Sandra V. Gallego

Development and validation of a real-time PCR assay for a novel HTLV-1 tax sequence detection and proviral load quantitation

A quantitative real-time PCR (qPCR) assay using SYBR Green dye was established in order to detect and quantify the proviral DNA of HTLV-1 in peripheral blood mononuclear cells (PBMCs). Primers were designed, and the assay was standardized to amplify a novel, conserved HTLV-1 tax region. Proviral load was normalized to the amount of cellular DNA by quantitation of the human albumin gene. Firstly, the qPCR was assessed determining the specificity, sensitivity, dynamic range and intra- and inter-assay reproducibility of the technique. The limit of detection as determined by PROBIT analysis using dilutions of the standard was 2.97 copies. The assay had an excellent dynamic range from 10⁵ to 10¹ copies per reaction and good intra- and inter-assay reproducibility, CVs less than 2%. Secondly, the performance of the qPCR was tested on 40 HTLV-1 seropositive individuals. Proviral load for HTLV-1 carriers ranged from 2.2×10² to more than 8.3×10⁴ copies/10⁶ PBMCs. The high sensitivity and wide dynamic range allowed the determination of a broad range of HTLV-1 proviral loads in infected individuals. This assay is a valuable alternative diagnostic tool when current available serological assays are insufficient. In addition, it will facilitate the study of the relationship between proviral load and pathogenesis.

Caracterización de la infección por Bordetella pertussis, Bordetella spp. y coqueluche en la provincia de Córdoba, Argentina

INTRODUCTION Whooping cough is a re-emerging infection in the world and Latin America. OBJECTIVE It was considered relevant to investigate the clinical and epidemiological profile of Bordetella spp. and Bordetella pertussis infection in Cordoba province, Argentina; evaluating, at the same time, the co-infection with virus producing respiratory infections that may be confused with whooping cough. MATERIAL AND METHODS All whooping cough suspected cases were studied by Polimerase Chain Reaction, amplifying the repeated insertion sequence (IS) 481 and the promoter gene encoding pertussis toxin, between 2011 and 2013. The data were obtained from the clinical and epidemiological records. RESULTS From 2,588 whooping cough suspected cases, 11.59% was infected by Bordetella spp. and 9.16% was confirmed as Bordetella pertussis infection. The rate of infection was 7.22 and 1.84 per 100,000 for 2011 and 2012, respectively. The infection presented a seasonal tendency and it was mainly found on the group of children between 13 and 24 months old. The co-infection with virus producing respiratory infections, were uncommon. Paroxysmal cough, cyanosis and/or vomiting were predictors of the infection for Bordetella pertussis. DISCUSSION AND CONCLUSIONS To deal with the re-emergence of whooping cough is important the knowledge of the regional epidemiological situation. This paper shows the situation of these infections in the regional clinical and epidemiological context, and makes the information available for health decision-making.

Usefulness of nucleic acid testing to reduce risk of hepatitis B virus transfusion-transmitted infection in Argentina: high rate of recent infections.

Results from 10‐year experience using nucleic acid test (NAT) screening in a blood bank of Córdoba are presented, showing the first data on prevalence of recent hepatitis B virus (HBV) infections and occult HBV infections (OBIs) in Argentina.

Informe breveSensibilidad del equipo Cobas AmpliScreen™ HIV-1 Test, v1.5, para la detección de HIV-1Sensitivity of the COBAS AmpliScreen™ HIV-1 Test v1.5 for HIV-1 detection

The introduction of nucleic acid amplification techniques (NAT) in blood banks was intended to reduce the residual risk of transfusion-transmitted infections. Co-circulation of a great diversity of HIV-1 variants in Argentina portrays the need to assess the sensitivity of serological and molecular assays available for their detection. In this study, we evaluated the sensitivity of the COBAS AmpliScreen™ HIV-1 Test, version 1.5 (Roche) for the detection of HIV-1 RNA in plasma samples of infected individuals from Argentina. The results of this study reveal that this technique has high sensitivity for the detection of HIV-1 RNA under assay conditions: using mini-pool testing, pools ≥ 50 RNA copies per ml achieved ≥ 92 % sensitivity, whereas in the standard procedure, samples ≥ 207 RNA copies/ml achieved 100 % sensitivity. Moreover, the COBAS AmpliScreen™ HIV-1 Test, version 1.5 (Roche) is suitable for detecting prevailing HIV-1 variants.Resumen Las tecnicas de amplificacion de acidos nucleicos (NAT) se incorporaron en los bancos de sangre para reducir el riesgo residual de transmision de infecciones por via transfusional. La cocirculacion de distintas variantes del HIV-1 en Argentina indica la necesidad de evaluar la sensibilidad de los ensayos serologicos y moleculares disponibles para su deteccion. En este trabajo se evaluo la sensibilidad del equipo COBAS AmpliScreen™ HIV-1 Test, version 1.5 (Roche), para detectar ARN viral en plasmas de individuos infectados con HIV-1 de Argentina. Los resultados demuestran que esta tecnica tiene una alta sensibilidad para detectar ARN de HIV-1 en las condiciones ensayadas: para ensayo de mini-pooles (pooles ≥ 50 copias de ARN/ml), la sensibilidad fue ≥ 92 %, y para procedimiento estandar (plasmas ≥ 207 copias de ARN/ml), la sensibilidad fue 100 %. Ademas, la tecnica COBAS AmpliScreen™ HIV-1 Test, version 1.5 (Roche), es adecuada para la deteccion de las variantes de HIV-1 prevalentes.

Sensibilidad del equipo Cobas AmpliScreen™ HIV-1 Test, v1.5, para la detección de HIV-1

The introduction of nucleic acid amplification techniques (NAT) in blood banks was intended to reduce the residual risk of transfusion-transmitted infections. Co-circulation of a great diversity of HIV-1 variants in Argentina portrays the need to assess the sensitivity of serological and molecular assays available for their detection. In this study, we evaluated the sensitivity of the COBAS AmpliScreen™ HIV-1 Test, version 1.5 (Roche) for the detection of HIV-1 RNA in plasma samples of infected individuals from Argentina. The results of this study reveal that this technique has high sensitivity for the detection of HIV-1 RNA under assay conditions: using mini-pool testing, pools ≥ 50 RNA copies per ml achieved ≥ 92 % sensitivity, whereas in the standard procedure, samples ≥ 207 RNA copies/ml achieved 100 % sensitivity. Moreover, the COBAS AmpliScreen™ HIV-1 Test, version 1.5 (Roche) is suitable for detecting prevailing HIV-1 variants.Resumen Las tecnicas de amplificacion de acidos nucleicos (NAT) se incorporaron en los bancos de sangre para reducir el riesgo residual de transmision de infecciones por via transfusional. La cocirculacion de distintas variantes del HIV-1 en Argentina indica la necesidad de evaluar la sensibilidad de los ensayos serologicos y moleculares disponibles para su deteccion. En este trabajo se evaluo la sensibilidad del equipo COBAS AmpliScreen™ HIV-1 Test, version 1.5 (Roche), para detectar ARN viral en plasmas de individuos infectados con HIV-1 de Argentina. Los resultados demuestran que esta tecnica tiene una alta sensibilidad para detectar ARN de HIV-1 en las condiciones ensayadas: para ensayo de mini-pooles (pooles ≥ 50 copias de ARN/ml), la sensibilidad fue ≥ 92 %, y para procedimiento estandar (plasmas ≥ 207 copias de ARN/ml), la sensibilidad fue 100 %. Ademas, la tecnica COBAS AmpliScreen™ HIV-1 Test, version 1.5 (Roche), es adecuada para la deteccion de las variantes de HIV-1 prevalentes.