Latin American-Mediterranean Family Research Articles

GENOMIC ANALYSIS OF LAM FAMILY OF MYCOBACTERIUM TUBERCULOSIS CLINICAL STRAINS

Drug-resistant tuberculosis is a global problem for humanity, especially in the countries of Southeast Asia, Eastern Europe and the CIS. In Kazakhstan, the rate of drug-resistant tuberculosis among newly diagnosed patients is more than 20%. Moreover, the Republic of Kazakhstan ranks second among the thirty countries with a high disease burden according to the World Health Organization. In this regard, it becomes obvious that it is necessary to study the nature of resistance of locally circulating strains of mycobacteria. The use of genetic typing methods will allow tracking the spread of tuberculosis pathogens of any family, which, in turn, will provide a personalized approach to treatment. The strains of the Mycobacterium tuberculosis complex capable of infecting humans include seven phylogenetic lineages, initially associated with their geographical distribution. In this study, the genomes of three drug-resistant clinical isolates of the Latin American Mediterranean family (LAM) circulating in Kazakhstan were studied. We used whole genome sequencing to study the distribution and drug resistance of isolates. Phylogenetic analysis grouped the genomes described in this study with sequences from Russia, Uzbekistan and Kazakhstan, also belonging to the LAM family. One isolate was identified as having extensive drug resistance to seven anti-TB drugs. Our results suggest that at least two drug-resistant genotypes of the LAM family are circulating in Kazakhstan. Thus, the use of genetic methods is becoming more and more popular and effective in combating the spread of tuberculosis infection.

Genomic analysis of Latin American-Mediterranean family of Mycobacterium tuberculosis clinical strains from Kazakhstan.

The human-adapted strains of the Mycobacterium tuberculosis complex (MTBC) comprise seven phylogenetic lineages originally associated with their geographical distribution. Here, we report the genomes of three drug-resistant clinical isolates of the Latin American-Mediterranean (LAM) family collected in Kazakhstan. We utilised whole-genome sequencing to study the distribution and drug resistance of these isolates. Phylogenetic analysis grouped the genomes described in this study with the sequences from Russia, Uzbekistan, and Kazakhstan belonging to the LAM family. One isolate has acquired extensive drug resistance to seven antituberculosis drugs. Our results suggest at least two multi-drug resistant (MDR)/extensively drug-resistant (XDR)-associated genotypes of the LAM family circulate in Kazakhstan.

Exploring the \u201cLatin American Mediterranean\u201d family and the RDRio lineage in Mycobacterium tuberculosis isolates from Paraguay, Argentina and Venezuela

BackgroundThe Latin American & Mediterranean (LAM) spoligotype family is one of the most successful genotype of Mycobacterium tuberculosis worldwide and particularly prevalent in South-America. Within this family, a sublineage named Region of Difference Rio (RDRio) was reported initially in Brazil and is characterized by a genomic deletion of about 26.3 kb. This lineage seems to show a specific adaptation to the Euro-Latin American population. In this context, we sought to evaluate the LAM family and the presence of the RDRio genotype in samples from three Latin American countries including Paraguay, Venezuela and Argentina. To detect LAM strains reliably we applied a typing scheme using spoligotyping, 12 loci MIRU-VNTR, the Ag85C103 SNP and the regions of difference RDRio and RD174. IS6110-RFLP results were also used when available.ResultsGenotyping of 413 M. tuberculosis isolates from three Latin-American countries detected LAM (46%) and the ill-defined T clade (16%) as the most frequent families. The highest clustering rate was detected in the sample population from the city of Caracas in Venezuela. We observed considerable differences in the presence of the RDRio lineage, with high frequency in Caracas-Venezuela (55%) and low frequency in Buenos Aires-Argentina (11%) and Paraguay (10%). The molecular markers (RD174, Ag85C103, MIRU02-MIRU40 signature) of the RDRio lineage were essentially confirmed. For the LAM family, the most polymorphic loci were MIRU40, MIRU31, MIRU10, MIRU26, MIRU16 and the least polymorphic MIRU24, MIRU20, MIRU04, MIRU23.ConclusionsOur results suggest a differential adaptation of LAM-sublineages in neighboring populations and that RDRio strains spread regionally with different rates of distribution. The Ag85C SNP and RDs (RD174, RDRio) tested in this study can in fact facilitate molecular epidemiological studies of LAM strains in endemic settings and low-income countries.

ON IMPORTANCE OF USING EVOLUTIONARILY ROBUST MARKERS FOR DETECTION OF MYCOBACTERIUM TUBERCULOSIS STRAINS OF LAM GENETIC FAMILY

Aim. The clinical and epidemiological significance of the Latin American Mediterranean (LAM) genetic family of Mycobacterium tuberculosis determines the importance of the correct detection of LAM strains. In this study, a complex of molecular methods was used to analyze LAM strains in the population of M. tuberculosis in the Omsk region of Western Siberia, which is characterized by a high incidence of drug-resistant tuberculosis. Materials and methods. The collection included 207 strains of M. tuberculosis, isolated in the Omsk region in 2015 — 2016. The strains were subjected to spoligotyping, analysis of LAM-specific SNP Rv0129c 309GA, and whole genome sequencing followed by bioinformatics analysis. Results. A comparison of the obtained CRISPR-spoligotyping profiles with the international SITVIT_WEB database, assigned 11 strains (5.3%) to the LAM genotype. At the same time, based on analysis of phylogenetic SNP in the gene Rv0129c, 30 isolates (14.5%) were assigned to LAM. Whole genome sequencing was performed for 4 isolates with different spoligotyping profiles. Conclusion. The results of this study show the limited utility of the decision rules implemented in SITVIT_WEB to define LAM family for isolates with long deleted blocks of spacers or abridged spoligoprofiles. The following approach can be recommended for detection of LAM isolates (1) primary spoligotyping, comparison with SITVIT_WEB, and mandatory interpretation in the light of expert knowledge; (2) detection of LAM-specific SNP (e.g., using PCR-RFLP).

Tuberculosis associated factors caused by Mycobacterium tuberculosis of the RDRio genotype.

BACKGROUND Tuberculosis (TB) continues to be a disease that affects many countries around the world, including Brazil. Recently, a subtype of Latin American-Mediterranean family strain was identified and characterised by RDRio. The strain has been associated with different characteristics of the disease.OBJECTIVES In the present study we investigated the association of epidemiological, clinical, radiological and bacteriological variables with pulmonary tuberculosis caused by RDRio Mycobacterium tuberculosis strain in large regions of São Paulo.METHODS We conducted a cross-sectional study in 530 patients with pulmonary tuberculosis, diagnosed using sputum culture, from two regions of the São Paulo state in Brazil. The samples were brought to São Paulo reference laboratories for epidemiological, clinical, radiological and bacteriological analyses, and the data were obtained from a TB notification system. RDRio genotyping and Spoligotyping of the samples were performed. For the analysis of the categorical variables we used the chi-square test or the Fisher’s exact test, and for the continuous variables, the Mann-Whitney test. In addition, a logistic regression was used for multivariate analysis. Differences with p < 0.05 were considered significant.FINDINGS The RDRio deletion was identified in 152 (28.7%) samples. In the univariate analysis, both the age groups above 25 years and alcohol consumption were associated with the RDRio deletion. The multivariate analysis confirmed the association of the RDRio deletion with the age groups: 25-35 years old [OR: 2.28 (1.02-5.07; p = 0.04)] and 36-60 years old (OR: 2.36 (1.11-5.05); p = 0.03], and also with alcohol consumption [OR: 1.63 (1.05-2.54); p = 0,03].MAIN CONCLUSIONS In this study, we identified new factors associated with the M. tuberculosis of the RDRio deletion strains infection.

Latin-American-Mediterranean lineage of Mycobacterium tuberculosis: Human traces across pathogen’s phylogeography

Currently, Mycobacterium tuberculosis isolates of Latin-American Mediterranean (LAM) family may be detected far beyond the geographic areas that coined its name 15years ago. Here, we established the framework phylogeny of this geographically intriguing and pathobiologically important mycobacterial lineage and hypothesized how human demographics and migration influenced its phylogeography. Phylogenetic analysis of LAM isolates from all continents based on 24 variable number of tandem repeats (VNTR) loci and other markers identified three global sublineages with certain geographic affinities and defined by large deletions RD115, RD174, and by spoligotype SIT33. One minor sublineage (spoligotype SIT388) appears endemic in Japan. One-locus VNTR signatures were established for sublineages and served for their search in published literature and geographic mapping. We suggest that the LAM family originated in the Western Mediterranean region. The most widespread RD115 sublineage seems the most ancient and encompasses genetically and geographically distant branches, including extremely drug resistant KZN in South Africa and LAM-RUS recently widespread across Northern Eurasia. The RD174 sublineage likely started its active spread in Brazil; its earlier branch is relatively dominated by isolates from South America and the derived one is dominated by Portuguese and South/Southeastern African isolates. The relatively most recent SIT33-sublineage is marked with enigmatic gaps and peaks across the Americas and includes South African clade F11/RD761, which likely emerged within the SIT33 subpopulation after its arrival to Africa. In addition to SIT388-sublineage, other deeply rooted, endemic LAM sublineages may exist that remain to be discovered. As a general conclusion, human mass migration appears to be the major factor that shaped the M. tuberculosis phylogeography over large time-spans.

Molecular profiling of drug resistant isolates of Mycobacterium tuberculosis in the state of Santa Catarina, southern Brazil.

Drug resistance is a global threat and one of the main contributing factors to tuberculosis (TB) outbreaks. The goal of this study was to analyse the molecular profile of multidrug-resistant TB (MDR-TB) in the state of Santa Catarina in southern Brazil. Fifty-three MDR Mycobacterium tuberculosisclinical isolates were analysed by spoligotyping and a partial region of therpoB gene, which is associated with rifampicin resistance (RMP-R), was sequenced. Some isolates were also distinguished by their mycobacterial interspersed repetitive units (MIRU). S531L was the most prevalent mutation found within rpoB in RMP-R isolates (58.5%), followed by S531W (20.8%). Only two MDR isolates showed no mutations withinrpoB. Isolates of the Latin American Mediterranean (LAM) family were the most prevalent (45.3%) found by spoligotyping, followed by Haarlem (9.4%) and T (7.5%) families. SIT106 was found in 26.4% of isolates and all SIT106 isolates typed by MIRU-12 (5 out of 14) belong to MIT251. There was a high correlation between the S531W mutation and the LAM family mainly because all SIT2263 (LAM9) isolates carry this mutation. Among isolates with the S531W mutation in rpoB MIRU demonstrates a cluster formed by four isolates (SIT2263 and MIT163) and very similar profiles were observed between eight of the nine isolates. Better characterisation of TB isolates may lead to new ways in which to control and treat TB in this region of Brazil.

Mycobacterium tuberculosis Latin–American–Mediterranean family in Bulgaria

IntroductionTuberculosis (TB) remains an important public health issue for Bulgaria. Although a number of new cases are showing a steady decline (44/100,000 in 2000, 40/100,000 in 2005), the TB incidence rate in Bulgaria is still sufficiently high (26.7/100,000 in 2013). The current population structure of Mycobacterium tuberculosis is clonal. Certain genetic families of this species have justly attracted more attention due to their global dissemination and/or remarkable pathogenic properties. Beijing, Haarlem, and LAM are the most known examples. The latter family LAM (Latin–American–Mediterranean) was shown in an increasing number of studies to possess increased transmissibility, hence the importance of its rapid detection and correct estimation of its prevalence in a population. Spoligotyping signature of LAM is absence of signals 21–24 and 33–36, although abridged spoligo-profiles with long blocks of deleted spacers result in an uncertain definition of such strains. The use of other molecular markers may be helpful. This study aimed to evaluate the prevalence of LAM strains among M. tuberculosis strains in Bulgaria based on the use of different molecular markers. Materials and methodsM. tuberculosis isolates were randomly selected among M. tuberculosis strains isolated from newly diagnosed, adult, pulmonary TB patients in different regions of Bulgaria from December 2004 to March 2006. Spoligotyping was used to analyze a variation in the DR locus. The spoligotyping patterns were compared with the international database SITVIT_WEB (Institut Pasteur de Guadeloupe). Analysis of the IS6110 element specific for the LAM genetic family was performed as described previously (Marais et al., 2006). ResultsA study sample included 133 strains from different regions of the country and characterized in previous publications (Valcheva et al., 2008, 2010). Application of the published rules for the definition of the major spoligotype clades and comparison with SITVIT_WEB global database permitted this study to assign most of the 133 strains to the known spoligotype families. All available strains (n=101) were tested for LAM-specific IS6110 insertion. Comparison of results by different methods identified 3 groups of strains. The first group included 11 strains with 2 amplified bands which present an apparent discrepancy; further study is warranted. The second group included 86 strains with a single amplified band, specific for the absence of the IS6110 insertion in the target locus, hence indicative of the other than LAM family. The third group included 4 strains with a LAM-specific band only. ConclusionsApplication of the LAM-specific PCR revealed double-sided discrepancies when the obtained results were compared with those obtained by spoligotyping. As a whole, a phylogenetic family of 38 strains was revised or questioned: 27 strains were shown not to belong to LAM, while 11 more strains showed apparently discrepant results that question the global utility of such PCR or at least highlight an importance of using multiple markers for molecular detection of the LAM family of M. tuberculosis. AcknowledgementsDr. Nadya Markova, Prof. Olga Narvskaya, and Prof. Nalin Rastogi are gratefully acknowledged for kind support and guidance.

Stranger in a strange land: Ibero-American strain of Mycobacterium tuberculosis in Tibet, China

The Latin-American-Mediterranean (LAM) family is among the largest, diverse and intriguing within Mycobacterium tuberculosis. Widespread in some world regions, as justly reflected by its name, LAM remains in the shadow of the Beijing and East-African Indian families in East Asia. Here, we applied evolutionarily robust markers to the spoligotyped collection of 699 M. tuberculosis clinical isolates recovered from tuberculosis (TB) patients in different provinces across China between 2005 and 2013. As a result, only two isolates were assigned to LAM, hence we conclude that this family has a negligible impact on TB transmission in China. Interestingly, these two isolates represented two grand sublineages within LAM: RD115 and RD-Rio/RD174.Our results highlight the great caution to be taken when interpreting spoligotyping profiles with large deleted blocks. Here, three geographically distant Chinese isolates of spoligotype SIT803 were excluded from LAM. This is in clear contrast with described SIT803 isolates of the LAM family in Russia and Georgia (Mokrousov et al., 2014; Niemann et al., 2010).Interestingly, a drug resistant isolate of the Ibero-American LAM RD-Rio sublineage was recovered from TB patient of Tibetan ethnicity, a resident in the Lhasa district in Tibet. An additional in-depth analysis by whole genome sequencing might help to understand the evolutionary history of this isolate and its possible route to the rural setting of the Tibetan highlands.

Draft Genome Sequence of the Mycobacterium tuberculosis Clinical Isolate C2, Belonging to the Latin American-Mediterranean Family.

Tuberculosis remains a major infectious disease in Taiwan. Here we present the draft genome sequence of the Mycobacterium tuberculosis C2 strain, belonging to the Latin American–Mediterranean lineage. The draft genome sequence comprises 4,453,307 bp with a G+C content of 65.6%, revealing 4,390 coding genes and 45 tRNA genes.

Mycobacterium tuberculosis Latin American-Mediterranean Family and Its Sublineages in the Light of Robust Evolutionary Markers

Mycobacterium tuberculosis has a clonal population structure, and the Latin American-Mediterranean (LAM) family is one of the largest and most widespread within this species, showing evidence for remarkable pathobiology and a confusing phylogeny. Here, we applied robust phylogenetic markers to study the evolution of the LAM family and its major sublineages circulating in Russia and neighboring countries. A total of 250 M. tuberculosis isolates were confirmed to belong to the LAM family based on the analysis of the LAM-specific single-nucleotide polymorphisms (SNPs) in the Rv3062 and Rv0129c genes. At this stage, the family status was rectified for 121 isolates misleadingly assigned by CRISPR spoligotyping to non-LAM families (T1- or T5-RUS1). Consequently, the reestimated LAM prevalence rate increased 2-fold in Russia and Kazakhstan and 4-fold in Belarus. The majority (91.8 to 98.7%) of the LAM isolates from all three countries belonged to the LAM-RUS sublineage. In contrast, the Ibero-American LAM RD-Rio sublineage was identified in only 7 Russian isolates. Taken together, our findings and further analyses suggest a monophyletic origin of LAM-RUS: at a historically distant time, in Russia, in a small founding bacterial/human population. Its dissemination pattern and high prevalence rate in Northern Eurasia may indicate a long-term coexistence of the LAM-RUS sublineage and local human populations hypothetically leading to coadaptation and reduced pathogenicity of the relatively more ancient clones, such as spoligotype international type 254 (SIT254), compared to the more recent SIT252 and SIT266 clones. In contrast, rare LAM RD-Rio isolates were likely brought to Russia through occasional human contact. The spread of RD-Rio strains is not as global as commonly claimed and is determined largely by human migration flows (rather than by pathobiological properties of these strains). Consequently, a host population factor appears to play a major role in shaping the in situ dissemination pattern of the imported strains in an autochthonous population.

Extrapulmonary Tuberculosis: Mycobacterium tuberculosis Strains and Host Risk Factors in a Large Urban Setting in Brazil

BackgroundFactors related to the development of extrapulmonary forms of tuberculosis (EPTB) are still poorly understood, particularly in high-endemic countries like Brazil. The objective of the paper is to determine host and Mycobacterium tuberculosis (MTB) strain-related factors associated with the development of EPTB in Espírito Santo state, Brazil.Methods and FindingsWe conducted a retrospective laboratory-based surveillance study of new tuberculosis (TB) cases diagnosed in Espírito Santo state, Brazil between 1998 and 2007. We genotyped 612 isolates of MTB from 606 TB patients using spoligotyping and IS6110-restriction fragment length polymorphism (RFLP) typing and compared sociodemographic and clinical characteristics of patients with pulmonary TB (PTB) and EPTB. Among 606 patients, 464 (77%) had PTB, 79 (13%) had EPTB, 51 (8%) had both, and 12 (2%) had miliary TB. The IS6110 RFLP analysis demonstrated that 250 (41%) isolates belonged to clustered RFLP patterns, 27 (11%) of which were from EPTB. We identified 73 clusters including 35 (48%) composed of 2 isolates each. By spoligotyping, 506 (83%) MTB isolates fell into known patterns and 106 (17%) fell into patterns with no family assignment; 297 (48%) isolates belonged to the Latin-American Mediterranean family. Higher school level (4-7 years OR: 0.16 95% CI 0.34-0.73 and > 8 years of education, OR 0.06 95% CI 0.009-0.50) white ethnicity (OR: 2.54 95% CI 1.03-6.25) and HIV infection (OR: 16.83 95% CI 5.23-54.18) were associated with EPTB. No specific strain lineage or percentage of clustering was associated with EPTB.ConclusionsThese results demonstrate that risk factors for EPTB are related more to host than to MTB strain lineage characteristics.

Multidrug-Resistant Tuberculosis in Panama Is Driven by Clonal Expansion of a Multidrug-Resistant Mycobacterium tuberculosis Strain Related to the KZN Extensively Drug-Resistant M. tuberculosis Strain from South Africa

Multidrug-resistant tuberculosis (MDR-TB) is a significant health problem in Panama. The extent to which such cases are the result of primary or acquired resistance and the strain families involved are unknown. We performed whole-genome sequencing of a collection of 66 clinical MDR isolates, along with 31 drug-susceptible isolates, that were isolated in Panama between 2001 and 2010; 78% of the MDR isolates belong to the Latin American-Mediterranean (LAM) family. Drug resistance mutations correlated well with drug susceptibility profiles. To determine the relationships among these strains and to better understand the acquisition of resistance mutations, a phylogenetic tree was constructed based on a genome-wide single-nucleotide polymorphism analysis. The phylogenetic tree shows that the isolates are highly clustered, with a single strain (LAM9-c1) accounting for nearly one-half of the MDR isolates (29/66 isolates). The LAM9-c1 strain was most prevalent among male patients of working age and was associated with high mortality rates. Members of this cluster all share identical mutations conferring resistance to isoniazid (KatG S315T mutation), rifampin (RpoB S531L mutation), and streptomycin (rrs C517T mutation). This evidence of primary resistance supports a model in which MDR-TB in Panama is driven by clonal expansion and ongoing transmission of several strains in the LAM family, including the highly successful MDR strain LAM9-c1. The phylogenetic analysis also shows that the LAM9-c1 strain is closely related to the KwaZulu-Natal (KZN) extensively drug-resistant TB strain identified in KwaZulu-Natal, South Africa. The LAM9-c1 and KZN strains likely arose from a recent common ancestor that was transmitted between Panama and South Africa and had the capacity to tolerate an accumulation of multiple resistance mutations.

Distinct clinical and epidemiological features of tuberculosis in New York City caused by the RDRioMycobacterium tuberculosis sublineage

BackgroundGenetic tracking of Mycobacterium tuberculosis is a cornerstone of tuberculosis (TB) control programs. The RDRioM. tuberculosis sublineage was previously associated with TB in Brazil. We investigated 3847 M. tuberculosis isolates and registry data from New York City (NYC) (2001–2005) to: (1) affirm the position of RDRio strains within the M. tuberculosis phylogenetic structure, (2) determine its prevalence, and (3) define transmission, demographic, and clinical characteristics associated with RDRio TB. MethodsIsolates classified as RDRio or non-RDRioM. tuberculosis by multiplex PCR were further classified as clustered (⩾2 isolates) or unique based primarily upon IS6110-RFLP patterns and lineage-specific cluster proportions were calculated. The secondary case rate of RDRio was compared with other prevalent M. tuberculosis lineages. Genotype data were merged with the data from the NYC TB Registry to assess demographic and clinical characteristics. ResultsRDRio strains were found to: (1) be restricted to the Latin American–Mediterranean family, (2) cause approximately 8% of TB cases in NYC, and (3) be associated with heightened transmission as shown by: (i) a higher cluster proportion compared to other prevalent lineages, (ii) a higher secondary case rate, and (iii) cases in children. Furthermore, RDRio strains were significantly associated with US-born Black or Hispanic race, birth in Latin American and Caribbean countries, and isoniazid resistance. ConclusionsThe RDRio genotype is a single M. tuberculosis strain population that is emerging in NYC. The findings suggest that expanded RDRio case and exposure identification could be of benefit due to its association with heightened transmission.

Fitness study of the RDRiolineage and Latin American–Mediterranean family ofMycobacterium tuberculosisin the city of Rio Grande, Brazil

RD(Rio) is a novel Mycobacterium tuberculosis lineage of the Latin American-Mediterranean (LAM) family. LAM has been found worldwide but is more predominant in South America. The aim of this study was to assess the presence of the RD(Rio) lineage and LAM family in the city of Rio Grande, Brazil, and to investigate the fitness of these strains based on determination of their growth rate. Fifty clinical isolates of M. tuberculosis were genotyped and 43 different patterns were found by spoligotyping and mycobacterial interspersed repetitive units-variable number of tandem repeats. The predominant genotypes belonged to the LAM family (54% of the strains) followed by clade T (22%) and Haarlem (16%). The RD(Rio) lineage represented 38% of the total strains and 70.4% of the LAM strains found in this study. Strains belonging to the LAM family showed a fitness advantage when comparing their rate of growth with that of non-LAM strains, but a significant difference between RD(Rio) and non-RD(Rio) strains was not confirmed.

Application of Sensitive and Specific Molecular Methods To Uncover Global Dissemination of the Major RD Rio Sublineage of the Latin American-Mediterranean Mycobacterium tuberculosis Spoligotype Family

The Latin American-Mediterranean (LAM) family of Mycobacterium tuberculosis is believed to be the cause of approximately 15% of tuberculosis cases worldwide. Previously, we defined a prevalent sublineage of the LAM family in Brazil by a single characteristic genomic deletion designated RD(Rio). Using the Brazilian strains, we pinpoint an Ag85C(103) single nucleotide polymorphism (SNP) (screened by restriction fragment length polymorphism [RFLP] analysis) that correctly identified all LAM family strains. Importantly, all RD(Rio) strains concomitantly possessed the RD174 deletion. These genetic signatures, along with a newly developed multiplex PCR for rapid differentiation between "wild-type" and RD(Rio) strains, were then used to analyze an international collection of M. tuberculosis strains. RD(Rio) M. tuberculosis was identified from four continents involving 11 countries. Phylogenetic analysis of the IS6110-RFLP patterns from representative RD(Rio) and LAM strains from Brazil, along with all representative clusters from a South African database, confirmed their genetic relatedness and transcontinental transmission. The Ag85C(103) SNP RFLP, as compared to results obtained using a PCR method targeting a LAM-restricted IS6110 element, correctly identified 99.8% of LAM spoligotype strains. Together, these tests were more accurate than spoligotyping at categorizing strains with indefinable spoligotypes and segregated true LAM strains from those with convergent spoligotypes. The fact that RD(Rio) strains were identified worldwide highlights the importance of this LAM family sublineage and suggests that this strain is a global threat that should be specifically targeted by public health resources. Our provision of simple and robust molecular methods will assist the evaluation of the LAM family and the RD(Rio) sublineage.

Molecular Characteristics of the Mycobacterium tuberculosis LAM-RUS Family Prevalent in Central Russia

We analyzed IS6110-associated polymorphisms in the phospholipase C genes of 107 isolates of Mycobacterium tuberculosis selected to be representative of isolates circulating in central Russia. We found that the majority of Latin American-Mediterranean family strains contained an insertion in a unique position in the plcA gene, suggesting a common ancestor. This insertion can serve as a specific genetic marker for this group, which we designate the LAM-RUS family.

Contribution of spoligotyping to the characterization of the population structure of Mycobacterium tuberculosis isolates in Portugal

Tuberculosis is a major health problem in Portugal. To begin characterizing the population structure of Mycobacterium tuberculosis, spoligotyping was used for the systematic typing, through consecutive sampling, of patient isolates from the Amadora-Sintra area of Greater Lisbon. Distribution amongst major spoligotype families, including the Latin American Mediterranean (LAM), T, Haarlem and Beijing, was compared to that of the international spoligotype database SpolDB4 and to the European countries of traditional Portuguese immigration represented in SpolDB4. Spoligotypes from 665 isolates were analyzed and 97 shared international types (SITs) identified. In SpolDB4 Portugal is represented by part of the spoligotypes from this study explaining the reduced number of unidentified patterns. The importance of the LAM family, and especially of LAM1 and LAM9 sub-families that alone represented 38% of all the isolates in this study as compared to 8% relative to the European sub group, led us to believe that at least in this respect the population structure was closer to that of Africa and South America than to Europe. Spoligotypes characteristic of Portugal or Portuguese related settings were identified. These included SIT244 a T1 sub-family predominant in Portugal and Bangladesh, SIT64 a LAM 6 sub-family common to Portugal and Brazil, and SIT1106 a LAM 9 sub-family. These studies were the first in Portugal stressing the importance of monitoring the population structure of M. tuberculosis isolates, an important step towards gaining an understanding of tuberculosis and the dynamics of this disease.