Molecular epidemiology of tuberculosis in Prague and South Moravia, Czech Republic: genetic analysis of Mycobacterium tuberculosis isolates by IS6110-RFLP fingerprinting and spoligotyping

OBJECTIVES: To genetically characterize and compare Mycobacterium tuberculosis isolates among culture-confirmed TB cases in two regions in the Czech Republic in 1998. METHODS: Consecutive M. tuberculosis isolates from 111 TB patients in Prague and 120 patients in the South Moravia region were genotyped using the standardized IS6110 Southern blot hybridization method and by spoligotyping. RESULTS: Eighty of the Prague patients (72.1%) had isolates with unique RFLP patterns, while 31 (27.9%) had isolates which belonged to 10 clusters. Seventy-eight (64.7%) of the South Moravia strains displayed unique RFLP pattern and 42 (35.3%) were assigned into 15 clusters. The spoligotype profiles previously identified in the U.S. were found in 69 (33%) samples and newly identified Czech spoligotypes in 24 (11.4%) of the total number of examined strains. CONCLUSIONS: The present population-based molecular epidemiological study performed in two regions of the Czech Republic in 1998 demonstrated the distribution of individual genotypes as well as clustered strains of M. tuberculosis isolated from TB patients, and confirmed the similarity between the Czech strain collection and the European Community TB Database, that includes countries with low TB rate. The sporadic import of TB cases from foreign countries and recent transmission events probably do not play significant roles in the epidemiological situation in the Czech Republic.     

Interpreting genotype cluster sizes of Mycobacterium tuberculosis isolates typed with IS6110 and spoligotyping

Molecular techniques such as IS6110-RFLP typing and spacer oligonucleotide typing (spoligotyping) have aided in understanding the transmission patterns of Mycobacterium tuberculosis. The degree of clustering of isolates on the basis of genotypes is informative of the extent of transmission in a given geographic area. We analyzed 130 published data sets of M. tuberculosis isolates, each representing a sample of bacterial isolates from a specific geographic region, typed with either or both of the IS6110-RFLP and spoligotyping methods. We explored common features and differences among these samples. Using population models, we found that the presence of large clusters (typically associated with recent transmission) as well as a large number of singletons (genotypes found exactly once in the data set) is consistent with an expanding infectious population. We also estimated the mutation rate of spoligotype patterns relative to IS6110 patterns and found the former rate to be about 10-26% of the latter. This study illustrates the utility of examining the full distribution of genotype cluster sizes from a given region, in the light of population genetic models.     

Role of spoligotyping and IS6110-RFLP in assessing genetic diversity of Mycobacterium tuberculosis in India

In the present study, genetic diversity analysis of Mycobacterium tuberculosis isolated from patients attending a tertiary care hospital, North India, has been attempted. Eighty three isolates of M. tuberculosis were subjected to DNA fingerprinting using spoligotyping and IS6110-RFLP techniques. Spoligotype patterns showed that central Asian (32.5%), ill defined T (13.2%) and Beijing (10.8%) families were predominant in ongoing transmission of the bacterium. Two STs; ST26 (CAS_Delhi) and ST1 (Beijing) represented 36.1% of the total M. tuberculosis population in eastern Uttar Pradesh, North India. IS6110 RFLP analysis showed that isolates having low and zero copy number of the IS element were 15.6% and 19.2%, respectively. Out of the 47 isolates clustered by spoligotyping, 40 could be further differentiated as unique strains by IS6110-RFLP. Therefore, this study recommends that both the techniques be used simultaneously for DNA fingerprinting of M. tuberculosis in India.     

Typing of drug resistant isolates of Mycobacterium tuberculosis from India using the IS6110 element reveals substantive polymorphism.

We investigated IS6110 polymorphism in clinical isolates of Mycobacterium tuberculosis from patients attending the outpatient department at various hospitals in northern India. DNA fingerprinting of 126 clinical isolates of M. tuberculosis was carried out using restriction fragment length polymorphism (RFLP) associated with the IS6110 element in M. tuberculosis genomes. A substantive degree of polymorphism was evident in the MDR M. tuberculosis isolates. The number of bands in the fingerprints varied from 0 to 19. However, the lack of common bands made it difficult to cluster the majority of these isolates. We were also unable to associate drug resistance with IS6110 copy number. Specific regions of the gyrA and katG genes from a representative number of these isolates were sequenced to determine the genotype. The majority of the isolates analyzed were found to belong to Group 1, indicating that these strains were evolutionarily older. We find no evidence of the W strain, prevalent in the US, in our study. The epidemiological patterns of the various strains in India seem to be very complex, as reflected by the presence of a large number of different strains (types) within north India.     

Molecular differentiation of Mycobacterium tuberculosis strains without IS6110 insertions

By using standard restriction fragment length polymorphism, 6 zero-copy IS6110 Mycobacterium tuberculosis isolates were identified from 1180 Maryland isolates as part of the National Tuberculosis Genotyping and Surveillance Network Project. By using various genotyping methods, we demonstrated that this zero band cluster can be differentiated into six genotypes.     

Molecular typing of Mycobacterium tuberculosis strains with a common two-band IS6110 pattern

We conducted a program of population-based molecular typing of all Mycobacterium tuberculosis isolates obtained in Alabama since 1994. Of 2452 isolates, 1013 (41%) had fewer than 6 bands of IS6110; 348 (14%) had a single two-band pattern (JH2). With conventional epidemiologic methods, we identified three groups of related patients with JH2 isolates. Spoligotyping and pattern of variable number of tandem repeats identified 10 molecular groups; two found by conventional methods were subdivided.     

IS6110限制性片段多态性分析标准方法的建立及其在结核分枝杆菌分子分型中的应用

目的 建立IS6110限制性片段多态性分析(IS6110-RFLP)标准方法 并评价该方法 的分型能力.方法 采用核酸提取、PCR、限制性内切酶分析、Southern杂交、琼脂糖凝胶电泳等技术,结合Gel-Pro analyzer 3.1和BioNumeries(Version 5.0)软件,对78株结核分枝杆菌插入序列IS6110-RFLP进行分析.结果 确定标准化的IS6110-RFLP技术,包括核酸提取、PCR、限制性内切酶分析、Southern杂交、琼脂糖凝胶电泳等实验步骤及标化参数的相关数据分析软件的使用;采用该技术,将78株结核分枝杆菌分为75个不同的基因型,分别归属于11个基因簇,其中有52株归属于同一个基因簇,占菌株总数的66.7%(52/78).结论 建立标准化的IS6110-RFLP技术方案,该方法 具有很强的基因分型和株水平鉴定能力,可用于结核病的病原学监测.     

Molecular epidemiology of Mycobacterium tuberculosis clinical isolates in Southwest Ireland

Tuberculosis has had significant effects on Ireland over the past two centuries, causing persistently higher morbidity and mortality than in neighbouring countries until the last decade. This study describes the results of genotyping and drug susceptibility testing of 171 strains of Mycobacterium tuberculosis complex isolated between January 2004 and December 2006 in a region of Ireland centred on the city of Cork. Spoligotype comparisons were made with the SpolDB4 database and clustered 130 strains in 23 groups, forty-one strains showed unique Spoligotyping patterns. The commonest spoligotypes detected were ST0137 (X2) (16.9%), and ST0351 (15.8%) (‘U’ clade). The major spoligotype clades were X (26.2%), U (19.3%), T (15.2%), Beijing (5.9%), Haarlem (4.7%), LAM (4.1%), BOVIS (1.75%), with 12.9% unassigned strains. A 24-locus VNTR genotyping produced 15 clusters containing 49 isolates, with high discrimination index (HGDI > 0.99). A combination of Spoligotyping and VNTR reduced the number of clustered isolates to 47 in 15 clusters (27.5%). This study identified ST351 as common among Irish nationals, and found a low rate of drug resistance with little evidence of transmission of drug resistant strains. Strain clustering was significantly associated with age under 55 years and Irish nationality. Only strains of Euro-American lineage formed clusters. Molecular typing did not completely coincide with the results of contact investigations.     

Instability of IS6110 patterns in multidrug-resistant strains of Mycobacterium tuberculosis

The stability of IS6110 restriction fragment length polymorphism (RFLP) pattern was determined in 31 isolates from patients with multidrug-resistant tuberculosis (MDR-TB). These patients were in actual chains of transmission and they referred to the National Institute of Tuberculosis and Lung Diseases, Tehran, Iran. Susceptibility testing against first- and second-line drugs were performed by the proportional method on Lowenstein-Jensen culture media. Thereafter, DNA fingerprinting by IS6110 with direct repeat (DR) region as a probe was performed by standard protocols. The rate of IS6110 changes was 16%, although, no variation was found in the DR region, in a time-span of 1-63 months. The strains with unstable IS6110 patterns were resistant to all drugs tested, and the majority of them (60%) were collected from HIV-positive patients. The results demonstrated that for a reliable interpretation of strain typing, it is better to use an additional marker along with IS6110 RFLP.     

Molecular epidemiology of Mycobacterium tuberculosis, Buenos Aires, Argentina

To analyze the molecular epidemiology of Mycobacterium tuberculosis strains at a hospital in Buenos Aires, Argentina, and mutations related to multidrug-resistant and extensively drug-resistant tuberculosis, we conducted a prospective case-control study. Our findings reinforce the value of incorporating already standardized molecular methods for rapidly detecting resistance.     

IS6110 Restriction Fragment Length Polymorphism Typing of Drug-resistant Mycobacterium tuberculosis Strains from Northeast South Africa

Tuberculosis (TB) remains a deadly infectious disease affecting millions of people worldwide; 95% of TB cases, with 98% of death occur in developing countries. The situation in South Africa merits special attention. A total of 21,913 sputum specimens of suspected TB patients from three provinces of South Africa routinely submitted to the TB laboratory of Dr. George Mukhari (DGM) Hospital were assayed for Mycobacterium tuberculosis (MTB) growth and antibiotic susceptibility. The genetic diversity of 338 resistant strains were also studied. DNA isolated from the strains were restricted with Pvu II, transferred on to a nylon membrane and hybridized with a PCR-amplified horseradish peroxidase 245 bp IS6110 probe. Of the 338 resistant strains, 2.09% had less than 5 bands of IS6110, and 98% had 5 or more bands. Unique restriction fragment length polymorphism (RFLP) patterns were observed in 84.3% of the strains, showing their epidemiological independence, and 15.7% were grouped into 22 clusters. Thirty-two strains (61.5%) from the 52 that clustered were from Mpumalanga, 16/52 (30.8%) from Gauteng, and 4/52 (9.6%) from Limpopo province. Clustering was not associated with age. However, strains from male patients in Mpumalanga were more likely to be clustered than strains from male patients in Limpopo and/or Gauteng province. The minimum estimate for the proportion of resistant TB that was due to transmission is 9.06% (52-22=30/331). Our results indicate that transmission of drug-resistant strains may contribute substantially to the emergence of drug-resistant tuberculosis in South Africa.Key words: Drug resistance, Epidemiology, IS6110, M. tuberculosis, PCR-RFLP, South Africa     

Molecular characterization of INH-resistant Mycobacterium tuberculosis isolates by PCR-RFLP and multiplex-PCR in North India

In the present study, among 327 Mycobacterium tuberculosis (MTB) isolates collected from patients attending three different centres of North India, we attempted to find out the most common mutations occurring both at the Ser315 codon of katG and at the regulatory region of the mabA-inhA operon to evaluate their role for INH drug resistance in India. Out of 121 phenotypically INH-resistant MTB isolates, 88 (72.7%) were resistant to INH by genotypic methods viz., PCR-RFLP with MspI and SatI digestion and multiplex-PCR. PCR-RFLP results showed that 67 (55.4%) isolates had mutation in codon 315 of katG by SatI endonuclease. Among these, eight isolates that were found resistant by SatI PCR-RFLP were found to be sensitive by MspI PCR-RFLP. By multiplex-PCR we found 49 (40.5%), 21 (17.4%) and 10 (8.3%) isolates having AGC → ACC substitution in katG only, mutation in inhA^C–15T only and mutation in both respectively.Simultaneous use of both PCR-RFLP and multiplex-PCR can improve the detection rate of INH-resistant strains and may have an advantage over the liquid culture system of detecting drug resistance. These findings also enhanced our understanding about potential of resistance-related mutations in M. tuberculosis clinical isolates in India and could help in development and designing of molecular methods for revealing the drug susceptibility profiles of M. tuberculosis clinical isolates.     

Genetic biodiversity of Mycobacterium tuberculosis isolates from patients with pulmonary tuberculosis in India.

Spoligotyping was performed on 540 Mycobacterium tuberculosis isolates in order to evaluate the genetic biodiversity of tubercle bacilli in India. One hundred and forty seven patterns were unique and 393 were grouped in 48 clusters. Comparison with an international spoligotype database showed that the most predominant clades among tuberculosis (TB) isolates were Central Asian (CAS) and East-African Indian (EAI) with shared-types (ST) ST26 and ST11 alone being responsible for 34% of all TB cases. Twenty one (3.8%) isolates belonged to the Beijing genotype. Marked variations were observed among circulating strains, STs belonging to CAS family predominated in the North, whereas the EAI family was more common in the Southern India. TB in India is predominantly caused by strains belonging to the principal genetic group 1 (PGG1), suggesting that most of the TB burden in India may be traced to ancestral clones of the tubercle bacilli. This study gives an insight into the global M. tuberculosis genetic biodiversity in India, the predominant spoligotypes and their impact on disease transmission.     

Molecular typing and drug susceptibility of Mycobacterium tuberculosis isolates from Chongqing Municipality,China

China’s tuberculosis (TB) burden is second only to that of India worldwide. In Chongqing, the largest municipality in southwestern China, although the prevalence of both TB and drug-resistant TB is higher than in other municipalities, the molecular characteristics and drug susceptibility phenotypes are poorly known. In this study, 297 Mycobacterium tuberculosis isolates from Chongqing were genotyped with spacer oligonucleotide typing (spoligotyping) and 28-locus MIRU-VNTR (24-locus MIRU-VNTR scheme and 4 other loci). Spoligotyping results were compared with drug-resistant profiles. Patients who showed clustering by both spoligotyping and 28-locus MIRU-VNTR were interviewed to investigate their detailed contact history. Our data demonstrated that the Beijing genotype was the most prevalent genotype, and ST1 was the most predominant lineage in Chongqing. The Beijing genotype was significantly associated with ethambutol resistance and multidrug-resistant phenotypes. A combination of the 10 most polymorphic loci permitted to achieve higher discriminatory power than 24-VNTR. In addition, a presumed transmission pathway was observed in a cluster of patients with the same MIRU-VNTR profile. The 10-VNTR locus set is suitable for genotyping of Mycobacterium tuberculosis in Chongqing.     

Differentiation of highly prevalent IS6110 single-copy strains of Mycobacterium tuberculosis from a rural community in South India with an ongoing DOTS programme.

We have prospectively analysed the DNA fingerprinting of Mycobacterium tuberculosis strains in a rural community from high prevalence area in South India with an ongoing DOTS programme. Strains from 451 culture-positive cases, diagnosed during July 1999-December 2000, were fingerprinted initially by both IS6110 and DR probes followed by polymorphic GC-rich repeat sequences (PGRS) typing only on low-copy strains. The results were correlated with selected epidemiological and clinical data. Forty one percent of strains showed single copy of IS6110, which further got differentiated into 62 DR and 27 PGRS patterns. One predominant DR pattern (5B/2) was found in 20% of the low-copy strains and was also involved in clusters. In all, 183 patients out of 451 (40%) were clustered in total 44 clusters when analysed by IS6110 and DR probes. With additional PGRS typing, the number of patients clustered was further reduced to 106 (23%). More number of patients (131) were clustered in IS6110 single-copy group. The maximum number of clusters was found with two or three patients. Only a small percentage (16%) of the patients reported direct epidemiological links while remaining patients might have had indirect links or casual contacts. Thus, a combination of two to three genetic markers is able to differentiate the most endemic strains of M. tuberculosis in areas with a high incidence of tuberculosis. The epidemiological data do not suggest any major outbreaks or a hot-spot hypothesis of transmission in this region. Phylogenetic analysis using IS6110, DR and PGRS RFLP (restriction fragment length polymorphism, RFLP) fingerprints showed that isolates exhibited clonal evolutionary pattern. The predominance of certain genotypes and agreement between the phylogenetic trees indicated that these strains were closely related and might have evolved or propagated from the common ancestor.     

Molecular typing of Mycobacterium tuberculosis isolates from a rural area of Kanpur by spoligotyping and mycobacterial interspersed repetitive units (MIRUs) typing.

Molecular typing of Mycobacterium tuberculosis isolates has greatly facilitated the understanding of epidemiology of tuberculosis (TB). This study was done to characterize prevalent genotypes of M. tuberculosis on a collection of 97 isolates based on spoligotyping and mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing in rural area of Kanpur, North India. In this area different types of interventions are being undertaken and follow-up studies are progressing. Predominant spoligotypes prevalent in this region belonged to Central Asian-Delhi family (CAS1_Del) (37%), East African-Indian family (11%), T1 family (8%) and Beijing (4%) family. Highly distinct MIRU-VNTR genotypes were obtained. Significant spoligotypes such as Beijing and CAS1_Del type were further divided into subtypes with MIRU-VNTR. This preliminary study reveals that CAS is the most predominant family in this rural area of Kanpur. If confirmed in other areas, this combined approach of molecular typing can be preferably be used as first line tool for studying linkage and transmission dynamics of TB in India.     

Genotyping of the Mycobacterium tuberculosis complex using MIRUs: association with VNTR and spoligotyping for molecular epidemiology and evolutionary genetics.

The recent introduction of molecular methods has gained increased acceptance as a powerful tool for epidemiology and phylogeny of tuberculosis (TB). In this investigation, the efficiency of molecular typing using mycobacterial interspersed repetitive units (MIRUs) was assessed on a set of 116 Mycobacterium tuberculosis complex clinical isolates from 11 different geographic origins. The results obtained were compared with spoligotyping and variable number of tandem DNA repeats (VNTRs) typing data. Eighty-nine different MIRU profiles were obtained on the sample studied. Spoligotyping- or VNTR-defined clusters were split into subclusters by MIRU typing. Conversely, almost all of the clinical isolates clustered by MIRUs were shown to belong to spoligotyping-based defined clusters. The calculation of the discriminative power by the Hunter-Gaston index (HGI) for VNTR, spoligotyping and MIRU typing gave the values of, respectively, 0.959, 0.965 and 0.988, showing the high discriminative power of the MIRUs. The allelic diversity of the sample was calculated for each of the MIRU-VNTR loci; five MIRU loci (MIRU nos. 10, 23, 26, 31 and 40) were "highly discriminant", four (MIRU nos. 4, 16, 24 and 39) were "moderately discriminant", and three (MIRU nos. 2, 20 and 27) were "poorly discriminant". Among the three complementary VNTRs (exact tandem repeats ETR-A, ETR-B and ETR-C), ETR-A was the most discriminant locus. A combined numerical analysis of spoligotyping, VNTR and MIRU typing results partly corroborated a recently hypothesized evolutionary scenario for the M. tuberculosis complex. M. canettii would be the first branch to have diverged from a common M. tuberculosis complex ancestor. The East-African Indian (EAI) clade could be the first family to have diverged thereafter. A third branching separated a M. africanum-M. bovis clade, followed by a node separating Beijing versus non-Beijing M. tuberculosis. The Beijing clade was distinct from the Central Asian 1 (CAS1) family. Among non-Beijing strains, branches such as the Latin-American and Mediterranean (LAM), X and Haarlem clades diverged later. In conclusion, the results obtained show the congruence between clades defined by spoligotyping, and MIRU-VNTR, and underline the potential of these methods for M. tuberculosis phylogeny reconstruction. We also conclude that MIRU typing is a very promising method that may be used in a "two PCR-based" genotyping strategy, in conjunction to conventional epidemiological investigations.     

Molecular characterization of multidrug-resistant Mycobacterium tuberculosis isolates from high prevalence tuberculosis states in Mexico

Mexico is one of the most important contributors of multidrug resistance tuberculosis (MDR-TB) in Latin-America, however little is known about the molecular characteristics of these strains. For this reason, the objective of this work was to determine the genotype and characterize polymorphisms in genes associated with resistance to rifampicin, isoniazid, and second-line drugs in isolates from two regions of Mexico with high prevalence of drug resistant tuberculosis.Clinical isolates from individuals with confirmed MDR-TB were genotyped using MIRU-VNTR 12 loci. To characterize the polymorphisms in genes associated with resistance to rifampicin, isoniazid and second-line drugs; rpoB, katG, inhA, rrs, eis, gyrA, gyrB and tlyA were sequenced.22 (41%) of the 54 MDR-TB isolates recovered were from the state of Baja California, while 32 (59%) were from Veracruz. The results show the katGS315T mutation was observed in 20% (11/54) of the isolates, while rpoBS315L was present in 33% (18/54). rrs had three polymorphisms (T1239C, ntA1401C and ntA1401G), gyrB presented no modifications, whereas gyrA showed five (S95T, F60Y, A90V, S91P and P124A), eis two (G-10A and A431G) and tlyA one (insertion at codon 67). Only 20% (11/54) of isolates were confirmed as MDR-TB by sequencing, and no mutations at any of the genes sequenced were observed in 43% (23/54) of the strains. Two isolates were recognized with the proper set of mutations like pre-XDR and one was XDR-TB. Eighteen isolates were classified as orphans and the remaining thirty-six were distributed in fourteen lineages, the most frequent were S (11%), Haarlem (9%), Ghana (9%) and LAM (7%). Out of the fourteen clusters identified, seven included unknown genotypes and nine had lineages.This is one of the most detailed analyses of genotypic characteristics and mutations associated with drug resistance to first and second-line drugs in MDR-TB isolates from Mexico. An important genetic variability and significant discrepancy between phenotypic tests and polymorphisms was observed. Our results set the need to screen additional loci as well as implement a molecular epidemiological surveillance system of MDR-TB in the country.     

Molecular diversity of Mycobacterium tuberculosis isolates from patients with pulmonary tuberculosis in Sri Lanka

The strain diversity of 100 Mycobacterium tuberculosis isolates collected over a period of 18 months from tuberculosis (TB) cases in Sri Lanka was studied by spoligotyping. When compared to the international spoligotyping database, 43 spoligotype patterns were identified, of which 20 were previously described. The majority of isolates (72.45%) were clustered into major genetic group 1, and the most common spoligotype pattern belonged to the Beijing (ST1) strain family. All the Beijing strain isolates belonged to more recently evolved sublineages of M. tuberculosis. The characterization of Sri Lankan M. tuberculosis isolates by spoligotyping shows a heterogeneous pattern. The physical separation from the main Indian peninsula may be responsible for the different patterns observed between the two countries. An in-depth field study is needed to understand the spread and the true epidemiology of this infection.