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.

Controle de qualite inter-laboratoires centraux pour le diagnostic microscopique de la tuberculose a Madagascar en 2000

"Tuberculosis diagnosis by microscopy in Madagascar : quality control comparison of the two central laboratories"" : 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 Hygiene 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."

Un cas de tuberculose pulmonaire a Mycobacterium bovis multiresistant a Madagascar

"A case of pulmonary multiresistant tuberculosis (Mycobacterium bovis) in 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."

[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.

Validity of intradermal tuberculin testing for the screening of bovine tuberculosis in Madagascar.

A sample survey with the objective of determining the prevalence of bovine tuberculosis by means of an intradermal tuberculin test was conducted in Madagascar and it was found that the prevalence rate varied from 0-30% by veterinary district. In order to estimate the true prevalence, the validity of the test was investigated by assessing its sensitivity and specificity in two groups of animals from two different regions, which were destined for slaughter. In the first group where the probability of non-infected animals should have been the highest, sensitivity was estimated at 0.52 (n = 21) and specificity at 0.99 (n = 79). In the second group selected on the basis of apparent ill health of the animals in a high-prevalence bovine tuberculosis area, sensitivity was estimated at 0.8 (n = 10) and specificity at 1 (n = 12). The results obtained from both groups of cattle were not combined for statistical purposes because the sensitivity of the skin test seemed to fluctuate in relation to the chronicity of the disease. These fluctuations are discussed. However, since the first group of zebu cattle was more representative of the cattle population across the country as a whole, its results were retained as operational parameters for further screening.

45/47 kilodalton (APA) antigen capture and antibody detection assays for the diagnosis of tuberculosis.

SETTING: APA complex (45/47 kDa) is an antigen specifically excreted by Mycobacterium tuberculosis and could therefore be a good candidate for diagnosis. OBJECTIVES: To develop three APA immunocapture ELISA assays using monoclonal antibodies (Mabs) and one IgG anti-APA ELISA test, and to determine their usefulness for the diagnosis of tuberculosis in Madagascar. DESIGN: For the Ag assays, 23 negative sputum and serum samples and 64 pairs of sputum and serum from active smear-positive patients (PTM+) were tested. For antibody assay, 116 negative controls, 143 PTM+ and 54 extra-pulmonary tuberculosis patients were tested. RESULTS: The sensitivities of the APA antigen detection assays were low (less than 40%) for a specificity of 95.6%, using either monoclonal antibodies or clinical specimens. The anti-APA serology was more sensitive (76.9% for PTM+ patients) but less specific (73.2%). Due to their poor predictive values, these tests cannot be recommended for the routine diagnosis of tuberculosis in Madagascar.

Predictive values of the ICT Tuberculosis test for the routine diagnosis of tuberculosis in Madagascar.

The rapid commercialised ICT Tuberculosis test has been tested in Madagascar for the diagnosis of smear-positive pulmonary (SM+) and extra-pulmonary tuberculosis (EPT), using microscopy, culture and histopathology as reference tests. Specificity was 83.3% and sensitivity 68.2% for SM+ patients and 65.2% for EPT patients. With a prevalence of 22.2% for SM+ patients and 52.4% for confirmed EPT among consulting patients in the National Reference Laboratory, the ICT Tuberculosis assay was not sufficiently predictive for application in the tuberculosis control programme.

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.

Anti-tuberculosis drug resistance in Madagascar in 1994-1995.

SETTING: A new tuberculosis control programme has been implemented in Madagascar since 1991. A survey on Mycobacterium tuberculosis resistance to the major drugs was conducted between August 1994 and December 1995. OBJECTIVE: To determine primary and acquired resistance in pulmonary tuberculosis patients in four main cities. DESIGN: Were included 401 randomly sampled new smear positive patients (36.2% of declared new patients) and 137 recurrent cases (72.9% of declared cases) from 8 centres. Drug susceptibility testing was performed on Löwenstein Jensen medium according to the proportion method. RESULTS: The male to female ratio was 1.35:1 in new patients (age range 11-74 years) and 1.98:1 in recurrent patients (age range 16-76 years). The primary resistance rate to any drug was 20% (95% Confidence Interval [CI] 16-23) and the acquired resistance rate 40% (95% CI 32-48, P < 2.10(-7). Primary resistance to one drug was 18% (95% CI 15-22), mainly attributable to streptomycin resistance (14.5%). Multidrug resistance (MDR) to isoniazid and rifampicin was 0.25% (95% CI 0-0.7) for primary resistance and 5% (95% CI 2.6-10.6) for secondary resistance. No difference was observed between sexes or ages. CONCLUSION: This survey conducted in big cities gives a very negative picture of resistance in Madagascar.

[Evolution of the Mycobacteria Laboratory of the Pasteur Institute of Madagascar from 1991 to 1994]. / Evolution du Laboratoire des Mycobactéries de l'Institut Pasteur de Madagascar de 1991 à 1994.

In 1991, the Laboratory of Mycobacteria was a small laboratory, part of the Clinical Biology Centre (CBC) of the Institut Pasteur de Madagascar: 656 pathological samples have been analysed for the account of the CBC and the National Control Programme activities. Within 4 years, the number of samples tested increased by more than threefold and the technical ability has evolved in an important way, specially for the identification and the antibiotic sensitivity testing. The scientific equipment have been modernized and the rooms surface increased by fourfold. In 1995, this laboratory was officially designated as the National Reference Laboratory for the culture, the identification and antibiogramme for the account of the National Control programme and for the private clinicians. It also participates to the tuberculosis research programmes of Institut Pasteur de Madagascar. It is associated to the Laboratory of Mycobacteria in the Institut d'Hygiène Sociale of Antananarivo which is the National Reference Laboratory for the bacilloscopy, the teaching and the supervision of the peripheral laboratories.