First national survey of Mycobacterium tuberculosis drug resistance, Madagascar, 2005-2006.

SETTING: A national survey of Mycobacterium tuberculosis resistance was conducted for the first time in Madagascar between October 2005 and July 2007. OBJECTIVE: To determine resistance rates among new and previously treated cases of pulmonary tuberculosis. METHODS: In a cluster sampling representative of the general population of the country, 1275 smear-positive tuberculosis patients recruited at 34 sites, 926 new patients and 87 previously treated patients underwent drug susceptibility testing against rifampicin (RMP), isoniazid (INH), streptomycin and ethambutol on Löwenstein-Jensen medium using the indirect proportion method. RESULTS: Resistance among new cases was 6.5% (95%CI 4.9-8) and among previously treated cases it was 11.5% (95%CI 4.8-18.2). Monoresistance among new cases was 5.8% (95%CI 4.2-7.3), mainly to INH (3.7%). Multiresistance to INH and RMP was 0.2% (95%CI 0-0.5) among new cases and 3.4% (95%CI 0-7.2) among previously treated cases. No significant difference was noted with regard to sex or age. CONCLUSION: The rates of resistance among new and previously treated cases remain relatively low in Madagascar.

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."

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."

Bacteriological follow-up of tuberculosis treatment: a comparative study of smear microscopy and culture results at the second month of treatment.

SETTING: Significance of a positive bacillary examination of sputum at 2 months of treatment in relation to the viability of the bacilli and the final treatment result. OBJECTIVE: To compare the results of smear microscopy and sputum culture at the second month of tuberculosis treatment and to follow the progress of the patients. METHODS: Follow-up of 297 patients with smear-positive pulmonary tuberculosis in Madagascar, 152 of whom were smear-positive at 2 months of treatment and 145 smear-negative. The number of bacilli was recorded, as were the culture results and the final outcome of treatment. RESULTS: Among the 152 patients who were smear-positive at the second month, 77 (51%) were culture-negative; there were 12 (8%) treatment failures and four relapses (4.6%). Among the 145 smear-negative patients, 22 (15%) were culture-positive, of which one was a treatment failure (1%). CONCLUSION: The majority of failures and relapses were observed in the group of smear-positive patients. It is important to reinforce the surveillance of these patients in order to reduce the number lost to follow-up. Furthermore, a positive smear microscopy at the end of the second month is not sufficiently specific for early identification of treatment failures. It is preferable to wait until the fifth month, as the great majority of patients who are positive at 2 months achieve cure. The treatment strategy currently recommended in Madagascar is satisfactory.

[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.

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.

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.

[Laboratory network for the microscopic diagnosis of tuberculosis in Madagascar: quality control of slides]. / Réseau des laboratoires de microscopie pour le diagnostic de la tuberculose à Madagascar: contrôle de qualité des lames en 1999.

As part of the National Tuberculosis Program (NTP), a quality control of the slides (search of acido-fast bacilli in the sputa) of the Treatment and Diagnosis Centers (TDC) forming the National Laboratory Network is carried out each year. In 1999, 76 TDC out of the 174 (44%) had been controlled using the method of double reading of the smears. The global concordance of the results in the 76 TDC is satisfactory (98%). Reability was 91% for the positive smears and 92% for the negative smears. A good quality of smears was observed in 53% of the centers. The TDC reliable at 100% for both positive and negative smears were 51 (67%) of which 36 (47%) had also a good quality of smears. Those later were mainly found in Toamasina, Fianarantsoa, Antananarivo and Mahajanga.

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.

Replacement of streptomycin by ethambutol in the intensive phase of tuberculosis treatment: no effect on compliance.

SETTING: Seven tuberculosis clinics in the National Tuberculosis Programme of Madagascar. OBJECTIVE: To compare the treatment efficacy and tolerance of regimens including either streptomycin or ethambutol for patient compliance during initial treatment of smear-positive tuberculosis. DESIGN: The 1023 patients included in the study were randomly divided into two treatment groups-one to receive streptomycin (S), isoniazid (H), rifampicin (R) and pyrazinamide (Z) (SHRZ), and the other to receive EHRZ, where streptomycin was replaced by ethambutol (E). During the 2-month intensive phase, drug delivery was completely supervised. The same 6-month continuation regimen was then given in both groups. Follow-up consisted of a clinical and bacteriological examination at the end of the second, fifth and eighth months. RESULTS: There was no significant difference between the two regimens as regards compliance with treatment, the number of patients lost or who died, or for bacteriological response during the intensive phase. EHRZ was better tolerated. During the continuation phase, the results of the two groups remained comparable, but treatment failures occurred earlier in the patients who had received streptomycin. CONCLUSION: Patient compliance was not better with streptomycin. The ethambutol-containing regimen was as efficient as the other, and better tolerated. There is no argument for preferring streptomycin in the intensive phase of treatment of smear-positive tuberculosis.