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Sensitive and specific genotyping of human papillomaviruses (HPVs) is critical for the surveillance and monitoring of the vaccine effectiveness. Here, HPV genotypes were identified in 137 cervical samples with different histology (79 ≤CIN1 and 58 CIN3+) using Nested-PCR followed by Next-Generation sequencing (NGS) and relative proportions for each genotype in multiple infections were computed. All samples had been previously genotyped by PCR-Reverse Blotting Hybridization (PCR-RBH) thus allowing for a concordance analysis between both techniques. Multiple infections were present in 85% of ≤CIN1 cases compared to only 41% in CIN3+ cases (p

The proportion of HPV16 and 18-associated cervical cancer (CC) appears rather constant worldwide (≥70%), but the relative importance of the other HR-HPV differs slightly by geographical region. Here, we studied the HPV genotype distribution of HPV positive Latin American (LA) women by histological grade, in a sub-cohort from the ESTAMPA study; we also explored the association of age-specific HPV genotypes in severe lesions. Cervical samples from 1,252 participants (854 ≤CIN1, 121 CIN2, 194 CIN3 and 83 CC) were genotyped by two PCRs-Reverse Blotting Hybridization strategies: i) Broad-Spectrum General Primers 5+/6+ and ii) PGMY9/11 PCRs. HPV16 was the most frequently found genotype in all histological grades, and increased with the severity of lesions from 14.5% in ≤ CIN1, 19.8% in CIN2, 51.5% in CIN3 to 65.1% in CC (p < 0.001). For the remaining HR-HPVs their frequency in CC did not increase when compared to less severe categories. The nonavalent vaccine HR-types ranked at the top in CC, the dominant ones being HPV16 and HPV45. HR-HPV single infection occurs, respectively, in 57.1% and 57.0% of ≤CIN1 and CIN2, increasing to 72.2% and 91.6% in CIN3 and CC (p

Our aim was to evaluate the analytical and clinical performance of the SARS-CoV-2 molecular detection kits used in Argentina. Nine real-time reverse-transcription polymerase chain reaction (RT-qPCR) and three reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assays were evaluated using the World Health Organization (WHO) recommended test as reference method. A secondary standard calibrated for the E, N and RdRp genes against the Pan American Health Organization—World Health Organization—International Standard was used to calculate the limit of detection (LoD). A panel of artificial clinical samples, 32 positive and 30 negative for SARS-CoV-2, were analyzed to estimate the kappa concordance (κ) and the diagnostic performance. Differences among the LoD values for the target genes amplified by each kit were &gt, 1 log copies/reaction. The κ for the RT-qPCR kits was greater than 0.9, whereas that for the RT-LAMP assays ranged from 0.75 to 0.93. The clinical performance of RT-qPCR kits showed 100% specificity and high sensitivity, although with variations according to the gene analyzed. The E and N genes provided greater clinical sensitivity, whereas the RdRp gene increased the clinical specificity. The RT-LAMP assays revealed a variable diagnostic performance. The information provided can be useful to choose the most appropriate diagnostic test and may contribute to the establishment of a consensus in the diagnosis of SARS-CoV-2 in Argentina and the region.

Highlights • HPV16/18 decreased by >93% in vaccinated sexually active Argentine girls. • Detected reduction of HPV31 and 45 would add to the success of immunization. • No genotype replacement was observed. • First HPV vaccination monitoring data reported from a Latin American country.

High levels of circulating EBV load are used as a marker of post-transplant lymphoproliferative disorders (PTLD). There is no consensus regarding the threshold level indicative of an increase in peripheral EBV DNA. The aim of the study was to clinically validate a developed EBV quantification assay for early PTLD detection.Transversal study: paired peripheral blood mononuclear cells (PBMC), plasma and oropharyngeal lymphoid tissue (OLT) from children undergoing a solid organ transplant with (n=58) and without (n=47) PTLD. Retrospective follow-up: 71 paired PBMC and plasma from recipients with (n=6) and without (n=6) PTLD history. EBV load was determined by real-time PCR. The diagnostic ability to detect all PTLD (categories 1–4), advanced PTLD (categories 2–4) or neoplastic PTLD (categories 3 and 4) was estimated by analyzing the test performance at different cut-off values or with a load variation greater than 0.5log units.The higher diagnostic performance for identifying all, advanced or neoplastic PTLD, was achieved with cut-off values of 1.08; 1.60 and 2.47log EBVgEq/105 PBMC or 2.30; 2.60; 4.47loggEq/105 OLT cells, respectively. EBV DNA detection in plasma showed high specificity but low (all categories) or high (advanced/neoplastic categories) sensitivity for PTLD identification. Diagnostic performance was greater when: (1) a load variation in PBMC or plasma was identified; (2) combining the measure of EBV load in PBMC and plasma.The best diagnostic ability to identify early PTLD stages was achieved by monitoring EBV load in PBMC and plasma simultaneously; an algorithm was proposed.

INTRODUCTION: The quantification of circulating Epstein-Barr virus (EBV) DNA is used to monitor transplant patients as an early marker of Post-Transplant Lymphoproliferative Disorders (PTLD). So far no standardized methodology exists for such determination. OBJECTIVE: Our purpose was to develop and validate a real-time PCR assay to quantify EBV DNA in clinical samples from transplant recipients. METHODS: A duplex real-time PCR method was developed to amplify DNA from EBV and from a human gene. The EBV load was determined in peripheral blood mononuclear cells (PBMC), plasma and oropharyngeal tissue from 64 non-transplanted patients with lymphoid-hypertrophy (Non-Tx), 47 transplant recipients without PTLD (Tx), 54 recipients with PTLD (Tx-PTLD), and 66 blood donors (BD). WinPEPI, version 11.14 software was used for statistical analysis. RESULTS: Analytical validation: the intra and inter-assays variation coefficients were less than 4.5% (EBV-reaction) and 3% (glyceraldehyde 3-phosphate dehydrogenase - GAPDH reaction). Linear ranges comprised 10(7)-10 EBV genome equivalents (gEq) (EBV-reaction) and 500,000-32 human gEq (GAPDH-reaction). The detection limit was 2.9 EBV gEq (EBV-reaction). Both reactions showed specificity. Application to clinical samples: higher levels of EBV were found in oropharyngeal tissue from transplanted groups with and without PTLD, compared to Non-Tx (p

Two Epstein Barr virus (EBV) genotypes: EBV-1 and EBV-2 have been described. A 30-bp deletion in latent membrane protein-1 gene (del-LMP-1) has been identified in various pathologies. The aim of this study was to determine EBV genotypes and 30-bp deletion frequency in HIV-infected patients from Argentina. The study was performed on 258 individuals: Cases: 144 HIV-infected patients that included: (a) 7 AIDS patients with primary central nervous system lymphoma (PCNSL), (b) 62 AIDS patients, and (c) 75 asymptomatic HIV-infected patients. Controls: 114 HIV-negative individuals. EBV genotypes and variants in LMP-1 gene were detected by polymerase chain reaction (PCR)-Southern blot on DNA extracted from peripheral blood mononuclear cells and brain biopsies. In PCNSL, the presence of EBV was confirmed by EBER RNA in situ hybridization, and DNA sequencing of 3′ end LMP-l gene of PCR products was performed. In HIV-infected patients, EBV-1 was detected in 48.6%, EBV-2 in 18.8%, and co-infection with both genotypes in 32.6%. In control group, EBV-1 was present in 74.3%, EBV-2 in 12.4%, and co-infection in 13.3%. Del-LMP-1 was found in 44.4% of HIV-infected patients samples (20.7% alone and 23.7% co-infection with non-deleted form) while it was found in 25.3% (6.3% alone and 19% with co-infection) in HIV-negative individuals. In HIV-infected patients EBV-2, co-infection and 30-bp deletion are more prevalent than in control group. In all, PCNSL brain biopsies samples, del-LMP-1 always was detected with EBV-2, but more cases would have to be included to draw definitive conclusions. J. Med. Virol. 79:401–407, 2007. © 2007 Wiley-Liss, Inc.

There are two types of Epstein Barr virus (EBV): EBV-1 and EBV-2, distinguished by genomic polymorphism in the genes encoding the nuclear antigens (EBNA-2, -3A, -3B, -3C). Latent membrane protein 1 (LMP-1) is an EBV protein with known oncogenic properties. Different variants had been described; among them, a 30 base pair (bp) deletion (del-LMP-1) had been reported in benign and malignant pathologies, but there is little information about its frequency in healthy populations. The aim of this study was to determine the distribution of the EBV genotypes and the 30 bp deletion frequency, in EBV healthy carriers from Argentina. Analysis of EBNA-3C and LMP-1 genes were done by polymerase chain reaction (PCR) followed by Southern blot hybridization on DNA of peripheral blood mononuclear cells (PBMCs) from blood bank donors. EBV-1 was present in 75.9% of samples, EBV-2 in 14.6%, and co-infections with both types in 6.5%. The deleted LMP-1 variant was found in 7.4% of analyzed samples, corresponding 3.2% to deleted variant alone and 4.2% to co-infections with non-deleted form. The non-deleted variant was found in 64.6% whereas in the remaining 28%, no PCR product was detected. These results showed that EBV-1 was the more prevalent type in healthy carriers of Argentina, similar to reports from others countries. A predominance of the non-deleted LMP-1 variant was observed. The presence of co-infections with both types and variants demonstrated that healthy individuals may also harbor multiple EBV infections.

High Epstein-Barr virus load has been related to an increased risk of Posttransplant Lymphoproliferative Disorders (PTLD) in transplant recipients.Development of a method to quantitate EBV DNA levels in peripheral blood mononuclear cells (PBMC) and evaluate its usefulness in transplant patients.We designed a semiquantitative nested PCR based on a limiting dilution analysis to detect high viral loads in PBMC. This method was applied to 25 healthy carriers, and 85 solid organ transplant recipients as follows: (A) 53 asymptomatic patients; (B) 24 symptomatic patients; (C) eight patients with PTLD.In healthy carriers the reciprocal of the limiting dilution (RLD) ranged between non-detected (ND) and 1, the median RLD was ND, which is equivalent to a viral load of1 copy per 10(5) PBMC. In the transplant population the medians RLD (range) were: (A) asymptomatic group: ND (ND-64), median equivalent to a viral load of1 copy per 10(5) PBMC; (B) symptomatic group: 4 (ND-256), median equivalent to a range of viral load of 4-64 copies per 10(5) PBMC. (C) PTLD group: 256 (16-16384), median equivalent to a range of viral load of 256-4096 copies per 10(5) PBMC. Statistically significant differences were found between all groups: A+B vs. C (P0.0001); A vs. B (P0.0001); A vs. C (P0.0001), B vs. C (P0.0001). We also observed a good correlation between viral loads and clinical findings in four follow-up patients. Considering the RLD=256 as a cutoff point to detect transplant patients with PTLD, resulted in sensitivity 75%, specificity 96.7%, positive predictive value 60%, negative predictive value 98.3%.This SQ-PCR method enables us to differentiate between transplant patients with and without PTLD; therefore, it could be applied as a marker for early detection of this pathology.