Mycobacterial Interspersed Repetitive Unit-variable Number Tandem Repeat Research Articles

Molecular characteristics of drug-resistance Mycobacterium tuberculosis strains isolated from extra pulmonary tuberculosis sites

ObjectivesChina is the second high tuberculosis (TB) burden country in the world. This article was to determinate the molecular characteristic of drug resistance Mycobacterium tuberculosis (DRTB) strains from extra pulmonary tuberculosis (EPTB). MethodsThe medical records of patients with EPTB were reviewed and collected from 2006 to 2016. The drug sensitivity of all samples was studied. All multiple drug resistance (MDR) and extensive drug resistance (XDR) strains were included. The detection of the deletion of region of difference 105 (RD105) and mycobacterial interspersed repetitive-unit variable-number tandem-repeat (MIRU-VNTR) were used to discriminate the molecular type of EPTB strains. Results162 DRTB isolates were from patients with EPTB including 104 male and 58 female. Beijing genotype had a significant correlation with the patterns of DR (P<0.05), re-treatment patients (P<0.05) and gender (P<0.05). The history of treatment had a statistically significant correlation with patterns of DR (P<0.05) and gender (P<0.05). Patterns of DR had no correlation with gender (P>0.05). Of 162 strains Beijing family strains represented 91.4%. The cluster rate was 17.9% and clustering ratio was 11.1%. Beijing family genotype is predominant in the patients with EPTB. The cluster rate and clustering ratio was low. ConclusionsBeijing family genotype is predominant and highly epidemic in the patients with drug resistance extra pulmonary tuberculosis (DR-EPTB). The cluster rate and clustering ratio was low. Genotype of re-treatment male patient with DR-EPTB is more likely Beijing family genotype.

Possible Transmission Mechanisms of Mixed Mycobacterium tuberculosis Infection in High HIV Prevalence Country, Botswana.

Tuberculosis caused by concurrent infection with multiple Mycobacterium tuberculosis strains (i.e., mixed infection) challenges clinical and epidemiologic paradigms. We explored possible transmission mechanisms of mixed infection in a population-based, molecular epidemiology study in Botswana during 2012–2016. We defined mixed infection as multiple repeats of alleles at >2 loci within a discrete mycobacterial interspersed repetitive unit–variable-number tandem-repeat (MIRU-VNTR) result. We compared mixed infection MIRU-VNTR results with all study MIRU-VNTR results by considering all permutations at each multiple allele locus; matched MIRU-VNTR results were considered evidence of recently acquired strains and nonmatched to any other results were considered evidence of remotely acquired strains. Among 2,051 patients, 34 (1.7%) had mixed infection, of which 23 (68%) had recently and remotely acquired strains. This finding might support the mixed infection mechanism of recent transmission and simultaneous remote reactivation. Further exploration is needed to determine proportions of transmission mechanisms in settings where mixed infections are prevalent.

Accuracy of the QIAxcel Automated System for MIRU-VNTR Genotyping of Mycobacterium tuberculosis in Two Limited Resource Settings.

Mycobacterial interspersed repetitive units variable number tandem repeat (MIRU-VNTR) typing of Mycobacterium tuberculosis complex (MTBC) isolates, based on 24 loci, is still widely used as the standard for routine molecular surveillance of tuberculosis (TB). QIAxcel system is proposed as an affordable tool that could replace conventional gel electrophoresis and provide high concordance with the reference methods regarding MIRU-VNTR typing of MTBC. We aimed to evaluate the QIAxcel accuracy for allele calling of MIRU-VNTR loci in two regional reference laboratories. A total of 173 DNA were used for the study. Results obtained with QIAxcel were compared to the reference results obtained with an ABI 3730 DNA analyzer. In Albania, the overall agreement with the reference method was 97.92%. A complete agreement result was obtained for 17 loci. In Tunisia, the overall agreement with the reference method was 98.95%. A complete agreement result was obtained for 17 loci. Overall agreement in both centers was 98.43%. In our opinion, use of QIAxcel technology has the potential to be reliable, given an optimized algorithm. Inaccuracies in sizing of long fragments should be solved, especially regarding locus 4052.

Application of MIRU-VNTR on smear slides: a shortcut for detection of polyclonal infections in tuberculosis patients.

Mixed (polyclonal) infections are one of the main problems in tuberculosis (TB) management. The best available method for detecting polyclonal infections in TB is mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR). According to multiple studies, MIRU-VNTR method can be applied to detect TB-related polyclonal infections in sputum samples or cultures. Setup of MIRU-VNTR on smear slides can be an efficient approach, regardless of the limitations of cultures and sputum samples in many laboratories. The present study aimed at investigating the diagnostic potential of MIRU-VNTR on smear slides in detecting mixed infections. Ziehl-Neelsen-stained microscopic slides were prepared from 14 clinical specimens. For amplifying 24 MIRU-VNTR loci, PCR assay was performed on the smear slides, clinical specimens, and cultures. Based on the 24-locus MIRU-VNTR analysis, polyclonal infections were reported in 42.85% of smear slides, while the corresponding rate was estimated at 57.1% (8/14) in the clinical samples. In the corresponding cultures, the rate of mixed infection was 7.14% (1/14). Use of smear slides can be a safe option for transferring clinical specimens between environmental and reference laboratories. Considering their significant impact on TB treatment, it is essential to diagnose mixed infections in low-resource countries with a high prevalence of mixed infections. The present findings show that direct MIRU-VNTR on smear slides can be conveniently used for the detection of mixed infections.

Characteristics of compensatory mutations in the rpoC gene and their association with compensated transmission of Mycobacterium tuberculosis.

The aim of this study was to characterize rpoC gene mutations in Mycobacterium tuberculosis (MTB) and investigate the factors associated with rpoC mutations and the relation between rpoC mutations and tuberculosis (TB) transmission. A total of 245 MTB clinical isolates from patients with TB in six provinces and two municipalities in China were characterized based on gene mutations through DNA sequencing of rpoC and rpoB genes, phenotyping via standard drug susceptibility testing, and genotypic profiling by mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing. Approximately 36.4% of the rifampin-resistant isolates harbored nonsynonymous mutations in the rpoC gene. Twenty-nine nonsynonymous single mutations and three double mutations were identified. The rpoC mutations at locus 483 (11.3%) were predominant, and the mutations at V483G, W484G, I491V, L516P, L566R, N698K, and A788E accounted for 54.5% of the total detected mutations. Fifteen new mutations in the rpoC gene were identified. Rifampin resistance and rpoB mutations at locus 531 were significantly associated with rpoC mutations. MIRU-VNTR genotype results indicated that 18.4% of the studied isolates were clustered, and the rpoC mutations were not significantly associated with MIRU-VNTR clusters. A large proportion of rpoC mutation was observed in the rifampicin-resistant MTB isolates. However, the findings of this study do not support the association of rpoC mutation with compensated transmissibility.

Characterization of Mycobacterium tuberculosis heteroresistance by genotyping

Heteroresistance is the coexistence of susceptible and resistant strains in the same individual, considered the preliminary step for total resistance, and can stem from mixed infection or clonal heterogeneity. The aim of this study was to evaluate the heteroresistance of Mycobacterium tuberculosis to rifampicin and isoniazid and its characterization. GenoType MTBDRplus®; Sanger sequencing of the rpoB, katG, and inhA genes; and Mycobacterial Interspersed Repetitive Unit - Variable Number Tandem Repeat (MIRU-VNTR) were performed. In a total of 654 isolates, 530 were resistant, 124 were susceptible, and 29 were heteroresistant to a first-line drug. GenoType MTBDRplus® detected heteroresistance in the rpoB gene in 26/29 (89.6%), as compared to 5/29 (17.2%) in the katG gene and 2/29 (6.8%) in the inhA gene. Four isolates showed heteroresistance in these genes. The Sanger sequencing detected heteroresistance in the rpoB gene in 7/29 (24.1%), as compared to 3/29 (10.3%) in the katG gene. In one isolate, heteroresistance was concomitant in both the rpoB and katG genes. MIRU-VNTR detected mixed infection in three heteroresistant isolates, while four isolates showed clonal heterogeneity. GenoType MTBDRplus® detected more cases of heteroresistance when compared to sequencing. It was also possible to characterize mixed infection and clonal heterogeneity by MIRU-VNTR.

Genomic diversity and transmission dynamics of M. tuberculosis in Africa: a systematic review and meta-analysis.

BACKGROUND: Mycobacterium tuberculosis complex (MTBC) and its human host are the most competent organisms with co-evolutionary trajectory. This review determined the phylogeography, clinical phenotype-related genotype and transmission dynamics of MTBC in Africa.METHODS: Spoligotyping and mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTR) based articles from Africa published in the English language were included. Articles were retrieved from PubMed and Scopus on 12 May 2018.RESULTS: In Africa, respectively 92% and 7% of tuberculosis (TB) cases were caused by M. tuberculosis and M. africanum. Among M. tuberculosis lineages (L), L4 was the predominant, at 67%, followed by L3/Central Asian (CAS; 10%). L7/ETH1 and L5/6/Maf were restricted to the Horn and Western Africa, respectively. L4.6/SIT37, H37Rv like, L4.1.2/Haarlem and H3-Ural were proportionally more frequent among tuberculous lymphadenitis (TBLN) than among pulmonary tuberculosis (PTB) cases. On 24-locus MIRU-VNTR, clustering rate was 31%; the secondary case rate from a single primary source case was 20%.CONCLUSION: Africa in general, and the east-west pole of Africa in particular, harboured a genetically diverse population of MTBC, with characteristics of geographic segregation. Both generalist and specialist genotypes are circulating in the region. L4 is dominant across the continent, while M. bovis is rarely detected as a cause for human TB. The clinical significance of genetic diversity of MTBC in the different geographic and population groups of Africa is not fully understood. Both person-to-person transmission and reactivation mode of TB is significant in Africa. Prevention and control strategies should therefore envisage these two scenarios.

Enhancing the differentiation of specific genotypes in Mycobacterium tuberculosis population

Today, significant attention is directed towards the global lineages and sublineages of Mycobacterium tuberculosis (Mtb). NEW-1 (SIT 127) and CAS1-Delhi (SIT 26) strains are recognized as growing and circulating Mtb genotypes, especially in Asian countries. It is crucial to develop or enhance Mtb genotyping methods for a more accurate and simple differentiation of these families. We used 24-loci mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing for genotyping 217 Mtb isolates. To select the optimal MIRU-VNTR loci, we calculated the Hunter-Gaston discriminatory index (HGDI), allelic diversity, and accumulation of percentage differences (APDs) between the strains among different groups of genotypes (NEW-1 and non-NEW-1; CAS1-Delhi and non-CAS). Finally, the minimum spanning tree was constructed for clustering analysis. In the NEW-1 population, loci with APD > 60% were found to have a high discriminatory power. VNTR loci with APD > 50% showed high discrimination power for the CAS population. Our findings suggest that APDs, which are valuable for the selection of VNTR loci sets, may improve the discriminatory power of MIRU-VNTR typing for identification of Mtb genotypes in specific regions.

Applicability of using 15 MIRU-VNTR loci for genotyping of Mycobacterium avium subsp. paratuberculosis from two cattle farms in Egypt.

Mycobacterium avium subspecies paratuberculosis (MAP) is a notorious infectious agent that causes Johne's disease which leads to serious economic losses in cattle farms all over the world. The Lack of accurate epidemiological and molecular data is a major barrier to the implementation of disease control strategies. Basically, the tracing of infections requires rapid detection of the widely spreading genotypes with the ability to determine isolates from common and different sources. This study aimed to evaluate the applicability and discriminatory power of 15 mycobacterial interspersed repetitive unit (MIRU)-variable number tandem repeat (VNTR) loci of M. tuberculosis for MAP genotyping. Additionally, detection of the most efficient loci combinations for molecular epidemiological investigations of MAP isolates. The discriminatory capacity and applicability of 15 known loci [2 exact tandem repeat (ETR) loci, 6 MIRU loci, 4 Mtub loci, and 3 Queen's University of Belfast (QUB) group loci] were assessed using 26 isolates from two cattle herds (Holstein Frisian) in El buhaira and Giza Governorates at north of Egypt. The results proved the presence of 12 different genotypes. All the used loci gave Hunter-Gaston discrimination index of DI = 0.963 while the ten loci (Mtub04, MIRU10, QUB11b, MIRU26, QUB26, QUB4156, MIRU04, ETRC, Mtub30, and Mtub39) were highly discriminating with DI = 0.956. Moreover, the five loci (Mtub21, MIRU31, MIRU16, MIRU40, and ETRA) gave moderate discriminatory power with DI = 0.839. The MIRU31 locus expressed no polymorphism among strains. MIRU-VNTR typing generally proved applicability and high discriminatory power with DI = 0.963. The ten highly discriminating DI = 0.956 proved to be the most suitable for the first-line genotyping of MAP from cattle, with nearly similar resolving ability as all the 15 loci. MIRU-VNTR proved fastness, efficiency, and feasibility in genotyping of MAP from cattle in Egypt.

Genotypes and genetic characters of Mycobacterium tuberculosis from Myanmar using three typing methods

Knowledge on basic characteristics of Mycobacterium tuberculosis (MTB) is helpful to understand the disease epidemiology and support the prediction of clinical outcome of the disease. The aim of this study was to detect the genotypes and genotypic characters of clinical Mycobacterium tuberculosis (MTB) isolates from new and retreatment rifampicin-resistant patients using three different genotyping methods. Mycobacterial interspersed repetitive units-variable number tandem repeat (MIRU-VNTR) typing was used to determine the diversity of 222 clinical isolates. Spoligotyping and IS6110-restriction fragment length polymorphism (RFLP) typing were also used to investigate the genetic characters of 105 MTB strains. Among the 15 genotypes detected by MIRU-VNTR, Beijing strains were the most prevalent of all strains (54.8%); new cases (40.5%) and retreatment cases (69.4%), followed by EAI strain. Spoligotyping categorized the strains into 11 lineages and 13 orphans whereas 96 different IS6110 patterns were identified using RFLP method. The mode number of IS6110 was 18 and 20. Higher band numbers were found in Beijing genotype (p < 0.001). Clustering rates by spoligotyping, MIRU-VNTR and IS6110-RFLP typing were 0.714, 0.004 and 0.085, respectively. Discriminatory powers of spoligotyping, MIRU-VNTR typing and IS6110-RFLP typing were 0.637, 1.000 and 0.997, respectively. Dominant Beijing genotype in both new and retreatment cases denoting that prevailing tuberculosis in Myanmar changed from EAI to Beijing lineage.

Insight into genetic diversity of Mycobacterium tuberculosis in Kandy, Sri Lanka reveals predominance of the Euro-American lineage

ObjectiveSri Lanka is a country where the molecular epidemiology of Mycobacterium tuberculosis (MTB) is poorly explored. Therefore, this study was performed to identify circulating lineages/sub-lineages of MTB and their transmission patterns. MethodsDNA was extracted from 89 isolates of MTB collected during 2012 and 2013 from new pulmonary tuberculosis patients in Kandy, Sri Lanka and analyzed by spoligotyping, large sequence polymorphism (LSP), mycobacterial interspersed repetitive unit–variable number tandem repeat (MIRU-VNTR) typing, and drug resistance-associated gene sequencing. ResultsThe predominant lineage was lineage 4 (Euro-American, 45.9%), followed by lineage 1 (Indo-Oceanic, 29.4%), lineage 2 (East-Asian, 23.5%), and lineage 3 (Central-Asian, 1.2%). Among 26 spoligotype patterns, eight were undesignated or new types and seven of these belonged to lineage 4. Undesignated lineage 4/SIT124 (n=2/8) and SIT3234 (n=8/8) clustered together based on 24-locus MIRU-VNTR typing. The dominant sub-lineage was Beijing/SIT1 (n=19), with the isoniazid resistance katG G944C mutation (Ser315Thr) detected in two of them. ConclusionsThe population structure of MTB in Kandy, Sri Lanka was different from that in the South Asian region. The clonal expansion of locally evolved lineage 4/SIT3234 and detection of the pre-multidrug resistant Beijing isolates from new tuberculosis patients is alarming and will require continuous monitoring.

Severe Clinical Outcomes of Tuberculosis in Kharkiv Region, Ukraine, Are Associated with Beijing Strains of Mycobacterium tuberculosis.

Genotypic variation in Beijing lineages of Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB), has been associated with hyper virulence and the spread of extensively and multiple drug (X/MDR) resistant MTB strains in Eastern Europe, Central Asia, and East Asia. The clinical outcomes of 215 new cases of TB among the population of the Kharkiv region of Eastern Ukraine were analyzed to uncover factors associated with severe infection. Infecting MTB strains were profiled by 5 locus exact tandem repeats (ETRs) and 15 locus mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) genotyping. Among diverse MTB genotypes discovered in Ukraine, the Beijing genotype (MIRU-VNTR 42425) was significantly associated with risk factors for severe outcomes of disease in the study population, including TB/HIV co-infection and treatment failure. Strain replacement (superinfection) was observed in 10 patients, suggesting repeated exposure to novel MTB strains in hospital or community settings. Inclusion of MTB genotyping data may identify at-risk patients and improve treatment adherence to prevent X/MDR development for effective public health response against tuberculosis in Ukraine.

First genetic characterisation of multidrug-resistant Mycobacterium tuberculosis isolates from Algeria.

To characterise the genotypes of multidrug-resistant (MDR) Mycobacterium tuberculosis (MTB) isolated in Algeria, where there is a low MDR-MTB incidence rate. Ten MDR isolates and one resistant to isoniazid were investigated by PCR-Sanger sequencing for 10 loci involved in resistance. Amplicon-based next generation sequencing (NGS) of 15 loci was additionally performed on isolates harbouring novel mutations. Sanger and amplicon-NGS provided the same results as with GenoType kits. Mutations known to be associated with resistance were described for most isolates: rpoB S531L in seven of 10 rifampicin-R MTB isolates, katG S315T in nine of 11 isoniazid-R, and promoter inhA c-15t in three of 11, embB M306V or M306I in two of two ethambutol-R, rpsL K43R in four of eight or rrs a514c associated with gidB L16R in streptomycin-R, gyrA A90V in the ofloxacin-R pre-XDR isolate. New and rare mutations were also described in rpoB (deletion 512-513-514), katG (S315R, M126I/ R496L), gidB (V124G, E92A, V139A, G37V), and gyrA (P8A). Mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) profiles were similar for three isolates (lineage Cameroon), indicating a possible clonal diffusion in epidemiologically unrelated patients. Resistant MTB isolates in Algeria harbour resistance genotypes similar to other countries, but some rare patterns may result from selection and transmission processes inherent to the country.

Spoligotyping analysis of Mycobacterium tuberculosis in Khyber Pakhtunkhwa area, Pakistan.

Background: Spoligotyping is a reproducible, reverse hybridization approach for genotyping of Mycobacterium tuberculosis complex (MTBC). Molecular typing of MTBC is helpful for understanding and controlling tuberculosis epidemics.Methods: Spoligotyping was performed on 166 clinical isolates of Mycobacterium tuberculosis (MTB) collected from 25 districts of Khyber Pakhtunkhwa, Pakistan. Results were compared to SITVIT2, an online database developed by the Institut Pasteur de la Guadeloupe, France.Results: Spoligotyping results showed that 145 strains (88%) displayed known patterns while 21 (12%) were new. Lineage 3/Central Asian strain (L3/CAS) was the predominant family (73%, χ2=19.9, P=0.001), followed by L2/Beijing (5.4%) and L4 (4.2%). L3/CAS1-Delhi was the major sublineage (82%) among the L3/CAS family (χ2=664, P=0.0001). Analysis showed that the majority of the clinical isolates with an unknown pattern had an evolutionary link with the L3/CAS strain, and nine (5.4%) of the unknown strains were epidemiologically linked and were tentatively named L3/CAS-KP (Khyber Pakhtunkhwa).Conclusion: The present study demonstrated that L3/CAS is the predominant lineage of MTB, widely distributed in different areas of the Khyber Pakhtunkhwa province of Pakistan. Spoligotyping patterns of some clinical isolates could not be matched to other reported patterns in an international database. Other tools, such as mycobacterial interspersed repetitive unit–variable number tandem repeat (MIRU-VNTR), will be helpful in future investigations into the epidemiological characteristics of clinical isolates in the Khyber Pakhtunkhwa province.

A first insight into the application of high discriminatory MIRU-VNTR typing using QIAxcel technology for genotyping Mycobacterium bovis isolated from the Delta area in Egypt

Mycobacterium bovis is a notorious infectious agent leading to serious economic losses for cattle farms worldwide. Analysis of the widely spreading genotypes is vital for tracing infections, understanding transmission dynamics, and controlling the cluster growth. This study aimed to evaluate the discrimination ability of 15 mycobacterial interspersed repetitive unit-variable number tandem repeats (MIRU-VNTR) loci and to assess the extremely efficient loci subset for molecular epidemiological investigations of M.bovis from farms in the Delta area of Egypt. The discriminating ability of MIRU-VNTR genotyping using 15 loci {2 exact tandem repeat (ETR) loci, 6 MIRU loci, 4 Mtub loci, and 3 Queen's University of Belfast (QUB) group loci)} were evaluated on 61 M.bovis isolates from cattle (Holstein Frisian) and buffaloes. The results indicate that there are 48 genotypes: 3 unique genotypes and 45 genotypes with shared similarities. Using the MIRU-VNTRplus database, M.bovis ID 7540/01 and ID 5346/02 were the nearest lineages to both groups. Six loci (MIRU10, QUB11b, QUB26, ETRA, Mtub30, and Mtub39) were highly discriminating, seven other loci (Mtub21, MIRU26, QUB4156, MIRU04 (ETRD), MIRU16, MIRU 40, and ETRC) gave moderate discriminatory power, and the last two loci (Mtub04 and MIRU31) were poorly discriminative. MIRU-VNTR typing generally proved efficacy and high discriminatory power, with a collective allele's diversification of 0.9641. Both the six highly discriminating (DI = 0.9492) and the seven moderately discriminating loci (DI = 0.9269) evidenced to be suitable for M.bovis first-step initial genotyping from cattle herds in Egypt. MIRU-VNTR is rapid and effective in the genotyping of M.bovis from cattle and buffaloes in Egypt.

Prevalence, Drug Resistance, and Genotypic Diversity of the Mycobacterium tuberculosis Beijing Family in Ecuador.

The Beijing family, the most successful Mycobacterium tuberculosis lineage, is considered hypervirulent, associated with clustering and has a strong association with multidrug-resistant tuberculosis. The Beijing strains have spread worldwide and also to Latin America. Genotyping of a countrywide collection of 380 M. tuberculosis strains from Ecuador, with 24-loci mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTR), revealed only six Beijing strains, but four of these were MDR-TB. There was no clustering as all six strains had very distinct MIRU-VNTR profiles that have not been reported in the rest of Latin America. Although active transmission for Beijing has been described for the neighboring countries Peru and Colombia, there is no evidence that Beijing strains in Ecuador are more frequently transmitted than other strains. Moreover, the low prevalence (1.6%) of the Beijing sublineage in Ecuador challenges the concept of hyperadaptability and transmissibility of the Beijing strains in our country.

Simplified Model to Survey Tuberculosis Transmission in Countries Without Systematic Molecular Epidemiology Programs.

Systematic molecular/genomic epidemiology studies for tuberculosis surveillance cannot be implemented in many countries. We selected Panama as a model for an alternative strategy. Mycobacterial interspersed repetitive unit–variable-number tandem-repeat (MIRU-VNTR) analysis revealed a high proportion (50%) of Mycobacterium tuberculosis isolates included in 6 clusters (A–F) in 2 provinces (Panama and Colon). Cluster A corresponded to the Beijing sublineage. Whole-genome sequencing (WGS) differentiated clusters due to active recent transmission, with low single-nucleotide polymorphism–based diversity (cluster C), from clusters involving long-term prevalent strains with higher diversity (clusters A, B). Prospective application in Panama of 3 tailored strain–specific PCRs targeting marker single-nucleotide polymorphisms identified from WGS data revealed that 31.4% of incident cases involved strains A–C and that the Beijing strain was highly represented and restricted mainly to Colon. Rational integration of MIRU-VNTR, WGS, and tailored strain–specific PCRs could be a new model for tuberculosis surveillance in countries without molecular/genomic epidemiology programs.

Clonality and genetic profiles of drug-resistant Mycobacterium tuberculosis in the Eastern Cape Province, South Africa.

In this study, we investigated the diversity of drug‐resistant Mycobacterium tuberculosis isolates from families who own cattle in the Eastern Cape Province of South Africa using spoligotyping and mycobacterial interspersed repetitive‐unit‐variable number tandem repeat (MIRU‐VNTR) typing. The Mycobacterium tuberculosis was investigated using MIRU‐VNTR and the Mycobacterium tuberculosis families were evaluated using spoligotyping. Spoligotyping grouped 91% of the isolates into seven clusters, while 9% of the deoxyribonucleic acid (DNA) from TB isolates were unclustered from a total of 154 DNA used. Previously described shared types were observed in 89.6% of the isolates, with the Beijing family, SIT1, the principal genotype in the province, while the families T, SIT53 and X1, SIT1329 were the least detected genotypes. MIRU‐VNTR grouped 81% of the isolates in 23 clusters while 19% were unclustered. A combination of the VNTR and spoligotyping grouped 79% of the isolates into 23 clusters with 21% unclustered. The low level of diversity and the clonal spread of drug‐resistant Mycobacterium tuberculosis isolates advocate that the spread of TB in this study may be instigated by the clonal spread of Beijing genotype. The results from this study provide vital information about the lack of TB control and distribution of Mycobacterium tuberculosis complex strain types in the Eastern Cape Province of South Africa.

Detecting tuberculosis clusters in urban neighborhoods, Taipei, Taiwan: Linking geographic and genotyping evidence

Tuberculosis (TB) clusters, defined as two or more active TB cases with epidemiological links and the same genotype of Mycobacterium tuberculosis strains, are direct evidence of recent TB transmission. We analyzed TB cases newly diagnosed in 2006–2008, confirmed by the diagnostic laboratory, at a university-affiliated hospital in Taipei metropolitan area where TB is endemic in elderly populations. Numbers of randomly selected non-TB patients (as controls) from each district were proportional to the district population (Pearson's correlation coefficient = 0.8), which supports that patients who visited the hospital represent a spatially independent sample of total population in this area. We used the nearest neighbor hierarchical (NNH) clustering method and the spatial scan statistics to identify spatial TB clusters among 969 cases within the 10-km buffer, adjusting for the elderly population. One cluster with 10 TB cases from different households in geographic proximity to each other was detected by both NNH clustering and spatial scan statistics. Spoligotyping and mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) genotyping showed that four of the six M. tuberculosis strains from this spatial cluster were identical. Three of those four patients were older than 70 years, but none were nursing home residents. The identical genotypes of M. tuberculosis strains in clustered elderly TB patients living in neighborhoods indicate that recent transmission plays a significant role in TB endemicity among elderly populations in Taiwan.

Genetic diversity and drug resistance of Mycobacterium tuberculosis in Ecuador.

The genetic diversity of Mycobacterium tuberculosis in Quito, Ecuador is not well known. To investigate mutations related to drug resistance and bacterial genotypes in M. tuberculosis strains in Ecuador. This was a retrospective study of M. tuberculosis isolates from 104 patients. Isolates were phenotypically resistant to rifampicin (RMP) and/or isoniazid (INH). The genotype was determined using 24-locus mycobacterial interspersed repetitive units-variable-number tandem repeats (MIRU-VNTR). Isolates showed mutations in the rpoB and katG genes, and the inhA promoter. In rpoB, we found 13 genetic alterations at codons 511, 513, 514, 515, 516, 526 and 531. Forty-six (44.2%) RMP-resistant isolates belonged to codon 531. In katG, there were nine genetic alterations at codons 296, 312, 314, 315, 322, 324 and 351. Fifty-three (51%) INH-resistant isolates belonged to codon 315. Five mutations not previously described were identified in katG: Thr324Ser, Thr314Ala, Ala312Pro, Trp351Stop and deleted G at 296 codon. The Latin American Mediterranean (LAM) (33.7%) and Ghana (30.8%) lineages presented most of the main mutations observed. This is the first report from Ecuador; it describes five new mutations in katG and indicates that LAM is the most prevalent lineage.