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.

Frequency and type-distribution of human papillomavirus from paraffin-embedded blocks of high grade cervical intraepithelial neoplasia lesions in Thailand.

Cervical cancer is the most important female gynecological cancer, the second leading cause of cancer mortality in women worldwide and the second most common cancer in Thai women. The major cause of cervical cancer is persistent infection of human papillomavirus (HPV), leading to abnormal epithelial lesions, with progression to precancerous and invasive cancer. This study was conducted to investigate the frequency and type distribution of HPV in Thai women who had abnormal cytology. HPV detection from FFPE confirmed abnormal of high grade cervical intraepithelial lesions were for SPF-10-Innogenic Line Probe Assay. HPV-positivity was detected in 320/355 cases (90.14%) and HPV-negativity in 35/355 (9.86%). HPV-positive was found 147/320 cases (41.4%) of single infection, whereas 173/320 cases (48.7%) showed the multiple HPV infection. The most common seven types were HPV-16, -52, -18, -11, -51, -31 and -33, in that order. HPV 16 and 18, the important oncogenic HPV type, were observed in 64.8% of HSIL cases. Interestingly, a high proportion of multiple infections was found in this study and more than ten types could be detected in one case. Therefore, HPV infection screening program in women is essential, particularly in Thailand. Effective primary and secondary prevention campaigns that reinforce HPV screening for HPV detection and typing may be decrease the incidence and mortality of cervical cancer in the future and may lead to significantly improve the quality of life in Thai women.

Prevalence of high-risk human papillomavirus infection and cytologic results in Thailand.

Cervical cancer is the second most common female cancer with a high mortality rate. The established cause is high-risk types of human papillomavirus (HPV) and a new modality for cervical cancer screening is the combination of cervical cytology with HPV testing. The aim of present study was to identify the prevalence of high-risk HPV infection, and cervical cytological profiles of healthy Thai women. This largest cross-sectional investigation of HPV testing so far with cytology screening in Thailand was conducted between April 2009 and March 2010, covering a total of 14,747 women. The correlation between HPV viral load and cytology was also assessed. The mean age of the study group was 46.4 years (range 20-77 years) and the prevalence of high risk HPV infection was 8.23%. In positive women, negative cytology was observed in 72.9% , and cytology abnormalities in 27.1%, as compared to 1.57% in HPV negative women. The highest prevalence of HPV infection was identified in the youngest age group (≤30 years). The mean viral load was 6.06x105 (range 5,040.13 to 1.05x107) and HPV viral load titers were higher among in women with abnormal cytology.