Clinically important mycobacteria in Guinea-Bissau, West Africa : phenotypic and genetic diversity

Abstract

Tuberculosis (TB) is a major and still increasing health problem in West Africa. In Guinea-Bissau, TB has an estimated annual incidence of 1501100.000. Knowledge on infections caused by nontuberculous mycobacteria, such as Mycobacterium avium complex (MAC), is very limited in Africa, including Guinea-Bissau. M tuberculosis complex isolates (n=229) and MAC isolates (n=28) collected in Guinea-Bissau during 1989 to 1996, from sputum samples from approximately 1000 patients with clinical diagnosis of pulmonary TB, were analysed for phenotypical and genotypical characteristics.

There was a high degree of heterogeneity in the M tuberculosis complex isolates in terms of biochemical properties as compared to what is normal in European isolates. Phenotypically, these isolates were assigned to one of five biovars, ranging within a spectrum of classical M. tuberculosis (biovar 5) to classical M. bovis (biovar 1). Genotypically, the strains could be divided into three groups (A-C), of which group A isolates are proposed to be imported and of European descent, while groups B and C isolates are unique and proposed to originate from West Africa (the Guinea-Bissau family of strains). A detailed genotypic analysis was carried out on 35 isolates from the Guinea-Bissau family. Based on the data obtained, and by comparing corresponding genes in mycobacteria outside the M. tuberculosis complex, it is proposed that the Guinea-Bissau family of strains is a unique branch of the M tuberculosis complex tree in between classical M. tuberculosis and classical M. bovis.

Drug susceptibility testing demonstrated a low rate of resistance to drugs used for TB treatment in Guinea-Bissau. However, studies in vitro of thiacetazone showed that the minimum inhibitory concentration of this drug on M africanum subtype 11 strains (biovar 4) was significantly higher than that for other strains studied. It is concluded that in areas where M africanum subtype II is a prevalent cause of TB, thiacetazone should not be considered for treatment.

M. bovis is known to lack the mtp40 gene. However, other investigators earlier reported two strains of M. bovis possessing the mtp40 gene. A detailed genotypic re-examination of these strains was performed showing that the two isolates, in fact, should be classified as M. africanum rather than M. bovis. This finding further strengthens the proposed evolutionary scenario of the M tuberculosis complex.

The finding of MAC in sputum samples is one of the first reports of MAC in patients from Africa. The results demonstrate the importance of adequate laboratory diagnosis of mycobacteria. In geographic areas where MAC pulmonary infections are common, it is of significance to identify MAC, especially so in patients often incorrectly thought to suffer from drug-resistant TB. The MAC isolates were studied by several molecular methods. The highest discriminatory power was obtained with 16S rRNA sequencing. By this method most of the Guinea-Bissau strains were found to belong to the M. intracellulare branch of the phylogenetic tree of MAC.

In summary, the phenotypic heterogeneity, and the genetic clustering revealed three major clusters allowing the identification of the new Guinea-Bissau family of M tuberculosis complex strains. This will contribute to a better understanding of the epidemiology of these organisms and also their evolution. The incidence of pulmonary MAC infections in patients in Guinea-Bissau, underlines the importance of correct laboratory diagnostic methods, not only for correct treatment, but also for epidemiological surveillance - frequently such MAC infections are incorrectly judged to be treatment failures caused by drug resistant TB complex organisms.