Archives of Medicine and Health Sciences

ORIGINAL ARTICLE
Year
: 2021  |  Volume : 9  |  Issue : 2  |  Page : 209--215

Detection and Genotyping of Human Papillomavirus among HIV-Infected Women from Belagavi: A District Place from the Southwest Indian State of Karnataka


Vinay Pala1, Chidanand Patil2, Mahantesh B Nagmoti3, Anita Dalal4, Arati Mane5,  
1 Departement of Microbiology, KAHER, Belagavi, Karnataka, India
2 Department of Microbiology, USM-KLE International Medical Programme, KAHER, Belagavi, Karnataka, India
3 Department of Microbiology, J N Medical College, KAHER, Belagavi, Karnataka, India
4 Department of Obstetrics and Gynecology, J N Medical College, KAHER, Belagavi, Karnataka, India
5 Division of Microbiology, National AIDS Research Institute, Pune, Maharashtra, India

Correspondence Address:
Mr. Vinay Pala
Department of Microbiology, KAHER, Belagavi, Karnataka
India

Abstract

Background and Aim: Human papillomavirus (HPV) infection is the established cause of cervical cancer. There is sparse literature with regard to HPV infection from the southern Belagavi region of India. This study was aimed to detect the HPV genotype distribution, the associated risk factors, and relation with cervical precancerous lesions among HIV-infected women from Belagavi, India. Materials and Methods: In this prospective observational study, a total of 214 HIV-infected women aged 18–45 years were recruited. Cervical samples were subjected to the Roche Linear Array assay for HPV detection and genotyping. Cervical status was determined by composite assessment of cytology, colposcopy, and histology. Data were analyzed using Software R version 3.6.0. Results: Of the 197/214 women with the adequate cervical sample, 86 (43.6%) were HPV positive, and 111 (56.3%) were HPV negative cases. A total of 132 (69.1%) women had normal cervical status, 26 (13.6%) had CIN1 lesions, 1 (0.5%) had CIN2 lesions, and 12 (6.3%) had CIN3 lesions. Single HPV infection was detected in 47 (54.6%) women and multiple (≥2) HPV genotypes were detected in 39 (45.3%). The HPV genotypes detected in descending order of frequency were HPV 16, HPV 33, HPV 35, HPV 52, and HPV 58. Ever pregnant (parous) women were 4.47 more likely to have HPV infection. Conclusion: A high prevalence of HPV infection, with a wide diversity of HPV genotypes and a greater prevalence of HPV 16 among HIV-positive women from Belagavi, India, was observed. Parity was the independent factor associated with HPV detection.



How to cite this article:
Pala V, Patil C, Nagmoti MB, Dalal A, Mane A. Detection and Genotyping of Human Papillomavirus among HIV-Infected Women from Belagavi: A District Place from the Southwest Indian State of Karnataka.Arch Med Health Sci 2021;9:209-215


How to cite this URL:
Pala V, Patil C, Nagmoti MB, Dalal A, Mane A. Detection and Genotyping of Human Papillomavirus among HIV-Infected Women from Belagavi: A District Place from the Southwest Indian State of Karnataka. Arch Med Health Sci [serial online] 2021 [cited 2022 Jan 29 ];9:209-215
Available from: https://www.amhsjournal.org/text.asp?2021/9/2/209/334024


Full Text



 Introduction



Human papillomavirus (HPV), a DNA virus, affects the basal epithelial cells, particularly of the skin and mucosal regions.[1] Among the identified HPV genotypes namely 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 66 are implicated in the causation of various types of cancer, including cervical, mucosal anogenital and head and neck cancers.[1] Furthermore, the chances of unidentified genotypes and possible chances of devastating ill effects exist.[2] Depending on their ability to cause cancers and lesions, the identified genotypes were broadly classified as high risk (HR) (genotype 16,18, 26,31,33,35,39,45,51,52,56,58,59, 66,68, 73,82) and low risk types (genotype 6,11,40,42,43,44,53,54,61,72,73,81).[3]

The transmission of HR HPV is directly attributed to several factors such as anatomical locations, HPV genotype, viral load, cofactors such as oral contraceptives, high parity, smoking, and sexually transmitted co-infections such as HIV and syphilis. In addition, being sexually transmitted infections, several studies have shown that HIV acts as an enhancer or co-factor of HPV infections.[4],[5] Highly comprised host immune systems of the HIV-infected patients are highly susceptible to infections. Many investigations elucidated that HIV-positive individuals are more likely to have multiple HPV infections and a high HPV viral load than their HIV-negative counterparts.[6],[7] In this regard, CDC has listed cervical cancer as one of the AIDS-defining illness. Meanwhile, several investigations have also reported HPV acts as a cofactor in HIV acquisition.[8]

In developed countries, the incidence of cervical cancer cases varies from 10 to 30 per 100,000 women.[9] The regional disparity in the incidence in India is due to a lack of necessary infrastructure and quality control of high-quality cytology screening that is not feasible for wide-scale implementation. It is reported that about 5% of the women in the general population in India harbor in the cervix.[10] Despite being the gold standard, Pap smear screening is not done routinely in Indian scenarios due to unawareness and casual attitude in women, lack of sensitivity and specificity, and regular follow-up visits.[11] Eventually, diagnosis of cancer is made only during advanced stages. Furthermore, the availability of HPV vaccines makes it essential to understand the geographical distribution of HPV genotypes.[2],[9],[10],[11],[12] Application HPV vaccines are effective in epidemiological studies in India will let public health to know about the vaccine used to protect Indian women. The older quadrivalent HPV vaccine covers five oncogenic types (31, 33, 45, 52, and 58). The new nine-valent vaccine added extra four genotype (6, 11, 16, and 18).[13] Currently three HPV vaccines–9-valent HPV vaccine (Gardasil® 9, 9 vHPV), quadrivalent HPV vaccine (Gardasil®, 4 vHPV), and bivalent HPV vaccine (Cervarix®, 2 vHPV) are available. All three HPV vaccines protect against HPV types 16 and 18 that cause most HPV cancers.

In another study from the Karnataka state, a comparative estimation of the virus infections of the cervix among women from the general and tribal population which revealed a different type-specific pattern of viral infection.[14] Studies showing HPV genotype distribution and their association with cervical disease status among HIV-infected women from the southern region of Belagavi, A distict place from southwest Indian state of Karnataka spare. Thus, this study was aimed to detect the HPV genotype distribution among HIV-infected women and to determine their association with cervical precancerous lesions.

 Materials and Methods



The study was approved by the Ethical Committee of KLE University Belagavi, India (Ref No. KLEU/Ethic/2012-13/D-4573). This prospective observational study was carried out for 1 year from June 2013 to June 2014 in the Obstetrics and Gynecology Clinic of KLE's Prabhakar Kore Hospital and Medical Research Center, Belagavi, India. By convenient sampling technique, consecutive confirmed HIV-1 positive women, regardless of their current status of CD4+ count and antiretroviral treatment (ART) usage attending the clinic were recruited. After obtaining a written informed consent form, a total of 214 HIV-positive women aged 18–45 years, were enrolled in the study. Patients with a history of hysterectomy, abnormal bleeding who were pregnant and in active labor were excluded from the study. Other factors like hormonal contraceptives and tobacco smoking might influence the HPV infection, however, these factors are not included in the study.

Demographic data of all the participants were collected and recorded in a precoded pro forma. All patients underwent routine gynecological examination and were counseled on the risk of sexual behavior and provided with contraceptives if needed. A standardized noninvasive colposcopy examination was performed by a trained gynecologist and confirmatory procedures about histology were performed in participants with clinical evidence of cervical abnormalities. The colposcopy and histology results were reported as per Richart cervical intraepithelial neoplasia (CIN) system. Cervical smears were collected with the help of Ayer's spatula and cytobrush from the squamocolumnar junction. The glass slides were then fixed by using 95% ethyl alcohol. The smears were stained with Papanicolaou stain, and findings were reported according to the revised Bethesda system (2001). Cervical status for the participants was classified as “normal” based on normal cytology, “low grade” based on either low-grade squamous intraepithelial lesions on cytology or CIN1/2 on histology, and “high grade” based on high-grade squamous intraepithelial lesions on cytology or CIN3 and severe/invasive cancer on histology.[15]

Human papillomavirus genotyping

Cervical samples were collected from the transformation zone of the cervix immediately stored in the PreservCyt Solution at 4³C and transported to the laboratory at the Department of Microbiology where they were further stored at −20³C until DNA extraction. HPV detection and genotyping were done with the Linear array HPV genotyping test (Roche, Branchburg, NJ, USA). HPV and cellular DNA was released by lysing cervical cell specimens using lysis buffer under denaturing conditions at elevated temperatures in the presence of proteinase K followed by DNA purification in columns with a silica-based membrane using vacuum processing. All the pre- and post-DNA extractions were carried out in a separate room to avoid errors due to contamination. The polymerase chain reaction (PCR) amplicons were denatured and subjected to hybridization on linear array HPV genotyping strips coated with HPV type-specific and human beta-globin probes according to the manufacturer's instructions. The biotin-labeled amplicons hybridized to the probes only if the type-specific sequence matched those of the amplicons. The biotin-labeled amplicons were detected by colorimetric development, and the results were read visually by comparing the pattern of colored lines to the provided reference guide. Each run was performed with negative and positive controls provided by the manufacturer to monitor the quality and performance of the assay.

Statistical analysis

Statistical analysis was performed using software R version 3.6.0. please change to software R version 3.6.0 (R Core Team, Vienna, Austria). The normality of the data was determined using the Shapiro–Wilk test. Continuous variables with normal distribution were presented as mean ± standard deviation and compared using paired t-test, whereas Chi-square test was employed for dichotomous data. Mann–Whitney U test was performed for variables without normal distribution. Categorical variables were presented as frequencies and percentages. Factors influencing the HPV were analyzed logistic regression test. Risk was also evaluated by the prevalence odds ratio and their 95% confidence interval (CI). A P < 0.05 was considered statistically significant at 95% CI.

 Results



Out of 214, 197 samples were considered for further analysis, as the remaining 17 samples were contaminated. The mean age of the women was 33.93 ± 5.7 years and the median CD4 + count was 468 cells/mm3 (interquartile range: 344–629). Majority of the women were receiving ART (96.26%). Sixty-nine (32.2%) patients are married and cohabiting with their husbands, 59 (27.6%) were illiterate, 28 (59.8%) reported total family income < 2500 INR monthly. A total of 20.09% of the study participants reported age at first sexual intercourse as 18 years. Twenty-five (11.7%) patients are reported to have ≥2 lifetime sexual partners.

A total of 132 (69.1%) women had normal cervical status, 26 (13.6%) had CIN1 lesions, 1 (0.5%) had CIN2 lesions, 12 (6.3%) had CIN3 lesions, while the rest had inadequate colposcopy. None of the women had invasive cervical cancer.

Out of 197 participants, 86 (43.6%) cases were detected with any HPV genotype; single HPV genotypes were found in 47 (54.6%) and multiple (≥2) HPV genotypes were found in 39 (45.3%) cases. 111 women were negative for HPV infection. The HPV genotypes detected in descending order of frequency were HPV 16, HPV 33, HPV 35, HPV 52, and HPV 58. The distribution of HPV genotypes by cervical status, among HIV-infected women is presented in [Table 1]. HPV 16 and HPV 72 were the significant genotypes detected in women with CIN lesions as compared to those with normal cervical status [Table 1].{Table 1}

The association of participant characteristics with HPV positivity is depicted in [Table 2]. By logistic regression model, ever pregnant women or parity was found to be at the significant risk factor of getting any HPV infection. By multivariable logistic regression, the presence of non/un carcinogenic, possibly carcinogenic, carcinogenic, and single and multiple HPV showed no significant association with CIN2+ lesions [Table 3].{Table 2}{Table 3}

 Discussion



A high percentage of research indicated that HIV-infected women are at higher risk of developing cervical cancer due to the persistence of immunosuppression. Furthermore, higher prevalence of multiple opportunistic infections and a broad range of HPV genotypes were reported in HIV-infected women.[12] To the best of our knowledge, this cross-sectional study is the largest one to date to determine the prevalence and distribution of specific HPV genotypes among HIV-infected women in Belagavi, A distict place from the southwest Indian state of Karnataka.

The present study documented an overall higher prevalence of HPV infection (43.6%) with multiple genotypes indicating that HIV-positive women are indeed at greater risk for the development of cervical cancer. Recent research has demonstrated different findings for HPV prevalence among HIV-infected women; furthermore, the tendency to a higher prevalence among HIV-infected cohort is invariably identified.[6],[8] Globally, the estimates of the prevalence of HPV among these cohorts varied by the level of the HIV epidemic and region.[5] This variation of HPV prevalence in various geographical locales could be to differing immunological and behavioral status of the participants, as well as the differences in sensitivity of the assays and primers used for PCR.[14]

In the present study, diverse HPV genotypes were detected in women with both normal services as well as with cervical precancerous disease. This diversified HPV genotypes in HIV-infected women are more frequently associated with the activation or persistence of pre-existing infections, due to reduced immunity.[15] Minkoff et al. and Sun et al. reported HPV DNA prevalence in their studies as 73% and 60% of HIV-infected women, respectively. Studies conducted in Sao Paulo and Brazil also showed 98% and 100% of HPV among HIV-infected women, respectively.[16],[17],[18],[19]

In the present study, HPV 16 was the commonest genotype found among HIV-infected women, followed by HPV 33, HPV 52, and HPV 58. Similar to our study findings, Mane et al.[15] also reported that HPV 16, HPV 52, and HPV 58 as the most commonly encountered genotypes in their study conducted in HIV-infected women in the Western part of the country.[14] In contrast, Luque et al. and Badial et al. reported a greater prevalence of other genotypes among HIV-positive women.[20],[21] Generally, HPV 16 has good evolutionary ability to escape the immune surveillance effects, while genotypes other than HPV 16 are often controlled by immunity.[22],[23] Research also indicated that increased HPV prevalence are in direct proportion with the severity of immunosuppression.[14] We found that parity as the only factor is significantly associated with any HPV infection. This can be attributed to the changed hormonal milieu due to pregnancy and immune response might favor the presence or persistence of HPV infection.[24],[25] According to Liu et al., 2018 HIV-positive women have higher risk of acquiring HPV, with risk inversely associated with CD4 cell count.[26] According to Levi et al., 2002 HIV-positive women have higher risk of acquiring HPV, with risk inversely associated with CD4 cell count.[19]

Our study had its limitations, and first we did not consider the ART duration for further evaluation of differences between the immunocompetent and immune-replete status of women on ART. Second, we did not follow-up the patients after hospital discharge. Also, we acknowledge that we will not be able to generalize the findings due to the small sample size and single-centered nature of the study, which emphasizes the need for larger and prospective cohort studies further to elucidate the association of immunosuppression with HPV risk. Our study definitely provide incident data, however, it requires further screening at primary health centers to have the effective study of incidence and/or prevalence

 Conclusion



We observed a high prevalence of HPV infection among HIV-positive women from Belagavi, India, with a wide diversity of HPV genotypes and a greater prevalence of HPV 16. Being pregnant (parity) was the only independent factor associated with HPV detection, warranting further studies to determine the correlation with pregnancy outcomes. In Cosmo Politian cities/3 tier cities there is a gap in the knowledge which might be essential for the policymakers, and also present publication might help treating clinicians towards incidence data. The present finding is also having relevance to cancer prevention programs early screening of the HPV can definitely help in early diagnosis and treatment, which in turn might reduce mortality. However, our study might not directly associated with HVP vaccine.

Acknowledgments

We are grateful to the National AIDS Research Institute (NARI) for supporting our study during the processing and testing of samples at NARI, Pune, India.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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