Local adaptive evolution of two distinct clades of Beijing and T families of Mycobacterium tuberculosis in Chongqing: a Bayesian population structure and phylogenetic study.

BACKGROUND: Beijing sub-pedigree 2 (BSP2) and T sub-lineage 6 (TSL6) are two clades belonging to Beijing and T family of Mycobacterium tuberculosis (MTB), respectively, defined by Bayesian population structure analysis based on 24-loci mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTR). Globally, over 99% of BSP2 and 89% of TSL6 isolates were distributed in Chongqing, suggesting their possible local adaptive evolution. The objective of this paper is to explore whether BSP2 and TSL6 originated by their local adaptive evolution from the specific isolates of Beijing and T families in Chongqing. METHODS: The genotyping data of 16 090 MTB isolates were collected from laboratory collection, published literatures and SITVIT database before subjected to Bayesian population structure analysis based on 24-loci MIRU-VNTR. Spacer Oligonucleotide Forest (Spoligoforest) and 24-loci MIRU-VNTR-based minimum spanning tree (MST) were used to explore their phylogenetic pathways, with Bayesian demographic analysis for exploring the recent demographic change of TSL6. RESULTS: Phylogenetic analysis suggested that BSP2 and TSL6 in Chongqing may evolve from BSP4 and TSL5, respectively, which were locally predominant in Tibet and Jiangsu, respectively. Spoligoforest showed that Beijing and T families were genetically distant, while the convergence of the MIRU-VNTR pattern of BSP2 and TSL6 was revealed by WebLogo. The demographic analysis concluded that the recent demographic change of TSL6 might take 111.25 years. CONCLUSIONS: BSP2 and TSL6 clades might originate from BSP4 and TSL5, respectively, by their local adaptive evolution in Chongqing. Our study suggests MIRU-VNTR be combined with other robust markers for a more comprehensive genotyping approach, especially for families of clades with the same MIRU-VNTR pattern.

Genetic diversity of Mycobacterium tuberculosis clinical isolates from HIV-TB patients from two public hospitals at Bogotá, Colombia.

The co-infection of TB/HIV is an increasing problem for public health worldwide. In Colombia, of 13.871 confirmed cases of TB in 2016 (prevalence of 0,028%) 14% correspond to HIV co-infection. However, we have scarce information regarding genetic diversity of strains infecting HIV patients. In this study, we carried-out an active search of cases of TB in 356 HIV-infected individuals, who were enrolled in two Public Hospitals at Bogotá-Colombia, between 2014 and 2015. We found 49 patients with HIV-TB co-infection. Genetic characterization of Mycobacterium tuberculosis (Mtb) isolates from these patients showed a predominance of three major sub-lineages: Haarlem (n = 26), LAM (n = 12) and T (n = 11). Remarkably, the most predominant pattern in the present study (SIT62/H1, n = 11) is very specific to this country. Indeed, taking in account distribution in countries with at least 3% of SIT62/H1, 36% of all such patterns collected worldwide were from Colombia. Furthermore, Colombia alone is responsible for almost all the SIT62/H1 strains in South America, suggesting a successful transmission of this genotype inside TB/HIV population from Colombia.

Macro-geographical specificities of the prevailing tuberculosis epidemic as seen through SITVIT2, an updated version of the Mycobacterium tuberculosis genotyping database.

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.

Molecular epidemiology of Mycobacterium tuberculosis strains from prison populations in Santa Catarina, Southern Brazil.

The Tuberculosis (TB) notification rates are 5 to 81 times higher in prisons worldwide when compared to the general population. The state of Santa Catarina (SC) has few epidemiological data regarding TB in prisons. The aim of this study was to evaluate the molecular epidemiology of circulating strains in prisons of SC. The study comprised 95 clinical samples from six prisons. Among the cases included, all subjects were male, predominantly caucasians, and young adults, with low education level. The positive smear in the TB diagnosis comprised 62.0% of cases. About 50% of subjects had some condition associated with TB. The Spoligotyping results showed that the most frequent lineages were LAM (50.7%), T (22.2%) and S (11.6%). The 12-loci MIRU generated 62 different genotypes. The MSTs showed evolutionary relationships between Mycobacterium tuberculosis spoligotypes from SC and evolutionary relationships between the prison isolates and studied parameters. This first study on TB in prison units of SC highlighted the predominance of SIT216/LAM5, and SIT34/S. Interestingly, his profile was found to be different from that observed in a previous study performed with the state's general population. This data shows the need for continued surveillance of episodes of TB occurring among prison inmates in an emerging country like Brazil.

New Mycobacterium tuberculosis Beijing clonal complexes in China revealed by phylogenetic and Bayesian population structure analyses of 24-loci MIRU-VNTRs.

Beijing lineage of Mycobacterium tuberculosis constitutes the most predominant lineage in East Asia. Beijing epidemiology, evolutionary history, genetics are studied in details for years revealing probable origin from China followed by worldwide expansion, partially linked to higher mutation rate, hypervirulence, drug-resistance, and association with cases of mixed infections. Considering huge amount of data available for 24-loci Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats, we performed detailed phylogenetic and Bayesian population structure analyses of Beijing lineage strains in mainland China and Taiwan using available 24-loci MIRU-VNTR data extracted from publications or the SITVIT2 database (n = 1490). Results on genetic structuration were compared to previously published data. A total of three new Beijing clonal complexes tentatively named BSP1, BPS2 and BSP3 were revealed with surprising phylogeographical specificities to previously unstudied regions in Sichuan, Chongqing and Taiwan, proving the need for continued investigations with extended datasets. Such geographical restriction could correspond to local adaptation of these "ecological specialist" Beijing isolates to local human host populations in contrast with "generalist pathogens" able to adapt to several human populations and to spread worldwide.

SpolSimilaritySearch - A web tool to compare and search similarities between spoligotypes of Mycobacterium tuberculosis complex.

Spoligotyping is one of the most commonly used polymerase chain reaction (PCR)-based methods for identification and study of genetic diversity of Mycobacterium tuberculosis complex (MTBC). Despite its known limitations if used alone, the methodology is particularly useful when used in combination with other methods such as mycobacterial interspersed repetitive units - variable number of tandem DNA repeats (MIRU-VNTRs). At a worldwide scale, spoligotyping has allowed identification of information on 103,856 MTBC isolates (corresponding to 98049 clustered strains plus 5807 unique isolates from 169 countries of patient origin) contained within the SITVIT2 proprietary database of the Institut Pasteur de la Guadeloupe. The SpolSimilaritySearch web-tool described herein (available at: http://www.pasteur-guadeloupe.fr:8081/SpolSimilaritySearch) incorporates a similarity search algorithm allowing users to get a complete overview of similar spoligotype patterns (with information on presence or absence of 43 spacers) in the aforementioned worldwide database. This tool allows one to analyze spread and evolutionary patterns of MTBC by comparing similar spoligotype patterns, to distinguish between widespread, specific and/or confined patterns, as well as to pinpoint patterns with large deleted blocks, which play an intriguing role in the genetic epidemiology of M. tuberculosis. Finally, the SpolSimilaritySearch tool also provides with the country distribution patterns for each queried spoligotype.

A snapshot of the predominant single nucleotide polymorphism cluster groups of Mycobacterium tuberculosis clinical isolates in Delhi, India.

Several attempts have been made to associate phylogenetic differences among Mycobacterium tuberculosis strains to variations in the clinical outcome of the disease and to drug resistance. We genotyped 139 clinical isolates of M. tuberculosis obtained from patients of pulmonary tuberculosis in North Delhi region. The isolates were analyzed using nine Single nucleotide polymorphism (SNP) markers, spoligotyping and MIRU-VNTRs; and the results were correlated with their drug susceptibility profile. Results of SNP cluster group (SCG) analysis (available for 138 isolates) showed that the most predominant cluster was SCG 3a, observed in 58.7% (81/138) of the isolates with 44.4% (36/81) of these being drug susceptible, while 16% (13/81) were multidrug resistant (MDR). Of the ancestral cluster SCG 1 observed in 19.5% (27/138) of the isolates, 14.8% (4/27) were MDR while 44.4% (12/27) were drug susceptible. SCG 2 formed 5.79% (8/138) of the isolates and 50% (4/8) of these were multidrug resistant (MDR). Spoligotyping subdivided the strains into 45 shared types (n = 125) and 14 orphan strains. The orphan strains were mostly associated with SCG 3a or SCG 1, reflecting the principal SCGs found in the Indian population. SCG 1 and SCG 2 genotypes were concordant with the East African Indian (EAI) and Beijing families respectively. Central Asian (CAS) clade and its sublineages were predominantly associated with SCG 3a. No consistent association was seen between the SCGs and Harlem, T or X clades. The 15 loci MIRU-VNTR typing revealed 123/136 isolates to be unclustered, while 13 isolates were present in 6 clusters of 2-3 isolates each. However, correlating the cluster analysis with patient details did not suggest any evidence of recent transmission. In conclusion, though our study revealed the preponderance of SCG 1 and 3a in the M. tuberculosis population circulating in the region, the diversity of strains highlights the changes occurring within lineages and reemphasizes the importance of cluster investigations in extended studies.

Contribution of spoligotyping and MIRU-VNTRs to characterize prevalent Mycobacterium tuberculosis genotypes infecting tuberculosis patients in Morocco.

In the present study, Mycobacterium tuberculosis complex (MTBC) clinical isolates from culture-positive TB patients in Morocco were studied by spoligotyping and 12-loci MIRU-VNTR typing methods to characterize prevalent genotypes (n = 219 isolates from 208 patients). Spoligotyping resulted in 39 unique patterns and 167 strains in 30 clusters (2-50 strains per cluster). Comparison with international database showed that 29 of 39 unique patterns matched existing shared spoligotype international types (SITs). Nine shared types containing 10 strains were newly created (SIT 2891 to SIT 2899); this led to the description of 69 SITs with 206 strains and two orphan patterns. The most prevalent spoligotype was SIT42 (LAM; n = 50 or 24% of isolates). The repartition of strains according to major MTBC clades was as follows LAM (46.1%)> Haarlem (26%) >ill-defined T superfamily (22.6%) and S clade (0.96%). On the other hand, Beijing, CAS (Central Asian) and EAI (East-African Indian) strains were absent in this setting. Subsequent 12-Loci MIRU typing resulted in a total of 25 SIT/MIT clusters (n = 66 isolates, 2-6 isolates per cluster), with a resulting recent transmission rate of 22.3%. The MIRU-VNTR patterns corresponded to 69 MITs for 138 strains and 46 orphan patterns. The most frequent patterns were MIT43 (n = 8), MIT9 (n = 7) and MIT42 (n = 7). HGDI analysis of the 12 MIRU loci showed that loci 10, 23 and 40 were highly discriminative in our setting. The results also underlined the usefulness of spoligotyping and MIRU-VNTR to detect mixed infections among certain of our TB patients. Globally, the results obtained showed that TB is almost exclusively transmitted in Morocco through evolutionary-modern MTBC lineages belonging to principal genetic groups 2/3 strains (Haarlem, LAM, T), with a high level of biodiversity seen by MIRU typing. This study provides with a 1st global snapshot of MTBC population structure in Morocco, and validates the potential use of spoligotyping in conjunction with minisatellites for future investigations in Morocco that should in future ideally include optimized 15- or 24-loci MIRU-VNTRs.

Epidemic spread of multidrug-resistant tuberculosis in Johannesburg, South Africa.

Numerous reports have documented isolated transmission events or clonal outbreaks of multidrug-resistant Mycobacterium tuberculosis strains, but knowledge of their epidemic spread remains limited. In this study, we evaluated drug resistance, strain diversity, and clustering rates in patients diagnosed with multidrug-resistant (MDR) tuberculosis (TB) at the National Health Laboratory Service (NHLS) Central TB Laboratory in Johannesburg, South Africa, between March 2004 and December 2007. Phenotypic drug susceptibility testing was done using the indirect proportion method, while each isolate was genotyped using a combination of spoligotyping and 12-MIRU typing (12-locus multiple interspersed repetitive unit typing). Isolates from 434 MDR-TB patients were evaluated, of which 238 (54.8%) were resistant to four first-line drugs (isoniazid, rifampin, ethambutol, and streptomycin). Spoligotyping identified 56 different strains and 28 clusters of variable size (2 to 71 cases per cluster) with a clustering rate of 87.1%. Ten clusters included 337 (77.6%) of all cases, with strains of the Beijing genotype being most prevalent (16.4%). Combined analysis of spoligotyping and 12-MIRU typing increased the discriminatory power (Hunter Gaston discriminatory index [HGDI] = 0.962) and reduced the clustering rate to 66.8%. Resolution of Beijing genotype strains was further enhanced with the 24-MIRU-VNTR (variable-number tandem repeat) typing method by identifying 15 subclusters and 19 unique strains from twelve 12-MIRU clusters. High levels of clustering among a variety of strains suggest a true epidemic spread of MDR-TB in the study setting, emphasizing the urgency of early diagnosis and effective treatment to reduce transmission within this community.

MLVA based classification of Mycobacterium tuberculosis complex lineages for a robust phylogeographic snapshot of its worldwide molecular diversity.

Multiple-locus variable-number tandem repeat analysis (MLVA) is useful to establish transmission routes and sources of infections for various microorganisms including Mycobacterium tuberculosis complex (MTC). The recently released SITVITWEB database contains 12-loci Mycobacterial Interspersed Repetitive Units--Variable Number of Tandem DNA Repeats (MIRU-VNTR) profiles and spoligotype patterns for thousands of MTC strains; it uses MIRU International Types (MIT) and Spoligotype International Types (SIT) to designate clustered patterns worldwide. Considering existing doubts on the ability of spoligotyping alone to reveal exact phylogenetic relationships between MTC strains, we developed a MLVA based classification for MTC genotypic lineages. We studied 6 different subsets of MTC isolates encompassing 7793 strains worldwide. Minimum spanning trees (MST) were constructed to identify major lineages, and the most common representative located as a central node was taken as the prototype defining different phylogenetic groups. A total of 7 major lineages with their respective prototypes were identified: Indo-Oceanic/MIT57, East Asian and African Indian/MIT17, Euro American/MIT116, West African-I/MIT934, West African-II/MIT664, M. bovis/MIT49, M.canettii/MIT60. Further MST subdivision identified an additional 34 sublineage MIT prototypes. The phylogenetic relationships among the 37 newly defined MIRU-VNTR lineages were inferred using a classification algorithm based on a bayesian approach. This information was used to construct an updated phylogenetic and phylogeographic snapshot of worldwide MTC diversity studied both at the regional, sub-regional, and country level according to the United Nations specifications. We also looked for IS6110 insertional events that are known to modify the results of the spoligotyping in specific circumstances, and showed that a fair portion of convergence leading to the currently observed bias in phylogenetic classification of strains may be traced back to the presence of IS6110. These results shed new light on the evolutionary history of the pathogen in relation to the history of peopling and human migration.

SITVITWEB--a publicly available international multimarker database for studying Mycobacterium tuberculosis genetic diversity and molecular epidemiology.

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.

Distribution of spoligotyping defined genotypic lineages among drug-resistant Mycobacterium tuberculosis complex clinical isolates in Ankara, Turkey.

BACKGROUND: Investigation of genetic heterogeneity and spoligotype-defined lineages of drug-resistant Mycobacterium tuberculosis clinical isolates collected during a three-year period in two university hospitals and National Tuberculosis Reference and Research Laboratory in Ankara, Turkey. METHODS AND FINDINGS: A total of 95 drug-resistant M. tuberculosis isolates collected from three different centers were included in this study. Susceptibility testing of the isolates to four major antituberculous drugs was performed using proportion method on Löwenstein-Jensen medium and BACTEC 460-TB system. All clinical isolates were typed by using spoligotyping and IS6110-restriction fragment length polymorphism (RFLP) methods. Seventy-three of the 95 (76.8%) drug resistant M. tuberculosis isolates were isoniazid-resistant, 45 (47.4%) were rifampicin-resistant, 32 (33.7%) were streptomycin-resistant and 31 (32.6%) were ethambutol-resistant. The proportion of multidrug-resistant isolates (MDR) was 42.1%. By using spoligotyping, 35 distinct patterns were observed; 75 clinical isolates were grouped in 15 clusters (clustering rate of 79%) and 20 isolates displayed unique patterns. Five of these 20 unique patterns corresponded to orphan patterns in the SITVIT2 database, while 4 shared types containing 8 isolates were newly created. The most prevalent M. tuberculosis lineages were: Haarlem (23/95, 24.2%), ill-defined T superfamily (22/95, 23.2%), the Turkey family (19/95, 20%; previously designated as LAM7-TUR), Beijing (6/95, 6.3%), and Latin-America & Mediterranean (LAM, 5/95 or 5.3%), followed by Manu (3/95, 3.2%) and S (1/95, 1%) lineages. Four of the six Beijing family isolates (66.7%) were MDR. A combination of IS6110-RFLP and spoligotyping reduced the clustering rate from 79% to 11.5% among the drug resistant isolates. CONCLUSIONS: The results obtained showed that ill-defined T, Haarlem, the Turkey family (previously designated as LAM7-TUR family with high phylogeographical specifity for Turkey), Beijing and LAM were predominant lineages observed in almost 80% of the drug-Resistant M. tuberculosis complex clinical isolates in Ankara, Turkey.

Spoligotypes of Mycobacterium tuberculosis complex isolates from patients residents of 11 states of Brazil.

One of the high tuberculosis (TB) incidence countries in the world, Brazil is characterized by considerable differences in TB incidence on regional and state level. In the present study, we describe Brazilian spoligotypes of 1991 Mycobacterium tuberculosis complex (MTC) clinical isolates from patients residents of 11 states from different regions of the country, diagnosed between 1996 and 2005. By performing spoligotyping on a large number of M. tuberculosis clinical isolates, one of the main objectives of this study was to determine the major genotype families causing TB in Brazil and to verify the region-associated genotype distribution. We observed a total of 577 distinct spoligopatterns, 12.6% of these corresponded to orphan patterns while 87.4% belonged to 326 shared-types (SITs). Among the latter, 86 SITs (isolated from 178 patients) had been observed for the first time in this study, the most frequent being SIT2517 which belonged to the T3-ETH lineage and was exclusively found among patients residents of Belém, the capital of the state of Pará (n=8 isolates). Irrespective of shared-type labeling, a total of 19.5% strains were unique (unclustered) in our study as opposed to 80.5% clustered isolates (189 clusters, size range from 2 to 205 isolates). The three largest clusters were SIT42 of the Latin-America & Mediterranean (LAM) 9 clade (10.3%), SIT53 of the T clade (7.6%), and SIT50 of the Haarlem clade (5.4%). The predominant MTC lineages in Brazil in decreasing order belonged to the LAM (46%); the ill-defined T (18.6%); the Haarlem (12.2%), the X (4.7%), the S (1.9%), and the East African Indian (EAI) (0.85%) families. The rest of clades grouped together as Mycobacterium africanum, Mycobacterium bovis, Beijing, Central Asian (CAS), and the Manu types, represented less than 1% of the strains. Finally, about 15% of the isolates showed spoligotype signatures that were not yet classified among well-defined lineages. In conclusion, we provide hereby a first insight into the population structure of MTC isolates in Brazil, showing the predominance of both LAM and T family and the existence of region-associated genotypes.

A first insight on the population structure of Mycobacterium tuberculosis complex as studied by spoligotyping and MIRU-VNTRs in Bogotá, Colombia.

With an incidence of 25.6/100,000 in 2008, tuberculosis (TB) remains an important public health problem in Colombia. In this study, a total of 152 Mycobacterium tuberculosis complex strains isolated in Bogotá, Colombia between years 1995 and 2007 were genotyped by spoligotyping and 12-loci MIRU-VNTRs. The various spoligotyping-based genotypic lineages in our sample were: Latin American & Mediterranean (LAM) n=75, 49.34%; Haarlem, n=38, 25.0%; ill-defined T group, n=21, 13.82%; S family, n=5, 3.29%; X clade, n=2, 1.32%; Beijing, n=1, 0.65%, while strains with unknown signatures (n=10) represented 6.58% of isolates. Using spoligotyping as a first molecular marker and MIRU-VNTRs as second marker, we obtained 102 single patterns and 14 clustered patterns (n=52 strains from 49 patients, 2-8 strains per cluster). The MIRU-VNTRs patterns corresponded to 50 MITs for 109 strains and 43 orphan patterns. The most frequent patterns were MIT190 (n=12), MIT45 (n=10), and MIT25 (n=9). The Hunter & Gaston discriminatory index (HGDI) of both methodologies used together showed a value of 0.992. In our setting, the HGDI of five loci subset (MIRU10, 16, 23, 26 and 40) contributed most to the discriminatory power of 12-loci format used (HGDI=0.977). The lineage distribution of M. tuberculosis showed that more than 3/4 of strains in Bogotá are commonly found in Latin America, Caribbean, and Europe. This observation might reflect the shared post-Columbus history of Colombia and its Latin-American neighbors as well as strains brought in by 20th century immigrants from Europe. We also demonstrate the usefulness of MIRU-VNTR to detect suspected links among patients and polyclonal infections.

Molecular epidemiology and genetic diversity of Mycobacterium tuberculosis complex in the Cross River State, Nigeria.

This study provides with a first insight on Mycobacterium tuberculosis complex epidemiology and genetic diversity in the Cross River State, Nigeria. Starting with 137 smear positive patients recruited over a period of 12months (June 2008 to May 2009), we obtained 97 pure mycobacterial isolates out of which 81 (83.5%) were identified as M. tuberculosis complex. Genotyping revealed a total of 27 spoligotypes patterns with 10 clusters (n=64% or 79% of clustered isolates, 2-32 isolates/cluster), with patients in the age group range 25-34 years being significantly associated with shared-type pattern SIT61 (p=0.019). Comparison with SITVIT2 database showed that with the exception of a single cluster (SIT727/H1), all other clusters observed were representative of West Africa; the two main lineages involved were LAM10-CAM (n=42/81% or 51.8%) of M. tuberculosis and AFRI_2 sublineage of Mycobacterium africanum (n=27/81% or 33.3%). Subsequent 12-loci MIRU typing resulted in a total of 13 SIT/MIT clusters (n=52 isolates, 2-9 isolates per cluster), with a resulting recent n-1 transmission rate of 48.1%. Available drug-susceptibility testing (DST) results for 58/81 clinical isolates revealed 6/58% or 10.4% cases of multiple drug-resistance (MDR); 5/6 MDR cases were caused by strains belonging to LAM10-CAM lineage (a specific cluster SIT61/MIT266 in 4/6 cases, and an orphan spoligotype pattern in 1/6 case). Additionally, MIT266 was associated with streptomycin resistance (p=0.016). All the six MDRTB isolates were concomitantly resistance to streptomycin and ethambutol; however, 4/6 MDR strains with identical MIRU patterns were characterized by consecutive strain numbers hence the possibility of laboratory cross contamination could not be excluded in 3/4 serial cases. The present preliminary study underlines the usefulness of spoligotyping and 12-loci MIRU-VNTRs to establish a baseline of circulating genotypic lineages of M. tuberculosis complex in Nigeria.

Comparison of spoligotyping, mycobacterial interspersed repetitive units typing and IS6110-RFLP in a study of genotypic diversity of Mycobacterium tuberculosis in Delhi, North India.

The aim of the present study was to compare polymerase chain reaction (PCR)-based methods--spoligotyping and mycobacterial interspersed repetitive units (MIRU) typing--with the gold-standard IS6110 restriction fragment length polymorphism (RFLP) analysis in 101 isolates of Mycobacterium tuberculosis to determine the genetic diversity of M. tuberculosis clinical isolates from Delhi, North India. Spoligotyping resulted in 49 patterns (14 clusters); the largest cluster was composed of Spoligotype International Types (SITs)26 [Central-Asian (CAS)1-Delhi lineage], followed by SIT11 [East-African-Indian (EAI) 3-Indian lineage]. A large number of isolates (75%) belonged to genotypic lineages, such as CAS, EAI and Manu, with a high specificity for the Indian subcontinent, emphasising the complex diversity of the phylogenetically coherent M. tuberculosis in North India. MIRU typing, using 11 discriminatory loci, was able to distinguish between all but two strains based on individual patterns. IS6110-RFLP analysis (n = 80 strains) resulted in 67 unique isolates and four clusters containing 13 strains. MIRUs discriminated all 13 strains, whereas spoligotyping discriminated 11 strains. Our results validate the use of PCR-based molecular typing of M. tuberculosis using repetitive elements in Indian isolates and demonstrate the usefulness of MIRUs for discriminating low-IS6110-copy isolates, which accounted for more than one-fifth of the strains in the present study.

Comparison of spoligotyping, mycobacterial interspersed repetitive units typing and IS6110-RFLP in a study of genotypic diversity of Mycobacterium tuberculosis in Delhi, North India

The aim of the present study was to compare polymerase chain reaction (PCR)-based methods - spoligotyping and mycobacterial interspersed repetitive units (MIRU) typing - with the gold-standard IS6110 restriction fragment length polymorphism (RFLP) analysis in 101 isolates of Mycobacterium tuberculosis to determine the genetic diversity of M. tuberculosis clinical isolates from Delhi, North India. Spoligotyping resulted in 49 patterns (14 clusters); the largest cluster was composed of Spoligotype International Types (SITs)26 [Central-Asian (CAS)1-Delhi lineage], followed by SIT11 [East-African-Indian (EAI) 3-Indian lineage]. A large number of isolates (75 percent) belonged to genotypic lineages, such as CAS, EAI and Manu, with a high specificity for the Indian subcontinent, emphasising the complex diversity of the phylogenetically coherent M. tuberculosis in North India. MIRU typing, using 11 discriminatory loci, was able to distinguish between all but two strains based on individual patterns. IS6110-RFLP analysis (n = 80 strains) resulted in 67 unique isolates and four clusters containing 13 strains. MIRUs discriminated all 13 strains, whereas spoligotyping discriminated 11 strains. Our results validate the use of PCR-based molecular typing of M. tuberculosis using repetitive elements in Indian isolates and demonstrate the usefulness of MIRUs for discriminating low-IS6110-copy isolates, which accounted for more than one-fifth of the strains in the present study.

Spoligotyping of clinical Mycobacterium tuberculosis isolates from the state of Minas Gerais, Brazil.

We performed spoligotyping on 114 strains of the Mycobacterium tuberculosis (Mtb) complex that had been isolated from patients in Minas Gerais Health Units during 2004. A total of 82/114 (72%) clinical isolates were clustered and 32/114 (28%) were unique. Seven shared types containing nine strains were newly created. A total of nine patterns corresponded to unreported orphan strains, as evaluated against all of the strains recorded in the SITVIT2 proprietary database in the Institut Pasteur de la Guadeloupe. The major clades were composed of isolates that belong to the following genotypes: Latin-America and Mediterranean (63/114, 55.3%) (the ill-defined T superfamily) (12/114, 10.5%), Haarlem (8/114, 7%), X clade (6/114, 5.3%), S clade (3/114, 2.6%) and the East-African Indian and Manu types, each with 1/114 (0.9%) isolates. A considerable number of strains (n = 20, 17.5%) showed patterns that did not fall within any of the previously described major clades. We conclude the bulk of tuberculosis (TB) (92/114, 80.7%) in our location is recent evolutionary strains that belong to the principal genetic groups 2/3. Further studies on epidemiology of TB are required to understand Mtb biodiversity and TB transmission in this region.

Finer snapshot of circulating Mycobacterium tuberculosis genotypes in Guadeloupe, Martinique, and French Guiana.

This study shows the benefit of spoligotyping coupled to mycobacterial interspersed repetitive-unit (MIRU) typing to pinpoint circulating Mycobacterium tuberculosis genotypes in Guadeloupe, Martinique, and French Guiana. We hereby propose reduced 4-locus and 6-locus subsets for LAM and Haarlem lineage strains that predominate in South America and the Caribbean, retaining 99.35% and 99.64% of the total discriminatory power of the 12-locus scheme, respectively.

Spoligotyping of clinical Mycobacterium tuberculosis isolates from the state of Minas Gerais, Brazil

We performed spoligotyping on 114 strains of the Mycobacterium tuberculosis (Mtb) complex that had been isolated from patients in Minas Gerais Health Units during 2004. A total of 82/114 (72 percent) clinical isolates were clustered and 32/114 (28 percent) were unique. Seven shared types containing nine strains were newly created. A total of nine patterns corresponded to unreported orphan strains, as evaluated against all of the strains recorded in the SITVIT2 proprietary database in the Institut Pasteur de la Guadeloupe. The major clades were composed of isolates that belong to the following genotypes: Latin-America and Mediterranean (63/114, 55.3 percent) (the ill-defined T superfamily) (12/114, 10.5 percent), Haarlem (8/114, 7 percent), X clade (6/114, 5.3 percent), S clade (3/114, 2.6 percent) and the East-African Indian and Manu types, each with 1/114 (0.9 percent) isolates. A considerable number of strains (n = 20, 17.5 percent) showed patterns that did not fall within any of the previously described major clades. We conclude the bulk of tuberculosis (TB) (92/114, 80.7 percent) in our location is recent evolutionary strains that belong to the principal genetic groups 2/3. Further studies on epidemiology of TB are required to understand Mtb biodiversity and TB transmission in this region.