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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 12  |  Issue : 4  |  Page : 159-164

Association of human papillomavirus with other coinfections prevailing in abnormal cervical lesions


1 Department of Oncogene Regulation, Chittaranjan National Cancer Institute, Kolkata, West Bengal, India
2 Department of Pathology, IPGME and R and SSKM Hospital, Kolkata, West Bengal, India

Date of Submission03-Sep-2021
Date of Acceptance11-Sep-2021
Date of Web Publication09-Dec-2021

Correspondence Address:
Dr. Chinmay Kumar Panda
Department of Oncogene Regulation, Chittaranjan National Cancer Institute, Kolkata, West Bengal
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jrcr.jrcr_32_21

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  Abstract 


Purpose: Human papillomavirus (HPV) is a common and necessary cause for cervical cancer development. However, association of other microbial infections such as bacterial vaginosis (BV), trichomonas vaginalis (TV), and hepatitis B virus (HBV) with HPV in development of the disease is not clear. This study aimed to evaluate the association of HPV with the other coinfections with the development of cervical lesions. Materials and Methods: Cervical smears of 120 women (age range 20–70 years) were screened by cytology/serology for detection of BV, TV, and HBV. The prevalence of HPV and its copy number were detected by polymerase chain reaction-based methods. Results: HPV infection was infrequent (4%) in normal cytology samples followed by increase in atypical squamous cell of undetermined significance (ASCUS) (60.4%), low-grade squamous intraepithelial lesion (LSIL) (94.1%), high-grade squamous intraepithelial lesion (HSIL) (100.0%), and squamous cell carcinoma (SCC) (100.0%). HPV 16 infection was prevalent than HPV 18 along their coinfections in some samples. The other microbial infections were comparatively less frequent (28.0%) in normal cytology samples than ASCUS (83.3%), LSIL (44.1%), HSIL (58.3%), and SCC (100.0%). BV infection was found in normal cytology as well as in ASCUS, LSIL, and HSIL samples, whereas TV infection was absent in normal cytology though prevalent in other cervical lesions. HBV infection was seen in only one ASCUS sample. Conclusions: Significant high HPV 16 copy number was seen to be associated with other microbial infections than the infection negative samples. Coinfection of HPV with other microbes has association with severity of cervical lesions.

Keywords: Atypical squamous cell of undetermined significance, cervical cancer, human papillomavirus, infection of bacterial vaginosis/trichomonas vaginalis/hepatitis B virus, low-grade squamous intraepithelial lesion


How to cite this article:
Mohanty G, Singha P, Datta C, Dutta S, Panda CK. Association of human papillomavirus with other coinfections prevailing in abnormal cervical lesions. J Radiat Cancer Res 2021;12:159-64

How to cite this URL:
Mohanty G, Singha P, Datta C, Dutta S, Panda CK. Association of human papillomavirus with other coinfections prevailing in abnormal cervical lesions. J Radiat Cancer Res [serial online] 2021 [cited 2022 May 16];12:159-64. Available from: https://www.journalrcr.org/text.asp?2021/12/4/159/332109




  Introduction Top


Cervical cancer is the fourth most common cancer worldwide and human papillomavirus (HPV) is a major etiological agent during its development.[1] Globally, the prevalence of cervical HPV infection is about 20%, although wide variations in its prevalence and type distributions can be seen.[2] It has been suggested that persistent infection of high-risk HPVs in cervix might develop cytological abnormalities leading to the development of premalignant lesions (low-grade squamous intraepithelial lesion [LSIL] and high-grade squamous intraepithelial lesion [HSIL]) followed by malignant transformations.[2] Apart from HPV infection, several sexually transmitted infections (STIs) such as Chlamydia spp., herpes simplex virus (HSV), trichomonas vaginalis (TV), and bacterial vaginosis (BV) might have important role in cervical carcinogenesis.[3],[4] The association of TV with HPV in cervical cancer was reported.[3] However, the association of BV with HPV in cervical carcinogenesis is not well studied.[3] Apart from the STIs, infection of hepatitis B virus (HBV) in cervical cancer has been reported though its association is not clear.[5],[6],[7] This study aimed to evaluate the association of HPV with the other coinfections (BV/TV/HBV) with severity of cervical lesions.


  Materials and Methods Top


Collection of cervical swabs and blood samples

About 3 ml cervical swabs were collected in BD SurePathTM vials (Becton, Dickinson and Co.) from 120 women visiting outpatient department (OPD) of IPGME and R and SSKM, Kolkata, having age between 20 and 70 years. The study was on hospital patients-based samples. Written informed consent was obtained from the respective women. Samples were collected for 1 year between March 2019 and 2020, and the study was approved by the Research Ethics Committee memo No. IPGME and R/IEC/2019/043. The inclusion criteria for the study were all women must attained the age of 20, were married and coming to the gynecological OPD for treatment with symptoms such as vaginal whitish discharge, pain during intercourse, abnormal menstrual bleeding after menopause, and backache. Women undergoing hysterectomy or no evidence of neoplasia in cervix or with preinvasive disease in cervix were excluded from the study.

An enrolment questionnaire containing demographic characteristics was filled up for analysis. After collection, one portion of the swab samples was taken for liquid-based cytology (LBC). Remaining swab samples were stored at −80°C for HPV DNA testing. In addition, 5 ml of blood from the women was collected, serum was isolated and then stored at 4°C for HBV testing.

Evaluation of cervical cytology

Cervical swabs samples that were collected for LBC were processed according to the standardized procedure.[8],[9] The pathologist interpreted and reported the slides. Cytologic diagnosis of normal, atypical squamous cell of undetermined significance (ASCUS), LSIL, HSIL, and squamous cell carcinoma (SCC) was done.

Detection of other coinfections

The BV/TV infection in the cervical swabs was detected by LBC through PAP staining.[9] The detection of HBV in serum was done by the method of HBsAg detection kit (VIRUCHECK, manufactured by VIOLA Diagnostic Systems, India). The other diagnostic methods for detection of BV may include real-time clinical/microbiological diagnosis or Gram stain analysis.

Detection of human papillomavirus and human papillomavirus 16/18

DNA was isolated from the cervical swabs preserved in BD SurePathTM according to the procedure described by Singh et al.[10] Briefly, the cervical swab that was immersed in 1 mL of phosphate-buffered saline transferred to 2 ml. eppendorf. Then, the suspension was centrifuged at 12,000 rpm for 15 min at room temperature, and the pellet was treated with lysis buffer (10 mM Tris–HCl, pH 7.4; 5 mM ethylenediaminetetraacetic acid (EDTA) and and kept at 37°C for 15 min. The lysate was further treated with proteinase K at 37°C for overnight followed by phenol–chloroform extraction and ethanol precipitation of DNA. The DNA precipitate was dissolved in 40-μL Tris-EDTA buffer (10 mM Tris–HCl, pH 7.4 + 1 mM EDTA), and the concentration of DNA was measured using an UV spectrophotometer (Cary 100, Middleburg, The Netherlands). The isolated DNA samples were tested for the presence of HPV by polymerase chain reaction (PCR)-based method using L1 consensus region primers (MY 09/11) and AptaTaq Fast DNA Polymerase (Roche, Basel, Switzerland) according to the standard procedure.[11],[12] The PCR products were electrophoresed in 2% agarose gel and stained with ethidium bromide for visualization of 450 bp amplicons of HPV under ultraviolet light and photographed.

The HPV-positive samples were then tested for the presence of HPV type 16/18 by PCR using HPV 16 type-specific primers from the E6 region and HPV 18 specific primers from the long control region.[13] The PCR products were electrophoresed in 2% agarose gel and stained with ethidium bromide for visualization under ultraviolet light and photographed.

Estimation of human papillomavirus copy number

HPV copy numbers in the HPV-positive samples were determined by semiquantitative PCR-based method using the primers from the E6 region of HPV 16.[2] The PCR products were electrophoresed in 2% agarose gel, stained by ethidium bromide, and photographed. The intensity of the bands in the gel was measured by gel documentation system. (BIO-RAD, serial no. 721BRO3502). For standard calibration using the PCR-based method, 10-fold serial dilutions of HPV-16 plasmid at concentrations of 500 pg/μL, 50 pg/μL, 5 pg/μL, 500fg/μL, 50fg/μL, and 5fg/μL were used.

Statistical analysis

HPV prevalence along with other coinfections was calculated. The x2 for linear trend was calculated for risk of HPV+/infection+ and HPV+/infection women by Epi Info software.


  Results Top


Human papillomavirus profile in cervical swabs

Among the collected cervical swabs (n = 120), normal cytology (age range 30–60, median 45) in 25 samples, ASCUS (age range 26–62, median 45) in 48 samples, LSIL (age range 26–62, median 45) in 34 samples, HSIL (age range 36–64, median 45) in 12 samples, and 1 SCC (age 60) sample were seen. HPV prevalence in the samples was detected by MY 09/11 primers followed by HPV 16/18 prevalence by type-specific primers [Figure 1]. The HPV prevalence was low (4%, 1/25) in normal samples followed by gradual increase in ASCUS (60.4%, 29/48), LSIL (94.1%, 32/34), HSIL (100%, 12/12), and SCC (100%, 1/1) [Figure 2]. Among the HPV-positive samples, variable frequencies of HPV 16 (25%–100%) and HPV 18 (6.3%–16.6%) were seen [Figure 2]. Both HPV 16 and 18 prevalence was gradually increased with severity of the lesions, namely ASCUS (13.8%, 4/29), LSIL (37.5%, 12/32), and HSIL (58.4%, 7/12).
Figure 1: Detection of HPV and HPV 16/18 from cervical swab samples. (a) Detection of HPV from swab samples by using primers MY 09/11. Lane-1: PUC 19 digested by MSP 1 as marker; Lane-2: Negative control; Lane-3:HPV 16 plasmid as Positive control; Lane-4,5,6,7(#123/c/2, 126/c/4, 123/c/10, 29/c/11): HPV positive swab samples. (b) Detection of HPV 16 by using primers HPV 16 E6. Lane-1:PUC 19 digested by MSP 1 as Marker; Lane-2: Negative control; Lane-3, 4, 6, 7 (#123/c/2, 126/c/4, 123/c/10,616):HPV16 positive swab samples; Lane-5(#29/c/11):HPV 16 negative sample; Lane-8:HPV 16 plasmid as Positive control. (c) Detection of HPV 18 by using primers of LCR region. Lane-1: PUC 19 digested by MSP 1 as Marker; Lane-2: Negative control; Lane-3, 5 (#29/c/11,126/c/1):HPV18 positive swab samples; Lane-4, 6 (#600,601): HPV 18 negative swab samples; Lane-7: HPV 18 plasmid as positive control

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Figure 2: HPV profile in the cervical swabs showing HPV profile in the cervical swabs. ASCUS: Atypical squamous cell of undetermined significance; LSIL: Low-grade Squamous Intraepithelial Lesion; HSIL: High-grade squamous intraepithelial lesion, SCC: Squamous cell carcinoma

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Coinfection profile in cervical swabs

The microbial infection other than HPV was comparatively low (28.0%, 7/25) in normal cytology samples than ASCUS (83.3%, 40/48), LSIL (44.1%, 15/34), HSIL (58.3%, 7/12), and SCC (100.0%, 1/1) [Figure 3]. Among the microbial infection samples, differential infection of BV and TV was seen in different cervical lesions. Variable frequencies of BV infection were seen in normal cytology (100.0%, 7/7), ASCUS (7.5%, 3/40), LSIL (53.3%, 8/15), and HSIL (28.5%, 2/7). On the other hand, frequent TV infection was seen ASCUS (90.0%, 36/40), LSIL (46.7%, 7/15), HSIL (71.5%, 5/7), and SCC (100.0%, 1/1). HBV infection was seen in only 1 ASCUS sample.
Figure 3: Coinfection profile in the cervical swabs showing coinfection profile in the cervical swabs. BV: Bacterial Vaginosis, TV: Trichomonas vaginalis, HBV: Hepatitis B virus

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HPV and other coinfection profile in the cervical swabs

In normal cytology samples, the absence of infection of HPV and other microbes as well as their coinfection was seen in majority (68.0%, 17/25) of the samples. However, the absence of any infection was infrequent (0%–10.4%) in other lesions. High coinfection was found in, ASCUS (54.1%, 26/48), LSIL (44.1%, 15/34), HSIL (58.4%, 7/12), and SCC (100.0%, 1/1) samples [Table 1]. Among the coinfected samples, TV infection was high (53.4%–100.0%) among the lesions than BV (7.7%-46.6%) and HBV (3.8%) infections [Table 2].
Table 1: Human papillomavirus and other infection profile in the cervical swabs

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Table 2: Coinfection profile of human papillomavirus and other infections

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Human papillomavirus copy number variation analysis

HPV copy number was comparable (1025–1753 copies) in HPV+/coinfection ASCUS, LSIL, and HSIL samples [Figure 4]. On the other hand, significantly high copy number of HPV was seen in HPV+/coinfection+ ASCUS samples compare to HPV+/infection ASCUS samples and became comparable in LSIL followed by significant increase in HSIL [Figure 4]. A significant linear trend (x2 for trend 1.96, P = 0.05) of increasing risk of infection with increasing grades of cytological abnormalities was observed.
Figure 4: Hepatitis B virus copy number analysis. Box plot showing distribution of human papillomavirus (HPV) copy number in HPV+/Co infection + samples (n = 48) having atypical squamous cell of undetermined significance (n = 26), LSIL (n = 15) and high-grade squamous intraepithelial lesion (n = 7) and HPV+/Co infection-samples (n = 25) having atypical squamous cell of undetermined significance (n = 3), LSIL (n = 17), and HSIL (n = 5).2 number of samples (1 normal + 1 squamous cell carcinoma) were excluded from analysis due to its less number. Copy numbers were plotted in the graph as log (HPV copies/100 ng of cellular gDNA). The median value of each sample group connected through a polynomial trend line, showed gradual increase in HPV copy number with progressive cytological severity

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  Discussion Top


In this study, we have analyzed the prevalence of HPV and infection of BV/TV/HBV in cervical swabs (n = 120) with different cytology to understand the importance of the infections with severity of the cervical lesions. Low prevalence of HPV was seen in normal cytology samples with gradual increase in severity of the lesions with prevalent infection of HPV 16 than 18. Similar to our data, low frequencies (4.1%–14.2%) of HPV prevalence were seen in normal cervical smears.[2],[14],[15],[16],[17] On the other hand, variable frequencies of HPV prevalence had been seen in ASCUS (3.2%–50.9%), LSIL (10.0%–71.2%), HSIL (53.3%–100.0%), and SCC (70.5%–100.0%) samples.[2],[6],[18],[19] This might be due to variation in sample size and methodologies used in analysis. Like our data, frequent HPV 16 infection than HPV 18 in different types of cervical lesions was reported in different populations.[16],[18],[19],[20] The gradual increase in HPV 16/18 prevalence with severity of the cervical lesions[2],[19] as seen in our analysis indicates the importance of the infection with development of the disease.

The microbial infections (BV/TV/HBV) other than HPV were found to be low in normal cytology samples compared to the abnormal cytology. Similar phenomenon has also been reported in other studies with low frequency of infection in normal cervix (14.7%–16.1%) than ASCUS (59.0%–67.9%), LSIL (32.0%–44.1%), and SCC (100.0%) except HSIL (19.0%).[21] In our analysis, infection with TV was prevalent than BV and HBV. Unlike us, low frequency (2.8%) of BV infection was reported in normal cytology samples.[21] In normal, cervical cytology coinfection of HPV with the other microbes was very infrequent. However, with progression of disease considerable coinfection with BV/TV/HBV particularly prevalent TV infection was seen in variable frequencies. Similar phenomenon has also been reported in other studies.[22],[23],[24] It seems that the coinfection of HPV and other microbes might have important role with development of the disease. There is evidence of an association between BV and HPV in cervical lesions.[25] BV may increase the risk of infection with or reactivation of HPV.[26] Like that, TV showed association with HPV in cervical carcinogenesis.[3] It seems that TV could modulate the host metabolism system for its survival and could enhance HPV infection ability.[27],[28] In conclusion, the increase in HPV copy number with other infections and severity of the lesions suggests their association with development of the disease.[2],[29] The limitation of our study is that we looked only for types 16 and 18 as they are the most oncogenic types. We propose to analyze stored positive samples to identify genotypes other than types 16 and 18. Hence, coinfection of HPV16/18 with other microbes such as BV, TV, and HBV has association with severity of cervical lesions and plays an important role in the progression of cervical cancer as HPV copy number increases with increasing grades of cervical abnormality. Thus, the infections may develop high grade of cancer if left untreated.


  conclusion Top


In cervical lessions high HPV copy number was associated with microbial co-infection.

Acknowledgment

We are thankful to Director, Chittaranjan National Cancer Institute, Kolkata, India, for providing laboratory support during this work. I am also thankful to Council of Scientific and Industrial Research (CSIR), Government of India to providing Research Associateship for financial support.

Financial support and sponsorship

Financial support for this work was provided by Research Associateship grants from the Council of Scientific and Industrial Research (CSIR), Government of India, to Dr Gangotree Mohanty (file No. 09/030 (0079)/2016-EMR-I).

Conflicts of interest

There are no conflicts of interest.



 
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