Mycobacterium Tuberculosis Complex Lineages Research Articles

Bacterial diversity dominates variable macrophage responses of tuberculosis patients in Tanzania

The Mycobacterium tuberculosis complex (MTBC) comprises nine human-adapted lineages that differ in their geographical distribution. Local adaptation of specific MTBC genotypes to the respective human host population has been invoked in this context. We aimed to assess if bacterial genetics governs MTBC pathogenesis or if local co-adaptation translates into differential susceptibility of human macrophages to infection by different MTBC genotypes. We generated macrophages from cryopreserved blood mononuclear cells of Tanzanian tuberculosis patients, from which the infecting MTBC strains had previously been phylogenetically characterized. We infected these macrophages ex vivo with a phylogenetically similar MTBC strain (“matched infection”) or with strains representative of other MTBC lineages (“mismatched infection”). We found that L1 infections resulted in a significantly lower bacterial burden and that the intra-cellular replication rate of L2 strains was significantly higher compared the other MTBC lineages, irrespective of the MTBC lineage originally infecting the patients. Moreover, L4-infected macrophages released significantly greater amounts of TNF-α, IL-6, IL-10, MIP-1β, and IL-1β compared to macrophages infected by all other strains. While our results revealed no measurable effect of local adaptation, they further highlight the strong impact of MTBC phylogenetic diversity on the variable outcome of the host–pathogen interaction in human tuberculosis.

A first insight into tuberculosis transmission at the border of Ecuador and Colombia: a retrospective study of the population structure of Mycobacterium tuberculosis in Esmeraldas province.

Tuberculosis (TB) is a major public health concern in Ecuador and Colombia, considering that both countries are high-burden TB settings. Molecular epidemiology is crucial to understand the transmission dynamics of Mycobacterium tuberculosis complex (MTBC) and to identify active transmission clusters of regional importance. We studied the potential transmission of TB between Colombia and Ecuador through the analysis of the population structure of MTBC lineages circulating in the Ecuadorian province of Esmeraldas at the border with Colombia. A total of 105 MTBC strains were characterized by 24-loci MIRU-VNTR and spoligotyping. MTBC lineage 4 is only present in Esmeraldas; no MTBC strains belonging to Lineage 2-sublineage Beijing were found despite its presence in other provinces of Ecuador and, in Colombia. Genotyping results revealed a high degree of diversity for MTBC in Esmeraldas: Neither active transmission clusters within this province nor including MTBC strains from Colombia or other provinces of Ecuador were found. Our data suggest that tuberculosis dynamics in this rural and isolated area may be not related to highly transmitted strains but could be influenced by other health determinants that favor TB relapse such as poverty and poor health system access. Further studies including a larger number of MTBC strains from Esmeraldas are necessary to test this hypothesis.

Molecular determinants of multidrug-resistant tuberculosis in Sierra Leone.

A substantial proportion of MDR-TB strains in Sierra Leone were resistant against all first line drugs; however this makes the all-oral-six-month BPaLM regimen or other 6-9 months all oral regimens still viable, mainly because there was no FQ resistance.Resistance to BDQ was detected, as well as RR, due to mutations outside of the hotspot region. While the prevalence of those resistances was low, it is still cause for concern and needs to be closely monitored.

The association between phylogenetic lineage and the subclinical phenotype of pulmonary tuberculosis: A retrospective 2-cohort study

Subclinical pulmonary tuberculosis (PTB) is an asymptomatic disease state between established TB infection and symptomatic (clinical) TB disease. It is present in 20-25% of PTB patients in high-income countries. Mycobacterium tuberculosis complex (MTBC) genetic heterogeneity, and differential host immunological responses, have been implicated in its pathogenesis. To determine the association between MTBC lineage and PTB disease phenotype, we used two retrospective cohorts of PTB patients in Canada and two independent lineage attribution methods (DNA fingerprinting and genome sequencing). The first cohort, Cohort 1, consisted of consecutively diagnosed PTB patients between 2014 and 2020. The second, Cohort 2, consisted of newly-arrived foreign-born PTB patients who either were or were not referred for post-landing medical surveillance between 2004 and 2017. Univariable and multivariable logistic regression models were sequentially fitted to both cohorts, adjusting for age, sex, disease type, drug resistance and HIV. Evolution of radiographic features was correlated to lineage in Cohort 2. Cohort 1 and 2 included 874 (209 subclinical) and 111 (44 subclinical) patients, respectively. In both cohorts, subclinical patients were more likely than clinical patients to have relapse/retreatment disease, be smear-negative, have longer times-to-culture positivity and to harbor an ancestral MTBC lineage (Indo-Oceanic or Mycobacterium africanum). Relapse/retreatment disease and ancestral MTBC lineage were independent predictors of subclinical disease (ORs and 95% CIs in Cohort 1, 1.85 [1.07,3.28], p<0.029 and 2.30 [1.66,3.18], p<0.001, respectively, and Cohort 2, 5.74 [1.37-24.06], p<0.017 and 3.21 (1.29,7.97], p<0.012, respectively). The geographic distribution of Indo-Oceanic strains causing subclinical disease was uneven. Non-progressive lung disease was more common in patients infected with ancestral than modern lineages in Cohort 2, 56.0% vs 25.4%, p<0.005. MTBC lineage is a strong predictor of PTB disease phenotype. The genetic drivers of this association, and the relative contribution of other explanatory variables, are unknown.

Fastlin: an ultra-fast program for Mycobacterium tuberculosis complex lineage typing.

Fastlin is a bioinformatics tool designed for rapid MTBC lineage typing. It utilises an ultra-fast alignment-free approach to detect previously identified barcode single nucleotide polymorphisms (SNPs) associated with specific MTBC lineages. In a comprehensive benchmarking against existing tools, fastlin demonstrated high accuracy and significantly faster running times. fastlin is freely available at https://github.com/rderelle/fastlin and can easily be installed using Conda. Supplementary data are available at Bioinformatics online.

Perception and Risk Factors Associated with Tuberculosis in the Manyara Region, Tanzania

Tuberculosis (TB) results from infection with members of the Mycobacterium tuberculosis complex (MTBC) and represents a major global public health concern. We here sought to assess the perceptions of human and animal TB and the prevalence of circulating MTBCs lineages and associated risk factors through a cross-sectional survey of 335 individuals presenting with symptoms of pulmonary or extrapulmonary TB in the Manyara region of Tanzania. After the enrollment of participants, a questionnaire survey was conducted, samples were collected for bacterial culture, and real-time multiplex PCR was performed to differentiate amongst primary animal and human MTBC lineages. The results show poor TB awareness: 31.6% of the participants were not aware of human TB; 82.4% were unaware of animal TB and 95.2% lacked awareness of zoonotic TB (zTB) transmission. A total of 18 recovered specimens (5%; 95% CI: 3–8%) were positive by culture, all of which were typed as M. tuberculosis sensu stricto using a lineage-specific PCR assay. While no single risk factor was significantly associated with MTBC culture positivity, the survey revealed considerable self-reported high-risk practices for contracting zTB. Together, the results show that Manyara residents have poor knowledge of diseases caused by MTBCs and high evidence of risky practices for contracting zTB.

Macrophage susceptibility to infection by Ghanaian Mycobacterium tuberculosis complex lineages 4 and 5 varies with self-reported ethnicity.

The epidemiology of Mycobacterium tuberculosis complex (MTBC) lineage 5 (L5) infections in Ghana revealed a significantly increased prevalence in Ewes compared to other self-reported ethnic groups. In that context, we sought to investigate the early phase of tuberculosis (TB) infection using ex vivo infection of macrophages derived from the blood of Ewe and Akan ethnic group volunteers with MTBC L4 and L5 strains. The study participants consisted of 16 controls, among which self-reported Akan and Ewe ethnicity was equally represented, as well as 20 cured TB cases consisting of 11 Akans and 9 Ewes. Peripheral blood mononuclear cells were isolated from both healthy controls and cured TB cases. CD14+ monocytes were isolated and differentiated into monocyte-derived macrophages (MDMs) before infection with L4 or L5 endemic strains. The bacterial load was assessed after 2 hours (uptake) as well as 3 and 7 days post-infection. We observed a higher capacity of MDMs from Ewes to phagocytose L4 strains (p < 0.001), translating into a higher bacillary load on day 7 (p < 0.001) compared to L5, despite the higher replication rate of L5 in Ewe MDMs (fold change: 1.4 vs. 1.2, p = 0.03) among the controls. On the contrary, within macrophages from Akans, we observed a significantly higher phagocytic uptake of L5 (p < 0.001) compared to L4, also translating into a higher load on day 7 (p = 0.04). However, the replication rate of L4 in Akan MDMs was higher than that of L5 (fold change: L4=1.2, L4=1.1, p = 0.04). Although there was no significant difference in the uptake of L4 and L5 among cured TB cases, there was a higher bacterial load of both L4 (p = 0.02) and L5 (p = 0.02) on day 7 in Ewe MDMs. Our results suggest that host ethnicity (driven by host genetic diversity), MTBC genetic diversity, and individual TB infection history are all acting together to modulate the outcome of macrophage infections by MTBC.

Lack of methoxy-mycolates characterizes the geographically restricted lineage 7 of Mycobacterium tuberculosis complex.

Lineage 7 (L7) emerged in the phylogeny of the Mycobacterium tuberculosis complex (MTBC) subsequent to the branching of 'ancient' lineage 1 and prior to the Eurasian dispersal of 'modern' lineages 2, 3 and 4. In contrast to the major MTBC lineages, the current epidemiology suggests that prevalence of L7 is highly confined to the Ethiopian population, or when identified outside of Ethiopia, it has mainly been in patients of Ethiopian origin. To search for microbiological factors that may contribute to its restricted distribution, we compared the genome of L7 to the genomes of globally dispersed MTBC lineages. The frequency of predicted functional mutations in L7 was similar to that documented in other lineages. These include mutations characteristic of modern lineages - such as constitutive expression of nitrate reductase - as well as mutations in the VirS locus that are commonly found in ancient lineages. We also identified and characterized multiple lineage-specific mutations in L7 in biosynthesis pathways of cell wall lipids, including confirmed deficiency of methoxy-mycolic acids due to a stop-gain mutation in the mmaA3 gene that encodes a methoxy-mycolic acid synthase. We show that the abolished biosynthesis of methoxy-mycolates of L7 alters the cell structure and colony morphology on selected growth media and impacts biofilm formation. The loss of these mycolic acid moieties may change the host-pathogen dynamic for L7 isolates, explaining the limited geographical distribution of L7 and contributing to further understanding the spread of MTBC lineages across the globe.

A multiplex PCR assay for the differentiation of Mycobacterium tuberculosis complex reveals high rates of mixed-lineage tuberculosis infections among patients in Ghana.

In low-resource settings with high tuberculosis (TB) burdens, lack of rapid diagnostic methods for detection and differentiation of Mycobacterium tuberculosis complex (MTBC) is a major challenge affecting TB management. This study utilized comparative genomic analyses of MTBC lineages; M. tuberculosis, M. africanum Lineages 5/6 and M. bovis to identify lineage-specific genes. Primers were designed for the development of a Multiplex PCR assay which was successful in differentiating the MTBC lineages. There was no cross-reaction with other respiratory pathogens tested. Validation of the assay using clinical samples was performed with sputum DNA extracts from 341 clinically confirmed active TB patients. It was observed that 24.9% of cases were caused by M. tuberculosis, while M. africanum L5 & L6 reported 9.0% and 14.4%, respectively. M. bovis infection was the least frequently detected lineage with 1.8%. Also, 27.0% and 17.0% of the cases were PCR negative and unspeciated, respectively. However, mixed-lineage TB infections were recorded at a surprising 5.9%. This multiplex PCR assay will allow speciation of MTBC lineages in low-resource regions, providing rapid differentiation of TB infections to select appropriate medication at the earliest possible time point. It will also be useful in epidemiological surveillance studies providing reliable information on the prevalence of TB lineages as well as identifying difficult to treat cases of mixed-lineage tuberculosis infections.

CRISPR-Based Detection, Identification and Typing of Mycobacterium tuberculosis Complex Lineages.

The polymerase chain reaction (PCR)-based detection of Mycobacterium tuberculosis (M. tuberculosis) complex (MTC) in clinical samples is a first-line approach by which to diagnose tuberculosis in clinical microbiology laboratories. In this study, the genome-wide profiling of 3,156 mycobacterial genomes using Roary determined the CRISPR-csm4 gene as specific for MTB. Real time (RT)-PCR and the PCR-sequencing of CRISPR-csm4, tested on a collection of 20 MTC and 5 nontuberculous mycobacteria, confirmed the 20 MTC isolates, whereas the 5 nontuberculous isolates were not detected. Further, 65 of the leftover clinical samples, including 25 GeneXpert-positive and 40 GeneXpert-negative samples, that were used to evaluate the CRISPR-csm4-MTB assay in the clinical microbiology laboratory setting yielded expected results in every case, further allowing for the identification of the M. tuberculosis Beijing lineage. RT-PCR and the PCR-sequencing of CRISPR-csm4 could be implanted in the clinical microbiology laboratory to complement the currently used assays, with the potential of increasing the specification of the MTC pathogens responsible for tuberculosis. IMPORTANCE The whole-genome sequence comparison of the Mycobacterium tuberculosis complex (MTC) genomic sequences that are available in the NCBI database identified a unique, specific gene to be used directly on clinical diagnostic samples to detect MTC against all species of mycobacteria and to differentiate between MTC species, lineages, and sublineages.

Strain diversity and gene mutations associated with presumptive multidrug-resistant Mycobacterium tuberculosis complex isolates in Northwest Ethiopia

In this study, we assessed the genetic diversity and gene mutations that confer resistance to rifampicin (RIF), isoniazid (INH), fluoroquinolone (FQ), and second-line injectable (SLI) drugs in RIF-resistant (RR)/multidrug-resistant tuberculosis (MDR-TB) isolates in Northwest Ethiopia. Spoligotyping was used to assign isolates to TB lineages (Ls), and Hain line probe assays were used to detect resistance to RIF, INH, and FQs, and SLIs. Among 130 analyzed strains, 68.5% were RR, and four major Mycobacterium tuberculosis complex lineages (L1, L3, L4, and L7) were identified with a predominance of the Euro-American L4 (72, 54.7%), while L7 genotypes were less common (3, 2.3%). Overall, the L4-T3-ETH (41, 32.0%), L3-CAS1-Delhi (29, 22.7%), and L3-CAS1-Killi (19, 14.8%) families were most common. Line probe analysis showed that among rpoB mutants, 65.2% were S450L, while 87.8% of katG mutants were S315T. Only three isolates showed mutation (c-15t) at the inhA gene, and no double mutation with katG and inhA genes was found. Six strains, two each of L1, L3, and L4, were resistant to FQs, having gyrA mutations (D94G, S91P), of which three isolates had additional resistance to SLI (rrs A1401G or C1402T mutations) including one isolate with low-level kanamycin (KAN) resistance. This study showed a predominance of L4-T3-ETH, L3-CAS1-Delhi, and L3-CAS1-Killi families, with a high rate of rpoB_S450L and katG_S315T mutations and a low proportion of gyrA and rrs mutations. L7 was less frequently observed in this study. Further investigations are, therefore, needed to understand L7 and other lineages with undefined mutations.

Molecular epidemiology of bovine tuberculosis in Northern Ghana identifies several uncharacterized bovine spoligotypes and suggests possible zoonotic transmission.

ObjectiveWe conducted an abattoir-based cross-sectional study in the five administrative regions of Northern Ghana to determine the distribution of bovine tuberculosis (BTB) among slaughtered carcasses and identify the possibility of zoonotic transmission.MethodsDirect smear microscopy was done on 438 tuberculosis-like lesions from selected cattle organs and cultured on Lowenstein-Jensen media. Acid-fast bacilli (AFB) isolates were confirmed as members of the Mycobacterium tuberculosis complex (MTBC) by PCR amplification of IS6110 and rpoß. Characterization and assignment into MTBC lineage and sub-lineage were done by spoligotyping, with the aid of the SITVIT2, miruvntrplus and mbovis.org databases. Spoligotype data was compared to that of clinical M. bovis isolates from the same regions to identify similarities.ResultsA total of 319/438 (72.8%) lesion homogenates were smear positive out of which, 84.6% (270/319) had microscopic grade of at least 1+ for AFB. Two hundred and sixty-five samples (265/438; 60.5%) were culture positive, of which 212 (80.0%) were MTBC. Approximately 16.7% (34/203) of the isolates with correctly defined spoligotypes were negative for IS6110 PCR but were confirmed by rpoß. Spoligotyping characterized 203 isolates as M. bovis (198, 97.5%), M. caprae (3, 1.5%), M. tuberculosis (Mtbss) lineage (L) 4 Cameroon sub-lineage, (1, 0.5%), and M. africanum (Maf) L6 (1, 0.5%). A total of 53 unique spoligotype patterns were identified across the five administrative regions (33 and 28 were identified as orphan respectively by the SITVIT2 and mbovis.org databases), with the most dominant spoligotype being SIT1037/ SB0944 (77/203, 37.93%). Analysis of the bovine and human M. bovis isolates showed 75% (3/4) human M. bovis isolates sharing the same spoligotype pattern with the bovine isolates.ConclusionOur study identified that approximately 29% of M. bovis strains causing BTB in Northern Ghana are caused by uncharacterized spoligotypes. Our findings suggest possible zoonotic transmission and highlight the need for BTB disease control in Northern Ghana.

Genetic diversity and drug resistance profiles of Mycobacterium tuberculosis complex isolates from patients with extrapulmonary tuberculosis in Ghana and their associated host immune responses

ObjectivesThis study sought to determine the genetic diversity and drug resistance profiles of Mycobacterium tuberculosis complex (MTBC) isolates from extrapulmonary tuberculosis (EPTB) patients in Ghana, and their associated immune responses. MethodsSpoligotyping was performed on 102 MTBC isolates from EPTB patients. Lineages/sub-lineages were assigned by comparing spoligotyping patterns primarily with the SITVIT2 database and subsequently with the TB-Lineage online tool for unknown isolates in SITVIT2. Drug susceptibility testing was performed using MGIT (BD BACTEC 960), Lowenstein-Jensen media (indirect proportion method), and GenoType MTBDRplus/MTBDRsl assays. Differential cytokine levels in the serum of 20 EPTB patients infected with MTBC lineage 4 were determined using the Luminex multiplex immunoassay. ResultsAround 95% (97/102) of isolates were Mycobacterium tuberculosis, predominantly lineage 4 (95%; 92/97). Of the lineage 4 isolates, the majority were sub-lineage Cameroon (37%, 34/92). Prevalence was significantly higher in the 15–34 years age group among EPTB patients infected with lineage 4 strains (p = 0.024). Fifteen isolates were resistant to at least one anti-TB drug tested. Decreased levels of IL-1β, IL-17A, and IFN-α were observed in individuals infected with Cameroon sub-lineages compared with other lineage 4 sub-lineages. ConclusionsOur study confirms Cameroon (SIT61) as the most common spoligotype causing human EPTB in Ghana, and that it is associated with decreased serum IL-1β, IL-17A, and IFN-α.

Origin and Global Expansion of Mycobacterium tuberculosis Complex Lineage 3.

Simple SummaryTuberculosis still causes 1.5 million deaths annually and is mainly caused by Mycobacterium tuberculosis complex strains belonging to three evolutionary modern lineages (Lineages 2–4). While Lineage 2 and Lineage 4 virtually conquered the world, Lineage 3 is particularly successful in Northern and Eastern Africa, as well as in Southern Asia, the suspected evolutionary origin of these strains. Here, we sought to understand how Lineage 3 strains came to the African continent. To this end, we performed routine genotyping to characterize over 2500 clinical isolates from 38 countries. We then selected a representative collection of 373 isolates for a whole-genome analysis and a modeling approach to infer the geographic origin of different sublineages. In fact, the origin of Lineage 3 could be located in India, and we found evidence for independent introductions of four distinct sublineages into North/East Africa, in line with known ancient exchanges and migrations between both world regions. Our study illustrates that the evolutionary history of humans and their pathogens are closely connected and further provides a systematic understanding of the genomic diversity of Lineage 3, which could be important for the development of new tuberculosis vaccines or new therapeutics.Mycobacterium tuberculosis complex (MTBC) Lineage 3 (L3) strains are abundant in world regions with the highest tuberculosis burden. To investigate the population structure and the global diversity of this major lineage, we analyzed a dataset comprising 2682 L3 strains from 38 countries over 5 continents, by employing 24-loci mycobacterial interspersed repetitive unit-variable number of tandem repeats genotyping (MIRU-VNTR) and drug susceptibility testing. We further combined whole-genome sequencing (WGS) and phylogeographic analysis for 373 strains representing the global L3 genetic diversity. Ancestral state reconstruction confirmed that the origin of L3 strains is located in Southern Asia and further revealed multiple independent introduction events into North-East and East Africa. This study provides a systematic understanding of the global diversity of L3 strains and reports phylogenetic variations that could inform clinical trials which evaluate the effectivity of new drugs/regimens or vaccine candidates.

Defining the pknH Transcriptional Network in the Animal Tuberculosis bacillus, Mycobacterium bovis

The Mycobacterium tuberculosiscomplex (MTBC) is a group of bacteria that show more than 99% genetic identity, yet they diverge in their host preference and the severity of associated disease. Mycobacterium bovis causes the disease in cattle and poses an economic challenge, with the cost of Irelands TB Eradication Scheme predicted to exceed €90 million in 2020. The aim was to focus on the RD900 and pknH gene network in MTBC members. ThepknH gene encodes a transmembrane serine threonine protein kinase, that plays a role in the regulation of signalling pathways in the mycobacterial cell and has been linked to virulence. The RD900 or “region of difference 900” contains the tbD2 and pknH2 genes. The part of the genome that contains this region varies substantially across MTBC members. The results of RNA-seq analysis showed that there are statistically significant levels of differential gene expression in wildtype and knock-in M. bovis strains. The RD900 analysis across the MTBC showed that there is variation in this important genomic region. Analysis carried out by Mata et al. 2020, showed that this RD900 region had been independently lost in different MTBC lineages and strains. M. bovis has retained the RD900 region and this is believed to be linked to its increased virulence. The majority of the M . africanum strains were found to possess the standard pknH, tbD2, pknH2, embR gene arrangement as is found in M. africanum GM041182, the strain that is typically used as a reference. Based on work of Mata et al., 2020.

Relevance of genomic diversity of Mycobacterium tuberculosis complex in Africa

BackgroundThe diversity in the lineages of Mycobacterium tuberculosis complex (MTBC) was initially considered insignificant. However, comparative genomics analysis of MTBC have found genomic variation among the genotypes with potential phenotypic implications. ObjectiveTherefore, this viewpoint seeks to discuss the impact of the identified genotypic diversity on the physiology of MTBC and the potential implications on TB control. ResultsStudies conducted in West Africa and other parts of Africa have unravelled the implications of the genomic diversity on phenotypes such as disease outcome, transmission dynamics and host immune response. The understanding of the phenotypic diversity among the different lineages of MTBC may be an important key to the fight against TB. ConclusionThe relevance of these differences has been observed in the design of new control tools such as diagnostics and anti-TB drugs/vaccines. This only points to the fact that the diversity in MTBC cannot be ignored in future studies especially clinical trials for new vaccines and new anti-TB drugs.

Sub-Lineage Specific Phenolic Glycolipid Patterns in the Mycobacterium tuberculosis Complex Lineage 1.

“Ancestral” Mycobacterium tuberculosis complex (MTBC) strains of Lineage 1 (L1, East African Indian) are a prominent tuberculosis (TB) cause in countries around the Indian Ocean. However, the pathobiology of L1 strains is insufficiently characterized. Here, we used whole genome sequencing (WGS) of 312 L1 strains from 43 countries to perform a characterization of the global L1 population structure and correlate this to the analysis of the synthesis of phenolic glycolipids (PGL) – known MTBC polyketide-derived virulence factors. Our results reveal the presence of eight major L1 sub-lineages, whose members have specific mutation signatures in PGL biosynthesis genes, e.g., pks15/1 or glycosyltransferases Rv2962c and/or Rv2958c. Sub-lineage specific PGL production was studied by NMR-based lipid profiling and strains with a completely abolished phenolphthiocerol dimycoserosate biosynthesis showed in average a more prominent growth in human macrophages. In conclusion, our results show a diverse population structure of L1 strains that is associated with the presence of specific PGL types. This includes the occurrence of mycoside B in one sub-lineage, representing the first description of a PGL in an M. tuberculosis lineage other than L2. Such differences may be important for the evolution of L1 strains, e.g., allowing adaption to different human populations.

Molecular detection of mycobacterium tuberculosis complex species in triatomines found in urban households and Peridomiciles in a city with high prevalence of tuberculosis in Northeastern Brazil.

Triatomine insects, native to northeastern Brazil, have been found in the urban peridomicile. The city of Sobral has a high number of tuberculosis cases and several triatomine species. This study investigates the presence of mycobacteria, particularly Mycobacterium tuberculosis complex (MTBC) species, in triatomines captured in the urban perimeter of Sobral. We analyzed 167 triatomines captured in urban households and peridomiciles of Sobral. Mycobacteria were identified by the PRA-hsp65 method followed by partial sequencing of the hsp65 and rpoB genes. The sequences confirmed as MTBC were also typed by mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTR) and spoligotyping. Triatoma brasiliensis (38.6%), Triatoma pseudomaculata (32.9%), Panstrongylus lutzi (24.3%) were the most frequently identified. In 51.1% (70/167) of them, species of the Mycobacteriaceae family were detected by PRA-hsp65; of these, 31.4% (22/70) were identified as belonging to MTBC species. Nine (12.9%) of the triatomine samples were confirmed by sequencing as belonging to MTBC species. MIRU-VNTR genotyping suggests that the presence of different MTBC sublines in the triatomines should be investigated. This is the first report of MTBC lineages in triatomine insects. These results indicate the migration and adaptation of these insects in an urban setting.

Bacterial Load Comparison of the Three Main Lineages of Mycobacterium tuberculosis Complex in West Africa

Studies have shown an association between bacterial load and virulence; however, not much is known about the diversity in this phenotypic characteristic of Mycobacterium tuberculosis complex (MTBC). This study was therefore aimed to determine the differences in bacterial load of the three most prevalent MTBC genotypes (L4, L5, and L6) in West Africa at the time of diagnosis. A total of 170 paired fresh sputum samples were collected; one part in guanidinium thiocyanate (GTC) was used for RNA extraction and tuberculosis molecular bacterial load assay (TB-MBLA), and the other part without GTC was confirmed for TB positivity using GeneXpert MTB/RIF, smear microscopy grading, and culture on Löwenstein–Jensen media slants. The 170 sputum samples comprised 155 new cases, three follow-up cases, and 12 TB negative sputum samples. The time-to-culture positivity (TTP) and degree of culture positivity (DCP) were recorded. All 122 isolates obtained were spoligotyped for lineage (L) classification, but spoligotypes were obtained from 120 isolates. Of the typed isolates, 70.0, 10.8, 10.8, 4.2, 2.5, 0.8, and 0.8% were lineages 4, 5, 6, 2, 3, 1, and Mycobacterium bovis, respectively. Further analysis of the three most prevalent lineages showed significantly shorter TTP and higher DCP by L4 compared to L5 and L6, respectively: TTP 20.8, vs. 26.5, and 28.2 days; p-value = 0.005 and DCP 1.27, vs. 0.81 and 0.29, p < 0.001. The average TB-MBLA measured bacterial load of L4 was 3.82 Log10eCFU/ml which was not significantly different from 3.81 and 3.80 Log10eCFU/ml of L5 and L6, respectively, p = 0.84. Degrees of smear microscopy L4 = 1.20, L5 = 1.20, and L6 = 0.92 and GeneXpert Cq values L4 = 17.08, L5 = 18.37, and L6 = 17.59 showed no significant difference between the lineages, p = 0.72 and p = 0.48, respectively. Retrospective analysis of a larger sample confirmed the difference in TTP, p < 0.001. In conclusion, the observed shorter TTP and high DCP of L4 could signify high growth rate in culture that is independent of total bacterial load at diagnosis.

Molecular epidemiology and multidrug resistance of Mycobacterium tuberculosis complex from pulmonary tuberculosis patients in the Eastern region of Ghana

BackgroundTuberculosis (TB) and drug-resistant TB (DR-TB) continue to persist as a serious public health challenges in Ghana. Although several research has evaluated the drug resistance of Mycobacterium tuberculosis complex (MTBc) strains across the country, there is a paucity of data on its magnitude as well as the various lineages circulating in the Eastern region of Ghana. ObjectiveThis study therefore evaluated the distribution of the various lineages of MTBc in the Eastern region of the country and the associated drug resistance. Materials and methodsOne hundred and forty-three (143) patients with pulmonary TB attending the Eastern Regional Hospital, Koforidua/Ghana were included in the study. The BACTEC MGIT 960 tube media was used for both sputum culture and drug susceptibility of streptomycin (STR), isoniazid (INH), rifampicin (RIF) and Ethambutol (ETH). Isolates were initially typed using IS6110, followed by large sequence polymorphisms analysis and spoligotyping. ResultsThe majority [108 (75.5%)] of the 143 patients were male gender and the 45–54 years [46 (32.2%)] age range had the highest frequency. Forty-one (28.7%) of the 143 isolates were IS6110 negative. Of the 102 spoligotyped isolates, the main sub-lineages included 45 (44.1%) Cameroon and 23 (22.5%) Ghana. SITs 61 and 53 represented the major cluster with 22/102 (21.6%) and 13/102 (12.7%) isolates respectively, while 59/65 (90.8%) isolates belonged to Lineage 4 with 27/65 (41.5%) LAM10_CAM. MDR-TB occurred in 26/79 (32.9%) isolates, and was not associated with neither gender [20/58 (34.5%) male vs 6/21 (28.6%) female, OR = 1.31; 95%CI, 0.44–3.92; p = 0.624)] nor age. No association was found between MDR-TB and the major sub-lineages [8/25 (32%) Cameroon (OR = 0.94; 95%CI, 0.34–2.59; p = 0.920) and 5/11 (45.5%) Ghana (OR = 1.87; 95%CI, 0.51–6.80; p = 0.489)], or previously treated [8/23 (34.8%), OR = 0.89; 95%CI, 0.32–2.48; p = 0.823)] patients. ConclusionDespite the serious threat posed by MDR in the study area, no sub-lineage was shown to be associated with drug resistance. Nonetheless, a sustained surveillance of drug resistance pattern is advocated. A lower proportion of M. africanum was observed in the Eastern region of Ghana and will require further evaluation.