Simultaneous Detection and Viral DNA Load Quantification of Different Human Papillomavirus Types in Clinical Specimens by the High Analytical Droplet Digital PCR Method

Abstract

Human papillomaviruses (HPVs) are small DNA tumor viruses that mainly infect mucosal epithelia of anogenital and upper respiratory tracts. There has been progressive demand for more analytical assays for HPV DNA quantification. A novel droplet digital PCR (ddPCR) method was developed to simultaneously detect and quantify HPV DNA from different HPV types. DdPCR was initially tested for assay sensitivity, accuracy, specificity as well as intra-and inter-run assay variation employing four recombinant plasmids containing HPV16, HPV18, HPV11, and HPV45 DNAs. The assay was extended to investigate/quantify HPV DNA in Cervical Intraepithelial Neoplasia (CIN, n = 45) specimens and human cell lines (n = 4). DdPCR and qPCR data from clinical samples were compared. The assay showed high accuracy, sensitivity and specificity, with low intra-/inter-run variations, in detecting/quantifying HPV16/18/11/45 DNAs. HPV DNA was detected in 51.1% (23/45) CIN DNA samples by ddPCR, whereas 40% (18/45) CIN tested HPV-positive by qPCR. Five CIN, tested positive by ddPCR, were found to be negative by qPCR. In CIN specimens, the mean HPV DNA loads determined by ddPCR were 3.81 copy/cell (range 0.002-51.02 copy/cell), whereas 8.04 copy/cell (range 0.003-78.73 copy/cell) by qPCR. DdPCR and qPCR concordantly detected HPV DNA in SiHa, CaSki and Hela cells, whereas HaCaT tested HPV-negative. The correlation between HPV DNA loads simultaneously detected by ddPCR/qPCR in CINs/cell lines was good (R 2 = 0.9706, p < 0.0001). Our data indicate that ddPCR is a valuable technique in quantifying HPV DNA load in CIN specimens and human cell lines, thereby improving clinical applications, such as patient management after primary diagnosis of HPV-related lesions with HPV-type specific assays.

Association between oncogenic human papillomavirus type 16 and Killian polyp

“…For HPV DNA detection the universal primers GP5+/GP6+ (Table 1) were used, as previously reported [33,34]. These primers allow simultaneous amplification of several HPV types [35,36], including those frequently detected in KP, such as HPV6/11/16/18 [27,28]. QPCR reactions included 2x of the SsoAdvanced Universal SYBR Green Supermix, Bio-Rad (Hercules, CA, USA) and a final concentration of 0.5 μM for each GP5+/GP6+ primer.…”

Leptomeningeal Carcinomatosis of a Poorly Differentiated Cervical Carcinoma Caused by Human Papillomavirus Type 18

“…In the patient of our case report, the positivity for HPV18 added additional risk for the presence of a (pre)neoplastic lesion. This HPV type is detected in a significant proportion of invasive carcinomas (and particularly of adenocarcinomas), but is much less frequent in the preneoplastic high-grade lesions [ 15 , 16 , 17 , 18 , 19 ]. Although it is not yet clear whether this might be linked to a different natural history of cell transformation or to a high rate of progression to invasion of such lesions, it implies a peculiar difficulty in the prevention of HPV18-associated lesions, particularly by cytological screening [ 20 ].…”

Methylation of SERPINA1 gene promoter may predict chronic obstructive pulmonary disease in patients affected by acute coronary syndrome

“…Continuous data were tested for normal distribution with the Kolmogorov–Smirnov test. Normally distributed values were presented as mean ± SD and compared by t test [ 30 ]. Otherwise, median [interquartile range] and Mann–Whitney U were used.…”

“…Lymphocyte values were analyzed with D'Agostino-Pearson test for normality, and groups were compared employing the nonparametric Mann–Whitney U test [ 32 , 33 ]. Spearman correlation analysis was applied to evaluate age-related variations in SERPINA1 promoter methylation by matching the level of methylation to the age of each individual [ 30 ]. Multiple linear regression model has been used to estimate the associations between SERPINA1 epigenotypes and lymphocytes count.…”