Roberto Zenteno-Cuevas

Mutations in rpoB and katG genes in Mycobacterium isolates from the Southeast of Mexico

The most frequent mutations associated with rifampin and isoniazid resistance in Mycobacterium are the substitutions at codons 531 and 315 in the rpoB and katG genes, respectively. Hence, the aim of this study was to characterize these mutations in Mycobacterium isolates from patients suspected to be infected with drug-resistant (DR) pulmonary tuberculosis (TB) in Veracruz, Mexico. Drug susceptibility testing of 25 clinical isolates revealed that five were susceptible while 20 (80%) were DR (15% of the annual prevalence for Veracruz). Of the DR isolates, 15 (75%) were resistant to rifampin, 17 (85%) to isoniazid and 15 (75%) were resistant to both drugs (MDR). Sequencing analysis performed in the isolates showed that 14 (93%) had mutations in the rpoB gene; seven of these (47%) exhibited a mutation at 531 (S-->L). Ten (58%) of the 20 resistant isolates showed mutations in katG; nine (52%) of these 10 exhibited a mutation at 315 (S-->T). In conclusion, the DR profile of the isolates suggests a significant number of different DR-TB strains with a low frequency of mutation at codons 531 and 315 in rpoB and katG, respectively. This result leads us to consider different regions of the same genes, as well as other genes for further analysis, which is important if a genetic-based diagnosis of DR-TB is to be developed for this region.

rrs and rpsL mutations in streptomycin-resistant isolates of Mycobacterium tuberculosis from Mexico.

BACKGROUND/PURPOSE Mutations in rpsL and rrs genes are associated with resistance to streptomycin in tuberculosis, but important geographical variation exists in these mutations. The goal of this study was to characterize these mutations in isolates of streptomycin-resistant mycobacteria originating from southeast Mexico. METHODS Mycobacteria were isolated from patients with suspected drug-resistant tuberculosis. Susceptibility tests were carried out using the fluorometric method, and rrs and rpsL DNA sequencing was performed by capillary electrophoresis. RESULTS Some 136 drug-resistant isolates were recovered, of which 91(67%) exhibited resistance to streptomycin. Mutations in rpsL were observed in 18 isolates (19%) in codons 43 (A→G, K/R, n = 12) and 88 (A→G, K/R, n = 4; A→C, K/Q, n = 2). Mutations in rrs were observed in 26 isolates (28%). These were at nucleotides 513 (A→C, n = 8) and 516 (C→T, n = 6), and six novel mutations at nucleotides 483 (A→T, n = 2), 485 (A→G, n = 2), 496 (G→A, n = 2), 795 (C→T, n = 6), 870 (C→T, n = 3), and 907 (A→C, n = 3), with some isolates showing more than one mutation. Finally, 47 (52%) of the isolates showed no mutation. CONCLUSION The variety and presence or absence of the mutations found suggest the circulation of an important diversity of strains and the existence of additional mechanisms contributing to streptomycin resistance in the region.

Type 2 diabetes mellitus and its influence in the development of multidrug resistance tuberculosis in patients from southeastern Mexico

AIMS To determine the factors associated with the presence of pulmonary tuberculosis in patients with type 2 diabetes mellitus and the effect in the development of drug and multi-drug resistance, in a population with tuberculosis from the southeast of Mexico. METHODS This is a case-control study including 409 individuals, 146 with the binomial tuberculosis-type 2 diabetes mellitus and 263 individuals with tuberculosis. Demographic, epidemiological and outcome variables were collected. Risks were calculated. RESULTS The factors associated with the presence of type 2 diabetes mellitus were age ≥35years, (OR=9.7; CI: 5.2-17.8), previous contact with a person infected with tuberculosis (OR=1.7; CI: 1.1-3.1). Body mass index ≥25 kg/m(2) (OR=2.2; CI: 1.1-4.3), and inherited family history of diabetes (OR=5.4; CI: 3.2-9.2). It was also found that patients with tuberculosis-type 2 diabetes mellitus presented a 4.7-fold (CI: 1.4-11.3) and 3.5-fold (CI: 1.1-11.1) higher risk of developing drug- and multidrug resistance tuberculosis, respectively. By last, individuals with tuberculosis-type 2 diabetes had a 2.3-fold (CI: 1.5-4.1) greater chance of persisting as tuberculosis-positive by the second month of treatment, delaying the resolution of the tuberculosis infection. CONCLUSIONS Type 2 diabetes exerts a strong influence on the presentation and evolution of tuberculosis within the analyzed population and displays remarkable particularities, necessitating the development of dedicated tuberculosis-diabetes surveillance systems that consider the particular epidemiological characteristics of the population affected.

Characterization of pncA gene mutations in pyrazinamide-resistant Mycobacterium tuberculosis isolates from Mexico.

Numerous studies have linked mutations in the pncA gene with resistance to pyrazinamide (Z) in Mycobacterium tuberculosis. However, variations in these mutations are specific to the country of origin of the isolate. The aim of this study was to characterize changes in pncA gene sequence in isolates of M. tuberculosis with resistance to Z, from patients in Mexico. M. tuberculosis isolates were recovered from individuals suspected of carrying drug resistant tuberculosis and respective susceptibility tests were developed. In isolates with resistance to pyrazinamide the pncA gene and its promoter were analyzed by capillary sequencing. From 127 drug-resistant isolates collected, 42 (33%) were resistant to pyrazinamide. The pncA sequences showed 26 changes in 34 (81%) isolates: 18 SNPs (n=26, 62%), four insertions (n=4, 9.5%) and four deletions (n=4, 9.5%). Absence of modifications was observed in eight (19%) sequences/isolates. The most frequent changes were the mutations L120P (n=7) and K96R (n=4). Twelve changes found are reported for the first time. This is the first description of pncA gene modifications in pyrazinamide resistant isolates originating in Mexico. We conclude that the diversity of changes in pncA indicates the presence of a noteworthy variety of pyrazinamide resistant strains occurring in the area.

Assessing the utility of three TaqMan probes for the diagnosis of tuberculosis and resistance to rifampin and isoniazid in Veracruz, México

Mutations at codons 526 and 531 in the rpoB gene and at 315 in the katG gene are considered diagnostic markers for resistance to rifampin and isoniazid in tuberculosis. The aim of this study was to design and evaluate three TaqMan probes for the identification of these mutations in 138 respiratory samples positive for acid-fast bacilli, and 32 clinical isolates from a region with considerable levels of drug resistance. The specificities of the probes for the diagnosis of resistance to both drugs were 100%; however, the sensitivities were calculated to be 50% for isoniazid and 56% for rifampin. DNA sequencing of rpoB and katG; and the spoligotyping assay of the clinical isolates, confirmed the diversity of the mutations and the presence of 11 spoligotypes with a shared international type and eight unique spoligotypes. Analysis of the respiratory samples identified 22 (16%) as drug-resistant and 4 (3%) as multidrug-resistant tuberculosis. The diagnostic value of the TaqMan probes was compromised by the diversity of mutations found in the clinical isolates. This highlights the need for better understanding of the molecular mechanisms responsible for drug resistance prior to the use of molecular probes, especially in regions with significant levels of drug-resistant tuberculosis.

Mutation at embB Codon 306, a Potential Marker for the Identification of Multidrug Resistance Associated with Ethambutol in Mycobacterium tuberculosis

ABSTRACT Ethambutol inhibits arabinogalactan and lipoarabinomannan biosynthesis in mycobacteria. The occurrence of mutations in embB codon 306 in ethambutol-susceptible isolates and their absence in resistant isolates has raised questions regarding the utility of this codon as a potential marker for resistance against ethambutol. The characterization of mutations on embB 306 will contribute to a better understanding of the mechanisms of resistance to this drug; therefore, the purpose of this study was to investigate the association between embB 306 mutations and first-line drug resistance profiles in tuberculosis isolates. We sequenced the region surrounding the embB 306 codon in 175 tuberculosis clinical isolates, divided according to drug sensitivity, in three groups: 110 were resistant to at least one first-line drug, of which 61 were resistant to ethambutol (EMBr), 49 were sensitive to ethambutol (EMBs) but were resistant to another drug, and 65 were pansensitive isolates (Ps). The associations between embB 306 mutations and phenotypic resistance to all first-line drugs were determined, and their validity and safety as a diagnostic marker were assessed. One of the Ps isolates (1/65), one of the EMBs isolates (1/49), and 20 of the EMBr isolates (20/61) presented with an embB 306 mutation. Four different single-nucleotide polymorphisms (SNPs) at embB 306 were associated with simultaneous resistance to ethambutol, isoniazid, and rifampin (odds ratio [OR], 17.7; confidence interval [CI], 5.6 to 56.1) and showed a positive predictive value of 82%, with a specificity of 97% for diagnosing multidrug resistance associated with ethambutol, indicating its potential as a molecular marker for several drugs.

Co-infection and risk factors of tuberculosis in a Mexican HIV+ population

INTRODUCTION The situation of tuberculosis (TB) is being modified by the human immunodeficiency virus (HIV), which is increasing the occurrence of new cases and the generation of drug resistant strains, affecting not only the people infected with HIV, but also their close contacts and the general population, conforming a serious public health concern.However, the magnitudes of the factors associated to this co-infection differ considerably in relation to the population groups and geographical areas. METHODS In order to evaluate the prevalence and risk factors for the co-infection of tuberculosis (TB) in a population with human immunodeficiency virus (HIV+) in the Southeast of Mexico, we made the analysis of clinical and epidemiological variables and the diagnosis of tuberculosis by isolation of mycobacteria from respiratory samples. RESULTS From the 147 HIV+ individuals analyzed, 12 were culture positive; this shows a prevalence of 8% for the co-infection. The only variable found with statistical significance for the co-infection was the number of CD4-T < 200 cells/mm3, OR 13(95%, CI 2-106 vs 12-109). CONCLUSIONS To our knowledge this is the first report describing the factors associated with tuberculosis co-infection with HIV in a population from Southern Mexico. The low number of CD4 T-cells was the only variable associated with the TB co-infection and the rest of the variables provide scenarios that require specific and particular interventions for this population group.

Epidemiologic investigation of tuberculosis in a Mexican population from Chihuahua State, Mexico: a pilot study

Tuberculosis (TB) and its co-morbid conditions have become a burden on global health economies. It is well understood that susceptibility of the host to TB infection/disease is influenced by both genetic and environmental factors and their interactions. The aims of this pilot case-control study are to characterize the sociodemographic and environmental factors related to active TB disease (TB/case) and latent TB infection (LTBI/control) status, and to identify risk factors associated with progression from LTBI to TB. We recruited 75 cases with TB (mean age=46.3y; females=41%) and 75 controls with LTBI (mean age=39.0y; females=37%), from the Mestizo population of Cuidad Juárez, Mexico. In addition to the determination of case/control status, information on environmental variables was collected (e.g., socioeconomic status, smoking, alcohol consumption, substance abuse, nutritional status, household demographics, medical histories and presence of type 2 diabetes [T2DM]). The data were analyzed to identify the environmental correlates of TB and LTBI using univariate and multivariate statistical approaches. Following multivariate logistic regression analysis, TB was associated with poor nutrition, T2DM, family history of TB, and non-Chihuahua state of birth. These preliminary findings have relevance to TB control at the Mexico-United States border, and contribute to our future genetic study of TB in Mexicans.

Viral coinfection in acute respiratory infection in Mexican children treated by the emergency service: A cross-sectional study

BackgroundAcute respiratory infections (ARIs) cause illness. Children under five years of age are highly vulnerable to these infections. Viral coinfection or multiple viral infection is a variable that can have a significant impact on the evolution of these diseases.MethodsThis cross-sectional study was carried out in Mexican children (under five years of age) who had an ARI and who were treated by an emergency service in a hospital in Guadalajara, Jalisco, Mexico. The viral etiology, as well as the presence of multiple viral infections, was determined. A structured questionnaire was used to obtain demographic and clinical information. Odds ratio (OR) was calculated, and univariate and multivariate analyses using logistic regression were performed.ResultsIn the study population, metapneumovirus (hMPV) was the most frequent virus (22%), followed by adenovirus (hAD) (16%), respiratory syncytial virus (RSV) (14%), rhinovirus (hRV) (12%), bocavirus (hBoV) (9%), influenza virus (IF) (7%), and parainfluenza (PIF) (4%). The frequency of viral coinfections was 31.62%, and multiple logistic regression analysis revealed that hMPV, RSV, PIF, and hBoV were independently associated with multiple viral infection. No difference was found in the clinical manifestation of children with simple and multiple infections. Simple hMPV infection was associated with patients who presented with severe ARI. Using a multivariate analysis, we found that overcrowding is associated with coinfection when the viral etiology was hRV (OR = 2.56, 95% confidence interval (CI) 1.07 to 6.13), IF (OR = 2.56, 95% CI 1.07 to 6.13), PIF (OR = 2.96, 95% CI 1.15 to 7.65), hAD (OR = 2.56, 95% CI 1.07 to 6.13), and hBoV (OR = 2.9, 95% CI 1.14 to 7.34).ConclusionsViral coinfections are frequent in children requiring treatment by an emergency service. However, the severity of ARI is similar to that of children with a simple infection. The hMPV is common and may confer a significant disease burden in the Mexican population. Finally, overcrowding is a housing characteristic that favors the development of coinfections.

A first insight into the genetic diversity of Mycobacterium Tuberculosis in Veracruz, Mexico

Objective/Background: Tuberculosis (TB) remains one of the most important infectious diseases. Although Mexico is one of the Latin American countries with the largest contribution to these statistics, there are few reports that describe the genotypic characteristics of TB. The aim of this study was to use the MIRU-VNTR-24 loci to analyze the genetic diversity of M. tuberculosis circulating in the state of Veracruz, Mexico. Methods: Here, we analyze by MIRU-VNTR-24 loci 80 clinical isolates from individuals with confirmed TB from Veracruz México, also clinical and epidemiological variables were recovered and analyzed. Results: Of the individuals included in the analyses 65% were from men with an average age of 42 (± 17) years, 17% and 6% were drug and multi-drug resistant. 88% of the isolates were included in 20 clusters, of which 52% were classified into twelve orphan clusters and the remaining 37% were distributed among eight lineages: LAM (10%), EAI (9%), Haarlem (8%), H37Rv (4%), S (4%) and TUR (2%). Conclusion: An important diversity of lineages and unknown genotypes was identified; however, more studies are necessary in order to understand the characteristics of the genotypes displayed in the region. There is no doubt regarding the need for a molecular epidemiological surveillance system that can help to evaluate the dynamics of genotypes circulating in the country and support strategies for the prevention and management of populations affected by TB.