Evaluation of a rapid culture method on liquid Bio FM (BIO-RAD) medium for the isolation of mycobacteria.

SETTING: The search continues for a simple, rapid culture system for the isolation of Mycobacterium tuberculosis in low-income countries. Bio FM (BIO-RAD) medium is an enriched 7H9 medium optimised for the growth of mycobacteria and contains a chromogenic indicator. OBJECTIVE: To evaluate the detection rate and time to detection of mycobacteria using the Bio FM system in comparison with the Löwenstein-Jensen (LJ) medium method routinely used in our laboratory. MATERIALS AND METHODS: A total of 270 pulmonary and 178 extra-pulmonary samples were cultured in parallel on Bio FM and LJ media. The mycobacterial detection rate and time were compared. RESULTS: The mycobacteria detection rate on Bio FM and LJ were not significantly different (respectively 97.9% and 93.15%, P > 0.05). The growth of M. tuberculosis was faster on Bio FM (mean 12.42 days [3-41] vs. 20.7 [10-48] days for LJ, P < 10(-6)). CONCLUSION: In our study, the culture method on liquid Bio FM medium was faster, but the detection rate was not better than with solid LJ medium.

Evaluation of the resazurin assay for the detection of multidrug-resistant Mycobacterium tuberculosis in Madagascar.

SETTING: Multidrug-resistant (MDR) tuberculosis (TB) can jeopardise the success of national TB control programmes. Rapid, simple drug susceptibility tests applicable in developing countries would allow earlier treatment of patients with MDR infections. OBJECTIVE: To test the feasibility and performance of the resazurin microtitre assay (REMA) as an indirect test for detecting isoniazid (INH) and rifampicin (RMP) resistance of Mycobacterium tuberculosis strains in Madagascar. DESIGN: Study comparing the sensitivity and specificity of the REMA plate test with the Löwenstein-Jensen proportion method for determining the resistance of M. tuberculosis strains to INH and RMP. RESULTS: The sensitivity and specificity of the resazurin test were studied in 77 strains and were respectively 95% and 97.3% for the detection of INH resistance, and 95% and 100% for the detection of RMP resistance. The sensitivity and specificity for the identification of MDR strains were respectively 89% and 100%. CONCLUSION: The resazurin test is sensitive and specific enough for the detection of INH- and RMP-resistant strains. It is also easy to use, rapid and inexpensive, making it suitable for developing countries. Its usefulness for national drug resistance surveys should be assessed.

[A case of pulmonary multiresistant Mycobacterium bovis tuberculosis in Madagascar]. / Un cas de tuberculose pulmonaire à Mycobacterium bovis multirésistant à Madagascar.

We report a chronic case of pulmonary tuberculosis in a Malagasy citizen from Antsohihy (West of Madagascar), who was infected with a multi-drug resistant Mycobacterium bovis strain. This is the first case reported of the isolation of such a strain in Madagascar.

La tuberculose chez les enfants de moins de 11 ans : resistance primaire et variants genetiques dominants de Mycobacterium tuberculosis a Antananarivo

"Childhood tuberculosis : primary resistance and genotypes of dominant Mycobacterium tuberculosis in Antananarivo"" : Tuberculosis during childhood is often due to Mycobacterium tuberculosis primo-infection. Tuberculosis is highly prevalent in Madagascar and most people are infected during childhood. Our objectives were to evaluate the primary resistance of M. tuberculosis and to determine the genotypes responsible for recent infection in the population. Thus we studied 142 isolated strains from 97 children (66 with pulmonary tuberculosis and 31 with extra-pulmonary tuberculosis) recruited in different health centers in Antananarivo from 1997 to 2000. Excepting one strain resistant to isoniazide; all strains were susceptible to the four antibiotics (streptomycin; isoniazid; ryfampicin and ethambutol). This result confirms the low rate of primaryresistance reported during the two surveys in 1994-1995 and 1999-2000. 67 strains of 1997-2000 were typed with the genetic marker IS6110; 44 has been assigned to 13clusters containing each 2 to 8 similar strains. Some IS6110 clusters have already been reported in 1994-1995. Some genotypes observed in 1994-1995 seemed to have disappeared in 1997-2000. (As the rate of the frequency of some genetic variants according to the period are more likely due to a difference in strain virulence). Since there is minimal antibiotic resistance versus M. tuberculosis in Madagascar; one can not explain the appearence or disappearence of certain variants because of drug resistance. Rather; this is due to the virulence of the various M. tuberculosis strains."

[Tuberculosis in children less than 11 years old: primary resistance and dominant genetic variants of Mycobacterium tuberculosis in Antananarivo]. / La tuberculose chez les enfants de moins de 11 ans: résistance primaire et variants génétiques dominants de Mycobacterium tuberculosis à Antananarivo.

Tuberculosis during childhood is often due to Mycobacterium tuberculosis primo-infection. Tuberculosis is highly prevalent in Madagascar and most people are infected during childhood. Our objectives were to evaluate the primary resistance of M. tuberculosis and to determine the genotypes responsible for recent infection in the population. Thus we studied 142 isolated strains from 97 children (66 with pulmonary tuberculosis and 31 with extra-pulmonary tuberculosis) recruited in different health centers in Antananarivo from 1997 to 2000. Excepting one strain resistant to isoniazide, all strains were susceptible to the four antibiotics (streptomycin, isoniazid, ryfampicin and ethambutol). This result confirms the low rate of primary resistance reported during the two surveys in 1994-1995 and 1999-2000. 67 strains of 1997-2000 were typed with the genetic marker IS6110, 44 has been assigned to 13 clusters containing each 2 to 8 similar strains. Some IS6110 clusters have already been reported in 1994-1995. Some genotypes observed in 1994-1995 seemed to have disappeared in 1997-2000. (As the rate of the frequency of some genetic variants according to the period are more likely due to a difference in strain virulence). Since there is minimal antibiotic resistance versus M. tuberculosis in Madagascar, one can not explain the appearance or disappearance of certain variants because of drug resistance. Rather, this is due to the virulence of the various M. tuberculosis strains.

[Central inter-laboratory quality control for microscopic diagnosis of tuberculosis in Madagascar in 2000]. / Contrôle de qualité inter-laboratoires centraux pour le diagnostic microscopique de la tuberculose à Madagascar en 2000.

Since June 1997, a quarterly quality control of sputum smear exam for the tubercle diagnosis, depending on double reading of slides, was implemented between both central laboratories of the Mycobacteria National Reference Centre in Madagascar (mycobactoria laboratories of Institut Pasteur Madagascar [IPM] and Institut Hygiène Sociale [IHS]--Health Ministry). In 2000, four controls were done, in the course of which 240 slides were coloured by auramine, coming both from IPM and IHS, and another 80 slides from IHS were coloured by Ziehl-Neelsen. All the results were in agreement for the samples stained with auramine, while two false negatives were found for the samples stained with Ziehl-Neelsen. The maintenance of this quality control between the two laboratories is necessary to insure the reliability of their results and the controls that they make for the peripheral laboratories.

[Mycobacterium tuberculosis resistance to antitubercular agents in Antananarivo in 2000]. / Résistance de Mycobacterium tuberculosis aux antituberculeux à Antananarivo en 2000.

In 1991, the National Tuberculosis control Program (NTP) of Madagascar adopted the short treatment course and the Directly Observed Treatment Strategy (DOTS), according to the recommendations of the OMS/UICTMR. Development of M. tuberculosis primary resistance to the four antituberculosis drugs (streptomycin [S], rifampicine [R], isoniazid [H], ethambutol [E]) is an indicator of the NTP efficiency. We report results from a five-year survey among patients with new smear positive pulmonary tuberculosis. Acquired resistance is assessed among recurrent cases. During the first survey, carried out in 1994-1995 in four large cities, multidrug resistance (MDR) rate to the major antituberculosis drug H and R was low, 0.25% for primary MDR and 5% for acquired MDR. No primary MDR was found in Antananarivo; on the other hand, acquired resistance rate was the highest there (22%). Because of logistical reasons, the second survey (1999-2000) was only carried out in the capital, Antananarivo. Results obtained among 789 new patients with smear positive pulmonary tuberculosis and 79 recurrents cases in 9 diagnostic centres showed low primary and acquired resistance of 11.1% to any drug. Primary resistance to one drug was 10.6%, mainly due to streptomycin 8.5%. MDR rates are comparable with those observed in 1994-1995: 0.1% for primary MDR and 4% for acquired MDR. These results show that ten years after the new NTP implementation, only a few MDR strains are circulating in Antananarivo, which suggests that NTP has been effective.

Predominance of serotype-specific mucosal antibody response in Shigella flexneri-infected humans living in an area of endemicity.

The mucosal humoral immune response elicited following Shigella flexneri infection in patients living in Antananarivo districts (Madagascar Island) was evaluated by measuring the gut-derived, circulating immunoglobulin A (IgA) antibody-secreting cells (ASC) specific for the major bacterial antigen lipopolysaccharide (LPS). Fifty, 34, 11, and 5% of the S. flexneri-positive patients were infected with serotypes 2a, 1a, 4a, and 3a, respectively. The total number of IgA ASC in infected patients increased significantly, compared to the number in healthy controls, early after the onset of disease. The number of anti-homologous LPS IgA ASC varied among individuals and peaked between days 5 and 10 after the onset of the disease. In the S. flexneri 1a- and 2a-infected patients, the level of IgA ASC cross-reactivity to heterologous S. flexneri serotypes was weak. These data indicate that S. flexneri 2a and 1a are the predominant strains responsible for shigellosis in this area of endemicity and that the anti-LPS antibody response following natural infection is mainly directed against serotype-specific determinants.

A combination of two genetic markers is sufficient for restriction fragment length polymorphism typing of Mycobacterium tuberculosis complex in areas with a high incidence of tuberculosis.

The incidence of tuberculosis (TB) in Madagascar is 150 cases per 100,000 people. Because of this endemicity, we studied the genetic diversity of Mycobacterium tuberculosis strains isolated in four big cities in 1994 to 1995 with the aim of monitoring TB transmission. Isolates from 316 cases of pulmonary TB (PTM(+)) were typed by Southern hybridization with genetic markers IS6110 and DR. Of the 316 PTM(+) strains, 66 (20.8%) had a single IS6110 band and were differentiated by the DR marker into 33 profiles. Using both markers, 37.7% (119) of the patients were clustered, a proportion similar to that in countries with a high prevalence of TB. There was no significant difference between clustered and nonclustered patients in age, sex, Mycobacterium bovis BCG status, and drug susceptibility of strains. Clustering was significantly greater in the capital, Antananarivo, than in the other cities, suggesting a higher rate of transmission. However, most of the patients in clusters were living in different areas, and, within a distance of 0.7 km, we did not find epidemiologically unrelated strains with the same restriction fragment length polymorphism profile. Despite an apparently low polymorphism, genetic markers such as IS6110 are potentially valuable for monitoring TB transmission. However, the high proportion of Malagasy isolates with a single IS6110 copy makes this marker alone unsuitable for typing. Additional markers such as DR are necessary for the differentiation of the isolates and for epidemiological surveys.

[Detection of leptospirosis reservoirs in Madagascar using the polymerase chain reaction technique]. / Recherche de réservoirs de la leptospirose à Madagascar par la technique d'amplification génique.

A polymerase chain reaction (PCR) technique was used for detection of the Leptospira interrogans rrs gene in kidney tissue from 115 rats, 50 zebu cattles and 13 pigs in an attempt to identify a possible animal reservoir of leptospirosis in Madagascar. In addition, serological testing of 105 individuals in close contact with animals was carried out. The PCR analysis was negative for all the samples tested and only one person was found seropositive at a low titer. The findings suggest that leptospirosis, if prevalent in Madagascar, is likely rare.

[Diagnosis of tuberculosis by immunocapture of the tuberculous bacillus (using magnetic beads)]. / Diagnostic de la tuberculose par immunocapture du bacille tuberculeux (sur billes magnétiques).

Tuberculosis is worldwide considered as a major health problem with high morbidity and mortality rates. Diagnosis of tuberculosis can be problematic. Microscopy, as the basic diagnostic method, stands inadequately alone due to a low sensitivity, and culture suffers from being time-consuming. A rapid, sensitive and simple diagnostic test, applicable in the field is therefore highly needed. A diagnostic method for the detection of M. tuberculosis by immunocapture technique has been developed using magnetic beads coated with polyclonal anti-M. tuberculosis. The detection of captured bacilli using biotinylated anti-APA monoclonal antibody (APA is a minor secreted antigen) was found more sensitive than microscopy. The results suggest that the development of a rapid strip test to detect major antigen could be a useful tool for the control of tuberculosis.

Transmission of tuberculosis in the prison of Antananarivo (Madagascar).

The prevalence of tuberculosis in the Antananarivo prison is 16 times higher than that in the general population of Madagascar. We compared the clustering of Mycobacterium tuberculosis strains within and outside the prison and studied the transmission of strains in the prison. M. tuberculosis strains isolated in 1994 to 1995 from 146 prisoners and from 260 nonprisoner patients from Antananarivo were typed using the genetic markers IS6110 and direct repeat. We compared the strains isolated from prisoners and nonprisoners and found that the clustering rate was higher within (58.9%) than outside the prison (40%) suggesting that the transmission rate was higher in prison. Of the 146 incarcerated patients, 82 were grouped into 22 clusters. We checked for possible tuberculosis transmission between prisoners with identical strains by epidemiological investigation of the various prison clusters. We found that 9.5% of the incarcerated patients could have been sources of infection and that only 15.1% could have been infected in the prison. One hundred and twenty-seven prison patients were new cases. Epidemiological data suggested that 37% of them resulted from a reactivation of an old infection, due to poor living conditions or recent transmission from an index case outside the prison.

Prevalence of Mycobacterium bovis in human pulmonary and extra-pulmonary tuberculosis in Madagascar.

The prevalence of human tuberculosis (TB) due to Mycobacterium bovis was determined in Madagascar in 1994-1995. A prevalence of M. bovis of 1.25% was observed among sputum smear-positive patients and 1.3% among extra-pulmonary TB patients. This study was conducted in urban areas and will be extended to rural zones, where the majority of the population lives.

[Genetic polymorphism of M. tuberculosis strains in Antananaviro]. / Polymorphisme génetique des souches du complexe M. tuberculosis à Antananarivo.

The genetic polymorphism of the mycobacteria of the tuberculosis complex in the city of Antananarivo was studied on 126 strains isolated from positive microscopy pulmonary tuberculosis patients. The genetic profiles established using the RFLP technic and the IS6110 marker yielded 83 clusters of 1 to 29 strains. There were 34 strains with a IS6110 unique band profile of which 29 had a band located at 1.4-1.5 kb. These strains could be differentiated using a second marker, the DR marker. 3 strains with an unique IS6110 band located at 1.8-1.9 kb were identified as M. bovis. In general, there was no evident epidemiological relationship between the patients presenting with identical profiles. In the prison of Antananarivo, the IS6110 typing of 36 strains yielded 28 clusters of 1 to 3 strains. Excepting 2 clusters showing an internal contamination, the absence of profiles specific to the jail suggests that the patients were probably contaminated before their entrance. This preliminary study shows that the RFLP profiles of M. tuberculosis, using the IS6110 and the DR markers, were polymorphic enough for using this method to study the transmission in Antananarivo.