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We present a short summary of recent observations on the global distribution of the major clades of the Mycobacterium tuberculosis complex, the causative agent of tuberculosis. This global distribution was defined by data-mining of an international spoligotyping database, SpolDB3. This database contains 11,708 patterns from as many clinical isolates originating from more than 90 countries. The 11,708 spoligotypes were clustered into 813 shared types. A total of 1,300 orphan patterns (clinical isolates showing a unique spoligotype) were also detected.

Background

We present a picture of the biodiversity of Mycobacterium tuberculosis in Paraguay, an inland South American country harboring 5 million inhabitants with a tuberculosis notification rate of 38/100,000.

Results

A total of 220 strains collected throughout the country in 2003 were classified by spoligotyping into 79 different patterns. Spoligopatterns of 173 strains matched 51 shared international types (SITs) already present in an updated version of SpolDB4, the global spoligotype database at Pasteur Institute, Guadeloupe. Our study contributed to the database 13 new SITs and 15 orphan spoligopatterns. Frequencies of major M. tuberculosis spoligotype lineages in our sample were as follows: Latin-American & Mediterranean (LAM) 52.3%, Haarlem 18.2%, S clade 9.5%, T superfamily 8.6%, X clade 0.9% and Beijing clade 0.5%. Concordant clustering by IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping identified transmission in specific settings such as the Tacumbu jail in Asuncion and aboriginal communities in the Chaco. LAM genotypes were ubiquitous and predominated among both RFLP clusters and new patterns, suggesting ongoing transmission and adaptative evolution in Paraguay. We describe a new and successfully evolving clone of the Haarlem 3 sub-lineage, SIT2643, which is thus far restricted to Paraguay. We confirmed its clonality by RFLP and mycobacterial interspersed repetitive unit (MIRU) typing; we named it "Tacumbu" after the jail where it was found to be spreading. One-fifth of the spoligopatterns in our study are rarely or never seen outside Paraguay and one-tenth do not fit within any of the major phylogenetic clades in SpolDB4.

Conclusion

Lineages currently thriving in Paraguay may reflect local host-pathogen adaptation of strains introduced during past migrations from Europe.

This investigation describes drug resistance patterns and genotyping data on a total of 145 Mycobacterium tuberculosis strains isolated between 2000 and 2004 in Malatya, Turkey. Drug susceptibility results indicated a total of 20% resistant and 4.8% of multidrug-resistant isolates. Spoligotyping resulted in 25 unique patterns and 120 strains in 19 clusters (2 to 33 strains per cluster). When the results were compared to an international spoligotyping database, 19 of 25 unique patterns matched existing shared spoligotype international types (SITs). This led to the description of 38 SITs with 139 strains and 6 orphan patterns (not previously reported). Five of the SITs (SIT759, SIT1936, SIT1937, SIT1938, and SIT2285) were newly created. The most prevalent spoligotype was SIT41 (LAM7-TUR) with 33 (23.9%) isolates. The repartition of strains according to major M. tuberculosis clades (in decreasing order) was as follows: ill-defined T clade (45.7%) > Latin American and Mediterranean (LAM; 29%) > Haarlem (15.9%). Strains belonging to Central Asian (CAS), East-African Indian (EAI), Beijing, and Africanum clades were absent in this setting. IS6110-restriction fragment length polymorphism (RFLP) resulted in 19 clusters (52 strains), with a final clustering rate of 35.9% and a recent transmission rate of 22.8%. Typing based on mycobacterial interspersed repetitive units (MIRUs) permitted us to identify 65 patterns (23 orphan patterns and 42 patterns that matched existing MIRU international types in an updated database). The combination of the three typing methods allowed us to calculate a final clustering rate of 22% and a significantly lower transmission rate of 13.1%. The discrimination achieved by IS6110-RFLP/MIRUs was not significantly improved by adding spoligotyping results (1.4%). We conclude that our patient population is infected by diverse M. tuberculosis populations; however, the majority of the ongoing transmission is due to “evolutionary recent” tuberculosis lineages belonging to principal genetic group 2 (PGG2; Haarlem and LAM) and PGG3 (ill-defined T clade), and most of it is attributable to the LAM7-TUR sublineage with an enhanced phylogeographical specificity for Turkey. An absence of lineages belonging to PGG1 clones (EAI, CAS, and Beijing, essentially found in Central, South, and Southeast Asia), is noteworthy.

We give an update on the worldwide spoligotype database, which now contains 3,319 spoligotype patterns of Mycobacterium tuberculosis in 47 countries, with 259 shared types, i.e., identical spoligotypes shared by two or more patient isolates. The 259 shared types contained a total of 2,779 (84%) of all the isolates. Seven major genetic groups represented 37% of all clustered isolates. Two types (119 and 137) were found almost exclusively in the USA and accounted for 9% of clustered isolates. The remaining 1,517 isolates were scattered into 252 different spoligotypes. This database constitutes a tool for pattern comparison of M. tuberculosis clinical isolates for global epidemiologic studies and phylogenetic purposes.

Spacer oligonucleotide (spoligotyping) analysis is a rapid polymerase chain reaction–based method of DNA fingerprinting the Mycobacterium tuberculosis complex. We examined spoligotype data using a bioinformatic tool (sequence logo analysis) to elucidate undisclosed phylogenetic relationships and gain insights into the global dissemination of strains of tuberculosis. Logo analysis of spoligotyping data provides a simple way to describe a fingerprint signature and may be useful in categorizing unique spoligotypes patterns as they are discovered. Large databases of DNA fingerprint information, such as those from the U.S. National Tuberculosis Genotyping and Surveillance Network and the European Concerted Action on Tuberculosis, contain information on thousands of strains from diverse regions. The description of related spoligotypes has depended on exhaustive listings of the individual spoligotyping patterns. Logo analysis may become another useful graphic method of visualizing and presenting spoligotyping clusters from these databases.

Although tuberculosis is still a public health problem in Mexico, there is little information about the genetic characteristics of the isolates. In the present study, we analyzed by spoligotyping 180 Mycobacterium tuberculosis clinical isolates from the urban area of Monterrey, Mexico, including drug-susceptible and drug-resistant isolates. The spoligotype patterns were compared with those in the international SITVIT2 spoligotyping database. Four isolates presented spoligotype patterns not found in the database (orphan types); the rest were distributed among 44 spoligo international types (SITs). SIT53 (clade T1) and SIT119 (clade X1) were predominant and included 43 (23.8%) and 28 (15.5%) of the isolates, respectively. In order to determine if there was a dominant spoligotype in the group of multidrug-resistant isolates, 37 of them were analyzed by IS6110-based restriction fragment length polymorphism assays, and scarce clustering of strains with more than five bands was observed. Fourteen isolates of this multidrug-resistant group presented four bands or less and were distributed in four SITs: SIT53 (n = 8), SIT92 (n = 3), SIT70 (n = 2), and SIT3038 (n = 1). When the molecular detection of mutations in the katG and rpoB genes were analyzed in these isolates with low copy numbers of IS6110, only two isolates shared the same IS6110, spoligotyping, and mutations patterns. When the distribution of the spoligotypes was analyzed by age cohort, SIT119 was predominantly found in patients 0 to 20 years old, especially in males, accounting for up to 40% of the isolates. In contrast, SIT53 was more prevalent in older females. This analysis demonstrates the variability of M. tuberculosis isolates in Monterrey and the partial dominance of SIT53 and SIT119 in that area of Mexico.

Background

Tuberculosis persists as a public health problem in Honduras. A better knowledge of the molecular characteristics of Mycobacterium tuberculosis strains will contribute to understand the transmission dynamics of the disease within the country. The aim of this study was to provide an insight of the genetic biodiversity of M. tuberculosis clinical isolates collected in Honduras between 1994 and 2002. Genotyping was performed using spoligotyping and RFLP. The spoligotypes obtained were compared with the SITVIT2 proprietary database of the Pasteur Institute of Guadeloupe.

Results

Spoligotyping grouped 84% of the isolates into 27 clusters (2 to 43 strains per cluster). Of the 44 shared international types (SITs) identified among the Honduran stains, 8 SITs were newly identified either within the present study or after match with an orphan type previously identified in the SITVIT2 database. In addition, 16 patterns corresponded to orphan, previously unreported isolates.

The Latin American Mediterranean (LAM) lineage was the most common in this study; 55% of the strains belonged to this family. Other genotypes found were Haarlem (16%), T (16%), X-clade (6%), Unknown signature (5%) and S (1%). Only one Beijing strain was identified (0.5%).

We observed a high degree of diversity after characterizing the 43 isolates belonging to the main spoligotyping cluster (SIT 33, LAM3) with IS6110-RFLP. A total of 35 different RFLP-fingerprints were detected, of which 6 patterns corresponded to the same number of clusters comprising 14 strains.

Conclusions

The findings obtained in this study show that tuberculosis transmission in Honduras is due to modern M. tuberculosis lineages with high level of biodiversity.

SUMMARY

Spoligotyping is used in molecular epidemiological studies, and signature patterns have identified strain families. However, homoplasy occurs in the markers used for spoligotyping, which could lead to identical spoligotypes in phylogenetically unrelated strains. We determined the accuracy of strain classification based on spoligotyping using the six large sequence and single nucleotide polymorphisms-defined lineages as a gold standard. Of 919 Mycobacterium tuberculosis isolates, 870 (95%) were classified into a spoligotype family. Strains from a particular spoligotype family belonged to the same lineage. We did not find convergence to the same spoligotype. Spoligotype families appear to be sub-lineages within the main lineages.

Objectives

To compare genotypes of Mycobacterium bovis strains from humans in Southern California with genotypes of M. bovis strains in cattle in Mexico and the USA to explore the possible origins of human infections.

Methods

We conducted a descriptive analysis of M. bovis genotypes from a binational population of humans and cattle using spacer oligonucleotide typing (spoligotyping).

Results

One hundred six human M. bovis spoligotypes were compared to spoligotypes from 496 Mexican cattle and 219 US cattle. Twelve spoligotype patterns were identified among human cases and 126 spoligotype patterns were detected in cattle. Over 91% (97/106) of the human M. bovis isolates had spoligotypes that were identical to those found in Mexican cattle. Four human cases had spoligotypes that matched both cattle born in Mexico and in the USA. Nine human cases had spoligotypes that did not match cattle born in Mexico or the USA.

Conclusions

Our data indicate that the population of M. bovis strains causing human TB disease in Southern California is closely related to the M. bovis strain population found in Mexican cattle and supports existing epidemiological evidence that human M. bovis disease in San Diego likely originated from Mexican cattle.

The genetic diversity of Mycobacterium tuberculosis isolates among patients from Sweden was determined by a combination of two PCR-based techniques (spoligotyping and variable number of tandem repeats analysis). It resulted in a clustering of 23.6% of the isolates and a rate of recent transmission of 14.1%. The clustered isolates mainly belonged to the Haarlem family (23.2%), followed by the Beijing (9.8%), Latin American and Mediterranean (LAM; 8%), and East African-Indian (EAI; 6.2%) families. A comparison of the spoligotypes with those in the international spoligotyping database showed that 62.5% of the clustered isolates and 36.6% of all isolates typed were grouped into six major shared types. A comparison of the spoligotypes with those in databases for Scandinavian countries showed that 33% of the isolates belonged to an ill-defined T family, followed by the EAI (22%), Haarlem (20%), LAM (11%), Central Asian (5%), X (5%), and Beijing (4%) families. Both the highest number of cases and the proportion of clustered cases were observed in patients ages 15 to 39 years. Nearly 10% of the isolates were resistant to one or more drugs (essentially limited to isoniazid monoresistance). However, none of the strains were multidrug resistant. Data on the geographic origins of the patients showed that more than two-thirds of the clustered patients with tuberculosis were foreign-born individuals or refugees. These results are explained on the basis of both the historical links within specific countries and recently imported cases of tuberculosis into Sweden.

Understanding the genetic diversity of Mycobacterium tuberculosis is needed for a better understanding of the epidemiology of TB and could have implications for the development of new diagnostics, drugs, and vaccines. M. tuberculosis isolates were characterized using spoligotyping and were compared with the SpoIDB4 database of the Pasteur Institute of Guadeloupe. A total of 53 different patterns were identified among 192 isolates examined. 169 of the isolates were classified into one of the 33 shared SITs, whereas the remaining 23 corresponded to 20 orphan patterns. 54% of the isolates were ascribed to the T family, a family which has not been well defined to date. Other prominent families were CAS, Haarlem, LAM, Beijing, and Unknown comprising 26%, 13%, 2.6%, 0.5%, and 2.1%, respectively. Among HIV-positive patients, 10 patterns were observed among 25 isolates. The T (38.5%), H (26.9%), and CAS (23.1%) families were the most common among HIV-positive individuals. The diversity of the M. tuberculosis strains found in this study is very high, and there was no difference in the distribution of families in HIV-positive and HIV-negative TB patients except the H family. Tuberculosis transmission in Addis Ababa is due to only the modern M. tuberculosis families (CAS, LAM, T, Beijing, Haarlem, and U).

Background

Tuberculosis is a major health problem in São Paulo, Brazil, which is the most populous and one of the most cosmopolitan cities in South America. To characterize the genetic diversity of Mycobacterium tuberculosis in the population of this city, the genotyping techniques of spoligotyping and MIRU were applied to 93 isolates collected in two consecutive years from 93 different tuberculosis patients residing in São Paulo city and attending the Clemente Ferreira Institute (the reference clinic for the treatment of tuberculosis).

Findings

Spoligotyping generated 53 different spoligotype patterns. Fifty-one isolates (54.8%) were grouped into 13 spoligotyping clusters. Seventy- two strains (77.4%) showed spoligotypes described in the international databases (SpolDB4, SITVIT), and 21 (22.6%) showed unidentified patterns. The most frequent spoligotype families were Latin American Mediterranean (LAM) (26 isolates), followed by the T family (24 isolates) and Haarlem (H) (11 isolates), which together accounted for 65.4% of all the isolates. These three families represent the major genotypes found in Africa, Central America, South America and Europe. Six Spoligo-International-types (designated SITs by the database) comprised 51.8% (37/72) of all the identified spoligotypes (SIT53, SIT50, SIT42, SIT60, SIT17 and SIT1). Other SITs found in this study indicated the great genetic diversity of M. tuberculosis, reflecting the remarkable ethnic diversity of São Paulo city inhabitants. The MIRU technique was more discriminatory and did not identify any genetic clusters with 100% similarity among the 93 isolates. The allelic analysis showed that MIRU loci 26, 40, 23 and 10 were the most discriminatory. When MIRU and spoligotyping techniques were combined, all isolates grouped in the 13 spoligotyping clusters were separated.

Conclusions

Our data indicated the genomic stability of over 50% of spoligotypes identified in São Paulo and the great genetic diversity of M. tuberculosis isolates in the remaining SITs, reflecting the large ethnic mix of the São Paulo city inhabitants. The results also indicated that in this city, M. tuberculosis isolates acquired drug resistance independently of genotype and that resistance was more dependent on the selective pressure of treatment failure and the environmental circumstances of patients.

Spoligotyping, a method based on the variability of distribution of the 43 inter-direct repeat (DR) spacers of Mycobacterium tuberculosis and Mycobacterium bovis BCG, is useful to study the molecular epidemiology of bovine and human tuberculosis. Recently, a major family of M. tuberculosis clinical isolates named the Haarlem family, which did not contain spacers 31 and 33 to 36, was reported in a multicenter study. Independently, a data bank containing all the published spoligotypes showed that the two most prevalent spoligotypes in the world differed only by the presence or absence of spacer 31. A careful analysis of the DR locus sequence led us to hypothesize that spacer 31 may not have been amplified in some isolates with the primer sets DRa and DRb currently used for spoligotyping. Consequently, a modified spoligotyping method based on different combinations of the 36-bp DR and IS6110 primers was devised that was able to discriminate between the left and the right parts of the DR locus and demonstrated the presence of the previously unamplified spacer 31 for some of the clinical isolates. By analogy, we suggest that a single-spacer difference in some epidemiologically linked cases of tuberculosis may simply arise due to the insertion of an extra copy of IS6110 within the DR locus, leading to its asymmetrical disruption and subsequent lack of the DRa or DRb targets. The influence of the IS6110 preferential insertion sites within the DR locus on spoligotyping results should be further investigated.

The Guinea-Bissau family of strains is a unique group of the Mycobacterium tuberculosis complex that, although genotypically closely related, phenotypically demonstrates considerable heterogeneity. We have investigated 414 M. tuberculosis complex strains collected in Guinea-Bissau between 1989 and 2008 in order to further characterize the Guinea-Bissau family of strains. To determine the strain lineages present in the study sample, binary outcomes of spoligotyping were compared with spoligotypes existing in the international database SITVIT2. The major circulating M. tuberculosis clades ranked in the following order: AFRI (n = 195, 47.10%), Latin-American-Mediterranean (LAM) (n = 75, 18.12%), ill-defined T clade (n = 53, 12.8%), Haarlem (n = 37, 8.85%), East-African-Indian (EAI) (n = 25, 6.04%), Unknown (n = 12, 2.87%), Beijing (n = 7, 1.68%), X clade (n = 4, 0.96%), Manu (n = 4, 0.97%), CAS (n = 2, 0.48%). Two strains of the LAM clade isolated in 2007 belonged to the Cameroon family (SIT61). All AFRI isolates except one belonged to the Guinea-Bissau family, i.e. they have an AFRI_1 spoligotype pattern, they have a distinct RFLP pattern with low numbers of IS6110 insertions, and they lack the regions of difference RD7, RD8, RD9 and RD10, RD701 and RD702. This profile classifies the Guinea-Bissau family, irrespective of phenotypic biovar, as part of the M. africanum West African 2 lineage, or the AFRI_1 sublineage according to the spoligtyping nomenclature. Guinea-Bissau family strains display a variation of biochemical traits classically used to differentiate M. tuberculosis from M. bovis. Yet, the differential expression of these biochemical traits was not related to any genes so far investigated (narGHJI and pncA). Guinea-Bissau has the highest prevalence of M. africanum recorded in the African continent, and the Guinea-Bissau family shows a high phylogeographical specificity for Western Africa, with Guinea-Bissau being the epicenter. Trends over time however indicate that this family of strains is waning in most parts of Western Africa, including Guinea-Bissau (p = 0.048).

A total of 2,346 Mycobacterium tuberculosis isolates from 13 provinces in China were genotyped by spoligotyping. Two hundred seventy-eight spoligotypes were identified: 2,153 isolates were grouped into 85 clusters, and the remaining 193 isolates were orphans. Comparison with the SpolDB4.0 database revealed that 118 spoligotypes had shared international type numbers in the database and the other 160 were novel. These 160 novel spoligotypes were assigned to families and subfamilies using the SpotClust program. The most prevalent family was the Beijing family (74.08%), followed by the T family (14.11%). CAS family strains were found only in the Xinjiang and Tibet regions, while EAI family strains were found only in Fujian Province. In conclusion, the present study of the M. tuberculosis population in China demonstrated that Beijing family isolates are the most prevalent strains in China and that they exhibit geographical variation. Furthermore, many new spoligotypes were found in this study.

Mycobacterium tuberculosis isolates from different regions of Bulgaria were studied by a variety of molecular typing tools. Based on spacer oligonucleotide typing (spoligotyping), the 113 strains were subdivided into 35 spoligotypes: 5 unique profiles and 15 profiles shared by two to 29 strains; the Hunter-Gaston diversity index (HGI) was 0.9. Comparison with the international database SITVIT2 at the Institut Pasteur de Guadeloupe showed the presence of two globally distributed shared types, ST53 (25.7%) and ST47 (6.2%). Nineteen (16.8%) and six (5.3%) strains belonged to the ST125 (LAM/S subfamily) and ST41 (LAM7_TUR subfamily) types described in SITVIT2 as ubiquitous/rare and ubiquitous/common types, respectively. Seven spoligoprofiles (12 strains) were not found in the database; two of them constituted new shared types. The Beijing genotype strains were not found in the studied collection in spite of close contacts with Russia in the recent and historical past. Additional subtyping by IS6110-restriction fragment length polymorphism (RFLP) and 12-locus mycobacterial interspersed repetitive unit (MIRU)-variable number of tandem repeat analyses were performed within selected spoligotypes. In particular, MIRU typing showed better discrimination within ST125 than IS6110-RFLP typing (HGI = 0.83 versus 0.39). A high gradient for ST125 in Bulgaria compared to its negligible presence in the global database and neighboring countries leads us to suggest a Bulgarian phylogeographic specificity of this spoligotype. To conclude, this first study of the Bulgarian M. tuberculosis population demonstrated its heterogeneity and predominance of several worldwide-distributed and Balkan-specific spoligotypes.

Tuberculosis is one of the important public health problems in Egypt. However, limited information on the Mycobacterium tuberculosis genotypes circulating in Egypt is available. A total of 151 M. tuberculosis strains were characterized by spoligotyping. The results revealed that 74.8% of M. tuberculosis isolates grouped into 13 different clusters, while 25.2% had unique spoligotype patterns. Comparison with an international spoligotyping database (the SITVIT2 database) showed that types SIT53 (T1 variant) and SIT54 (Manu2 variant) were the most common types between cluster groups. In addition, new shared types SIT2977, SIT2978, and SIT2979 were observed. The results identified for the first time an unusually high proportion of ancestral Manu strains of M. tuberculosis from patients in Egypt. The percentage of the Manu clade in this study (27.15%) was significantly higher than its overall representation of 0.4% in the SITVIT2 database. We show that in Egypt tuberculosis is caused by a predominant M. tuberculosis genotype belonging to the ancestral Manu lineage which could be a missing link in the split between ancestral and modern tubercle bacilli during the evolution of M. tuberculosis.

Background

Mozambique is one of the countries with the highest burden of tuberculosis (TB) in Sub-Saharan Africa, and information on the predominant genotypes of Mycobacterium tuberculosis circulating in the country are important to better understand the epidemic. This study determined the predominant strain lineages that cause TB in Mozambique.

Results

A total of 445 M. tuberculosis isolates from seven different provinces of Mozambique were characterized by spoligotyping and resulting profiles were compared with the international spoligotyping database SITVIT2.

The four most predominant lineages observed were: the Latin-American Mediterranean (LAM, n = 165 or 37%); the East African-Indian (EAI, n = 132 or 29.7%); an evolutionary recent but yet ill-defined T clade, (n = 52 or 11.6%); and the globally-emerging Beijing clone, (n = 31 or 7%). A high spoligotype diversity was found for the EAI, LAM and T lineages.

Conclusions

The TB epidemic in Mozambique is caused by a wide diversity of spoligotypes with predominance of LAM, EAI, T and Beijing lineages.

We present the first comprehensive analysis of Mycobacterium tuberculosis isolates circulating in the Kaohsiung region of southern Taiwan. The major spoligotypes found in the 224 isolates studied were Beijing lineages (n = 97; 43.3%), EAI lineages (n = 72; 32.1%) and Haarlem lineages (n = 18; 8.0%). By 24 MIRU-VNTR typing, 174 patterns were identified, including 24 clusters of 74 isolates and 150 unique patterns. The combination of spoligotyping and 12-MIRU-VNTR revealed that 129 (57.6%) of the 224 isolates were clustered in 18 genotypes. Moreover, 63.6% (7/11) of infected persons younger than 30 years had a Beijing strain, which could suggest recent spread among younger persons by this family of TB strains in Kaohsiung. Among the 94 Beijing family (SIT1, SIT250 and SIT1674) isolates further analyzed for SNPs by mass spectrometry, the most frequent strain found was ST10 (n = 49; 52%), followed by ST22 (n = 17; 18%) and ST19 (n = 11; 12%). Among the EAI-Manila family isolates analyzed by region deletion-based subtyping, the most frequent strain found was RD type 1 (n = 63; 87.5%), followed by RD type 2 (n = 9; 12.5%). In our previous study, the proportion of modern Beijing strains (52.5%) in northern Taiwan was significantly higher than the proportion of EAI strains (11%). In contrast, in the present study, EAI strains comprised up to 32% of Beijing strains in southern Taiwan. In conclusion, both ‘modern’ (Beijing) and ‘ancient’ (EAI) M. tuberculosis strains are prevalent in the Kaohsiung region, perhaps suggesting that both strains are somehow more adapted to southern Taiwan. It will be interesting to investigate the dynamics of the lineage composition by different selection pressures.

Background

Molecular typing of Mycobacterium tuberculosis strains has become a valuable tool in the epidemiology of tuberculosis (TB) by allowing detection of outbreaks, tracking of epidemics, identification of genotypes and transmission events among patients who would have remained undetected by conventional contact investigation. This is the first genetic biodiversity study of M. tuberculosis in Venezuela. Thus, we investigated the genetic patterns of strains isolated in the first survey of anti-tuberculosis drug-resistance realised as part of the Global Project of Anti-tuberculosis Drug Resistance Surveillance (WHO/IUATLD).

Results

Clinical isolates (670/873) were genotyped by spoligotyping. The results were compared with the international spoligotyping database (SpolDB4). Multidrug resistant (MDR) strains (14/18) were also analysed by IS6110-RFLP assays, and resistance to isoniazid and rifampicin was characterised.

Spoligotyping grouped 82% (548/670) of the strains into 59 clusters. Twenty new spoligotypes (SITs) specific to Venezuela were identified. Eight new inter-regional clusters were created. The Beijing genotype was not found. The genetic network shows that the Latin American and Mediterranean family constitutes the backbone of the genetic TB population-structure in Venezuela, responsible of >60% of total TB cases studied. MDR was 0.5% in never treated patients and 13.5% in previously treated patients. Mutations in rpoB gene and katG genes were detected in 64% and 43% of the MDR strains, respectively.

Two clusters were found to be identical by the four different analysis methods, presumably representing cases of recent transmission of MDR tuberculosis.

Conclusion

This study gives a first overview of the M. tuberculosis strains circulating in Venezuela during the first survey of anti-tuberculosis drug-resistance. It may aid in the creation of a national database that will be a valuable support for further studies.

An unusually high prevalence of pyrazinamide (PZA) monoresistance in Mycobacterium tuberculosis has been observed in Quebec. In the absence of a recognized outbreak, we hypothesized that these isolates most likely represented reactivation of an old endemic strain in this low-incidence area. A case-control study of 77 PZA-resistant isolates with a specific Quebec mutation and 253 PZA-susceptible control M. tuberculosis isolates was undertaken. By molecular analysis, all 77 case isolates shared a unique mutation profile in the pncA gene which was not present in control isolates. While control isolates manifested diverse IS6110 restriction fragment length polymorphism (RFLP) patterns, spoligotypes, and major genetic groups, case isolates had similar but nonidentical IS6110 RFLP patterns, had common spoligotypes, and were confined to one major genetic group, suggesting a common clonal ancestor. By epidemiologic and geographic analyses, however, there were no significant differences between the cases and the controls. We conclude that a clonally related family of PZA-monoresistant M. tuberculosis isolates in Quebec represents historic rather than recent transmission.

Background

Mycobacterium tuberculosis has a global population structure consisting of six main phylogenetic lineages associated with specific geographic regions and human populations. One particular M. tuberculosis genotype known as “Beijing” has repeatedly been associated with drug resistance and has been emerging in some parts of the world. “Beijing” strains are traditionally defined based on a characteristic spoligotyping pattern. We used three alternative genotyping techniques to revisit the phylogenetic classification of M. tuberculosis complex (MTBC) strains exhibiting the typical “Beijing” spoligotyping pattern.

Methods and Findings

MTBC strains were obtained from an ongoing molecular epidemiological study in Switzerland and Nepal. MTBC genotyping was performed based on SNPs, genomic deletions, and 24-loci MIRU-VNTR. We identified three MTBC strains from patients originating from Tibet, Portugal and Nepal which exhibited a spoligotyping patterns identical to the classical Beijing signature. However, based on three alternative molecular markers, these strains were assigned to Lineage 3 (also known as Delhi/CAS) rather than to Lineage 2 (also known as East-Asian lineage). Sequencing of the RD207 in one of these strains showed that the deletion responsible for this “Pseudo-Beijing” spoligotype was about 1,000 base pairs smaller than the usual deletion of RD207 in classical “Beijing” strains, which is consistent with an evolutionarily independent deletion event in the direct repeat (DR) region of MTBC.

Conclusions

We provide an example of convergent evolution in the DR locus of MTBC, and highlight the limitation of using spoligotypes for strain classification. Our results indicate that a proportion of “Beijing” strains may have been misclassified in the past. Markers that are more phylogenetically robust should be used when exploring strain-specific differences in experimental or clinical phenotypes.

The variations in biochemical as well as molecular characteristics among several members of the Mycobacterium tuberculosis complex that are not M. tuberculosis have been assessed to facilitate an unambiguous species identification. Altogether, 96 M. tuberculosis complex strains including 52 M. bovis isolates and 44 M. africanum isolates were analyzed by spoligotyping. The strains could be clustered into five spoligotype groups. All M. bovis isolates showed the typical absence of the spacers 39 to 43 and typical biochemical properties. However, within these strains we found a group of strains that had a spoligotype pattern which is clearly defined by the additional absence of spacers 3 to 16 and that were uncommonly susceptible to pyrazinamide (PZA). This spoligotype pattern has previously been described as being typical for a caprine genotype because of its predominant isolation from sheep and goats. Due to the clinical importance of PZA resistance, we propose two M. bovis subtypes: M. bovis subtype bovis, which is resistant to PZA, and M. bovis subtype caprae, which is susceptible to PZA. Two additional strains that clustered in group 3 showed biochemical and genetic properties typical for M. bovis and were also sensitive to PZA; thus, they may represent a third PZA-susceptible M. bovis subtype. The M. africanum isolates could be clustered into two spoligotype groups which can be differentiated from M. bovis by hybridization to spacers 39 to 43. These groups correspond to the previously described M. africanum subtypes I and II and can be clearly distinguished from each other by spoligotyping and resistance to thiophen-2-carboxylic acid hydrazide. Our results demonstrate that spoligotyping is a useful tool for differentiation of M. bovis and M. africanum. Moreover, we describe two PZA-susceptible M. bovis subtypes and describe a method that facilitates an unambiguous differentiation of the two M. africanum subtypes.

Spoligotyping is a major tool for molecular typing of Mycobacterium tuberculosis complex organisms. For epidemiological purposes, strains are considered clonal only when their spoligotyping patterns are identical. We report a change in the spoligotyping profiles of truly isogenic strains (a clinical isolate and a subculture derived in the laboratory) caused by deletion of a direct variable repeat. Without the information about the relationship between them, a link between these strains would have gone unnoticed. Evolutionary events should be taken into account in the interpretation of spoligotyping results and in the design of databases.

Background

Globally, China is the second place with high burden of tuberculosis (TB). To explore the characteristics of the pathogens of Mycobacterium tuberculosis (MTB) circulating in this area is helpful for understanding and controlling the spread of the strains. Recent developments in molecular biology have allowed prompt identification and tracking specific strains of MTB spreading through the population.

Methods

Spacer-oligonucleotide typing (spoligotyping) and mycobacterial interspersed repetitive units variable number tandem repeat (MIRU-VNTR) were performed in combination to yield specific genetic profiles of 260 MTB strains isolated from 30 counties of Jiangsu province in China between June and July 2010. The spoligotyping results were in comparison to the world Spoligotyping Database of Institute Pasteur de Guadeloupe (SpolDB4). Drug susceptibility test (DST) was performed on all strains by proportion method on Lowenstein-Jensen (LJ) culture media.

Results

Based on the spoligotyping method, 246 strains displayed known patterns and 14 were absent in the database. Predominant spoligotypes belonged to the Beijing family (80.4%). By using the 24-loci VNTR typing scheme, 224 different patterns were identified, including 20 clusters and 204 unique patterns. The largest clade comprised 195 strains belonging to the Beijing family. The combination of spoligotyping and 24-loci MIRU-VNTR demonstrated maximal discriminatory power. Furthermore, we observed a significant association between Beijing family strains and drug-resistant phenotypes. The Beijing family strains presented increased risks for developing multi-drug resistant TB, with the OR (95% CI) of 11.07(1.45-84.50).

Conclusions

The present study demonstrated that Beijing family isolates were the most prevalent strains circulating in Jiangsu province of China. The utility of spoligotyping in combination with 24-loci MIRU-VNTR might be a useful tool for epidemiological analysis of MTB transmission.