Herimanana Ramarokoto

African 1, an Epidemiologically Important Clonal Complex of Mycobacterium bovis Dominant in Mali, Nigeria, Cameroon, and Chad

We have identified a clonal complex of Mycobacterium bovis present at high frequency in cattle in population samples from several sub-Saharan west-central African countries. This closely related group of bacteria is defined by a specific chromosomal deletion (RDAf1) and can be identified by the absence of spacer 30 in the standard spoligotype typing scheme. We have named this group of strains the African 1 (Af1) clonal complex and have defined the spoligotype signature of this clonal complex as being the same as the M. bovis BCG vaccine strain but with the deletion of spacer 30. Strains of the Af1 clonal complex were found at high frequency in population samples of M. bovis from cattle in Mali, Cameroon, Nigeria, and Chad, and using a combination of variable-number tandem repeat typing and spoligotyping, we show that the population of M. bovis in each of these countries is distinct, suggesting that the recent mixing of strains between countries is not common in this area of Africa. Strains with the Af1-specific deletion (RDAf1) were not identified in M. bovis isolates from Algeria, Burundi, Ethiopia, Madagascar, Mozambique, South Africa, Tanzania, and Uganda. Furthermore, the spoligotype signature of the Af1 clonal complex has not been identified in population samples of bovine tuberculosis from Europe, Iran, and South America. These observations suggest that the Af1 clonal complex is geographically localized, albeit to several African countries, and we suggest that the dominance of the clonal complex in this region is the result of an original introduction into cows naïve to bovine tuberculosis.

Variation in gamma interferon responses to different infecting strains of Mycobacterium tuberculosis in acid-fast bacillus smear-positive patients and household contacts in Antananarivo, Madagascar.

ABSTRACT The majority of healthy individuals exposed to Mycobacterium tuberculosis will not develop tuberculosis (TB), though many may become latently infected. More precise measurement of the human immune response to M. tuberculosis infection may help us understand this difference and potentially identify those subjects most at risk of developing active disease. Gamma interferon (IFN-γ) production has been widely used as a proxy marker to study infection and to examine the human immune response to specific M. tuberculosis antigens. It has been suggested that genetically distinct M. tuberculosis strains may invoke different immune responses, although how these differences influence the immune responses and clinical outcome in human tuberculosis is still poorly understood. We therefore evaluated the antigen-specific IFN-γ production responses in peripheral blood mononuclear cells from two cohorts of subjects recruited in Antananarivo, Madagascar, from 2004 to 2006 and examined the influence of the infecting M. tuberculosis strains on this response. The cohorts were sputum-positive index cases and their household contacts. Clinical strains isolated from the TB patients were typed by spoligotyping. Comparison of the IFN-γ responses with the spoligotype of the infecting clinical strains showed that “modern” M. tuberculosis strains, like Beijing and Central Asian (CAS) strains, tended to induce lower IFN-γ responses than “ancient” strains, like East African-Indian (EAI) strains, in index cases and their household contacts. These results suggest that new strains may have evolved to induce a host response different from that of ancient strains. These findings could have important implications in the development of therapeutic and diagnostic strategies.

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

ABSTRACT 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.

Validation of an immunochromatographic assay kit for the identification of the Mycobacterium tuberculosis complex

The performance of the immunochromatographic assay, SD BIOLINE TB Ag MPT64 RAPID®, was evaluated in Madagascar. Using mouse anti-MPT64 monoclonal antibodies for rapid discrimination between the Mycobacterium tuberculosis complex and nontuberculous mycobacteria, the kit was tested on mycobacteria and other pathogens using conventional methods as the gold standard. The results presented here indicate that this kit has excellent sensitivity (100%) and specificity (100%) compared to standard biochemical detection and can be easily used for the rapid identification of M. tuberculosis complex.

Tuberculin reactivity in first‐year schoolchildren in Madagascar

Objective  The tuberculin skin test (TST) is an important tool in the diagnosis of tuberculosis infection in children. However, the interpretation of TST may be complicated by prior Bacillus Calmette‐Guerin (BCG) vaccination. We evaluated the effect of vaccination with BCG on TST reactivity in first‐year pupils attending state schools in Antananarivo.

RFLP clusters of Mycobacterium tuberculosis strains from the Indian Ocean Region: local and South Asian characteristics

This is the first study describing the genetic polymorphism of Mycobacterium tuberculosis strains in the Indian Ocean Region. Using IS6110 RFLP analysis, 475 M. tuberculosis isolates from Madagascar, Comoros, Mauritius, Mozambique and La Reunion were compared. Of the 332 IS6110 profiles found, 43 were shared by clusters containing 2-65 strains. Six clusters were common to at least two countries. Of 52 families of strains with similar IS6110 profiles, 10 were common to at least two countries. Interestingly, another characteristic was the frequency (16.8%) of IS6110 single-copy strains. These strains could be distinguished using the DR marker. This preliminary evaluation suggests genetic similarity between the strains of the Indian Ocean Region. However, additional markers would be useful for epidemiological studies and to assess the ancient transmission of strains between countries of this region.

Increase in the number of tuberculosis cases treated following tuberculin skin testing in first-year schoolchildren in Madagascar.

Background Tuberculosis continues to cause unacceptably high levels of disease and death worldwide. Active preventive strategies are required to improve tuberculosis control and to increase the number of cases treated in developing countries. The aim of this study was to evaluate the utility of the tuberculin skin test (TST) in first-year schoolchildren as a means of increasing the number of tuberculosis cases detected through the screening of close contacts. Methods All members of the households of 90 schoolchildren assigned to three groups on the basis of TST category (≤5 mm, [5–15)mm, ≥15 mm) were screened for sputum smear-positive pulmonary tuberculosis. The percentage detection of tuberculosis in close contacts was compared between TST categories. Results We identified 433 close contacts of the 90 schoolchildren, who were then evaluated for tuberculosis. We identified 11 cases of pulmonary tuberculosis among the close contacts (7 already on treatment and 4 previously undiagnosed): 0 in TST category ≤5 mm, 3 in TST category [5–15) mm and 8 in TST category ≥15 mm). This approach increased the detection of tuberculosis cases by a factor of 1.6 in first-year schoolchildren of the TST ≥5 mm group. Conclusion TST in first-year schoolchildren is a potentially effective method for improving the detection of tuberculosis in close contacts.