RrpoB, katG and inhA mutations in multi-drug resistant strains of Mycobacterium tuberculosis clinical isolates from southeast Mexico / Mutaciones en rpoB, katG e inhA en cepas clínicas de Mycobacterium tuberculosis multi-farmacorresistentes del sureste de México

INTRODUCTION: Previous knowledge of molecular mechanisms related with multi-drug resistances in tuberculosis is important if molecular diagnostic procedures want to be used in specific geographical regions. For that reason, the aim of this study was to investigate the mutations at rpoB, katG and inhA in multi-drug resistant tuberculosis isolates from Southeast Mexico. METHODS: Isolates of tuberculosis with a confirmed resistance against rifampicin and isoniazid were collected and sequencing analysis was performed of the rpoB rifampicin resistance-determining region, the katG and the encoding region of inhA. RESULT: Of 74 isolates with multidrug resistance, 34 (46%) presented six mutations in katG; the most abundant was katG315 in 29 (39%) isolates. At inhA, nine (11%) isolates presented three mutations; the most frequent was inhA21, located in five (6%) strains. Eleven polymorphisms were observed at rpoB in 61 (82%) isolates, prevailing rpoB531 and rpoB 526 in 48 (64%) and ten (12%) isolates, respectively. Eleven double combinations were observed in 39 (52%) isolates, the most common of which was rpoB531 + katG315, found in 22 (29%) strains. CONCLUSION: This study provides valuable information on the diversity of polymorphisms in genes related to multidrug-resistant tuberculosis, as well as the presence of new mutations not previously described; this information should be considered in the implementation of molecular diagnostic tests

INTRODUCCIÓN: El conocimiento previo de los mecanismos moleculares relacionados con las multi-farmacorresistencia es de suma importancia si se desean emplear procedimientos de diagnóstico molecular en regiones geográficas específicas. Por esa razón, el objetivo de este estudio fue investigar las mutaciones en rpoB, katG e inhA en cepas aisladas de tuberculosis multirresistente circulantes en el sureste de México. MÉTODOS: Se recuperaron aislados de tuberculosis con una resistencia confirmada a rifampicina e isoniazida, y se realizó la secuenciación y posterior análisis de la región determinante de la resistencia a rifampicina en el gen rpoB, así como del gen katG y la región codificante de inhA. RESULTADO: De 74 cepas con multirresistencia, solo 34 (46%) presentaron 6 mutaciones en katG; la más abundante fue katG315 presente en 29 (39%) aislados. En inhA, solo 9 (11%) aislados presentaron 3 mutaciones; la más frecuente fue inhA21, localizada en 5 (6%) cepas. Se observaron en 61 (82%) cepas 11 polimorfismos en rpoB, siendo los más frecuentes rpoB531 en 48 (64%) y rpoB526 en 10 (12%) aislados, respectivamente. Se observaron 11 combinaciones dobles en 39 (52%) cepas, la más común fue rpoB531+katG315, presente en 22 (29%) cepas. CONCLUSIÓN: Este estudio proporciona información valiosa sobre la diversidad de polimorfismos en genes relacionados con la tuberculosis multirresistentes, así como la presencia de nuevas mutaciones no descritas previamente, esta información deberá ser considerar en la implementación de pruebas de diagnóstico molecular

rpoB, katG and inhA mutations in multi-drug resistant strains of Mycobacterium tuberculosis clinical isolates from southeast Mexico.

INTRODUCTION: Previous knowledge of molecular mechanisms related with multi-drug resistances in tuberculosis is important if molecular diagnostic procedures want to be used in specific geographical regions. For that reason, the aim of this study was to investigate the mutations at rpoB, katG and inhA in multi-drug resistant tuberculosis isolates from Southeast Mexico. METHODS: Isolates of tuberculosis with a confirmed resistance against rifampicin and isoniazid were collected and sequencing analysis was performed of the rpoB rifampicin resistance-determining region, the katG and the encoding region of inhA. RESULT: Of 74 isolates with multidrug resistance, 34 (46%) presented six mutations in katG; the most abundant was katG315 in 29 (39%) isolates. At inhA, nine (11%) isolates presented three mutations; the most frequent was inhA21, located in five (6%) strains. Eleven polymorphisms were observed at rpoB in 61 (82%) isolates, prevailing rpoB531 and rpoB 526 in 48 (64%) and ten (12%) isolates, respectively. Eleven double combinations were observed in 39 (52%) isolates, the most common of which was rpoB531+katG315, found in 22 (29%) strains. CONCLUSION: This study provides valuable information on the diversity of polymorphisms in genes related to multidrug-resistant tuberculosis, as well as the presence of new mutations not previously described; this information should be considered in the implementation of molecular diagnostic tests.

Genetic diversity of drug and multidrug-resistant Mycobacterium tuberculosis circulating in Veracruz, Mexico.

BACKGROUND: Mexico is one of the most important contributors of drug and multidrug-resistant tuberculosis in Latin America; however, knowledge of the genetic diversity of drug-resistant tuberculosis isolates is limited. METHODS: In this study, the genetic structure of 112 Mycobacterium tuberculosis strains from the southeastern Mexico was determined by spoligotyping and 24-loci MIRU-VNTRs. FINDINGS: The results show eight major lineages, the most of which was T1 (24%), followed by LAM (16%) and H (15%). A total of 29 (25%) isolates were identified as orphan. The most abundant SITs were SIT53/T1 and SIT42/LAM9 with 10 isolates each and SIT50/H3 with eight isolates. Fifty-two spoligotype patterns, twenty-seven clusters and ten clonal complexes were observed, demonstrating an important genetic diversity of drug and multidrug-resistant tuberculosis isolates in circulation and transmission level of these aggravated forms of tuberculosis. Being defined as orphan or as part of an orphan cluster, was a risk factor for multidrug resistant-tuberculosis (OR 2.5, IC 1.05-5.86 and OR 3.3, IC 1-11.03, respectively). Multiple correspondence analyses showed association of some clusters and SITs with specific geographical locations. CONCLUSIONS: Our study provides one of the most detailed description of the genetic structure of drug and multidrug-resistant tuberculosis strains in southeast Mexico, establishing for the first time a baseline of the genotypes observed in resistant isolates circulating, however further studies are required to better elucidate the genetic structure of tuberculosis in region and the factors that could be participating in their dispersion.

PhyloFlu, a DNA microarray for determining the phylogenetic origin of influenza A virus gene segments and the genomic fingerprint of viral strains.

Recent evidence suggests that most influenza A virus gene segments can contribute to the pathogenicity of the virus. In this regard, the hemagglutinin (HA) subtype of the circulating strains has been closely surveyed, but the reassortment of internal gene segments is usually not monitored as a potential source of an increased pathogenicity. In this work, an oligonucleotide DNA microarray (PhyloFlu) designed to determine the phylogenetic origins of the eight segments of the influenza virus genome was constructed and validated. Clades were defined for each segment and also for the 16 HA and 9 neuraminidase (NA) subtypes. Viral genetic material was amplified by reverse transcription-PCR (RT-PCR) with primers specific to the conserved 5' and 3' ends of the influenza A virus genes, followed by PCR amplification with random primers and Cy3 labeling. The microarray unambiguously determined the clades for all eight influenza virus genes in 74% (28/38) of the samples. The microarray was validated with reference strains from different animal origins, as well as from human, swine, and avian viruses from field or clinical samples. In most cases, the phylogenetic clade of each segment defined its animal host of origin. The genomic fingerprint deduced by the combined information of the individual clades allowed for the determination of the time and place that strains with the same genomic pattern were previously reported. PhyloFlu is useful for characterizing and surveying the genetic diversity and variation of animal viruses circulating in different environmental niches and for obtaining a more detailed surveillance and follow up of reassortant events that can potentially modify virus pathogenicity.

Expression, purification, and evaluation of diagnostic potential and immunogenicity of a recombinant NS3 protein from all serotypes of dengue virus.

Dengue is one of the major public health concerns in the world. Since all the four serotypes are actively circulating in Mexico, there is a need to develop an efficient diagnosis system to improve case management of the patients. There exist few studies evaluating the use of the NS3 protein as a protective antigen against dengue virus (DENV). In this paper we show the expression of a recombinant NS3 protein from all serotypes of dengue virus (GST-DVNS3-1-4) and report a reliable "in-house detection system" for the diagnosis of dengue infection which was field-tested in a small village (Tezonapa) in the state of Veracruz, Mexico. The fusion proteins were immunogenic, inducing antibodies to be able to recognize to antigens up to a 1 : 3200 dilution. The purified proteins were used to develop an in-house detection system (ELISA) and were further tested with a panel of 239 serum samples. The in-house results were in excellent agreement with the commercial kits with κ = 0.934 ± 0.064 (95% CI = 0.808-1.061), and κ = 0.872 ± 0.048 (95% CI = 0.779-0.965) for IgM and IgG, respectively. The agreement between the NS1 antigen detection versus the rNS3 ELISA, κ = 0.837 ± 0.066 (95% CI = 0.708-0.966), was very good. Thus, these results demonstrate that recombinant NS3 proteins have potential in early diagnosis of dengue infections.

Characterization of an influenza A virus in Mexican swine that is related to the A/H1N1/2009 pandemic clade.

In the spring of 2009, swine-origin influenza H1N1pdm09 viruses caused the first influenza pandemic of this century. We characterized the influenza viruses that circulated early during the outbreak in Mexico, including one newly sequenced swine H1N1pdm09 virus and three newly sequenced human H1N1pdm09 viruses that circulated in the outbreak of respiratory disease in La Gloria, Veracruz. Phylogenetic analysis revealed that the swine isolate (A/swine/Mexico/4/2009) collected in April 2009 is positioned in a branch that is basal to the rest of the H1N1pdm09 clade in two (NP and PA) of the eight single-gene trees. In addition, the concatenated HA-NA and the complete whole-genome trees also showed a basal position for A/swine/Mexico/4/2009. Furthermore, this swine virus was found to share molecular traits with non-H1N1pdm09 H1N1 viral lineages. These results suggest that this isolate could potentially be the first one detected from a sister lineage closely related to the H1N1pdm09 viruses.

[Dengue fever during pregnancy. Cases report]. / Dengue durante el embarazo. Comunicación de casos.

Dengue is known as an endemic disease of tropical and subtropical regions. It was considered a disease very frequent on kids, but recently an increase was reported on adult people. Some of these cases were related to pregnant women, for that reason, we decided to check eight cases, including just the mothers who presented dengue virus infection through ELISA IgM. IgG and ELISA IgM studies. Five products were determined between 3 and 9-born-babies. Eight cases of dengue were analyzed during pregnancy, three cases of fever dengue and five cases of hemorrhagic dengue; main complications detected were threat of abortion, and premature labour, postsurgical bleeding with desiccant haematoma of uterine artery, oligohydramnios, as well as pleural effusion, two of the neonates were classified as septic for presenting fever. In no case, IgG or IgM for fever dengue was detected in neonates.