Evaluación del método de Petroff modificado con solución salina para el diagnóstico de micobacterias en el sistema Bact/Alert 3D / Assessment of Petroff method modified with saline solution for the diagnosis of mycobacteria in Bact/Alert 3D system

Introducción: Existen diferentes métodos de descontaminación de muestras pulmonares para el diagnóstico de micobacterias. El Programa Nacional de Control de Tuberculosis recomienda el método de Petroff modificado con solución salina, pero no existen evidencias documentadas que avalen este método. Objetivo: Evaluar el método de Petroff modificado con solución salina para el diagnóstico de micobacterias en el sistema Bact/Alert 3D. Métodos: Se realizó un estudio observacional analítico de pruebas diagnósticas utilizando 100 muestras pulmonares recibidas en el Laboratorio Nacional de Referencia e Investigaciones de Tuberculosis, Lepra y Micobacterias del Instituto Pedro Kourí, abril 2016 enero 2017. La muestra se dividió en 3 alícuotas y se descontaminaron mediante 3 métodos; luego se inocularon en los medios de cultivo sólido y líquido. Se compararon los resultados del cultivo en cuanto: tiempo de detección de crecimiento, tasa de contaminación, por ciento de positividad, además se calcularon indicadores de desempeño. Resultados: Al comparar el método Petroff modificado con solución salina con el Petroff modificado con solución fosfato en Löwenstein Jensen, el tiempo de detección de crecimiento, por ciento de positividad y la tasa de contaminación se comportaron de forma similar y la sensibilidad (93,75 por ciento), concordancia (96,47 por ciento) e índice de Youden (0,91) fueron elevadas. Al compararlo el Petroff modificado con solución salina con el N-Acetil-L-Cisteína, las variables no mostraron diferencias significativas y los Indicadores de Desempeño se comportaron por encima del 93 por ciento, para el medio sólido y líquido. Conclusiones: Los resultados avalan la continuidad del uso del Petroff modificado con solución salina como método de descontaminación de las muestras pulmonares en la red de laboratorios de Cuba y como alternativa en el pretratamiento de las muestras para el medio líquido (Bact/Alert 3D), además constituye un soporte para el Programa Nacional de Control de Tuberculosis(AU)

Introduction: There are different decontamination methods of pulmonary samples for the diagnosis of mycobacteria. The National Program for the Control of Tuberculosis recommends Petroff method modified with saline solution; but there are not documented evidences that endorse it. Objective: Assess Petroff method modified with saline solution for the diagnosis of mycobacteria in Bact / Alert 3D system. Methods: An observational analytic study of diagnostic tests was conducted; there were used 100 pulmonary samples received in the National Laboratory of References and Researches of Tuberculosis, Leprosy and Mycobacteria of Pedro Kourí Institute, from April 2016 to January 2017. The sample was divided in 3 aliquots and those were decontaminated using 3 methods; then, they were inoculated in the solid and liquid culture means. Cultures´ results were compared according to: growth's detection time, contamination rate, percent of positivity; in addition, performance indicators were calculated. Results: When comparing Petroff method modified with saline solution with Petroff method modified with phosphate solution in Löwenstein Jensen, the growth's detection time, the percent of positivity and the rate of contamination behaved similarly, and sensitivity (93,75percent), concordance (96,47percent) and Youden´s index (0,91) were high. When the Petroff method modified with saline solution was compared with N-Acetil-L- Cisteina, the variables did not show significative differences and the behaviour indicators were over 93percent for the solid and liquid mean. Conclusions: The results endorse the continuity of the use of Petroff method modified with saline solution as a decontamination method of pulmonary samples in the network of Cuban laboratories and as alternative to the pre-treatment of the samples for the liquid mean (Bact/Alert 3D); it also constitutes a support for the National Program for the Control of Tuberculosis(AU)

Isolation of Nontuberculous Mycobacteria from the Environment of Ghanian Communities Where Buruli Ulcer Is Endemic.

UNLABELLED: This study aimed to isolate nontuberculous mycobacterial species from environmental samples obtained from some selected communities in Ghana. To optimize decontamination, spiked environmental samples were used to evaluate four decontamination solutions and supplemented media, after which the best decontamination solution and media were used for the actual analysis. The isolates obtained were identified on the basis of specific genetic sequences, including heat shock protein 65, IS2404, IS2606, rpoB, and the ketoreductase gene, as needed. Among the methods evaluated, decontamination with 1 M NaOH followed by 5% oxalic acid gave the highest rate of recovery of mycobacteria (50.0%) and the lowest rate of contamination (15.6%). The cultivation medium that supported the highest rate of recovery of mycobacteria was polymyxin B-amphotericin B-nalidixic acid-trimethoprim-azlocillin-supplemented medium (34.4%), followed by isoniazid-supplemented medium (28.1%). Among the 139 samples cultivated in the main analysis, 58 (41.7%) yielded mycobacterial growth, 70 (50.4%) had no growth, and 11 (7.9%) had all inoculated tubes contaminated. A total of 25 different mycobacterial species were identified. Fifteen species (60%) were slowly growing (e.g., Mycobacterium ulcerans, Mycobacterium avium, Mycobacterium mantenii, and Mycobacterium malmoense), and 10 (40%) were rapidly growing (e.g., Mycobacterium chelonae, Mycobacterium fortuitum, and Mycobacterium abscessus). The occurrence of mycobacterial species in the various environmental samples analyzed was as follows: soil, 16 species (43.2%); vegetation, 14 species (38.0%); water, 3 species (8.0%); moss, 2 species (5.4%); snail, 1 species (2.7%); fungi, 1 species (2.7%). This study is the first to report on the isolation of M. ulcerans and other medically relevant nontuberculous mycobacteria from different environmental sources in Ghana. IMPORTANCE: Diseases caused by mycobacterial species other than those that cause tuberculosis and leprosy are increasing. Control is difficult because the current understanding of how the organisms are spread and where they live in the environment is limited, although this information is needed to design preventive measures. Growing these organisms from the environment is also difficult, because the culture medium becomes overgrown with other bacteria that also live in the environment, such as in soil and water. We aimed to improve the methods for growing these organisms from environmental sources, such as soil and water samples, for better understanding of important mycobacterial ecology.

Distribution of environmental mycobacteria in Karonga District, northern Malawi.

The genus Mycobacterium includes many species that are commonly found in the environment (in soil and water or associated with plants and animals), as well as species that are responsible for two major human diseases, tuberculosis (Mycobacterium tuberculosis) and leprosy (Mycobacterium leprae). The distribution of environmental mycobacteria was investigated in the context of a long-term study of leprosy, tuberculosis, Mycobacterium bovis BCG vaccination, and the responses of individuals to various mycobacterial antigens in Karonga District, northern Malawi, where epidemiological studies had indicated previously that people may be exposed to different mycobacterial species in the northern and southern parts of the district. A total of 148 soil samples and 24 water samples were collected from various locations and examined to determine the presence of mycobacteria. The detection method involved semiselective culturing and acid-fast staining, following decontamination of samples to enrich mycobacteria and reduce the numbers of other microorganisms, or PCR with primers specific for the mycobacterial 16S rRNA gene, using DNA extracted directly from soil and water samples. Mycobacteria were detected in the majority of the samples, and subsequent sequence analysis of PCR products amplified directly from soil DNA indicated that most of the products were related to known environmental mycobacteria. For both methods the rates of recovery were consistently higher for dry season samples than for wet season samples. All isolates cultured from soil appeared to be strains of Mycobacterium fortuitum. This study revealed a complex pattern for the environmental mycobacterial flora but identified no clear differences between the northern and southern parts of Karonga District.