Species and genotypic diversity of non-tuberculous mycobacteria isolated from children investigated for pulmonary tuberculosis in rural Uganda

Abstract

BackgroundSmear microscopy, a mainstay of tuberculosis (TB) diagnosis in developing countries, cannot differentiate M. tuberculosis complex from NTM infection, while pulmonary TB shares clinical signs with NTM disease, causing clinical and diagnostic dilemmas. This study used molecular assays to identify species and assess genotypic diversity of non-tuberculous mycobacteria (NTM) isolates from children investigated for pulmonary tuberculosis at a demographic surveillance site in rural eastern Uganda.MethodsChildren were investigated for pulmonary tuberculosis as part of a TB vaccine surveillance program (2009–2011). Two cohorts of 2500 BCG vaccinated infants and 7000 adolescents (12–18 years) were recruited and followed up for one to two years to determine incidence of tuberculosis. Induced sputum and gastric aspirates were processed by the standard N-acetyl L-cysteine (NALC)-NaOH method. Sediments were cultured in the automated MGIT (Becton Dickson) liquid culture system and incubated at 37°C for at least six weeks. Capilia TB assay was used to classify mycobacteria into MTC and NTM. The GenoType CM/AS assays were performed to identify species while Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR genotyping was used to assess genetic diversity of the strains within each species.ResultsAmong 2859 infants and 2988 adolescents screened, the numbers of TB suspects were 710 and 1490 infants and adolescents respectively. The prevalence of NTM in infant suspects was 3.7% (26/710) (95% CI 2.5–5.2) while that in adolescent suspects was 4.6% (69/1490) (95% CI 3.6–5.8). On culture, 127 isolates were obtained, 103 of which were confirmed as mycobacteria comprising of 95 NTM and eight M. tuberculosis complex. The Genotype CM/AS assay identified 63 of the 95 NTM isolates while 32 remained un-identified. The identified NTM species were M. fortuitum (40 isolates, 63.5%), M. szulgai (9 isolates, 14.3%), M. gordonae (6 isolates, 9.5%), M. intracellulare (3 isolates, 4.7%), M. scrofulaceum (2 isolates, 3.2%), M. lentiflavum (2 isolates, 3.2%), and M. peregrinum (1 isolate, 1.6%). Genotyping did not reveal any clustering in M. intracellulare, M. gordonae and M. szulgai species. M. fortuitum, on the other hand, had two clusters, one with three isolates of M. fortuitum 1 and the other with two isolates of M. fortuitum 2 subspecies. The remaining 35 of the 40 isolates of M. fortuitum had unique fingerprint patterns.ConclusionM. fortuitum is the most common cause of infection by NTM among Infants and adolescents in rural Uganda. There is a varied number of species and genotypes, with minimal clustering within species, suggesting ubiquitous sources of infection to individuals in this community.

Multidrug-Resistant Nontuberculous Mycobacteria Isolated from Cystic Fibrosis Patients

“…ERIC-PCR is considered a fast and economic alternative to PFGE. In a study with 40 Ugandan children and teenagers with pulmonary infection caused by M. fortuitum, ERIC-PCR revealed the presence of a clonal group of three isolates, another group comprised of two isolates, and also 35 distinct profiles (32).…”

Epidemiology of Human Pulmonary Infection with Nontuberculous Mycobacteria

“…Of 1,603 TB suspects, 444 (28%) had pulmonary mycobacteria identified, of whom 375 (85%) had M. tuberculosis complex species and 69 (15%) had NTM (56). Finally, in a surveillance study of children and adolescents in rural Eastern Uganda, including 2,200 individuals identified as pulmonary TB suspects, only eight patients (0.36%) had M. tuberculosis complex species isolated, while 95 patients (4.3%) had NTM isolated (57). Although the above studies did not provide adequate details regarding the proportion of patients who fulfilled criteria for NTM disease, the results suggest that a potentially significant proportion of African TB suspects may have NTM disease, but may not be detected in routine care.…”

Methodological and Clinical Aspects of the Molecular Epidemiology of Mycobacterium tuberculosis and Other Mycobacteria

“…Their presence in the genome of M. tuberculosis was shown to be restricted to transcribed regions of the genome, either intergenic regions of polycistronic operons or nontranslated regions upstream or downstream of open reading frames (388). The variability of ERIC sequences allowed the proposal of a new typing method, ERIC-PCR typing, which has been applied for estimating the genetic diversity of many mycobacterial species, including M. tuberculosis (388), M. gordonae (389,390), M. intracellulare, M. szulgai, M. fortuitum (173,390), M. chelonae, and M. abscessus (391).…”