Mycobacteria mobility shift assay: a method for the rapid identification of Mycobacterium tuberculosis and nontuberculous mycobacteria

The identification of mycobacteria is essential because tuberculosis (TB) and mycobacteriosis are clinically indistinguishable and require different therapeutic regimens. The traditional phenotypic method is time consuming and may last up to 60 days. Indeed, rapid, affordable, specific and easy-to-perform identification methods are needed. We have previously described a polymerase chain reaction-based method called a mycobacteria mobility shift assay (MMSA) that was designed for Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) species identification. The aim of this study was to assess the MMSA for the identification of MTC and NTM clinical isolates and to compare its performance with that of the PRA-hsp65 method. A total of 204 clinical isolates (102 NTM and 102 MTC) were identified by the MMSA and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing fragments of the 16S rRNA and hsp65 genes. Both methods correctly identified all MTC isolates. Among the NTM isolates, the MMSA alone assigned 94 (92.2%) to a complex or species, whereas the PRA-hsp65 method assigned 100% to a species. A 91.5% agreement was observed for the 94 NTM isolates identified by both methods. The MMSA provided correct identification for 96.8% of the NTM isolates compared with 94.7% for PRA-hsp65. The MMSA is a suitable auxiliary method for routine use for the rapid identification of mycobacteria.

Evaluación de una técnica de hibridación reversa para identificación rápida de micobacterias en Chile / Evaluation of a reverse hybridization assay for the identification of Mycobacterium in Chile

Objective: Identification for Mycobacterium assay based in the new technology of reverse hybridization DNA probe assay was evaluated (Line Probe Assays-LiPAs). Methods: 74 strains belonging to 23 mycobacterial species or complex classified previously by classical biochemical methods, genetic probes and PRA (patterns of restriction analysis), with and without specific pattern expected to be identified at specie level were analysed.The utilized test, GenoType CM (Hain Lifescience, Nehren, Alemania), is able of identifying 14 of the most common mycobacterial species after a multiplex PCR technique targeting a 23S rRNA gene region followed by reverse hybridization technology. Results: Sensitivity of 94.0 percent (95 percent CI: 84.4-98.0 percent) and specificity of 88.0 percent (95 percent CI:46.7-99.3 percent) were obtained with the assay. Conclusion: GenoType CM is an appropriated tool for the identification of Mycobacteria, rapid, sensitive, operational in the current working conditions of the National Reference Laboratory of Mycobacteria in Chile and it might constitute a real breakthrough for shortening the time delay in the procedure, providing a better opportunity to use treatment only in cases where it is required.

Objetivos: Se evalúa una técnica para la identificación de micobacterias basada en la nueva tecnología de hibridación en tiras con sondas (Line Probe Assays-LiPAs). Métodos: Se analizaron 74 cepas, correspondientes a 23 especies y/o complejos, preclasificadas mediante pruebas bioquímicas tradicionales, sondas genéticas y PRA (análisis de patrones de restricción), identificables y no identi-ficables a nivel de especie por el kit utilizado. El kit evaluado, GenoType CM (Hain Lifescience, Nehren, Alemania), permite la identificación genética molecular de 14 de las especies micobacterianas más comunes, mediante una PCR múltiple e hibridación reversa del producto en tiras con sondas de regiones genéticas de ARNr de 23S. Resultados: Con la utilización de este ensayo para identificación de Micobacterias se obtuvo 94,0 por ciento(CI95 por ciento 84,4-98,0) de sensibilidad y 88,0 por ciento (CI95 por ciento 46,7-99,3) de especificidad totales. Conclusiones: Se concluye que GenoType CM constituye una herramienta adecuada para la identificación de micobacterias, rápida, sensible, operativa en las actuales condiciones de trabajo del Laboratorio de Referencia Nacional de Micobacterias en Chile y que podría constituir un avance para el acortamiento en los tiempos que demora el proceso, lo que implica una mejor oportunidad de aplicación de tratamiento sólo en los casos en que éste sea requerido.

Distribution and Clinical Significance of Nontuberculous Mycobacteria Identified by High Performance Liquid Chromatography in Clinical Specimens / 대한임상미생물학회지

BACKGROUND: Infections caused by nontuberculous mycobacteria (NTM) are significantly increasing over the last decade. Due to the uncertainty in the clinical significance of these organisms, their effective diagnosis and treatment has been challenging. The purpose of this study was to investigate the distribution and clinical significance of NTM in clinical specimens. METHODS: Acid-fast culture positive 3,107 clinical specimens were identified by mycolic acid analysis using high performance liquid chromatography (HPLC.) The HPLC patterns of 384 NTM strains were compared with those of standard mycobacterium species. Clinical significance of NTM was investigated by a retrospective study including acid-fast stain and culture, medical history, symptoms and signs, radiological and other laboratory findings, pathologic findings, response to treatment, and follow-up study, and was confirmed according to the guideline of American Thoracic Society. RESULTS: Among the 3,107 Mycobacterium-positive specimens, 384 (12.4%) were found to be positive for NTM. Of these, 367 (95.6%) were successfully identified by HPLC as 19 different species, each of which comprising 0.3% to 15.9% of the total NTM, Studies on the pathogenic role of NTM showed that 0~79.6% of each species or 0~100% of isolates from each specimen could be considered clinically significant. CONCLUSION: HPLC method is highly discriminative for the identification of NTM in clinical specimens. When NTM is isolated from clinical specimens in the Ulsan area, the findings from this study could serve as a database on which to determine its clinical significance depending on species type and also specimen type.

Identification of Mycobacteria using High Performance Liquid Chromatography in Clinical Specimens / 대한임상미생물학회지

BACKGROUND: As tuberculous and nontuberculous mycobacterial infections are increasing, it is very important to differentiate the myobacterial species. High performance liquid chromatography (HPLC) method has been proven to be a useful technique for the identification of mycobacteria. The purpose of this study was to investigate the identification rate using HPLC and to know nontuberculous mycobacterial distribution in Ulsan University Hospital. METHOD: Mycobacteria grew in 959 clinical specimens, which were analyzed by HPLC, and their distribution was reviewed by retrospective studies. RESULTS: The patterns of HPLC were divided into single, double, and triple cluster groups which consist of 9, 20, and 4 species of mycobacteria respectively. The identification rate of mycobacteria by HPLC was 98.9%, And the rate of nontuberculous mycobacteria in mycobacterial culture positive specimens was 12.2%. CONCLUSION: HPLC is an excellent tool for mycobacterial identification. And the culture rate of nontuberculous mycobacteria in clinical specimens is increasing in Korea.