Rapid Identification of Drug Resistance and Phylogeny in M. tuberculosis, Directly from Sputum Samples.

Tuberculosis (TB) remains one of the most important infectious diseases globally. Establishing a resistance profile from the initial TB diagnosis is a priority. Rapid molecular tests evaluate only the most common genetic variants responsible for resistance to certain drugs, and Whole Genome Sequencing (WGS) needs culture prior to next-generation sequencing (NGS), limiting their clinical value. Targeted sequencing (TS) from clinical samples avoids these drawbacks, providing a signature of genetic markers that can be associated with drug resistance and phylogeny. In this study, a proof-of-concept protocol was developed for detecting genomic variants associated with drug resistance and for the phylogenetic classification of Mycobacterium Tuberculosis (Mtb) in sputum samples. Initially, a set of Mtb reference strains from the WHO were sequenced (WGS and TS). The results from the protocol agreed >95% with WHO reported data and phenotypic drug susceptibility testing (pDST). Lineage genetics results were 100% concordant with those derived from WGS. After that, the TS protocol was applied to sputum samples from TB patients to detect resistance to first- and second-line drugs and derive phylogeny. The accuracy was >90% for all evaluated drugs, except Eto/Pto (77.8%), and 100% were phylogenetically classified. The results indicate that the described protocol, which affords the complete drug resistance profile and phylogeny of Mtb from sputum, could be useful in the clinical area, advancing toward more personalized and more effective treatments in the near future. IMPORTANCE The COVID-19 pandemic negatively affected the progress in accessing essential Tuberculosis (TB) services and reducing the burden of TB disease, resulting in a decreased detection of new cases and increased deaths. Generating molecular diagnostic tests with faster results without losing reliability is considered a priority. Specifically, developing an antimicrobial resistance profile from the initial stages of TB diagnosis is essential to ensure appropriate treatment. Currently available rapid molecular tests evaluate only the most common genetic variants responsible for resistance to certain drugs, limiting their clinical value. In this work, targeted sequencing on sputum samples from TB patients was used to identify Mycobacterium tuberculosis mutations in genes associated with drug resistance and to derive a phylogeny of the infecting strain. This protocol constitutes a proof-of-concept toward the goal of helping clinicians select a timely and appropriate treatment by providing them with actionable information beyond current molecular approaches.

Whole genomic sequencing as a tool for diagnosis of drug and multidrug-resistance tuberculosis in an endemic region in Mexico.

BACKGROUND: Whole genome sequencing (WGS) has been proposed as a tool for diagnosing drug resistance in tuberculosis. However, reports of its effectiveness in endemic countries with important numbers of drug resistance are scarce. The goal of this study was to evaluate the effectiveness of this procedure in isolates from a tuberculosis endemic region in Mexico. METHODS: WGS analysis was performed in 81 tuberculosis positive clinical isolates with a known phenotypic profile of resistance against first-line drugs (isoniazid, rifampin, ethambutol, pyrazinamide and streptomycin). Mutations related to drug resistance were identified for each isolate; drug resistant genotypes were predicted and compared with the phenotypic profile. Genotypes and transmission clusters based on genetic distances were also characterized. FINDINGS: Prediction by WGS analysis of resistance against isoniazid, rifampicin, ethambutol, pyrazinamide and streptomycin showed sensitivity values of 84%, 96%, 71%, 75% and 29%, while specificity values were 100%, 94%, 90%, 90% and 98%, respectively. Prediction of multidrug resistance showed a sensitivity of 89% and specificity of 97%. Moreover, WGS analysis revealed polymorphisms related to second-line drug resistance, enabling classification of eight and two clinical isolates as pre- and extreme drug-resistant cases, respectively. Lastly, four lineages were identified in the population (L1, L2, L3 and L4). The most frequent of these was L4, which included 90% (77) of the isolates. Six transmission clusters were identified; the most frequent was TC6, which included 13 isolates with a L4.1.1 and a predominantly multidrug-resistant condition. CONCLUSIONS: The results illustrate the utility of WGS for establishing the potential for prediction of resistance against first and second line drugs in isolates of tuberculosis from the region. They also demonstrate the feasibility of this procedure for use as a tool to support the epidemiological surveillance of drug- and multidrug-resistant tuberculosis.

Hsp65 phylogenetic assay for molecular diagnosis of nontuberculous mycobacteria isolated in Mexico.

BACKGROUND AND AIMS: Nontuberculous mycobacteria (NTM) are mainly distributed as important emerging pathogens in patients with chronic or immunosuppressive diseases. Accurate identification of causative species is crucial for proper treatment and patient follow-up. However, several difficulties are associated with phenotypic and molecular diagnostic methods for precise identification at the species level due to shared metabolic and genetic characteristics. We undertook this study to evaluate the application of the phylogenetic method based on hsp65 gene into Telenti's PCR-restriction enzyme analysis (PRA) for molecular identification of NTM. METHODS: The study population was comprised of 1646 Mycobacterium clinical isolates (AFB positive) collected from 2008-2011, of which 537 (32.6%) were MNT identified by PRA analysis. DNA sequencing of hsp65 in 53 isolates (10%) was performed. Sequence identification through NCBI-Basic Local Alignment Search Tool (BLAST) achieved correct identification in 23 isolates. Phylogenetic trees including hsp65 available GenBank sequences for all described genres of NTM and hsp65 obtained sequences were constructed using Mega 5.05 software. We compared sequence identification based on phylogenetic clustering and BLAST similarity search. RESULTS: Phylogenetic clustering allowed more specific differentiation of closely related species and clearer identification in comparison with BLAST; 30 Mycobacterium species (this is the first report of isolation of some of these from clinical samples in Mexico) were identified in this way. CONCLUSIONS: The proposed 440 bp hsp65 phylogenetic method allows a better identification tool to differentiate Mycobacterium species and is useful to complement diagnosis and epidemiological surveillance of NTM.

Multidrug-resistant tuberculosis among patients in Baja California, Mexico, and Hispanic patients in California.

OBJECTIVES: We sought to compare prevalence and determinants of multidrug-resistant tuberculosis (MDR-TB) between tuberculosis patients in Baja California, Mexico, and Hispanic patients in California. METHODS: Using data from Mexico's National TB Drug Resistance Survey (2008-2009) and California Department of Public Health TB case registry (2004-2009), we assessed differences in MDR-TB prevalence comparing (1) Mexicans in Baja California, (2) Mexico-born Hispanics in California, (3) US-born Hispanics in California, and (4) California Hispanics born elsewhere. RESULTS: MDR-TB prevalence was 2.1% in Baja California patients, 1.6% in Mexico-born California patients, 0.4% in US-born California patients, and 2.7% in Hispanic California patients born elsewhere. In multivariate analysis, previous antituberculosis treatment was associated with MDR-TB (odds ratio [OR] = 6.57; 95% confidence interval [CI] = 3.34, 12.96); Mexico-born TB patients in California (OR = 5.08; 95% CI = 1.19, 21.75) and those born elsewhere (OR = 7.69; 95% CI = 1.71, 34.67) had greater odds of MDR-TB compared with US-born patients (reference category). CONCLUSIONS: Hispanic patients born outside the US or Mexico were more likely to have MDR-TB than were those born within these countries. Possible explanations include different levels of exposure to resistant strains and inadequate treatment.

[The effects of a rural environment on the prevalence of allergic rhinitis among schoolchildren in Mexicali, Baja California, Mexico]. / Efecto del ambiente rural sobre la prevalencia de rinitis alérgica en escolares de Mexicali, Baja California, México.

OBJECTIVES: To assess the prevalence of allergic rhinitis symptoms among schoolchildren in Mexicali, Baja California, Mexico, and determine what impact a rural environment might have on this condition. METHODS: A population-based, cross-sectional study using a standardized questionnaire administered to a random sample of 2087 schoolchildren 6-7 years of age (1078 girls and 1009 boys) living in the city of Mexicali, Baja California, Mexico. The study analyzed associations between exposure variables and personal and family health history, and two dependent variables-nasal symptoms and nasal and ocular symptoms, in the absence of a cold or flu-using odd ratios (OR) and 95% Confidence Intervals (95%CI). The study took place in February-July 2004 and followed the methodology of the International Study of Asthma and Allergies in Childhood (ISAAC). RESULTS: The general prevalence of nasal symptoms and nasal and ocular symptoms was 25.0% and 10.5%, respectively. The boys and girls who had ever lived on a farm or in a rural area had a lower probability, adjusted for confounding variables, of presenting nasal symptoms (OR=0.43; 95%CI: 0.24-0.77), or nasal and ocular symptoms (OR=0.39; 95%CI: 0.16-0.93). CONCLUSIONS: In the study population, exposure to a rural environment in early childhood decreased the risk of developing allergic rhinitis, regardless of a family history of asthma.

Efecto del ambiente rural sobre la prevalencia de rinitis alérgica en escolares de Mexicali, Baja California, México / The effects of a rural environment on the prevalence of allergic rhinitis among schoolchildren in Mexicali, Baja California, Mexico

OBJETIVOS: Determinar la prevalencia de síntomas de rinitis alérgica en escolares de Mexicali, Baja California, México, y evaluar la influencia del medio ambiente rural sobre esta afección. MÉTODOS: Estudio transversal poblacional mediante la aplicación de un cuestionario estandarizado en una muestra aleatoria de 2 087 escolares de 6 a 7 años (1 078 niñas y 1 009 niños) residentes en la ciudad de Mexicali, Baja California, México. Se evaluó la asociación entre variables de exposición y antecedentes clínicos personales y familiares, y dos variables dependientes: síntomas nasales y síntomas nasales y oculares, en ausencia de catarro o gripe, con sus razones de posibilidades (OR, odds ratios) e intervalos de confianza de 95 por ciento (IC95 por ciento). El estudio, realizado entre febrero y julio de 2004, utilizó la metodología del Estudio Internacional de Asma y Alergia en la Infancia (ISAAC). RESULTADOS: Las prevalencias generales de síntomas nasales y síntomas nasales y oculares fueron de 25,0 por ciento y 10,5 por ciento, respectivamente. Los niños y las niñas que habían vivido en el campo o zona rural en algún momento presentaron menor probabilidad de padecer síntomas nasales (OR = 0,43; IC95 por ciento: 0,24 a 0,77) y síntomas nasales y oculares (OR = 0,39; IC95 por ciento: 0,16 a 0,93), después de ajustar por factores de confusión. CONCLUSIONES: El contacto con el medio rural en los primeros años de vida disminuyó el riesgo de presentar rinitis alérgica en la población estudiada, independientemente de los antecedentes familiares de asma.

OBJECTIVES: To assess the prevalence of allergic rhinitis symptoms among schoolchildren in Mexicali, Baja California, Mexico, and determine what impact a rural environment might have on this condition. METHODS: A population-based, cross-sectional study using a standardized questionnaire administered to a random sample of 2 087 schoolchildren 6-7 years of age (1 078 girls and 1 009 boys) living in the city of Mexicali, Baja California, Mexico. The study analyzed associations between exposure variables and personal and family health history, and two dependent variables-nasal symptoms and nasal and ocular symptoms, in the absence of a cold or flu-using odd ratios (OR) and 95 percent Confidence Intervals (95 percentCI). The study took place in February-July 2004 and followed the methodology of the International Study of Asthma and Allergies in Childhood (ISAAC). RESULTS: The general prevalence of nasal symptoms and nasal and ocular symptoms was 25.0 percent and 10.5 percent, respectively. The boys and girls who had ever lived on a farm or in a rural area had a lower probability, adjusted for confounding variables, of presenting nasal symptoms (OR = 0.43; 95 percentCI: 0.24-0.77), or nasal and ocular symptoms (OR = 0.39; 95 percentCI: 0.16-0.93). CONCLUSIONS: In the study population, exposure to a rural environment in early childhood decreased the risk of developing allergic rhinitis, regardless of a family history of asthma.