SITVIT2 Database Research Articles

Genetic diversity of Mycobacterium tuberculosis strains isolated from spiritual holy water site attendees in Northwest Ethiopia. A cross-sectional study

BackgroundThe genetic diversity of Mycobacterium tuberculosis complex (MTBC) strains was characterized among isolates from individuals with pulmonary tuberculosis (PTB) symptoms attended holy water sites (HWSs) in the Amhara region, Ethiopia. MethodsA cross-sectional study was done from June 2019 to March 2020 to describe the genetic diversity and drug-resistance profiles of MTBC isolates. Sputum specimens were collected and cultured in the Löwenstein-Jensen culture medium. Line Probe Assay, MTBDRplus VER 2.0, and MTBDRsl VER 2.0 were used to detect first-and second-line anti-TB drug-resistance patterns. A spoligotyping technique was utilized to characterize the genetic diversity. Statistical analysis was performed using STATA 15. ResultsOf 560 PTB-symptomatic participants, 122 (21.8%) were culture-positive cases. Spoligotyping of 116 isolates revealed diverse MTBC sublineages, with four major lineages: Euro-American (EA) (Lineage 4), East-African-Indian (EAI) (Lineage 3), Ethiopian (ETH) (Lineage 7), East Asian (EA) (Lineage 2). The majority (96.6%) of the isolates were EA (lineage 4) and EAI, with proportions of 54.3% and 42.2%, respectively. A total of 31 spoligotype patterns were identified, 26 of which were documented in the SITVIT2 database. Of these, there were 15 unique spoligotypes, while eleven were grouped with 2-17 isolates. SIT149/T3-ETH (n = 17), SIT26/CAS1-DELHI (n = 16), SIT25/CAS1-DELHI (n = 12), and SIT52/T2 (n = 11) spoligotypes were predominant. A rare spoligotype pattern: SIT41/Turkey and SIT1/Beijing, has also been identified in North Shewa. The overall clustering rate of sub-lineages with known SIT was 76.4%.Of the 122 culture-positive isolates tested, 16.4% were resistant to rifampicin (RIF) and/or isoniazid (INH). Multidrug-resistant TB (MDR-TB) was detected in 12.3% of isolates, five of which were fluoroquinolones (FLQs) resistant. SIT149/T3-ETH and SIT21/CAS1-KILI sublineages showed a higher proportion of drug resistance. ConclusionsDiverse MTBC spoligotypes were identified, with the T and CAS families and EA (lineage 4) predominating. A high prevalence of drug-resistant TB, with SIT149/T3-ETH and CAS1-KILI sublineages comprising a greater share, was observed. A study with large sample size and a sequencing method with stronger discriminatory power is warranted to understand better the genetic diversity of circulating MTBC in this cohort of study, which would help to adopt targeted interventions.

Genetic diversity of Mycobacterium tuberculosis isolates from the central, eastern and southeastern Ethiopia

IntroductionThe population structure of Mycobacterium tuberculosis complex (MTBC) in Ethiopia is diverse but dominated by Euro-American (Lineage 4) and East-African-Indian (Lineage 3) lineages. The objective of this study was to describe the genetic diversity of MTBC isolates in Central, Eastern and Southeastern Ethiopia. MethodsA total of 223 MTBC culture isolates obtained from patients referred to Adama and Harar TB reference laboratories were spoligotyped. Demographic and clinical characteristics were collected. ResultsSix major lineages: Euro-American (Lineage 4), East-African-Indian (Lineage 3), East Asian (Lineage 2), Indo-Oceanic (Lineage 1), Mycobacterium africanum (Lineage 5 and Lineage 6) and Ethiopian (Lineage 7) were identified. The majority (94.6 %) of the isolates were Euro-American and East-African-Indian, with proportions of 75.3 % and 19.3 %, respectively. Overall, 77 different spoligotype patterns were identified of which 42 were registered in the SITVIT2 database. Of these, 27 spoligotypes were unique, while 15 were clustered with 2–49 isolates. SIT149/T3_ETH (n = 49), SIT53/T1 (n = 33), SIT21/CAS1_Kili (n = 24) and SIT41/Turkey (n = 11) were the dominant spoligotypes. A rare Beijing spoligotype pattern, SIT541, has also been identified in Eastern Ethiopia. The overall clustering rate of sub-lineages with known SIT was 71.3 %. Age group (25–34) was significantly associated with clustering. ConclusionWe found a heterogeneous population structure of MTBC dominated by T and CAS families, and the Euro-American lineage. The identification of the Beijing strain, particularly the rare SIT541 spoligotype in Eastern Ethiopia, warrants a heightened surveillance plan, as little is known about this genotype. A large-scale investigation utilizing a tool with superior discriminatory power, such as whole genome sequencing, is necessary to gain a thorough understanding of the genetic diversity of MTBC in the nation, which would help direct the overall control efforts.

Spoligotyping of Mycobacterium tuberculosis – Comparing in vitro and in silico approaches

Spoligotyping is one of the molecular typing methods widely used for exploring the genetic variety of Mycobacterium tuberculosis. The aim of this study was to compare the spoligoprofiles of M. tuberculosis clinical isolates, obtained using in vitro and in silico approaches.The study included 230 M. tuberculosis isolates, recovered from Poland and Lithuania between 2018 and 2021. Spoligotyping in vitro was performed with a commercially available kit. Whole genome sequencing (WGS) was done with Illumina NovaSeq 6000 sequencer. Spoligotype International Types (SITs) were assigned according to the SITVIT2 database or using three different in silico tools, and based on WGS data, namely SpoTyping, SpolPred, and lorikeet.Upon in vitro spoligotyping, the isolates produced 65 different spoligotypes. Spoligotypes inferred from the WGS data were congruent with in vitro generated patterns in 81.7% (188/230) for lorikeet and 81.3% (187/230) for SpolPred and SpoTyping. Spacers 18 and 31 produced the highest ratio of discrepant results between in vitro and in silico approaches, with their signals discordantly assigned for 15 (6.5%) and 9 (3.9%) isolates, respectively.All three in silico approaches used were similarly efficient for M. tuberculosis spoligotype prediction. However, only SpoTyping could predict spoligotypes without a need for manual curation. Thus, we consider it as the most accurate tool. Its use is further advocated by the shortest time of analysis. A relatively high (ca. 20%) discordance between in vitro and in silico spoligotyping results was observed. While we discourage comparing conventional spoligotyping with in silico equivalents, we advise the use of the latter, as it improves the accuracy of spoligopatterns, and thus depicts the relatedness between the isolates more reliably.

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

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

Spoligotyping of Clinical Isolates of Mycobacterium tuberculosis Complex Species in the Oromia Region of Ethiopia.

BackgroundTuberculosis (TB) is a leading cause of morbidity and mortality in Ethiopia. Investigation of the Mycobacterium tuberculosis complex (MTBC) species circulating in the Ethiopian population would contribute to the efforts made to control TB in the country. Therefore, this study was conducted to investigate the MTBC species and spoligo patterns in the Oromia region (central) of Ethiopia.MethodsA cross-sectional study design was used to recruit 450 smear positive pulmonary TB (PTB) cases from the Oromia region between September 2017 and August 2018. Mycobacteria were isolated from sputum samples on the Lowenstein Jensen (LJ) medium. Molecular identification of the isolates was performed by spoligotyping. The results of spoligotyping were transferred into a query box in the SITVIT2 database and Run TB-Lineage in the TB Insight website for the identification of spoligo international type (SIT) number and linages of the isolates, respectively. Statistical Product and Service Solutions (SPSS) 20 was applied for statistical analysis.ResultsThree hundred and fifteen isolates were grouped under 181 different spoligotype patterns. The most dominantly isolated spoligotype pattern was SIT149 and it consisted of 23 isolates. The majority of the isolates were grouped under Euro-American (EA), East-African-Indian (EAI), and Indo-Oceanic (IO) lineages. These lineages consisted of 79.4, 9.8, and 9.8% of the isolates, respectively. One hundred and sixty-five of the isolates were classified under 31 clustered spoligotypes whereas the remaining 150 were singleton types. Furthermore, 91.1% of the total isolates were classified as orphan types. Clustering of spoligotypes was associated (p < 0.001) with EAI lineage.ConclusionSIT149 and EA lineage were predominantly isolated from the Oromia region substantiating the findings of the similar studies conducted in other regions of Ethiopia. The observation of significant number of singleton and orphan spoligotypes warrants for additional genetic typing of the isolates using method(s) with a better discriminatory power than spoligotyping.

Genotypic diversity of multi- and pre-extremely drug-resistant Mycobacterium tuberculosis isolates from Morocco.

In Morocco, the prevalence of multidrug resistant tuberculosis (MDR-TB) continues to increase especially within previously treated cases; these MDR cases may evolve to extensively drug resistant tuberculosis (XDR-TB) raising major concern to TB control programs. From an epidemiological window, scarce informations are available about the genetic diversity of Mycobacterium tuberculosis (MTB) strains fueling these forms of resistance. The aim of this study was to assess to genetic diversity of MDR-MTB strains. Hence, this prospective study was conducted on patients diagnosed with MDR-TB at Pasteur Institute of Casablanca from 2010 to 2013. A total of 70 MDR-MTB isolates were genotyped by spoligotyping and 15-loci MIRU-VNTR methods. Spoligotyping generated four orphan patterns, five unique profiles whereas 61 strains were grouped in nine clusters (2 to 25 strains per cluster), the clustering rates being 87.1%. Subtyping by 15 loci MIRU-VNTR splitted all clusters already established by spoligotyping and generated 70 unique profiles not recognized in SITVIT2 database; clustering rate was equal to zero. HGDI analysis of 15 loci MIRU demonstrated that eight out of 15 loci were highly discriminant. Of note, all pre-XDR strains belongs to many clades, meaning that there no association between gyrA mutants and particular clade. Overall, the data generated by this study (i) describe the population structure of MDR MTBC in Morocco which is highly homogenous, (ii) confirm that TB in Morocco is almost exclusively transmitted by modern and evolutionary lineages with high level of biodiversity seen by MIRU, and (iii) validate the use of optimized 15-loci MIRU-VNTR format for future investigations in Morocco.

One-step melting curve analysis-based McSpoligotyping reveals genotypes of Mycobacterium tuberculosis in a coastal city, China.

A one-step 3h melting curve analysis-based technology for spacer oligonucleotide typing (McSpoligotyping) analyzed with the SITVIT2 database was applied to detect epidemiological events of Mycobacterium tuberculosis (MTB) in a coastal city of China from 2016 to 2018. 306 MTB isolates were identified by Fuzhou tuberculosis designated hospitals between 2016 and 2018. The results showed that the MTB isolates were divided into Beijing family and non-Beijing families, accounting for 45.42% (139/306) and 54.58% (167/306), respectively. H and T families were the prevalent genotypes in non-Beijing families. Herein, the spoligotyping technology has practical application for the classification and tracing of tuberculosis in the public.

Ubiquitous and multifaceted: SIT53 spoligotype does not correlate with any particular family of Mycobacterium tuberculosis

Euro-American lineage (Lineage 4) of Mycobacterium tuberculosis comprises genetically and geographically diverse families that differ in their clinical and/or epidemiological capacities. Due to the characteristic structure of the CRISPR locus (presence of almost all 43 classical spacers except for deleted signals 33–36), spoligotype SIT53 takes the basal position in the evolution of this lineage. In the SITVIT database, it is assigned to the “ill-defined” T family and T1 subfamily. Here, I analyzed the phylogenetic diversity of SIT53 isolates and discussed interconnected terminological issues concerning M. tuberculosis population structure. The 24-MIRU-VNTR profiles of 266 SIT53 isolates from Europe, Asia, Africa, and South America were submitted to the phylogenetic analysis jointly with reference profiles of different families from MIRU-VNTRplus database. Under this analysis, SIT53 isolates were clustered within different and distant families such as Ghana, NEW-1 (L4.5), TUR (L4.2.2.1), etc whereas many remained unclassified within L4. This confirms the evolutionarily basal position of this spoligotype and in its turn, this demonstrates that SIT53 does not correspond to any particular family of M. tuberculosis. Instead, different SIT53 subpopulations with evolutionarily stable and unchanged CRISPR locus gave rise to different and distant families that in many instances evolved through long-term allopatric evolution.

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

BackgroundBeijing 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.MethodsThe 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.ResultsPhylogenetic 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.ConclusionsBSP2 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.

Molecular epidemiological study of multidrug-resistant tuberculosis isolated from sputum samples in Eastern Cape, South Africa

Drug-resistant tuberculosis prevalence is still a global challenge. Making it imperative to examine the molecular epidemiology of drug resistant tuberculosis. Molecular epidemiology methods can evaluate transmission patterns and risk factors, ascertain transmission cases of multidrug-resistant tuberculosis (MDR-TB) and furthermore determine transmission patterns in a human populace. This work focuses on MDR-TB isolates in distinguishing them into several species and genotyping the MDR-TB isolates, mainly for epidemiological studies using the genomic regions of difference and the spoligotyping techniques. A total of 184 deoxyribonucleic acid isolated from sputum samples that showed resistance against the two major first-line anti-tuberculosis drugs (Rifampicin and Isoniazid) were examined. The deoxyribonucleic acid samples were amplified with primers specific for each flanking region of the genomic regions of difference for the identification of different MTBC species. Isolates were further characterized into different lineages using the spoligotyping commercial kit. The M. tuberculosis species was detected in 83.7% (154/184) of the deoxyribonucleic acid isolates, followed by the M. caprae in 8.7% (16/184) and the least detected species was the M. africanum in 2.2% (4/184). Nineteen spoligotype international types (SITs) were identified in this study. The pre-existing shared types were from 94.6% (174/184) isolates with 1.1% (2/184) isolates recognized as orphans and 4.3% (8/184) isolates were not found in the SITVIT database. The predominant family (spoligotype) was the Beijing with 67.4% (124/184) strains. This study gives a general overview of drug resistant strains and the circulating strains in the Eastern Cape, South Africa and it shows that the common Mycobacteria in the province is the Beijing strain.

Genetic Diversity Using Spoligotyping Method in the Case of Pulmonary Tuberculosis in Certain Localities of West Region of Cameroon

Tuberculosis (TB) remains a major global health problem. In 2016, an estimated 10.4 million people developed TB and 1.8 million died from the disease. TB epidemiology in Cameroon has so far largely consisted of reporting the number of cases detected and their demographic data. The present study investigates the genetic diversity among <i>Mycobacterium tuberculosis</i> complex circulating in the west region of Cameroon and analyzes the relationship between genotypes and epidemiology situation of patient. Spoligotyping was performed by PCR-amplification followed by the reverse hybridization of 120 cultured specimens and were identified by comparison to reference strains in SITVIT database. <i>Mycobacterium tuberculosis</i> was the only species identified. LAM10_CAM and T1 families were the most prevalent genetic families with 43.33% and 31.66% respectively. No association was found between genotypes with HIV serostatus. However, there was a significant association between genotypes and the gender also between genotypes and Patients age. Patients with age above 35 were more likely infected by LAM10_CAM strains, and belong genotypes and locality where the patients of Dschang were more likely infected by T1 strain. Population structure of <i>Mycobacterium tuberculosis</i> complex strains from the West region of Cameroon was found to be diverse and the Share Type 61 from the LAM10_CAM family was highly predominant.

Genetic variability and consequence of Mycobacterium tuberculosis lineage 3 in Kampala-Uganda.

BackgroundLimited data existed exclusively describing Mycobacterium tuberculosis lineage 3 (MTB-L3), sub-lineages, and clinical manifestations in Kampala, Uganda. This study sought to elucidate the circulating MTB-L3 sub-lineages and their corresponding clinical phenotypes.MethodA total of 141 M. tuberculosis isolates were identified as M. tuberculosis lineage 3 using Single nucleotide polymorphism (SNP) marker analysis method. To ascertain the sub-lineages/sub-strains within the M. tuberculosis lineage 3, the direct repeat (DR) loci for all the isolates was examined for sub-lineage specific signatures as described in the SITVIT2 database. The infecting sub-strains were matched with patients’ clinical and demographic characteristics to identify any possible association.ResultThe data showed 3 sub-lineages circulating with CAS 1 Delhi accounting for 55% (77/141), followed by CAS 1-Kili 16% (22/141) and CAS 2/CAS 8% (12/141). Remaining isolates 21% (30/141) were unclassifiable. To explore whether the sub-lineages differ in their ability to cause increased severe disease, we used extent of lung involvement as a proxy for severe disease. Multivariable analysis showed no association between M. tuberculosis lineage 3 sub-lineages with severe disease. The risk factors associated with severe disease include having a positive smear (OR = 9.384; CI 95% = 2.603–33.835), HIV (OR = 0.316; CI 95% = 0.114–0.876), lymphadenitis (OR = 0. 171; CI 95% = 0.034–0.856) and a BCG scar (OR = 0.295; CI 95% = 0.102–0.854).ConclusionIn Kampala, Uganda, there are three sub-lineages of M. tuberculosis lineage 3 that cause disease of comparable severity with CAS-Dehli as the most prevalent. Having HIV, lymphadenitis, a BCG scar and a smear negative status is associated with reduced severe disease.

Usefulness of 3'- 5' IS6110-RFLP genotyping and spoligotyping of Mycobacterium tuberculosis isolated in a tertiary hospital: a retrospective study detecting unsuspected epidemiological events.

ABSTRACTA drug resistance survey involving Mycobacterium tuberculosis isolated from patients of a tertiary Hospital in the Rio de Janeiro city (RJ), Brazil, between the years 1996 and 1998 revealed a high frequency of isoniazid (HR) resistance. These isolates were revisited and genotyped. Patients came from different RJ neighborhoods and municipalities, and 70% were outpatients. Applying the 3’ and 5’ IS 6110 -RFLP and the Spoligotype genotyping methods, the clonal structure of this population was investigated obtaining a snapshot of past epidemiological events. The 3’ clusters were subsequently 5’ IS 6110 -RFLP typed. Spoligotyping was analyzed in the SITVIT2 database. Epidemiological relationships were investigated. The major lineage was T (54.4%), and SIT 53/T1 and SIT 535/T1 were the most frequent. The T1 sublineage comprises 12.8% of resistant strains and SIT 535 were assigned for 31.8% of them. Orphan patterns corresponded to 12% and 73.3% and belonged to the T lineage. One pattern was unlisted in the SITVIT2. The 5’ IS 6110 -RFLP did not confirm 3/12 of the 3’ IS 6110 -RFLP clusters. A combination of all methods decreased the number of clusters to three. Nosocomial transmission was associated with one cluster involving a hospital cupbearer. This event was suspected in a multidrug resistant-TB inpatient caregiver who harbored a mixed infection. The 3’ IS 6110 clusters were associated with HR (p=0.046). These genotypic retrospective data may reflect a fraction of more extensive recent transmission in different communities that may be corroborated by the concentration of HR patients, and may serve as a database for further evolutionary and characterization evaluation of circulating strains and together with epidemiological data favors a more effective transmission control.

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.

Genetic Diversity of Mycobacterium tuberculosis Isolates in the Metropolitan Area of Rome

Background: The presence in a geographic area of Mycobacterium tuberculosis (Mtb) strains belonging to different phylogeographic lineages and showing different drug susceptibility patterns may suggest recent transmission, with implications in terms of patient clinical management and disease control. The aim of this study was to carry out a preliminary epidemiological investigation of tuberculosis (TB) cases in Rome. Methods: A total of 232 Mtb isolates, collected from new or previously treated patients, admitted between 2008 and 2014 at 2 hospital settings in Rome with a diagnosis of TB, were analyzed by spoligotyping and analyzing 24 variable-number tandem repeats (VNTR) mycobacterial interspersed repetitive-unit (MIRU) loci. The SITVIT2 database and the MIRU-VNTRplus web applications were used to identify the strain genotypes and to generate phylogenetic trees. Results: Based on the position on the phylogenetic tree, 97.4% of the strains were associated with 1 of the 7 main lineages. The Euro-American lineage was the most commonly represented (81.9%) within both Italian and foreign-born populations, although all main lineages were present. The highest frequency of drug-resistant strains was found among the East-Asian lineage (Beijing genotype) isolated from foreign-born patients. Conclusions: Dynamics of TB transmission in Rome indicate recent spread of Mtb strains belonging to phylogeographic lineages and clades usually found in countries and geographic areas with a high incidence of TB, similarly to what is observed in most metropolitan areas in Western Europe. Knowledge from molecular and classical epidemiology provides an important tool for disease control.

Genotypic diversity of Mycobacterium tuberculosis in Buenos Aires, Argentina

Buenos Aires is an overpopulated port city historically inhabited by people of European descent. Together with its broader metropolitan area, the city exhibits medium tuberculosis rates, and receives migrants, mainly from tuberculosis highly endemic areas of Argentina and neighboring countries. This work was aimed to gain insight into the Mycobacterium tuberculosis population structure in two suburban districts of Buenos Aires which are illustrative of the overall situation of tuberculosis in Argentina. The Lineage 4 Euro-American accounted for >99% of the 816 isolates analyzed (one per patient). Frequencies of spoligotype families were T 35.9%, LAM 33.2%, Haarlem 19.5%, S 3.2%, X 1.5%, Ural 0.7%, BOV 0.2%, Beijing 0.2%, and Cameroon 0.2%. Unknown signatures accounted for 5.3% isolates. Of 55 spoligotypes not matching any extant shared international type (SIT) in SITVIT database, 22 fitted into 15 newly-issued SITs. Certain autochthonous South American genotypes were found to be actively evolving. LAM3, which is wild type for RDrio, was the predominant LAM subfamily in both districts and the RDrio signature was rare among autochthonous, newly created, SITs and orphan patterns. Two genotypes that are rarely observed in neighboring countries ̶ SIT2/H2 and SIT159/T1 Tuscany ̶ were conspicuously represented in Argentina. The infrequent Beijing patterns belonged to Peruvian patients. We conclude that the genotype diversity observed reflects the influence of the Hispanic colonization and more recent immigration waves from Mediterranean and neighboring countries. Unlike in Brazil, the RDrio type does not play a major role in the tuberculosis epidemic in Buenos Aires.

Molecular epidemiology and drug sensitivity pattern of Mycobacterium tuberculosis strains isolated from pulmonary tuberculosis patients in and around Ambo Town, Central Ethiopia.

IntroductionTuberculosis (TB) is caused by M. tuberculosis complex and remains a major global public health problem. The epidemic remains a threat to sub-Saharan Africa, including Ethiopia, with further emergence of drug resistant TB. We investigated the drug sensitivity pattern and molecular epidemiology of mycobacterial strains isolated from pulmonary TB patients in and around Ambo town in Oromia Region, Central Ethiopia.MethodsA cross-sectional study was conducted involving 105 consecutive new smear positive pulmonary TB patients diagnosed at Ambo Hospital and surrounding Health Centers between May 2014 and March 2015 upon informed consent. Sputum samples were cultured on Löwenstein-Jensen (LJ) media using standard techniques to isolate mycobacteria. Region of difference 9 (RD9)-based polymerase chain reaction (PCR) and spoligotyping was employed for the identification of the isolates at species and strain levels. The spoligotype patterns were entered into the SITVIT database to determine Octal and SIT (Spoligotyping International Typing) numbers for each strain. The sensitivity of the isolates to isoniazid (INH), rifampicin (RIF), ethambutol (ETB) and streptomycin (STM) was evaluated on LJ-medium with the indirect proportion method.ResultsCultures were positive in 86/105 (82%) of newly diagnosed smear positive pulmonary TB cases. All of the 86 isolates were confirmed as M. tuberculosis. The majority (76.7%) of them were clustered into seven groups while the rest (23.3%) appeared unique. The most predominant Spoligotypes were SIT53 and SIT149, consisting of 24.4% and 20.9% of the isolates, respectively. Assigning of the isolates to family using SPOTCLUST software revealed that 45.3% of the isolates belonged to T1, 23.3% to T3 and 13% to CAS family. The majority (76.7%) of the M. tuberculosis isolates were susceptible to all the four drugs. Any resistance to any one of the four drugs was detected in 23.3% of the isolates. The highest proportion of any resistance was observed against isoniazid (9.3%) and ethambutol (7%). There was only a single case (1.2%) of multidrug resistant/rifampicin resistant (MDR/RR) TB.ConclusionThe majority of the isolates were clustered suggesting on-going active transmission in the study area. Mono resistance is relatively prevalent while the magnitude of MDR/RR-TB was found to be lower than in previous studies.

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.

Mycobacterium tuberculosis genotypes and predominant clones among the multidrug-resistant isolates in Spain 1998–2005

Although the incidence of tuberculosis (TB) is gradually decreasing in Spain, there is an increase in the proportion of foreign-born cases. This changing scenario is slowly shifting the local TB epidemiology from endemic to imported cases with an increased risk for multidrug-resistant (MDR) and extensively drug resistant (XDR) strains of Mycobacterium tuberculosis complex. MDR/XDR strains from Spain (n=366 MTBC isolates, 1 strain per patient) isolated between 1998 and 2005 were retained for this retrospective analysis. All strains were analyzed by spoligotyping, while 12-loci MIRU-VNTR data were available for 106 isolates from 2003 to 2005. Demographic, phylogenetic, and epidemiologic analyses using anonymized data were collected and analyzed using the SITVIT2 database. Our study provides with a first snapshot of genetic diversity of MDR/XDR-TB in several autonomous regions of Spain. It highlights significantly more of SIT1/Beijing and SIT66/BOV MDR isolates (5.7% and 7.38% respectively) and increasingly more foreign-born cases from Eastern Europe. Future studies should focus on shared genotypes between Spanish and foreign-born patients to decipher the modes of transmission and risk factors involved, and decipher the proportion of imported cases of active disease versus cases of reactivation of latent TB infection among foreign-born individuals.

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.