ABSTRACT Background: We aimed to evaluate the costs of GenoType® MTBDRplus and MTBDRsl incurred during the diagnosis of first- and second-line drug-resistant tuberculosis (TB) in São Paulo, Brazil. Methods: Mean and activity-based costs of GenoType® were calculated in a referral laboratory for TB in Brazil. Results: The mean cost value and activity-based cost of GenoType® MTBDRplus were USD 19.78 and USD 35.80 and those of MTBDRsl were USD 54.25 and USD 41.85, respectively. Conclusions: The cost of GenoType® MTBDRplus was reduced owing to the high number of examinations performed and work optimization.

ABSTRACT Objective To present the frequency and species diversity of non-tuberculous mycobacteria, estimate the prevalence of non-tuberculous mycobacterial pulmonary disease, describe the epidemiological profile, and determine the follow-up of patients with non-tuberculous mycobacterial pulmonary disease living in a region with a high burden of tuberculosis. Methods This a retrospective cohort observational study using data records obtained from the Instituto Adolfo Lutz - Santos and from the São Paulo Sistema de Vigilância de Tuberculose do Estado de São Paulo in the period between 2000 and 2009. The studied variables were: socio-demographic characteristics, current and past history of tuberculosis, aspects related to diagnosis, and treatment and associated diseases. Results We included 319 non-tuberculous mycobacteria isolates in the study, corresponding to 257 patients. The species Mycobacterium kansasii (28.5%) and Mycobacterium fortuitum (16.6%) presented the higher occurrence. In 10.9% (24) of the patients, there was a criterion for confirming a case of pulmonary disease due to non-tuberculous mycobacteria. In relation to gender and age, male and individuals over 50 years old were the most frequent. Considering the confirmed cases, 47.8% had a past history of tuberculosis. Conclusion The lack of information about the cases is evident, since pulmonary disease due to non-tuberculous mycobacteria is not mandatory. The therapeutic regimen according to the identified species is fundamental for success in combating the infections caused by non-tuberculous mycobacteria. Besides that, information about the regional epidemiology of pulmonary disease caused by non-tuberculous mycobacteria and the search for associations with other comorbidities are important to establish the correct treatment. In order to improve surveillance of pulmonary diseases by non-tuberculous mycobacteria, we suggest the implantation of a sentinel surveillance and of population-based studies.

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.

Abstract Brazil is one of the high burden countries for tuberculosis, and a rapid diagnosis is essential for effective control of the disease. In the present study, an in-house real-time polymerase chain reaction (PCR) assay targeting the mpt64 gene for identification of Mycobacterium tuberculosis complex isolates was evaluated under routine diagnosis conditions in a reference laboratory. From May 2011 to July 2012, 1,520 isolates of mycobacteria were prospectively submitted for phenotypic and/or PRA-hsp65 identification and to real-time PCR. The mpt64 real-time PCR showed 99.7% sensitivity and 96% specificity and detected 79.4% of the cases missed by phenotypic and PRA-hsp65 identification. The in-house real-time PCR assay showed high sensitivity and specificity and was successfully implemented in the routine diagnosis of tuberculosis in a reference laboratory from a high burden setting.

Around 50 mycobacteria species cause human disease. Immunosuppressive states predispose to non-tuberculous mycobaterium infection, such as Mycobacterium chelonae: AFB, non-tuberculous, fast growth of low virulence and uncommon as a human pathogen. It may compromise the skin and soft tissues, lungs, lymph nodes and there is also a disseminated presentation. The diagnosis involves AFB identification and culture on Agar and Lowenstein-Jensen medium base. A 41-year-old female with MCTD (LES predominance) is reported, presenting painless nodules in the right forearm. She denied local trauma. Immunosuppressed with prednisone and cyclophosphamide for 24 months. Lesion biopsy has demonstrated positive bacilloscopy (Ziehl-Neelsen stain) and M.chelonae in culture (Lowenstein-Jensen medium base), therefore clarithromycin treatment has been started (best therapy choice in the literature).

Novas metodologias têm sido desenvolvidas para a identificação de Nocardia spp. mas o diagnóstico inicial ainda necessita de método rápido e preciso, principalmente devido à similaridade com o gênero Mycobacterium, clínica e bacteriologicamente. O crescimento em meio de Löwenstein Jensen (LJ), a presença de bacilos corados pela coloração de Ziehl Neelsen e colônias com características diferentes podem ser fatores de confusão entre nocardias e micobactérias. Este estudo descreve a ocorrência de Nocardia spp. em laboratório de referência em micobacteriologia, observando-se as principais dificuldades em diferenciar Nocardia spp. e Mycobacterium spp., correlacionando isolados com casos de nocardiose. Os registros laboratoriais dos anos 2008 a 2012 foram analisados e os isolados identificados como Nocardia sp. ou como bacilos não álcool - ácido resistentes (NBAAR) foram selecionados. Os dados epidemiológicos e bacteriológicos foram analisados. Trinta e três isolados identificados como Nocardia sp. e 22 como NBAAR foram selecionados para este estudo, perfazendo 0,12% do total de isolados identificados no período estudado. A identificação presuntiva foi baseada na morfologia macroscópica e microscópica, resistência à lisozima e perfis de restrição pelo método PRA-hsp65. Nocardia spp. pode crescer em meios de isolamento para micobactérias (LJ e BBL MGIT™) e microscopia de morfologia e as colônias são muito semelhantes a algumas espécies de micobactérias. Dezessete pacientes (54,8%) foram notificados e tratados para tuberculose, mas apresentaram sinais e sintomas para nocardiose. Concluimos que a ocorrência de Nocardia sp. no período estudado foi de 0,12%. Os isolados com características de bacilos filamentosos, formadores de hifas aéreas, com colônias que podem ter pigmento, rugosas e que não possuem padrão de digestão para BstEII no método PRA-hsp65 são sugestivos de Nocardia spp. Para um laboratório de rotina de Micobactérias, um fluxo de identificação presuntiva para Nocardia spp. é essencial para permitir que esses isolados sejam identificados com técnicas mais precisas, para que seja oferecido o tratamento adequado e qualidade de vida aos pacientes.

New methodologies were developed for the identification of Nocardia but the initial diagnosis still requires a fast and accurate method, mainly due to the similarity to Mycobacterium, both clinical and bacteriologically. Growth on Löwenstein-Jensen (LJ) medium, presence of acid-fast bacilli through Ziehl-Neelsen staining, and colony morphology can be confusing aspects between Nocardia and Mycobacterium. This study describes the occurrence of Nocardia spp. in a mycobacterial-reference laboratory, observing the main difficulties in differentiating Nocardia spp. from Mycobacterium spp., and correlating isolates with nocardiosis cases. Laboratory records for the period between 2008 and 2012 were analyzed, and the isolates identified as Nocardia sp. or as non-acid-fast filamentous bacilli were selected. Epidemiological and bacteriological data were analyzed as well. Thirty-three isolates identified as Nocardia sp. and 22 as non-acid-fast bacilli were selected for this study, and represented 0.12% of isolates during the study period. The presumptive identification was based on macroscopic and microscopic morphology, resistance to lysozyme and restriction profiles using the PRA-hsp65 method. Nocardia spp. can grow on media for mycobacteria isolation (LJ and BBL MGIT™) and microscopy and colony morphology are very similar to some mycobacteria species. Seventeen patients (54.8%) were reported and treated for tuberculosis, but presented signs and symptoms of nocardiosis. It was concluded that the occurrence of Nocardia sp. during the study period was 0.12%. Isolates with characteristics of filamentous bacilli, forming aerial hyphae, with colonies that may be pigmented, rough and without the BstEII digestion pattern in PRA-hsp65 method are suggestive of Nocardia spp. For a mycobacterial routine laboratory, a flow for the presumptive identification of Nocardia is essential, allowing the use of more accurate techniques for the correct identification, proper treatment and better quality of life for patients.

This study investigated biological characteristics of recovered stressed M. tuberculosis isolates that failed to grow in differential culture media for phenotypic identification and in culture media containing anti-tuberculosis drugs for drug-susceptibility testing, despite of having grown in primary culture. It represents an improvement in the diagnosis of MDR tuberculosis and tuberculosis control.

A single strain of Mycobacterium abscessus subsp. bolletii, characterised by a particular rpoB sequevar and two highly related pulsed field gel electrophoresis patterns has been responsible for a nationwide outbreak of surgical infections in Brazil since 2004. In this study, we developed molecular tests based on polymerase chain reaction restriction-enzyme analysis (PRA) and sequencing for the rapid identification of this strain. Sequences of 15 DNA regions conserved in mycobacteria were retrieved from GenBank or sequenced and analysed in silico. Single nucleotide polymorphisms specific to the epidemic strain and located in enzyme recognition sites were detected in rpoB, the 3' region of the 16S rDNA and gyrB. The three tests that were developed, i.e., PRA-rpoB, PRA-16S and gyrB sequence analysis, showed 100%, 100% and 92.31% sensitivity and 93.06%, 90.28% and 100% specificity, respectively, for the discrimination of the surgical strain from other M. abscessus subsp. bolletii isolates, including 116 isolates from 95 patients, one environmental isolate and two type strains. The results of the three tests were stable, as shown by results obtained for different isolates from the same patient. In conclusion, due to the clinical and epidemiological importance of this strain, these tests could be implemented in reference laboratories for the rapid preliminary diagnosis and epidemiological surveillance of this epidemic strain.

OBJETIVO: A diferenciação rápida entre Mycobacterium tuberculosis e micobactérias não-tuberculosas é fundamental para os pacientes coinfectados com tuberculose e HIV. Para tanto, utilizamos duas metodologias em nosso laboratório: detecção do fator corda e PCR-restriction enzyme analysis (PRA). O objetivo do estudo foi avaliar a acurácia desse teste de triagem em meio sólido como um método rápido para a identificação presuntiva do complexo M. tuberculosis, considerando custos e tempo de resultado. MÉTODOS: Foram processadas 152 cepas pelo teste de triagem combinado, que consistiu da detecção do fator corda por microscopia (esfregaço corado por Ziehl-Neelsen) e avaliação do aspecto macroscópico das colônias, e PRA (padrão ouro). Os custos foram estimados através da obtenção dos preços dos insumos necessários para a realização de cada teste. RESULTADOS: A acurácia da detecção do fator corda foi de 95,4% (IC95%: 90,7-98,1%) e a do teste de triagem combinado foi de 99,3% (IC95%: 96,4-100%). O custo da detecção do fator corda foi de R$ 0,60 e do PRA de R$ 16,00. Os resultados da detecção do fator corda estão prontos em 2 dias, ao passo que os de PRA necessitam de 4 dias. CONCLUSÕES: A identificação presuntiva de M. tuberculosis usando o aspecto macroscópico das colônias em conjunto com a detecção de fator corda por microscopia é um teste simples, rápido e de baixo custo. Recomendamos o teste de triagem combinado para rapidamente identificar M. tuberculosis em sítios com poucos recursos financeiros e em laboratórios menos equipados, enquanto se aguarda a identificação definitiva por métodos moleculares ou bioquímicos.

OBJECTIVE: The rapid differentiation between Mycobacterium tuberculosis and nontuberculous mycobacteria is fundamental for patients co-infected with tuberculosis and HIV. To that end, we use two methods in our laboratory: detection of cord factor and PCR-restriction enzyme analysis (PRA). The objective of this study was to evaluate the accuracy of a screening test on solid medium as a rapid method for the presumptive identification of M. tuberculosis complex, considering costs and turnover time. METHODS: A total of 152 strains were submitted to a combined screening test, consisting of the detection of cord factor under microscopy (Ziehl-Neelsen staining) and evaluation of the macroscopic aspect of colonies, as well as to PRA, which was used as the gold standard. Costs were estimated by calculating the price of all of the materials needed for each test. RESULTS: The overall accuracy of cord factor detection alone was 95.4% (95% CI: 90.7-98.1%), and that of the combined screening test was 99.3% (95% CI: 96.4-100%). Cord factor detection costs US$ 0.25, whereas the PRA costs US$ 7.00. Results from cord factor detection are ready in 2 days, whereas PRA requires 4 days to yield results. CONCLUSIONS: The presumptive identification of M. tuberculosis using the macroscopic evaluation of colonies combined with cord factor detection under microscopy is a simple, rapid and inexpensive test. We recommend the combined screening test to rapidly identify M. tuberculosis in resource-poor settings and in less well-equipped laboratories while awaiting a definite identification by molecular or biochemical methods.

OBJETIVO: Estudar a ocorrência de micobactérias não-tuberculosas e a variabilidade das espécies isoladas na região atendida pelo Instituto Adolfo Lutz-Regional de São José do Rio Preto-no período entre 1996 e 2005, assim como mostrar a importância do diagnóstico laboratorial. MÉTODOS: A partir de amostras pulmonares e extrapulmonares, foi realizado o isolamento de micobactérias, e estas foram identificadas por métodos fenotípicos e pelo método molecular polymerase chain reaction-restriction enzyme analysis. RESULTADOS: Foram isoladas 317 cepas de micobactérias não-tuberculosas: complexo Mycobacterium avium, 182 (57,4%); M. gordonae, 33 (10,4%); M. fortuitum, 25(7,9%); M. chelonae, 8 (2,5%); complexo M. terrae, 8 (2,5%); M. kansasii, 7 (2,2%); e espécies menos freqüentes, 54 (17%). No período, foram caracterizados 72 casos (33,3%) de micobacterioses, de acordo com os critérios bacteriológicos estabelecidos pela American Thoracic Society (2007).Desses, complexo M. avium foi responsável por 56 casos, sendo que 29 (51,8%) foram caracterizados como doença disseminada. M. fortuitum foi responsável por 6 casos; M. gordonae, 3; M. chelonae, 2; M. abscessus, 1; M. kansasii, 1; M. intracellulare, 1; M. malmoense, 1; e Mycobacterium ssp., 1. CONCLUSÕES: Os resultados obtidos mostraram a importância do diagnóstico bacteriológico das micobacterioses, pois a identificação das espécies possibilita a introdução de um tratamento adequado precocemente.

OBJECTIVE: To study the incidence of nontuberculous mycobacteria and the range of species isolated between 1996 and 2005 at a regional branch of the Adolfo Lutz Institute-located in the city of São José do Rio Preto, Brazil-and to show the importance of laboratory testing. METHODS: Mycobacteria were isolated from pulmonary and extrapulmonary specimens and identified through phenotyping and molecular methods (polymerase chain reaction-restriction enzyme analysis). RESULTS: We isolated 317 nontuberculous mycobacterium strains: Mycobacterium avium complex, 182 (57.4%); M. gordonae, 33 (10.4%); M. fortuitum, 25 (7.9%); M. chelonae, 8 (2.5%); M. terrae complex, 8(2.5%); M.kansasii, 7 (2.2%); and less frequent species, 54 (17%). During this period, 72 cases (33.3%) were characterized as mycobacteriosis, according to bacteriological criteria established by the American Thoracic Society in 2007. Of those 72 cases, 56 were attributed to M.avium complex. Of those 56, 29 (51.8%) were characterized as disseminated disease. Six cases were attributed to M. fortuitum, 3 to M. gordonae, 2 to M. chelonae, 1 to M. abscessus, 1 to M. kansasii, 1 to M. intracellulare, 1 to M. malmoense and 1 to Mycobacterium ssp. CONCLUSIONS: These results show the importance of the bacteriological diagnosis, since identification of the species enables early and appropriate treatment.

OBJETIVOS: Verificar evidências da formação de aerossóis durante a manipulação de cepas de micobactérias para teste de sensibilidade às drogas (ANT) e identificação (TIP) e o efeito da descontaminação com solução de álcool 70% e luz ultravioleta (UV) na cabine de segurança biológica (CSB), após os procedimentos laboratoriais. MÉTODOS: Uma placa foi exposta na CSB durante os procedimentos de ANT e TIP. Ao término, a CSB foi limpa e descontaminada com álcool 70% e exposta à luz UV por 15 minutos. Após esse tempo outra placa foi exposta por duas horas, somente com a ventilação da CSB ligada. As placas foram incubadas a 37°C e observadas por 30 dias. Os esfregaços das colônias isoladas foram corados pelas técnicas de Ziehl Neelsen e Gram, e as colônias de bacilo álcool-ácido-resistente (BAAR) foram identificadas pelos métodos tradicionais. RESULTADOS: Nas 38 placas expostas durante o ANT, cresceram micobactérias em 10 placas (26,3%), fungos em uma (2,6%) e outros bacilos em duas (5,3%). Das placas com micobactérias, oito (80%) foram identificadas como M. tuberculosis e duas (20%) tiveram identificação inconclusiva. Mesmo após a descontaminação com álcool 70% e uso de UV, cresceram fungos em duas placas (5,3%) e cocos em outras duas (5,3%). Nas 30 placas colocadas nas CSB durante a TIP, cresceram micobactérias em 10 placas (33,3%), fungos em duas (6,6%), cocos em uma (3,4%) e uma mistura de micobactérias e outro bacilo em uma (3,4%). Não houve crescimento nas placas expostas após descontaminação das CSB com álcool a 70% e uso de UV ao término da TIP. CONCLUSÃO: Durante os procedimentos houve formação de aerossóis contendo micobactérias, fato que ficou comprovado pelo crescimento de colônias de micobactérias nas placas expostas. Técnicas laboratoriais adequadas devem ser respeitadas para minimizar a formação de aerossóis como também observar a experiência dos profissionais, a manutenção de um programa de capacitação e a manutenção periódica da CSB.

OBJECTIVES: To verify the evidence of aerosol formation during the manipulation of mycobacteria strains for susceptibility (ST) and identification tests (IT) as well as the decontamination effect of alcohol solution 70% and ultraviolet (UV) radiation in biological safety cabinets (BSC) after laboratory procedures. METHODS: One plate was exposed in a BSC during ST and IT procedures. Afterwards, the BSC was cleaned and decontaminated with alcohol solution 70% and exposed to UV radiation for 15 minutes. After that, another plate was exposed for two hours, only with the BSC ventilation on. Both plates were incubated at 37°C and observed for 30 days. The smears from the isolated colonies were stained with Ziehl Neelsen and Gram techniques, and acid fast bacilli (AFB) were identified by conventional methods. RESULTS: In 38 plates exposed during ST, there was mycobacteria growth in 10 plates (26.3%), fungi in one (2.6%) and bacilli in two (5.3%). Among those plates that presented mycobacteria growth, eight (80%) were identified as M. tuberculosis and two (20%) had inconclusive identification. Even after decontamination with alcohol solution 70% and UV radiation, two plates presented fungi growth (5.3%) and other two presented cocci growth (5.3%). Among 30 plates exposed during IT procedures, there was mycobacteria growth in 10 of them (33.3%), fungi in two (6.6%), cocci in one (3.4%) and one (3.4%) mixed mycobacteria and another bacillus. No growth was observed when alcohol solution 70% and UV radiation were used for decontamination after IT procedures. CONCLUSION: During the procedures there was aerosol formation with mycobacteria, which was proved by mycobacteria growth on the exposed plates. Not only should adequate laboratory techniques be respected to minimize aerosol formation, but professional expertise, the continuity of capacity programs and periodic BSC maintenance should also be observed.

OBJETIVO: Este estudo teve por objetivo descrever a freqüência das espécies de micobactérias não tuberculosas (MNT) identificadas laboratorialmente a partir do isolamento de sítios não estéreis (escarro) de indivíduos infectados ou não pelo vírus HIV na Baixada Santista (SP), período de 2000 a 2005. MÉTODOS: Foi realizada análise retrospectiva dos resultados de baciloscopia e cultura, disponíveis nos registros do laboratório regional de tuberculose, Instituto Adolfo Lutz-Santos. RESULTADOS: Analisou-se 194 cepas de MNT correspondentes a 125 indivíduos, sendo 73 (58,4%) HIV negativos e 52 (41,6%) HIV positivos. Foram identificadas 13 diferentes espécies: Mycobacterium kansasii; complexo M. avium; M. fortuitum; M. peregrinum; M. gordonae; M. terrae; M. nonchromogenicum; M. intracellulare; M. flavescens; M. bohemicum; M. chelonae; M. shimoidei; e M. lentiflavum. Em 19,2% dos casos obteve-se diagnóstico bacteriológico confirmado pelo isolamento da mesma espécie em no mínimo duas amostras consecutivas. CONCLUSÕES: Os resultados mostram a importância da realização sistemática da identificação de MNT na rotina laboratorial e sua integração com a clínica, podendo contribuir na caracterização da doença e ações de efetivo controle, como nas populações co-infectadas tuberculose e HIV.

OBJECTIVE: The present study aims at describing the frequency of nontuberculous mycobacteria (NTM) species identified through laboratory testing of samples collected from non-sterile sites (sputum), as well as its frequency in HIV-infected and non-HIV-infected individuals in the Baixada Santista region of the state of São Paulo, Brazil, in the period from 2000 to 2005. METHODS: Retrospective analysis of sputum smear microscopy results and culture was conducted based on the records on file at the Instituto Adolfo Lutz-Santos, the regional tuberculosis laboratory. RESULTS: We analyzed 194 NTM strains isolated from 125 individuals, of whom 73 (58.4%) were HIV-negative and 52 (41.6%) were HIV-positive. Thirteen different species were identified: Mycobacterium kansasii; M. avium complex; M. fortuitum; M. peregrinum; M. gordonae; M. terrae; M. nonchromogenicum; M. intracellulare; M. flavescens; M. bohemicum; M. chelonae; M. shimoidei; and M. lentiflavum. In 19.2% of the cases, the bacteriological diagnosis was confirmed by isolation of the same species in at least two consecutive samples. CONCLUSIONS: Our results show the importance of including systematic identification of NTM in the laboratory routine, and that its integration into the clinical routine could improve the characterization of the disease, thereby informing the planning of effective control measures in specific populations, such as individuals presenting tuberculosis/HIV co-infection.

Micobactérias não-tuberculosas isoladas no Laboratório Central de Saúde Pública de Mato Grosso do Sul em 2003 e 2004 foram identificadas usando métodos fenotípicos convencionais (TI) e PCR-Restriction Enzyme Analysis (PRA) tendo o gene hsp65 como alvo (PRA-hsp65). Em 15 dos 32 isolados analisados os resultados obtidos com ambos métodos foram concordantes, sendo 8 Mycobacterium avium, 3 M. fortutium, 1 M. kansasii, 1 M. flavescens, 1 M. peregrinum e 1 Nocardia brasiliensis. TI de 12 isolados não foi conclusiva. Perfis não descritos de PRA-hsp65 foram observados com 11 isolados. Dados dos prontuários médicos foram avaliados para inferir a relevância clínica dos isolados.

Non-tuberculous mycobacteria isolated at the Central Public Health Laboratory from Mato Grosso do Sul in 2003 and 2004 were identified by conventional phenotypic methods (TI) and by PCR-Restriction Enzyme Analysis (PRA) using the hsp65 gene as target (PRA-hsp65). With 15 of the 32 analysed isolates, results of both methods were concordant, being 8 Mycobacterium avium, 3 M. fortutium, 1 M. kansasii, 1 M. flavescens, 1 M. peregrinum and 1 Nocardia brasiliensis. TI of 12 isolates was inconclusive. Novel PRA-hsp65 patterns were observed with 11 isolates. Medical data were evaluated for inference of clinical relevance of these isolates.