Overview: Detection of Human Papillomavirus in Clinical Samples.

Human papillomavirus (HPV) is the most common sexually-transmitted virus and it is known that persistent infection by high-risk HPV is a necessary factor for cervical carcinogenesis. Although cytological screening has decreased the incidence of cervical cancer, the sensitivity and specificity of testing is limited. To date, HPV-driven molecular techniques have provided a number of potential biomarkers for both diagnostic and prognostic use in clinical management. In addition, they can provide insights into the biology of HPV-induced cancers leading to non-surgical therapy. This review summarizes current knowledge of detection methods for HPV and related biomarkers that can be used to discriminate lesions with a high risk of progression of cervical cancer.

Prognostic factors of human papillomavirus genotypes of invasive cervical carcinoma: an analytical cross-sectional study in lower north-east Thailand.

BACKGROUND: Cervical cancer (CXCA) caused by persistent infections by high-risk human papillomavirus (HR-HPV) can lead to multi-step carcinogenesis. The best management strategy and significant prognosis for cervical cancer patients remain unclear. OBJECTIVE: To investigate the associations of the two most common HR-HPVs with clinical outcomes of progression and recurrence status as well as prognosis outcomes of patients. MATERIAL AND METHOD: An analytical cross-sectional study of patients registered at Ubon Ratchathani Cancer Hospital was conducted from 2007 to 2010. Clinical data, histopathological features, and clinical outcomes of progression and recurrence status were recorded. HPV type-specific E6/E7 nested multiplex polymerase chain reaction (NMPCR) was performed to identify HR-HPV16 and 18 using extracted deoxyribonucleic acid (DNA) from embedded paraffin. Clinical findings and HPV genotypes were analyzed using Fisher's exact test. Association studies of crucial factors and HR-HPV genotypes were performed using logistic regression analysis (odds ratio [OR]) and 95% confidence interval [CI]). A p-value of less than 0.05 was considered statistically significant. RESULTS: The study found single HPV16 infection in 57.3%, single HPV18 in 17.3%, mixed HR-HPV16/18 in 13.1%, and non-HPV16, 18, or 16/18 in 12.3%. The findings showed significant association among their genotypes and histopathological types and grading (p < 0.0001 and p = 0.014). Clinical outcomes of progression and recurrence status with increased severity of clinical staging were associated significantly (p = 0.001 and p = 0.002). HPV18 type-specific was shown as a poor prognostic type with its relevance to the severity of disease higher than that of HPV16. CONCLUSION AND DISCUSSION: HPV16 and 18 remain the major type-specifics especially in relation to invasive CXCA, requiring further therapeutic vaccination study and proper prognosis. HR-HPV type-specific is very important during cervical carcinogenesis but other crucial contributing factors for prognostic outcomes should be further elucidated.

Effect of single-visit VIA and cryotherapy cervical cancer prevention program in Roi Et, Thailand: a preliminary report.

AIM: To assess the effect of introducing visual inspection with acetic acid and cryotherapy on cervical cancer incidence rates in Roi Et province over time, between 1997 and 2006, and compare this with two nearby provinces. METHODS: Data from two cancer registration units, one in Srinagarind Hospital and another in Ubon Ratchathani Cancer Center (to which all cervical cancer patients were referred from the three study provinces) were registered, extracted, combined and analyzed using a generalized estimation equation. RESULTS: Cervical cancer detection rates improved. These are represented by the apparent increased incidence rates in Roi Et province during the study period compared with two nearby provinces (P = 0.01), equivalent to a doubling of the previously reported age-standardized incidence ratio and three times its baseline in 2006. CONCLUSIONS: A single-visit approach to cervical cancer prevention in Roi Et province using visual inspection with acetic acid and cryotherapy appeared to have an effect in revealing an increased cervical cancer incidence rate by achieving higher coverage, resulting in increased case finding.

Comparative accuracy of Pap smear and HPV screening in Ubon Ratchathani in Thailand.

We evaluate the potential for using high-risk human papillomavirus (hr-HPV) testing-based screening for cervical intraepithelial neoplasia (CIN) in routine health services in Thailand; its accuracy in comparison to that of conventional cytology (CC); and the utility of HPV16/18 positive results and liquid-based cytology (LBC) triage for HPV-positive women in the detection of high-grade CIN. Women aged 30-60 years in Ubon Ratchathani province, Thailand were screened with CC and hr-HPV testing and those abnormal on either tests were referred for colposcopy and/or directed biopsies. The final diagnosis using COBAS was based on histology or colposcopy when histology was not available. Estimation of test accuracy parameters was done using latent class analysis using Bayesian models. Of the 5004 women were enrolled, 20 (0.4%) had abnormal CC and 174 (3.5%) women were HPV-positive. Among 185 women abnormal on CC or HPV-positive, 176 (95.1%) underwent colposcopy, of whom 101 (57.4%) had abnormal colposcopy findings. Ninety-seven women with abnormal and 69 with normal colposcopy had biopsies performed. All 21 women with histological CIN2 or worse had hr-HPV and none were abnormal on CC. The estimated sensitivity, specificity and positive predictive value were respectively 71.8%, 97.0% and 13.0% of HPV testing; 53%, 98.7% and 20.3% for triage of HPV-positive women with LBC; and 70.4%, 98.2% and 16.9% when test positivity was taken as HPV16/18 irrespective of LBC result or positive for hr-HPV non 16/18 types and LBC triage. Our study findings indicate poor performance of cytology screening and demonstrate the potential and utility of using HPV testing in public health services in Thailand as well as the utility of primary HPV testing and LBC triage in screening for cervical neoplasia.

Detection assay for HPV16 and HPV18 by loop­mediated isothermal amplification with lateral flow dipstick tests.

Cervical cancer is the third highest cause of death in developing countries and most commonly results from high­risk human papillomavirus (HR­HPV) infection. Among HR­HPV genotypes, HPV16 and HPV18 are the most prevalent in cervical cancers. Therefore, the present study aimed to develop a detection assay for HPV16 and HPV18 infection using loop­mediated isothermal amplification (LAMP) with lateral flow dipstick (LFD) tests. This assay is a simplified, user­friendly method for the visual detection of HPV genotypes. DNA was extracted from clinical tissue samples, and HPV genotyping was performed using nested polymerase chain reaction (PCR). The clinical samples were demonstrated to include 44 HPV16­positive, 18 HPV18­positive and 80 HPV­negative samples. All DNA samples were also used as templates for a LAMP reaction (30 min at 65˚C), and subsequently, a fluorescein isothiocyanate­labelled probe was hybridized with the reaction product. Finally, the LFD test was performed. The sensitivity of the LAMP­LFD test was higher than LAMP­turbidity, exhibiting up to 100­fold higher sensitivity for HPV16 and 10­fold higher sensitivity for HPV18. All HPV16 and HPV18­positive samples generated positive results in both assays; however, 22 samples detected as HPV­negative by LAMP­turbidity exhibited positive results by LAMP­LFD test (22 of 80 samples). Therefore, these samples were further examined using quantitative (q)PCR. The results demonstrated that 20 out of the 22 samples designated positive by LAMP­LFD, but negative by LAMP turbidity, gave a positive result with qPCR, while the remaining 2 samples were negative by qPCR. The present results suggested that LAMP­LFD provided higher sensitivity than LAMP­turbidity and nested PCR. Thus, the LAMP­LFD test developed in the present study might be useful for the detection of HPV16 and HPV18 in local hospitals.

High sensitivity, loop-mediated isothermal amplification combined with colorimetric gold-nanoparticle probes for visual detection of high risk human papillomavirus genotypes 16 and 18.

High-risk human papillomavirus (HR-HPV) causes cervical cancer. HPV16 and HPV18 are the most prevalent strains of the virus reported in women worldwide. Loop-mediated isothermal amplification (LAMP) is an alternative method for DNA detection under isothermal conditions. However, it results in a turbid amplified product which is not easily detected by the naked eye. This study aimed to develop an improved technique by using gold nanoparticles (AuNPs) attached to a single-stranded DNA probe for the detection of HPV16 and HPV18. Detection of the LAMP product by AuNP color change was compared with detection by visual turbidity. The optimal conditions for this new LAMP-AuNP assay were an incubation time of 20min and a temperature of 65°C. After LAMP amplification was complete, its products were hybridized with the AuNP probe for 5min and then detected by the addition of magnesium salt. The color changed from red to blue as a result of aggregation of the AuNP probe under high ionic strength conditions produced by the addition of the salt. The sensitivity of the LAMP-AuNP assay was greater than the LAMP turbidity assay by up to 10-fold for both HPV genotypes. The LAMP-AuNP assay showed higher sensitivity and ease of visualization than did the LAMP turbidity for the detection of HPV16 and HPV18. Additionally, AuNP-HPV16 and AuNP-HPV18 probes were stable for over 1year. The combination of LAMP and the AuNP-probe colorimetric assay offers a simple, rapid and highly sensitive alternative diagnostic tool for the detection of HPV16 and HPV18 in district hospitals or field studies.

Prevalence of high risk human papillomavirus infection with different cervical cytological features among women undergoing health examination at the National Cancer Institute, Thailand.

High-risk (HR) human papillomavirus (HPV) testing is important in cervical cancer screening for triage colposcopy. The objective of the study was to evaluate the prevalence of HR HPV infection with different cervical cytological features among women undergoing health examination. A total of 2,897 women were retrospectively evaluated between May 2011 to December 2011. DNA was extracted from residual specimens collected during routine liquid-based cytology tests at the National Cancer Institute. Overall, HR HPV prevalence was 9.3% including 1.6% of HPV-16 and 0.4% of HPV-18. Of all 270 HPV positive samples, 211 (78.1% were HR-HPV non 16/18; 47 (17.4%) were HPV-16 and 12 (4.4%) were HPV-18. The prevalence of HPV infection was similar in all age groups, although a higher rate was observed in women age 31-40 years. Among women with normal cytology, HR HPV positive were found in 6.7%. In abnormal cytology, HR HPV were found 46.7% in atypical squamous cells (ASC), 54.8% in low-grade squamous intraepithelial lesions (LSIL) and 80.0% in high-grade squamous intraepithelial lesions (HSIL). HPV-16 was detected in 8.6%, 6.4% and 12.0% of ASC, LSIL and HSIL, respectively. The results of this study provide baseline information on the HPV type distribution, which may be useful for clinicians to decide who should be monitored or treated more aggressively.