Resistencia cruzada entre isoniacida y etionamida y su alta correlación con la mutación C-15T en aislamientos de Mycobacterium tuberculosis de Perú / Cross-resistance between isoniazid and ethionamide and its strong association with mutation C-15T in Mycobacterium tuberculosis isolates from Peru

Resumen Diversos estudios han evidenciado una resistencia cruzada entre isoniacida y etionamida, 2 de los fármacos utilizados en el tratamiento de la tuberculosis multirresistente.El objetivo del presente estudio fue determinar la resistencia cruzada entre ambos fármacos en aislados de Mycobacterium tuberculosis obtenidos en un hospital de Lima (Perú), conalta proporción de pacientes con tuberculosis. Se calculó la frecuencia de mutaciones asociadas con la resistencia a la isoniacida (INH) evaluando el gen katG y la región promotorainhA mediante la prueba molecular Genotype MTBDRplus v2.0. El método gold standard conocido como agar proporciones en placa (APP) permitió la identificación de resistencia a INH yetionamida. De 107 aislamientos resistentes a INH, 54 fueron multirresistentes (identificadosmediante la prueba Genotype MTBDRplus) y 49 (es decir, el 45,8% del total) también fueronresistentes a etionamida por el método APP. En los aislamientos resistentes a INH, se encontraron mutaciones en el gen katG en el 50,5% (54/107); en la región promotora inhA en el23,3% (25/107), y un 14,0% (15/107) presentaron mutaciones en ambos. Un 12,1% (13/107)fueron resistentes a INH por ausencia de banda wild type y banda de mutación. La mutaciónC-15T en la región promotora inhA presentó una fuerte asociación con la resistencia a etionamida y alcanzó el 73,4% (36/49) de los aislamientos resistentes a dicho fármaco. Los resultadosdel presente estudio sugieren que la identificación de mutaciones relacionadas con resistenciaa INH, sobre todo en la región promotora inhA, podría ser de gran utilidad para identificarla resistencia cruzada a etionamida y mejorar el tratamiento de las personas afectadas portuberculosis.© 2019 Asociacion Argentina de Microbiolog´ía. Publicado por Elsevier Espana, S.L.U. Este es unart´ículo Open Access bajo la licencia CC BY-NC-ND (https://creativecommons.org/licenses/by-nc-nd/4.0/).

Abstract Several studies have shown cross-resistance between isoniazid and ethionamide, 2of the drugs used in the treatment of multidrug-resistant tuberculosis. The objective of this study was to determine the cross-resistance between both drugs in Mycobacterium tuberculosis isolates from a hospital with high incidence of tuberculosis in Lima, Peru. The frequency of mutations to isoniazid in the katG gene and the inhA promoter region was identified by the Genotype MTBDRplus v2.0 molecular test. The gold standard Agar Proportion method (APM) allowed todetect resistance to isoniazid and ethionamide. Of 107 isoniazid-resistant isolates (54 multidrug-resistant isolates identified by the Genotype MTBDRplus test, 45.8% (49/107) were also resistant to ethionamide by the APM. Mutations were found in the katG gene in 50.5% (54/107), in the promoter region inhA in 23.3% (25/107) and 14.0% (15/107) that share both mutations in the resistant isolates to INH. The absence of the wild type and mutation bands indicated that 12.1% (13/107) of the isolates were resistant to INH. The mutation C-15T in the inhA promoter region showed a strong association with resistance to ethionamide in 73.4% (36/49) of the isolates analyzed. The results of the present study suggest that the identification of mutations related to resistance to isoniazid, especially in the inhA promoter region, could be very useful to identify cross-resistance to ethionamide and improve the treatment of individuals suffering from this disease.

Genomic analysis of Latin American-Mediterranean family of Mycobacterium tuberculosis clinical strains from Kazakhstan

The human-adapted strains of the Mycobacterium tuberculosis complex (MTBC) comprise seven phylogenetic lineages originally associated with their geographical distribution. Here, we report the genomes of three drug-resistant clinical isolates of the Latin American-Mediterranean (LAM) family collected in Kazakhstan. We utilised whole-genome sequencing to study the distribution and drug resistance of these isolates. Phylogenetic analysis grouped the genomes described in this study with the sequences from Russia, Uzbekistan, and Kazakhstan belonging to the LAM family. One isolate has acquired extensive drug resistance to seven antituberculosis drugs. Our results suggest at least two multi-drug resistant (MDR)/extensively drug-resistant (XDR)-associated genotypes of the LAM family circulate in Kazakhstan.

Evaluation of drug susceptibility profile of Mycobacterium tuberculosis Lineage 1 from Brazil based on whole genome sequencing and phenotypic methods

BACKGROUND The evaluation of procedures for drug susceptibility prediction of Mycobacterium tuberculosis based on genomic data against the conventional reference method test based on culture is realistic considering the scenario of growing number of tools proposals based on whole-genome sequences (WGS). OBJECTIVES This study aimed to evaluate drug susceptibility testing (DST) outcome based on WGS tools and the phenotypic methods performed on isolates of M. tuberculosis Lineage 1 from the state of Pará, Brazil, generally associated with low levels of drug resistance. METHODOLOGY Culture based DST was performed using the Proportion Method in Löwenstein-Jensen medium on 71 isolates that had been submitted to WGS. We analysed the seven main genome sequence-based tools for resistance and lineage prediction applied to M. tuberculosis and for comparison evaluation we have used the Kappa concordance test. FINDINGS When comparing the WGS-based tools against the DST, we observed the highest level of agreement using TB-profiler. Among the tools, TB-profiler, KvarQ and Mykrobe were those which identified the largest number of TB-MDR cases. Comparing the four most sensitive tools regarding resistance prediction, agreement was observed for 43 genomes. MAIN CONCLUSIONS Drug resistance profiling using next-generation sequencing offers rapid assessment of resistance-associated mutations, therefore facilitating rapid access to effective treatment.

Tuberculosis associated factors caused by Mycobacterium tuberculosis of the RDRio genotype

BACKGROUND Tuberculosis (TB) continues to be a disease that affects many countries around the world, including Brazil. Recently, a subtype of Latin American-Mediterranean family strain was identified and characterised by RDRio. The strain has been associated with different characteristics of the disease. OBJECTIVES In the present study we investigated the association of epidemiological, clinical, radiological and bacteriological variables with pulmonary tuberculosis caused by RDRioMycobacterium tuberculosis strain in large regions of São Paulo. METHODS We conducted a cross-sectional study in 530 patients with pulmonary tuberculosis, diagnosed using sputum culture, from two regions of the São Paulo state in Brazil. The samples were brought to São Paulo reference laboratories for epidemiological, clinical, radiological and bacteriological analyses, and the data were obtained from a TB notification system. RDRio genotyping and Spoligotyping of the samples were performed. For the analysis of the categorical variables we used the chi-square test or the Fisher’s exact test, and for the continuous variables, the Mann-Whitney test. In addition, a logistic regression was used for multivariate analysis. Differences with p < 0.05 were considered significant. FINDINGS The RDRio deletion was identified in 152 (28.7%) samples. In the univariate analysis, both the age groups above 25 years and alcohol consumption were associated with the RDRio deletion. The multivariate analysis confirmed the association of the RDRio deletion with the age groups: 25-35 years old [OR: 2.28 (1.02-5.07; p = 0.04)] and 36-60 years old (OR: 2.36 (1.11-5.05); p = 0.03], and also with alcohol consumption [OR: 1.63 (1.05-2.54); p = 0,03]. MAIN CONCLUSIONS In this study, we identified new factors associated with the M. tuberculosis of the RDRio deletion strains infection.

A molecular platform for the diagnosis of multidrug-resistant and pre-extensively drug-resistant tuberculosis based on single nucleotide polymorphism mutations present in Colombian isolates of Mycobacterium tuberculosis

Developing a fast, inexpensive, and specific test that reflects the mutations present in Mycobacterium tuberculosis isolates according to geographic region is the main challenge for drug-resistant tuberculosis (TB) control. The objective of this study was to develop a molecular platform to make a rapid diagnosis of multidrug-resistant (MDR) and extensively drug-resistant TB based on single nucleotide polymorphism (SNP) mutations present in therpoB, katG, inhA,ahpC, and gyrA genes from Colombian M. tuberculosis isolates. The amplification and sequencing of each target gene was performed. Capture oligonucleotides, which were tested before being used with isolates to assess the performance, were designed for wild type and mutated codons, and the platform was standardised based on the reverse hybridisation principle. This method was tested on DNA samples extracted from clinical isolates from 160 Colombian patients who were previously phenotypically and genotypically characterised as having susceptible or MDR M. tuberculosis. For our method, the kappa index of the sequencing results was 0,966, 0,825, 0,766, 0,740, and 0,625 forrpoB, katG, inhA,ahpC, and gyrA, respectively. Sensitivity and specificity were ranked between 90-100% compared with those of phenotypic drug susceptibility testing. Our assay helps to pave the way for implementation locally and for specifically adapted methods that can simultaneously detect drug resistance mutations to first and second-line drugs within a few hours.

Phenotypic and genotypic characteristics of drug resistance in Mycobacterium tuberculosis isolates from pediatric population of Chennai, India.

Purpose: Multidrug-resistant TB (MDR-TB) has been reported in almost all parts of the world. Childhood TB is accorded low priority by national TB control programs. Probable reasons include diagnostic difficulties, limited resources, misplaced faith in BCG and lack of data on treatment. Good data on the burden of all forms of TB among children in India are not available. Objective: To study the drug sensitivity pattern of tuberculosis in children aged from 3 months to 18 years and the outcome of drug-resistant tuberculosis by BACTEC culture system and PCR-based DNA sequencing technique. Materials and Methods: This is a retrospective study. One hundred and fifty-nine clinical specimens were processed for Ziehl-Neelsen stain, Mycobacterial culture by BACTEC method, phenotypic DST for first-line drugs for Mycobacterium tuberculosis (M. tuberculosis) isolates and PCR-based DNA sequencing was performed for the M. tuberculosis isolates targeting rpoB, katG, inhA, oxyR-ahpC, rpsL, rrs and pncA. Results and Conclusion: Out of the 159 Mycobacterial cultures performed during the study period, 17 clinical specimens (10.7%) were culture positive for M. tuberculosis. Among the 17 M. tuberculosis isolates, 2 were multidrug-resistant TB. PCR-based DNA sequencing revealed the presence of many novel mutations targeting katG, inhA, oxyR-ahpC and pncA and the most commonly reported mutation Ser531Leu in the rpoB gene. This study underlines the urgent need to take efforts to develop methods for rapid detection and drug susceptibility of tubercle bacilli in the pediatric population.

Characterization of gyrA and gyrB mutations and fluoroquinolone resistance in Mycobacterium tuberculosis clinical isolates from Hubei Province, China

OBJECTIVE: The study aimed to investigate gyrA and gyrB mutations in Mycobacterium tuberculosis (MTB) clinical strains from 93 patients with pulmonary tuberculosis in Hubei Province, China, and analyze the association between mutation patterns of the genes and ofloxacin resistance level. RESULTS: Among 93 MTB clinical isolates, 61 were ofloxacin-resistant by the proportion method, and 32 were ofloxacin-susceptible MDR-TB. No mutation in the gyrB gene was found in any MTB strains. In the 61 ofloxacin-resistant isolates, 54 mutations were observed in the gyrA gene. Only one mutation in the gyrA gene was found in ofloxacin-susceptible MDR-TB isolates. In this study, the mutation patterns of gyrA involved seven patterns of single codon mutation (A90V, S91P, S91T, D94N, D94Y, D94G or D94A) and two patterns of double codons mutation (S91P & D94H, S91P & D94A). The ofloxacin minimal inhibitory concentrations (MICs) of three patterns of single codon mutations in the gyrA gene (codons 94, 90 and 91) showed a statistically significant difference (p < 0.0001). CONCLUSIONS: The gyrA mutations at codons 90, 91 and 94 constitute the primary mechanism of fluoroquinolone resistance in MTB, and mutations at codon 91 in the gyrA gene may be associated with low-level resistance to ofloxacin.

Multidrug-resistant tuberculosis in Port-au-Prince, Haiti / Tuberculosis multirresistente en Puerto Príncipe, Haití

OBJECTIVE: To determine the prevalence of multidrug-resistant tuberculosis (MDR-TB) among patients with new smear-positive pulmonary TB in Port-au-Prince, Haiti. METHODS: Sputum samples were cultured from 1 006 patients newly diagnosed with TB in 2008. The core region of the rpoB gene that is associated with resistance to rifampin was sequenced. All isolates with rpoB mutations were sent to the New York State reference laboratory for conventional drug susceptibility testing (DST). All isolates were also tested with the GenoType MTBDRplus line-probe assay. RESULTS: Mycobacterium tuberculosis was isolated from 906 patients. Twenty-six (2.9 percent) of the isolates had missense mutations or deletions in rpoB and were resistant to rifampin by DST. All 26 were also resistant to isoniazid and classified as MDR-TB. Forty-six control isolates without rpoB mutations were found to be rifampin sensitive by DST. The GenoType MTBDRplus line-probe assay correctly identified 26 MDR-TB strains. It misclassified one pansusceptible isolate as rifampin resistant. CONCLUSIONS: This study shows an MDR-TB prevalence of 2.9 percent in newly diagnosed TB patients in Haiti and suggests that rpoB sequencing and hybridization assays are good screening tools for early detection of MDR-TB.

OBJETIVO: Determinar la prevalencia de tuberculosis (TB) multirresistente en pacientes con TB pulmonar nueva con baciloscopia positiva en Puerto Príncipe, Haití. MÉTODOS: Se cultivaron muestras de esputo de 1 006 pacientes con diagnóstico reciente de tuberculosis efectuado durante el 2008. Se secuenció la región nuclear del gen rpoB, que se asocia con la resistencia a la rifampicina. Todos los aislados con mutaciones de rpoB se enviaron al laboratorio de referencia del estado de Nueva York para llevar a cabo un antibiograma convencional. Todos los aislados se estudiaron también con el ensayo de sonda lineal GenoType MTBDRplus. RESULTADOS: Se aisló Mycobacterium tuberculosis de 906 pacientes. Veintiséis (2,9 por ciento) de los aislados presentaban mutaciones de sentido erróneo o deleciones en rpoB y fueron resistentes a la rifampicina en el antibiograma. Los 26 aislados fueron resistentes también a la isoniacida y se clasificaron como TB multirresistente. Cuarenta y seis aislados de control sin mutaciones de rpoB resultaron sensibles a la rifampicina en el antibiograma. El ensayo de sonda lineal GenoType MTBDRplus identificó correctamente a las 26 cepas de TB multirresistente y clasificó de manera errónea un aislado sensible a múltiples fármacos como resistente a la rifampicina. CONCLUSIONES: Este estudio revela una prevalencia de TB multirresistente de 2,9 por ciento en los pacientes con TB recién diagnosticada en Haití e indica que los ensayos de secuenciación e hibridación de rpoB son estudios de detección sistemática adecuados para la detección temprana de la TB multirresistente.

Mycobacterial Interspersed Repetitive Unit typing in Mycobacterium tuberculosis isolates from Sichuan Province in China.

Background & objectives: Emergence and spread of drug resistant Mycobacterium tuberculosis is a serious threat to tuberculosis (TB) control programme. Therefore, the objective of this study was to genotype drug-resistant M. tuberculosis strains isolated from patients in Sichuan, China, using Mycobacterial Interspersed Repetitive Units (MIRU) for epidemiological analysis. Methods: Drug-resistance testing of M. tuberculosis isolates from pulmonary TB patients was confirmed by proportion method. Twelve MIRU loci were analyzed on 80 drug-resistant and 9 susceptible isolates by polymerase chain reaction and agarose gel electrophoresis. Hunter-Gaston discriminatory index (HGI) values were determined for each 12 MIRU loci for the evaluation of their discrimination power. Results: Among 12 MIRU loci examined, polymorphic bands could be generated on 11 loci. Sixty five isolates had distinct MIRU patterns, while other 24 belonged to 8 clusters and resistant to at least one anti-TB drug tested. The association between the MIRU patterns and the mutation patterns of drug-resistance relevant target genes was not significant among the drug-resistant isolates. Interpretation & conclusions: The results showed that with a satisfactory discrimination power exhibited, the 12 loci based MIRU typing could be a valuable tool for epidemiological studies in M. tuberculosis isolates from Sichuan.

Study of promoter and structural gene sequence of whiB7 in MDR and XDR forms of Mycobacterium tuberculosis / Estudio del promotor del ADN y la secuencia de genes estructurales de whiB7 en las formas MDR y XDR de Mycobacterium tuberculosis

Resistance phenomenon in M tuberculosis is mainly based on decreased permeability of the bacterial envelope and function of effluent pumps. The regulatory gene of the whiB7 transcription determines drug resistance in these bacteria. Increases in WhiB7 protein activity induce transcription of resistance genes leading to intrinsic multidrug resistance. The aim of this work was to evaluate the whiB7 gene sequence in susceptible, MDR and XDR clinical isolates of M tuberculosis in order to further design an inhibitor. Thirty-three clinical isolates of MTB identified as susceptible, MDR and XDR-TB were investigated by PCRfor sequencing of the entire promoter (429 bp), structural gene (279 bp) and the end of the upstream gene uvrD (265 bp). No differences were detected in the sequences of the structural gene in susceptible and MDR with XDR isolates and all of them terminated at TGA as stop codon. Examination of sequence profiles of the promoter part of whiB7 by several sets ofprimers proved that there were no differences between sequence ofsusceptible, MDR and XDR isolates by type strain (H37Rr). Furthermore, the structure of WhiB7 protein was studied in achieved sequences from clinical isolates. We found that the promoter and structural gene of whiB7 are highly conservative in clinical susceptible and resistant isolates. It is a key finding that would assist in the design ofan inhibitor for the WhiB7 protein in all clinical forms in further studies.

El fenómeno de resistencia en M tuberculosis se basa principalmente en la disminución de la permeabilidad de la envoltura bacterial y la función de las bombas efluentes. El gene regulador de la trascripción de whiB7 determina la resistencia al medicamento en estas bacterias. Los aumentos en la actividad de proteína de WhiB7 inducen la trascripción de genes de resistencia que llevan a la resistencia intrínseca de multimedicamentos. El objetivo de este trabajo fue evaluar la secuencia de genes de whiB7 en aislados clínicos susceptibles MDR y XDR de M tuberculosis para mejorar el diseño de un inhibidor. Treinta y tres aislados clínicos de MTB identificados como MDR y XDR-TB susceptibles, fueron investigados por PCR para la secuenciación del promotor entero (429 bp), el gene estructural (279 bp) y el extremo del uvrD gen arriba (265 bp). No se detectó diferencia alguna en las secuencias del gene estructural en aislados susceptibles, MDR y XDR, terminando todos ellos en TGA como codón de terminación. El examen de perfiles de la secuencia de la parte de promotor de whiB7 por varios conjuntos de iniciadores (primers), demostró que no había ninguna diferencia entre la secuencia de aislados susceptibles MDR y XDR por tipo de cepa (H37Rv). Además, la estructura de la proteína de WhiB7 se estudió en secuencias logradas de aislados clínicos. Se encontró que el promotor y el gene estructural whiB7 son muy conservadores en aislados clínicos susceptibles y resistentes. Se trata de un hallazgo clave que ayudaría a designar un inhibidor para la proteína WhiB7 en todas las formas de este patógeno en estudios ulteriores.

Detección de mutaciones asociadas a cepas multidrogo resistente de Mycobacterium tuberculosis en Chile / Detection of genes associated with drug resistance in Mycobacterium tuberculosis strains isolated in Chile

Background: The incidence of acquired resistance to antituberculous drugs of Mycobacterium tuberculosis in Chile is approximately 23 percent. Aim: To analyze the mutations associated with drug resistance in drug resistant strains of Mycobacterium tuberculosis. Material and Methods: In 28 drug resistant Mycobacterium tuberculosis strains isolated in Chile, genes leading to drug resistance were studied. DNA was amplifed by polymerase chain reaction (PCR) and sequencing was carried out using the ABI PRISM big dye terminator cycle sequencing ready reaction kit. Results: In rifampicin-resistant strains, the mutations in rpoβ gene were in the codons S531W/L (56 percent), D516Y (16 percent) and D516V (16 percent). The predominant mutation in katG gene was in the codon S315L (73 percent) in isoniazid-resistant strains. The mutation S95T was found in the 71 percent of ciprofoxacin resistant strains. Only one ethambutol resistant strain had the M306I mutation. Three unreported mutations in katG were identifed. Conclusions: Drug resistance associated mutations of Mycobacterium tuberculosis isolated in Chile were similar to those reported abroad.

[Correlation study of phospholipase C genes and pathogenicity of Mycobacterium tuberculosis strains of the Beijing family and non- Beijing family]

The Beijing strain of Mycobacterium tuberculosis is responsible for more than 25% of cases of Tuberculosis in the world, it has a high transmission potential and there is a significant correlation between Beijing strain and multidrug resistance. In this study we compared the presence of Phospholipase C genes in Beijing and Nonbeijing strains of Mycobacterium tuberculosis. CTAB method was used to extract DNA from positive culture specimens from patients with pulmonary tuberculosis. Spoligotyping was used to identify Beijing from non-Beijing strains. Presence of Phospholipase C genes was ascertained by using PCR. Of 200 specimens, 19 were Beijing strain, [9.5%] and 181 non Beijing strains, [90.5%]. In Beijing strains, 16 specimens, [84.2%]were positive for plcA,17 [89.5%] were positive for plcB and 17 [89.5%] were positive for plcC genes and in non Beijing strains 17 specimens, [9.4%], were positive for plcA, 18[9.9%], for plcB and 18[9.9%] for plcC. Majority of Beijing strains have phospholipase C genes that may be responsible for the virulence of this strain

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 percent) were DR (15 percent of the annual prevalence for Veracruz). Of the DR isolates, 15 (75 percent) were resistant to rifampin, 17 (85 percent) to isoniazid and 15 (75 percent) were resistant to both drugs (MDR). Sequencing analysis performed in the isolates showed that 14 (93 percent) had mutations in the rpoB gene; seven of these (47 percent) exhibited a mutation at 531 (S[L). Ten (58 percent) of the 20 resistant isolates showed mutations in katG; nine (52 percent) 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.

[Detection of mutation in codon 315 katG gene as a gene marker associated with isoniazid resistance, in mycobacterium tuberculosis strains isolated from patients in Isfahan and Tehran by PCR-RFLP method]

Drug resistance to tuberculosis is continuously increasing and is a significant threat to tuberculosis control programs because afew effective drugs are present against Mycobacterium tuberculosis. Although isoniazid [INH] is the most effective drug against tuberculosis, resistance to this drug also develops readily. Mutations in katG, specially the Ser315Thr substitution, are responsible for isoniazid resistance in a large proportion of patients with tuberculosis. However, the frequency of the katG Ser315Thr substitution varies among population samples. This study provided molecular characterization of isoniazid resistance of M. tuberculosis strains and extended our knowledge about molecular basis of M. tuberculosis drug resistance that is widely applicable for rapid drug resistance detection. Using 1% proportional method, the sensitivity of 126 strains isolated from patients in Isfahan and Tehran to isoniazid was determined. The katG mutations in codon 315 associated with isoniazid resistance among isoniazid resistant isolates was determined by PCR-RFLP. In this way, 355 bp PCR products were digested by MspI. Out of 126 isolates of M. tuberculosis, 32 [25.4%] strains were determined as INH resistant. Resistance rate was 22.6% [19 strains] in Isfahan and 31% [13 strains] in Tehran. Overall, 72% of isoniazidresistant isolates could be identified by analysis of just katG 315 loci. The PCR-RFLP using MspI restriction enzyme that detects katG Ser315Thr substitution could be identified in 72% of isoniazid-resistant strains. Elucidation of the molecular characterization of isoniazid resistance in M. tuberculosis has led to the development of different genotypic approaches to the rapid detection of isoniazid resistant in clinical isolates

Multiple Mycobacterium tuberculosis infections in an HIV-infected patient.

Mycobacterial colonies of two different morphologies were isolated from one sputum sample of a HIV-positive patient. One morphological type was resistant to streptomycin (STR) and susceptible to isoniazid (INH), while the other isolate with different colony morphology was resistant to both of these anti-TB drugs. A mycobacterial isolate of one pus from a lymph node sample was resistant to these two anti-TB drugs, while the other isolate from another pus sample was resistant to STR but susceptible INH. IS6110 RFLP based finger printing revealed that the HIV-positive patient was infected with different strains of Mycobacterium tuberculosis. A subculture of isolates on solid medium is useful to examine mixed infection.

Frequency and molecular characterization of rifampicin-resistance in rpoB region of multiple drug resistance [MDR] isolates from tuberculosis patients in southern endemic region of Iran

The aim of this study was to investigate the frequency, location and type of rpoB gene mutations in Mycobacterium tuberculosis [MTB] collected from patients in the southern endemic region of Iran. Drug susceptibility testing was determined by using the BACTEC system and the center for diseases controls [CDC] standard conventional proportional method. In 29 rifampicin-resistant MTB [85%] isolates, 60 mutations and 13 micro-deletions were identified. Missense mutations produced 23 types of amino acid substitutions. In five rifampicin-resistant MTB isolates [15%] no mutations were found in the core region of the rpoB gene. All silent mutations were localized in codon 507. Most frequent mutations detected in Iranian strains, were found in codons 523 and 526. Five alleles in codon 526 and three alleles occurring in triplets in each of the codons 507, 508, 513 were also found. Thus in Iran the highest frequency of common mutations shared between primary and secondary infections was found to occur in codons 523 and 526

Estimation of efflux mediated multi-drug resistance and its correlation with expression levels of two major efflux pumps in mycobacteria.

Multidrug resistance has been posing an increasing problem in the treatment of tuberculosis. Mutations in the genomic targets of drugs have been identified as the major mechanism behind this resistance. However, high degree of resistance in some isolates towards major drugs like rifampicin, isoniazid, ethambutol and streptomycin can not be explained solely on the basis of mutations. Besides this, certain other mechanisms like efflux pumps have also been considered as alternative mechanisms in the drug resistant isolates where there is no mutation and these mechanisms are specially important for drug resistance in non-tuberculous mycobacteria (NTM). In this study, we have estimated efflux pump mediated drug resistance in different mycobacterial species with the help of efflux pump inhibitors. All major anti-tuberculous drugs have been shown to be extruded by efflux pumps and the degree to which these drugs are extruded, vary in different mycobacterial species and isolates. The correlation of this resistance with functional activity of two major efflux pump genes pstB and Rv1258c was also assessed by reverse transcription PCR. Besides the significant role of these pumps observed, other efflux pumps, present in mycobacteria, may also be involved in drug resistance and need to be investigated.

Tuberculosis multirresistente en pacientes con SIDA a comienzos del milenio / Multidrug-resistant tuberculosis in AIDS patients at the beginning of the millennium

La tuberculosis multirresistente (TBMR) asociada al sida emergió durante los años 90 en varios países del mundo. En Argentina, el brote más importante se originó en el Hospital Muñiz y susconsecuencias persisten hasta ahora. Con el objeto de evaluar la situación de la TBMR en este hospital, analizamoslas características clínico-demográfico-epidemiológicas de los 53 pacientes masculinos con TBMR/sida internados por primera vez en el trienio 2001-2003 con relación al genotipo del polimorfismo de longitud de fragmentos de restricción (RFLP) IS6110 de los aislamientos. La edad promedio de los pacientes fue 32 años, 37 (70%) residían en el conurbano bonaerense, 36 (68%) eran usuarios de drogas ilícitas y 14 (26.4%) tenían antecedentes carcelarios. El 88% presentó grave inmunodepresión (CD4+<100/μl) y el 58.5% falleció. La mortalidadse asoció a baja adherencia al tratamiento y a comorbilidades, pero no a enfermedad por Mycobacteriumtuberculosis cepa “M”, causante del brote original. De los 40 casos analizados por RFLP, 29 (72.5%) conformaron clusters y 24 presentaban el genotipo “M”. La resistencia a 5 o 6 drogas resultó un indicador de enfermedad por esa cepa. El genotipo “M” se asoció significativamente a internaciones previas en el Hospital Muñiz oencarcelamiento. En síntesis, 14 años después de ocurrido el primer caso de TBMR/sida, se constata la persistenciay predominancia en el hospital de la cepa responsable del brote. Se requiere una intensificación de las medidas de control de la diseminación institucional de la tuberculosis para consolidar la tendencia decrecientede la TBMR observada en el país en la última década

Aids-related multidrug-resistant tuberculosis (MDRTB) emerged during the 90s in several countries aroundthe world. In Argentina, the most notorious outbreak was documented in the Hospital Muñiz, which is still undergoing its aftermaths. In order to evaluate the situation in this hospital regarding MDRTB, we analysed clinical,demographic and epidemiological traits of the 53 male MDRTB-aids patients admitted during 2001-2003 at award especially dedicated to their isolation. Patients’ mean age was 32 years, 70% lived in Buenos Aires suburbs. A history of illicit drug users or imprisonment was recorded in 68% and 26% of the patients, respectively.Severe immunodepression (CD4+ count <100/μl) was found in 88% of the patients and 58% died. Mortality wasassociated with non-adherence to treatment and co-morbidity, but not with the genotype of the “M” strain, responsible for the original outbreak. Of 40 cases available for restriction fragment length polymorphism (RFLP),29 (72.5%) resulted in cluster. RFLP patterns of 24 matched the “M” genotype. In this study, resistance to 5 or 6 drugs was found to be an indicator of disease due to the “M” strain. The “M” genotype associated significantlyto previous admission at the Hospital Muñiz or imprisonment. In brief, 14 years after the detection of the firstMDRTB-aids case, we report here the persistence and predominance of the original outbreak strain at the hospital.Stronger TB infection control measures are urgently needed in hospitals and jails in order to strengthenthe declining trend of the MDRTB observed in our country towards the end of the last decade