Culturing Mycobacteria.

Building upon the foundational research of Robert Koch, who demonstrated the ability to grow Mycobacterium tuberculosis for the first time in 1882 using media made of coagulated bovine serum, microbiologists have continued to develop new and more efficient ways to grow mycobacteria. Presently, all known mycobacterial species can be grown in the laboratory using either axenic culture techniques or in vivo passage in laboratory animals. This chapter provides conventional protocols to grow mycobacteria for diagnostic purposes directly from clinical specimens, as well as in research laboratories for scientific purposes. Detailed protocols used for production of M. tuberculosis in large scale (under normoxic and hypoxic conditions) in bioreactors and for production of obligate intracellular pathogens such as Mycobacterium leprae and "Mycobacterium lepromatosis" using athymic nude mice and armadillos are provided.

Comparación de la técnica serológica ML flow y frotis cutáneas para evaluar la carga bacteriana en pacientes nuevos diagnosticados de Lepra / No disponible

Introducción: La técnica ML Flow es un ensayo inmunocromatográfico que detecta anticuerpos de tipo IgM glicolípido fenólico-I (PGL-1) M. leprae específico. Además de los frotis cutáneos teñidos mediante Ziehl-Neelsen puede ser útil en la clasificación de los pacientes de lepra para administrar el correspondiente tratamiento farmacológico. Objetivo: Este estudio revisa la relación entre los niveles de anticuerpos detectables mediante la técnica serológica semi-cuantitativa ML Flow y la carga bacteriana cuantificada mediante frotis cutáneo. Pacientes y métodos: Se obtuvieron frotis cutáneos de 135 pacientes nuevos diagnosticados de lepra en un servicio dermatológico de referencia en Brasil (registrados como índice bacteriológico – IB) y un resultado con el ML Flow (registrado cuantitativa y semi-cuantitativamente) en el momento de ser admitidos al estudio. Se calcularon la regresión logística y concordancia (índice kappa). Resultados: Los frotis cutáneos resultaron positivos en el 35.9% de los pacientes y el 57% de los pacientes eran seropositivos para el PGL-1. De entre los seropositivos, el 41.6% presentaban 5 o menos de 5 lesiones y el 65.8% presentaba la implicación de más de un nervio periférico. Los frotis resultaron positivos en sólo tres casos seronegativos (5.6%) y negativos en el 41.9% de pacientes seropositivos. Los pacientes con un IB+4 presentaban un OR de 3.3 de ser seropositivos en comparación con los de IB más bajo. Conclusiones: Hay una correlación entre el test serológico y los frotis cutáneos. Por tanto, un test ML Flow puede resultar útil para la clasificación clínica de la lepra, además de los frotis cutáneos que requieren una infraestructura de laboratorio y personal con experiencia (AU)

Introduction. The ML Flow test is an immunochromatographic assay that detects IgM antibodies against M. leprae-specific anti-phenolic glycolipid I (PGL-I). In addition to slit skin smears stained by the Ziehl-Neelsen technique, it can be helpful in the operational classification of leprosy patients for treatment purposes. Objective. This work studied the relationship between antibody levels as detected by semi-quantitative ML Flow serologic test and bacterial load as quantified by slit skin smear. Patients and methods. 135 patients with newly detected leprosy at the reference service in Sanitary Dermatology in Brazil had slit skin smears (registered as bacillary index – BI) and an ML Flow test (registered qualitatively and semi-quantitatively) performed at admission. A logistic regression and agreement measures (kappa index) were calculated. Results. Slit skin smears were positive in 35.9% of patients and 57% of patients were seropositive for PGL-1 antibodies. Among the seropositive patients, 41.6% had five or fewer skin lesions, and 65.8% had more than one peripheral nerve involved. Slit skin smears were positive in only three seronegative patients (5.6%), and negative in 41.9% of seropositive patients. Patients with a BI of 4had an OR of 3.3 for being seropositive in comparison to those with a low BI. Conclusions. There is a correlation between serologic test and slit skin smear results. Therefore, an ML Flow test may become a useful tool in the clinical classification of leprosy, besides slit skin smears, which require a proper laboratory infrastructure and experienced personnel (AU)

Intracellular fate of Mycobacterium leprae in cultured murine macrophages in 24-well trays.

Mycobacterium leprae is an obligate intracellular parasite which grows within mononuclear phagocytes. In clinical cases, M. leprae reaches enormous numbers in the macrophage-rich granulomas of leprosy. Peritoneal macrophages from CBA mice were cultured in Waymouth medium containing fresh horse serum in Costar 3424 trays (24 wells, 16 mm in diameter) each containing 9 x 12 mm cover slips. This medium was supplemented with 0.5 micrograms/ml of cycloheximide. These cells were infected with M. leprae Thai-53 strain obtained by nude mice inoculation. Significant multiplication of the acid-fast bacilli in the macrophages was observed three weeks after inoculation. This experiment showed M. leprae mainly multiplied in cells and not by rephagocytization of M. leprae derived from destroyed cells.

Use of a unit gravity sedimentation chamber for the purification of Mycobacterium leprae.

The present paper summarizes results concerning a mild isolation method of Mycobacterium lepraemurium and M. bovis-BCG from host tissues, and describes the application of such a method in purifying M. leprae (M1) from either infected armadillo tissues or human skin biopsies. This isolation method consists of homogenization, two-phase partition in dextranpolyethylene glycol and finally sedimentation in sucrose gradient using a unit gravity chamber. Such a purified M1 preparation appears to be devoid of host-tissue contaminants as examined by light and electron microscopy as well as by a radioimmuno spot test. The results indicate that the present method is mild enough to allow the purification of M1 from infected host tissues with in vivo conservation of antigenicity and viability of the bacilli.

Energetics (adenosine 5'-triphosphate) of Mycobacterium lepraremurium in diffusion chambers incubated in vitro and in mice.

Adenosine 5'-triphosphate (ATP) measurements and the processing of samples have been refined to a point where the energetics and growth potential of microscopic samples of unwashed host-grown, host-dependent microbes can be investigated. Mycobacterium lepraemurium, the noncultivated agent of murine leprosy, was employed to examine three reports of the slow microscopic growth of this organism in the absence of host cells. A few million bacterial cells were enclosed in Rightsel- and Ito-type diffusion chambers, which were incubated in vitro and in the peritoneal cavities of mice. In the in vitro experiments, a complex medium containing bovine serum and mouse brain extracts, renewed three times a week, did not sustain the energetics of the bacilli. The microscopic counts declined to 72% and the ATP per culture to 9% of the original values. Very different results were obtained from chambers incubated in the peritoneal cavities of mice. The bacterial biomass increased 2.7-fold and the ATP per culture increased 2.5-fold. Because the ATP per cell was 93% of the original, this system is regarded as the first to permit the extracellular growth of a so-called "obligate intracellular microbe." The results obtained with only 1 x 10(6) host-grown cells per assay demonstrate a significant biochemical tool for investigating the growth potential of host-grown microbes during the progression, regression, and therapy of disease.