Nontuberculous Mycobacteria Isolated from Tuberculosis Suspects in Ibadan, Nigeria

Hindawi Publishing Corporation Journal of Pathogens Volume 2016, Article ID 6547363, 5 pages http://dx.doi.org/10.1155/2016/6547363 Research Article Nontuberculous Mycobacteria Isolated from Tuberculosis Suspects in Ibadan, Nigeria Simeon Idowu Cadmus,1 Bassirou Diarra,2 Brehima Traore,3 Mamoudou Maiga,2 Sophia Siddiqui,2 Anatole Tounkara,2 Olutayo Falodun,4 Wole Lawal,5 Isaac Folurunso Adewole,6 Rob Murphy,7 Dick van Soolingen,8 and Babafemi Taiwo7 1 Tuberculosis and Brucellosis Research Laboratories, Department of Veterinary Public Health & Preventive Medicine, University of Ibadan, Ibadan 200005, Nigeria 2 Project SEREFO (Centre de Recherche et de Formation sur le VIH/Sida et la Tuberculose)/University of Sciences, Technics and Technologies of Bamako (USTTB), Bamako, Mali 3 Centre d’Infectiologie Charles Mérieux, rue Dr. Charles Mérieux, ex-base aérienne, BP E2283, Bamako, Mali 4 Department of Microbiology, University of Ibadan, Ibadan 200005, Nigeria 5 Tuberculosis and Leprosy Division, Oyo State Ministry of Health, Ibadan 200005, Nigeria 6 Department of Obstetrics and Gynaecology, University College Hospital, Ibadan 200005, Nigeria 7 Division of Infectious Disease and Center for Global Health, Northwestern University, 645 North Michigan Avenue, Chicago, IL 60611, USA 8 Diagnostic Laboratory for Bacteriology and Parasitology (BPD), Center for Infectious Disease Research, Diagnostics and Perinatal Screening (IDS), National Institute for Public Health and the Environment (RIVM), P.O. Box 1, 3720 BA Bilthoven, Netherlands Correspondence should be addressed to Simeon Idowu Cadmus; Received 31 October 2015; Accepted 6 March 2016 Academic Editor: Abhineet S. Sheoran Copyright © 2016 Simeon Idowu Cadmus et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In Nigeria, one of the highest tuberculosis (TB) burdened nations, sputum smear microscopy is routinely employed for TB diagnosis at Directly Observed Treatment Short-Course (DOTS) Centers. This diagnostic algorithm does not differentiate Mycobacterium tuberculosis complex (MTC) from nontuberculous mycobacteria (NTM). Between December 2008 and January 2009, consecutive patients diagnosed with TB were screened for inclusion at 10 DOTS centers in Ibadan, Nigeria. To verify Mycobacterium species in patients diagnosed, we cultured and identified mycobacterial isolates using PCR, line probe assay, and spoligotyping techniques. From 48 patients screened, 23 met the inclusion criteria for the study. All the 23 study patients had a positive culture. Overall, we identified 11/23 patients (48%) with MTC only, 9/23 (39%) with NTM only, and 3/23 (13%) with evidence of both MTC and NTM. Strains of MTC identified were Latin American Mediterranean (LAM) genotype (𝑛 = 12), M. africanum (𝑛 = 1), and the genotype family T (𝑛 = 1). Four M. avium-intracellulare-M. scrofulaceum complexes, one M. chelonae complex, one M. abscessus, and one M. intracellulare were identified. Our findings underscore the need to incorporate molecular techniques for more precise diagnosis of TB at DOTS centers to improve clinical outcomes and safe guard public health, particularly in TB endemic countries. 1. Introduction For several years, Nigeria has remained in the league of the highest TB burdened nations of the world and is currently ranked 4th globally [1]. Sputum smear microscopy is routinely employed for TB diagnosis at Directly Observed Treatment Short-Course (DOTS) Centers in Nigeria [2]. The diagnostic algorithm entails interpreting presence of acid fast bacilli in sputum smear microscopy as TB. This algorithm does not differentiate Mycobacterium tuberculosis complex (MTC) from nontuberculous mycobacteria (NTM) [3], which are ubiquitous environmental mycobacteria [4–6], 2 Journal of Pathogens Table 1: Epidemiological profile of patients with positive mycobacterial culture. Number of patients Clinical history ZiehlNeelsen staining Culture on selective agar MTC/NTM (phenotypic speciation) INNO-LiPA test NTM (molecular speciation) Spoligotyping MTC (molecular speciation) 10 New + M. tuberculosis NR + 1 Relapse + NR + 1 New + MAIS complex + 2 1 Relapse + MAIS complex M. intracellulare + 11 1 New + M. tuberculosis M. tuberculosis Unclassified NTM M. tuberculosis Unclassified NTM M. tuberculosis Unclassified NTM 1, 3, 4, 6, 7, 8, 10, 12, 13, 14 5 ND + 9 6 New + Unclassified NTM ND NR 15, 16, 17, 18, 19, 21 1 New + Unclassified NTM NR 20 1 New + Unclassified NTM NR 22 1∗ Relapse − Unclassified NTM NR 23 MAIS complex M. chelonae complex MAIS complex M. abscessus MAIS complex Identity of patients ∗ refers to patient with negative sputum smear but positive X-ray; +/− positive or negative; NR: not required; ND: not determined. MAIS complex: Mycobacterium avium-intracellulare-Mycobacterium scrofulaceum complex. increasingly isolated from immune competent and immune compromised hosts [2, 7, 8]. Also other acid fast bacilli, like Rhodococcus spp. cannot be identified using this approach. Since clinical implications and therapeutic options for NTM differ markedly from those for TB, accurate discrimination of TB from NTM infection is essential to avoid underor overtreatment of either condition, bearing in mind the potential patient care and economic consequences [6, 8]. To explore whether misdiagnosis is a problem in Nigeria, we cultured sputum and conducted molecular characterization of acid fast isolates in patients already diagnosed with TB based on the local diagnostic algorithm at DOTS centers in Ibadan, Southwestern Nigeria. 2. Materials and Methods and examined by fluorescent microscopy. Pellets from each sample were cultured simultaneously on Mycobacteria growth indicator (MGIT) tubes containing oleic acid-albumin-dextrose-catalase and polymyxin-amphotericin B-nalidixic acid-trimethoprim-azlocillin and Middlebrook 7H11 agar medium plates (for morphological identification). The inoculated media were incubated at 37∘ C with 7% CO2 for 6 weeks as previously described [9]. Positive cultures were screened by Ziehl-Neelsen and Gram stain in order to exclude other positive Gram staining bacteria. Observation of colony morphology, recognition of mixed infections (more than one Mycobacterium species), and differentiation of the patients’ cultures into MTC and NTM was carried out through microscopic examination of the growths on the Middlebrook 7H11 culture plates [5]. 2.1. Patient Cohort. This study was conducted at 10 DOTS centers in Ibadan, Southwestern Nigeria, between December 2008 and January 2009. Consecutive patients diagnosed with TB (based on positive smear microscopy or symptoms plus chest X-ray) were screened for inclusion. Those on TB therapy for more than 3 days were excluded. Demographic, clinical, and radiolo (...truncated)