Effectiveness and safety of clofazimine in multidrug-resistant tuberculosis: a nationwide report from Brazil.

Although clofazimine is used to treat multidrug-resistant tuberculosis (MDR-TB), there is scant information on its effectiveness and safety. The aim of this retrospective, observational study was to evaluate these factors as well as the tolerability of clofazimine in populations in Brazil, where it was administered at a daily dose of 100 mg·day-1 (body weight ≥45 kg) as part of a standardised MDR-TB treatment regimen until 2006 (thereafter pyrazinamide was used).All MDR-TB patients included in the Sistema de Informação de Tratamentos Especiais da Tuberculose (SITETB) individual electronic register were analysed. The effectiveness of clofazimine was assessed by comparing the treatment outcomes of patients undergoing clofazimine-containing regimens against those undergoing clofazimine-free regimens and its safety by describing clofazimine-attributed adverse events. A total of 1446 patients were treated with clofazimine-containing regimens and 1096 with pyrazinamide-containing regimens.Although success rates were similar in patients treated with clofazimine versus those treated with pyrazinamide (880 out of 1446, 60.9%, versus 708 out of 1096, 64.6%; p=0.054), clofazimine-treated cases exhibited higher death rates due to tuberculosis than pyrazinamide-treated ones (314 out of 1446, 21.7%, versus 120 out of 1096, 10.9%) but fewer failures (78 out of 1446, 5.4%, versus 95 out of 1096, 8.7%) and less loss to follow-up (144 out of 1446, 10.0%, versus 151 out of 1096, 13.8%). No relevant differences were detected when comparing adverse events in patients treated with clofazimine-containing regimens to those treated with clofazimine-free regimens. However, the incidence of side-effects was less than previously reported (gastro-intestinal complaints: 10.5%; hyper-pigmentation: 50.2%; neurological disturbances: 9-13%).

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.

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.

Global expansion of Mycobacterium tuberculosis lineage 4 shaped by colonial migration and local adaptation.

On the basis of population genomic and phylogeographic analyses of 1669 Mycobacterium tuberculosis lineage 4 (L4) genomes, we find that dispersal of L4 has been completely dominated by historical migrations out of Europe. We demonstrate an intimate temporal relationship between European colonial expansion into Africa and the Americas and the spread of L4 tuberculosis (TB). Markedly, in the age of antibiotics, mutations conferring antimicrobial resistance overwhelmingly emerged locally (at the level of nations), with minimal cross-border transmission of resistance. The latter finding was found to reflect the relatively recent emergence of these mutations, as a similar degree of local restriction was observed for susceptible variants emerging on comparable time scales. The restricted international transmission of drug-resistant TB suggests that containment efforts at the level of individual countries could be successful.

Low incidence of colonization and no cases of disseminated Mycobacterium avium complex infection (DMAC) in Brazilian AIDS patients in the HAART era.

OBJECTIVE: Evaluate the incidence of mycobacterial disease and the colonization of the respiratory and gastrointestinal tracts by Mycobacterium avium complex (MAC) bacteria in AIDS patients. INCLUSION CRITERIA: HIV-positive individuals with at least one CD(4)(+) count < 100 cells/mm(3). EXCLUSION CRITERIA: Mycobacterial disease and MAC prophylaxis. Stool, sputum, and blood cultures were prospectively obtained every month from September, 1997, to December, 1999. The incidence was calculated using Poisson regression. Survival was estimated by the Kaplan Meier method and the Cox proportional hazard model. RESULTS: We followed-up 79 patients during a median period of 428 days. Blood cultures (n = 742) were negative for all mycobacteria. Positive cultures (25 samples) were obtained from non-sterile sites: Stools (19/703 specimens = 2.7%) and sputum (14/742 specimens = 1.9%). MAC was isolated in 7/703 stool samples (1%) and 1/32 sputum specimens (0.1%). The incidence of patient colonization with MAC was 0.09 /year (CI=0.05-0.18). CD4 counts in patients colonized with MAC were below 100 cells/mm(3) in only 2 out of 8 cases. Restoration of CD(4)(+) counts >100 cells/mm(3) (HR = 0.18; CI = 0.05-0.70) predicted a lower risk of death (P<0.05) but was not protective for MAC colonization (HR=0.52;CI =0.62-4.35, P=0.55). CONCLUSION: The absence of DMAC infection in colonized individuals argues in favor of a HAART protective effect against; DMAC; however, restoration of CD(4) counts did not protect patients against MAC colonization.

Multidrug resistant Mycobacterium tuberculosis: a retrospective katG and rpoB mutation profile analysis in isolates from a reference center in Brazil.

BACKGROUND: Multidrug resistance is a critical factor in tuberculosis control. To gain better understanding of multidrug resistant tuberculosis in Brazil, a retrospective study was performed to compare genotypic diversity and drug resistance associated mutations in Mycobacterium tuberculosis isolates from a national reference center. METHODS AND FINDINGS: Ninety-nine multidrug resistant isolates from 12 Brazilian states were studied. Drug-resistance patterns were determined and the rpoB and katG genes were screened for mutations. Genotypic diversity was investigated by IS6110-RFLP and Luminex 47 spoligotyping. Mutations in rpoB and katG were seen in 91% and 93% of the isolates, respectively. Codon 315 katG mutations occurred in 82.8% of the isolates with a predominance of the Ser315Thr substitution. Twenty-five isolates were clustered in 11 groups with identical IS6110-RFLP patterns while 74 showed unique patterns with no association between mutation frequencies or susceptibility profiles. The most prevalent spoligotyping lineages were LAM (47%), T (17%) and Haarlen (12%). The Haarlen lineage showed a higher frequency of codon 516 rpoB mutations while codon 531 mutations prevailed in the other isolates. CONCLUSIONS: Our data suggest that there were no major multidrug resistant M. tuberculosis strains transmitted among patients referred to the reference center, indicating an independent acquisition of resistance. In addition, drug resistance associated mutation profiles were well established among the main spoligotyping lineages found in these Brazilian multidrug resistant isolates, providing useful data for patient management and treatment.

Low incidence of colonization and no cases of disseminated Mycobacterium avium complex infection (DMAC) in Brazilian AIDS patients in the HAART era

OBJECTIVE: Evaluate the incidence of mycobacterial disease and the colonization of the respiratory and gastrointestinal tracts by Mycobacterium avium complex (MAC) bacteria in AIDS patients. METHODS: Inclusion criteria: HIV-positive individuals with at least one CD4+ count < 100 cells/mm³. Exclusion criteria: Mycobacterial disease and MAC prophylaxis. Stool, sputum, and blood cultures were prospectively obtained every month from September, 1997, to December, 1999. The incidence was calculated using Poisson regression. Survival was estimated by the Kaplan Meier method and the Cox proportional hazard model. RESULTS: We followed-up 79 patients during a median period of 428 days. Blood cultures (n = 742) were negative for all mycobacteria. Positive cultures (25 samples) were obtained from non-sterile sites: Stools (19/703 specimens = 2.7 percent) and sputum (14/742 specimens = 1.9 percent). MAC was isolated in 7/703 stool samples (1 percent) and 1/32 sputum specimens (0.1 percent). The incidence of patient colonization with MAC was 0.09 /year (CI=0.05 - 0.18). CD4 counts in patients colonized with MAC were below 100 cells/mm³ in only 2 out of 8 cases. Restoration of CD4+ counts >100 cells/mm³ (HR = 0.18; CI = 0.05 - 0.70) predicted a lower risk of death (P<0.05) but was not protective for MAC colonization (HR=0.52;CI =0.62 - 4.35, P=0.55). CONCLUSION: The absence of DMAC infection in colonized individuals argues in favor of a HAART protective effect against; DMAC; however, restoration of CD4 counts did not protect patients against MAC colonization

Epidemiologia molecular da tuberculose / Molecular epidemiology of tuberculosis

A tuberculose continua a figurar como uma das principais enfermidades que afetam a humanidade. A Organizaçäo Mundial de Saúde OMS) estima que cerca de 1/3 da populaçäo mundial esteja infectada pelo Mycobacterium tuberculosis. A pandemia causada pelo HIV contribui muito para o agravamento da situaçäo epidemiológica da tuberculose em todo o mundo. Além disso, o controle da tuberculose também está ameaçado pela emergência de cepas multirresistentes às drogas antituberculostáticas. Assim, estudos epidemiológicos que permitam a diferenciaçäo de cepas de M. tuberculosis säo importantes para o controle da doença por meio da detecçäo de casos índices. Neste trabalho, descrevemos as técnicas de biologia molecular mais utilizadas para a epidemiologia molecular da tuberculose, assim como fazemos uma breve revisäo da literatura com os resultados obtidos na aplicaçäo destas técnicas.

Esterilizaçäo a frio através de agentes químicos líquidos: avaliaçäo de formulaçöes à base de formaldeído / Cold sterilization with liquid chemical agents: evaluation of formaldehyde preparations

O efeito esporocida de preparaçöes contendo formaldeído nas concentraçöes de 4, 5, 6 e 8%, juntamente com álcool e quaternários de amônio, foi avaliado pela técnica da AOAC (The Association of Official Analytical Chemists). O tempo necessário para a destruiçäo dos esporos de Bacillus subtilis e Clostridium sporogenes foi, respectivamente, de 8 e 18 horas. É importante assinalar que as soluçöes contendo 4 e 5% de formaldeído näo apresentam os vapores irritantes täo característicos deste desinfetante quando utilizado em concentraçöes mais altas

Lise-centrifugaçäo e semeadura direta como métodos de detecçäo de micobacteriemia em pacientes com AIDS / Lysis-Centrifuge and direct inoculation as methods of mycobacteremia detection in patients with AIDS

Foram utilizados dois métodos para detectar micobacteriemia em 50 pacientes com AIDS. 1.inoculaçäo direta em meio bifásico; 2.um sistema näo comercial de lise-centrifugaçäo. Os métodos demonstraram uma positividade de 50 por cento e 56 por cento, respectivamente. Os resultados demonstram que a cultura de sangue pode ser executada utilizando-se método näo comercial de lise-centrifugaçäo o qual é sensível, barato e pode ser um método alternativo confiável para o diagnóstico de micobacteriemia em países em desenvolvimento.