Introduction
Women infected with HIV are at a higher risk for cervical neoplasia than HIV-uninfected women due to a high frequency of incident, persistent, and progressive human papillomavirus (HPV) infection [1–4]. On the one hand, antiretroviral therapy (ART) has increased life expectancy and lowered the risk of various opportunistic diseases in HIV-infected women, whereas on the other hand, ART-associated immune restoration seems to have little impact on the high risk of HPV infection and the cumulative incidence of cervical cancer among HIV-infected women [3–7]. Increased survival of HIV-infected women in a moderately immuno-compromised state through widespread use of ART seems to increase the risks of persistent HPV infection and the development of high-grade cervical intraepithelial neoplasia (CIN2 and 3) and cervical cancer [8,9]. The current recommendations call for intensive screening of HIV-infected women based on cervical cytology 6 months apart after the initial HIV diagnosis and, if results of both are normal, annual cytological screening [10–12]. This approach is neither feasible nor realistic in many low and medium-resource countries, given the challenges and limitations in offering frequently repeated high quality cytology screening. Considering the need for affordable, feasible, and effective strategies to prevent cervical cancer in these high-risk women, we evaluated the concurrent performance of cytology, HPV testing using Hybrid Capture 2 (HC2), visual inspection with acetic acid (VIA) and with Lugol's iodine (VILI) in detecting CIN2 and 3 lesions, and the feasibility and efficacy of treatment of CIN using cold coagulation among HIV-infected women in Maharashtra state, India.
Methods
Study design
We conducted a cross-sectional study in which all women received screening with cytology, HPV testing, VIA, and VILI; and colposcopy by doctors blinded to the screening test results. Women with cervical abnormalities on colposcopy had directed biopsies and immediate treatment using cold coagulation (if eligible).
The scientific and ethical review committees of the International Agency for Research on Cancer (IARC) of the WHO, Lyon, France, and the ethics committees of Hirabai Cowasji Jehangir Medical Research Institute (HCJMRI), and Prayas Health Group, Pune, India approved the study. Screening was initiated on 9 September 2010 and completed on 3 November 2011.
Eligible women
Eligible participants were HIV-infected women (with serological evidence) and aged between 21 and 60 years, with no debilitating illness as assessed by the study clinician. All the women were not pregnant at recruitment, had intact uterus with no prolapse, and had no previous history of cervical neoplasia. After explaining the study procedures, a written informed consent was obtained and they were interviewed for socio-demographic, sexual, reproductive, medical, and HIV infection-related characteristics using a structured questionnaire by a female social worker.
Screening
Women were screened in a designated cervical cancer screening clinic in Pune. Screening tests were performed by three nurses who had been trained in a 2-week course in the collection of cervical cells for HPV testing and cytology, preparation, and fixation of cervical smears, and in performing VIA and VILI. The IARC manual on visual screening was used to train the nurses [9].
Speculum examination of the cervix was performed by a nurse with women in a modified lithotomy position. Cervical cell samples were collected using a ‘Christmas tree’ brush and placed in a specimen transport medium (STM) for HC2 assay; a second sample of cervical cells was then collected using a ‘Cervex-brush’ to prepare a cervical smear and fixed in 70% ethyl alcohol for cervical cytology. Subsequently, 5% dilute acetic acid was applied on the cervix using a cotton swab and women with well defined, dense acetowhite lesions close to the squamocolumnar junction (SCJ) or the os or with aceto-whitening of a cervical growth 1 min after the application of acetic acid were categorized as VIA-positive [13]. Following VIA, colposcopy was carried out by a doctor. When the doctor applied Lugol's iodine as part of colposcopic assessment, the nurse assessed the cervix independently with the naked eye and recorded the VILI findings. Women with well defined saffron or mustard-yellow noniodine uptake areas close to the SCJ or a cervical growth turning mustard-yellow were categorized as VILI-positive [13]. During colposcopy, if SCJ was not seen, visualization of the endocervical canal was carefully attempted with the help of a cotton-tipped applicator to push the anterior or posterior lip of the cervix or a long artery forceps was used to open up the endocervical canal. Women presenting with any reproductive tract infection or sexually transmitted infection were offered treatment following WHO guidelines for the management of sexually transmitted infections [14].
The STM-containing cervical cells were tested by the HC2 assay for 13 high-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) as per the manufacturer's instructions (Qiagen) at the Nargis Dutt Memorial Cancer Hospital (NDMCH), Barshi. A positive result was recorded for specimens with a ratio of relative light unit to a positive control (RLU/PC) of 1 or more, corresponding to 5000 or more viral copies. Cervical smears were stained and were reported by experienced cytotechnologists at the NDMCH according to the revised Bethesda system [15]. Cytology positivity was defined at two thresholds: at the level of atypical squamous cells of undetermined significance or worse lesions (ASCUS+); low-grade squamous intraepithelial or worse lesions (LSIL+).
Reference investigations for final diagnosis and treatment
Following VIA, all the screened women had immediate colposcopy by a trained study clinician blinded to the VIA/VILI findings by the nurse. Colposcopy was performed as described in the IARC manual and the colposcopic findings were reported as normal, inflammation, probable low or high-grade precancerous lesions, or suspected invasive cancer [16]. The colposcopic findings were explained to the woman and multiple punch biopsies were directed from abnormal areas. Based on colposcopic impression, immediate ablative therapy with cold coagulation was advised if the following criteria were met: the lesion involved three quadrants or less of the cervix with no extension into the endocervix or vagina, the SCJ was fully visible, and there was no clinically suspected invasive cancer. Cold coagulation involved the application of an electrically heated probe (105°C) for 45 s to ablate the entire cervical transformation zone bearing the lesion. If a single application did not cover the entire transformation zone, then two or more additional, overlapping applications of 45 s each were carried out. No local anaesthesia was given prior to cold coagulation. Women with lesions not suitable for cold coagulation (large lesions, lesions involving endocervix or when occult invasion was suspected) and having histologically diagnosed CIN2 and 3 lesions on directed biopsy underwent loop electrosurgical excision procedure (LEEP) at a later date. Women diagnosed with invasive cancers were referred to cancer hospitals in Pune and Barshi for appropriate cancer management. Women with normal colposcopic findings were advised to revisit after 6 months for further testing and then every year thereafter.
Biopsy specimens were processed at a local nationally accredited laboratory and the histological findings were reported according to the CIN terminology [17]. All the histology slides were re-reviewed by another experienced external pathologist from the Regional Cancer Centre, Trivandrum and, in the case of nonagreement, a consensus diagnosis was reached. The final diagnosis was based on histopathology findings for those who had biopsies or excised LEEP cervical specimens and on colposcopy in women who had no colposcopic abnormalities. CIN2 or worse lesions, diagnosed by histopathology based on colposcopically directed biopsies or excision specimens, constituted the true disease; true negatives were those with no evidence of disease on colposcopy, colposcopically directed biopsy, excision specimens, or histologically diagnosed CIN1 cases.
Data management and statistical analysis
Data were entered using Access 2000 software and statistical analysis was carried out using STATA 11 software. Study participant characteristics, screening test positivity rates, women detected with CIN and invasive cancer lesions, and women treated for lesions were presented as numbers and proportions. Standard formulae were used to calculate the sensitivity, specificity, positive and negative predictive values, and their exact 95% confidence intervals (CIs) for the screening tests to detect CIN2 and 3 lesions and for CIN3 lesions alone. The above were also calculated for simulated combinations of screening tests as ‘sequential’ or ‘parallel’ tests to address whether adding a second test would have a more acceptable balance of sensitivity and specificity than single tests in the early detection of high-grade CIN. Sequential testing implies screen positivity only when both the tests are positive (i.e. a negative result by either test is considered as a negative screening result) and parallel testing implies screen positivity if either test is positive (i.e. a negative results in both screening tests is considered as a negative screening result). Since disease ascertainment was done for all women included in the final analysis based on histology and negative colposcopy, we minimized verification bias in our test performance parameters.
Results
The total number of women recruited, number of women screened with VIA, cytology, and HPV testing, number of women excluded, and number of women included in the final analysis are given in Fig. 1. The characteristics of the 1128 women included in the analysis are given in Table 1. Four-fifths of the women were between 21 and 40 years of age, half were widowed or separated, more than 90% were premenopausal, half had CD4 cell counts more than 500, three-quarters were on ART, one-third on ART had CD4 cell counts more than 200, and the vast majority had not had prior cervical screening.
;)
Fig. 1: Screening process of the study.CIN, cervical intraepithelial neoplasia; HC2, Hybrid Capture 2; VIA, visual inspection with acetic acid; VILI, visual inspection with Lugol's iodine.
Table 1-a: Patient characteristics.
Table 1-b: Patient characteristics.
Of the 1128 women tested with VIA, 169 (15.0%) were positive; of the 1127 who had VILI, 167 (14.8%) were positive; of the 1081 women who had cytology, 88 (8.1%) were positive at ASCUS threshold and 69 (6.4%) were positive at the LSIL threshold; of the 1128 women tested with HC2, 298 (26.4%) were positive (Fig. 1; Table 2). Of the 1128 women who had colposcopy, 149 (13.2%) had abnormalities suggestive of cervical neoplasia; the number of screen-negative and screen-positive women who had colposcopic abnormalities and directed biopsies are given in Fig. 1. Among 169 VIA-positive women, 106 (62.7%) had colposcopic abnormalities and had biopsy; of the 88 cytology positive (ASCUS +) women, 30 (34.1%) had directed biopsies whereas 76 (25.5%) of 298 HPV-positive women had directed biopsies.
Table 2: Distribution of final disease status by results from different screening tests.
Histopathology was based on punch biopsies in 141 women and on LEEP in eight women. Among the screened women 4.9% (55/1128) had high-grade CIN lesions. The distribution of women with histologically detected CIN1 (N = 45), CIN2 (N = 21), CIN3 (N = 34), and invasive cancer (N = 5) according to screening test results are given in Table 2. The number of women diagnosed with high-grade CIN or invasive cancer was six times higher among cytology-negative women as compared to HPV-negative women (Fig. 1).
The sensitivity, specificity, and positive predictive values for VIA to detect CIN2 and 3 lesions were 83.6, 88.8, and 27.7%, respectively; the corresponding values for VILI were 89.1, 89.3, and 30.1%; for cytology at ASCUS threshold were 63.3, 94.5, and 35.2%, and for HPV testing were 94.6, 77.4, and 17.8%, respectively (Table 3). Cytology had significantly higher specificity than VIA, VILI, and HPV testing and had significantly lower sensitivity than VILI and HPV testing. Although VIA had a higher sensitivity than cytology, it did not reach statistical significance. HPV testing was 100% sensitive in detecting CIN3 lesions; however it had significantly lower specificity than VIA, VILI, and cytology (P < 0.001).
Table 3: Accuracy of the different screening tests for detection of cervical intraepithelial neoplasia.
Sequential combinations of VIA and VILI; HPV testing and VIA; HPV testing and VILI; and HPV testing and both visual tests substantially improved the specificity with minimal loss in sensitivity; parallel testing with the above tests resulted in substantial loss in specificity as compared to the use of single tests (Table 4). Sequential testing with VIA and cytology; VILI and cytology; and HPV testing and cytology resulted in substantial loss in sensitivity.
Table 4: Accuracy of the different screening test combinations to detect cervical intraepithelial neoplasia 2–3.
Of the 149 women with colposcopic abnormalities suggestive of cervical neoplasia, 124 received cold coagulation. Based on the biopsy reports, eight had LEEP and six women got simple hysterectomy done outside the study referral system. Of the 124 women who had cold coagulation on the basis of colposcopic abnormalities, 40 (32.3%) had no histological evidence of CIN. More than 95% of women with a final diagnosis of CIN2 and 3 lesions received treatment. During cold coagulation treatment, 31 (25.0%) complained of mild pain or cramps, one had mild bleeding, and one had a fainting attack. Among eight treated with LEEP, three had mild pain or cramps and one had a fainting attack during the procedure. Within 2 weeks of cold coagulation treatment, three reported having foul smelling discharge and one reported lower abdominal pain; one patient reported foul smelling vaginal discharge following LEEP. Of the 39, 19, and 25 patients with CIN1, 2, and 3 lesions who had cold coagulation, 22, 9, and 14 reported for follow-up between 6 and 9 months from treatment; 20 (90.9%), 9 (100%), and 11 (78.6%) of those followed-up, respectively, had no evidence of disease at follow-up. Of the eight women who were treated with LEEP, for the follow-up period between 6 and 9 months from treatment, four had no disease, three have not yet come for any follow-up, and one was diagnosed with invasive cancer based on the LEEP specimen and was referred for appropriate anticancer treatment.
Discussion
The improved survival of HIV-infected women following increasing availability and uptake of ART, the lack of impact of ART on the HPV-related disease, and the limited resources call for pragmatic, affordable yet effective screening practices to control the high risk of cervical cancer among those women in low and medium-resource countries. Those regions with a high burden of HIV/AIDS have also a high burden of cervical cancer [18,19] and a striking lack of cervical cancer prevention programmes.
Our study findings, based on the evaluation of four screening tests with minimal verification bias, provide important leads to feasible and effective screening strategies that yield high detection of true-positive disease with less frequent false-positive tests. We studied different combinations of tests to assess whether sequential or parallel testing with two tests resulted in more balanced detection of precursor lesions while avoiding false-positive outcomes as compared to screening with a single test. To our knowledge, very few studies have directly compared the performance of more than one screening test among HIV-infected women in terms of sensitivity, specificity, and predictive values in detecting CIN2 and 3 lesions with minimal verification bias; [20,21] and no study has yet compared three or four screening tests concurrently in such populations.
Our results indicate that HPV testing, VILI, and VIA have a higher sensitivity in detecting high-grade CIN than that of conventional cytology. Although cytology had a significantly higher specificity, it missed a higher proportion of CIN2–3 lesions. A higher sensitivity and lower specificity of VIA as compared to cytology has been reported in a previous study with minimal verification bias among HIV infected women in India [20]. As a single test, HPV testing had the highest sensitivity to detect CIN2–3 lesions and it detected 100% of CIN3 lesions in our study. It had significantly lower specificity than VIA, VILI, and cytology due to the high prevalence of HPV infection among HIV-infected women. The low specificity of HPV testing is a major limitation for its use as a sole screening test; hence adding a second test to avoid as many false-positive HPV tests without substantially affecting its sensitivity improves its utility and saves costs, particularly when a ‘screen-and-treat’ approach [22–24] is used to treat positive lesions without colposcopy/biopsy triage.
Visual inspection with acetic acid and VILI had similar test characteristics in our study and it is not surprising given that both tests were assessed by the same nurse. Both the visual tests are subjective, less reproducible and provider-dependent, and were found to have a wide range in performance in sensitivity and specificity among women in the general population in different settings [25–29]. The visual tests seem to perform better in HIV-infected women than in the general population due to the increased prevalence of high-grade lesions and the possibility of large lesions in the former [20].
Our findings confirm that parallel testing with two tests is not a suitable screening strategy in low-resource settings, given the high costs associated with co-testing all women, the low specificity (Table 4) and increased false-positive results leading to unnecessary investigations, treatments and anxiety. Parallel testing will result in substantial overtreatment in ‘screen-and-treat’ settings. However, sequential testing of VIA-positive women with VILI or HPV-positive women with VIA or VILI or both visual tests resulted in significantly increased specificity with negligible loss in sensitivity. Given the objectivity and high reproducibility of HPV testing, primary HPV testing followed by triaging HPV-positive women with VIA or VILI or both seems to be the most effective approach to detect high-grade CIN and to apply a ‘screen-and-treat’ strategy in HIV-infected women. Nevertheless, the high costs, infrastructure and consumable requirements make HPV testing less feasible in many low-resource settings at least for the time being. In such settings where HPV testing is not feasible, sequential testing with VIA and VILI is a suitable alternative option. Implementing sequential VIA-VILI-based screening within HIV/AIDS care programmes may facilitate the eventual introduction of the highly anticipated low-cost HPV-based rapid screening tests [30].
The high risk of cervical neoplasia in HIV-infected women necessitates repeated screening at intervals shorter than 3 years, although a single round of screening has been shown to reduce cervical cancer deaths significantly in the general population [31,32]. We plan to follow-up these women in our study with repeated screening at yearly intervals to further evaluate intensively repeated screening in HIV-infected women.
Prevalence of CIN2/3 lesions in our study was 4.9% and it is similar to some of the recent cross-sectional studies among HIV-infected women. In a study in Brazil, prevalence of CIN2/3 was 6% [33], in a study in Kenya it was 5.4% [34], in southwestern China it was 8.4% [35]; however, it was comparatively higher (16%) in a study in the same region of Maharashtra, India with much smaller sample size of 303 women [20].
It may be necessary to initiate screening at younger ages in HIV-infected women than in the general population and the optimum age of screening may be addressed in future studies. In our cohort; two women of 25 or less age had CIN3 lesions.
A novel feature of our study is that we used cold coagulation for ablative treatment of lesions colposcopically suggestive of CIN; directing biopsies before treatment proved to be well tolerated which allowed the establishment of histological diagnosis of treated lesions and the extent of overtreatment and reduced the chances of missing any preclinical early invasive cancer. This ‘see-and-treat’ approach resulted in overtreatment of 32% of women who had no histological evidence of CIN. Similar rates of unnecessary treatment following ablative treatment using cryotherapy in ‘see-and-treat’ settings have been previously documented and such overtreatment has not been associated with any severe adverse events during follow-up [36,37].
The challenges in maintaining cryotherapy services due to the high-cost refrigerant gas prompted us to evaluate the safety and effectiveness of cold coagulation as an alternative ablative treatment. Cold coagulation has been found to be a well tolerated and acceptable treatment in previous studies in general populations in the UK where it was shown to restore cervical cytology to normal in 1518 of 1628 (93%) women with CIN3 at 1 year from treatment, and among 1165/1205 (96.7%) women with CIN1 and 2 at 1 year from treatment [38–41]. We are not aware of any reports on the use, safety, and efficacy of cold coagulation in the treatment of high-grade CIN among HIV-infected women or in the general population in low and medium-resource countries, and we plan to evaluate this method of treatment by long-term follow-up of our HIV-infected women's cohort. Our preliminary findings indicate that it is a well tolerated and safe out-patient treatment for CIN and 6-month cure rates exceeded 80% which is encouraging.
Although our study design maximized detection of true-positive disease in the study population by offering colposcopy to all participants and directed biopsies in all cases of colposcopic abnormalities, the possibility of misclassification of disease outcomes due to subjective interpretations in colposcopy and histopathology cannot be entirely ruled out. We did not direct biopsies in women with colposcopically normal cervix due to ethical concerns and the harms associated with unnecessary invasive procedures in HIV-infected women. Another limitation in our study is simulation of sequential testing for different combinations of tests; in a real setting, the second test is provided with the knowledge on the first test and thus overestimation of specificity of sequential testing cannot ruled out.
We believe that HIV/AIDS care programmes are a suitable platform to provide cervical screening services not only to HIV-infected women but also the general population. The utility of providing VIA screening through such platforms have been demonstrated in some sub-Saharan African countries [24,42]. Our results provide compelling evidence to introduce sequential screening with VIA followed by VILI in HIV/AIDS clinics in India and in low-resource African settings rather than trying to introduce cytology screening as is done in some ART centres. HPV testing followed by visual screening may be considered when affordable, rapid HPV tests such as careHPV [30] become commercially available in low-resource countries.
Acknowledgements
We thank Dr Sanjay Pujari, Director of Institute for Infectious Diseases, Pune; Dr Shrinivas Natekar, Miraj and many nongovernmental organizations in Maharashtra for referring study participants. We thank Dr Sylla G Malvi, Consultant Cytologist, Nargis Dutt Memorial Cancer Hospital, Barshi; Mr. Madan K. Chauhan, Chief Coordinator of Projects, Nargis Dutt Memorial Cancer Hospital, Barshi and Dr Radhakrishna Pillai, Director, Rajiv Gandhi Centre for Biotechnology, Trivandrum. We thank Dr Jyothi Unni of Jehangir Hospital and Medical Centre, Pune for surgical treatment of study participants requiring hysterectomy or LEEP without any operating charges. We thank Mrs Evelyn Bayle, Ms Sandrine Montigny, and Mrs Krittika Guinot, IARC, for their help in the preparation of the manuscript.
Authors’ contributions: S.J. was responsible for the conduct of the study, and participated in its conception and design, monitoring, supervision, acquisition, and interpretation of the data and the provision of clinical services in the study.
R.S. had the initial idea and was responsible for the conception, study design, and the conduct, monitoring and supervision of the study, acquisition, analysis, and interpretation of the data.
R.M. was responsible for the statistical analysis of the data, monitoring the study, and the interpretation of the data.
V.K. participated in the conduct of the study and the acquisition and interpretation of the data.
T.S. was responsible for histopathology reporting, analysis, and interpretation of the data.
U.D. participated in the conception and study design, conduct and monitoring of the study, acquisition and interpretation of data.
All authors were involved in drafting the manuscript and have read and approved the text as submitted to AIDS.
R.S., as corresponding author, confirms that he had access to all data and had final responsibility for the decision to submit for publication.
Funding support: We gratefully acknowledge the funding support provided by the Union for International Cancer Control (UICC), Geneva under the UICC Cervical Cancer Initiative. We gratefully acknowledge Qiagen for the donation of HPV testing kits that facilitated the HPV testing in this study. We thank our study participants. We thank Dr Chaitali Gore, Dr Pratima Sheth, and Dr Trupti Darak for clinical assessment of participants and Mr Sandeep Bhingardiwe for data management.
Conflicts of interest
We declare that we have no conflicts of interest.
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