Deciphering the growth behaviour of Mycobacterium africanum.

BACKGROUND: Human tuberculosis (TB) in West Africa is not only caused by M. tuberculosis but also by bacteria of the two lineages of M. africanum. For instance, in The Gambia, 40% of TB is due to infections with M. africanum West African 2. This bacterial lineage is associated with HIV infection, reduced ESAT-6 immunogenicity and slower progression to active disease. Although these characteristics suggest an attenuated phenotype of M. africanum, no underlying mechanism has been described. From the first descriptions of M. africanum in the literature in 1969, the time to a positive culture of M. africanum on solid medium was known to be longer than the time to a positive culture of M. tuberculosis. However, the delayed growth of M. africanum, which may correlate with the less virulent phenotype in the human host, has not previously been studied in detail. METHODOLOGY/PRINCIPAL FINDINGS: We compared the growth rates of M. tuberculosis and M. africanum isolates from The Gambia in two liquid culture systems. M. africanum grows significantly slower than M. tuberculosis, not only when grown directly from sputa, but also in growth experiments under defined laboratory conditions. We also sequenced four M. africanum isolates and compared their whole genomes with the published M. tuberculosis H37Rv genome. M. africanum strains have several non-synonymous SNPs or frameshift mutations in genes that were previously associated with growth-attenuation. M. africanum strains also have a higher mutation frequency in genes crucial for transport of sulphur, ions and lipids/fatty acids across the cell membrane into the bacterial cell. Surprisingly, 5 of 7 operons, recently described as essential for intracellular survival of H37Rv in the host macrophage, showed at least one non-synonymously mutated gene in M. africanum. CONCLUSIONS/SIGNIFICANCE: The altered growth behaviour of M. africanum might indicate a different survival strategy within host cells

Evaluation of the Kudoh method for mycobacterial culture: Gambia experience

AbstractObjective/backgroundTo evaluate the Kudoh swab method for improving laboratory diagnosis of tuberculosis (TB) in Gambia.MethodsA total of 75 sputa (50 smear positive and 25 smear negative) were examined. Sputum samples were collected from leftover routine samples from the Medical Research Council Unit, Gambia TB Diagnostic Laboratory. The samples were processed using the standard N-acetyl-l-cysteine-NaOH (NALC-NaOH) methods currently used and Kudoh swab method. These were cultured on standard Lowenstein Jensen (LJ) and Modified Ogawa media, respectively, and incubated aerobically at 36±1°C for mycobacterial growth. To determine if the decontamination and culture methods compared could equally detect the Mycobacterium tuberculosis complex (MTBC) highly commonly isolated in Gambia, spoligotyping was done.ResultsIn total, 72% (54/75) of MTBC were recovered by both LJ and Modified Ogawa methods. The LJ method recovered 52% (39/75) and Modified Ogawa recovered 56% (42/75) of the MTBC, respectively. Spoligotyping showed Euro-American 35% (19/54), Indo-Oceanic 35% (19/54), Mycobacterium africanum (West African type 2) 26% (14/54), Beijing 2% (1/54), and M. africanum (West African type 1) 2% (1/54).ConclusionThe Kudoh method is simpler and cheaper than the NALC-NaOH method. There was no significant difference in recovery between the methods. The Kudoh method is ideal in overburdened TB laboratories with poor resources in developing countries. The predominant lineages were Euro-American and Indo-Oceanic, followed by M. africanum (West African type 2)

Learning from epidemiological, clinical, and immunological studies on Mycobacterium africanum for improving current understanding of host-pathogen interactions, and for the development and evaluation of diagnostics, host-directed therapies, and vaccines for tuberculosis

Mycobacterium africanum comprises two phylogenetic lineages within the Mycobacterium tuberculosis complex (MTBC). M. africanum was first described and isolated in 1968 from the sputum of a Senegalese patient with pulmonary tuberculosis (TB) and it has been identified increasingly as an important cause of human TB, particularly prevalent in West Africa. The restricted geographical distribution of M. africanum, in contrast to the widespread global distribution of other species of MTBC, requires explanation. Available data indicate that M. africanum may also have important differences in transmission, pathogenesis, and host-pathogen interactions, which could affect the evaluation of new TB intervention tools (diagnostics and vaccines)-those currently in use and those under development. The unequal geographical distribution and spread of MTBC species means that individual research findings from one country or region cannot be generalized across the continent. Thus, generalizing data from previous and ongoing research studies on MTBC may be inaccurate and inappropriate. A major rethink is required regarding the design and structure of future clinical trials of new interventions. The West, Central, East, and Southern African EDCTP Networks of Excellence provide opportunities to take forward these pan-Africa studies. More investments intomolecular, epidemiological, clinical, diagnostic, and immunological studies across the African continent are required to enable further understanding of host-M. africanum interactions, leading to the development of more specific diagnostics, biomarkers, host-directed therapies, and vaccines for TB. (C) 2016 The Author(s). Published by Elsevier Ltd on behalf of International Society for Infectious Diseases. This is an open access article under the CC BY-NC-ND license

Pulmonary non-tuberculous mycobacteria in colonisation and disease in The Gambia

The clinical relevance of pulmonary non-tuberculous mycobacteria (PNTM) in The Gambia is unknown. The aim of this study was to estimate the prevalence of non-tuberculous mycobacteria (NTM) in colonisation, and the burden of clinically relevant pulmonary NTM (PNTM) disease in The Gambia. This was a cross-sectional study of the prevalence of NTM in participants aged ≥ 15 years, in a nationwide tuberculosis (TB) prevalence survey between December 2011 and January 2013. We enrolled 903 participants with suspected NTM and NTM cultures were confirmed by 16S rRNA gene sequencing analyses. We applied the American Thoracic Society/Infectious Disease Society of America (ATS/IDSA) diagnostic criteria to determine clinical relevance of NTM. A total of 575 participants had acid-fast bacilli (AFB) positive Mycobacterial Growth Indicator Tube (MGIT) cultures and 229 (39.8%) were NTM. M. avium complex was by far the most isolated NTM (71.0%), followed by M. fortuitum (9.5%) and M. nonchromogenicum (2.9%). Older participants (> 24 years old) were four times more likely to have NTM in their sputa. Only 20.5% (9/44) NTM cases met the ATS/IDSA criteria for NTM disease. This study provides important data on the prevalence of NTM in pulmonary samples of suspected TB cases with AFB positive cultures from a nationally representative population in The Gambia. Enhanced PNTM surveillance is recommended to better understand the contribution of NTM to pulmonary disease

Pulmonary non-tuberculous mycobacteria in colonisation and disease in The Gambia

The clinical relevance of pulmonary non-tuberculous mycobacteria (PNTM) in The Gambia is unknown. The aim of this study was to estimate the prevalence of non-tuberculous mycobacteria (NTM) in colonisation, and the burden of clinically relevant pulmonary NTM (PNTM) disease in The Gambia. This was a cross-sectional study of the prevalence of NTM in participants aged ≥ 15 years, in a nationwide tuberculosis (TB) prevalence survey between December 2011 and January 2013. We enrolled 903 participants with suspected NTM and NTM cultures were confirmed by 16S rRNA gene sequencing analyses. We applied the American Thoracic Society/Infectious Disease Society of America (ATS/IDSA) diagnostic criteria to determine clinical relevance of NTM. A total of 575 participants had acid-fast bacilli (AFB) positive Mycobacterial Growth Indicator Tube (MGIT) cultures and 229 (39.8%) were NTM. M. avium complex was by far the most isolated NTM (71.0%), followed by M. fortuitum (9.5%) and M. nonchromogenicum (2.9%). Older participants (> 24 years old) were four times more likely to have NTM in their sputa. Only 20.5% (9/44) NTM cases met the ATS/IDSA criteria for NTM disease. This study provides important data on the prevalence of NTM in pulmonary samples of suspected TB cases with AFB positive cultures from a nationally representative population in The Gambia. Enhanced PNTM surveillance is recommended to better understand the contribution of NTM to pulmonary disease

A cross-sectional study of tuberculosis drug resistance among previously treated patients in a tertiary hospital in Accra, Ghana: public health implications of standardized regimens.

BACKGROUND: Mycobacterium tuberculosis drug resistance is a major challenge to the use of standardized regimens for tuberculosis (TB) therapy, especially among previously treated patients. We aimed to investigate the frequency and pattern of drug resistance among previously treated patients with smear-positive pulmonary tuberculosis at the Korle-Bu Teaching Hospital Chest Clinic, Accra. METHODS: This was a cross-sectional survey of mycobacterial isolates from previously treated patients referred to the Chest Clinic Laboratory between October 2010 and October 2013. The Bactec MGIT 960 system for mycobactrerial culture and drug sensitivity testing (DST) was used for sputum culture of AFB smear-positive patients with relapse, treatment failure, failure of smear conversion, or default. Descriptive statistics were used to summarize patient characteristics, and frequency and patterns of drug resistance. RESULTS: A total of 112 isolates were studied out of 155 from previously treated patients. Twenty contaminated (12.9%) and 23 non-viable isolates (14.8%) were excluded. Of the 112 studied isolates, 53 (47.3%) were pan-sensitive to all first-line drugs tested Any resistance (mono and poly resistance) to isoniazid was found in 44 isolates (39.3%) and any resistance to streptomycin in 43 (38.4%). Thirty-one (27.7%) were MDR-TB. Eleven (35.5%) out of 31 MDR-TB isolates were pre-XDR. MDR-TB isolates were more likely than non-MDR isolates to have streptomycin and ethambutol resistance. CONCLUSIONS: The main findings of this study were the high prevalence of MDR-TB and streptomycin resistance among previously treated TB patients, as well as a high prevalence of pre-XDR-TB among the MDR-TB patients, which suggest that first-line and second-line DST is essential to aid the design of effective regimens for these groups of patients in Ghana

Whole-genome sequencing illuminates the evolution and spread of multidrug-resistant tuberculosis in Southwest Nigeria.

Nigeria has an emerging problem with multidrug-resistant tuberculosis (MDR-TB). Whole-genome sequencing was used to understand the epidemiology of tuberculosis and genetics of multi-drug resistance among patients from two tertiary referral centers in Southwest Nigeria. In line with previous molecular epidemiology studies, most isolates of Mycobacterium tuberculosis from this dataset belonged to the Cameroon clade within the Euro-American lineage. Phylogenetic analysis showed this clade was undergoing clonal expansion in this region, and suggests that it was involved in community transmission of sensitive and multidrug-resistant tuberculosis. Five patients enrolled for retreatment were infected with pre-extensively drug resistant (pre-XDR) due to fluoroquinolone resistance in isolates from the Cameroon clade. In all five cases resistance was conferred through a mutation in the gyrA gene. In some patients, genomic changes occurred in bacterial isolates during the course of treatment that potentially led to decreased drug susceptibility. We conclude that inter-patient transmission of resistant isolates, principally from the Cameroon clade, contributes to the spread of MDR-TB in this setting, underscoring the urgent need to curb the spread of multi-drug resistance in this region

Evolution of Mycobacterium tuberculosis complex lineages and their role in an emerging threat of multidrug resistant tuberculosis in Bamako, Mali

In recent years Bamako has been faced with an emerging threat from multidrug resistant TB (MDR-TB). Whole genome sequence analysis was performed on a subset of 76 isolates from a total of 208 isolates recovered from tuberculosis patients in Bamako, Mali between 2006 and 2012. Among the 76 patients, 61(80.3%) new cases and 15(19.7%) retreatment cases, 12 (16%) were infected by MDR-TB. The dominant lineage was the Euro-American lineage, Lineage 4. Within Lineage 4, the Cameroon genotype was the most prevalent genotype (n = 20, 26%), followed by the Ghana genotype (n = 16, 21%). A sub-clade of the Cameroon genotype, which emerged ~22 years ago was likely to be involved in community transmission. A sub-clade of the Ghana genotype that arose approximately 30 years ago was an important cause of MDR-TB in Bamako. The Ghana genotype isolates appeared more likely to be MDR than other genotypes after controlling for treatment history. We identified a clade of four related Beijing isolates that included one MDR-TB isolate. It is a major concern to find the Cameroon and Ghana genotypes involved in community transmission and MDR-TB respectively. The presence of the Beijing genotype in Bamako remains worrying, given its high transmissibility and virulence

Phenotypic and genotypic monitoring of Schistosoma mansoni in Tanzanian schoolchildren five years into a preventative chemotherapy national control programme

We conducted combined in vitro PZQ efficacy testing with population genetic analyses of S. mansoni collected from children from two schools in 2010, five years after the introduction of a National Control Programme. Children at one school had received four annual PZQ treatments and the other school had received two mass treatments in total. We compared genetic differentiation, indices of genetic diversity, and estimated adult worm burden from parasites collected in 2010 with samples collected in 2005 (before the control programme began) and in 2006 (six months after the first PZQ treatment). Using 2010 larval samples, we also compared the genetic similarity of those with high and low in vitro sensitivity to PZQ

A Mycobacterial Perspective on Tuberculosis in West Africa: Significant Geographical Variation of M. africanum and Other M. tuberculosis Complex Lineages.

BACKGROUND: Phylogenetically distinct Mycobacterium tuberculosis lineages differ in their phenotypes and pathogenicity. Consequently, understanding mycobacterial population structures phylogeographically is essential for design, interpretation and generalizability of clinical trials. Comprehensive efforts are lacking to date to establish the West African mycobacterial population structure on a sub-continental scale, which has diagnostic implications and can inform the design of clinical TB trials. METHODOLOGY/PRINCIPAL FINDINGS: We collated novel and published genotyping (spoligotyping) data and classified spoligotypes into mycobacterial lineages/families using TBLineage and Spotclust, followed by phylogeographic analyses using statistics (logistic regression) and lineage axis plot analysis in GenGIS, in which a phylogenetic tree constructed in MIRU-VNTRplus was analysed. Combining spoligotyping data from 16 previously published studies with novel data from The Gambia, we obtained a total of 3580 isolates from 12 countries and identified 6 lineages comprising 32 families. By using stringent analytical tools we demonstrate for the first time a significant phylogeographic separation between western and eastern West Africa not only of the two M. africanum (West Africa 1 and 2) but also of several major M. tuberculosis sensu stricto families, such as LAM10 and Haarlem 3. Moreover, in a longitudinal logistic regression analysis for grouped data we showed that M. africanum West Africa 2 remains a persistent health concern. CONCLUSIONS/SIGNIFICANCE: Because of the geographical divide of the mycobacterial populations in West Africa, individual research findings from one country cannot be generalized across the whole region. The unequal geographical family distribution should be considered in placement and design of future clinical trials in West Africa