In order to provide a global overview of genotypic, epidemiologic, demographic, phylogeographical, and drug resistance characteristics related to the prevailing tuberculosis (TB) epidemic, we hereby ...report an update of the 6th version of the international genotyping database SITVIT2. We also make all the available information accessible through a dedicated website (available at http://www.pasteur-guadeloupe.fr:8081/SITVIT2). Thanks to the public release of SITVIT2 which is currently the largest international multimarker genotyping database with a compilation of 111,635 clinical isolates from 169 countries of patient origin (131 countries of isolation, representing 1032 cities), our major aim is to highlight macro- and micro-geographical cleavages and phylogeographical specificities of circulating Mycobacterium tuberculosis complex (MTBC) clones worldwide. For this purpose, we retained strains typed by the most commonly used PCR-based methodology for TB genotyping, i.e., spoligotyping based on the polymorphism of the direct repeat (DR) locus, 5-loci Exact Tandem Repeats (ETRs), and MIRU-VNTR minisatellites used in 12–, 15–, or 24–loci formats. We describe the SITVIT2 database and integrated online applications that permit to interrogate the database using easy drop-down menus to draw maps, graphics and tables versus a long list of parameters and variables available for individual clinical isolates (year and place of isolation, origin, sex, and age of patient, drug-resistance, etc.). Available tools further allow to generate phylogenetical snapshot of circulating strains as Lineage-specific WebLogos, as well as minimum spanning trees of their genotypes in conjunction with their geographical distribution, drug-resistance, demographic, and epidemiologic characteristics instantaneously; whereas online statistical analyses let a user to pinpoint phylogeographical specificities of circulating MTBC lineages and conclude on actual demographic trends. Available associated information on gender (n = 18,944), age (n = 16,968), drug resistance (n = 19,606), and HIV serology (n = 2673), allowed to draw some important conclusions on TB geo-epidemiology; e.g. a positive correlation exists between certain Mycobacterium tuberculosis lineages (such as CAS and Beijing) and drug resistance (p-value<.001), while other lineages (such as LAM, X, and BOV) are more frequently associated with HIV-positive serology (p-value<.001). Besides, availability of information on the year of isolation of strains (range 1759–2012), also allowed to make tentative correlations between drug resistance information and lineages – portraying probable evolution trends over time and space. To conclude, the present approach of geographical mapping of predominant clinical isolates of tubercle bacilli causing the bulk of the disease both at country and regional level in conjunction with epidemiologic and demographic characteristics allows to shed new light on TB geo-epidemiology in relation with the continued waves of peopling and human migration.
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•SITVIT2, an international Mycobacterium tuberculosis genotyping database is described (http://www.pasteur-guadeloupe.fr:8081/SITVIT2).•It contains available genotyping (spoligotyping, MIRU-VNTRs), demographic and epidemiologic information on 111,635 clinical isolates.•Integrated applications allow to draw maps, graphics and tables versus a long list of parameters and variables available for individual isolates.•It allows to generate geographical mapping of predominant clinical isolates of tubercle bacilli at country, regional and local level.•A snapshot of prevailing disparities may be drawn by superimposing maps with available data thanks to Geographic Information Systems.•A comprehension of prevailing drawbacks will allow to take appropriate actions to monitor, understand and control the tuberculosis epidemic.
The East African Indian (EAI) and Central Asian (CAS) lineages of Mycobacterium tuberculosis complex (MTBC) mainly infect tuberculosis (TB) patients in the eastern hemisphere which contains many of ...the 22 high TB burden countries including China and India. We investigated if phylogeographical, epidemiological and demographical characteristics for these 2 lineages differed in SITVIT2 database. Genotyping results and associated data (age, sex, HIV serology, drug resistance) on EAI and CAS lineages (n = 10,974 strains) were extracted. Phylogenetic and Bayesian, and other statistical analyses were used to compare isolates. The male/female sex ratio was 907/433 (2.09) for the EAI group vs. 881/544 (1.62) for CAS (p-value<0.002). The proportion of younger patients aged 0-20 yrs. with CAS lineage was significantly higher than for EAI lineage (18.07% vs. 10.85%, p-value<0.0001). The proportion of multidrug resistant and extensively drug resistant TB among CAS group (30.63% and 1.03%, respectively) was significantly higher than in the EAI group (12.14% and 0.29%, respectively; p-value<0.0001). Lastly, the proportion of HIV+ patients was 20.34% among the EAI group vs. 3.46% in the CAS group (p-value<0.0001). This remarkable split observed between various parameters for these 2 lineages was further corroborated by their geographic distribution profile (EAI being predominantly found in Eastern-Coast of Africa, South-India and Southeast Asia, while CAS was predominantly found in Afghanistan, Pakistan, North India, Nepal, Middle-east, Libya, Sudan, Ethiopia, Kenya and Tanzania). Some geo-specificities were highlighted. This study demonstrated a remarkable cleavage for aforementioned characteristics of EAI and CAS lineages, showing a North-South divide along the tropic of cancer in Eastern hemisphere-mainly in Asia, and partly prolonged along the horn of Africa. Such studies would be helpful to better comprehend prevailing TB epidemic in context of its historical spread and evolutionary features, and provide clues to better treatment and patient-care in countries and regions concerned by these lineages.
Molecular typing has revolutionized epidemiological studies of infectious diseases, including those of a mycobacterial etiology. With the advent of fingerprinting techniques, many traditional ...concepts regarding transmission, infectivity, or pathogenicity of mycobacterial bacilli have been revisited, and their conventional interpretations have been challenged. Since the mid-1990s, when the first typing methods were introduced, a plethora of other modalities have been proposed. So-called molecular epidemiology has become an essential subdiscipline of modern mycobacteriology. It serves as a resource for understanding the key issues in the epidemiology of tuberculosis and other mycobacterial diseases. Among these issues are disclosing sources of infection, quantifying recent transmission, identifying transmission links, discerning reinfection from relapse, tracking the geographic distribution and clonal expansion of specific strains, and exploring the genetic mechanisms underlying specific phenotypic traits, including virulence, organ tropism, transmissibility, or drug resistance. Since genotyping continues to unravel the biology of mycobacteria, it offers enormous promise in the fight against and prevention of the diseases caused by these pathogens. In this review, molecular typing methods for Mycobacterium tuberculosis and nontuberculous mycobacteria elaborated over the last 2 decades are summarized. The relevance of these methods to the epidemiological investigation, diagnosis, evolution, and control of mycobacterial diseases is discussed.
Among various genotyping methods to study Mycobacterium tuberculosis complex (MTC) genotypic polymorphism, spoligotyping and mycobacterial interspersed repetitive units–variable number of DNA tandem ...repeats (MIRU–VNTRs) have recently gained international approval as robust, fast, and reproducible typing methods generating data in a portable format. Spoligotyping constituted the backbone of a publicly available database SpolDB4 released in 2006; nonetheless this method possesses a low discriminatory power when used alone and should be ideally used in conjunction with a second typing method such as MIRU–VNTRs for high-resolution epidemiological studies. We hereby describe a publicly available international database named SITVITWEB which incorporates such multimarker data allowing to have a global vision of MTC genetic diversity worldwide based on 62,582 clinical isolates corresponding to 153 countries of patient origin (105 countries of isolation). We report a total of 7105 spoligotype patterns (corresponding to 58,180 clinical isolates) – grouped into 2740 shared-types or spoligotype international types (SIT) containing 53,816 clinical isolates and 4364 orphan patterns. Interestingly, only 7% of the MTC isolates worldwide were orphans whereas more than half of SITed isolates (n=27,059) were restricted to only 24 most prevalent SITs. The database also contains a total of 2379 MIRU patterns (from 8161 clinical isolates) from 87 countries of patient origin (35 countries of isolation); these were grouped in 847 shared-types or MIRU international types (MIT) containing 6626 isolates and 1533 orphan patterns. Lastly, data on 5-locus exact tandem repeats (ETRs) were available on 4626 isolates from 59 countries of patient origin (22 countries of isolation); a total of 458 different VNTR patterns were observed – split into 245 shared-types or VNTR International Types (VIT) containing 4413 isolates) and 213 orphan patterns. Datamining of SITVITWEB further allowed to update rules defining MTC genotypic lineages as well to have a new insight into MTC population structure and worldwide distribution at country, sub-regional and continental levels. At evolutionary level, the data compiled may be useful to distinguish the occasional convergent evolution of genotypes versus specific evolution of sublineages essentially influenced by adaptation to the host. This database is publicly available at: http://www.pasteur-guadeloupe.fr:8081/SITVIT_ONLINE.
Tuberculosis (TB) remains broadly present in the Americas despite intense global efforts for its control and elimination. Starting from a large dataset comprising spoligotyping (n = 21183 isolates) ...and 12-loci MIRU-VNTRs data (n = 4022 isolates) from a total of 31 countries of the Americas (data extracted from the SITVIT2 database), this study aimed to get an overview of lineages circulating in the Americas. A total of 17119 (80.8%) strains belonged to the Euro-American lineage 4, among which the most predominant genotypic family belonged to the Latin American and Mediterranean (LAM) lineage (n = 6386, 30.1% of strains). By combining classical phylogenetic analyses and Bayesian approaches, this study revealed for the first time a clear genetic structuration of LAM9 sublineage into two subpopulations named LAM9C1 and LAM9C2, with distinct genetic characteristics. LAM9C1 was predominant in Chile, Colombia and USA, while LAM9C2 was predominant in Brazil, Dominican Republic, Guadeloupe and French Guiana. Globally, LAM9C2 was characterized by higher allelic richness as compared to LAM9C1 isolates. Moreover, LAM9C2 sublineage appeared to expand close to twenty times more than LAM9C1 and showed older traces of expansion. Interestingly, a significant proportion of LAM9C2 isolates presented typical signature of ancestral LAM-RDRio MIRU-VNTR type (224226153321). Further studies based on Whole Genome Sequencing of LAM strains will provide the needed resolution to decipher the biogeographical structure and evolutionary history of this successful family.
Ancient sublineage of the Mycobacterium tuberculosis Beijing genotype is endemic and prevalent in East Asia and rare in other world regions. While these strains are mainly drug susceptible, we ...recently identified a novel clonal group Beijing 1071-32 within this sublineage emerging in Siberia, Russia and present in other Russian regions. This cluster included only multi/extensive drug resistant (MDR/XDR) isolates. Based on the phylogenetic analysis of the available WGS data, we identified three synonymous SNPs in the genes Rv0144, Rv0373c, and Rv0334 that were specific for the Beijing 1071-32-cluster and developed a real-time PCR assay for their detection. Analysis of the 2375 genetically diverse M. tuberculosis isolates collected between 1996 and 2020 in different locations (European and Asian parts of Russia, former Soviet Union countries, Albania, Greece, China, Vietnam, Japan and Brazil), confirmed 100% specificity and sensitivity of this real-time PCR assay. Moreover, the epidemiological importance of this strain and the newly developed screening assay is further stressed by the fact that all identified Beijing 1071-32 isolates were found to exhibit MDR genotypic profiles with concomitant resistance to additional first-line drugs due to a characteristic signature of six mutations in rpoB450, rpoC485, katG315, katG335, rpsL43 and embB497. In conclusion, this study provides a set of three concordant SNPs for the detection and screening of Beijing 1071-32 isolates along with a validated real-time PCR assay easily deployable across multiple settings for the epidemiological tracking of this significant MDR cluster.
Tuberculosis affects vulnerable groups to a greater degree, indigenous population among them.
To determine molecular epidemiology of clinical isolates of Mycobacterium tuberculosis circulating in an ...indigenous population through Spoligotyping and 24-loci MIRU-VNTR.
A descriptive cross-sectional study was conducted in 23 indigenous communities of Puerto Nariño-Amazonas, Colombia. Recovered clinical isolates were genotyped. For genotyping analyzes global SITVIT2 database and the MIRU-VNTRplus web portal were used.
74 clinical isolates were recovered. Genotyping of clinical isolates by spoligotyping determined 5 different genotypes, all of them belonged to Euro-American lineage. By MIRU-VNTR typing, a total of 14 different genotypes were recorded. Furthermore, polyclonal infection was found in two patients from the same community. The combination of the two methodologies determined the presence of 19 genotypes, 8 formed clusters with 63 clinical isolates in total. Based on epidemiological information, it was possible to establish a potential chain of active transmission in 10/63 (15.9%) patients.
High genomic homogeneity was determined in the indigenous population suggesting possible chains of active transmission. The results obtained showed that specific genotypes circulating among the indigenous population of Colombia are significantly different from those found in the general population.
Nontuberculous mycobacterial pulmonary disease (NTM-PD) has become an emerging infectious disease and is responsible for more deaths than tuberculosis in industrialized countries. NTM-PD mortality ...remains high in some series reportedly ranging from 25% to 40% at five years and often due to unfavorable evolution of NTM-PD despite established treatment. The purpose of our study was to search for early factors that could predict the favorable or unfavorable evolution of NTM-PD at the first year of treatment.
In this retrospective and multicenter study, we selected 119 patients based on clinical, radiological and microbiological data from 2002 to 2012 from three French university hospitals (Guadeloupe, Martinique, Montpellier) with definite (meeting the criteria of the American Thoracic Society and the Infectious Disease Society of America in 2007; ATS/IDSA) or probable (one positive sputum culture) NTM-PD. We compared two patient groups: those who improved at one year (clinical symptoms, radiological lesions and microbiology data) and those who did not improve at one year. The data were analyzed for all patients as well as for subgroups by gender, HIV-positive patients, and Mycobacterium avium complex (MAC) infection.
The average patient age was 50 years ± 19.4; 58% had respiratory comorbidities, 24% were HIV positive and 19% had cystic fibrosis. Coughing concerned 66% of patients and bronchiectasis concerned 45%. The most frequently isolated NTM were MAC (46%). 57% (n = 68) of patients met the ATS criteria and improved status concerned 38.6% (n = 46). The improvement factors at one year of NTM-PD were associated with the duration of ethambutol treatment: (Odds ratio adjusted ORa: 2.24, 95% Confidence interval CI; 2.11-3.41), HIV-positive status: (ORa: 3.23, 95% CI; 1.27-8.45), and male gender: (ORa: 2.34, 95% CI; 1.26-8.16). For the group with NTM-PD due to MAC, improvement was associated with the duration of macrolide treatment (ORa: 3.27, 95% CI; 1.88-7.30) and an age <50 years (ORa: 1.88, 95% CI; 1.55-8.50).
In this retrospective multicenter study, improvement at one year in patients with definite or probable NTM-PD was associated with the duration of ethambutol treatment, HIV-positive status and male gender. For the group of patients infected with MAC, improvement was associated with the duration of macrolide treatment and an age <50 years. Identifying predictors of improvement at one year of NTM-PD is expected to optimize the management of the disease in its early stages.
Molecular typing based on 12 loci containing variable numbers of tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTRs) has been adopted in combination with spoligotyping as the ...basis for large-scale, high-throughput genotyping of Mycobacterium tuberculosis. However, even the combination of these two methods is still less discriminatory than IS6110 fingerprinting. Here, we define an optimized set of MIRU-VNTR loci with a significantly higher discriminatory power. The resolution and the stability/robustness of 29 loci were analyzed, using a total of 824 tubercle bacillus isolates, including representatives of the main lineages identified worldwide so far. Five loci were excluded for lack of robustness and/or stability in serial isolates or isolates from epidemiologically linked patients. The use of the 24 remaining loci increased the number of types by 40%--and by 23% in combination with spoligotyping--among isolates from cosmopolitan origins, compared to those obtained with the original set of 12 loci. Consequently, the clustering rate was decreased by fourfold--by threefold in combination with spoligotyping--under the same conditions. A discriminatory subset of 15 loci with the highest evolutionary rates was then defined that concentrated 96% of the total resolution obtained with the full 24-locus set. Its predictive value for evaluating M. tuberculosis transmission was found to be equal to that of IS6110 restriction fragment length polymorphism typing, as shown in a companion population-based study. This 15-locus system is therefore proposed as the new standard for routine epidemiological discrimination of M. tuberculosis isolates and the 24-locus system as a high-resolution tool for phylogenetic studies.
Molecular epidemiological studies of Mycobacterium tuberculosis (MTB) are the core of current research to find out the association of the M. tuberculosis genotypes with its outbreak and transmission. ...The high prevalence of the Beijing genotype strain among multidrug resistance (MDR) TB has already been reported in various studies around India. The overall objective of this study was to detect the prevalence of Beijing genotype strains of MDR M. tuberculosis and their association with the clinical characteristics of TB patients. In this study 381 M. tuberculosis clinical isolates were obtained from sputum samples from 2008 to 2014. The multiplex-PCR and Spoligotyping (n = 131) methods were used to investigate the prevalence of the Beijing genotype strain by targeting the Rv2820 gene and their association with drug resistance and clinical characteristics of TB patients. The drug susceptibility testing of first-line anti-TB drugs was performed by using the proportion method and MGIT960. A collection of isolates having Beijing and non-Beijing strains were also characterized to see if Beijing genotype strains had a higher rate of mutations at codons 516, 526 and 531 of the 81-bp region of the rpoB gene, codon 315 of the katG gene, and codon 306 of the embB gene. The sensitivities and specificities of multiplex-PCR assay compared to that of standard Spoligotyping was detected to be 100%. Further, we observe that the multi drug-resistance was significantly associated with Beijing genotype strains (p = 0.03) and a strong correlation between Beijing genotype strains and specific resistance mutations at the katG315, rpoB531, and embB306 codons (p = < 0.0001, < 0.0001 & 0.0014 respectively) was also found. This rapid, simple, and cost-effective multiplex PCR assay can effectively be used for monitoring the prevalence of Beijing genotype strains in low resource settings. Findings of this study may provide a scientific basis for the development of new diagnostic tools for detection and effective management of DR-TB in countries with a higher incidence rate of Beijing genotype strains.
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