Genetic Characterization and Population Structure of Drug-Resistant Mycobacterium tuberculosis Isolated from Brazilian Patients Using Whole-Genome Sequencing.

The present study aimed to determine the genetic diversity of isolates of Mycobacterium tuberculosis (Mtb) from presumed drug-resistant tuberculosis patients from several states of Brazil. The isolates had been submitted to conventional drug susceptibility testing for first- and second-line drugs. Multidrug-resistant (MDR-TB) (54.8%) was the most frequent phenotypic resistance profile, in addition to an important high frequency of pre-extensive resistance (p-XDR-TB) (9.2%). Using whole-genome sequencing (WGS), we characterized 298 Mtb isolates from Brazil. Besides the analysis of genotype distribution and possible correlations between molecular and clinical data, we determined the performance of an in-house WGS pipeline with other online pipelines for Mtb lineages and drug resistance profile definitions. Sub-lineage 4.3 (52%) was the most frequent genotype, and the genomic approach revealed a p-XDR-TB level of 22.5%. We detected twenty novel mutations in three resistance genes, and six of these were observed in eight phenotypically resistant isolates. A cluster analysis of 170 isolates showed that 43.5% of the TB patients belonged to 24 genomic clusters, suggesting considerable ongoing transmission of DR-TB, including two interstate transmissions. The in-house WGS pipeline showed the best overall performance in drug resistance prediction, presenting the best accuracy values for five of the nine drugs tested. Significant associations were observed between suffering from fatal disease and genotypic p-XDR-TB (p = 0.03) and either phenotypic (p = 0.006) or genotypic (p = 0.0007) ethambutol resistance. The use of WGS analysis improved our understanding of the population structure of MTBC in Brazil and the genetic and clinical data correlations and demonstrated its utility for surveillance efforts regarding the spread of DR-TB, hopefully helping to avoid the emergence of even more resistant strains and to reduce TB incidence and mortality rates.

Detection of Drug Resistant Mycobacterium Tuberculosis Strains Using Kit SIRE Nitratase®: a Multicenter Study

Abstract (1) Background: The Commercial Kit SIRE Nitratase® PlastLabor, is a drug susceptibility test kit used to detect Mycobacterium tuberculosis resistance to first-line TB treatment drugs. The present study aimed at evaluating its performance in a multicenter study. (2) Methods: To determine its accuracy, the proportion methods in Lowenstein Jensen medium or the BACTECTMMGITTM960 system was used as a gold standard. (3) Results: The study revealed that the respective accuracies of the kit with 190 M. tuberculosis clinical isolates, using the proportion methods in Lowenstein Jensen medium or BACTECTMMGITTM960 system as a gold standard, were 93.9% and 94.6%, 96.9% and 94.6%, 98.0% and 97.8%, and 98.0% and 98.9%, for streptomycin, isoniazid, rifampicin, and ethambutol, respectively. (4) Conclusion: Thus, the kit can rapidly screen resistance to streptomycin, isoniazid, rifampicin, and ethambutol. Additionally, it does not require sophisticated equipment; hence, it can be easily used in the laboratories of low and middle income countries.

Description of a new Mycobacterium intracellulare pattern of PCR restriction enzyme analysis of hsp65 gene.

This work comprises 9 pulmonary nontuberculous mycobateria isolates obtained from sputum of 4 different patients from Brazil. The sequencing and phylogenetic analysis allowed their accurate identification as Mycobacterium intracellulare. We report a mutation at position 453 creating a new HaeIII cutting site and, therefore, a new PRA-hsp65 M. intracellulare profile.

Mycobacterium fragae sp. nov., a non-chromogenic species isolated from human respiratory specimens.

Three isolates of a slow-growing, non-chromogenic mycobacterium were grown from three sputum samples of a patient from the north-eastern Ceará state in Brazil. Identification at species level could not be obtained with PCR restriction analysis of the hsp65 gene. In order to characterize the isolates we carried out phenotypic and genotypic tests. We sequenced the nearly complete 16S rRNA gene and obtained partial sequences of the hsp65 (encoding the hypervariable region of the 65 kDa heat-shock protein) and rpoB (encoding the beta-subunit of RNA polymerase) genes. The three isolates turned out to be identical and most closely related to the species Mycobacterium celatum and Mycobacterium kyorinense. The results, however, showed significant differences between these species and the isolates studied, which led us to consider them members of a novel species for which we propose the name Mycobacterium fragae. The type strain is HF8705(T) ( = Fiocruz-INCQS/CMRVS P4051(T) = DSM 45731(T)).

First isolation of Mycobacterium kyorinense from clinical specimens in Brazil.

In this article, the first isolation of Mycobacterium kyorinense specimens in Brazil is described. M. kyorinense is a recently identified species, with a few strains reported only in Japan. The Brazilian isolates were initially identified as Mycobacterium celatum by PCR restriction enzyme pattern analysis (PRA) with hsp65. However, biochemical tests indicated the same profile of M. kyorinense and distinguished them from M. celatum and Mycobacterium branderi. The sequencing of the hsp65, rpoB, and 16S rRNA genes allowed the accurate identification of isolates as M. kyorinense.

Evaluation of indirect susceptibility testing of Mycobacterium tuberculosis to the first- and second-line, and alternative drugs by the newer MB/BacT system

In order to evaluate the Organon Teknika MB/BacT system used for testing indirect susceptibility to the alternative drugs ofloxacin (OFLO), amikacin (AMI), and rifabutin (RIF), and to the usual drugs of standard treatment regimes such as rifampin (RMP), isoniazid (INH), pyrazinamide (PZA), streptomycin (SM), ethambutol (EMB), and ethionamide (ETH), cultures of clinical specimens from 117 patients with pulmonary tuberculosis under multidrug-resistant investigation, admitted sequentially for examination from 2001 to 2002, were studied. Fifty of the Mycobacterium tuberculosis cultures were inoculated into the gold-standard BACTEC 460 TB (Becton Dickinson) for studying resistance to AMI, RIF, and OFLO, and the remaining 67 were inoculated into Lowenstein Jensen (LJ) medium (the gold standard currently used in Brazil) for studying resistance to RMP, INH, PZA, SM, EMB, and ETH. We observed 100 percent sensitivity for AMI (80.8-100), RIF (80.8-100), and OFLO (78.1-100); and 100 percent specificity for AMI (85.4-100), RIF (85.4-100), and OFLO (86.7-100) compared to the BACTEC system. Comparing the results obtained in LJ we observed 100 percent sensitivity for RMP (80-100), followed by INH - 95 percent (81.8-99.1), EMB - 94.7 percent (71.9-99.7), and 100 percent specificity for all drugs tested except for PZA - 98.3 (89.5-99.9) at 95 percent confidence interval. The results showed a high level of accuracy and demonstrated that the fully automated, non-radiometric MB/BacT system is indicated for routine use in susceptibility testing in public health laboratories

Direct sensitivity test of the MB/BacT system.

In order to evaluate the direct-method test of sensitivity to drugs used in the principal tuberculosis treatment regimes, in the Organon Teknika MB/BacT system, we tested 50 sputum samples positive to microscopy taken from patients with pulmonary tuberculosis and with clinical indications for an antibiogram, admitted sequentially for examination during the routine of the reference laboratory. The material was treated v/v with 23% trisodium phosphate solution, incubated for 24 h at 35 degrees C, and neutralized v/v with 20% monosodium phosphate solution. The material was then centrifuged and the sediment inoculated into flasks containing Rifampin - 2 micro g/ml, Isoniazid - 0.2 micro g/ml, Pyrazinamide - 100 micro g/ml, Ethambutol - 2.5 micro g/ml, Ethionamide - 1.25 micro g/ml, and Streptomycin - 2 micro g/ml. The tests were evaluated using the indirect method in the BACTEC 460 TB (Becton Dickinson) system as the gold standard. The results showed that the Rifampin test performed best, i.e., 100% sensitivity at 95% Confidence Interval (82.2-100) and 100% specificity at 95% Confidence Interval (84.5-100), followed by Isoniazid and Pyrazinamide. In this experiment, 92% of the materials showed a final reading in 30 days; this period represents the time for primary isolation as well as the results of the sensitivity profile, and is within Centers for Disease Control and Prevention recommendations regarding time for performance of the antibiogram. The inoculated flasks showed no contamination during the experiment. The MB/BacT is shown to be a reliable, rapid, fully automated nonradiometric system for the tuberculosis antibiogram.

Direct sensitivity test of the MB/BacT system

In order to evaluate the direct-method test of sensitivity to drugs used in the principal tuberculosis treatment regimes, in the Organon Teknika MB/BacT system, we tested 50 sputum samples positive to microscopy taken from patients with pulmonary tuberculosis and with clinical indications for an antibiogram, admitted sequentially for examination during the routine of the reference laboratory. The material was treated v/v with 23 percent trisodium phosphate solution, incubated for 24 h at 35ºC, and neutralized v/v with 20 percent monosodium phosphate solution. The material was then centrifuged and the sediment inoculated into flasks containing Rifampin - 2 æg/ml, Isoniazid - 0.2 æg/ml, Pyrazinamide - 100 æg/ml, Ethambutol - 2.5 æg/ml, Ethionamide - 1.25 æg/ml, and Streptomycin - 2 æg/ml. The tests were evaluated using the indirect method in the BACTEC 460 TB (Becton Dickinson) system as the gold standard. The results showed that the Rifampin test performed best, i.e., 100 percent sensitivity at 95 percent Confidence Interval (82.2-100) and 100 percent specificity at 95 percent Confidence Interval (84.5-100), followed by Isoniazid and Pyrazinamide. In this experiment, 92 percent of the materials showed a final reading in 30 days; this period represents the time for primary isolation as well as the results of the sensitivity profile, and is within Centers for Disease Control and Prevention recommendations regarding time for performance of the antibiogram. The inoculated flasks showed no contamination during the experiment. The MB/BacT is shown to be a reliable, rapid, fully automated nonradiometric system for the tuberculosis antibiogram