PCR: Polymerase chain reaction; HPV: Human papilloma virus

• A real time PCR based test and an automated testing platform for HPV detection
• Includes all the reagents required to perform sample-to-result analysis
• PCR components in a lyophilized format
• Inclusion of controls for quality check, ensure result integrity and result reliability
• Analytical sensitivity of 50 IU/mL for HPV16 and HPV18
• Storage and transport at ambient temperature (15 to 30 ⁰C)

Globally, cervical cancer is the third leading cause of deaths among women [1]. According to recent estimations, Asia has the highest rate of cervical cancer cases (58.2%), whereas Northern America has the lowest rate (2.5%) [2]. In India, in 2020, it accounted for 18.3% (123,907) of total new cancer cases [3]. The human papillomavirus (HPV) is an etiologic agent of cervical cancer that belongs to the family of Papillomaviridae. According to the Center for Disease Control and prevention (CDC), HPV accounts for more than 90% of cervical and anal cancers, about 70% of vaginal cancers, and 60% of penile cancers is due to HPV [4].

Most women and men are reported to develop HPV infection at least once in their lifetime, even in the absence of any noticeable signs or symptoms. Although the majority of HPV infections are cleared naturally by the immune system, in some women, HPV infection may persist over time, which can cause the cells of the cervix to change to cancerous cells [5-7]. More than 100 types of HPV have been identified to date, of which HPV 16 and 18 are known to be the most prevalent cancer-causing genotypes (high-risk) [8].

In view of the WHO global strategy for cervical cancer elimination, the World Health Assembly in 2020 suggested that 70% of women globally should be screened regularly for cervical disease and it is recommended that an HPV DNA-based test be used as the preferred method for screening [9]. However, there are still some challenges including lack of screening facilities or transportation particularly in rural settings, inconvenient sampling procedures, with perceptions of fear and embarrassment. These factors, along with the high cost, could make it difficult to achieve the WHO-recommended screening coverage goal of 70%. As India is on the way to universalizing the national-level screening program for cervical cancer, it is crucial to investigate the areas vulnerable to poor screening.

A few commercial industries like GeneProof (Czechia, Europe), Roche (Indiana, United States), TaKaRa (Manufacturing unit at New Delhi, India), HELINI (Tamil Nadu, India), CIK Biotech (Poway, United States) have developed and commercialized HPV Screening PCR Kits. The Roche Cobas 4800 HPV / Cobas 6800 tests (Roche Molecular Systems, Pleasanton, USA) have been approved by the FDA for cervical cancer screening. However, the sample transportation in cold chain to rural areas, accessibility, cost are the major hurdles associated with these solutions and direct reporting systems to the national health grid is also lacking.

The HPV 16 and 18 subtypes are more crucial for diagnosis since they are more common in India (36%) and have a stronger (50%) correlation with cervical cancer rates. The present study describes the development and validation of an indigenously developed solution that comprises a test named "HPV 16 and 18 detection kit" and a testing platform known as the "Compact series automated system" for HPV 16 and 18 screening.

Description of the test

The nucleic acid isolation process for cervicovaginal specimens was optimized using magnetic bead binding technology. Input volumes from 200 µL to 500 µL were tested to identify the most efficient extraction conditions. The protocol was refined to allow for the simultaneous isolation of both viral and genomic DNA through sequential lysis, washing, and elution steps. For cell lysis, different combinations of guanidine isothiocyanate and various detergents were optimized, and the effects of pH variations were carefully assessed. Paramagnetic magnetic beads were used at optimal concentrations to ensure effective binding. The washing steps utilized alcohol-based buffers to remove any remaining contaminants, while elution was conducted at an acidic pH to maximize nucleic acid recovery efficiency. The effectiveness of these procedures was validated to ensure the highest possible yield of nucleic acids. The isolated viral nucleic acids were subsequently tested using real-time PCR for HPV16 and HPV18.

The "HPV 16 and 18 detection kit" is based on real-time PCR technology for the amplification of a specific conserved target sequence of the E6/E7 region of HPV 16 as well as 18, and detection by a target-specific probe. The PCR reagents were lyophilized to ensure stability at room temperature. The test includes all essential components, such as PCR tubes containing oligonucleotides, fluorescent probes, and an enzyme mix in lyophilized form. It also includes separate quality controls, including positive and negative template controls, for the detection of HPV 16/18. The RnaseP gene was used as housekeeping gene to check for extraction efficiency and PCR inhibition.

A run method optimization was carried out with temperature gradient PCR at 58°C, 60°C, and 62°C using positive controls, clinical samples and performance was verified using international standards (NIBSC standards, 19/224). The targets and reporters include HPV 16 (VIC), HPV 18 (FAM) and IC (RNaseP) (Cy5). The thermal cycling conditions were: polymerase activation (Hold) at 95 °C for up to 1 min, PCR stage (40 cycles) with denaturation at 95°C at 5 sec and annealing and extension at 60°C for up to 30 secs. The PCR formulation was optimized in such a way that it gives similar performance on various available real time PCR platforms. Further comparative analysis of liquid PCR mix and lyophilized PCR mix were determined for analytical performance and stability.

The test was performed using either a compact series automated platform or a manual open real time PCR platform. The compact platform automates sample to-result analysis using sealed prefilled reagent cartridges. For sample processing, magnetic bead-based extraction protocol optimized initially (Maverick nucleic acid extraction kit,1001A050) was used to isolate the viral nucleic acid followed by PCR amplification and detection by HPV16 & HPV18 detection kit. Additionally, auto-reporting software was integrated for positive and negative sample reporting based on CT cut-off values; eliminating the raw data analysis and manual interpretation. On the open platform, the test is compatible with commercially available real time PCR platforms including ABI7500, QuantStudio5, Biorad CFX 96, Rotor-Gene Q, MyLab Compact Q PCR Detection System.

Analytical and clinical performance

Analytical sensitivity, also called as Limit of Detection (LoD) of the test was determined by analyzing a logarithmic dilution series of the HPV NIBSC control (19/224 for HPV16 and HPV18). The NIBSC standard was diluted in known negative clinical specimen matrix prepared by collecting cervical swabs in transport medium. The tentative LoD was determined by screening the 10-fold dilutions of the viral stock to give an idea about the sensitivity at cut-off. These tests were performed in triplicates for each dilutions. The lowest dilution which gave the 100% positive results in the tentative LoD was further diluted in a series of 2-fold dilutions to give confirmatory LoD. A total of 20 replicates per concentration were tested in 5 independent runs.

Cross reactivity and microbial interference were evaluated by testing commensal and pathogenic microorganisms that may be present in the cervical specimens. Each of the organism, viruses, and yeast were tested in triplicates in the absence and in the presence of HPV 16 and HPV 18 respectively. In silico analysis using BLAST was used to assess the degree of nucleotide sequence homology.

Precision (Repeatability and reproducibility) of the assay was verified by intra-assay and inter-assay testing of low positive and moderate positive specimens with different operators, days and instruments.

Inhouse clinical testing was carried out retrospectively where known positive and negative specimens were tested using Mylab HPV 16 and 18 test and comparator CE-IVD approved test. Further, the test was also enrolled under QCMD 2023 Human Papillomavirus (Surepath) EQA Programme - QAV184204_1.

In independent external evaluation, a total of 42 confirmed positive cases of HPV Infection (35-57 years old female) and 75 negative samples (33-57 years old female) were studied at NABL accredited pathology lab (Dhruv Pathology Labs, Nagpur, India) and concordance was calculated with comparator kits (TRUPCR HPV HR With 16 and 18 Genotyping Kit).

Statistical analysis

Each assay was performed in triplicate and Ct values were obtained. The SD and percentage coefficient of variation (% CV) was calculated to assess the precision of the replicates as well as the assay performance.

Test performance

The extraction efficiency for nucleic acid recovery was determined to be 80% or higher. The lyophilized real-time PCR test for HPV16 and HPV18 demonstrated comparable analytical performance to the liquid real-time PCR test, with extended stability of up to 2 years at room temperature when following the claimed shelf-life stability protocol.

Analytical sensitivity

Analytical sensitivity/ Limit of Detection (LOD) of HPV 16 and 18 Detection Test was 50 IU/mL at 95% confidence interval (Table 1).

Cross-reactivity

The cross reactivity with similar micro-organisms obtained from ATCC or NIBSC source were checked. The microorganisms (n-41) (human immunodeficiency virus, hepatitis B, hepatitis C, adenovirus , cytomegalovirus, herpes simplex virus 1, herpes simplex virus 2, Lactobacillus acidophilus, Staphylococcus epidermidis, Staphylococcus aureus, Enterococcus faecalis, Streptococcus pyogenes, Streptococcus agalactiae, Corynebacterium spp., Escherichia coli, Enterococcus spp., Clostridium spp., Peptostreptococcus spp., Klebsiella spp., Enterobacter spp., Proteus spp., Pseudomonas spp., Bacteroides spp., Bifidobacterium spp., Fusobacterium spp., Chlamydia trachomatis, Neisseria gonorrhoeae, Candida albicans, Trichomonas vaginalis, other HPV high risk genotypes(n-12)) were tested in an in silico analysis where no cross reactivity was detected. Additional in vitro testing was carried out according to the availability of the pathogens where no cross reactivity was detected in the absence of HPV 16 & HPV 18 where each pathogen was spiked with HPV negative clinical matrix and tested using real time PCR in triplicate. No PCR amplification was observed for either pathogen indicating no cross reactivity in the absence of HPV16 and HPV18.

Microbial interference study was carried out using 7XLOD HPV16 & HPV18 specimens spiking with similar microorganisms at high concentration (up to 10^5 copies/ml) and tested in triplicate using real time PCR. Results demonstrated that false negatives were not detected when HPV 16 and/or HPV 18 was present in a specimen with other microorganisms.

Precision

Real time PCR amplification for HPV16 and HPV18 were analyzed and compared for Ct values for intra-assay and inter-assays. The coefficients of variation (%CV) was detected <20% for both the targets (Table 2).

Clinical evaluation

During an internal evaluation, a total of 50 clinical samples (15 known positive and 35 negative) were analyzed. The results showed >98% sensitivity, 100% clinical specificity with Positive Predictive Value (PPV) of 100% and Negative Predictive Value (NPV) of 97.22%. One sample was false negative during testing out of 15 true positives which could be attributed to transport of clinical samples and long storage after sample collection. Even the comparator test did not show reproducible results upon re-testing at original site.

External evaluation of the test was performed with three evaluation batches at Dhruv Pathology Labs, Nagpur. The comparative analysis of performance was carried out using commercially available kits such as TRUPCR HPV HR with 16 and 18 Genotyping Kit and a 100% concordance was observed (Table 3).

The present study offers a unique, regulatory approved diagnostic solution for HPV screening with the novel features of high throughput RT-PCR test, complete automation without the need for pipette handling, transportation and storage at ambient temperatures, with implications for early detection for mass screening and clinical management.

In India, cancer of the cervix is the 3^rd most common cancer and the second leading cause of death with a mortality rate of 9.1% as per GLOBOCAN 2020. Around 440 million women in India are aged 25 to 60, an age at which cervical cancer screening is important. For cervical and/or vaginal screening, low-cost procedures like acetic acid visual inspection or PAP smears from the surface of the cervix are frequently used in India. However, these techniques are not substantially effective in detecting HPV invasion in the body because there are no symptoms at the early stages of infection. The highly accurate screening test currently available for early cervical cell changes is the HPV DNA test, where vaginal and cervical cells are tested using a real-time polymerase chain reaction. Further, WHO has recommended real time-PCR based testing for HPV screening.

The developed “HPV 16 and 18 detection assay” is a multiplex, real time PCR based test (hydrolysis probe technology) directed against the E6/E7 gene for HPV16 & 18 high risk HPV types and an internal control that uses fluorescent probes for the detection of one or more accumulating PCR products. The oncoproteins, E6 and E7 bind to various gene products particularly the tumor suppressors p53 and pRb [10]. The degradation of tumor suppressors p53 by E6 and retinoblastoma protein (Rb) by E is reported to increase the risk of malignant transformation [11] and serve as a better risk evaluation factor to confirm the presence of HPV E6 and E7 mRNA in cervical carcinomas.

The HPV assay has been designed for the detection of HPV 16 and 18 in cervical specimens. The automated system (Mylab developed Compact series platform), comprising a compact analyzer and cartridges, can automatically process sample-to-result analysis. This platform includes real time PCR preparation and enables amplification and detection on the real-time PCR machine and data analysis by software. During each PCR cycle, the fluorescent signal increases in a logarithmic manner, resulting in an amplification curve. As soon as the amplification curve of the target surpasses its threshold, the sample is considered positive for that target. The multiplex format allows the simultaneous detection of all three different fluorescent dyes per reaction, with each fluorescent dye representing different targets. By employing a unique and proprietary PCR design, the system delivers significantly faster results in determining the presence of HPV subtypes infection. Some recent studies by Ali et al. have reported the prevalence and risk factors in the sexually inactive and sexually active women populations of Bihar, India [12] and symptomatic women with a negative inflammatory Pap smear [13] using Mylab’s “PathoDetect HPV 16 &18 detection assay”.

Automation of the entire process from extraction to detection or even to reporting of results helps to reduce the manual errors and shortens the timepoint required for end-to-end processing of the samples. The platform enables testing of single sample to up to 32 samples in one go, covering low to high-throughput. The device can be utilized as a POC using mobile van facility for remote setting. There is also a provision of software for tracking and direct reporting to the Government portal as per the user’s or government’s demand.

Some of the commercially available molecular tests based on real time PCR include TRUPCR® HPV 16 and 18 Detection Kit, AccuPower® HPV 16 and 18 Real-Time PCR Kit and Cobas HPV assay. The Sensitivity of TRUPCR® kit is reported to be 100 IU/mL for HPV16 and HPV18 while that of AccuPower® is 13.18 copies/test for HPV 16 and 10.18 copies/test for HPV 18. The PCR time for the above assays is reported as 2.0 hours and ≤ 1.5 hours respectively. Our test provides a sensitivity of 50 IU/mL with a turnaround time of 1.5 hours. TRUPCR® HPV 16 and 18 Detection kit involves 2 tube assays with the reagents in liquid form. In contrast, the composition of Mylab’s HPV test enables simultaneous detection and discrimination of HPV 16 and 18 subtypes in a single reaction tube with a freeze-dried premix that allows room temperature storage. Multiplex detection of these three targets (HPV 16, 18, IC) in a single assay enables reduction in cost, time and labor as compared to separate detection methods.

Given the various barriers to regular screening (fear of sampling procedure, financial constraint, cultural issues, or personal modesty), the approach of self-sampling will be beneficial. In order to improve HPV screening uptake in low resource setting, we have designed a self-sampling device. The device involves specimen collection and transportation media that are stable at room temperature. Using self-sampled swabs, we conducted internal validation and obtained trustworthy results. However, in-depth clinical investigations with self-collected cervico-vaginal specimens are necessary to validate the test. A preliminary study was also carried out using urine samples spiked with HPV to establish if urine can serve as an alternative clinical material. However, a thorough investigation and clinical assessment are being planned. Future studies are underway to validate tests for the detection of HPV 14 high-risk genotypes for wider coverage.

In conclusion, the HPV 16 and 18 detection test with a decentralized and load-and-go diagnostic platform would enable timely and accurate diagnosis of HPV genotypes. The test with innovative features of automation, algorithm-based analysis tool would enable its use as a near point-of-care system. This sensitive, economical, high-throughput will have implications for National Cervical Screening Program. Routine cervical screenings would help greatly to reduce both the number of cervical cancer cases and deaths from the disease. Such a complete platform would also simplify analysis of patient response to surgical procedures and for routine monitoring of vaccine efficacy.

Conflict of interest

None.

Data availability

The data are available from the corresponding authors on reasonable request

Funding

This research received funding from Mylab Discovery Solutions Pvt. Ltd.

Author contributions

Conceptualization: Minal Dakhave. Execution and formal analysis: Shruti Gadekar, Amrita Khaire, Kamini Patil. Writing-original draft preparation: Amrita Khaire, Minal Dakhave. Writing-review and editing: Amrita Khaire, Minal Dakhave. Approval of final manuscript: All authors.

Acknowledgement

We thank the Mylab Discovery Solutions for funding the project. We thank the R&D team for their contribution in executing the projects till completion.