ORIGINAL ARTICLE |
https://doi.org/10.5005/jacm-11020-0001 |
Nested Multiplex Endpoint Polymerase Chain Reaction: An Alternative Method for Human Papilloma Virus Genotyping in Resource-limited Settings
1Department of Gynecologic Oncology and Microbiology, Tata Medical Center, Kolkata, West Bengal, India
2–4Department of Gynecologic Oncology, Tata Medical Center, Kolkata, West Bengal, India
5Department of Microbiology, Tata Medical Center, Kolkata, West Bengal, India
6Department of Medical Administration, Tata Medical Center, Kolkata, West Bengal, India
Corresponding Author: Sanjay Bhattacharya, Department of Microbiology, Tata Medical Center, Kolkata, West Bengal, India, e-mail: sanjay.bhattacharya@tmckolkata.com
Received: 10 April 2024; Accepted: 28 May 2024; Published on: 26 July 2024
ABSTRACT
Purpose: Cervical cancer caused by human papillomavirus (HPV) is a public health problem.
The aim of the study was to develop a low-cost and reliable genotyping test of HPV for resource-limited settings.
Materials and methods: This observational study was done in a cancer hospital in eastern India.
Nonpregnant women with intact uteri (age: 25–65 years) were screened by testing their cervical swabs using the hybrid capture 2 (HC2) assay (HC2; Qiagen). HPV genotyping was done using a laboratory-developed test [LDT-nested multiplex endpoint polymerase chain reaction (PCR) assay]. The results of LDT were verified using HC2 genotypic assay (Qiagen). The t-test was used to ascertain statistical significance.
Results: A total of 253 out of 2,502 (10.11%) individuals tested positive for HPV by the HC2 assay. Out of these 253 HC2-positive samples, genotyping was successful by nested multiplex end point PCR in 182 (72%) of the samples. The commonest genotypes were the type 16 in 21.3% of samples and type 18 in 14.6% of samples. Among the non-16/18 genotypes, type 68 was the most prevalent (15.4% of samples). We detected single HPV genotypes in 123 out of 182 samples (68%), two genotypes in 44 out of 182 (24%), and three genotypes in 13 out of 182 (7%) samples. Two samples yielded four genotypes (1%). The cost of HPV genotyping by this nested multiplex endpoint PCR was Rs. 840 ($10.51). The cost of HPV genotyping by Sanger-based deoxyribonucleic acid (DNA) sequencing was Rs. 4,456 ($55.76), next-generation sequencing (NGS) Rs. 5,000 ($62.56), and that of HC2 test-based genotyping (using Qiagen kits) was Rs. 1,120 ($14.01) per positive sample.
Conclusion: The study provides important epidemiological information and shows that nested multiplex endpoint PCR-based methods may be used in HPV genotyping in resource-limited settings. The results are significant for surveillance and vaccination strategies.
Keywords: Cost, End point nested multiplex polymerase chain reaction, Genotyping, Human papillomavirus, Hybrid capture
How to cite this article: SYMEC Research Group, Mukhopadhyay A, Mathai S, et al. Nested Multiplex Endpoint Polymerase Chain Reaction: An Alternative Method for Human Papilloma Virus Genotyping in Resource-limited Settings. J Acad Clin Microbiol 2024;26(1):1–6.
Source of support: Integrated cervical cancer prevention and treatment stratification study—Systems Medicine Cluster approach (ICC SyMeC) funded by Department of Biotechnology (DBT), India: BT/ Med-II/NIBMG/SyMeC/2014/Vol. II, dated 9th January 2017.
Conflict of interest: None
INTRODUCTION
Cervical cancer, caused by human papillomavirus (HPV), is the third-ranked cancer in India with 123,907 new cases and 77,348 deaths in 2020, with a 5-year prevalence of 42.82/100,000 population.1,2 Screening methods have been developed to detect HPV infection and precancerous lesions at an early stage. These approaches include Pap smear, visual inspection of the cervix with acetic acid application (VIA), colposcopy followed by biopsy, and molecular methods [polymerase chain reaction (PCR), hybrid capture 2 (HC2), transcription-mediated amplification] on cervical swabs for the detection of various high-risk genotypes of HP.3
Human papillomavirus is a double-stranded, nonenveloped DNA virus with >200 different genotypes.4-6 Among the genotypes, there are low-risk (nononcogenic) genotypes such as 6, 11, 42, 43, and 44, as well as 14 high-risk genotypes such as 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68.
The genome of HPV is divided into three regions—(1) an upstream regulatory region (URR); (2) an early region, encoded by six genes (E1, E2, E4, E5a/5b, E6, E7); and (3) a late region, encoded by two genes for capsid proteins (L1, L2). The tests designed for HPV detection target L1 and L2 regions or the E6 and E7 genes for the oncoproteins.4 Laboratory methods for HPV detection include DNA detection (PCR, HC2, and fluorescence in situ hybridization (FISH) in formalin-fixed paraffin-embedded tissue) and RNA detection [E6, E7 messenger RNA (mRNA) PCR, and transcription-mediated amplification using Aptima assay].7,8
The detection of only low- and high-risk HPV as a group is not sufficient for disease management, epidemiological studies, vaccine preparation strategies, and patient counseling. The genotypes of HPV differ in oncogenic potential and prevalence.7 HPV genotypes 16 and 18 contribute to 70% of cervical cancers, whereas HPV genotypes 31, 33, 35, 45, 52, and 58 contribute to another 20%.9,10
The current methods available for HPV genotyping include real-time PCR, cartridge-based nucleic acid amplification test (CBNAAT), HC2 assay, Sanger sequencing, line probe assay, and next-generation sequencing (NGS). The cost of performing these tests varies with the method used and the number of samples per run and may range between Rs. 1,000 and Rs. 5,000 per sample. They also require expensive equipment and trained human resources. Therefore, there is a need for exact typing of HPV genotypes using a relatively simple, cost-effective, rapid, and reliable technique. Real-time PCR is one of the most common methods used for HPV screening and genotyping. However, we preferred to use an alternative method for the following reasons—(1) The real-time PCR system requires a more expensive thermal cycler than the conventional endpoint PCR system (approximately Rs. 1 million vs Rs. 2 million); (2) the reagent cost of real-time PCR is higher than that of endpoint PCR (real-time PCR uses dyes or probes which are more expensive and not used in conventional endpoint PCR systems); and (3) some of the real-time PCR machines are closed systems (using cartridges or kits from specific companies only). The objective of the current study was to develop a method that enables laboratories engaged in HPV diagnostics to develop and evaluate such a test for use in clinical and epidemiological contexts applicable in low- and middle-income countries (LMICs).
MATERIALS AND METHODS
Patient Selection
Nonpregnant women giving informed consent, aged between 25 and 65 years with intact uteri, were screened. Women with nongynecological cancer treatment and female relatives of cancer patients were included. Additionally, women from around the city of Kolkata who were motivated for screening by these sensitized women and their relatives were also included.
Sampling
Samples were collected between January 2018 and August 2019. Physicians collected cervical scrapes with a cytobrush (Rovers Cervex-Brush, Netherlands; catalog number 380100431), which were placed into transport medium PreservCyt solution (Hologic Inc., United States of America; catalog number 70097-005). Samples were transported to the testing laboratory in a cold box and stored at 2–8°C. Long-term storage of samples was done at –80°C.
Screening for Human Papillomavirus
Hybrid capture 2 technology (digene® HC2 High-Risk HPV DNA Test; Qiagen; catalog number: 5197-1330) was used. The digene HC2 High-Risk HPV DNA Test utilizes Hybrid Capture® 2 (HC2) technology, a nucleic acid hybridization assay with signal amplification. It employs microplate chemiluminescence for the qualitative detection of 13 high-risk types of HPV DNA in cervical specimens. This kit detects the following HPV genotypes—16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. The HC2 assay utilizes specific RNA probes, RNA–DNA hybridization, antibody capture, signal amplification, and qualitative chemiluminescent signal detection.
Human Papillomavirus Genotyping by Laboratory-developed Test
Nested multiplex end point PCR (nested multiplex end point PCR) was performed on samples that tested positive for HPV by the HC2 assay. It involved the following steps:
Deoxyribonucleic acid extraction: Around 1.5 mL of HPV-positive cervical sample in PreservCyt was centrifuged at 13,000 g for 10 minutes, and the cell pellets were resuspended in 500 µL of phosphate-buffered saline. They were then centrifuged again at 13,000 g for 5 minutes. The washed cell pellets were resuspended in 200 µL of sterile water, and DNA extraction was performed using the QIAmp DNA Mini Kit from Qiagen (catalogue number 51304) according to the manufacturer’s instructions. The 200 μL of HPV positive sample was added to a microcentrifuge tube containing 20 μL Qiagen Proteinase K. Next, 200 μL of Buffer AL was mixed by pulse-vortex with sample-protease mixture and incubated at 56°C for 10 minutes. After the addition of 200 μL of 100% ethanol, the mixture was applied to the QIAamp Mini spin column and centrifuged at 8000 rpm for 1 minute. The column was washed with 500 μL of Buffer AW1 and AW2. Finally, the DNA was eluted with 50 μL Buffer AE.
Type-specific nested multiplex end point PCR: The assay was designed to allow specific detection of thirteen high-risk HPV genotypes—16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 by nested multiplex endpoint PCR.11 First-round PCR was performed with GP-E6/E7 primers that hybridize to E6 and E7 oncogenes.11 The PCR amplifications using the GP-E6/E7 primers were carried out in a total volume of 25 µL. The enzyme activation was performed at 95°C for 4 minutes, followed by 40 amplification cycles of 60 seconds at 94°C, 60 seconds at 42°C, and 120 seconds at 72°C, with a final extension step of 10 minutes at 72°C. PCR products from the first round with GP-E6/E7 consensus primers were used as templates for the second-round type-specific PCR. The primers (Table 1) were used in three cocktails (Table 2), each containing four to five different primer pairs. The type-specific PCR amplifications were carried in a total volume of 20 µL. The activation of the enzyme was carried out at 95°C for 4 minutes, followed by 35 amplification cycles of 30 seconds at 94°C, 30 seconds at 56°C, and 45 seconds at 72°C, and a final extension of 4 minutes at 72°C. The identification of each HPV type present was achieved by determining the size of the nested PCR amplification product by gel electrophoresis (Table 2 and Fig. 1).
| HPV genotypes | Number of samples positive by HC2 assay |
|---|---|
| HPV 16 | 7 |
| HPV 18 | 7 |
| HPV16 + 18 | 3 |
| HPV 45 | 2 |
| HPV 16/18/45 negative | 22 |
| Total genotyped by HC2 assay | 41 |
| Type of genotype | Number of women with HPV subtypes |
|---|---|
| Total number of HPV positives | 253 |
| HPV 16 | 54 (21.3%) |
| HPV 18 | 37 (14.6%) |
| HPV 31 | 11 (4.3%) |
| HPV 33 | 7 (2.7%) |
| HPV 35 | 4 (1.5%) |
| HPV 39 | 4 (1.5%) |
| HPV 45 | 10 (3.9%) |
| HPV 51 | 22 (8.6%) |
| HPV 52 | 14 (5.5%) |
| HPV 56 | 20 (7.9%) |
| HPV 58 | 27 (10.6%) |
| HPV 59 | 9 (3.5%) |
| HPV 68 | 39 (15.4%) |
| HPV 16 and others | 22 (8.6%) |
| HPV 18 and others | 11 (4.3%) |
| Non-16/18 HPV type | 21 (8.3%) |
| Undetermined | 67 (26.4%) |
Fig. 1: Agarose gel electrophoresis record depicting HPV genotyping by nested multiplex end point PCR
Validation of Nested Multiplex Endpoint Polymerase Chain Reaction Method
This was done by next-generation DNA sequencing and by the HC2 genotyping assay kit, Qiagen digene® HPV Genotyping PS Test (catalogue number 613615). The digene HPV Genotyping PS Test is a reflex test intended for the qualitative detection of high-risk HPV types 16, 18, and 45 following a positive digene HC2 High-Risk HPV DNA Test result (which qualitatively detects 13 high-risk types). The HC2 method was performed internally using the same system but a different kit than that used for the HPV screening assay.
Quality Control
The following quality control (QC) strains were used:
-
First WHO International Standard for human papillomavirus (HPV) type 16 DNA, NIBSC code: 06/202 (https://www.nibsc.org/documents/ifu/06-202.pdf).
-
First WHO International Standard for anti-human papillomavirus 18, NIBSC code: 10/140 (https://www.nibsc.org/documents/ifu/10-140.pdf).
Each of the assays (digene HC2 screening assay, digene HC2 genotyping assay, and LDT, the nested multiplex endpoint PCR test) were tested on the QC strains to verify their ability to detect these QC strains and type them.
Cost Calculation
The cost of testing by various methods (HC2, PCR, Sanger sequencing, NGS) was calculated, taking into account the consumable costs.
Statistical Test
The t-test was used to calculate the statistical significance of the relative light unit (RLU) values of the genotypable and nongenotypable positive HPV-positive samples. https://www.graphpad.com/quickcalcs/ttest1/?format=C
RESULTS
Hybrid Capture Assay Result
A total of 253 individuals tested positive from a total of 2,502 tested (10.11% test positivity rate) by HC2 assay (Fig. 1 and Fig. 2). The signal in RLU from the HC2 assay for the positive samples varied from 1.03 to 16131.38 (median RLU was 6.71; interquartile range was 3.06–63.33). Around 67 samples failed genotyping by the in-house nested multiplex endpoint PCR assay. The median RLU value of these samples, which failed to genotype, was 5.17 (range: 1.03–9403.92; IQR: 2.67–10.03). The median RLU value of the samples which were successfully genotyped by the nested multiplex end point PCR assay (n = 182) was 9.82 (range: 1.04–16131.38; IQR: 3.12–116.25). The difference in the RLU values of the positive samples that were successfully genotyped by nested multiplex end point PCR and those that were untypable by this method was not statistically significant (p = 0.46; t-test).
Fig. 2: Consolidated Standards of Reporting Trials flowchart of HPV screening and genotyping
Results from the Validation
The results of the nested multiplex end point PCR method had a complete correlation with the HC2 method in 32/41 samples (78.15%) (Table 1).
Genotyping Results from Nested Multiplex Endpoint Polymerase Chain Reaction
Altogether, genotyping was attempted in 253 HPV-positive samples. Out of these 253 positive samples, genotyping was successful by type-specific nested PCR in 182 (71.94%) of the samples. Single HPV genotypes were detected in 123 out of the 182 successfully typed samples (68%), two genotypes were detected in 44 out of 182 (24%), and three genotypes were detected in 13 out of 182 (7%) samples. Two samples yielded four genotypes (1%) (Table 2). The commonest genotypes were the type 16 in 21.3% of samples and type 18 in 14.6% of samples. Among the non-16/18 genotypes, type 68 was the most prevalent (15.4% of samples).
Cost of Human Papillomavirus Genotyping
The cost of HPV genotyping by the LDT (multiplex endpoint PCR) was Rs. 840 ($10.51 USD). Compared to that, the cost of HPV genotyping by Sanger-based DNA sequencing was estimated to be Rs. 4,456 ($55.76 USD), by NGS was about Rs. 5,000 ($62.56 USD), and by HC2 test-based genotyping (using Qiagen kits) was Rs. 1,120 ($14.01 USD) per positive sample (Table 3).
| Consumables | COST (in INR Rs.) processing 25 samples |
|---|---|
| HPV control | 100 |
| 10 µL tips—filter barrier | 891.4 |
| 1.75 mL microcentrifuge tube | 350 |
| DNA isolation kit (Qiagen) | 2,750 |
| 1250 µL tips—filter barrier | 855.60 |
| 200 µL tips—filter barrier | 454.70 |
| Qiagen multiplex PCR mix | 15,500.00 |
| PCR tube | 300.00 |
| Agarose gel | 400.00 |
| Ethidium bromide | 300.00 |
| Gloves | 100.00 |
| Overheads (includes manpower, electricity, water, depreciation costs of pipettes, wastage) | 1,500.00 |
| Total expenditure incurred for 25 samples | 21,010.3 |
| Cost per sample using this LDT (lab developed test) | Rs. 840 ($10.51 USD) |
| Qiagen HC2 genotyping test kit cost | 1,120 ($ 14.01) |
| DNA sequencing cost | 4,456 ($55.76) |
| Viral DNA extraction (Qiagen): 180.00 INR | |
| Target DNA amplification (SYBR Green master mix + PCR primers): 200.00 INR | |
| Target DNA cleanup by Magbio kit: 36.00 INR | |
| Cycle sequencing: 4000.00 INR | |
| Postcleanup by Magbio kit: 20.00 INR | |
| Plastic consumables (tips + tubes + PCR plate): 20.00 INR | |
| Total: 180 + 200 + 36 + 4000 + 20 + 20 = 4456.00 INR (approx.) | |
| HPV genotyping by NGS | Rs. 5,000 ($62.56) |
DISCUSSION
Persistent HPV infection significantly contributes to cervical cancer. The distribution of different HPV types can vary by age and geographical region. Therefore, accurately determining predominant HPV genotypes in each region using sensitive, specific, cost-effective, rapid, and user-friendly methods is crucial for epidemiological analysis, genotype-specific disease prognosis, clinical pathways, patient counseling, and development of appropriate vaccines. Among high-risk HPV types, HPV 16 is the most common worldwide, and HPV type 18 is the second most common genotype.10,12,13 Previous studies have indicated that in eastern India (Kolkata), the high-risk genotypes 16 and 18 are commonly found.14PCR-sequencing of HPV may be done targeting the E6, E7, L1, and the long control region genes.11,15 It has been reported that the E6 (447 bp), E7 (291 bp), and L1 (1512 bp) genes from the HPV62 reference genome (8092 bp) code for proteins with 148, 96, and 503 amino acids, respectively. A study from Mexico explored the genetic variation of the E6 and E7 open reading frames (ORFs) as well as that of the L1 ORF.16 A study from Thailand compared NGS, nested PCR, line probe assay (using INNO LiPA), and DNA chip for HPV typing.17
Senapati et al. reported that the most prevalent HPV genotype from Odisha was HPV16 in 82.28%, followed by HPV18 in 24.45%, and HPV51 in 3.46%. The prevalence of single genotypes was 76.58%. The most frequent genotype combination was HPV16 + 18 in 9.4%, followed by HPV16 + 66 + 68 in 2.7%. Genotyping in this study was conducted by sequencing the L1 region of the HPV genome using the Sanger sequencing method.18 In our study, we used the E6/E7 region for genotyping. A study from Chandigarh using PCR showed that HPV16 was the most common genotype in HPV-positive women with human immunodeficiency virus (HIV) infection, followed by HPV45 in 15%, HPV18/52/31/58 in 11.5%, and HPV33 in 7.6%.19
Another study from Pune, Maharashtra, by Mane et al., investigated HPV genotype distribution in HIV-infected women with cervical intraepithelial neoplasia (CIN). Two or more HPV genotypes were present in 50.7% of the women.20 Suresh et al., in a study from Kerala using the careHPV system and nested PCR (to amplify the L1 hypervariable region for Sanger sequencing), detected HPV16 and 33 as the commonest genotypes—52 and 40%, respectively.21
The bivalent HPV vaccine (Cervarix) targets HPV 16 and 18, while the quadrivalent vaccine (Gardasil) protects against HPV 6, 11, 16, and 18. These vaccines offer limited protection against nonvaccine serotypes. The nonavalent vaccine (Gardasil 9), which includes HPV 6, 11, 16, 18, 31, 33, 45, 52, and 58, is expected to be more effective in providing broader protection against a wider range of HPV types in such situations. In a study of oncogenic HPV among the HIV-infected female population in West Bengal, Sarkar et al. found that the prevalence of HPV 16 and 18 was higher among HIV-infected females compared to HIV-negative females (32.2 vs 9.1%). They noted that 53% (23 out of 43) of the oncogenic HPV infections were caused by types other than 16 and 18, either as single infections or in combination with other HPV types.14
Tests other than genotyping have been used as triage tests for cervical cytology. In a study from Italy, Benevolo et al. reported the use of E6/E7 mRNA assay for triage.22 The cost-effectiveness of HPV 16/18 genotype triage in cervical cancer screening was investigated by Vijayaraghavan et al. from the United States of America. The use of HPV genotyping had an incremental cost-effectiveness ratio of $34,074 per quality-adjusted life year (QALY) compared to liquid-based cytology (LBC) screening.23
The common genotyping methods include PCR, HC2, or line probe assay (PCR followed by hybridization). However, some investigators have reported HPV genotypes using DNA sequencing. Targeted NGS and whole genome sequencing using high throughput sequencing (HTS) have been utilized in some studies. Challenges associated with sequencing techniques include relatively higher costs, process complexity, longer turnaround times, the need for expertise in molecular biology and bioinformatics, and infrastructure requirements. The advantage of the nested multiplex end point PCR includes the fact it can be done by a basic molecular biology laboratory without the need for DNA sequencers or real-time PCR equipment. In this study, we found HPV 16 and HPV 18 to be the most common, with a prevalence of 29.12 and 17.5%, respectively. HPV 51, 58, and 68 were also frequently found in our study. Among them, HPV 68 was the third most prevalent at 10.9%, following HPV 16 and 18. It was noteworthy that HPV 68 was mostly detected as a single infection, whereas HPV 51 and 58 were mostly detected as coinfections. Current HPV vaccines do not provide protection against these other HPV types (68, 51) detected in this study. The efficiency of HPV vaccines in preventing HPV infection is affected by local epidemiological variation of HPV infections. The different genotypes of HPV reported in this study may have an impact on the design of new vaccines and assay designs in the future.
The limitations of our study include the following—(1) Not all positive samples could be genotyped using the nested multiplex end point PCR method. This may be attributed to the method’s relatively lower sensitivity, (2) there was a lack of correlation with the comparator assays (HC2) for some samples, possibly due to the lower sensitivity and specificity of the LDT. The reasons for failure of amplification and genotyping could be related to DNA degradation over time.24 However, other reasons, such as failure of DNA extraction methods, might also be responsible for genotyping failure in some samples.25
CONCLUSION
This study demonstrates the potential for a low-cost genotyping test suitable for LMICs, where expensive technologies like DNA sequencers and real-time PCR systems may not be readily available. However, the technique requires further standardization to enhance sensitivity and specificity.
SYMEC Research Group
Kamakshi Sureka
Anusha Harishankar
Sudipto Mandal
Subhanita Roy
Abhirupa Kar
Barnali Ghosh
Shrabanti Sarkar Ghosh
Ajit Mukhopadhyay.
ETHICAL APPROVAL
The study was approved by the Institutional Review Board (IRB) of Tata Medical Center, Kolkata, West Bengal, India (IRB ethical approval reference number EC/GOVT/17/17 dated 6th January 2018).
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