Distribution of human papillomavirus genotypes in women with high-grade cervical intraepithelial lesions and cervical carcinoma and analysis of human papillomavirus-16 genomic variants

Aim To analyze the distribution of high-risk human papil - lomavirus (HR-HPV) genotypes and the diversity of HPV16 genomic variants in Croatian women with high-grade squamous intraepithelial lesions (HSIL) and cervical carci - noma. Methods Tissue biopsy specimens were obtained from 324 women with histopathologically confirmed HSIL or cervical carcinoma, 5 women with low-grade SIL, and 49 women with negative histopathology. HR-HPV DNA was detected with Ampliquality HPV-type nucleic-acid hybrid - ization assay, which identifies 29 different HPV genotypes. HPV-16 genomic variants were analyzed by an in-house se - quencing. Results The most common HPV type in women with HSIL was HPV-16, detected in 127/219 (57.9%) specimens. HPV16 was also the dominant type in squamous cell cervical carcinoma (46/69 or 66.7%) and in adenocarcinoma (18/36 or 50.0%). Out of 378 patients, 360 had HR-HPV (282 sin - gle infections and 79 multiple infections), 3 (0.8%) patients had low-risk HPV, and 15 (4%) tested negative. HPV-16 vari - ants were determined in 130 HPV-16 positive specimens, including 74 HSIL and 46 carcinoma specimens. In HSIL specimens, 41 distinct variants were found, 98.6% belong - ing to the European branch and 1.4% belonging to the African branch. In cervical carcinoma specimens, 95% isolates grouped in 41 variants belonging to the European branch, one isolate (2.5%) belonged to the North American, and one (2.5%) to the Asian-American branch. Conclusion HPV-16, mainly belonging to the European branch, was the most frequent HPV genotype in women from Croatia with histologically confirmed HSIL and cervi - cal cancer

Sporadic hepatitis A virus PCR false-positive results observed during reflex testing of serum samples previously tested for Anti-HAV antibodies and caused by contamination with HAV RNA present in the reagents of the commercial Anti-HAV immunoassay

Hepatitis A diagnosis relies on serology and occasionally on hepatitis A virus (HAV) RNA detection. For timely diagnosis and the avoidance of drawing additional blood, molecular testing is often performed as reflex testing by using blood specimens that were initially sent for anti-HAV serology. Reflex molecular testing is preferably performed from different sample aliquots, but, for limited sample quantities, it uses samples that have been preprocessed in an immunoassay analyzer. In 2012, we first observed sporadic HAV RNA-positive cases that were inconsistent with patients’ serological profiles and/ or medical histories, suggesting that occasional laboratory contamination was causing false-positive PCR results. Multiple external quality assurance (EQA) and laboratory surface contamination checks were performed, questionable specimens were tested with various HAV RNA tests, and follow-up serum/stool samples were collected. All contamination-check samples and samples from healthy individuals tested HAV RNA-negative, and the laboratory successfully passed all EQAs. The HAV RNA-positive results were reproducible with various HAV RNA assays. No patients seroconverted, and their follow-up samples were consistently HAV RNA-negative. Finally, a detailed review of testing protocols revealed a correlation between HAV RNA false positivity and preceding anti-HAV testing with the Cobas-e411 automated immunoassay analyzer. HAV RNA was detected in the Cobas-e411 anti-HAV reagents, with the HAV sequences matching those from the false-positive samples. Preceding anti-HAV testing using two other immunoassay analyzers did not result in subsequent HAV RNA false positivity during reflex testing. The Cobas-e411 pipetting procedure with a single pipette tip collecting samples and anti-HAV reagents contaminated the original sample with the HAV RNA that was present in the immunoassay’s reagents, thereby resulting in HAV RNA false positivity during the reflex testing

An Improved Protocol for Comprehensive Etiological Characterization of Skin Warts and Determining Causative Human Papillomavirus Types in 128 Histologically Confirmed Common Warts

Human papillomaviruses (HPVs) are etiologically associated with various benign and malignant neoplasms of cutaneous and mucosal epithelia. We describe an improved diagnostic protocol for comprehensive characterization of causative HPV types in common warts, in which broad-spectrum PCRs followed by Sanger sequencing, two previously described and seven newly developed type-specific quantitative real-time PCRs (qPCRs) coupled with the human beta-globin qPCR were used for: (i) diagnosis of HPV infection in warts; (ii) estimation of cellular viral loads of all HPV types detected; and (iii) determination of their etiological role in 128 histologically confirmed fresh-frozen common wart tissue samples. A total of 12 different causative HPV types were determined in 122/126 (96.8%) HPV-positive warts, with HPV27 being most prevalent (27.0%), followed by HPV57 (26.2%), HPV4 (15.1%), HPV2 (13.5%), and HPV65 (7.9%). The cellular viral loads of HPV4 and HPV65 were estimated for the first time in common warts and were significantly higher than the viral loads of HPV2, HPV27, and HPV57. In addition, we showed for the first time that HPV65 is etiologically associated with the development of common warts in significantly older patients than HPV27 and HPV57, whereas HPV4-induced warts were significantly smaller than warts caused by HPV2, HPV27, HPV57, and HPV65

GeneXpert Enterovirus Assay: One-Year Experience in a Routine Laboratory Setting and Evaluation on Three Proficiency Panels▿

A prospective unblinded comparative evaluation of three assays for the detection of enteroviral RNA performed on 83 positive and 79 negative cerebrospinal fluid samples showed initial and resolved sensitivities of 90.4% and 98.8%, respectively, for the Cepheid GeneXpert enterovirus assay; 94.0% and 97.6%, respectively, for the Argene enterovirus consensus kit; and 100% and 100%, respectively, for an in-house real-time PCR. The initial and resolved specificities were 100% for all three assays

Real-life head-to-head comparison of performance of two high-throughput automated assays for the detection of SARS-CoV-2 RNA in nasopharyngeal swabs

The Alinity m (Abbott Molecular, Des Plaines, IL) automated molecular analyzer allows continuous loading of samples and sample-to-result molecular detection of several microorganisms. The detection of SARS-CoV-2 by the Alinity m was compared with that of the cobas 6800 (Roche Molecular Systems, Branchburg, NJstandard comparator) in a manufacturer-independent clinical evaluation on 2157 consecutive nasopharyngeal swab samples. Valid initial results on Alinity m and cobas 6800 were obtained from 2129 (98.7%) and 2157 (100%) samples, respectively. The overall percent agreement (95% CI) was 98.3% (2092/2129 [97.6%–98.7%])positive percent agreement, 100% (961/961 [99.6%–100%])negative percent agreement, 96.8% (1131/1168 [95.7%–97.7%])and high κ value, 0.965 (0.954–0.976). There were 37 discordant results on Alinity m and, based on discordant analyses, including previous and/or follow-up PCR results, 22 could be considered analytically true positive with high probability. Due to a lack of additional information and an inability to perform repeated/further testing, the status of the remaining 15 discordant results remained unresolved. The throughput of the two analyzers was compared using testing on 564 samples in parallel across two 8-hour shifts in clinical practice. The turnaround times were compared using processing of 94 routine samples in parallel on each working day for 5 consecutive days. The two analyzers showed similar performance, with certain differences that have potential importance in some laboratory settings

Assessing Gammapapillomavirus infections of mucosal epithelia with two broad-spectrum PCR protocols

Background: Human papillomaviruses (HPVs) have been divided into mucosal and cutaneous types according to their primary epithelial tissue tropism. However, recent studies showed the presence of several cutaneous types in mucosal lesions and healthy mucosa from different anatomical sites. Methods: Here, the HPV prevalence and type-specific distribution were assessed in a variety of mucosal samples from 435 individuals using a combination of two established broad-spectrum primer systems: Gamma-PV PCR and CUT PCR. Results: Overall HPV prevalence in anal canal swabs, cervical cancer biopsies, genital warts and oral swabs was 85, 47, 62 and 4%, respectively. In anal canal swabs, Alpha-PVs were most frequently found (59%), followed by Gamma-(37%) and Beta-PVs (4%). The prevalence and persistence of HPV infection in the anal canal of 226 individuals were further explored. Overall HPV, Gamma-PVs and multiple HPV infections were significantly higher in men vs. women (p = 0.034, p = 0.027 and p = 0.003, respectively); multiple HPV infections were more common in individuals ≤40 years (p = 0.05), and significantly higher prevalence of Gamma-PVs and multiple HPV infections was observed in HIV-1-positive vs. HIV-1-negative individuals (p = 0.003 and p = 0.04, respectively). Out of 21 patients with follow-up anal swabs, only one persistent infection with the same type (HPV58) was detected. Conclusions: Our findings suggest that Gamma-PVs (except species Gamma-6) are ubiquitous viruses with dual muco-cutaneous tissue tropism. Anal canal Gamma-PV infections may be associated with sexual behavior and the host immune status. This study expands the knowledge on Gamma-PVs’ tissue tropism, providing valuable data on the characteristics of HPV infection in the anal canal.Fil: Bolatti, Elisa María. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología Molecular y Celular de Rosario. Grupo Virología Humana (CONICET-IBR); Argentina.Fil: Bolatti, Elisa María. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Área Virología; Argentina.Fil: Hošnjak, Lea. University of Ljubljana. Faculty of Medicine. Institute of Microbiology and Immunology; Slovenia.Fil: Chouhy, Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología Molecular y Celular de Rosario. Grupo Virología Humana (CONICET-IBR); Argentina.Fil: Chouhy, Diego. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Área Virología; Argentina.Fil: Casal, Pablo E. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Área Virología; Argentina.Fil: Re Louhau, María F. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología Molecular y Celular de Rosario. Grupo Virología Humana (CONICET-IBR); Argentina.Fil: Bottai, Hebe. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Área Estadística y Procesamiento de Datos; Argentina.Fil: Komloš, Kristina Fujs. University of Ljubljana. Faculty of Medicine. Institute of Microbiology and Immunology; Slovenia.Fil: Poljak, Mario. University of Ljubljana. Faculty of Medicine. Institute of Microbiology and Immunology; Slovenia.Fil: Giri, Adriana Angélica. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología Molecular y Celular de Rosario. Grupo Virología Humana (CONICET-IBR); Argentina.Fil: Giri, Adriana Angélica. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Área Virología; Argentina