Non-tuberculous mycobacteria hybridisation profiles in the GenoType MTBDRplus assay: experience from a diagnostic routine of a high-throughput laboratory.

Introduction. Disease caused by non-tuberculous mycobacteria (NTM) is an emergent problem. Because NTM pulmonary disease and tuberculosis (TB) have similar clinical presentations, many cases of NTM may be misdiagnosed as TB before laboratory identification of the NTM species.Hypothesis/Gap Statement. Clinical laboratories should always perform differentiation between Mycobacterium tuberculosis complex (MTBC) and NTM to guide patients' correct treatment.Aim. To describe the characteristics and to identify mycobacterial isolates presumptively classified as MTBC by macroscopic characteristics in culture media that tested negative in GenoType MTBDRplus.Methodology. All cultures from February 2019 to December 2021 showing MTBC macroscopic characteristics were processed by GenoType MTBDRplus. MTBC-negative cultures underwent species identification by immunochromatography, line probe assays and PRA-hsp65. Patients' data were obtained from Brazilian surveillance systems.Results. Only 479 (3.1%) of 15 696 isolates presumptively identified as MTBC were not confirmed by GenoType MTBDRplus and were then subjected to identification. A total of 344 isolates were shown to be NTM, of which 309 (64.5%) and 35 (7.3%) were identified to the species and genus levels, respectively. Of the 204 NTM isolates with MTBC characteristics, the most frequent species were M. fortuitum (n=52, 25.5%), M. abscessus complex (MABC; n=27, 13.2%) and M. avium complex (MAC; n=26, 12.7%). Regarding the GenoType MTBDRplus results from NTM isolates, there were diverse hybridisation profiles with rpoB gene's different wild-type (WT) probes. Seventy-six (16.1%) of the 473 patients were classified as having NTM disease, the most frequent being MAC (n=15, 19.7%), MABC (n=13, 17.1%), M. kansasii (n=10, 13.2%) and M. fortuitum (n=6, 7.9%).Conclusion. Because the signs and symptoms of pulmonary TB are similar to those of pulmonary mycobacteriosis and treatment regimens for TB and NTM are different, identifying the disease-causing species is paramount to indicate the correct management. Thus, in the laboratory routine, when an isolate presumptively classified as MTBC is MTBC-negative, it is still essential to perform subsequent identification.

Use of an immunochromatographic assay for rapid identification of Mycobacterium tuberculosis complex clinical isolates in routine diagnosis.

Accurate identification of Mycobacterium tuberculosis complex (MTBC) isolates is essential for tuberculosis (TB) control, especially in a high-burden country such as Brazil. Conventional identification methods are laborious and time-consuming, while rapid molecular methods are expensive and require skilled personnel and appropriate physical laboratory infrastructure. Immunochromatographic assays (ICAs) have been shown to provide a rapid and reliable TB diagnosis at a low cost. The use of the SD Bioline TB Ag MPT64 ICA (MPT64 assay) for rapid identification of MTBC clinical isolates in the routine diagnosis of a large-volume reference TB laboratory was evaluated. We analysed 375 isolates on solid and liquid media concurrently with conventional phenotypic methods, the PRA-hsp65 molecular technique and the MPT64 assay. The sensitivity, specificity and accuracy of the ICA were 97.7, 100 and 98.1 %, respectively. The MPT64 assay yielded rapid and accurate results, enabling the treatment to be initiated early and also impacting on TB control.

Pseudooutbreak of rapidly growing mycobacteria due to Mycobacterium abscessus subsp bolletii in a digestive and respiratory endoscopy unit caused by the same clone as that of a countrywide outbreak.

BACKGROUND: The nontuberculous mycobacteria (NTM) are widely spread. In Brazil, 2,520 cases of rapidly growing mycobacteria (RGM) infections after medical procedures were reported, with 5.4% of cases related to nonsurgical invasive procedures and with an occurrence of 1 clone (BRA100) of Mycobacterium abscessus subsp bolletii. OBJECTIVE: To describe a pseudooutbreak of M abscessus subsp bolletii in an endoscopy and bronchoscopy unit. METHODS: The alert for a pseudooutbreak was given when 3 patients, in the same week, had a positive bronchoalveolar lavage culture for M abscessus subsp bolletii. The patients had no symptoms/signs of mycobacterial infection; thus, contamination of bronchoscopes was suspected. Samples for culturing were collected from bronchoscopes, digestive endoscopes, automated disinfection machines, and the water supply. Clinical samples were identified by polymerase chain reaction restriction-enzyme analysis (PRA) of the hsp65 gene and their pulsed-field gel electrophoresis pattern was compared with environmental samples. RESULTS: The investigation demonstrated a contamination of bronchoscopes, digestive endoscopes, and disinfection machines. Molecular typing demonstrated that all strains belonged to the same clone (MAB01), identical to clone BRA100. DISCUSSION: Cross-transmission due to poor disinfection as well as resistance to glutaraldehyde may play roles in the spread of MAB01 M abscessus subsp bolletii, which may have a unique resistance to the environment and adaption to human hosts. However the water supply may have played a role. Attention is needed to ensure the quality of water used to rinse disinfected equipment.

Cord factor detection and macroscopic evaluation of mycobacterial colonies: an efficient combined screening test for the presumptive identification of Mycobacterium tuberculosis complex on solid media / Detecção do fator corda e avaliação do aspecto macroscópico das colônias de micobactérias: um eficiente teste de triagem combinado para a identificação presuntiva do complexo Mycobacterium tuberculosis em meios só

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 percent (95 percent CI: 90.7-98.1 percent), and that of the combined screening test was 99.3 percent (95 percent CI: 96.4-100 percent). 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: 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 por cento (IC95 por cento: 90,7-98,1 por cento) e a do teste de triagem combinado foi de 99,3 por cento (IC95 por cento: 96,4-100 por cento). 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.

Cord factor detection and macroscopic evaluation of mycobacterial colonies: an efficient combined screening test for the presumptive identification of Mycobacterium tuberculosis complex on solid media.

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.

Monitoramento em cabine de segurança biológica: manipulação de cepas e descontaminação em um laboratório de micobactérias / Biological safety cabinet monitoring: strains manipulation and decontamination in a mycobacteria laboratory

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 por cento 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 por cento 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 37ºC 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 por cento), fungos em uma (2,6 por cento) e outros bacilos em duas (5,3 por cento). Das placas com micobactérias, oito (80 por cento) foram identificadas como M. tuberculosis e duas (20 por cento) tiveram identificação inconclusiva. Mesmo após a descontaminação com álcool 70 por cento e uso de UV, cresceram fungos em duas placas (5,3 por cento) e cocos em outras duas (5,3 por cento). Nas 30 placas colocadas nas CSB durante a TIP, cresceram micobactérias em 10 placas (33,3 por cento), fungos em duas (6,6 por cento), cocos em uma (3,4 por cento) e uma mistura de micobactérias e outro bacilo em uma (3,4 por cento). Não houve crescimento nas placas expostas após descontaminação das CSB com álcool a 70 por cento 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...

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 percent 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 percent 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 37ºC 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 percent), fungi in one (2.6 percent) and bacilli in two (5.3 percent). Among those plates that presented mycobacteria growth, eight (80 percent) were identified as M. tuberculosis and two (20 percent) had inconclusive identification. Even after decontamination with alcohol solution 70 percent and UV radiation, two plates presented fungi growth (5.3 percent) and other two presented cocci growth (5.3 percent). Among 30 plates exposed during IT procedures, there was mycobacteria growth in 10 of them (33.3 percent), fungi in two (6.6 percent), cocci in one (3.4 percent) and one (3.4 percent) mixed mycobacteria and another bacillus. No growth was observed when alcohol solution 70 percent 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...

Cabine de segurança biológica: efeito da luz ultravioleta nas micobactérias / Biological safety cabinet: utraviolet radiation effect on mycobacteria

A cabine de segurança biológica (CSB) é o principal equipamento para efetuar a contenção de aerossóis produzidos nos procedimentos laboratoriais e a descontaminação com lâmpada UV, 15 minutos antes do início das atividades e 15 minutos após utilização da cabine é parte das boas práticas de laboratório. O objetivo deste estudo foi avaliar a ação da lâmpada UV da CSB classe II B2, em diversas espécies de micobactérias e correlacionar com o tempo de exposição. Cepas de referência foram subcultivadas, semeadas e incubadas a 37ºC até produzir turvação compatível com o tubo 1 da escala de MacFarland. Foram semeados 100L de suspensão bacteriana em placas com meio 7H11; as placas foram cobertas parcialmente com papel alumínio e expostas à radiação UV durante 5 e 10 minutos. Após exposição, os papéis foram retirados e as placas incubadas a 37ºC por 30 dias. Todas as placas apresentaram inibição de crescimento de bactérias na porção da placa em que houve exposição direta à radiação UV. Os resultados obtidos mostraram que a prática de utilização da radiação UV por 15 minutos após o uso da cabine e antes de iniciar outra atividade técnica, garante descontaminação adequada da CSB. Esta prática de biossegurança é recomendável para descontaminar a própria CSB e os materiais que são retirados da cabine.