Bridging the gap between molecular and genomic epidemiology in tuberculosis: inferring MIRU-VNTR patterns from genomic data.

The transition from MIRU-VNTR-based epidemiology studies in tuberculosis (TB) to genomic epidemiology has transformed how we track transmission. However, short-read sequencing is poor at analyzing repetitive regions such as the MIRU-VNTR loci. This causes a gap between the new genomic data and the large amount of information stored in historical databases. Long-read sequencing could bridge this knowledge gap by allowing analysis of repetitive regions. However, the feasibility of extracting MIRU-VNTRs from long reads and linking them to historical data has not been evaluated. In our study, an in silico arm, consisting of inference of MIRU patterns from long-read sequences (using MIRUReader program), was compared with an experimental arm, involving standard amplification and fragment sizing. We analyzed overall performance on 39 isolates from South Africa and confirmed reproducibility in a sample enriched with 62 clustered cases from Spain. Finally, we ran 25 consecutive incident cases, demonstrating the feasibility of correctly assigning new clustered/orphan cases by linking data inferred from genomic analysis to MIRU-VNTR databases. Of the 3,024 loci analyzed, only 11 discrepancies (0.36%) were found between the two arms: three attributed to experimental error and eight to misassigned alleles from long-read sequencing. A second round of analysis of these discrepancies resulted in agreement between the experimental and in silico arms in all but one locus. Adjusting the MIRUReader program code allowed us to flag potential in silico misassignments due to suboptimal coverage or unfixed double alleles. Our study indicates that long-read sequencing could help address potential chronological and geographical gaps arising from the transition from molecular to genomic epidemiology of tuberculosis. IMPORTANCE: The transition from molecular epidemiology in tuberculosis (TB), based on the analysis of repetitive regions (VNTR-based genotyping), to genomic epidemiology transforms in the precision with which we track transmission. However, short-read sequencing, the most common method for performing genomic analysis, is poor at analyzing repetitive regions. This means that we face a gap between the new genomic data and the large amount of information stored in historical databases, which is also an obstacle to cross-national surveillance involving settings where only molecular data are available. Long-read sequencing could help bridge this knowledge gap by allowing analysis of repetitive regions. Our study demonstrates that MIRU-VNTR patterns can be successfully inferred from long-read sequences, allowing the correct assignment of new cases as clustered/orphan by linking new data extracted from genomic analysis to historical MIRU-VNTR databases. Our data may provide a starting point for bridging the knowledge gap between the molecular and genomic eras in tuberculosis epidemiology.

Molecular Characterisation of Mycobacterium bovis Isolates from Cattle Slaughtered in Adamawa and Gombe States, North-Eastern Nigeria.

Bovine tuberculosis is endemic in Nigeria with control measures as provided by the laws of the country being minimally enforced mostly at the abattoirs only. This study focused on bovine tuberculosis in Adamawa and Gombe States. Tuberculosis lesions were observed in 183 of 13,688 slaughtered cattle in the regions between June and December 2020. Analysis of tissue samples resulted in 17 Mycobacterium bovis isolates, predominantly from Gombe State. Spoligotyping identified four spoligotypes, including SB0944, SB1025, SB1104, and one novel pattern. MIRU-VNTR analysis further differentiated these spoligotypes into eight profiles. All isolates belonged to the Af1 clonal complex. The study emphasises the need for broader coverage and more isolates to comprehensively understand the molecular epidemiology of bovine tuberculosis in Nigeria. To enhance research and surveillance, a cost-effective approach is proposed, utilising a discriminatory VNTR panel comprising five or nine loci. The five-locus panel consists of ETR-C, QUB26, QUB11b, MIRU04, and QUB323. Alternatively, the nine-locus panel includes ETR-A, ETR-B, QUB11a, and MIRU26. Implementing this approach would provide valuable insights into the genetic diversity of M. bovis strains in Nigeria. These findings are crucial for developing effective control measures and minimising the impact of bovine tuberculosis on both animal and human health.

Mycobacterium tuberculosis genotypes in an ethnically diverse area with millions of pilgrims and thousands of immigrants.

BACKGROUND: Immigration is considered as a risk factor of tuberculosis (TB). Qom province receives millions of pilgrims and significant numbers of immigrants each year. Most of the immigrants to Qom, arrive from neighboring TB-endemic countries. This study aimed to identify the current circulating Mycobacterium tuberculosis genotypes in Qom province using 24-locus MIRU-VNTR genotyping. METHODS: Eighty six M. tuberculosis isolates were collected during 2018-2022 from patients referring to Qom TB reference laboratory. The DNA of isolates was extracted and followed by 24 loci MIRU-VNTR genotyping which performed using the web tools available on MIRU-VNTRplus. RESULTS: Of 86 isolates, 39 (45.3%) were of Delhi/CAS genotype, 24 (27.9%) of NEW-1, 6 (7%) of LAM, 6 (7%) of Beijing, 2 (2.3%) of UgandaII, 2 (2.3%) of EAI, 1 of S (1.2%) and 6 (7%) did not match with profiles present in MIRUVNTRplus database. CONCLUSIONS: About half of the isolates belong to Afghan immigrants; which warns health policy makers about the future situation of TB in Qom. Also, the similarity of Afghan and Iranian genotypes provides evidence that immigrants partake in the circulation of M. tuberculosis. This study underpin the studies about the circulating M. tuberculosis genotypes, their geographical distribution, the association of TB risk factors with these genotypes and the impact of immigration on the situation of TB in Qom province.

Association between Mycobacterium tuberculosis genotype and diabetes mellitus/hypertension: a molecular study.

BACKGROUND: A paucity of studies focused on the genetic association that tuberculosis (TB) patients with non-communicable diseases (NCDs) are more likely to be infected with Mycobacterium tuberculosis (MTB) with more potent virulence on anti-TB drug resistance than those without NCDs. The study aimed to document the predominant genotype, determine the association between MTB genotypes and NCD status and drug resistance. METHODS: We conducted a molecular study in 105 TB patients based on a cross-sectional study focused on the comorbid relationship between chronic conditions and TB among 1773 subjects from September 1, 2019 to August 30, 2020 in Guizhou, China. The participants were investigated through face-to-face interviews, followed by NCDs screening. The DNA of MTB isolates was extracted prior to genotyping using 24 loci MIRU-VNTR. The subsequent evaluations were performed by phylogenetic trees, combined with tests of statistical power, Chi-square or Fisher and multivariate logistic regression analysis. RESULTS: The Beijing family of Lineage 2 (East Asia) was the predominant genotype accounting for 43.8% (46/105), followed by Lineage 4 (Euro-America) strains, including Uganda I (34.3%, 36/105), and the NEW-1 (9.5%, 10/105). The proportion of Beijing strain in patients with and without NCDS was 28.6% (8/28) and 49.4% (38/77), respectively, with a statistical power test value of 24.3%. No significant association was detected between MTB genotype and NCD status. A low clustering rate (2.9%) was identified, consisting of two clusters. The rates of global, mono-, poly- and multi-drug resistance were 16.2% (17/105), 14.3% (15/105), 1.0% (1/105) and 4.8% (5/105), respectively. The drug-resistant rates of rifampicin, isoniazid, and streptomycin, were 6.7% (7/105), 11.4% (12/105) and 5.7% (6/105), respectively. Isoniazid resistance was significantly associated with the Beijing genotype of Lineage 2 (19.6% versus 5.1%). CONCLUSIONS: The Lineage 2 East Asia/Beijing genotype is the dominant genotype of the local MTB with endogenous infection preponderating. Not enough evidence is detected to support the association between the MTB genotype and diabetes/hypertension. Isoniazid resistance is associated with the Lineage 2 East Asia/Beijing strain.

Transmission of multidrug-resistant tuberculosis within family households by DTM-PCR and MIRU-VNTR genotyping.

BACKGROUND: Drug-resistant tuberculosis (TB) continues to be a public health threat. There are few studies on transmission and genotyping of MDR-TB family households in China. This study aimed to investigate transmission of multidrug-resistant tuberculosis (MDR-TB) within family households by deletion-targeted multiplex polymerase chain reaction (DTM-PCR), mycobacterial interspersed repetitive unit variable number tandem repeats (MIRU-VNTR) genotyping. METHODS: Among 993 MDR-TB patients registered from Wuhan Institute for Tuberculosis Control, drug resistance and the time interval between the index patients and secondary patients were analyzed in 49 MDR-TB patients from 23 families, in which 22 MDR-TB strains from 11 families who had matched strains were genotyped by DTM-PCR and standard 24-loci MIRU-VNTR genotyping method. RESULTS: The time interval between the index patients and the secondary patients ranged from half a month to 110 months. Thirteen secondary patients developed active MDR-TB within two years and accounted for 50% (13/26) of all secondary patients. Among eleven pairs of MDR-TB families, six pairs had identical genotypes, the cluster rate was 54.5% (12/22); three pairs had a single MIRU-VNTR locus variation. If a single MIRU-VNTR locus variation was tolerated in the cluster definition, the cluster rate raised to 81.8% (18/22). CONCLUSIONS: The family households of MDR-TB patients are at risk for infection of MDR-TB. To reduce transmission, MDR-TB patients should be diagnosed earlier and promptly treated in an effective manner, meanwhile, the close family contacts should be screened for TB infection.

Distribution and Clonality of drug-resistant tuberculosis in South Africa.

BACKGROUND: Studies have shown that drug-resistant tuberculosis (DR-TB) in South Africa (SA) is clonal and is caused mostly by transmission. Identifying transmission chains is important in controlling DR-TB. This study reports on the sentinel molecular surveillance data of Rifampicin-Resistant (RR) TB in SA, aiming to describe the RR-TB strain population and the estimated transmission of RR-TB cases. METHOD: RR-TB isolates collected between 2014 and 2018 from eight provinces were genotyped using combination of spoligotyping and 24-loci mycobacterial interspersed repetitive-units-variable-number tandem repeats (MIRU-VNTR) typing. RESULTS: Of the 3007 isolates genotyped, 301 clusters were identified. Cluster size ranged between 2 and 270 cases. Most of the clusters (247/301; 82.0%) were small in size (< 5 cases), 12.0% (37/301) were medium sized (5-10 cases), 3.3% (10/301) were large (11-25 cases) and 2.3% (7/301) were very large with 26-270 cases. The Beijing genotype was responsible for majority of RR-TB cases in Western and Eastern Cape, while the East-African-Indian-Somalian (EAI1_SOM) genotype accounted for a third of RR-TB cases in Mpumalanga. The overall proportion of RR-TB cases estimated to be due to transmission was 42%, with the highest transmission-rate in Western Cape (64%) and the lowest in Northern Cape (9%). CONCLUSION: Large clusters contribute to the burden of RR-TB in specific geographic areas such as Western Cape, Eastern Cape and Mpumalanga, highlighting the need for community-wide interventions. Most of the clusters identified in the study were small, suggesting close contact transmission events, emphasizing the importance of contact investigations and infection control as the primary interventions in SA.

Whole genome sequencing analysis of multidrug-resistant tuberculosis in Singapore, 2006-2018.

There were 290 multidrug-resistant (MDR)-TB cases diagnosed in Singapore from 2006 to 2018. Eighty-one percent were foreign-born. Spoligotyping and MIRU-VNTR methods identified 108 patients in 24 clusters. The Beijing spoligotype accounted for 22 clusters. Whole genome sequencing (WGS) analysis reduced the number of clustered patients and clusters to 43 and nine respectively. One MIRU cluster was redefined into three WGS clusters. All the clusters had foreign-born source cases. Forty percent of local-born, versus 9% of foreign-born, MDR-TB cases belonged to WGS clusters. WGS more accurately elucidated potential MDR-TB transmission which was overestimated by conventional genotyping methods in Singapore.

Genetic diversity and drug susceptibility of Mycobacterium tuberculosis in a city with a high prevalence of drug resistant tuberculosis from Southeast of Mexico.

BACKGROUND: Mexico is on the top five countries with the highest number of TB cases in America continent, nevertheless, information about genotypes circulating is practically unknown. Considering the above this study aims to characterize the genetic diversity of TB in the city of Veracruz, México. METHODS: A cross-sectional study was conducted among positive smear samples from patients living in Veracruz City, samples were cultured, and first-line drug profiles determined. Genotyping was made by spoligotyping and MIRU-VNTR 24 loci. Associations of lineages, clusters, and variables were also analyzed. RESULTS: Among the 202 isolates analyzed resistance to at least one drug was observed in 60 (30%) isolates and 41(20%) were multidrug-resistant. Three major lineages were identified: L4/Euro-American (88%), L1/Indo-Oceanic (9%), and L2/East Asian (3%). The Euro-American lineage included more than six sublineages, the most abundant were: H (32%), T (23%), LAM (18%), and X (12%). 140 isolates (70%) were placed in 42 SITs patterns. CONCLUSIONS: These results provide the first baseline data on the genetic structure of TB in the city of Veracruz. Sublineages H, X and LAM were predominant; however, it was founded an important diversity of genotypes that could contribute to the dispersion of TB and explain the high prevalence. This information might be useful for the development of further interventions to reduce impact of TB.

Mycobacterium avium subsp. paratuberculosis (MAP) molecular diversity in cattle, sheep, and goats from Latin America and the Caribbean: a systematic review.

This study aimed to systematically collect and appraise the scientific evidence to answer the research question: What MAP genotypes have been isolated from cattle, sheep, and goats in Latin America and the Caribbean? An electronic search was conducted on three platforms (i.e., OVID®, Web of Science®, SciELO) as well as on the proceedings of the International Colloquium on Paratuberculosis. Inclusion and exclusion criteria were defined a priori and conserved through the systematic process and only articles published in peer-reviewed journals were considered. A total of 26 articles met the definitive inclusion criteria. All were published in English, in 15 different journals, and between 1989 and 2020. The relevant articles reported the use of six different genotyping techniques (i.e., polymerase chain reaction-restriction endonuclease analysis, restriction fragment length polymorphism, type-specific-PCR, mycobacterial interspersed repetitive units-variable number of tandem repeats, multi-locus short sequence repeat, single nucleotide polymorphism) in isolates from seven countries. Genotypes found so far in the region using typing techniques were mainly C type. MIRU-VNTR mostly reported INMV 1, INMV 2, and INMV 11 subtypes, among others. MLSSR reported genotypes from four different countries, reporting nine different subtypes of which 7g-10g-4ggt was the most common for loci 1, 2, and 8, respectively. Regardless the high diversity of techniques used so far to genotype Latin American and Caribbean MAP isolates, the original question of this systematic review has been answered. In addition, a relative genetic similarity between MAP strains recovered from cattle, goats, and sheep unrelatedly of the matrix and geographic origin was identified.

MOLECULAR EPIDEMIOLOGY APPROACH TO TACKLE TREATMENT FAILURE IN DRUG RESISTANT TUBERCULOSIS (DR-TB).

OBJECTIVE: The aim: To analyze molecular epidemiology features of M. tuberculosis in Kyiv oblast; and to identify the safest mode of TB treatment, which will allow clinicians to minimize the risk of drug-resistant strains nosocomial transmission. PATIENTS AND METHODS: Materials and methods: 55 isolates of M. tuberculosis were collected in January-April, 2018 from 31 patients with new cases and 24 patients with re-treatment cases of sputum culture-positive pulmonary TB, in Kyiv oblast, Ukraine. DNA samples extracted from all the isolates were used for 15-loci MIRU-VNTR molecular typing with further M. tuberculosis strains comparison by means of MIRU-VNTRplus web tool (http://www.miru-vntrplus.org). RESULTS: Results: Phylogenetic tree that reflects strains interrelationship reveals four main clusters, the largest of which spans 34 isolates. The presence of two big subclusters with 10 and 7 identical genotypes inside the largest cluster strongly suggests their tight epidemiologic relationship. Smaller clusters consist of five (Harlem), three (URAL), and two unidentified isolates. 10 singletons were detected, among which LAM, URAL and Cameroon lineages were identified; in these cases, epidemiological connection was presumably ruled out. CONCLUSION: Conclusions: High clustering level of isolates of M. tuberculosis suggests possible contact between patients from whom these isolates were obtained. Predominance of Beijing family in the clusters is associated with high DR-TB level, at least in eastern European countries. Similarity of the clusters isolated from different patients in a household or a hospital suggests high probability of recent disease transmission. Clustering genotypes from households and hospital wards can be a surrogate criterion of infection control effectiveness.

The impact of HIV infection on tuberculosis transmission in a country with low tuberculosis incidence: a national retrospective study using molecular epidemiology.

BACKGROUND: HIV is known to increase the likelihood of reactivation of latent tuberculosis to active TB disease; however, its impact on tuberculosis infectiousness and consequent transmission is unclear, particularly in low-incidence settings. METHODS: National surveillance data from England, Wales and Northern Ireland on tuberculosis cases in adults from 2010 to 2014, strain typed using 24-locus mycobacterial-interspersed-repetitive-units-variable-number-tandem-repeats was used retrospectively to identify clusters of tuberculosis cases, subdivided into 'first' and 'subsequent' cases. Firstly, we used zero-inflated Poisson regression models to examine the association between HIV status and the number of subsequent clustered cases (a surrogate for tuberculosis infectiousness) in a strain type cluster. Secondly, we used logistic regression to examine the association between HIV status and the likelihood of being a subsequent case in a cluster (a surrogate for recent acquisition of tuberculosis infection) compared to the first case or a non-clustered case (a surrogate for reactivation of latent infection). RESULTS: We included 18,864 strain-typed cases, 2238 were the first cases of clusters and 8471 were subsequent cases. Seven hundred and fifty-nine (4%) were HIV-positive. Outcome 1: HIV-positive pulmonary tuberculosis cases who were the first in a cluster had fewer subsequent cases associated with them (mean 0.6, multivariable incidence rate ratio [IRR] 0.75 [0.65-0.86]) than those HIV-negative (mean 1.1). Extra-pulmonary tuberculosis (EPTB) cases with HIV were less likely to be the first case in a cluster compared to HIV-negative EPTB cases. EPTB cases who were the first case had a higher mean number of subsequent cases (mean 2.5, IRR (3.62 [3.12-4.19]) than those HIV-negative (mean 0.6). Outcome 2: tuberculosis cases with HIV co-infection were less likely to be a subsequent case in a cluster (odds ratio 0.82 [0.69-0.98]), compared to being the first or a non-clustered case. CONCLUSIONS: Outcome 1: pulmonary tuberculosis-HIV patients were less infectious than those without HIV. EPTB patients with HIV who were the first case in a cluster had a higher number of subsequent cases and thus may be markers of other undetected cases, discoverable by contact investigations. Outcome 2: tuberculosis in HIV-positive individuals was more likely due to reactivation than recent infection, compared to those who were HIV-negative.

Population Structure of Mycobacterium bovis in Germany: a Long-Term Study Using Whole-Genome Sequencing Combined with Conventional Molecular Typing Methods.

Mycobacterium bovis is the primary cause of bovine tuberculosis (bTB) and infects a wide range of domestic animal and wildlife species and humans. In Germany, bTB still emerges sporadically in cattle herds, free-ranging wildlife, diverse captive animal species, and humans. In order to understand the underlying population structure and estimate the population size fluctuation through time, we analyzed 131 M. bovis strains from animals (n = 38) and humans (n = 93) in Germany from 1999 to 2017 by whole-genome sequencing (WGS), mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing, and spoligotyping. Based on WGS data analysis, 122 out of the 131 M. bovis strains were classified into 13 major clades, of which 6 contained strains from both human and animal cases and 7 only strains from human cases. Bayesian analyses suggest that the M. bovis population went through two sharp anticlimaxes, one in the middle of the 18th century and another one in the 1950s. WGS-based cluster analysis grouped 46 strains into 13 clusters ranging in size from 2 to 11 members and involving strains from distinct host types, e.g., only cattle and also mixed hosts. Animal strains of four clusters were obtained over a 9-year span, pointing toward autochthonous persistent bTB infection cycles. As expected, WGS had a higher discriminatory power than spoligotyping and MIRU-VNTR typing. In conclusion, our data confirm that WGS and suitable bioinformatics constitute the method of choice to implement prospective molecular epidemiological surveillance of M. bovis The population of M. bovis in Germany is diverse, with subtle, but existing, interactions between different host groups.

Molecular epidemiology of clinical Mycobacterium tuberculosis complex isolates in South Omo, Southern Ethiopia.

BACKGROUND: Tuberculosis (TB) is caused by Mycobacterium tuberculosis complex (MTBC). Mapping the genetic diversity of MTBC in high TB burden country like Ethiopia is important to understand principles of the disease transmission and to strengthen the regional TB control program. The aim of this study was to investigate the genetic diversity of Mycobacterium tuberculosis complex (MTBC) isolates circulating in the South Omo, southern Ethiopia. METHODS: MTBC isolates (N = 156) were genetically analyzed using spacer oligotyping (spoligotyping) and mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) typing. Major lineages and lineages were identified using MTBC databases. Logistic regression was used to correlate patient characteristics with strain clustering. RESULTS: The study identified Euro-American (EA), East-African-Indian (EAI), Indo-Oceanic (IO), Lineage_7/Aethiops vertus, Mycobacterium bovis and Mycobacterium africanum major lineages in proportions of 67.3% (105/156), 22.4% (35/156), 6.4% (10/156), 1.9% (3/156), 1.3% (2/156) and 0.6% (1/156), respectively. Lineages identified were Delhi/CAS 23.9% (37/155), Ethiopia_2 20.6% (32/155), Haarlem 14.2% (22/155), URAL 14.2%(22/155), Ethiopia_3 8.4% (13/155), TUR 6.5% (10/155), Lineage_7/Aethiops vertus 1.9% (3/155), Bovis 1.3% (2/155), LAM 1.3% (2/155), EAI 0.6% (1/155), X 0.6% (1/155) and Ethiopia H37Rv-like strain 0.6% (1/155). Of the genotyped isolates 5.8% (9/155) remained unassigned. The recent transmission index (RTI) was 3.9%. Orphan strains compared to shared types (AOR: 0.09, 95% CI: 0.04-0.25) were associated with reduced odds of clustering. The dominant TB lineage in pastoral areas was EAI and in non-pastoral areas was EA. CONCLUSION: The epidemiological data, highly diverse MTBC strains and a low RTI in South Omo, provide information contributing to the TB Control Program of the country.

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.

Development of a hierarchical typing approach for Mycobacterium avium subsp. paratuberculosis (MAP) and characterization of MAP field cultures from Central Germany.

AIMS: Development of a novel hierarchical Mycobacterium avium subsp. paratuberculosis (MAP) typing approach and characterization of MAP field cultures in Central Germany. METHODS AND RESULTS: By combining single nucleotide polymorphisms (SNPs) and mycobacterial interspersed repetitive unit-variable number tandem repeat, we developed a highly discriminating and phylogenetically accurate hierarchical MAP typing approach. Moreover, a novel stepwise workflow was employed to reduce the number of SNP reactions required making the typing approach more affordable. MAP field cultures (n = 142) from dairy herds in Central Germany were classified as cattle type and showed a high level of heterogeneity. Intra-herd multiple genotypes were evident in (13-25%) of the investigated herds. CONCLUSIONS: The hierarchical MAP typing approach proved to be useful in fine discrimination between MAP cultures within limited geographical regions. This could potentially be used in unravelling MAP transmission chains in the respective regions. The observed heterogeneity in some herds is assumed to be due to either multiple introductions through inter-herd trade or intra-herd evolution over time. SIGNIFICANCE AND IMPACT OF THE STUDY: Future MAP epidemiological studies will benefit from the advantages of the novel hierarchical typing approach. The SNP number reduction approach employed here could be extrapolated for other analogous pathogens.

Epidemiological characterization of Mycobacterium caprae strains isolated from wildlife in the Bieszczady Mountains, on the border of Southeast Poland.

BACKGROUND: The majority of animal tuberculosis (TB) cases reported in wildlife in Poland over the past 20 years have concerned the European bison inhabiting the Bieszczady Mountains in Southeast Poland: an area running along the border of Southeast Poland. As no TB cases have been reported in domestic animals in this region since 2005, any occurrence of TB in the free-living animals inhabiting this area might pose a real threat to local livestock and result in the loss of disease-free status. The aim of the study was to describe the occurrence of tuberculosis in the wildlife of the Bieszczady Mountains and determine the microbiological and molecular characteristics of any cultured strains. Lymph node samples were collected for analysis from 274 free-living animals, including European bison, red foxes, badgers, red deer, wild boar and roe deer between 2011 and 2017. Löwenstein-Jensen and Stonebrink media were used for culture. Molecular identification of strains was performed based on hsp65 sequence analysis, the GenoType®MTBC (Hain Lifescience, Germany) test, spoligotyping and MIRU-VNTR analysis. RESULTS: Mycobacterium caprae was isolated from the lymph nodes of 21 out of 55 wild boar (38.2%; CI 95%: 26.5%, 51.4%) and one roe deer. Since 2014, no new TB cases have been reported in the Bieszczady European bison population. CONCLUSIONS: The identification of TB in wild boar in the Bieszczady is an alarming phenomenon, which requires further investigation. The Bieszczady mountains are a precious, unique area, home to many protected species. However, it is also the only area in Poland where TB cases have been reported in free-living animals. The occurrence of TB in wild boar inhabiting this area might pose a real threat to local livestock and many of the protected species (for example European bison that can share feeding places with wild boar). Given this situation, ongoing monitoring of the prevalence of TB should be conducted, and protective measures should be considered.

Exportation of MDR TB to Europe from Setting with Actively Transmitted Persistent Strains in Peru.

We performed a cross-border molecular epidemiology analysis of multidrug-resistant tuberculosis in Peru, Spain, and Italy. This analysis revealed frequent transmission in Peru and exportation of a strain that recreated similar levels of transmission in Europe during 2007-2017. Transnational efforts are needed to control transmission of multidrug-resistant tuberculosis globally.

Mycobacterium tuberculosis transmission in an ethnically-diverse high incidence region in England, 2007-11.

BACKGROUND: Transmission patterns in high tuberculosis incidence areas in England are poorly understood but need elucidating to focus contact tracing. We study transmission within and between age, ethnic and immigrant groups using molecular data from the high incidence West Midlands region. METHODS: Isolates from culture-confirmed tuberculosis cases during 2007-2011 were typed using 24-locus Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeats (MIRU-VNTR). We estimated the proportion of disease attributable to recent transmission, calculated the proportion of isolates matching those from the two preceding years ("retrospectively clustered"), and identified risk factors for retrospective clustering using multivariate analyses. We calculated the ratio (RCR) between the observed and expected proportion clustered retrospectively within or between age, ethnic and immigrant groups. RESULTS: Of the 2159 available genotypes (79% of culture-confirmed cases), 34% were attributed to recent transmission. The percentage retrospectively clustered decreased from 50 to 24% for 0-14 and ≥ 65 year olds respectively (p = 0.01) and was significantly lower for immigrants than the UK-born. Higher than expected clustering occurred within 15-24 year olds (RCR: 1.4 (95% CI: 1.1-1.8)), several ethnic groups, and between UK-born or long-term immigrants with the UK-born (RCR: 1.8 (95% CI: 1.1-2.4) and 1.6 (95% CI: 1.2-1.9) respectively). CONCLUSIONS: This study is the first to consider "who clusters with whom" in a high incidence area in England, laying the foundation for future whole-genome sequencing work. The higher than expected clustering seen here suggests that preferential mixing between some age, ethnic and immigrant groups occurs; prioritising contact tracing to groups with which cases are most likely to cluster retrospectively could improve TB control.

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.

MIRU-VNTR 15 loci capability in diagnosis of Beijing M. Tuberculosis strains in comparison with Real Time PCR.

Beijing strain has great importance among Mycobacterium tuberculosis (MTB) genotypes due to their drug resistance and pathogenicity. Determination of MTB genotypes and comparison of MIRU-VNTR 15 loci and real-time PCR methods for Beijing family identification was the main objective in this study. This study was conducted on 92 confirmed MTB isolates, 31 and 61 of which previously were determined by real- time PCR as Beijing and non-Beijing, respectively. Allelic diversity, clustering rate, phylogenetic tree, clonal complexes and molecular genotypes of isolates were determined by MIRU-VNTR 15 loci on its online software. In addition MIRU-VNTR 15 loci were performed for 16 non-tuberculosis isolates. Concordance between MIRU-VNTR 15 loci and real Time PCR in determination of Beijing genotype was 95.6(89.2-98.8). 74 different pattern of MIRU-VNTR were detected in 92 MTB isolates. 69 patterns were unique and 5 clusters were determined. Largest clusters contain 11 and 6 members. The clustering rate was 19.56%. Among 15 loci, Mtub04 and MIRU10 have the highest and MIRU04 had the lowest discriminatory power. In non-Beijing isolates, New1 and Delhi/CAS with 25% and 16.3% were the most prevalent MTB genotypes. None of the non-Tuberculosis isolates had the complete MIRU-VNTR 15 loci pattern. The results of this study showed that MIRU-VNTR 15 loci, in addition to being able to differentiate MTB genotypes, can distinguish non-tuberculosis species from MTB strains, but for the exact differentiation of Beijing MTB genotypes, it may be necessary to increase the patterns known in MIRU-VNTR data base.