Spatiotemporal co-existence of two Mycobacterium ulcerans clonal complexes in the Offin River Valley of Ghana

In recent years, comparative genome sequence analysis of African Mycobacterium ulcerans strains isolated from Buruli ulcer (BU) lesion specimen has revealed a very limited genetic diversity of closely related isolates and a striking association between genotype and geographical origin of the patients. Here, we compared whole genome sequences of five M. ulcerans strains isolated in 2004 or 2013 from BU lesions of four residents of the Offin river valley with 48 strains isolated between 2002 and 2005 from BU lesions of individuals residing in the Densu river valley of Ghana. While all M. ulcerans isolates from the Densu river valley belonged to the same clonal complex, members of two distinct clonal complexes were found in the Offin river valley over space and time. The Offin strains were closely related to genotypes from either the Densu region or from the Asante Akim North district of Ghana. These results point towards an occasional involvement of a mobile reservoir in the transmission of M. ulcerans, enabling the spread of bacteria across different regions

TB-diabetes co-morbidity in Ghana : the importance of Mycobacterium africanum infection

Diabetes Mellitus (DM) is a known risk factor for tuberculosis (TB) but little is known on TB-Diabetes Mellitus (TBDM) co-morbidity in Sub-Saharan Africa.; Consecutive TB cases registered at a tertiary facility in Ghana were recruited from September 2012 to April 2016 and screened for DM using random blood glucose and glycated hemoglobin (HbA1c) level. TB patients were tested for other clinical parameters including HIV co-infection and TB lesion location. Mycobacterial isolates obtained from collected sputum samples were characterized by standard methods. Associations between TBDM patients' epidemiological as well as microbiological variables were assessed.; The prevalence of DM at time of diagnosis among 2990 enrolled TB cases was 9.4% (282/2990). TBDM cases were significantly associated with weight loss, poor appetite, night sweat and fatigue (p<0.001) and were more likely (p<0.001) to have lower lung cavitation 85.8% (242/282) compared to TB Non-Diabetic (TBNDM) patients 3.3% (90/2708). We observed 22.3% (63/282) treatment failures among TBDM patients compared to 3.8% (102/2708) among TBNDM patients (p<0.001). We found no significant difference in the TBDM burden attributed by M. tuberculosis sensu stricto (Mtbss) and Mycobacterium africanum (Maf) and (Mtbss; 176/1836, 9.6% and Maf; 53/468, 11.3%, p = 0.2612). We found that diabetic individuals were suggestively likely to present with TB caused by M. africanum Lineage 6 as opposed to Mtbss (odds ratio (OR) = 1.52; 95% confidence interval (CI): 0.92-2.42, p = 0.072).; Our findings confirms the importance of screening for diabetes during TB diagnosis and highlights the association between genetic diversity and diabetes. in Ghana

Mycobacterium africanum is associated with patient ethnicity in Ghana

Mycobacterium africanum is a member of the Mycobacterium tuberculosis complex (MTBC) and an important cause of human tuberculosis in West Africa that is rarely observed elsewhere. Here we genotyped 613 MTBC clinical isolates from Ghana, and searched for associations between the different phylogenetic lineages of MTBC and patient variables. We found that 17.1% (105/613) of the MTBC isolates belonged to M. africanum, with the remaining belonging to M. tuberculosis sensu stricto. No M. bovis was identified in this sample. M. africanum was significantly more common in tuberculosis patients belonging to the Ewe ethnic group (adjusted odds ratio: 3.02; 95% confidence interval: 1.67-5.47, p>0.001). Stratifying our analysis by the two phylogenetic lineages of M. africanum (i.e. MTBC Lineages 5 and 6) revealed that this association was mainly driven by Lineage 5 (also known as M. africanum West Africa 1). Our findings suggest interactions between the genetic diversity of MTBC and human diversity, and offer a possible explanation for the geographical restriction of M. africanum to parts of West Africa

Schematic illustration of TSP assay performance.

<p>NAS and LS primer locations relative to the <i>M. ulcerans</i> DNA sequence surrounding a SNP locus are shown for the different PCR reaction phases. <b>A</b> Initial PCR conditions enable an amplification of the larger LS PCR product by applying an annealing temperature (Ta) of 58°C. <b>B</b> Reduction of Ta to 45°C facilitates a possible incorporation of the NAS primer into the enriched LS PCR product. <b>C</b> Competitive amplification of the larger LS and the smaller NAS PCR products are ensured by an increase of Ta to 53°C.</p

Summary of <i>M. ulcerans</i> genotyping in Ghana.

1<p>genotyping after comparison of 3 <i>M. ulcerans</i> genomes.</p>2<p>genotyping after comparison of 7 <i>M. ulcerans</i> genomes.</p

Optimization of TSP SNP typing assays.

<p>TSP endpoint detection by analysis of PCR product sizes on ethidium bromide-stained agarose gels for haplotypes 1–10 (lanes 1–10) at TSP assay locus 6. TSPs were performed using: <b>A</b> a four-primer system including both NAS and LS forward and reverse primers as well as a three-primer system with only one NAS primer and different NAS∶LS primer ratios of <b>B</b> 2.5∶1, <b>C</b> 5∶1, <b>D</b> 7.5∶1 and <b>E</b> 10∶1.</p

Application of TSP assay 16 in two different research laboratories.

<p>Comparison of TSP endpoint detection by analysis of PCR product sizes on ethidium bromide-stained agarose gels in two different laboratories in Basel, Switzerland and Accra, Ghana. PCR products are shown for haplotypes 1–10 (lanes 1–10).</p

Selection of TSP SNP typing assays.

<p><b>A</b> Linearized phylogenetic tree of the ten <i>M. ulcerans</i> haplotypes (HT1–10) detected in the Densu River Valley of Ghana (MEGA software version 4.1 (beta), scale: number of differences at the 89 SNP loci tested). <b>B</b> Schematic overview of reference (0) and SNP (1) alleles present in the sequence of the ten <i>M. ulcerans</i> haplotypes, which can be identified by TSP assays 1, 3, 4, 6, 8, 9, 15, 16, 17 and 18.</p

Isolation of nontuberculous Mycobacteria from the environment of Buruli ulcer endemic communities in Ghana

This study aimed to isolate nontuberculous mycobacterial species from environmental samples obtained from some selected communities in Ghana. To optimize decontamination, spiked environmental samples were used to evaluate four decontamination solutions and supplemented media, after which the best decontamination solution and media were used for the actual analysis. The isolates obtained were identified on the basis of specific genetic sequences, including heat shock protein 65, IS2404, IS2606, rpoB, and the ketoreductase gene, as needed. Among the methods evaluated, decontamination with 1 M NaOH followed by 5% oxalic acid gave the highest rate of recovery of mycobacteria (50.0%) and the lowest rate of contamination (15.6%). The cultivation medium that supported the highest rate of recovery of mycobacteria was polymyxin B-amphotericin B-nalidixic acid-trimethoprim-azlocillin-supplemented medium (34.4%), followed by isoniazid-supplemented medium (28.1%). Among the 139 samples cultivated in the main analysis, 58 (41.7%) yielded mycobacterial growth, 70 (50.4%) had no growth, and 11 (7.9%) had all inoculated tubes contaminated. A total of 25 different mycobacterial species were identified. Fifteen species (60%) were slowly growing (e.g., Mycobacterium ulcerans, Mycobacterium avium, Mycobacterium mantenii, and Mycobacterium malmoense), and 10 (40%) were rapidly growing (e.g., Mycobacterium chelonae, Mycobacterium fortuitum, and Mycobacterium abscessus). The occurrence of mycobacterial species in the various environmental samples analyzed was as follows: soil, 16 species (43.2%); vegetation, 14 species (38.0%); water, 3 species (8.0%); moss, 2 species (5.4%); snail, 1 species (2.7%); fungi, 1 species (2.7%). This study is the first to report on the isolation of M. ulcerans and other medically relevant nontuberculous mycobacteria from different environmental sources in Ghana.; Diseases caused by mycobacterial species other than those that cause tuberculosis and leprosy are increasing. Control is difficult because the current understanding of how the organisms are spread and where they live in the environment is limited, although this information is needed to design preventive measures. Growing these organisms from the environment is also difficult, because the culture medium becomes overgrown with other bacteria that also live in the environment, such as in soil and water. We aimed to improve the methods for growing these organisms from environmental sources, such as soil and water samples, for better understanding of important mycobacterial ecology

OMNIgene SPUTUM: A good transport and decontaminating reagent for tuberculosis testing

Background: Sputum culture is limited to centralized facilities. Thus, samples require transportation from peripheral laboratories to these facilities, compromising specimen quality since it is difficult to maintain cold chain. We evaluated OMNIgene SPUTUM Reagent (OMS) for transporting sputum samples for tuberculosis (TB) testing. The study was carried out at Noguchi Memorial Institute for Medical Research using sputa from Korle Bu Teaching Hospital and La General Hospital in Ghana. Methods: In a laboratory-based controlled experiment (CE), sputum contaminants were determined on blood agar before treatment with OMS and N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH). TB testing included smear microscopy, culture, and Xpert MTB/RIF. Afterward, two peripheral laboratories were trained to transport sputum samples with OMS without cold chain. Positivity, negativity, and contamination rates were compared between both methods using Chi-square and Fisher's exact tests. Cohen's Kappa was also used to determine agreements. Results: Among 104 sputum samples analyzed in the CE, 93 (89.4%) had bacterial growth on blood agar before decontamination, while 6 (5.8%) and 5 (4.8%) contaminated after NALC-NaOH and OMS treatment, respectively. Contamination was high with NALC-NaOH (12.8%) than OMS (4.3%) on Lowenstein–Jensen media (P < 0.001), but mycobacterial positivity was comparable: NALC-NaOH of 74.5% and OMS of 78.7%. Smear positivity after NALC-NaOH treatment was 89.4% and OMS was 75.9% (P = 0.491). All except one of the samples tested positive by Xpert MTB/RIF after both treatment. Sixteen samples were evaluated in the field experiment and 81.3% yielded positive culture, and no contamination on LJ was observed. Conclusion: Our findings indicate that OMS works well as a transport and decontaminating reagent of samples for TB testing