PCR-RFLP and FTIR-based detection of high-risk human papilloma virus for cervical cancer screening and prevention

Abstract

Background Approximately 70% of cervical carcinoma cases show the presence of high-risk Human Papilloma Virus (HPV), especially HPV-16 and HPV-18, and can be used to stratify high risk patients from low risk and healthy. Currently, molecular biology techniques such as polymerase chain reaction (PCR) are used to identify the presence of virus in patient samples. While the methodology is highly sensitive, it is labor intensive and time-consuming. Alternative techniques, such as vibrational spectroscopy, has been suggested as a possible rapid alternative. Therefore, in this study, we evaluate the efficiency of cervical fluid Fourier Transform Infrared spectroscopy (FTIR) in patient risk stratification informed by PCR. Methods Cervical fluid samples (n = 91) were obtained from patients who have undergone routine Papanicolaou (Pap) test. Viral genome was identified and classified as high/low-risk by PCR-Restriction Fragment Length Polymorphism (PCR-RFLP). FTIR spectra were acquired from samples identified by PCR-RFLP as No-HPV (n = 10), high-risk HPV (n = 7), and low-risk HPV (n = 7). Results Of the 91 samples, was detected the viral genome by PCR in 36 samples. Of these 36 samples, nine samples were identified to contain high-risk HPV (HR-HPV) and nine samples were found to have low-risk HPV (LR-HPV). The FTIR spectra acquired from No-HPV, LR-HPV, and HR-HPV showed differences in 1069, 1437, 1555, 1647, 2840, 2919, and 3287 cm -1 bands. Principal Component Analysis (PCA) showed distinct clusters for No-HPV and HR-HPV and No-HPV and LR-HPV, but there was significant overlap in the clusters of HR-HPV and LR-HPV. PCA-Linear Discriminant Analysis (PC-LDA) after Leave One Out Cross Validation (LOOCV) classified No-HPV from HR-HPV and No-HPV from LR-HPV with 100% efficiency in the 1400-1800 cm -1 spectral range. LOOCV classifications for LR-HPV and HR-HPV from each other were 71 and 75%, respectively, in the 2800-3400 cm -1 spectral range. Conclusions The results highlight the high sensitivity of PCR-RFLP in HPV identification and show that FTIR can classify samples identified as healthy, low, and high-risk samples by PCR-RFLP. General significance We show the possibility of using FTIR for initial cervical cancer risk stratification followed by detailed PCR-RFLP investigations for suspect cases.

Understanding viruses and viral infections by biophotonic methods

“…PCR-based methods are considered to be the current gold standard for virus detection [37,38]. Due to the amplification of specific nucleic acid sequences, viruses can be identified in a sensitive and highly specific manner [38,39].…”

“…Therefore, it is difficult to isolate them from the sample fluid and as described above they are often bound to nanoparticles or antibodies for isolation and quantification. In clinical surroundings, standardized tests based on PCR methods are used for viral load determination [37,39]. Since a higher viral load correlates with the severity of the infection, many different optical approaches-like fluorescence microscopy, vibrational spectroscopy-are being explored to detect viral particles [83].…”

“…FTIR was employed as a fast testing technique for COVID-19 detection, where SARS-CoV-2 RNA extracted from patients were screened [161]. Melo et al demonstrated how FTIR spectroscopy can be employed for risk assessment of HPV infections by investigating cervical fluid samples [37]. Depending on the HPV type the oncogenic risk can be high or low [37].…”

“…Melo et al demonstrated how FTIR spectroscopy can be employed for risk assessment of HPV infections by investigating cervical fluid samples [37]. Depending on the HPV type the oncogenic risk can be high or low [37]. Barauna et al utilized attenuated total reflection (ATR) FTIR spectroscopy for investigating both spiked and clinical saliva samples for differentiating between SARS‐CoV‐2 positive and negative samples [162].…”

Contributions of vibrational spectroscopy to virology: A review

“…Based on a patient cohort of 40 HIV positive pregnant women and 80 uninfected controls, they received a classification accuracy of 89% using a DA algorithm. In another study, it was shown how FTIR can detect HPV in cervical fluid samples with a classification of 100% efficiency within the spectral ranges 1400–1800 cm -1 [159] and furthermore could differentiate between low risk HPV and high risk HPV with 71–75% efficiency. ATR-FTIR has thus proven to be a very useful method for viral diagnosis, not only is it rapid and cost effective technique and coupled with only a minuscule amount of body fluid is needed for accurate results, making this method a good candidate to translate to the clinical benchtop [33] .…”