Merkel cell polyomavirus (MCPyV), a small DNA tumor virus, has been detected in Merkel cell carcinoma (MCC) and in normal tissues. Since MCPyV infection occurs in both MCC-affected patients and healthy subjects (HS), innovative immunoassays for detecting antibodies (abs) against MCPyV are required. Herein, sera from HS were analyzed with a novel indirect ELISA using two synthetic peptides mimicking MCPyV capsid protein epitopes of VP1 and VP2. Synthetic peptides were designed to recognize IgGs against MCPyV VP mimotopes using a computer-assisted approach. The assay was set up evaluating its performance in detecting IgGs anti-MCPyV on MCPyV-positive (n=65) and -negative (n=67) control sera. Then, the ELISA was extended to sera (n=548) from HS aged 18-65 yrs old. Age-specific MCPyV-seroprevalence was investigated. Performance evaluation indicated that the assay showed 80% sensitivity, 91% specificity and 83.9% accuracy, with positive and negative predictive values of 94.3% and 71%, respectively. The ratio expected/obtained data agreement was 86%, with a Cohen’s kappa of 0.72. Receiver-operating characteristic (ROC) curves analysis indicated that the areas under the curves (AUCs) for the two peptides were 0.82 and 0.74, respectively. Intra-/inter-run variations were below 9%. The overall prevalence of serum IgGs anti-MCPyV in HS was 62.9% (345/548). Age-specific MCPyV-seroprevalence was 63.1% (82/130), 56.7% (68/120), 64.5% (91/141), and 66.2% (104/157) in HS aged 18-30, 31-40, 41-50 and 51-65 yrs old, respectively (p>0.05). Performance evaluation suggests that our indirect ELISA is reliable in detecting IgGs anti-MCPyV. Our immunological data indicate that MCPyV infection occurs asymptomatically, at a relatively high prevalence, in humans.

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.

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SERine Protein INhibitor-A1 (SERPINA1) is an inducible blood cell gene coding for alpha1-antitrypsin (AAT), a plasma protease inhibitor whose circulating levels are raised during inflammation, infection and advanced pregnancy. DNA methylation has been suggested to play a role in SERPINA1 gene expression regulation in peripheral blood mononuclear cells (PBMCs). The methylation status of SERPINA1 in PBMCs is unknown. The aim of this study was to evaluate the methylation profile of the SERPINA1 promoter in PBMC. To this purpose PBMCs and serum were collected from healthy subjects (HS) (n = 75), including blood donors (BD) (n = 25), pregnant women at early pregnancy (EP) (n = 25), i.e., within the first trimester, and pregnant women at late pregnancy (LP) (n = 25), i.e., at the third trimester. DNA from PBMCs was treated with sodium bisulfite and PCR amplified for SERPINA1 gene promoter, followed by sequencing analyses. AAT serum levels were determined by ELISA test. SERPINA1 was found hypermethylated in 58.7% of HS. The prevalence of SERPINA1 hypermethylation was significantly higher in BD (68%) and EP (88%) than in LP (20%) (p < 0.01). The median serum AAT concentration was 1.07, 0.63, and 3.15 mg/ml in BD, EP, and LP, respectively (p < 0.05, BD and EP vs LP). This study indicates, for the first time, that SERPINA1 gene promoter is differentially methylated in PBMCs from HS. Likely, modulation of the methylation may be a novel epigenetic regulator mechanism of AAT expression in the PBMC of HS. Therefore, SERPINA1 gene promoter methylation may represent an epigenetic biomarker of PBMCs in healthy subjects.

Viral infections are considered to be risk factors for spontaneous abortion (SA). Conflicting results have been reported on the association between Human Papillomavirus (HPV) and SA. HPV DNA was investigated in matched chorionic villi tissues and peripheral blood mononuclear cells (PBMCs) from women who experienced SA (n = 80, cases) and women who underwent a voluntary interruption of pregnancy (VI; n = 80, controls) by qualitative PCR and quantitative droplet digital PCR (ddPCR). Viral genotyping was performed using real-time PCR in HPV-positive samples. Specific IgG antibodies against HPV16 were investigated in sera from SA (n = 80) and VI (n = 80) females using indirect ELISA assays. None of the DNA samples from SA subjects was HPV-positive (0/80), whilst HPV DNA was detected in 2.5% of VI women (p > 0.05), with a mean viral DNA load of 7.12 copy/cell. VI samples (n = 2) were found to be positive for the HPV45 genotype. The ddPCR assay revealed a higher number of HPV-positive samples. HPV DNA was detected in 3.7% and 5% of SA and VI chorionic tissues, respectively, with mean viral DNA loads of 0.13 copy/cell in SA and 1.79 copy/cell in VI (p >0.05) samples. All DNA samples from the PBMCs of SA and VI females tested HPV-negative by both PCR and ddPCR. The overall prevalence of serum anti-HPV16 IgG antibodies was 37.5% in SA and 30% in VI (p > 0.05) women. For the first time, HPV DNA was detected and quantitatively analyzed using ddPCR in chorionic villi tissues and PBMCs from SA and VI women. Circulating IgG antibodies against HPV16 were detected in sera from SA and VI females. Our results suggest that HPV infection in chorionic villi may be a rare event. Accordingly, it is likely that HPV has no significant role in SA.