Population structure and genetic diversity of Mycobacterium tuberculosis in Ecuador.

Tuberculosis (TB) is a significant public health problem in Ecuador with an incidence of 43 per 100,000 inhabitants and an estimated multidrug-resistant-TB prevalence in all TB cases of 9%. Genotyping of Mycobacterium tuberculosis (MTBC) is important to understand regional transmission dynamics. This study aims to describe the main MTBC lineages and sublineages circulating in the country. A representative sample of 373 MTBC strains from 22 provinces of Ecuador, with data comprising geographic origin and drug susceptibility, were genotyped using 24 loci-MIRU-VNTR. For strains with an ambiguous sublineage designation, the lineage was confirmed by Regions of Difference analysis or by Whole Genome Sequencing. We show that lineage 4 is predominant in Ecuador (98.3% of the strains). Only 4 strains belong to lineages 2-sublineage Beijing and two strains to lineage 3-sublineage Delhi. Lineage 4 strains included sublineages LAM (45.7%), Haarlem (31.8%), S (13.1%), X (4.6%), Ghana (0.6%) and NEW (0.3%). The LAM sublineage showed the strongest association with antibiotic resistance. The X and S sublineages were found predominantly in the Coastal and the Andean regions respectively and the reason for the high prevalence of these strains in Ecuador should be addressed in future studies. Our database constitutes a tool for MIRU-VNTR pattern comparison of M. tuberculosis isolates for national and international epidemiologic studies and phylogenetic purposes.

Fast, Simple, and Cheap: the Kudoh-Ogawa Swab Method as an Alternative to the Petroff-Lowenstein-Jensen Method for Culturing of Mycobacterium tuberculosis.

The objective of this study was to compare the diagnostic yield of the Kudoh-Ogawa (K-O) swab method for the culturing of Mycobacterium tuberculosis from clinical samples with the standard Petroff-Lowenstein-Jensen (P-LJ) procedure. A total of 2,287 sputum samples and 685 extrapulmonary clinical specimens were processed with both decontamination methods and compared for M. tuberculosis detection rate, recovery of M. tuberculosis colonies, and culture contamination. Overall, 23.9% and 23.5% of the samples, processed with, respectively, the K-O swab method and the P-LJ procedure, yielded M. tuberculosis after 8 weeks of incubation. The K-O swab method and the P-LJ procedure provided comparable diagnostic yields for extrapulmonary clinical specimens (P = 0.688), but the K-O method showed a slightly but statistically significantly higher diagnostic yield for pulmonary samples (P = 0.002). No significant difference for culture contamination or colony recovery was found for either method. The turnaround time for the isolation of M. tuberculosis was significantly shorter for the K-O swab method, with 77% of the M. tuberculosis cultures being positive within 3 weeks of incubation, and only 6.1% positivity for the P-LJ method. Concerning the workload, the K-O swab method needs a minimum sample manipulation and takes less than 4 min per sample, as the samples are not centrifuged in this procedure. The K-O swab method is an efficient and fast (in terms of sample processing and culture growth) alternative for culturing M. tuberculosis from either pulmonary or extrapulmonary clinical specimens. The method is particularly suitable for laboratories with a high workload and for laboratories lacking a special infrastructure.

Country-wide rapid screening for the Mycobacterium tuberculosis Beijing sublineage in Ecuador using a single-nucleotide polymorphism-polymerase chain reaction method.

Background: Strains of the Beijing sublineage of Mycobacterium tuberculosis have caused large outbreaks of tuberculosis, often involving multidrug resistance strains and this genetically highly conserved family of strains predominates in some geographic areas. For most of the countries of Latin America, no country-wide studies about the prevalence of the Beijing lineage are available. Methods: In this study, we determine the prevalence of the Beijing sublineage in Ecuador, using a large nation-wide sample of 991 isolates from the years 2014-2016 and with the strains, in case-related-proportional representation, emerging from most of the provinces of the country. The isolates were genotyped with asinglenucleotidespecific polymorphism (SNP) polymerase chain reaction for the Beijing sublineage. SNPpositive strains were confirmed as belonging to this lineage with 24 mycobacterial interspersed repetitive unitvariable number of tandem repeat and DNA sequencing. Results: We identified only four Beijing isolates in this collection of 991 strains and calculated a prevalence rate of 0.43%. Conclusions: Our study shows a limited dissemination of the Beijing strains in the Ecuadorian population. This in contrast with the neighbor countries of Peru and Colombia were locally a prevalence of up to 16% has been reported.

Prevalence, Drug Resistance, and Genotypic Diversity of the Mycobacterium tuberculosis Beijing Family in Ecuador.

The Beijing family, the most successful Mycobacterium tuberculosis lineage, is considered hypervirulent, associated with clustering and has a strong association with multidrug-resistant tuberculosis. The Beijing strains have spread worldwide and also to Latin America. Genotyping of a countrywide collection of 380 M. tuberculosis strains from Ecuador, with 24-loci mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTR), revealed only six Beijing strains, but four of these were MDR-TB. There was no clustering as all six strains had very distinct MIRU-VNTR profiles that have not been reported in the rest of Latin America. Although active transmission for Beijing has been described for the neighboring countries Peru and Colombia, there is no evidence that Beijing strains in Ecuador are more frequently transmitted than other strains. Moreover, the low prevalence (1.6%) of the Beijing sublineage in Ecuador challenges the concept of hyperadaptability and transmissibility of the Beijing strains in our country.

A First Insight into the katG and rpoB Gene Mutations of Multidrug-Resistant Mycobacterium tuberculosis Strains from Ecuador.

The aim of this study was to characterize the most frequent mutations associated with rifampicin (RIF) and isoniazid (INH) resistance of Mycobacterium tuberculosis isolates from Ecuador. Sequence analysis of 40 strains, resistant for the tuberculosis drugs INH, RIF, or for both showed that of the 31 strains with resistance to INH, 20 strains (64.5%) carried a mutation in the katG gene (codon 315). Eight INH-resistant strains carried a mutation in the katG gene at codon 463. This katG463 mutation, considered a phylogenetic marker, was exclusively found in INH-resistant strains and not in 121 INH-susceptible strains. Of the 35 strains resistant to RIF, 33 (93.9%) had mutations in the hot spot region of the rpoB gene, predominantly in codons 531, 516, and 526. Our results show that sequence-based detection for drug resistance of the katG will identify, respectively, 64.5% or, considering katG463 as a marker, 90.3% of the INH-resistant strains. Sequencing of the hot spot region of the rpoB gene will detect 94.3% of the RIF drug-resistant isolates in Ecuador. This is appropriate for fast screening for drug resistance with the GeneXpert MTB/RIF assay or by direct sequencing of a part of the genes katG and rpoB of PCR products obtained from DNA isolation from primary cultures.

Detección de genes asociados a resistencia para isoniacida y rifampicina en cepas de Mycobacterium tuberculosis en Ecuador / Detection of genes associated to resistance for isoniacida and rifampicin in cepas of Mycobacterium tuberculosis in Ecuador

Antecedentes: La incidencia de la multidrogorresistencia (MDR) adquirida a drogas antituberculosas de Mycobacterium tuberculosis en el Ecuador es aproximadamente del 27,83 %. Objetivo: Detectar las mutaciones asociadas con la resistencia a los fármacos en cepas de M. tuberculosis resistentes a fármacos utilizando la PCR-RFLP y PCR en tiempo real. Materiales y métodos: En cepas de M. tuberculosis drogorresistentes (DR) aisladas de muestras provenientes de 18 provincias del Ecuador (2006-2012), se analizaron mutaciones en los genes rpoB y KatG que conducen a la resistencia a rifampicina e isoniazida, respectivamente. EL ADN fue amplificado por Reacción en Cadena de la Polimerasa (PCR), siendo luego analizado por RFLP (40 cepas), qPCR (346 cepas). Resultados: Nuestros resultados demostraron una correlación entre la resistencia fenotípica a rifampicina e isoniazida y las mutaciones encontradas en los genes rpoß (91,6 %) y katG (90,3 %) en cepas ecuatorianas de M. tuberculosis. Conclusiones: Las mutaciones asociadas a la drogorresistencia de aislados del M. tuberculosis en el Ecuador fueron similares a las reportadas en otros países.

Background: The incidence of multidrug-resistance (MDR) acquired in Mycobacterium tuberculosis antituberculosis drugs in Ecuador is approximately 27,83 %. Objective: To detect mutations associated with drug resistance in drug-resistant M. tuberculosis using PCR-RFLP and real-time PCR. Materials and methods: Mutations in rpoB and KatG genes leading to resistance to rifampicin and isoniazid, respectively, were analyzed in isolates of M. tuberculosis (DR) isolated from samples from 18 provinces of Ecuador (2006-2012). The DNA was amplified by Polymerase Chain Reaction (PCR) and then analyzed by RFLP (40 strains), qPCR (346 strains) and Results: Our results demonstrated a correlation between the phenotypic resistance to rifampicin and isoniazid and the mutations found in the rpoß (91, 6 %) and katG (90,3%) genes in Ecuadorian M. tuberculosis strains. Conclusions: Mutations associated with the drug resistance of M. tuberculosis isolates in Ecuador were similar to those reported in other countries.