|Ahead of print publication
Detection of human papillomavirus high-risk genotypes in rural women of Lucknow, North India
Anand Narian Srivastava1, Jata Shanker Misra1, Saliha Rizvi2
1 Department of Pathology, Era's Lucknow Medical College and Hospital, Era University, Lucknow, Uttar Pradesh, India
2 Department of Biotechonology, Era's Lucknow Medical College and Hospital, Era University, Lucknow, Uttar Pradesh, India
|Date of Submission||21-Aug-2019|
|Date of Decision||30-Oct-2019|
|Date of Acceptance||30-Dec-2019|
|Date of Web Publication||07-Apr-2021|
Jata Shanker Misra,
Department of Pathology, Era's Lucknow Medical College and Hospital, Era University, Lucknow, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Background: Human papilloma virus (HPV) has been widely implicated in cervical carcinogenesis and 90% of carcinoma cervix cases are due to high-risk HPV infection. This study was done to find the high-risk HPV genotypes in the rural women of Lucknow, North India.
Materials and Methods: HPV-DNA testing has been carried out in 130 cases of squamous intraepithelial lesions (SILs) of the cervix to find HPV status and type of high-risk HPV genotype infecting the rural women. These cases were collected from the rural cervical cancer screening program carried out in the villages of West Lucknow, North India.
Results: HPV status in 130 SIL cases revealed HPV positivity in only 17 cases (13.1%), whereas the remaining 113 cases were HPV negative (86.9%). HPV genotypes detected in the study were HPV-18, HPV-31, HPV-33, and HPV-35. HPV positivity was found highly associated with the young and sexually active group of women complaining of vaginal discharge. High HPV infection rate was also seen with multiparity and illiteracy as majority of women attending the camps were multiparous and illiterate.
Conclusions: The present study revealed highly oncogenic HPV-18 alone or in combination with multiple infections of high-risk genotypes – 31, 33, and 35 – in the rural women of Lucknow, North India. Since HPV vaccine currently available in India is for HPV-16 and HPV-18 combined, efforts should be made to make region-specific vaccine according to their prevalence in that particular state of the country to provide effective HPV vaccination.
Keywords: Cervical cancer, human papillomavirus genotypes, multiparity, squamous intraepithelial lesion, vaginal discharge
| > Introduction|| |
Carcinoma cervix is a major public health problem worldwide. It is the major cause of cancer mortality in Indian women. Cytological screening is a standard method for control of carcinoma cervix in the rural population of India, but organized screening programs are rare. In the last few decades, human papilloma virus (HPV) has been implicated in the causation of cervical cancer, and it has been found that more than 90% of cervical malignancies are due to HPV infection of high-risk types. There are 184 different HPV genotypes and only 40 of them infect the anogenital region. These forty HPV types have been classified into three classes based on their oncogenic potential: high-risk, intermediate-, and low-risk types. Included in the high-risk types are 15 of which HPV-16 and HPV-18 are highly oncogenic. There is a wide variation in the prevalence of HPV infection and the genotype distribution which is attributed to the diversified socioeconomic and geoclimatic conditions.
HPV is a common sexually transmitted virus, and majority of these infections are benign and transient; persistent infection is associated with development of carcinoma cervix. Hence, high-risk HPV infection has been used as a great risk factor in cervical carcinogenesis. The cervical cancer screening program is in progress in rural women population of Lucknow, North India, since May 2013, and till May 2019, a total of 2776 women have been cytologically evaluated who attended camps organized in villages after proper counseling and motivation. A total of 488 squamous intraepithelial lesion (SIL) cases have been diagnosed (462 low-grade SIL [LSIL] and 26 high-grade SIL [HSIL]) in these 2776 women. Since we have performed HPV-DNA testing for high-risk types in 130 SIL cases (15 – HSIL and 115 – LSIL), we thought it interesting to report the prevalence and association of different high-risk HPV types detected with different risk factors of cervical cancer in this north region of India.
| > Materials and Methods|| |
For HPV-DNA testing, cervical scrapes were collected from the squamocolumnar junction of the cervix of the women attending rural camps for cervical cancer screening in the villages of Lucknow West. The samples were collected in saline and stored at −20° C before processing the material. The DNA was isolated from the collected material before HPV-DNA testing. Informed consent was obtained from the patients on the Pap smear form as thumb impression if the patient was illiterate or signature if educated.
HPV-DNA testing has been performed in 130 SIL cases using Gene Nav Q1 PCR kit which is manufactured by the GenomeMe, Canada. This kit was found to be more economical and effective and identifies 14 types of high-risk HPVs including HPV-16 and HPV-18. It is also capable of distinguishing HPV-16 and HPV-18 from all the other types. The internal control, human ACTIN, indicates the quality of extracted DNA. Four channels (FAM, HEX, ROX, and CY5) are used in this PCR assay.
Statistical analysis of the data was carried out by applying Chi-square test. The software used was SPSS and version was 22 (IBM North America, New York, USA). Ethical clearance was obtained from the Ethical Committee of the college for performing HPV-DNA testing in SIL cases detected during the cervical cancer screening.
| > Results|| |
HPV-DNA testing carried out in 130 SIL cases revealed HPV positivity in only 17 (13.1%) and the remaining 113 SIL cases were HPV negative (86.9%).
Of the 130 SIL cases, 15 were HSIL and the remaining 115 were LSIL. Only 5 of the HSIL cases were found HPV positive (33.3%), whereas the remaining 10 were HPV negative. Similarly, only 12 of the 115 LSIL cases were found HPV positive (10.4%), whereas the remaining 103 were HPV negative (89.6%). The difference in the HPV positivity rate in HSIL and LSIL cases was found to be statistically significant (χ2 = 6.12; P = 0.015).
In the five HSIL cases who were found HPV positive, the following strains of HPV were found.
- HPV-18 three cases
- HPV-31, HPV-33, and HPV-35 (multiple infections) two cases.
Thus, HPV-18 which is highly oncogenic was more common in HSIL cases (60%) than other HPV strains of 31, 33, and 35 (40%). On the other hand, in 12 LSIL cases who were HPV positive, only multiple strains of HPV were seen.
- HPV-31, HPV-33, and HPV-35 (including HPV-18) - 4 cases
- Only HPV-31, HPV-33, and HPV-35 - 8 cases.
Thus, in LSIL cases, multiple strains of HPV-31, HPV-33, and HPV-35 were more common (66.6%). In the remaining 33.3% of cases, it was also associated with HPV-18. Thus, on the whole, a single HPV type was seen in only 3 of the 17 SIL cases (17.6%), whereas in the remaining 14 cases (82.4%), the HPV positivity was multiple. The difference in the prevalence rate of high oncogenic HPV-18 in HSIL and LSIL cases was found to be statistically significant (χ2 = 8.74; P = 0.003).
Follow-up was available in 13 of the 17 HPV-positive cases, and progression or persistence of SIL was seen in 8 cases (61.5%). However, this was seen in only 9 cases out of 102 HPV-negative cases followed (8.8%). This shows that some other etiological factors other than HPV such as age and parity might play a role in the progression or persistence of SIL.
Different predisposing risk factors to cervical carcinogenesis were evaluated in 130 SIL cases according to HPV status. Gynecological symptoms are highly associated with the development of cervical cancer, and these were analyzed in HPV-positive and HPV-negative cases [Table 1]. As expected, HPV positivity was found associated maximum with vaginal discharge (52.9%), followed by pain in the lower abdomen (23.5%) and menstrual disorders (11.7%). Two of the 17 HPV-positive cases were asymptomatic. In HPV-negative cases, the maximum number of cases was associated with pain in the lower abdomen (38.1%). Thus, women complaining of vaginal discharge are at great risk of harboring HPV infection. However, difference in the HPV positivity rate in the three groups was found to be statistically insignificant (χ2 = 2.232; P = 0.135).
|Table 1: Relation between human papillomavirus status and different variables|
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HPV positivity was also analyzed in relation to age [Table 1]. The HPV infection was more common in younger sexually active women between 21 and 30 years of age (52.9%), followed by 31–40 years' age group (47.1%). No HPV-positive case was seen in women beyond 40 years. The maximum number of HPV-negative cases was seen in the age group of 31–40 years (39.8%), followed by younger women of 21–30 years, and was least in the old age group beyond 40 years (20.3%). Thus, HPV being a highly sexually transmitted agent was found highly associated with young and sexually active age group where chances of HPV infection are higher. However, HPV positivity rate was found to be statistically insignificant between the three age groups (χ2 = 0.153; P = 0.696).
HPV positivity was also analyzed in relation to parity [Table 1]. HPV infection was found higher in multiparous women (70.6%) than 29.4% seen in Para 1 and 2. However, a similar trend was also seen in HPV-negative cases. In the present rural cervical cancer screening, the women attending the camps were mostly multiparous, and hence, it is obvious that a high positivity rate was seen associated with multiparity. However, the difference in the HPV positivity rate between the two parity groups was found to be statistically insignificant (χ2 = 0.017; P = 0.897).
| > Discussion|| |
HPV-DNA testing carried out in 130 SIL cases revealed high-risk HPV positivity in only 17 cases (13.1%). In HSIL cases, the HPV positivity was 33.3%, and in LSIL cases, this was 10.4%. Chandana et al. have found HPV positivity in 32.9% of LSIL cases in rural women of Andhra Pradesh. However, Franceschi et al. have found a very high incidence of 64.9% of HPV positivity in cervical abnormality cases in rural women of Tamil Nadu. Senapati et al. have also found 54.3% HPV positivity under inflammatory conditions of the cervix in rural women of Orissa, East India.
The most common HPV high-risk type reported from different parts of rural India has been HPV 16 in addition to other high-risk genotypes (Chandana et al., Sureshkumar et al. Franceschi et al., and Sowjanya et al. in South India, Senapati et al. in East India, Singh et al. and Das et al. in North India). Nahar et al. have also found HPV-16 more common in rural women of Bangladesh. However, in our study, HPV-18 was more common followed by multiple infections of HPV-31, HPV-33, and HPV-35 genotypes. Hence, there is wide geographical variation in the occurrence of different HPV high-risk genotypes in India itself.
Further, as seen in the present study, where multiple HPV infection was seen in 82.6% of SIL cases, similar findings of high multiple HPV infections were also reported by Franceschi et al., Chandana et al., and Sureshkumar et al. and others. The findings of other investigators from different parts of India are given in [Table 2].
Internationally, HPV-16 and HPV-18 have also been found common in different countries. In the United States of America and Britain, both HPV-16 and HPV-18 were commonly reported, whereas in Italy and Asian countries, such as China, Thailand, Vietnam, and Pakistan, only HPV-16 was only commonly seen. The findings are summarized in [Table 3].
|Table 3: HPV findings reported from different Western and Asian Countries|
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In the present investigation, HPV positivity was found associated with vaginal discharge in 52.9% of cases, but this association was found statistically insignificant when compared to the prevalence of vaginal discharge in HPV-negative cases. Chandana et al. have also reported a high HPV incidence of 32.9% in rural women of Andhra Pradesh who complained of vaginal discharge. As regards age, the authors have found HPV more common in younger sexually active women between 21 and 30 years (52.9%) decreasing with rising age, and no HPV positivity was seen in older women beyond 40 years of age. However, no statistical correlation was found between the HPV positivity and the three age groups when the age pattern was analyzed in HPV-negative cases. Sharma et al. and Singh et al. have also found HPV positivity higher in early age below 30 years in rural women of North India and Nahar et al. in rural women of Bangladesh. However, studies from South India have shown a high positivity rate in middle and old age groups of rural women.
The present study revealed the correlation of HPV infection with high parity. Senapati et al., Chandana et al., and Nahar et al. have also found HPV infection higher in multiparous women. However, Franceschi et al. have not found any relation of HPV infection with parity in rural women of Tamil Nadu. The parity findings in our HPV-negative cases also revealed no correlation between parity and HPV positivity.
The HPV-16 and HPV-18 are the high-risk HPV types commonly reported from different parts of India, and HPV vaccine currently available and given to women is only for these two HPV genotypes. However, in the present study, multiple infections of HPV-31, HPV-33, and HPV-35 have been found in majority of the cases. A similar trend has also been reported from the other parts of India by different investigators. Gardasil, a bivalent vaccine, and Cervarix, a quadrivalent vaccine, are currently under Phase II and Phase III trials. The bivalent vaccine appears to be more effective against HPV types – 31, 33, and 45 – than the quadrivalent vaccine, but the differences were not significant which may be due to differences in the trial design. However, there are many concerns about the safety and efficacy of these vaccines which have forced the Government of India to suspend the research on HPV vaccination till a consensus is reached on its efficacy in long-term control of cervical cancer and safety from the side effects. In light of current findings and also other investigators in India, it is felt that stress should be made on the formulation of region-specific HPV vaccine effective in women of that particular region of the country. However, clinical implications of multiple HPV vaccines should be extensively evaluated regarding its safety and efficacy before taking a further step in its preparation and application to younger women if any meaningful protection from HPV infection is to be provided for the prevention of carcinoma cervix. All these facts should be considered by the government for implementation of an effective HPV vaccination program for control of cervical cancer.
The authors thank village Heads of Kakori and Malihabad blocks of Lucknow West for helping in organizing camps for cervical cancer screening.
Financial support and sponsorship
The authors sincerely thank Secretary, Era's Educational Trust of Era University, Lucknow, for providing funds for carrying out rural cervical cancer screening program in the form of Intramural Research Project.
Conflicts of interest
There are no conflicts of interest.
| > References|| |
Pal SK, Mittal B. Improving cancer care in India: Prospects and challenges. Asian Pac J Cancer Prev 2004;5:226-8.
Chhabra S, Sonak M, Prem V, Sharma S. Gynaecological malignancies in a rural institute in India. J Obstet Gynaecol 2002;22:426-9.
Basu P, Roychowdhury S, Bafna UD, Chaudhury S, Kothari S, Sekhon R, et al
. Human papillomavirus genotype distribution in cervical cancer in India: Results from a multi-center study. Asian Pac J Cancer Prev 2009;10:27-34.
Deodhar K, Gheit T, Vaccarella S, Romao CC, Tenet V, Nene BM, et al
. Prevalence of human papillomavirus types in cervical lesions from women in rural Western India. J Med Virol 2012;84:1054-60.
Chandana V, Gaguturu RB. Prevalence and determinants of high risk human papilloma virus in Hyderabad, India. Int J Reprod Contracept Obstet Gynecol 2018;7:1012-8.
Franceschi S, Rajkumar R, Snijders PJ, Arslan A, Mahé C, Plummer M, et al
. Papillomavirus infection in rural women in southern India. Br J Cancer 2005;92:601-6.
Senapati R, Nayak B, Kar SK, Dwibedi B. HPV Genotypes distribution in Indian women with and without cervical carcinoma: Implication for HPV vaccination program in Odisha, Eastern India. BMC Infect Dis 2017;17:30.
Sureshkumar BT, Shanmughapriya S, Das BC, Natarajaseenivasan K. A population-based study of the prevalence of HPV in three districts of Tamil Nadu, India. Int J Gynaecol Obstet 2015;129:58-61.
Sowjanya AP, Jain M, Poli UR, Padma S, Das M, Shah KV, et al
. Prevalence and distribution of high-risk human papilloma virus (HPV) types in invasive squamous cell carcinoma of the cervix and in normal women in Andhra Pradesh, India. BMC Infect Dis 2005;5:116.
Singh MP, Kaur M, Gupta N, Kumar A, Goyal K, Sharma A, et al
. Prevalence of high-risk human papilloma virus types and cervical smear abnormalities in female sex workers in Chandigarh, India. Indian J Med Microbiol 2016;34:328-34.
] [Full text]
Das BC, Gopalkrishna V, Sharma JK, Roy M, Luthra UK. Human papillomavirus DNA in urine of women with preneoplastic and neoplastic cervical lesions. Lancet 1992;340:1417-8.
Nahar Q, Sultana F, Alam A, Islam JY, Rahman M, Khatun F, et al
. Genital human papillomavirus infection among women in Bangladesh: Findings from a population-based survey. PLoS One 2014;9:e107675.
Tanton C, Mesher D, Beddows S, Soldan K, Clifton S, Panwar K, et al
. Human papillomavirus (HPV) in young women in Britain: Population-based evidence of the effectiveness of the bivalent immunisation programme and burden of quadrivalent and 9-valent vaccine types. Papillomavirus Res 2017;3:36-41.
Mix JM, Unger E, Querec T, Thompson T, Greek A, Tucker T, et al
. Abstract 4199: HPV prevalence of cervical cancer in the United States 2014-2015. Cancer Res 2019;68;724-8.
Verteramo R, Pierangeli A, Mancini E, Calzolari E, Bucci M, Osborn J, et al
. Human papillomaviruses and genital co-infections in gynaecological outpatients. BMC Infect Dis 2009;9:16.
Coscia MF, Monno R, Ballini A, Mirgaldi R, Dipalma G, Pettini F, et al
. Human papilloma virus (HPV) genotypes prevalence in a region of South Italy (Apulia). Ann Ist Super Sanita 2015;51:248-51.
Gul S, Murad S, Javed A. Prevalence of high risk human papillomavirus in cervical dysplasia and cancer samples from twin cities in Pakistan. Int J Infect Dis 2015;34:14-9.
Wang J, Tang D, Wang K, Wang J, Zhang Z, Chen Y, et al
. HPV genotype prevalence and distribution during 2009-2018 in Xinjiang, China: Baseline surveys prior to mass HPV vaccination. BMC Womens Health 2019;19:90.
Shen XH, Liu SH. Human papillomavirus genotypes associated with mucopurulent cervicitis and cervical cancer in Hangzhou, China. Asian Pac J Cancer Prev 2013;14:3603-6.
Natphopsuk S, Settheetham-Ishida W, Pientong C, Sinawat S, Yuenyao P, Ishida T, et al
. Human papillomavirus genotypes and cervical cancer in Northeast Thailand. Asian Pac J Cancer Prev 2013;14:6961-4.
Vu LT, Bui D. Prevalence of cervical human papilloma virus infection among married women in Vietnam, 2011. Asian Pac J Cancer Prev 2012;13:37-40.
Sharma V, Singh P, Sharma N, Pracheta, Paliwal R. Study of epidemiology of HPV infection in the uterine cervix of women's in Delhi/NCR regions, India. Int J Drug Dev Res 2012;4:311-5.
Nigam A, Saxena P, Acharya AS, Mishra A, Batra S. HPV Vaccination in India: Critical Appraisal. ISRN Obs Gynec; 2014. Available from: http://dx.org/10.1155/2014/394595
. [Last accessed on 2014 Mar 11].
[Table 1], [Table 2], [Table 3]