MIRU-VNTR Typing Research Articles

Description of the population structure and genetic diversity of tuberculosis in Estado de México, a low prevalence setting from Mexico.

In order to identify the genetic characteristics of the strains of mycobacteria circulating in the Estado de México, one of the states with the lowest prevalence of tuberculosis in Mexico, spoligotyping and 12-loci MIRU-VNTR typing were used to genotype tuberculosis clinical isolates. The average age of the 183 patients analyzed was 50 (± 17) years, drug resistance was noted in 57 (31%) and multidrug resistance in 22 (12%) individuals. The results from the isolates recovered showed that 80% were located in four major Euro-American lineages: Haarlem (17%), LAM (15%), T (20%) and X (29%). Other lineages found in lower proportions were: EAI, S, Beijing, West African, Turkey, Vole and Bovis. Eighteen isolates were orphans. Only 57 isolates were grouped in nine clusters and the SIT119 (X1) showed the highest number of members (23). The LAM lineage showed an increased risk for development of drug resistance (RR=4, IC: 95%: 1.05-14.2, p=0.03). Despite the important prevalence of four major lineages found and the diversity of strains circulating in the population, we found the presence of one of the largest populations of isolates clustered to the X lineage in a setting from a Latin American country.

First baseline of circulating genotypic lineages of Mycobacterium tuberculosis in patients from the brazilian borders with Argentina and Paraguay.

BackgroundAt the triple border Brazil/Paraguay/Argentina there is easy mobility from one city to another for economic and tourism activities. This constant and fast population mobility is mainly to visit Iguazu Falls, in the Iguazu River, on the border of the Brazilian state of Paraná and the Argentina. As the incidence of tuberculosis is high in this setting, our study aimed to establish a first baseline of circulating genotypic lineages of Mycobacterium tuberculosis.Methodology/Principal FindingsThis study included 120 patients from 10 cities in southwestern Paraná, Brazil with pulmonary symptoms, from July 2009 to July 2011. Information about sex, age, clinical features and address was collected by reviewing the national tuberculosis notification database. Of these, 96 (80%) isolates were identified as M. tuberculosis and 22 (22.9%) were drug resistant (20, 20.8% INH mono-resistant and 2, 2.1% multidrug-resistant). All isolates were subjected to genotyping by Spoligotyping and MIRU-VNTR typing. The distribution of the isolates analyzed by spoligotyping revealed 30 distinct patterns. The four mainly detected clades were Latin American and Mediterranean (LAM), ill-defined T, Haarlem (H) and S. The MIRU-VNTR showed 85 distinct patterns. Spoligotyping combined to MIRU-VNTR allowed 90 distinct patterns.Conclusions/SignificanceOur study demonstrated that there is significant molecular diversity in circulating M. tuberculosis, with predominance of the LAM and T clades in cities of southwestern Paraná, Brazil, bordering Argentina and Paraguay.

Genomic variability of Mycobacterium tuberculosis strains of the Euro-American lineage based on large sequence deletions and 15-locus MIRU-VNTR polymorphism.

A sample of 260 Mycobacterium tuberculosis strains assigned to the Euro-American family was studied to identify phylogenetically informative genomic regions of difference (RD). Mutually exclusive deletions of regions RD115, RD122, RD174, RD182, RD183, RD193, RD219, RD726 and RD761 were found in 202 strains; the RDRio deletion was detected exclusively among the RD174-deleted strains. Although certain deletions were found more frequently in certain spoligotype families (i.e., deletion RD115 in T and LAM, RD174 in LAM, RD182 in Haarlem, RD219 in T and RD726 in the “Cameroon” family), the RD-defined sublineages did not specifically match with spoligotype-defined families, thus arguing against the use of spoligotyping for establishing exact phylogenetic relationships between strains. Notably, when tested for katG463/gyrA95 polymorphism, all the RD-defined sublineages belonged to Principal Genotypic Group (PGG) 2, except sublineage RD219 exclusively belonging to PGG3; the 58 Euro-American strains with no deletion were of either PGG2 or 3. A representative sample of 197 isolates was then analyzed by standard 15-locus MIRU-VNTR typing, a suitable approach to independently assess genetic relationships among the strains. Analysis of the MIRU-VNTR typing results by using a minimum spanning tree (MST) and a classical dendrogram showed groupings that were largely concordant with those obtained by RD-based analysis. Isolates of a given RD profile show, in addition to closely related MIRU-VNTR profiles, related spoligotype profiles that can serve as a basis for better spoligotype-based classification.

MOLECULAR CHARACTERIZATION OF MYCOBACTERIUM TUBERCULOSIS STRAINS CIRCULATING IN THE URAL REGION, RUSSIA

Abstract. Overall 178 Mycobacterium tuberculosis isolates recovered in 2009–2011 from newly diagnosed epidemiologically unlinked to TB patients in the Ural region of Russia have been studied. The absolute concentration method was used for drug susceptibility testing. Mutations katG, inhA and rpoB associated with resistance to isoniazid and rifampicin were detected by microchip technology («TB-Biochip»). The isolates were genotyped by real-time PCR for the detection of Beijing/non-Beijing genotypes and 15-locus MIRU-VNTR typing according to «MIRU-VNTRplus» (http://www.miru-vntrplus.org). More than half (55.1%) of 178 isolates belonged to the Beijing family, 58.7% of them were multiple drug resistant (MDR) mostly due to rpoBSer531→Leu and katGSer315→Thr1 substitutions. Fifty VNTR profiles were found in 98 Beijing isolates; 57 of them grouped into 9 clusters. The largest VNTR cluster included 23 (23.5%) Beijing isolates and 21 of them were MDR. The 80 non-Beijing isolates showed 64 distinct VNTR patterns which belonged to 6 genetic families: LAM, Ural, Haarlem, etc. Among LAM and Ural isolates 30.4% and 28.6% were MDR, respectively. The 5 of 7 MDR LAM isolates had specific mutation profile: rpoBAsp516→Val substitution and mutations katGSer315→Thr1 and inhA_T15. The MDR Ural isolates showed the heterogeneity of mutations in rpoB gene compared to other genotypes. Taken together, these findings suggest the emergence and spread of MDR-TB in the Ural region which is characterized by circulation of MDR strains of different genotypes with the Beijing family genotype to be predominant.

High-throughput multiplex MIRU-VNTR typing of Mycobacterium bovis

Spoligotyping is the most widely used method for genotyping Mycobacterium bovis (M. bovis). However, its discriminatory power varies widely between countries. MIRU-VNTR typing could be a promising alternative, although it generally requires the time consuming and laborious simplex PCR assays using standard agarose gel electrophoresis. The accuracy of this approach depends on good standardization and a certain degree of expertise. This study presents a version of MIRU-VNTR based on three triplex PCRs with automatic allelic assignation of the products analyzed in capillary electrophoresis. The technique was prospectively applied to 44 M. bovis and two Mycobacterium caprae (M. caprae) isolates, and 22 different MIRU-VNTRtypes were obtained; with spoligotyping, only 14 different types were obtained. The proposal makes it possible to shorten response times, automate procedures, and increase accuracy, thus minimizing errors in assigning genotypes. It would enable the switch from a standard limited method of genotyping M. bovis to a high-throughput discriminatory fingerprinting approach.

Transmission of multidrug-resistant tuberculosis in the UK: a cross-sectional molecular and epidemiological study of clustering and contact tracing

Between 2000 and 2012 the number of multidrug-resistant (MDR) tuberculosis cases in the UK increased from 28 per year to 81 per year. We investigated the proportion of MDR tuberculosis cases arising from transmission in the UK and associated risk factors. We identified patients with MDR tuberculosis notified in England, Wales, and Northern Ireland between Jan 1, 2004, and Dec 31, 2007, by linking national laboratory and surveillance data. Data for laboratory isolates, including drug sensitivities and 24-mycobacterial interspersed repetitive-unit-variable-number tandem repeat (MIRU-VNTR) typing were obtained routinely from the National Tuberculosis Reference laboratories as part of national tuberculosis surveillance. We investigated clusters of cases with indistinguishable MIRU-VNTR profiles to identify epidemiological links. We calculated transmission using the n-1 method and established associated risk factors by logistic regression. We also assessed the likelihood of transmission to additional secondary active tuberculosis cases, identified through conventional contact tracing. 204 patients were diagnosed with MDR tuberculosis in the study period; 189 (92·6%) had an MIRU-VNTR profile. We identified 12 clusters containing 40 individuals and 149 unique strains. The proportion of cases attributable to recent transmission, on the basis of molecular data, was 15% (40 cases clustered-12 clusters/189 with a strain type). The proportion of cases attributable to recent transmission (ie, transmission within the UK) after adjustment for epidemiological links was 8·5% (22 cases with epidemiological links-six clusters/189 cases with a strain type). Being UK born (odds ratio 4·81; 95% CI 2·03-11·36, p=0·0005) and illicit drug use (4·75; 1·19-18·96, p=0·026) were significantly associated with clustering. The most common transmission setting was the household but 21 of 22 of epidemiological links were missed by conventional contact tracing. 13 secondary active tuberculosis cases identified by conventional contact tracing were mostly contacts of patients with MDR tuberculosis from countries of high tuberculosis burden. 11 (85%) of 13 shared the same country of birth as the index case, of whom ten did not share a strain type or drug resistance pattern. Transmission of MDR tuberculosis in the UK is low and associated with being UK born or illicit drug use. MIRU-VNTR typing with cluster investigation was more successful at identifying transmission events than conventional contact tracing. Individuals with tuberculosis who have had contact with a known MDR tuberculosis source case from a country of high tuberculosis burden should have drug-sensitivity testing on isolates to ensure appropriate treatment is given. Public Health England.

Optimization of Standard In-House 24-Locus Variable-Number Tandem-Repeat Typing for Mycobacterium tuberculosis and Its Direct Application to Clinical Material

Variable-number tandem-repeat (VNTR) typing with a panel of 24 loci is the current gold standard in the molecular typing of Mycobacterium tuberculosis complex isolates. However, because of technical problems, a part of the loci often cannot be amplified by multiplex PCRs. Therefore, a considerable number of single-locus PCRs have to be performed for the loci with missing results, which impairs the laboratory work flow. Therefore, the original in-house method described by Supply et al. in 2006 was reevaluated. We modified seven primers and the PCR master mixture and obtained a strongly optimized in-house 24-locus VNTR typing method. The percentage of instantly complete 24-locus VNTR patterns detected in the routine flow of typing activities increased to 84.7% from the 72.3% obtained with the typing conducted with the commercially available Genoscreen MIRU-VNTR typing kit. The analytical sensitivity of the optimized in-house method was assessed by serial dilutions of M. tuberculosis in bronchoalveolar lavage fluid. A 1:10 dilution of the different strains tested was the lowest dilution for the detection of a complete 24-locus VNTR pattern. The optimized in-house 24-locus VNTR typing method will reduce the turnaround time of typing significantly and also the financial burden of these activities.

Molecular Epidemiology of Mycobacterium tuberculosis Complex in Singapore, 2006-2012

BackgroundTuberculosis remains common in Singapore, increasing in incidence since 2008. We attempted to determine the molecular epidemiology of Mycobacterium tuberculosis complex (MTC) isolates locally, identifying major circulating genotypes and obtaining a glimpse of transmission dynamics.MethodologyNon-duplicate MTC isolates archived between 2006 and 2012 at the larger clinical tuberculosis laboratory in Singapore were sampled for spoligotyping and MIRU-VNTR typing, with case data obtained from the Singapore Tuberculosis Elimination Program registry database. Isolates between 2008 and 2012 were selected because of either multidrug-resistance or potential epidemiological linkage, whereas earlier isolates were randomly selected. Separate analyses were performed for the early (2006-2007) and later (2008-2012) study phases in view of potential selection bias.Principal FindingsA total of 1,612 MTC isolates were typed, constituting 13.1% of all culture-positive tuberculosis cases during this period. Multidrug-resistance was present in 91 (5.6%) isolates – higher than the national prevalence in view of selection bias. The majority of isolates belonged to the Beijing (45.8%) and EAI (22.8%) lineages. There were 347 (30.7%) and 133 (27.5%) cases clustered by combined spoligotyping and MIRU-VNTR typing from the earlier and later phases respectively. Patients within these clusters tended to be of Chinese ethnicity, Singapore resident, and have isolates belonging to the Beijing lineage. A review of prior contact investigation results for all patients with clustered isolates failed to reveal epidemiological links for the majority, suggesting either unknown transmission networks or inadequate specificity of the molecular typing methods in a country with a moderate incidence of tuberculosis.ConclusionOur work demonstrates that Singapore has a large and heterogeneous distribution of MTC strains, and with possible cross-transmission over the past few years based on our molecular typing results. A universal MTC typing program coupled with enhanced contact investigations may be useful in further understanding the transmission dynamics of tuberculosis locally.

Genotypic characteristics of Mycobacterium tuberculosis isolated from household contacts of tuberculosis patients in the Philippines

BackgroundThe Philippines has an extremely high rate of tuberculosis but little is known about M. tuberculosis genotypes and transmission dynamics in this country. The aim of this study was to determine the proportion of household contacts who develop active TB due to direct transmission from an index case in that household.MethodsMycobacterium tuberculosis isolates from household contacts of tuberculosis patients in the Philippines were characterized using restriction-fragment-length polymorphism analysis, spoligotyping, and mycobacterial interspersed repetitive units – variable number tandem repeats typing (12-loci) to determine their utility in elucidating transmission in an area of high tuberculosis prevalence. Drug susceptibility patterns for these isolates were also determined.ResultsSpoligotyping and MIRU-VNTR typing results matched in 10 (62.5%) of 16 index patient-household contact pairs while IS6110 fingerprints matched in only six (37.5%) pairs. Only 3/16 (18.8%) index patient-household contact pairs had identical drug susceptibility results.ConclusionsStrain typing of M. tuberculosis isolates from household contacts in the Philippines indicates that transmission of strains does not necessarily occur directly from the index patient living in close proximity in the same household but rather that community-based transmission also frequently occurs. Accurate susceptibility testing of all isolates is necessary to insure optimal care of both the index patients and any culture-positive household contacts.

Evaluation of the Tuberculosis Strain Typing Service (TB-STS) in England

BackgroundAfter increases in tuberculosis notification rates, the Chief Medical Officer's Tuberculosis Action Plan in 2004 led to the introduction of the national Tuberculosis Strain-Typing Service (TB-STS), using MIRU-VNTR typing, in England in 2010. In addition to reducing the proportion of individuals who are misdiagnosed as having tuberculosis, the TB-STS could reduce tuberculosis incidence by increasing the detection of latent infections and by accelerating diagnosis of active cases, through better-targeted cluster investigation. Complex public health interventions are often implemented without a prospective assessment. We describe a service-wide evaluation of a TB-STS using laboratory and contact tracing data, and mathematical modelling, to estimate effects on tuberculosis incidence and cost-effectiveness. MethodsThe proportion of false-positive Mycobacterium tuberculosis isolates, which had not been identified before strain-typing results became available, was estimated from laboratory data. The effect of cluster investigations on diagnostic delay (time between symptom onset and date of diagnosis) was estimated for pulmonary tuberculosis cases diagnosed in 2010–11. The contact tracing yield (the median number of individuals with tuberculosis infection or active disease identified per case), according to whether cases were in clusters that were investigated, was estimated from data from north central London and Leicester in 2011. With an age-structured deterministic compartmental model, we explored the effectiveness of the TB-STS in population groups in which the incidence is low (native UK population), medium (similar to the non-white UK-born population), and high (similar to the non-UK-born population). Cost-effectiveness was estimated for various scenarios, with use of outputs from the model and service assessment, costs of establishing and running the service, and estimated costs and quality-adjusted life-year (QALY) effects of tuberculosis infection and disease. FindingsFrom 11 059 isolates typed between June, 2010, and June, 2012, there were 30 incidents of false-positive M tuberculosis isolation, 17 (57%) of which had not been identified before strain-typing results were available. Cluster investigations had no significant effect on the diagnostic delay (median 85 days [IQR 47–155] vs 77 days [41–157]) or on contact tracing yield (mean number of cases with active tuberculosis 0·33, 95% CI 0·1 to 0·6 vs 0·2, −0·0 to 0·5) or latent infection (1·9, 0·3–3·2 vs 1·7, 0·8–2·5). Mathematical modelling suggested that increasing the proportion of infections that are detected would have little effect on tuberculosis incidence and the number of cases prevented in the native UK population. In medium-incidence or high-incidence settings, under base-case assumptions, increasing the proportion of infections detected by a small amount (eg, from 3% per year to 13% per year with strain typing) could lead to about an 11% reduction in tuberculosis incidence over 20 years, ranging between 4% and 12% for pessimistic and optimistic assumptions, respectively, relating to uptake and retention of preventive treatment. In medium-incidence settings and with all other factors remaining unchanged, reducing diagnostic delay by 1 week could lead to greater than 25% reductions in tuberculosis incidence over 20 years. These findings are sensitive to the assumed proportion of immigrants who enter the UK with tuberculosis. Under base case assumptions, the service was not estimated to be cost-effective over a 20-year period (£97 311 per QALY). InterpretationInsufficient baseline and post-implementation data limit direct estimation of public-health outcomes and interpretation of the model. Present models suggest that the TB-STS is not cost-effective. However, the TB-STS provides an invaluable resource for epidemiological and microbiological research and surveillance, informing national and local tuberculosis control efforts; targeting population-level interventions; and contributing to understanding molecular epidemiology in Europe and worldwide. FundingPublic Health England's TB Strain Typing Project Board. JM is funded by a University College London and Public Health England Impact Studentship. IA is funded by an NIHR Senior Research Fellowship.

Tuberculosis Transmission among Immigrants and Autochthonous Populations of the Eastern Province of Saudi Arabia

BackgroundEastern province of Saudi Arabia is an industrial zone with large immigrant population and high level of tuberculosis case notification among immigrants. The impact of immigration and current trends of tuberculosis transmission among immigrants and autochthonous population in the region had not been investigated so far using molecular tools.MethodologyDuring 2009- 2011, a total of 524 Mycobacterium tuberculosis isolates were collected from the central tuberculosis reference laboratory, representing an estimated 79.2% of the culture-positive tuberculosis cases over the study period in the province. These isolates were genotyped by using 24 locus-based MIRU-VNTR typing and spoligotyping followed by first line drug susceptibility testing. The molecular clustering profiles and phylogenetic diversity of isolates were determined and compared to the geographical origins of the patients.Principle FindingsGenotyping showed an overall predominance of Delhi/CAS (29.4%), EAI (23.8%) and Ghana (13.3%) lineages, with slightly higher proportions of Delhi/CAS among autochthonous population (33.3 %) and EAI (30.9%) among immigrants. Rate of any drug resistance was 20.2% with 2.5% of multi-drug resistance. Strain cluster analysis indicated 42 clusters comprising 210 isolates, resulting in a calculated recent transmission index of 32.1%. Overall shared cluster ratio was 78.6% while 75.8% were shared between autochthonous population and immigrant population with a predominance of immigrants from South east Asia (40.7%). In contrast, cross national transmission within the immigrant population was limited (24.2%). Younger age (15-30- p value-0.043, 16-45, p value 0.030), Saudi nationality (p value-0.004) and South East Asian origin (p value-0.011) were identified as significant predisposing factors for molecular strain clustering.ConclusionsThe high proportion of molecular clusters shared among the autochthonous and immigrant populations suggests a high permeability of tuberculosis transmission between both populations in the province. These results prompt for the need to strengthen the current tuberculosis control strategies and surveillance programs.

MDR TB Transmission, Singapore

MDR TB Transmission, Singapore

Contribution of spoligotyping and MIRU-VNTRs to characterize prevalent Mycobacterium tuberculosis genotypes infecting tuberculosis patients in Morocco

In the present study, Mycobacterium tuberculosis complex (MTBC) clinical isolates from culture-positive TB patients in Morocco were studied by spoligotyping and 12-loci MIRU-VNTR typing methods to characterize prevalent genotypes (n=219 isolates from 208 patients). Spoligotyping resulted in 39 unique patterns and 167 strains in 30 clusters (2–50 strains per cluster). Comparison with international database showed that 29 of 39 unique patterns matched existing shared spoligotype international types (SITs). Nine shared types containing 10 strains were newly created (SIT 2891 to SIT 2899); this led to the description of 69 SITs with 206 strains and two orphan patterns. The most prevalent spoligotype was SIT42 (LAM; n=50 or 24% of isolates). The repartition of strains according to major MTBC clades was as follows LAM (46.1%)> Haarlem (26%) >ill-defined T superfamily (22.6%) and S clade (0.96%). On the other hand, Beijing, CAS (Central Asian) and EAI (East-African Indian) strains were absent in this setting. Subsequent 12-Loci MIRU typing resulted in a total of 25 SIT/MIT clusters (n=66 isolates, 2–6 isolates per cluster), with a resulting recent transmission rate of 22.3%. The MIRU-VNTR patterns corresponded to 69 MITs for 138 strains and 46 orphan patterns. The most frequent patterns were MIT43 (n=8), MIT9 (n=7) and MIT42 (n=7). HGDI analysis of the 12 MIRU loci showed that loci 10, 23 and 40 were highly discriminative in our setting. The results also underlined the usefulness of spoligotyping and MIRU-VNTR to detect mixed infections among certain of our TB patients. Globally, the results obtained showed that TB is almost exclusively transmitted in Morocco through evolutionary-modern MTBC lineages belonging to principal genetic groups 2/3 strains (Haarlem, LAM, T), with a high level of biodiversity seen by MIRU typing. This study provides with a 1st global snapshot of MTBC population structure in Morocco, and validates the potential use of spoligotyping in conjunction with minisatellites for future investigations in Morocco that should in future ideally include optimized 15- or 24-loci MIRU-VNTRs.

Current trends of Mycobacterium tuberculosis molecular epidemiology in Saudi Arabia and associated demographical factors

Data are scarce on demographical factors related to the population structure of Mycobacterium tuberculosis in Saudi Arabia. A study was conducted on 902 clinical isolates to explore current trends in the phylogeography and associated demographical factors of tuberculosis by using spoligotyping and 24 loci based MIRU-VNTR typing. Young male patients (aged 16–29 and 30–44) were predominant in this cohort. The phylogenetic diversity among M. tuberculosis isolates was found high, as almost all known genetic lineages were identified. Delhi/CAS (26.4%), EAI (13.7%) and Haarlem (11.3%) were the most common lineages observed, particularly among the low age groups (16–29 and 30–44years), whereas elderly patients (>60years) showed a predominance in the lineages S, Ghana, TUR and Uganda-I. A statistically significant association was observed between gender of the patients and lineages of EAI (p value 0.026) and LAM (p value 0.005). Overall, molecular strain cluster rate was 34.4% with an elevated rate among patients aged below 15years (43.1%), while cases among the elderly (>60years) showed the lowest degree of clustering (12.5%). The largest level of clustering was noticed among cases caused by strains of the lineages Haarlem (59.8%), Beijing (55.8%) and LAM (42.8%). The current population structure of M. tuberculosis in Saudi Arabia is highly diverse with significant associations to demography, transmission dynamics and origin of the patients. The difference in genotype distributions among low and high aged patients reflects the ongoing change in the strain population structure in the country.

First Molecular Epidemiology Study of Mycobacterium tuberculosis in Kiribati

Tuberculosis incidence rates in Kiribati are among the highest in the Western Pacific Region, however the genetic diversity of circulating Mycobacterium tuberculosis complex strains (MTBC) and transmission dynamics are unknown. Here, we analysed MTBC strains isolated from culture positive pulmonary tuberculosis (TB) cases from the main TB referral centre between November 2007 and October 2009. Strain genotyping (IS6110 typing, spoligotyping, 24-loci MIRU-VNTR and SNP typing) was performed and demographic information collected. Among 73 MTBC strains analysed, we identified seven phylogenetic lineages, dominated by Beijing strains (49%). Beijing strains were further differentiated in two main branches, Beijing-A (n = 8) and -B (n = 28), that show distinct genotyping patterns and are characterized by specific deletion profiles (Beijing A: only RD105, RD207 deleted; Beijing B: RD150 and RD181 additionally deleted). Many Kiribati strains (59% based on IS6110 typing of all strains) occurred in clusters, suggesting ongoing local transmission. Beijing-B strains and over-crowded living conditions were associated with strain clustering (likely recent transmission), however little evidence of anti-tuberculous drug resistance was observed. We suggest enhanced case finding amongst close contacts and continued supervised treatment of all identified cases using standard first-line drugs to reduce TB burden in Kiribati. Beijing strains can be subdivided in different principle branches that might be associated with differential spreading patterns in the population.

Endogenous reactivation followed by exogenous re-infection with drug resistant strains, a new challenge for tuberculosis control in Saudi Arabia

Endogenous reactivation and exogenous reinfection of tuberculosis were studied for the first time in Saudi Arabia after enrolling a total of 39 patients with multiple episodes of tuberculosis between 2009 and 2010. All of the primary and subsequent isolates enrolled were subjected to spoligotyping, 24 loci based MIRU-VNTR typing and first line anti-tuberculosis drug susceptibility testing. The primary episode isolates from patients born outside Saudi Arabia were dominated by lineages which are prevalent in their country of origin (e.g. Ghana, Cameroon, Uganda-I, among African patients/Delhi/CAS and EAI among Asian patients). On the other hand, in Saudi Arabian patients, (median age of 67 years) Delhi/CAS, TUR and Slineages were predominant. The second episode of infection was mainly caused by the lineages Delhi/CAS, EAI, Uganda-I, Haarlem, and LAM which are currently disseminating in the country. Surprisingly, all of the first episode isolates were pan-susceptible, while 35.9% of the re-infected cases were drug resistant. Reactivation of a remote infection eventually followed by an exogenous reinfection was confirmed among patients, particularly those of African origin. Immediate actions to break the cycle of transmission of drug resistant tuberculosis are greatly needed in Saudi Arabia.

Application of FLiP Method for Differentiation of Mycobacterium tuberculosis Strains in Comparison to Commonly Used Methods, Spoligotyping and MIRU-VNTR Typing

The current "gold standard" in molecular epidemiological studies of Mycobacterium tuberculosis is IS6110 RFLP based on IS6110 polymorphism. However PCR-based methods are becoming increasingly important. Recently, fast ligation-mediated PCR (FLiP), based on IS6110 polymorphism was proposed. In this study, the discriminatory power of FLIP, spoligotyping and MIRU-VNTR typing, in differentiation of M. tuberculosis isolates was compared. The discriminatory index (HGI) of spoligotyping, MIRU-VNTR analysis, and FLiP was 0.653, 0.837, and 0.917, respectively. This indicates that FLiP allows a high level of differentiation among M. tuberculosis strains and it might be a valuable alternative to the other typing methods.

High Prevalence of Bovine Tuberculosis in Dairy Cattle in Central Ethiopia: Implications for the Dairy Industry and Public Health

BackgroundEthiopia has the largest cattle population in Africa. The vast majority of the national herd is of indigenous zebu cattle maintained in rural areas under extensive husbandry systems. However, in response to the increasing demand for milk products and the Ethiopian government's efforts to improve productivity in the livestock sector, recent years have seen increased intensive husbandry settings holding exotic and cross breeds. This drive for increased productivity is however threatened by animal diseases that thrive under intensive settings, such as bovine tuberculosis (BTB), a disease that is already endemic in Ethiopia.Methodology/Principal FindingsAn extensive study was conducted to: estimate the prevalence of BTB in intensive dairy farms in central Ethiopia; identify associated risk factors; and characterize circulating strains of the causative agent, Mycobacterium bovis. The comparative intradermal tuberculin test (CIDT), questionnaire survey, post-mortem examination, bacteriology, and molecular typing were used to get a better understanding of the BTB prevalence among dairy farms in the study area. Based on the CIDT, our findings showed that around 30% of 2956 tested dairy cattle from 88 herds were positive for BTB while the herd prevalence was over 50%. Post-mortem examination revealed gross tuberculous lesions in 34/36 CIDT positive cattle and acid-fast bacilli were recovered from 31 animals. Molecular typing identified all isolates as M. bovis and further characterization by spoligotyping and MIRU-VNTR typing indicated low strain diversity within the study area.Conclusions/SignificanceThis study showed an overall BTB herd prevalence of 50% in intensive dairy farms in Addis Ababa and surroundings, signalling an urgent need for intervention to control the disease and prevent zoonotic transmission of M. bovis to human populations consuming dairy products coming from these farms. It is suggested that government and policy makers should work together with stakeholders to design methods for the control of BTB in intensive farms in Ethiopia.

A whole-genome sequencing approach to targeting Mycobacterium tuberculosis outbreak management

BackgroundEpidemiological investigations into Mycobacterium tuberculosis outbreaks use 24-locus genotyping (MIRU-VNTR typing). Where no epidemiological link can be found between patients, the importance of shared genotypes remains unclear. This issue is especially problematic and time-consuming when tracing contacts within some social groups at high tuberculosis risk, in which unwillingness to volunteer information is common. We investigated whether whole-genome sequencing (WGS) could delineate outbreaks with greater resolution than MIRU-VNTR typing has done. MethodsWe sequenced 390 M tuberculosis isolates from 254 patients from the UK Midlands (1994–2011) using Illumina technology (appendix). We estimated the expected genomic diversity between isolates within a transmission chain by measuring pairwise nucleotide differences between genomes within hosts (79 individuals with pulmonary and extrapulmonary disease, or multiple pulmonary episodes) and between hosts within 25 household outbreaks (63 individuals). We then investigated 11 MIRU-VNTR-based community clusters (168 patients, 157 transmission events) to assess whether WGS could delineate outbreaks more effectively. For each cluster we reconstructed the most plausible transmission chain based on epidemiological data collected by tuberculosis nurses, pairwise nucleotide distances, and times of diagnosis, and compared the genomic diversity across these constructed links with that within individuals and within household outbreaks. Findings109 (96%) of 114 isolates were within five SNPs of another isolate taken from the same individual or from an individual in the same household outbreak. On the basis of longitudinal isolates from individuals or households, we estimated an evolutionary rate of 0·5 SNPs per genome per year, consistent with a maximum of five SNPs between related isolates 3 years or less apart. Using a greater than five SNP threshold to assess 11 MIRU-VNTR-based community clusters, we found that none of 69 epidemiologically related pairs of MIRU-VNTR-matched cases plausibly related by transmission, two of 13 possibly related pairs, and 13 of 75 pairs with no known epidemiological relation were separated by more than five SNPs (p<0·0001). Seven MIRU-VNTR-matched pairs with no epidemiological relation had more than 30 SNPs, five of seven belonging to the same immigrant community cluster. WGS also showed that 62 of 75 MIRU-VNTR-matched pairs for which no epidemiological relation had been identified from contact tracing were highly likely to indicate transmission: in one substance misuse cluster, 38 individuals were linked by five or fewer SNPs without a single epidemiological link having been established previously. Further analysis suggested that microevolutionary divergence of lineages within outbreaks could signal possible super-spreaders, corroborated by clinical and epidemiological data in two clusters. InterpretationWGS can delineate tuberculosis outbreaks with greater resolution than has previously been possible. These findings offer public health teams the potential to limit outbreak investigations to patients who are likely to be linked by recent transmission, irrespective of whether it has been possible to identify epidemiological links, and to save resources where they are not, even in the context of matched MIRU-VNTR genotypes. Uniquely, WGS also provides information about the genetic structure of outbreak clusters, thereby providing the potential to direct public health resources towards individuals most likely to have infected the largest number of secondary cases. As a consequence, the Health Protection Agency is considering introduction of WGS technology for routine tuberculosis public health practice in England. FundingNIHR Oxford Biomedical Research Centre and the UKCRC Modernising Medical Microbiology Consortium (UKCRC Translational Infection Research Initiative supported by MRC, Biotechnology and Biological Sciences Research Council, and NIHR on behalf of the Department of Health [grant G0800778] and the Wellcome Trust [087646/Z/08/Z]).

The genotypic population structure of Mycobacterium tuberculosis complex from Moroccan patients reveals a predominance of Euro-American lineages.

BackgroundTuberculosis (TB) remains a major health problem in Morocco. Characterization of circulating Mycobacterium tuberculosis genotypic lineages, important to understand the dynamic of the disease, was hereby addressed for the first time at a national level.Methodology/Principal FindingsSpoligotyping was performed on a panel of 592 M. tuberculosis complex strains covering a 2-year period (2004–2006). It identified 129 patterns: 105 (n = 568 strains) corresponded to a SIT number in the SITVIT2 database, while 24 patterns were labeled as orphan. A total of 523 (88.3%) strains were clustered vs. 69 or 11.7% unclustered. Classification of strains within 3 large phylogenetical groups was as follows: group 1– ancestral/TbD1+/PGG1 (EAI, Bovis, Africanum), group 2– modern/TbD1−/PGG1 group (Beijing, CAS), group 3– evolutionary recent/TbD1−/PGG2/3 (Haarlem, X, S, T, LAM; alternatively designated as the Euro-American lineage). As opposed to group 3 strains (namely LAM, Haarlem, and T) that predominated (86.5% of all isolates), 6 strains belonged to group 2 (Beijing n = 5, CAS n = 1), and 3 strains (BOV_1 n = 2, BOV_4-CAPRAE) belonged to ancestral group 1 (EAI and AFRI lineage strains were absent). 12-loci MIRU-VNTR typing of the Casablanca subgroup (n = 114 strains) identified 71 patterns: 48 MITs and 23 orphan patterns; it allowed to reduce the clustering rate from 72.8% to 29.8% and the recent transmission rate from 64% to 20.2%.ConclusionThe M. tuberculosis population structure in Morocco is highly homogeneous, and is characterized by the predominance of the Euro-American lineages, namely LAM, Haarlem, and T, which belong to the “evolutionary recent” TbD1−/PGG2/3 phylogenetic group. The combination of spoligotyping and MIRUs decreased the clustering rate significantly, and should now be systematically applied in larger studies. The methods used in this study appear well suited to monitor the M. tuberculosis population structure for an enhanced TB management program in Morocco.