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Year : 2019  |  Volume : 37  |  Issue : 4  |  Page : 557--562

Types of human papillomavirus observed in hospital-based population

Priyanka Wagh1, Priyanka Kulkarni1, Shilpa Kerkar1, Hemant Tongaonkar2, Kedar Deodhar3, Bharat Rekhi3, Vinita Salvi4, Hemangi Chaudhari4, Himangi Warke4, Jayanti Mania-Pramanik1,  
1 Department of Infectious Diseases Biology, ICMR-National Institute for Research in Reproductive Health, Mumbai, Maharashtra, India
2 Department of Urologic and Gynecologic Oncology, Hinduja Hospital, Mahim; Department of Urology and Gynecologic Oncology, Tata Memorial Hospital, Mumbai, Maharashtra, India
3 Department of Pathology, Tata Memorial Hospital, Mumbai, Maharashtra, India
4 Department of Obstetrics and Gynecology, Seth G. S. Medical College, KEM Hospital, Parel, Mumbai, Maharashtra, India

Correspondence Address:
Dr. Jayanti Mania-Pramanik
ICMR-National Institute for Research in Reproductive Health, Parel, Mumbai - 400 012, Maharashtra


Background and Objectives: Human papillomavirus (HPV) is the causative agent of cervical cancer, a major cause of cancer mortality in Indian women. The current study was undertaken to add information to the existing data on HPV type distribution in Indians, in an attempt to document HPV types for future vaccination programme, if any. Materials and Methods: HPV infection was screened in 223 cervical cancer cases and 2408 healthy women without cancer and cervical intraepithelial neoplasia (control). HPV was typed using polymerase chain reaction, Southern hybridisation using specific probes and HPV GenoArray (Hybribio) test. Results: HPV DNA was found in 92.8% of cases and 7.3% of controls. Of the 383 HPV-infected women, 30.0% had single infection; 50.9% had multiple infections (two or more types) and 19.1% were infected with HPV types other than HPV-16, -18, -6 and -11. Besides HPV-16, HPV-51 and HPV-33 were also seen as single infection in cases. In cases, HPV-18 or its homologous HPV-45 was always present as co-infection with HPV-16 or with other high-risk type. Binary logistic regression (backward) analysis highlighted significant association of age, parity and socioeconomic status with HPV infection. The present study highlighted the presence of multiple HPV infection (186 of 207, 89.9%) along with HPV-16 in women with cervical cancer. In control, 27.3% were co-infected with other sexually transmitted infections, while Chlamydia trachomatis infection was seen in 13% of cases. Conclusions: The study highlighted the type of HPV infection seen among the hospital-based population. For better screening, HPV tests available in the market should include all the types seen in the population.

How to cite this article:
Wagh P, Kulkarni P, Kerkar S, Tongaonkar H, Deodhar K, Rekhi B, Salvi V, Chaudhari H, Warke H, Mania-Pramanik J. Types of human papillomavirus observed in hospital-based population.Indian J Med Microbiol 2019;37:557-562

How to cite this URL:
Wagh P, Kulkarni P, Kerkar S, Tongaonkar H, Deodhar K, Rekhi B, Salvi V, Chaudhari H, Warke H, Mania-Pramanik J. Types of human papillomavirus observed in hospital-based population. Indian J Med Microbiol [serial online] 2019 [cited 2020 Oct 27 ];37:557-562
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Cervical cancer is the major cause of cancer mortality in Indian women, 60,078 die every year, with an incidence of 14.9/100,000.[1] High-risk human papillomavirus (HR-HPV) types are associated with cervical cancer. HPV infection is transient and persists only in a minority, leading to high-grade cervical intraepithelial neoplasia and invasive cancer. According to their oncogenic potential, HPV types are classified into low-risk (LR) types (HPV-6, -11, -40, -42, -43, -44, -54, -61, -70, -72, -81 and -89), probable HR types (HPV-26, -53, -66, -68, -73 and -82) and HR types such as HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58 and -59.[2] The International Agency for Research on Cancer confirmed HPV-16 as the most common type, followed by HPV-18, together accounting for 70% of the HPVs identified in invasive cervical cancer (ICC) specimens. Frequency of the next six most common HPV types (HPV-45, -31, -33, -52, -58 and -35) was similar, accounting for an additional 20% of cervical cancers worldwide.[1] Compared to the disease burden,information on HPV type distribution among Indian women is scanty and region specific.[3],[4] Most studies in India have assessed the presence of HPV-16 and -18 in cervical cancer tissues. A meta-analysis of HPV genotype distribution listed Indian studies on multiple HPV types in ICC.[5] These studies suggested that HPV-16 and -18 prevalence is higher in India to the tune of 82%. Knowledge of HPV infection is important in assessing the risk of a woman with abnormal cervical smears in developing cervical cancer. Vaccines targeting HPV-16 and -18 have been shown to prevent persistent infection with these types. The spectrum of HPV types targeted in current vaccine trials is based largely on data from developed world.[6] There are three prophylactic vaccines: Gardasil® (Merck Sharp and Dohme Corp., New Jersey, United States of America) is a quadrivalent vaccine against four HPV types (types 6, 11, 16 and 18) for the prevention of high-grade cervical lesions and benign warts. The most recent Gardasil 9 prevents infection with the same four HPV types plus five additional HR-HPV types (31, 33, 45, 52 and 58) and is therefore called a nonavalent, or 9-valent, vaccine. This is also in use to prevent infection and related external genital lesions/warts in males.[7] The other bivalent vaccine, Cervarix® (GlaxoSmithKline, Middlesex, United Kingdom), provides protection against only two HR-HPV types 16 and 18 although it has demonstrated cross-protection benefits. Depending on the age of the individual, all the three vaccines are given through a series of two to three intramuscular injections with an interval of 6-month period.[8] The Food and Drug Administration has approved Gardasil and Gardasil 9 for use in females aged 9 through 26 for the prevention of HPV-caused cervical, vulvar, vaginal and anal cancers; precancerous cervical, vulvar, vaginal and anal lesions and genital warts. Gardasil and Gardasil 9 are also approved for use in males for the prevention of HPV-caused anal cancer, precancerous anal lesions and genital warts. Gardasil is approved for use in males aged 9 through 26, whereas Gardasil 9 is approved for use in males aged 9 through 45.[9],[10]

Because India is yet to initiate a programme for the implementation of HPV vaccination, it becomes imperative to record the infections and their types in hospital-based population. Hence, we aimed to screen HPV infections and their type distribution in women with and without cervical cancer, in an attempt to gather more relevant information to support preventive programmes.

 Materials and Methods


The study was taken up as an exploratory study to evaluate the prevalence and types of HPV infection in hospital-based population. The study protocol was approved by both the participating institutional ethics committees (ethics approval no. of institute: 151/2009, 289/2016, ethical approval no. K.E.M Hospital : EC/GOVT-2/2011 and project ethics no. of Tata Memorial Hospital: 731). The sample size was determined on the basis of a meta-analysis report of 11.9% HPV prevalence in the general population,[5] hence, to screen at least 2000 women with Papanicolaou (Pap)-negative cytology report indicating a healthy cervix to get at least 200 of them with HPV infection. The feasibility of screening within a specific period was also taken into consideration. Accordingly, an equal number of cancer cases (n = 200, women with cervical cancer) attending the outpatient department (between 2010 and 2015) of a tertiary cancer referral Tata Memorial Hospital, were informed about the study. Another group of women without any cervical abnormality as per Pap-negative reports attending the outpatient department (between 2010 and 2017) of Obstetrics and Gynecology, Seth Gordhandas Sunderdas Medical College and King Edward Memorial Hospital, Mumbai, were informed about the study. To confirm the findings of HPV infection in fresh biopsy samples, another lot of paraffin-embedded tissue blocks (n = 39) containing a large part of the resected cervical tumour tissue, retrieved from the archives of the department of pathology, were also included for HPV typing. The archived samples were used after the permission from the ethical committees for waiver of consent.



Tissue biopsies from clinically diagnosed cervical cancer cases were collected in 0.1 M phosphate-buffered saline (PBS) solution. In controls, two endocervical specimens using cytobrush were collected; one in 1 ml PBS to detect HPV and Chlamydia trachomatis infection by polymerase chain reaction (PCR) and the other kept as dry swab for quality control analysis. Cervical smear was taken for Pap staining. Vaginal swab specimens were collected from the posterior fornix for smear preparation for the detection of Bacterial vaginosis (BV) and Candida infection and for wet mount to detect Trichomonas infection.

Polymerase chain reaction for human papillomavirus

DNA was isolated from specimens using non-enzymatic salting-out method manually. The cells present in the specimen were pelleted and resuspended in Tris-MgCl2-KCl buffer (pH 7.4) and treated with 10% sodium dodecyl sulphate at 55°C for 10 min to lyse the cells. The proteins were precipitated using saturated sodium chloride solution. DNA was precipitated using 100% ethanol and eluted in Tris ethylenediaminetetraacetic acid buffer.[11] The quality and quantity of DNA were estimated by electrophoresis and spectrophotometry, respectively. PCR for the β-globin gene was also performed for each sample as an internal control to rule out the presence of inhibitory factors in the extracted specimen.[12] PCR was performed in this extracted DNA using consensus primers (MY09/11) designed from L1 region of the capsid protein.[13] Presence of 450 bp indicated the presence of HPV infection. Further, to confirm this PCR product of 450 bp, standard protocol for Southern blotting was carried out using specific generic probes for HPV. Once the sample was positive for HPV by Southern blot, specific probes such as HPV-16, -18, -11 and -6 were used for their typing.[12] Other probes were not available for use in Southern blotting, hence could not be typed [Figure 1]. Cervical biopsy samples were processed by PCR-Southern blotting as well as by HPV GenoArray (GA) test (Hybribio Ltd., Hong Kong, China). It utilises L1 consensus primers to amplify 21 HPV genotypes, followed by flow-through hybridisation with immobilised genotype-specific probes. It is marked as Conformité Européenne (CE) for use in Europe and China. Both high- (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) and low-risk (6, 11, 42, 43 and 44) as well as the undetermined-risk HPV types (HPV-53 and CP8304/81) were identified. Positive and negative internal controls were included in each run. Randomly selected specimens (5% of the tested specimens) were cross checked to evaluate the reproducibility of these two typing methods by direct sequencing using DNA sequencer by Sanger method (ABI 3130-XL genetic analyser, Thermofisher Scientific, Waltham, MA USA 02451).{Figure 1}

Screening of other infections

Per-speculum examination recorded the signs of infection in the control group. Vaginal smear was used to detect BV and Candida using Gram staining. Vaginal secretion in normal saline was examined under microscope to detect Trichomonas infection. C. trachomatis was screened both in cases and controls using in-house PCR.[14]

Statistical analysis

Data were analysed with SPSS 19 software (SPSS Inc., Chicago, IL, USA). Univariate binary logistic regression analysis was performed to assess the association of HPV with different demographic factors. Odds ratio at 95% confidence interval was calculated. The tests were considered statistically significant if P ≤ 0.05.


Enrolment details of cases and controls and the infections detected are depicted in [Figure 2]. Quality control analysis in randomly selected 5% of samples matched to the original results. Among the 2408 women with healthy cervix (Pap negatives) enrolled as control for screening of HPV infection, 7.3% (176/2408) were HPV positive (mean age: 35.1 ± 3.8 years) and 27.3% (48/176) were co-infected with other infections, such as C. trachomatis, BV and Candida [Figure 2]. HPV type distribution revealed the presence of both high- and low-risk HPV infection in this group [Table 1]. Majority of the infected women (61.4%, 108 of 176) in the control group were asymptomatic. Co-infection of C. trachomatis and HPV was seen in 13.1% of cases and 15.3% of controls.{Figure 2}{Table 1}

Among the 223 enrolled cases (mean age: 48.6 ± 10.8 years), 184 were fresh cervical tissue biopsies and 39 were archived paraffin blocks of biopsy tissue. Squamous cell carcinoma, adenocarcinoma, adenosquamous cell carcinoma and vault recurrence were seen in 91.7%, 6.1%, 0.5% and 1.6% of women, respectively. Among the cases, 92.8% (n = 207) showed the presence of HPV infection. However, due to inadequate DNA sample in 64, only HPV-16, -18, -6 and -11 could be detected by Southern hybridisation, while flow-through hybridisation could be performed only in 143 cases [Table 1] and [Figure 1]. The most common five HPV types seen in these cases were HPV-16, HPV-51 and HPV-33 either as single or with other types, whereas HPV-18 and -45 were always found as a co-infection or with multiple HPV type [Table 1]. HPV-16 infection was detected in 89.9% of the cases. In cases, the percentage prevalence of HPV types in decreasing order was HPV-16 (89.9%), -18 (43.9%), -45 (43.9%), -51 (19.9%), -33 (9.9%), -31 (2.1%), -58 (2.1%), -52 (1.4%), -56 (0.7%) and HPV-59 (0.7%). HR-HPV types 39, 66 and 68 and the low or intermediate HPV-42, -43, -44, -53 and -81 were absent in the study population.

Thus, of the 383 HPV-infected women (207 cases and 176 controls), 30.0% had single infection, 50.9% had multiple infections (more than two types) and 19.1% were infected with HPV type other than HPV-16/18/6/11.

Univariate binary logistic regression analysis revealed that age, parity and socioeconomic status were significantly associated with HPV infection [Table 2].{Table 2}


The prevalence of HPV in cervical cancer cases (92.8%) was similar to that of a meta-analysis report[5] and a report from Solapur and Osmanabad districts of Maharashtra.[4] HPV-negative women with cervical cancer might have low viral load or absence of HPV DNA in the specific site of tumours or loss of viral genomes due to the tumour's own genetic instability.[15] The prevalence (7.3%) of HPV in the control group was similar to that of other reports,[1],[16] however comparatively low to the meta-analysis (11.9%) and region-specific reports.[3],[5] HPV typing for control samples was carried out using Southern blotting as viral DNA concentration is low in cytologically normal population, whereas in confirmed cervical cases with fresh biopsy samples, HPV typing was done by PCR-Southern blotting as well as by commercially available HPV typing Hybribio kit.

HPV-16 as single or co-infection with two or more HPV types in 186 (89.9%) cases, again confirmed HPV-16 as the major aetiological agent in the population. Single infection (HPV-16, -6, -33 and -51) was present only in 26.1% of cases, which is low compared to the meta-analysis report (81%) or in other studies.[5] This might be due to the use of advanced technique for the identification of more number of HPV types. The study also highlighted the association of multiple infections along with HPV-16 with cervical cancer. The meta-analysis report (1990–2010) also suggested an increase in the prevalence of multiple types of HPV in cancer cases over a period, while a decreased rate of single infection.[17] The biological significance of multiple HPV-type infection was not known. However, this is an important observation in cervical cancer cases that highlights the use of multivalent vaccine.[18]

In cases, the other most common HPV types were HPV-18 and -45. Frequent presence of HPV-16, -18 and -45 in cancer cases was similar to that of the reports from Delhi/North India.[19] The present study revealed the absence of HPV-18 as single infection in cases, indicating that HPV-18 might not be solely associated with cervical cancer. Absence of other HR-HPV types (HPV types 39 and 68), which are homologous to HPV-18, also supports this observation. A similar report from Chittaranjan National Cancer Institute, India, also observed that high-grade intraepithelial lesion cytology was more frequent in women with HPV-16, though the prevalence of HPV-18 was more in women without cervical cancer in the age group of 25–65 years.[16] This suggested that elderly women with HPV-18 did not show any cervical abnormality. The meta-analysis on HPV type also suggested an increased prevalence of HPV-16 over a period (1990–2010), while no significant change in HPV-18 prevalence. Another remarkable finding was the high prevalence of HPV-45 in cases, not reported in any Indian studies. Even among the cases with adenocarcinoma or adeno-squamous carcinoma, HPV-16 was present in all the cases, whereas only 72.7% had HPV-18.[18] In ten cases, HPV-16 was present with other HPV types (HPV types 33, 35, 45,51, 52, 56 and 58). A similar observation was reported from the same Cancer Hospital.[13] We also reported a high rate of HPV-51 (19.86%) in cases compared to that of an earlier report (0.9%–1.9%) from India.[3] HPV-35 was seen only in one case, which was more frequent in South Indian population.[20]

We had observed multiple HPV types in cancer cases; however, all were negative for HPV-11. In addition, not a single study reported the presence of HPV-11 even with HPV-16 in cervical cancer. It was interesting to note that women with HR-HPV types along with HPV-11 as co-infection, had normal cervix.

In the control population, the prevalence of HPV-16 and/or HPV-18 (3.6% vs. 4.7%) was comparable, while the prevalence of HPV-11 (1.7% vs. 0.1%) was higher compared to that of ICO/WHO report.[1] Presence of C. trachomatis in cervical cancer cases could be one of the causes of invasive squamous cell carcinoma.[21] The high frequency of sexually transmitted infections in HPV-infected controls highlighted the importance of their screening.

The current HPV Virus-like particles (VLPs) L1 vaccines offer type-specific protection and partial protection against the homologous types, but fail to cross protect against heterotypic infections. In the present study, the number of cases encountered with mixed infections was more, and there was a lot of difference in the frequencies of homologous versus heterologous HPV types. Hence, for a developing country like India, a multivalent vaccine to protect all HR-HPV genotypes prevalent in the region is in need. The results of the use of the most recent vaccine Gardasil 9 need to be established to document its effectiveness in the long run. Reports from India and World have indicated that other less prevalent HR-HPV types are evolving as a causative agent of cervical cancer.[5] HPV typing can manage further complications, when a single round of HPV testing in women is associated with a significant reduction in the number of advanced cervical cancers and death.[22] Although visual inspection with acetic acid for cervical cancer in low-income settings is recommended, HPV testing for cervical cancer has far-better outcome in preventing the incidence of cervical cancer. The HPV screening might reduce the cost of cervical cancer prevention programme.

In the current study, univariate binary logistic regression analysis revealed that age, parity and socioeconomic status were significantly associated with HPV infection. As reported previously, high parity maybe associated with cervical cancer, as it maintains transformation zone on the exocervix for many years, thereby facilitating the direct exposure to HPV.[23] Even significantly high rate of HPV infection was observed among post menopause women. The high incidence of HPV in older women may be due to the reactivation of latent HPV infection because of immune senescence and weakened immune response.[24]

This study has two limitations: (i) small number of samples tested and (ii) absence of long-term follow-up of control women with HR-HPV16. However, the significant findings outweigh these limitations. The present study reported the distribution of HPV types in hospital population. The study highlighted that besides HPV-16, -51 and -33, multiple types of HPV infection are seen in cervical cancer. These significant observations might help in the success of cervical cancer prevention programmes, including vaccination programme.


The study highlighted the association of multiple HPV types and cofactors with cervical cancer, type of HPV infection as well as co-infections seen among the hospital-based population. For better screening, HPV tests available in the market should include all the types seen in the population.


We thank all participants who agreed to enroll themselves in this study. We acknowledge the financial support provided by Indian Council of Medical Research (ICMR)-National Institute for Research in Reproductive Health to conduct this research project, TATA Memorial Hospital, Seth G.S. Medical College and KEM Hospitals, Mumbai, for their clinical support. We also acknowledge the ICMR for the Senior Research Fellowship to Mrs Priyanka Wagh.

Financial support and sponsorship

This study was financially supported by the ICMR-National Institute for Research in Reproductive Health.

Conflicts of interest

There are no conflicts of interest.


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