A Rapid, Sensitive and Type-Specific Detection of High-Risk HPV-16 and HPV-18

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Human papillomavirus (HPV) infection, particularly infection with HPVs 16 and 18, is a major cause of cervical cancer. The current high-risk HPV screening or diagnosis tests use cytological or molecular techniques that are primarily based on qualitative HPV DNA detection. Comparative studies, however, revealed that different assays have varying sensitivities for detecting specific HPV types. Here, we developed and optimized a sensitive PCR (Polymerase Chain Reaction) assay for detection of high-risk HPV-16 and HPV-18. The PCR parameters were optimized, and analytical specificities were validated. Performance of developed PCR assay was evaluated in clinical samples (n = 100) which showed 100% specificity for both the assays and 96.97% and 94.12% sensitivity for HPV-16 and HPV-18, respectively. The developed assay demonstrated high sensitivity and specificity for detection of high-risk HPV-16 and HPV-18, making it applicable to routine HPV detection practices.

Keywords : Human papillomavirus · PCR · Detection · Cervical cancer

Human papillomaviruses (HPVs) are non-enveloped, double- stranded, circular DNA viruses having a genome size close to 8 kilobases [1]. HPV infection causes cutaneous and genital warts [2]. More than 200 HPV types are currently described which are further classified in to two major groups namely low risk (LR) (e.g., types 6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81) and high risk (HR) (e.g., types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68) based on their ability to promote cell transformation, which leads to cancer development [3].

Cervical malignancy is the fourth most common malignancy in women worldwide and a significant cause of morbidity and mortality [4]. According to epidemiological studies, the leading etiological cause of cervical cancer is persistent infection with a high-risk HPV type. Among all HR HPV types, the most common is HPV 16 genotype (50–60%) responsible for causing cervical cancer followed by HPV 18 (11–15% of cervical cancer) [5]. Therefore, high-risk HPV testing may have implication for cervical cancer screening as well as management of women with cervical lesion. Early detection of the HR-HPVs using a simple and sensitive method is immensely important for therapeutic measure against cervical cancer particularly in areas where specialized equipment is not available.

There are several tests available for HPV detection based on either cytological examination or HPV genotyping. The most commonly used HPV detection test employs signal amplification techniques (e.g., Hybrid Capture 2) to detect HPV DNA via an RNA DNA hybridization probe with chemiluminescence or fluorescence signals [6]. However, this method of detection is associated with certain limitations of high running costs and also requires dedicated equipment. Whereas, PCR-based detection of specific target DNA is one of the most sensitive and cost-effective method for HPV detection. In this study, we have designed and evaluated a type-specific PCR assay targeting the E6/E7 regions for the sensitive detection of high-risk HPV-16 and HPV-18. The sensitivity and specificity of developed assay were also evaluated by testing clinical samples.

Cervical cancer is one of the most known causes of death among women in developing countries [8]. Human Papilloma Virus (HPV) is known to induce cervical cancer if left untreated [9]. High mortality rate due to poor diagnosis of HPV infection is major concern in developing as well as under developed nations. It is the need of the hour to have efficient diagnosis for early detection and treatment. Detection of viral DNA helps in diagnosis of the infection. Among the various HPV infection types, the high-risk types such as 16, 18, 33 and 58 are known to be most prevalent [10].

Initial on set and progression of the cancer are dependent on two main oncogenes E6 and E7 and are expressed constitutively [11]. As these oncogenes contribute as carcinogenic factors, targeting this conserved region would be effective in testing for HPV and subsequently in cervical screening. Targeting highly conserved regions in the viral genome enables to develop PCR primer sets such as MY09-MY11, PGMY09-MY11, GP5 + -GP6 + , SPF10, and LCR-E7 [12]. Typically, HPV16 and HPV18 are the subtypes with the highest infection rate worldwide and classified as “high-risk” type [13]. Globally HPV 16 and 18 affects between 60 to 90% people and which is also reason for HPV cancer [14]. Rather than considering all conserved regions, our study focuses on targeting the most prevalent region known for HPV 16 and 18. Targeting conserved regions allows in specific detection of various HPV genotypes.

We have developed and validated PCR-based assay which is type-specific and sensitive in detection of HPV 16 and 18 infections based on the amplification of conserved viral region of E6 and E7. This method combines the method of type-specific detection and sensitivity which was validated by testing on clinical samples. This assay helps in detection of high-risk types HPV-16 and HPV-18 by targeting E6/E7 region as primer binding site. The assay also shows sensitivity and specificity when performed with clinical samples. Because the developed assay demonstrated high sensitivity and specificity for detection of high-risk HPV-16 and HPV- 18, it is applicable to routine HPV detection practices.

Acknowledgements We would like to acknowledge Gupte Hospital, Pune, for providing clinical samples for this study

Declarations

Conflict of interest We have no conflicts of interest to disclose.

Ethical Approval We ensure that this work is original and has not been published elsewhere, nor it is currently under consideration for publication elsewhere. We also acknowledge that all authors have substantially contributed to the underlying research and drafting of this manuscript and agree with the content of the manuscript.

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