Rapid identification of Mycobacterium tuberculosis in cultures by molecular and immunological methods / Identificação rápida de Mycobacterium tuberculosis em culturas por método molecular e método imunológico

Objective: The study aimed to evaluate molecular and immunological methods and to propose a workflow using them for tuberculosis (TB) diagnosis routine. Methods: A cross-sectional retrospective study was performed, including 121 liquid cultures from a TB laboratory located in the extreme south of Brazil. All cultures were positive for Mycobacterium tuberculosis complex (MTBC) by in-house Polymerase Chain Reaction (PCR) using DNA extracted by the CTAB method (PCR-CTAB) for IS6110 detection. These cultures were subjected to faster tests than this one, the immunological MPT64 assay and the PCR using DNA extracted by thermal lysis method (PCR-TL), and these were evaluated for MTBC identification using PCR-CTAB as a reference method. Results: The sensitivity of MPT64 assay and PCR-TL to identify MTBC in positive cultures by PCR-CTAB were 73.6% (89/121) and 98.3% (119/121), respectively. We proposed a workflow based on the use of MPT64 assay in liquid cultures suggestive of MTBC, and in case of a negative result, we suggest the performance of PCR-TL. The PCR-CTAB is suggested only if faster tests are negative. Conclusions: Methods capable of confirming MTBC in cultures should continue to be standardized, tested, and optimized to meet the ideal requirements of simplicity, quickness, and effectiveness. The molecular and immunological methods evaluated have differences in the execution and detection of MTBC in cultures, but they are rapid tools for laboratory TB diagnosi

Objetivos: O estudo objetivou avaliar métodos molecular e imunológico e propor um fluxo de trabalho utilizando-os para a rotina de diagnóstico da tuberculose (TB). Métodos: Foi realizado um estudo transversal retrospectivo, incluindo 121 culturas líquidas de um laboratório de TB localizado no extremo sul do Brasil. Todas as culturas foram positivas para o complexo Mycobacterium tuberculosis (CMTB) por Reação em Cadeia da Polimerase (PCR) in-house para detecção do IS6110, usando DNA extraído pelo método CTAB (PCR-CTAB). Essas culturas foram submetidas a testes mais rápidos que este, o ensaio imunológico MPT64 e a PCR com DNA extraído pelo método de lise térmica (PCR-LT), e estas foram avaliadas para identificação de CMTB usando PCR-CTAB como método de referência. Resultados: A sensibilidade do ensaio MPT64 e da PCR-LT para identificar o CMTB em culturas positivas pela PCRCTAB foi de 73,6% (89/121) e 98,3% (119/121), respectivamente. Propusemos um fluxo de trabalho baseado no uso do ensaio MPT64 em culturas líquidas sugestivas de CMTB e, em caso de resultado negativo, sugerimos a realização de PCR-LT. Sugere-se a PCR-CTAB apenas se os testes mais rápidos forem negativos. Conclusões: Os métodos capazes de confirmar o CMTB em culturas devem continuar sendo padronizados, testados e otimizados para atender aos requisitos ideais de simplicidade, rapidez e eficácia. Os métodos molecular e imunológico avaliados apresentam diferenças na execução e detecção do CMTB em culturas, mas são ferramentas rápidas para o diagnóstico laboratorial da TB.

Performance of an in-house real-time polymerase chain reaction for identification of Mycobacterium tuberculosis isolates in laboratory routine diagnosis from a high burden setting

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.

Development of a Quantitative Sandwich Enzyme-Linked Immunosorbent Assay for Detecting the MPT64 Antigen of Mycobacterium tuberculosis

PURPOSE: Tuberculosis (TB) is a major infectious disease and is responsible for two million deaths annually. For the identification and quantitation of Mycobacterium tuberculosis (M. tuberculosis), a causative agent of TB, a sandwich enzyme-linked immunosorbent assay (ELISA) against the MPT64 protein of M. tuberculosis, an antigen marker of the M. tuberculosis complex, was developed. MATERIALS AND METHODS: The MPT64 protein was expressed, and anti-MPT64 monoclonal antibodies were prepared. A sandwich ELISA was established using recombinant MPT64 protein and anti-MPT64 monoclonal antibodies. The sandwich MPT64 ELISA was evaluated using reference and clinical mycobacterial strains. RESULTS: The sandwich MPT64 ELISA detected MPT64 protein from 2.1 ng/mL to 250 ng/mL (equivalent to 1.7x10(4) CFU/mL and 2.0x10(6) CFU/mL). All 389 clinical M. tuberculosis isolates tested positive in the sandwich MPT64 ELISA (sensitivity, 100%), and the assay showed no cross reactivity to any tested nontuberculous mycobacterial strain (specificity, 100%). CONCLUSION: The sandwich MPT64 ELISA is a highly sensitive and quantitative test for MPT64 protein, which can identify M. tuberculosis.

The methodology for selecting DNA aptamer to MPT164 protein from Mycobacterium tuberculosis with SELEX technology / 中华微生物学和免疫学杂志

Objective To obtain DNA oligonucleotide aptamers which can specifically bind to MPT64 protein from Mycobacterium tuberculosis by SELEX(systematic evolution of ligands by exponential enrichment)technology. Methods A random ssDNA library with in vitro synthesized 78 nucleotides in length was subiected to 12 rounds of selection by SELEX method against MPT64 protein. The binding ability of the aptamers to the protein was examined by biotin-streptavidin-horseradish peroxidase system. Results The selective system used was as follows:in PCR amplification,annealing temperature was 65℃ and the concentration of Mg2+ was 1.5 mmol/L in optimizing library, and when preparation of ssDNA with asymmetrical PCR amplification, 0.75 mmol/L of Mg2+ was used. When using the plate for ELISA as the substrate for the selection, the pattern of electrophoretic band of PCR product after the tenth round selection became unitary and denser than that of the first round. The binding assay demonstrated that A value at 450 nm of the tenth round increased by 9.18 times as compared with that of the first round. The results showed that the affinities of the aptamers were different. The highest A value at 450 nm was 1.606, and the lowest 0.572. Conclusion A set of aptamers with considerable binding affinity to MPT64 protein are successfully picked out from the initial random DNA library.