pap smear abnormality in population at high risk for acquisition of HIV in India gain significance. Identification of associated risk factors may help in planning and implementing appropriate intervention strategies.
In the present study, HIV seropositive women were more than twice likely to have squamous cell abnormality in Pap smears as compared to those who were HIV seronegative and this difference was statistically significant. Three to nearly 14 fold increased risk of abnormal Pap smears in HIV infected women has been reported in Kenya14 and Zaire 15.
It is of great concern that 10 per cent of the HIV seronegative female STD patients had abnormal Pap smears. Such pathology in women attending STD clinics could have resulted from associated STDs. We have previously documented the relationship between STDs and incident and prevalent HIV infections6. Thus STDs could not only be facilitating HIV transmission, but could also be responsible for cervical abnormality. This highlights the need to incorporate Pap smear screening of women in the STD clinics for early detection of cervical abnormalities and appropriate intervention. However, this association needs to be studied in prospective studies with control comparisons for more meaningful and generalizable results.
It was also found that HIV seropositive women presenting with STDs had 3.51 times more risk of having squamous cell abnormality compared to HIV seronegative women without STDs. This possibly suggests that STDs and HIV infection may have a synergistic effect resulting in Pap smear abnormalities. It was also observed that women at the age of 30 yr and above had nearly half the risk of having inflammatory cytology compared to those below 30 yr. However, comparison of base line characteristics indicated that women whose smears were not collected were younger as compared to those whose smears were collected. Hence, our estimates of inflammatory Pap smear changes probably underestimate the actual risk.
We did not find any statistical significance associated between sex work as an occupation and Pap smear results. This strengthens the case for a more direct relationship between HIV infection and Pap smear abnormalities.
In the best case multivariate stepwise logistic regression analysis, an independent association between Pap smear abnormality and life time partner more than I was observed. In the multivariate stepwise logistic regression worst case analysis, HIV serostatus and presence of STD were found to be independently associated with Pap smear abnormality. The assumption of the worst case analysis is supported by the fact that vaginal and cervical inflammations caused by sexually transmitted infections are suspected to promote development of cervical intraepithelial neoplasia (CIN)17. The initiation of the neoplastic process could be caused by some biochemical substances produced during metabolism in chronically inflammed tissues18. It may be necessary to treat STDs vigorously to prevent initiation of local inflammatory process in the vagina and cervix that could lead to CIN and eventually to cancer cervix.
Relation between HIV positive serostatus and Pap smear abnormality was retained in all three types of univariate analyses and this might be indicative of a strong association between the two.
This study represents a relationship between HIV infection and Pap smear abnormalities in women attending STD clinics in India and a need for periodic Pap smear screening of HIV infected as well as uninfected women. The prevalence of mild to moderate dysplasia in general population based screening programmes in Barsi tehsil in Maharashtra in 1982 and 1987 was found to be 0.9 per cent and 0.5 per cent respectively19. Cervical cancer burden in India alone is estimated as 100,000 in 2001 AD20 and it appears that HIV infection will significantly add further to this.
Cervical cytology appears to be adequate as a screening tool for detection of CIN in HIV sero positive women and many studies have stressed a need for careful interpretation, further evaluation with colposcopy and directed biopsy and follow up21-24.
Though preventable but not prevented, cancer cervix is diagnosed very late in most parts of the developing world. Early diagnosis of preneoplasic lesions on the relatively easily accessible uterine cervix was ushered in as early as 1950s by the use of smear examination introduced by Papanicolaou25. Our data provide additional justification for use of this simple, relatively inexpensive, reliable, less time consuming and generally acceptable investigational tool for screening of women attending STD clinic in an overall Indian scenario of existing high death rate from cervical cancer and high HIV seroprevalence.
The financial assistance from the National Institutes of Health (NIH), Bethesda, USA, the NIH-Fogarty International Center, Programme of International Training Grants in Epidemiology related to AIDS; Family Health International with funds from the National Institute of Allergy and Infectious Diseases, NIH; the Indian Council of Medical Research, New Delhi is acknowledged. Authors thank Dr A. Ravetkar from the Health Department of the Pune Municipal Corporation for his help. The contribution of the HIVNET group consisting of Shriyut H. Mahajani, R. Yelgate, S. Nawlakha, A. Parkhe, M. Chavan and R. Brahme in laboratory procedures and data management is acknowledged.
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S. Joshi, A. Chandorkar*, G. Krishnan**, A. Walimbe^, R. Gangakhedkar, A. Risbud, V. Jadhav^^, R. Bollinger *^ & S. Mehendale
National AIDS Research Institute (ICMR), *Sanjeevan Hospital. **Prerna Laboratories, ^National Institute of Virology (ICMR), ^^Dr Kotnis Hospital, Pune Municipal Corporation, Pune, India & *^John Hopkins University, Baltimore, USA
Reprint request: Dr S.M. Mehendale, Deputy Director, National AIDS Research Institute, G-73, MIDC, Post Box 1985, Bhosari, Pune 411026, India.
Copyright Indian Council of Medical Research May 2001
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