Development of a method to control the RNA extraction and reverse transcription steps for the detection of dengue virus present in human blood samples

AIMS: Molecular biology techniques based on the detection of genomic sequences by reverse transcription combined with polymerase chain reaction (PCR) have enabled the detection of different RNA viruses in serum or plasma samples. Since the dengue epidemic outbreak declared in Argentina in 2009, numerous patients´ samples were analyzed for the acute phase of infection. One of the main methodological drawbacks is the lack of internal control to measure the effectiveness of the viral extraction and reverse transcription process. In this article, we propose to standardize a molecular method to detect beta actin (â-Act) and glucose 6 phosphate dehydrogenase (G6PDH) complementary DNAs (cDNAs) present in patient´s plasma/serum, as a control process. RESULTS: RNA extraction, reverse transcription, and PCRs for human G6PDH, â-Act, and the dengue virus genome were performed. cDNA fragments for â-Act and G6PDH were amplified for all samples, regardless of the presence or absence of viral RNA. CONCLUSIONS: Amplification of â-Act and G6PDH cDNAs can be used as a control for the extraction and reverse transcription processes during dengue virus detection. This could also be a useful method for controlling the above steps when infections caused by other RNA viruses are studied, even if another methodology is employed, such as real-time PCR.Fil: Galván, Cristian Ariel. Universidad Católica de Córdoba. Facultad de Ciencias Químicas; ArgentinaFil: Elbarcha, Osvaldo C.. No especifíca;Fil: Fernández, Eduardo J.. No especifíca;Fil: Beltramo, Dante Miguel. Universidad Católica de Córdoba. Facultad de Ciencias Químicas; Argentina. Provincia de Córdoba. Ministerio de Ciencia y Técnica. Centro de Excelencia en Productos y Procesos de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Soria, Néstor Walter. Universidad Católica de Córdoba. Facultad de Ciencias Químicas; Argentin

Phylodynamics of Hepatitis C Virus Subtype 2c in the Province of Córdoba, Argentina

The Hepatitis C Virus Genotype 2 subtype 2c (HCV-2c) is detected as a low prevalence subtype in many countries, except in Southern Europe and Western Africa. The current epidemiology of HCV in Argentina, a low-prevalence country, shows the expected low prevalence for this subtype. However, this subtype is the most prevalent in the central province of Córdoba. Cruz del Eje (CdE), a small rural city of this province, shows a prevalence for HCV infections of 5%, being 90% of the samples classified as HCV-2c. In other locations of Córdoba Province (OLC) with lower prevalence for HCV, HCV-2c was recorded in about 50% of the samples. The phylogenetic analysis of samples from Córdoba Province consistently conformed a monophyletic group with HCV-2c sequences from all the countries where HCV-2c has been sequenced. The phylogeographic analysis showed an overall association between geographical traits and phylogeny, being these associations significant (α = 0.05) for Italy, France, Argentina (places other than Córdoba), Martinique, CdE and OLC. The coalescence analysis for samples from CdE, OLC and France yielded a Time for the Most Common Recent Ancestor of about 140 years, whereas its demographic reconstruction showed a “lag” phase in the viral population until 1880 and then an exponential growth until 1940. These results were also obtained when each geographical area was analyzed separately, suggesting that HCV-2c came into Córdoba province during the migration process, mainly from Europe, which is compatible with the history of Argentina of the early 20th century. This also suggests that the spread of HCV-2c occurred in Europe and South America almost simultaneously, possibly as a result of the advances in medicine technology of the first half of the 20th century

Comparison of two serologic methods for the diagnosis of hydatidosis

The sera of 176 patients with epidemiologic antecedents or radiologic and clinical signs of hydatidosis were tested by counterimmunoelectrophoresis (CIE) and enzyme-linked immunoassay (ELISA). A semipurified antigen from cysts of human origin was used for both techniques. The results were compared with those obtained from complementary radiologic studies and were confirmed by examination of excised cysts. Biopsy confirmed the diagnosis of hydatidosis in 65 patients (37%) and revealed the presence of other diseases in the remaining 111 (63%). Of the original 176 patients, 36 (20.4%) were positive by CIE and 62 (35.2%) by ELISA. Both techniques showed an excellent correlation with postsurgical diagnosis; neither produced any false positives, and the ELISA gave false negative results for only three patients (4.6%) with cysts that were infected, infertile, or calcified to some degree. The paper describes standardization of an inexpensive and easy-to-use microELISA

Distribution of polymorphisms in cytochrome P450 2B6, histocompatibility complex P5, chemokine coreceptor 5, and interleukin 28B genes in inhabitants from the central area of Argentina

The selection of the most appropriate treatment for several diseases relies on a number of factors such as environment, age, gender, and nutrition. Additionally, the contribution of different genetic polymorphisms to treatment efficacy has been largely recognized. The lack of information on the pharmacogenetic profile of our population prompted us to analyze the frequency of polymorphisms known to be relevant to achieve treatment efficacy with different therapeutic agents in viral infectious diseases, such as Hepatitis C and AIDS. Results: The allelic frequencies for the wild-type variant of the genes analyzed were cytochrome P450 2B6 (CYP2B6; rs3745274; 516G) 0.618 (95% confidence interval [CI]: 0.523, 0.711), chemokine coreceptor 5 (CCR5; rs333) 0.961 (95% CI: 0.942, 0.98), histocompatibility complex P5 (HCP5; rs2395029; 335T) 0.971 (95% CI: 0.937, 1), and interleukin 28B (IL28B; rs12979860; 12007005C) 0.656 (95% CI: 0.564, 0.747), respectively. Conclusions: Our data indicate that the genetic profile of the population studied is similar to that reported for other Caucasian populations, with only slight differences for CYP2B6. Noteworthy, the considerable number of patients carrying CYP2B6 (516T) and IL28B (12007005T) alleles underlies the importance of considering pharmacogenetic testing before starting drug therapy protocols to prevent toxicity and/or lack of effectiveness in AIDS or hepatitis C virus infections.Fil: Galván, Cristian A.. Universidad Católica de Córdoba; Argentina. Laboratorio de Análisis Clínicos Especializados; ArgentinaFil: Elbarcha, Osvaldo C.. Laboratorio de Análisis Clínicos Especializados; ArgentinaFil: Fernández, Eduardo J.. Laboratorio de Análisis Clínicos Especializados; ArgentinaFil: Beltramo, Dante Miguel. Universidad Católica de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Centro de Excelencia En Productos y Procesos de Cordoba; ArgentinaFil: Soria, Néstor Walter. Laboratorio de Análisis Clínicos Especializados; Argentina. Universidad Católica de Córdoba; Argentin

First detection of hepatitis E virus in Central Argentina: Environmental and serological survey

Background: The hepatitis E virus (HEV) is an emergent causative agent of acute hepatitis worldwide, transmitted by fecal-oral route. In Argentina it is considered rare, so differential laboratory testing is not routinely performed. Besides, in Argentina's central area epidemiological and molecular characteristics of HEV are still unknown. Objectives: Provide evidence of local circulation of HEV by molecular detection on environmental samples and by serological survey in healthy adult population of Córdoba city, Argentina. Study design: Environmental surveillance was conducted in river and sewage samples collected between 2007 and 2009–2011. Viral detection was performed by RT-Nested PCR of ORF-1 and ORF-2 partial regions. Anti-HEV IgG was determined by EIA in 433 serum samples collected between 2009 and 2010. Results: HEV was detected in 6.3% of raw sewage samples and in 3.2% of riverine samples. Nucleotide sequencing analyses revealed that all isolates belonged to genotype 3, subtypes a, b and c. The prevalence of IgG anti-HEV was 4.4%. Seroprevalence increased with the age of the individuals (OR: 3.50; 95% CI 1.39–8.87; p = 0.0065) and, although the prevalence was higher in low income population, no statistical relation was found between anti-HEV and socioeconomic level. Conclusions: The environmental findings added to serological results, demonstrate that HEV circulates in central Argentina. Contamination of water with HEV could represent a route of transmission for local populations, which have a high number of susceptible individuals. This fact alerts local health care systems in order to include detection of HEV in the diagnostic algorithm of viral hepatitis.Fil: Martinez Wassaf, Maribel Graciela. Universidad Nacional de Córdoba. Facultad de Medicina. Instituto de Virología “Dr. J. M. Vanella”; Argentina. Universidad Catolica de Córdoba. Facultad de Ciencias Químicas; Argentina. Laboratorio LACE; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pisano, María Belén. Universidad Nacional de Córdoba. Facultad de Medicina. Instituto de Virología “Dr. J. M. Vanella”; Argentina. Universidad Catolica de Córdoba. Facultad de Ciencias Químicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Barril, Patricia Angelica. Universidad Nacional de Córdoba. Facultad de Medicina. Instituto de Virología “Dr. J. M. Vanella”; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Elbarcha, Osvaldo C.. Laboratorio LACE; Argentina. Universidad Catolica de Córdoba. Facultad de Ciencias Químicas; ArgentinaFil: Pinto, Marcelo A.. Instituto Oswaldo Cruz; BrasilFil: Oliveira, Jaqueline Mendes de. Instituto Oswaldo Cruz; BrasilFil: DiGiusto, Pablo. Universidad Nacional de Córdoba. Facultad de Medicina. Instituto de Virología “Dr. J. M. Vanella”; ArgentinaFil: Nates, Silvia Viviana. Universidad Nacional de Córdoba. Facultad de Medicina. Instituto de Virología “Dr. J. M. Vanella”; ArgentinaFil: Ré, Viviana Elizabeth. Universidad Catolica de Córdoba. Facultad de Ciencias Químicas; Argentina. Universidad Nacional de Córdoba. Facultad de Medicina. Instituto de Virología “Dr. J. M. Vanella”; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Evaluation of five screening tests licensed in Argentina for detection of hepatitis C virus antibodies

This study was conducted to compare among the most recent generation of five screening tests licensed in Argentina, in order to evaluate which of the tests has the best sensitivity for detection of antibodies against hepatitis C virus (HCV). The tests analyzed were: Detect-HCV™ (3.0) Biochem ImmunoSystems, Canada; Hepatitis C EIA Wiener Lab., Argentina; Equipar HCV Ab, Italy; Murex HCV 4.0, UK and Serodia-HCV particles agglutination test, Japan. The results obtained showed high discrepancy between the different kits used and show that some of the tests assessed have a low sensitivity for anti-HCV detection in both chronic infections and early seroconversion, and indicate that among the commercially available kits in Argentina, Murex HCV 4.0 (UK) and Serodia-HCV particles agglutination test (Japan) have the best sensitivity for HCV screening. Although the sensitivity of the assays is the first parameter to be considered for blood screening, more studies should be carried out to assess the specificity of such assays