Phenotypic and Genotypic Drug Susceptibility Assessment of Mycobacterium bovis Bacillus Calmette-Guérin Clinical Strains.

PURPOSE: Mycobacterium bovis Bacillus Calmette-Guérin (BCG) is the only vaccine licensed against tuberculosis. Despite the protection offered by the vaccine, in some circumstances children and immunocompromised individuals can develop associated infections, known as BCGitis. Drug susceptibility patterns of BCG clinical strains have rarely been described. We aimed to describe the susceptibility pattern of BCG clinical strains isolated in two different countries. METHODS: We performed culture-based drug susceptibility testing of thirty one BCG strains isolated from patients in Brazil (n=5, 16%) and Argentina (n=26, 84%) using the broth micro-dilution method (phenotypic method). Final antibiotic concentrations for susceptibility testing ranged from 0.5 to 16 mg/L for amikacin, 0.3125 to 10 mg/L for ethambutol, 0.05 to 1.6 mg/L for isoniazid and 0.25 to 8 mg/L for rifampicin, streptomycin and ofloxacin. Additionally, we compared the results with genetic data obtained by whole genome sequencing. RESULTS: By using the phenotypic method we detected one strain resistant to ethambutol, three strains resistant to rifampicin and one resistant to isoniazid. Additionally, two strains that were phenotypically resistant to both isoniazid and rifampicin carried mutations in the katG and rpoB genes simultaneously. CONCLUSION: There is evidence of the emergence of BCG-resistant strains isolated from vaccine-related complications. We recommend drug susceptibility testing of the BCG strain causing the infection in order to prevent treatment failure.

Characterization of Mycobacterium tuberculosis var. africanum isolated from a patient with pulmonary tuberculosis in Brazil.

Human tuberculosis (TB) is caused by members of the Mycobacterium tuberculosis complex (MTBC), including Mycobacterium tuberculosis var. tuberculosis (MTB) and Mycobacterium tuberculosis var. africanum (MAF). While MTB is isolated worldwide, MAF is almost completely restricted to the African continent, and despite the historical proximity between Brazil and Africa during the slave trade, no case of TB being caused by MAF has been reported in Brazil to date. We hereby describe the first case of TB caused by MAF in Brazil comparing its genome against the published ones. A female patient who had never visited Africa presented with clinical symptoms typical of pulmonary TB. Based on 16S rRNA gene sequencing, the cultured isolate was identified as belonging to MTBC and partial sequence of the hsp65 gene was identical to that of MAF. This was confirmed by genotyping based on detection of Single Nucleotide Polymorphism (SNP), Region of Difference (RD) and spoligotyping. The isolate presented the Shared International Typing (SIT) 181. In the whole-genome comparison against MAF genomes available on published EMBL-EBI European Nucleotide Archive (ENA), the Brazilian genome (MAFBRA00707) was identified as belonging to Lineage 6 and clustered with isolates from The Gambia. This is the first report of the isolation of MAF from a patient from Brazil, without evidence of having any contact with an African index case.

Discovery of a novel Mycobacterium asiaticum PRA-hsp65 pattern.

Twenty-one pulmonary sputum samples from nine Brazilian patients were analyzed by the PRA-hsp65 method for identification of Mycobacterium species and the results were compared by sequencing. We reported a mutation at the position 381, that generates a suppression cutting site in the BstEII enzyme, thus leading to a new PRA-hsp65 pattern for M. asiaticum identification.

Drug susceptibility profile of Mycobacterium kansasii clinical isolates from Brazil.

OBJECTIVES: Mycobacterium kansasii (M. kansasii) pulmonary infection can cause disease with clinical and radiological features similar to tuberculosis. Failure to treat M. kansasii infection is usually associated with resistance; to increase the chance of successful treatment it is important to identify the species and know the susceptibility profile. This study aimed to evaluate the antimycobacterial susceptibility profiles of M. kansasii isolates from Brazil. METHODS: Sixty-nine M. kansasii isolates from 69 patients were identified by partial sequencing of the hsp65 gene, and their susceptibility profiles were analysed by minimal inhibitory concentration (MIC) assays. RESULTS: From 69 isolates, 68 showed susceptibility to clarithromycin, amikacin, and moxifloxacin. Most strains showed high rates of resistance to trimethoprim-sulfamethoxazole and ciprofloxacin. Resistance to rifampicin and ethambutol was found in 12% and 25% of isolates, respectively. CONCLUSIONS: Worrying results were found regarding susceptibility to some drugs used as first-line agents in the treatment of diseases caused by M. kansasii.

First detection of Mycobacterium triplex in Latin America.

In this study we describe the first isolation of Mycobacterium triplex in Latin America. This species causes infections in humans, with very few reports from around the world. We isolated two sputum specimens of a patient with a 6-year history of human immunodeficiency and tuberculosis treatment failure. All tests used confirmed M. triplex and the patient responded well to drug therapy for 18months.

Risk Factors for Mycobacterium abscessus subsp. bolletii infection after laparoscopic surgery during an outbreak in Brazil.

OBJECTIVE To identify risk factors related to Mycobacterium abscessus subsp. bolletii infection during an outbreak, associated with laparoscopic surgery and to propose recommendations for preventing new cases. DESIGN A retrospective cohort study. SETTING A private hospital in Manaus, Brazil. PATIENTS A cohort of 222 patients who underwent laparoscopic surgery between July 2009 and August 2010 by a single surgical team. METHODS We collected information about the patients and the surgical procedure using a standard form. We included sex, age, and variables with P≤0.2 in the bivariate analysis in a logistic regression model. Additionally, we reviewed the procedures for reprocessing the laparoscopic surgery equipment, and the strains obtained with culture were identified by molecular methods. RESULTS We recorded 60 (27%) cases of infection. After multivariate analysis, the duration of surgery beyond 1 hour (odds ratio [OR] 2.4; 95% confidence interval [CI] 1.2-4.5), not to have been the first operated patient on a given day (OR, 2.7; 95% CI, 1.4-5.2), and the use of permanent trocar (OR, 2.2; 95% CI, 1.1-4.2) were associated with infection. We observed that the surgical team attempted to sterilize the equipment in glutaraldehyde solution when sanitary authorities had already prohibited it. Eleven strains presented 100% DNA identity with a single strain, known as BRA100 clone. CONCLUSIONS Because contaminated material can act as vehicle for infection, ensuring adequate sterilization processing of video-assisted surgery equipment was crucial to stopping this single clonal outbreak of nonturbeculous mycobacteria in Brazil.

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% sensitivity for AMI (80.8-100), RIF (80.8-100), and OFLO (78.1-100); and 100% 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% sensitivity for RMP (80-100), followed by INH-95% (81.8-99.1), EMB-94.7% (71.9-99.7), and 100% specificity for all drugs tested except for PZA-98.3 (89.5-99.9) at 95% 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.