Carlos Eduardo Dias Campos

Nontuberculous mycobacteria in respiratory samples from patients with pulmonary tuberculosis in the state of Rondonia, Brazil

The main cause of pulmonary tuberculosis (TB) is infection with Mycobacterium tuberculosis (MTB). We aimed to evaluate the contribution of nontuberculous mycobacteria (NTM) to pulmonary disease in patients from the state of Rondônia using respiratory samples and epidemiological data from TB cases. Mycobacterium isolates were identified using a combination of conventional tests, polymerase chain reaction-based restriction enzyme analysis of hsp65 gene and hsp65 gene sequencing. Among the 1,812 cases suspected of having pulmonary TB, 444 yielded bacterial cultures, including 369 cases positive for MTB and 75 cases positive for NTM. Within the latter group, 14 species were identified as Mycobacterium abscessus, Mycobacterium avium, Mycobacterium fortuitum, Mycobacterium intracellulare, Mycobacterium gilvum, Mycobacterium gordonae, Mycobacterium asiaticum, Mycobacterium tusciae, Mycobacterium porcinum, Mycobacterium novocastrense, Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium phlei and Mycobacterium holsaticum and 13 isolates could not be identified at the species level. The majority of NTM cases were observed in Porto Velho and the relative frequency of NTM compared with MTB was highest in Ji-Paraná. In approximately half of the TB subjects with NTM, a second sample containing NTM was obtained, confirming this as the disease-causing agent. The most frequently observed NTM species were M. abscessus and M. avium and because the former species is resistant to many antibiotics and displays unsatisfactory cure rates, the implementation of rapid identification of mycobacterium species is of considerable importance.

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)).

Multilocus Sequence Typing Scheme versus Pulsed-Field Gel Electrophoresis for Typing Mycobacterium abscessus Isolates

ABSTRACT Outbreaks of infections by rapidly growing mycobacteria following invasive procedures, such as ophthalmological, laparoscopic, arthroscopic, plastic, and cardiac surgeries, mesotherapy, and vaccination, have been detected in Brazil since 1998. Members of the Mycobacterium chelonae-Mycobacterium abscessus group have caused most of these outbreaks. As part of an epidemiological investigation, the isolates were typed by pulsed-field gel electrophoresis (PFGE). In this project, we performed a large-scale comparison of PFGE profiles with the results of a recently developed multilocus sequence typing (MLST) scheme for M. abscessus. Ninety-three isolates were analyzed, with 40 M. abscessus subsp. abscessus isolates, 47 M. abscessus subsp. bolletii isolates, and six isolates with no assigned subspecies. Forty-five isolates were obtained during five outbreaks, and 48 were sporadic isolates that were not associated with outbreaks. For MLST, seven housekeeping genes (argH, cya, glpK, gnd, murC, pta, and purH) were sequenced, and each isolate was assigned a sequence type (ST) from the combination of obtained alleles. The PFGE patterns of DraI-digested DNA were compared with the MLST results. All isolates were analyzable by both methods. Isolates from monoclonal outbreaks showed unique STs and indistinguishable or very similar PFGE patterns. Thirty-three STs and 49 unique PFGE patterns were identified among the 93 isolates. The Simpsons index of diversity values for MLST and PFGE were 0.69 and 0.93, respectively, for M. abscessus subsp. abscessus and 0.96 and 0.97, respectively, for M. abscessus subsp. bolletii. In conclusion, the MLST scheme showed 100% typeability and grouped monoclonal outbreak isolates in agreement with PFGE, but it was less discriminative than PFGE for M. abscessus.

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.

Mycobacterium kyorinense Infection

To the Editor: Mycobacterium kyorinense is a nonpigmented, slowly growing mycobacterium that was initially isolated in 2007 from a patient with pneumonia in Japan (1,2). The sequences of the 16S rRNA, hsp65, and rpoB genes of M. kyorinense were closely related to, but different from, those of the type strains of M. celatum and M. branderi, the 2 most phylogenetically related species (1). Biochemical tests, such as those for arylsulfatase activity, tellurite reduction, and heat-stable catalase, also distinguish M. kyorinense from M. celatum and M. branderi. In our initial report, in which this species was first recognized, we described 3 strains isolated from Japanese patients (1). Recently, 1 additional case was reported in Brazil (3). Here we describe 7 newly identified patients whose infection may have been caused by M. kyorinense. In reviewing the characteristics of these 11 patients (10 from Japan and 1 from Brazil), we found no apparent contacts among them. Nine of the 11 patients had respiratory infections, 1 had lymphadenitis, and 1 had arthritis (Table). Of these, 9 patients fulfilled the criteria for infections of clinical significance (4) and were considered to harbor infection by M. kyorinense. Of the 9 patients with respiratory infections, 4 died as a result of the infection. These data suggest that M. kyorinense belongs to a class of nontuberculous mycobacteria that are pathogenic for humans and have substantial clinical effects. Table Clinical characteristics of patients infected with Mycobacterium kyorinense and antimicrobial susceptibility of the organism* Among the 10 patients for whom precise clinical records were available, 7 patients were treated with first-line tuberculosis drugs, mainly rifampin, isoniazid, and ethambutol, but these therapies were ineffective for all patients. Six patients received a combination of antimicrobial drugs, including macrolides and fluoroquinolones, as first- or second-line chemotherapy, and infection was subdued without recurrence in 5 patients. In contrast, 4 patients with pneumonia who did not receive sufficient therapy with the latter regimen eventually died of infection (3 patients) or breast cancer (1 patient). MICs of various antimicrobial drugs for the 9 strains of M. kyorinense were determined by the broth microdilution method as described (1). For most strains, the MICs of rifampin, ethambutol, and isoniazid were relatively high, and MICs of macrolides, aminoglycosides, and quinolones were relatively low. Notably, MICs of rifampin were remarkably high (>32 μg/mL) for all tested strains (Table). Direct sequencing of the 16S rRNA gene, performed as previously described, revealed that 8 of the 9 available M. kyorinense isolates were identical across the entire sequenced interval (1,470 bp). The sole exception was the strain from Brazil, which showed a 4-bp substitution that the other strains did not (3). Although the other 8 strains had identical 16S rRNA sequences, all showed heterogeneity at 9 positions that had not been observed to be heterogeneous in the previous investigation (1). This observation might reflect the presence of 2 copies of the 16S rRNA gene, as has been occasionally reported for other mycobacterial species, including M. celatum (5). Direct sequencing of the entire rpoB gene demonstrated that all strains had identical sequences for this locus. The strains differed from the sequence of M. tuberculosis at 15 nt within codons 511–533. At the amino acid level, these changes were synonymous for the 2 species, with the exception of amino acid residue 531. This residue, Ser531 in the M. tuberculosis RpoB protein, was replaced by an Asp in M. kyorinense. Notably, Ser531 is the most frequent location of substitutions in rifampin-resistant strains of M. tuberculosis (6). Why M. kyorinense has been isolated almost exclusively in Japan is not clear. This tendency may be largely caused by a reporting bias in Japan. However, M. kyorinense may have a particular geographic distribution. In this context, it is noteworthy that the sole strain from Brazil characterized in the current study differed slightly in 16S rRNA sequences from the strains isolated in Japan. It also is notable that the M. kyorinense strains isolated so far were invariably resistant to rifampin by in vitro susceptibility testing. Rifampin appeared to have been clinically ineffective in most patients, although definite efficacy of antimicrobial drugs cannot be evaluated by this retrospective type of study. Analysis of the rpoB gene sequence of M. kyorinense revealed the replacement of aa 531 when compared to the rpoB gene sequence of the M. tuberculosis protein. This finding suggests that M. kyorinense is inherently resistant to rifampin because of the structural features of its RpoB protein. Amino acid replacement at RpoB residue 531 also has been reported in other bacterial species resistant to rifampin, such as M. celatum, Borrelia burgdorferi, and Spiroplasma citri (7–9). In any case, understanding the intrinsic resistance of M. kyorinense to rifampin is critical for appropriately treating infection by this microorganism. On the basis of the results of our study, we recommend that a combination of fluoroquinolones and macrolides and/or aminoglycosides be used for the initial treatment of infection by M. kyorinense in most patients.

First isolation of Mycobacterium kyorinense from clinical specimens in Brazil.

ABSTRACT 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.

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 18 months.

Whole genome sequence of Mycobacterium kansasii isolates of the genotype 1 from Brazilian patients with pulmonary disease demonstrates considerable heterogeneity

Mycobacterium kansasii is an opportunistic pathogen and one of the most commonly encountered species in individuals with lung disease. We here report the complete genome sequence of 12 clinical isolates of M. kansasii from patients with pulmonary disease in Brazil.

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.