Higher COVID-19 pneumonia risk associated with anti-IFN-α than with anti-IFN-ω auto-Abs in children.

We found that 19 (10.4%) of 183 unvaccinated children hospitalized for COVID-19 pneumonia had autoantibodies (auto-Abs) neutralizing type I IFNs (IFN-α2 in 10 patients: IFN-α2 only in three, IFN-α2 plus IFN-ω in five, and IFN-α2, IFN-ω plus IFN-ß in two; IFN-ω only in nine patients). Seven children (3.8%) had Abs neutralizing at least 10 ng/ml of one IFN, whereas the other 12 (6.6%) had Abs neutralizing only 100 pg/ml. The auto-Abs neutralized both unglycosylated and glycosylated IFNs. We also detected auto-Abs neutralizing 100 pg/ml IFN-α2 in 4 of 2,267 uninfected children (0.2%) and auto-Abs neutralizing IFN-ω in 45 children (2%). The odds ratios (ORs) for life-threatening COVID-19 pneumonia were, therefore, higher for auto-Abs neutralizing IFN-α2 only (OR [95% CI] = 67.6 [5.7-9,196.6]) than for auto-Abs neutralizing IFN-ω only (OR [95% CI] = 2.6 [1.2-5.3]). ORs were also higher for auto-Abs neutralizing high concentrations (OR [95% CI] = 12.9 [4.6-35.9]) than for those neutralizing low concentrations (OR [95% CI] = 5.5 [3.1-9.6]) of IFN-ω and/or IFN-α2.

Vaccine breakthrough hypoxemic COVID-19 pneumonia in patients with auto-Abs neutralizing type I IFNs.

Life-threatening 'breakthrough' cases of critical COVID-19 are attributed to poor or waning antibody response to the SARS-CoV-2 vaccine in individuals already at risk. Pre-existing autoantibodies (auto-Abs) neutralizing type I IFNs underlie at least 15% of critical COVID-19 pneumonia cases in unvaccinated individuals; however, their contribution to hypoxemic breakthrough cases in vaccinated people remains unknown. Here, we studied a cohort of 48 individuals (age 20-86 years) who received 2 doses of an mRNA vaccine and developed a breakthrough infection with hypoxemic COVID-19 pneumonia 2 weeks to 4 months later. Antibody levels to the vaccine, neutralization of the virus, and auto-Abs to type I IFNs were measured in the plasma. Forty-two individuals had no known deficiency of B cell immunity and a normal antibody response to the vaccine. Among them, ten (24%) had auto-Abs neutralizing type I IFNs (aged 43-86 years). Eight of these ten patients had auto-Abs neutralizing both IFN-α2 and IFN-ω, while two neutralized IFN-ω only. No patient neutralized IFN-ß. Seven neutralized 10 ng/mL of type I IFNs, and three 100 pg/mL only. Seven patients neutralized SARS-CoV-2 D614G and the Delta variant (B.1.617.2) efficiently, while one patient neutralized Delta slightly less efficiently. Two of the three patients neutralizing only 100 pg/mL of type I IFNs neutralized both D61G and Delta less efficiently. Despite two mRNA vaccine inoculations and the presence of circulating antibodies capable of neutralizing SARS-CoV-2, auto-Abs neutralizing type I IFNs may underlie a significant proportion of hypoxemic COVID-19 pneumonia cases, highlighting the importance of this particularly vulnerable population.

HPV E6/E7mRNA association with interleukin 10 (rs1800872) polymorphism in a group of Macedonian women.

Interleukin 10 (IL-10) is an immunosuppressive cytokine and its genetic variants could have an indirect impact on viral biology and human papillomavirus (HPV) E6/E7 messenger RNA (mRNA) expression as well. This study evaluates the association between IL-10-592 C/A (rs1800872) single-nucleotide polymorphism and HPV E6/E7 mRNA expression in a group of women from the Republic of North Macedonia. Using a commercial test, 272 women's cervical samples were analyzed for HPV E6/E7 mRNA and HPV DNA presence. The cases were stratified into three groups: double-positive (n = 108, positive for both tests), negative (n = 51, negative for HPV E6/E7 mRNA and HPV DNA positive), and the control group (n = 113, negative for both tests). The IL-10-592 C/A polymorphism was analyzed using polymerase chain reaction-restriction fragment length polymorphism. The results showed the CC genotype and the C allele frequencies of IL-10-592C/A were significantly higher in double-positive (59.3% and 78.2%) compared to negative group (39.2% and 65.7%), (p = 0.018, confidence interval [CI] = 2.25; 1.14-4.45 and p = 0.016, CI = 1.88; 1.11-3.16, respectively). The CC genotype and C allele of rs1800872 polymorphism were shown to be associated with HPV E6/E7 mRNA but not with HPV DNA positivity, which implies a possible role of this polymorphism in the course of the infection only after HPV onset, and lack of association with the susceptibility to HPV.

Association of TNF-a (rs361525 and rs1800629) with susceptibility to cervical intraepithelial lesion and cervical carcinoma in women from Republic of North Macedonia.

Cervical cancer (CCa) is one of the most common malign diseases in women associated with human papillomavirus (HPV). The virus is an initiating factor, but not sufficient for the development of cervical intraepithelial lesions (CIN) and CCa. The disease might be a result of the influence of host's genetic factors and polymorphisms in inflammatory-related genes that modify the immune response to HPV and attribute to cancer susceptibility. We carried out a study to determine the association between TNF-a-238G/A and TNF-a-308 G/T polymorphisms with HPV-positive CIN and CCa in women living in the Republic of North Macedonia. Using multiplex SNaPshot analysis for single nucleotide polymorphisms (SNPs), we analysed the genotype and allele distributions of TNF-a-238G/A and TNF-a-308 G/T in 134 cases (HPV-positive and histologically confirmed CIN and CCa) and in 113 controls (cytological and HPV-negative women). For further analysis, the case group was stratified in three subgroups (all cases: CINs+ CCa- group; CIN2+ -group and CIN1- group). Data analysed using the odds ratio (OR) and chi-square test showed the frequency of AA genotypes and A alleles are not significantly higher in cases compared to the controls for both SNPs: AA of TNF-a-238 (0.7% versus 0%) and TNF-a-308 (1.5% versus 0.9%) as well as A allelic frequency (3.0% versus 1.7%) and (13.1% versus 10.6), respectively. The comparison of the case's subgroups with the control group did not show a statistically significant difference. Compared to controls, TNF-a-238G/A and TNF-a-308 G/T are not associated with the risk of HPV associated CIN or CCa in the studied women.

Association of IL-10 (rs1800872) and IL-4R (rs1805010) polymorphisms with cervical intraepithelial lesions and cervical carcinomas

PURPOSE: Genetic characteristic of cytokines may influence cervical intraepithelial neoplasia (CIN) and cervical cancer (CCa) susceptibility. We analysed an association of IL-10-592 A/C, IL-4R I75VA/G polymorphisms with susceptibility to human papillomavirus (HPV) positive CIN and CCa. METHODS: Using multiplex PCR- SNaPShot analysis, 134 cases (HPV positive CINs and CCa) and 113 controls (HPV negative NILM) were genotyped for these two cytokine variants. RESULTS: Data analyzed using odds ratio (OR) and chisquare (x2) test showed that the frequency of CC of IL-10-592 genotype was significantly higher in cases (67.2%) than in controls (49.6%) [CC vs CA+AA; p=0.005, OR=2.08 (95%CI:1.24-3.49)] as well as the allelic frequency of major C allele (82.1%) in cases than in controls (72.6%) [p=0.01, OR=1.73 (95%CI: 1.13-2.66)]. Furthermore, AA genotype of IL-4RI75V had significantly lower frequency in CIN1 (25.0%) comparedwith CIN2+ group (30.8%) (p=0.03, OR=0.39, 95%CI: 0.14- 1.11) after the stratifications of the cases in low grade and high grade with CCa as separate groups. CONCLUSION: We concluded that IL-10-592 A/AA variant indicates a protective role in cervical cancer development and the GG genotype of IL-4RI75V conferred protection against progression of CIN1 to CIN2+ or CCa among women from Republic of North Macedonia.

Comparison Between HPV DNA Testing and HPV E6/E7 MRNA Testing in Women with Squamous Cell Abnormalities of the Uterine Cervix.

INTRODUCTION: The aim of the study was to compare the results of two human papillomavirus (HPV) diagnostic techniques: human papillomavirus deoxyribonucleic acid (HPV DNA) testing and human papillomavirus E6/E7 messenger ribonucleic acid (HPV E6/E7 mRNA) testing in women with squamous cell abnormalities of the uterine cervix. MATERIAL AND METHODS: Comparative prospective study, conducted in the period from January 2016 to June 2017 of 128 sexually active women, age groups of 20 to 59 years (40.50 ± 10.85) with squamous cell abnormalities on the cervical cytology. All patients were subject to: HPV DNA testing, HPV E6/E7 mRNA testing and colposcopic cervical biopsy with endocervical curettage for histopathologycal analysis. HPV DNA testing was done using multiplex polymerase chain reaction (PCR) and reverse hybridization methods. HPV E6/E7 mRNA testing was done using real-time PCR method. RESULTS: Data analysis showed an association between the results of HPV DNA testing and HPV E6/E7 mRNA testing (p˂0.0001). The concordance between the results of both tests was moderate (55.47%). The results show that HPV E6/E7 mRNA testing had a higer specificity 88.89% and positive predictive value (PPV) 93.59% for HSIL + invasive squamous cell carcinoma compared to HPV DNA testing that had specificity of 55.56% and PPV 84.61%, respectively. CONCLUSION: The results of our study suggested that HPV E6/E7 mRNA testing is more specific and has a higher positive predictive value than HPV DNA testing and that viral oncoproteins E6 and E7 are superior biomarkers for the detection of high-risk HPV-associated squamous intraepithelial lesions of the uterine cervix.

Human Papillomavirus as a Potential Risk Factor for Oral Premalignant Lesions.

Oral premalignant lesions (OPLs) and numerous alterations of oral mucosa remain unsolved due to their complex etiopathogenesis. Human papillomaviruses (HPVs), in particular, have been reported as the possible risk factors or cofactors. The aim of the study was to determine the association of different HPV types with oral premalignant lesions, and the potential role of smoking and alcohol use. Eighty patients (mean age ± SD, 52.45±5.56) of both genders, 19 (23.75%) male and 61 (76.25%) female, were enrolled in the study. Study group included 40 patients diagnosed with OPLs (leukoplakia, erythroplakia, actinic keratosis and lichen planus), while control group included another 40 patients with healthy oral mucosa. Genotyping of the HPV types was performed by qualitative real-time HPV typing polymerase chain reaction test. HPV DNA was detected in 30% (12/40) of study group patients and 2.5% (1/40) of control group patients. The results revealed the presence of HPV16 in 15% (6/40), HPV56 in 10% (4/40), and HPV18 in 5% (2/40) of study group cases, and HPV31 in 1 (2.5%) control group patient. Th e association of oral HPV positivity and smoking/alcohol use in the study group was not statistically significant (p<0.05). In conclusion, high-risk HPV types are associated with oral premalignant disorders. However, it remains unknown whether HPV acts as an innocent bystander or it has a role in initiating development of premalignant lesions. Smoking and alcohol use were not associated with the existing oral HPV infection.

HPV E6/E7 mRNA versus HPV DNA biomarker in cervical cancer screening of a group of Macedonian women.

High risk types of human papillomaviruses E6/E7 oncogenes and their association with tumor suppressor genes products are the key factors of cervical carcinogenesis. This study proposed them as specific markers for cervical dysplasia screening. The aim of the study is to compare the clinical and prognostic significance of HPV E6/E7 mRNA as an early biomarker versus HPV DNA detection and cytology in triage of woman for cervical cancer. The study group consists of 413 women: 258 NILM, 26 ASC-US, 81 LSIL, 41 HSIL, and 7 unsatisfactory cytology. HPV4AACE screening, real-time multiplex PCR and MY09/11 consensus PCR primers methods were used for the HPV DNA detection. The real-time multiplex nucleic acid sequence-based assay (NucliSENS EasyQ HPV assay) was used for HPV E6/E7 mRNA detection of the five most common high risk HPV types in cervical cancer (16, 18, 31, 33, and 45). The results show that HPV E6/E7 mRNA testing had a higher specificity 50% (95% CI 32-67) and positive predictive value (PPV) 62% (95% CI 46-76) for CIN2+ compared to HPV DNA testing that had specificity of 18% (95% CI 7-37) and PPV 52% (95% CI 39-76) respectively. The higher specificity and PPV of HPV E6/E7 mRNA testing are valuable in predicting insignificant HPV DNA infection among cases with borderline cytological finding. It can help in avoiding aggressive procedures (biopsies and over-referral of transient HPV infections) as well as lowering patient's anxiety and follow up period.