Mycobacterium Tuberculosis Complex Genotypes 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.

Transmission Dynamics of a Mycobacterium tuberculosis Complex Outbreak in an Indigenous Population in the Colombian Amazon Region.

Whole genome sequencing (WGS) has become the main tool for studying the transmission of Mycobacterium tuberculosis complex (MTBC) strains; however, the clonal expansion of one strain often limits its application in local MTBC outbreaks. The use of an alternative reference genome and the inclusion of repetitive regions in the analysis could potentially increase the resolution, but the added value has not yet been defined. Here, we leveraged short and long WGS read data of a previously reported MTBC outbreak in the Colombian Amazon Region to analyze possible transmission chains among 74 patients in the indigenous setting of Puerto Nariño (March to October 2016). In total, 90.5% (67/74) of the patients were infected with one distinct MTBC strain belonging to lineage 4.3.3. Employing a reference genome from an outbreak strain and highly confident single nucleotide polymorphisms (SNPs) in repetitive genomic regions, e.g., the proline-glutamic acid/proline-proline-glutamic-acid (PE/PPE) gene family, increased the phylogenetic resolution compared to a classical H37Rv reference mapping approach. Specifically, the number of differentiating SNPs increased from 890 to 1,094, which resulted in a more granular transmission network as judged by an increasing number of individual nodes in a maximum parsimony tree, i.e., 5 versus 9 nodes. We also found in 29.9% (20/67) of the outbreak isolates, heterogenous alleles at phylogenetically informative sites, suggesting that these patients are infected with more than one clone. In conclusion, customized SNP calling thresholds and employment of a local reference genome for a mapping approach can improve the phylogenetic resolution in highly clonal MTBC populations and help elucidate within-host MTBC diversity. IMPORTANCE The Colombian Amazon around Puerto Nariño has a high tuberculosis burden with a prevalence of 1,267/100,000 people in 2016. Recently, an outbreak of Mycobacterium tuberculosis complex (MTBC) bacteria among the indigenous populations was identified with classical MTBC genotyping methods. Here, we employed a whole-genome sequencing-based outbreak investigation in order to improve the phylogenetic resolution and gain new insights into the transmission dynamics in this remote Colombian Amazon Region. The inclusion of well-supported single nucleotide polymorphisms in repetitive regions and a de novo-assembled local reference genome provided a more granular picture of the circulating outbreak strain and revealed new transmission chains. Multiple patients from different settlements were possibly infected with at least two different clones in this high-incidence setting. Thus, our results have the potential to improve molecular surveillance studies in other high-burden settings, especially regions with few clonal multidrug-resistant (MDR) MTBC lineages/clades.

Genotyping of Mycobacterium tuberculosis complex isolated from humans and animals in northeastern Iran

The objective of this study was to genotype Mycobacterium tuberculosis complex isolated from humans and cattle in northern Iran. Over the course of one year, a total of 120 human and 21 cattle isolates were tested using region of difference (RD)-based polymerase chain reaction (PCR) and mycobacterial interspersed repetitive unites-variable number tandem repeats (MIRU-VNTR). In M. tuberculosis, out of 120 isolates investigated, the most common genotype detected was NEW-1 (53.3%), followed by CAS/ Delhi (24.1%), Haarlem (5%), Beijing (4.16%), Uganda I (4.16%), S (3.3%), Ural (0.83%), TUR (0.83%), Uganda II (0.83%), Lam (0.83%) and Cameroon (0.83%). The HGDI rate was 0.9981 and the clustering rate was 10.83. Of the isolates, QUB26 had the highest allele diversity (h: 0.76), while the loci Mtub29 and MIRU24 had the lowest (h: 0). In M. Bovis, out of 123 collected tissue samples, 21 (17%) grew on culture media. The HGDI rate was 0.71 and clustering rate was 85.7%. The locus ETRC had the highest allele diversity (h: 0.45). The findings of this study suggest that there is high genetic diversity among M. tuberculosis isolates in Khorasan Razavi Province, which is consistent with similar results from other studies in other provinces in Iran and neighboring countries. This indicates that the prevalent genotypes in this study are spreading in the Middle East region. Furthermore, considering that M. Bovis isolates were identified in two clusters, it seems that all of them have a common origin and are circulating among the livestock farms in the province.

Back-to-Africa introductions of Mycobacterium tuberculosis as the main cause of tuberculosis in Dar es Salaam, Tanzania.

In settings with high tuberculosis (TB) endemicity, distinct genotypes of the Mycobacterium tuberculosis complex (MTBC) often differ in prevalence. However, the factors leading to these differences remain poorly understood. Here we studied the MTBC population in Dar es Salaam, Tanzania over a six-year period, using 1,082 unique patient-derived MTBC whole-genome sequences (WGS) and associated clinical data. We show that the TB epidemic in Dar es Salaam is dominated by multiple MTBC genotypes introduced to Tanzania from different parts of the world during the last 300 years. The most common MTBC genotypes deriving from these introductions exhibited differences in transmission rates and in the duration of the infectious period, but little differences in overall fitness, as measured by the effective reproductive number. Moreover, measures of disease severity and bacterial load indicated no differences in virulence between these genotypes during active TB. Instead, the combination of an early introduction and a high transmission rate accounted for the high prevalence of L3.1.1, the most dominant MTBC genotype in this setting. Yet, a longer co-existence with the host population did not always result in a higher transmission rate, suggesting that distinct life-history traits have evolved in the different MTBC genotypes. Taken together, our results point to bacterial factors as important determinants of the TB epidemic in Dar es Salaam.

Genotyping of Mycobacterium tuberculosis Complex Isolates from Patients with Tuberculosis by MIRU-VNTR and Spoligotyping Methods

Genotyping of Mycobacterium tuberculosis Complex Isolates from Patients with Tuberculosis by MIRU-VNTR and Spoligotyping Methods

Novel Mycobacterium tuberculosis Complex Genotype Related to M. caprae.

We report the unusual genotypic characterization of a bacterium isolated from a clinical sample of a patient who grew up in Bangladesh and lives in the United States. Using whole-genome sequencing, we identified the bacterium as a member of the Mycobacterium tuberculosis complex (MTBC). Phylogenetic placement of this strain suggests a new MTBC genotype. Even though it had the same spoligotype as M. caprae strains, single-nucleotide polymorphism–based phylogenetic analysis placed the isolate as a sister lineage distinct from M. caprae, most closely related to 5 previously sequenced genomes isolated from primates and elephants in Asia. We propose a new animal-associated lineage, La4, within MTBC.

Country-wide genotyping of Mycobacterium tuberculosis complex in Singapore, 2011–2017

ObjectiveTo describe the molecular epidemiology of Mycobacterium tuberculosis complex (MTBC) and factors associated with its transmission in Singapore. MethodsSpoligotyping, 24-loci mycobacterial interspersed repetitive units – variable number of tandem repeats (MIRU-VNTR) typing and demographic data from the national TB notification registry of MTBC culture-positive cases notified from January 2011 to December 2017 were analysed. ResultsOf the 12,046 culture-positive cases notified, complete spoligotyping and MIRU-VNTR typing results were available for 8690 (72.1%) belonging to 4950 (57.0%) local-born and 3740 (43.0%) foreign-born persons. From these, 4810 (55.3%) were identified in 883 clusters. The proportion of recent transmission was 45.2%. The East-Asian Lineage 2 accounted for 4045 (47.1%) of isolates, and disproportionately accounted for large clusters. Clustered cases were more likely to be older than 50 years, male, Malay, local-born, Singapore citizens or Permanent Residents, of lower socioeconomic status, imprisoned; to harbour East-Asian Lineage 2 strain; to have cavitary pulmonary TB, positive sputum smear or be recalcitrant treatment defaulters. They were less likely to have multidrug-resistant, or isoniazid or rifampicin mono-resistant TB. ConclusionWe demonstrated the diversity of MTBC strains and, notwithstanding the likely over-estimation of clustering using these genotyping methods, elucidated factors associated with TB transmission in Singapore.

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.

Genotypic and phenotypic diversity of Mycobacterium tuberculosis complex genotypes prevalent in West Africa.

Findings from previous comparative genomics studies of the Mycobacterium tuberculosis complex (MTBC) suggest genomic variation among the genotypes may have phenotypic implications. We investigated the diversity in the phenotypic profiles of the main prevalent MTBC genotypes in West Africa. Thirty-six whole genome sequenced drug susceptible MTBC isolates belonging to lineages 4, 5 and 6 were included in this study. The isolates were phenotypically characterized for urease activity, tween hydrolysis, Thiophen-2-Carboxylic Acid Hydrazide (TCH) susceptibility, nitric oxide production, and growth rate in both liquid (7H9) and solid media (7H11 and Löwenstein–Jensen (L-J)). Lineage 4 isolates showed the highest growth rate in both liquid (p = 0.0003) and on solid (L-J) media supplemented with glycerol (p<0.001) or pyruvate (p = 0.005). L6 isolates optimally utilized pyruvate compared to glycerol (p<0.001), whereas L5 isolates grew similarly on both media (p = 0.05). Lineage 4 isolates showed the lowest average time to positivity (TTP) (p = 0.01; Average TTP: L4 = 15days, L5 = 16.7days, L6 = 29.7days) and the highest logCFU/mL (p = 0.04; average logCFU/mL L4 = 5.9, L5 = 5.0, L6 = 4.4) on 7H11 supplemented with glycerol, but there was no significant difference in growth on 7H11 supplemented with pyruvate (p = 0.23). The highest release of nitrite was recorded for L5 isolates, followed by L4 and L6 isolates. However, the reverse was observed in the urease activity for the lineages. All isolates tested were resistant to TCH except for one L6 isolate. Comparative genomic analyses revealed several mutations that might explain the diverse phenotypic profiles of these isolates. Our findings showed significant phenotypic diversity among the MTBC lineages used for this study.

Broad diversity of Mycobacterium tuberculosis complex strains isolated from humans and cattle in Northern Algeria suggests a zoonotic transmission cycle.

Mycobacterium tuberculosis complex (MTBC) comprises closely related species responsible for human and animal tuberculosis (TB). Efficient species determination is useful for epidemiological purposes, especially for the elucidation of the zoonotic contribution. In Algeria, data on MTBC genotypes are largely unknown. In this study, we aimed to investigate the occurrence and diversity of MTBC genotypes causing human and bovine TB in Northern Algeria. During a two-year sampling period (2017–2019) in two regions of Northern Algeria, we observed an overall prevalence of 6.5% of tuberculosis (TB) among slaughtered cattle, which is higher than previous Algerian data yet comparable to neighboring countries. A total of 296 Mycobacterium tuberculosis complex (MTBC) isolates were genotyped by spoligotyping: 181 from tissues with TB-like lesions collected from 181 cattle carcasses and 115 from TB patients. In human isolates, we identified 107 M. tuberculosis, seven M. bovis and one “M. pinnipedii-like”, while for bovine samples, 174 isolates were identified as M. bovis, three as M. caprae, three as “M. pinnipedii-like” and one as “M. microti-like”. The majority of isolates (89.2%) belonged to 72 different known Shared International Types (SIT) or M. bovis spoligotypes (SB), while we also identified seven new SB profiles (SB2695 to SB2701). Twenty-eight of the SB profiles were new to Algeria. Our data suggest zoonotic transmission in Sétif, where significantly more TB was observed among cattle (20%) compared to the slaughterhouses from the three other regions (5.4%–7.3%) (p < 0.0001), with the isolation of the same M. bovis genotypes from TB patients. The present study showed a high genetic diversity of MTBC isolated from human and cattle in Northern Algeria. Even though relatively small in terms of numbers, our data suggest the zoonotic transmission of TB from cattle to humans, suggesting the need for stronger eradication strategies for bovine TB.

Development and application of affordable SNP typing approaches to genotype Mycobacterium tuberculosis complex strains in low and high burden countries

The Mycobacterium tuberculosis complex (MTBC) comprises the species that causes tuberculosis (TB) which affects 10 million people every year. A robust classification of species, lineages, and sub-lineages is important to explore associations with drug resistance, epidemiological patterns or clinical outcomes. We present a rapid and easy-to-follow methodology to classify clinical TB samples into the main MTBC clades. Approaches are based on the identification of lineage and sub-lineage diagnostic SNP using a real-time PCR high resolution melting assay and classic Sanger sequencing from low-concentrated, low quality DNA. Thus, suitable for implementation in middle and low-income countries. Once we validated our molecular procedures, we characterized a total of 491 biological samples from human and cattle hosts, representing countries with different TB burden. Overall, we managed to genotype ~95% of all samples despite coming from unpurified and low-concentrated DNA. Our approach also allowed us to detect zoonotic cases in eight human samples from Nigeria. To conclude, the molecular techniques we have developed, are accurate, discriminative and reproducible. Furthermore, it costs less than other classic typing methods, resulting in an affordable alternative method in TB laboratories.

MIRUReader: MIRU-VNTR typing directly from long sequencing reads.

Mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing is widely used to genotype Mycobacterium tuberculosis complex in epidemiological studies for tracking tuberculosis transmission. Recent long-read sequencing technologies from Pacific Biosciences and Oxford Nanopore Technologies can produce reads that are long enough to cover the entire repeat regions in each MIRU-VNTR locus which was previously not possible using the short reads from Illumina high-throughput sequencing technologies. We thus developed MIRUReader for MIRU-VNTR typing directly from long sequence reads. Source code and documentation for MIRUReader program is freely available at https://github.com/phglab/MIRUReader. Supplementary data are available at Bioinformatics online.

Spoligotyping analysis of Mycobacterium tuberculosis in Khyber Pakhtunkhwa area, Pakistan.

Background: Spoligotyping is a reproducible, reverse hybridization approach for genotyping of Mycobacterium tuberculosis complex (MTBC). Molecular typing of MTBC is helpful for understanding and controlling tuberculosis epidemics.Methods: Spoligotyping was performed on 166 clinical isolates of Mycobacterium tuberculosis (MTB) collected from 25 districts of Khyber Pakhtunkhwa, Pakistan. Results were compared to SITVIT2, an online database developed by the Institut Pasteur de la Guadeloupe, France.Results: Spoligotyping results showed that 145 strains (88%) displayed known patterns while 21 (12%) were new. Lineage 3/Central Asian strain (L3/CAS) was the predominant family (73%, χ2=19.9, P=0.001), followed by L2/Beijing (5.4%) and L4 (4.2%). L3/CAS1-Delhi was the major sublineage (82%) among the L3/CAS family (χ2=664, P=0.0001). Analysis showed that the majority of the clinical isolates with an unknown pattern had an evolutionary link with the L3/CAS strain, and nine (5.4%) of the unknown strains were epidemiologically linked and were tentatively named L3/CAS-KP (Khyber Pakhtunkhwa).Conclusion: The present study demonstrated that L3/CAS is the predominant lineage of MTB, widely distributed in different areas of the Khyber Pakhtunkhwa province of Pakistan. Spoligotyping patterns of some clinical isolates could not be matched to other reported patterns in an international database. Other tools, such as mycobacterial interspersed repetitive unit–variable number tandem repeat (MIRU-VNTR), will be helpful in future investigations into the epidemiological characteristics of clinical isolates in the Khyber Pakhtunkhwa province.

Development of a new High Resolution Melting (HRM) assay for identification and differentiation of Mycobacterium tuberculosis complex samples

The rapid identification and differentiation of members of the Mycobacterium tuberculosis complex (MTBC) is essential to assess the potential zoonotic risk. Different available molecular methods are time consuming since they depend on cultivation of mycobacteria. High Resolution Melting (HRM) is a low cost, rapid and easy to perform single-tube method not limited to cultured samples. In this study, a HRM assay specifically targeting gyrB was developed to simultaneously identify and differentiate Mycobacterium (M.) tuberculosis, M. microti and M. bovis/M. caprae. To evaluate the performance of this assay, 38 MTBC isolates and 25 directly extracted clinical specimens were analysed. HRM results of all 38 (100%) examined isolates correlated with the results obtained with the commercially available GenoType MTBC test (Hain Lifescience). From the 25 clinical specimens tested, species identification by HRM showed concordant results with the previously used identification methods in 23 samples (92%). The assay demonstrated a good analytical sensitivity, specificity and reproducibility and can be used directly on clinical specimens.

Mycobacterium tuberculosis complex genotypes circulating in Nigeria based on spoligotyping obtained from Ziehl-Neelsen stained slides extracted DNA.

Tuberculosis (TB) remains one of the most threatening diseases and Nigeria has one of the world’s largest burdens. This study performed high-throughput spoligotyping directly on sputum smears to describe the Mycobacterium tuberculosis strains circulating among new TB patients in Nigeria.MethodsAll State TB control programmes in Nigeria were requested to submit 25–50 smear-positive Ziehl-Neelsen (ZN) stained slides for screening during 2013–2014. DNA was extracted from 929 slides for spoligotyping and drug-resistance analysis using microbead-based flow-cytometry suspension arrays.ResultsSpoligotyping results were obtained for 549 (59.1%) of 929 samples. Lineage 4 Cameroon sublineage (L4.6.2) represented half of the patterns, Mycobacterium africanum (L5 and L6) represented one fifth of the patterns, and all other lineages, including other L4 sublineages, represented one third of the patterns. Sublineage L4.6.2 was mostly identified in the north of the country whereas L5 was mostly observed in the south and L6 was scattered. The spatial distribution of genotypes had genetic geographic gradients. We did not obtain results enabling the detection of drug-resistance mutations.Conclusion/SignificanceWe present the first national snapshot of the M. tuberculosis spoligotypes circulating in Nigeria based on ZN slides. Spoligotyping data can be obtained in a rapid and high-throughput manner with DNA extracted from ZN-stained slides, which may potentially improve our understanding of the genetic epidemiology of TB.

First Nationwide Survey on the Resistance to First Line Anti-Tuberculosis Drugs in Ghana

Background/objective: A nationwide survey on the resistance to first line anti-tuberculosis (anti-TB) drugs was conducted in Ghana from 2007-2008 by Noguchi Memorial Institute for Medical Research in collaboration with the National Tuberculosis Control Programme. We aimed to characterize mycobacterial species causing pulmonary tuberculosis (PTB) and determine the resistance pattern to first line anti-TB drugs among newly diagnosed and previously treated PTB patients in Ghana. Methods: Two sputum samples from consented new smear positive PTB patients who had never been treated for TB or had been on anti-TB treatment for less than a month and patients who had been treated for TB previously for more than a month in selected diagnostic centres nationwide were collected for culture, identification and drug susceptibility test. Culture positive isolates were tested against streptomycin (S), isoniazid (H), rifampicin (R) and ethambutol (E) using the simplified proportion method and line probe assay (LPA). The LPA was performed in mid-2017. Results: Among 410 samples, 345 positive cultures were obtained and identified as Mycobacterium tuberculosis complex (MTBC). Of the 345 isolates, 133 were further differentiated by GenoType MTBC® as M. tuberculosis, 126 (94.7%) and M. africanum 7 (5.3%). The overall drug resistance patterns were as follows: 43/345 (12.5%), 6/345 (1.7%), 9/345 (2.6%) and 71/345 (20.6%) were resistant to H, R, E and S respectively and 5/345 (1.4%) were multi-drug resistant (MDR). Conclusion: The results indicate high levels of resistance to S and H among new and previously treated TB patients. We recommend adequate surveillance systems including periodic national anti-TB drug resistance surveys.

Efficacy of multilevel functional surgery in the reduction of stiff knee gait in stroke patients. A retrospective study

We argue in favor of a concerted and coordinated response to stop tuberculosis (TB) by monitoring global TB spread, drug-resistance surveillance and populations at risk using available molecular and web tools to identify circulating clones of Mycobacterium tuberculosis complex (MTBC). We took specific example of the Beijing lineage associated with worldwide emergence of both multiple, and extensively drug resistant (MDR/XDR)-TB. The study dataset (n = 10,850 isolates, 92 countries of patient origin) was extracted from our multimarker SITVIT2 database on MTBC genotyping (n = 111,635 isolates, 169 countries of patient origin). Epidemiological and demographic information in conjunction with spoligotyping (n = 10,850), MIRU-VNTR minisatellites (n = 2896), and drug resistance (n = 2846) data was mapped at macro-geographical (United Nations subregions) and country level, followed by statistical, bioinformatical, and phylogenetical analysis. The global male/female sex ratio was 1.96, the highest being 4.93 in Russia vs. range of 0.8–1.13 observed in Central America, Caribbean, Eastern Africa and Northern Europe (p < 0.0001). The major patient age-group was 21–40 yrs worldwide except Japan (with majority of patients >60 yrs). Younger patients were more common in South America, South Asia, and Western Africa since 25–33% of TB cases due to Beijing genotype occurred in the age group 0–20 yrs. A continuous progression in the proportion of MDR and XDR strains is visible worldwide since 2003 and 2009 respectively. Pansusceptible TB mainly concerned older patients >60 yrs (44%) whereas Drug resistant, MDR and XDR-TB concerned patients preferentially aged 21–40 yrs (between 52 and 58%). Although the proportion of SIT1 pattern vs. other patterns was very high (93%); the proportion of MDR was highest for an emerging genotype SIT190 (p < 0.0001). Lastly, proportion of pansusceptible strains was highest in Japan, while MDR/XDR strains were most common in Russia and Northern Europe. We underline remarkable macro/micro-geographical cleavages in phylogenetic and epidemiologic diversity of Beijing genotype, with phylogeographical specificity of certain genotypes.

Spatiotemporal Clustering of Mycobacterium tuberculosis Complex Genotypes in Florida: Genetic Diversity Segregated by Country of Birth.

BackgroundTuberculosis (TB) is caused by members of the Mycobacterium tuberculosis complex (MTBC). Although the MTBC is highly clonal, between-strain genetic diversity has been observed. In low TB incidence settings, immigration may facilitate the importation of MTBC strains with a potential to complicate TB control efforts.MethodsWe investigated the genetic diversity and spatiotemporal clustering of 2,510 MTBC strains isolated in Florida, United States, between 2009 and 2013 and genotyped using spoligotyping and 24-locus MIRU-VNTR. We mapped the genetic diversity to the centroid of patient residential zip codes using a geographic information system (GIS). We assessed transmission dynamics and the influence of immigration on genotype clustering using space-time permutation models adjusted for foreign-born population density and county-level HIV risk and multinomial models stratified by country of birth and timing of immigration in SaTScan.Principal FindingsAmong the 2,510 strains, 1,245 were reported among foreign-born persons; including 408 recent immigrants (<5 years). Strain allelic diversity (h) ranged from low to medium in most locations and was most diverse in urban centers where foreign-born population density was also high. Overall, 21.5% of cases among U.S.-born persons and 4.6% among foreign-born persons clustered genotypically and spatiotemporally and involved strains of the Haarlem family. One Haarlem space-time cluster identified in the mostly rural northern region of Florida included US/Canada-born individuals incarcerated at the time of diagnosis; two clusters in the mostly urban southern region of Florida were composed predominantly of foreign-born persons. Both groups had HIV prevalence above twenty percent.Conclusions/SignificanceAlmost five percent of TB cases reported in Florida during 2009–2013 were potentially due to recent transmission. Improvements to TB screening practices among the prison population and recent immigrants are likely to impact TB control. Due to the monomorphic nature of available markers, whole genome sequencing is needed to conclusively delineate recent transmission events between U.S. and foreign-born persons.

Prevalence and Etiology of Tuberculosis from Sputum of Patients using Geno-Type Mycobacterium Tuberculosis Complex (MTBC) in Niger State, North Central, Nigeria.

This study aimed at the prevalence of Patients infected with Tuberculosis and their causative Organisms and this was carried out from Six General Hospitals of Niger State,, North Central, Nigeria. Three sputum samples and 2ml of blood were collected from 1920 Patients that have cough of two weeks and above. Their HIV status was determined and Viral antibody detection was carried out using World Health Organization approved kits called Determine, Unigold and Star pack. Their sputum samples were cultured after routine microscopy. Sputum samples were processed using Ziehl -Neelsen (ZN) reagents. The sputa were cultured on Loewenstein-Jensen egg based medium and incubated at 370C for eight weeks. All the Acid-fast bacill (AFB) positive smears and culture positive isolates were tested for genetic identification using Mycobacterium tuberculosis complex (MTBC) (GenoType MTBC (Hain Lifescience)) to confirmed the isolates to species level. The results reviewed that out of the one thousand Nine hundred and twenty (1920) Patients that were screened for HIV and TB, four hundred and sixteen (416) (23.1%) had MTB complex infection: four hundred and thirteen (413) (99.2%) had Mycobacterium tuberculosis ; two (2) (0.4%) had Mycobacterium africanum ; and one (1) (0.2%) had Mycobacterium bovis ; fourty six samples were contaminated while seventy four samples were non tuberculosis mycobacterium (NTM) and were removed from the research, one thousand eight hundred Patients were used for the research. The most affected age group was between elevem to fourty (11-40) years and male had the highest prevalence of tuberculosis infection with two hundred and twelve (212) (50.9%) compared to the female with two hundred and four (204) (49.0%), statistical analysis shows that there was an association between gender and also between the age group with P< 0.05. Five hundred and fifteen (515) (28.6%) patients were screened positive for HIV and one hundred and eighty six (186) (36.1%) Patients had HIV and TB co-infections.

A single-nucleotide-polymorphism real-time PCR assay for genotyping of Mycobacterium tuberculosis complex in peri-urban Kampala.

BackgroundAccurate and high-throughput genotyping of Mycobacterium tuberculosis complex (MTBC) may be important for understanding the epidemiology and pathogenesis of tuberculosis (TB). In this study, we report the development of a LightCycler® real-time PCR single-nucleotide-polymorphism (LRPS) assay for the rapid determination of MTBC lineages/sublineages in minimally processed sputum samples from TB patients.MethodGenotyping analysis of 70 MTBC strains was performed using the Long Sequence Polymorphism-PCR (LSP-PCR) technique and the LRPS assay in parallel. For targeted sequencing, 9 MTBC isolates (three isolates per MTBC lineage) were analyzed for lineage-specific single nucleotide polymorphisms (SNPs) in the following three genes to verify LRPS results: Rv004c for MTB Uganda family, Rv2962 for MTB lineage 4, and Rv0129c for MTB lineage 3. The MTBC lineages present in 300 smear-positive sputum samples were then determined by the validated LRPS method without prior culturing.ResultsThe LSP-PCR and LRPS assays produced consistent genotyping data for all 70 MTBC strains; however, the LSP-PCR assay was 10-fold less sensitive than the LRPS method and required higher DNA concentrations to successfully characterize the MTBC lineage of certain samples. Targeted sequencing of genes containing lineage-specific SNPs was 100 % concordant with the genotyping results and provided further validation of the LRPS assay. Of the 300 sputum samples analyzed, 58 % contained MTBC from the MTBC-Uganda family, 27 % from the MTBC lineage 4 (excluding MTBC Uganda family), 13 % from the MTBC lineage 3, and the remaining 2 % were of indeterminate lineage.ConclusionThe LRPS assay is a sensitive, high-throughput technique with potential application to routine genotyping of MTBC in sputum samples from TB patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-015-1121-7) contains supplementary material, which is available to authorized users.