MIRU VNTR Research Articles

Mycobacteriosis in slaughter pigs from South Africa from 1991 to 2002: Mycobacterium spp. diversity and Mycobacterium avium complex genotypes.

Mycobacterium avium complex (MAC) bacteria are the most prominent etiological agents of lymphadenitis in pigs. M. avium subspecies hominissuis (MAH) is a member of MAC and has been reported in many parts of the world to be the most prevalent non-tuberculous mycobacteria (NTM) to cause mycobacteriosis in humans, mainly in children. Thus, the economic and zoonotic impact of MAC species are increasingly being recognized. In South Africa, little is known about the distribution of NTM and the molecular epidemiology of M. avium in pigs. In this study, lymph nodes including mandibular, mesenteric, submandibular, and retropharyngeal, with tuberculosis-like lesions were collected during routine meat inspection of slaughter pigs with no disease symptoms (n = 132), between 1991 and 2002. These pigs were slaughtered at 44 abattoirs distributed across seven of the nine South African provinces. Mycobacterial culture, polymerase chain reaction (PCR), and sequencing of the Mycobacterium specific 577 bp 16S rRNA gene fragment were performed for species and subspecies identification. The majority of the isolates (each per sample); 114 (86.4%) were identified as MAH, 8 (6%) as MAA/M. avium subsp. silvaticum, 4 (3%) were Mycobacterium tuberculosis, 2 (1.5%) as Mycobacterium intracellulare, and 1 (0.75%) as Mycobacterium bovis. The other isolates were identified as Mycobacterium lentiflavum (0.75%), Mycobacterium novocastrense (0.75%), and a Micrococcus spp. (0.75%). Using an eight-marker MLVA typing tool, we deciphered at least nine MIRU VNTR INMV types of MAH and MAA. Identification of known zoonotic mycobacteria, including MAH, MAA, M. intracellulare, M. bovis, and M. tuberculosis, from slaughter pigs has a potential public health impact and also strengthens recognition of the potential economic impact of MAC. This study has also for the first time in South Africa, revealed MAC MIRU VNTR INMV genotypes which will aid in the future epidemiological investigation of MAC in South Africa.

Mycobacterium tuberculosis lineage 4 associated with cavitations and treatment failure

BackgroundMycobacterium tuberculosis genotyping has been crucial to determining the distribution and impact of different families on disease clinical presentation. The aim of the study was to evaluate the associations among sociodemographic and clinical characteristics and M. tuberculosis lineages from patients with pulmonary tuberculosis in Orizaba, Veracruz, Mexico.MethodsWe analyzed data from 755 patients whose isolates were typified by 24-loci mycobacterial interspersed repetitive unit–variable number of tandem repeats (MIRU–VNTR). The associations among patient characteristics and sublineages found were evaluated using logistic regression analysis.ResultsAmong M. tuberculosis isolates, 730/755 (96.6%) were assigned to eight sublineages of lineage 4 (Euro-American). Alcohol consumption (adjusted odds ratio [aOR] 1.528, 95% confidence interval (CI) 1.041–2.243; p = 0.030), diabetes mellitus type 2 (aOR 1.625, 95% CI 1.130–2.337; p = 0.009), sputum smear positivity grade (3+) (aOR 2.198, 95% CI 1.524–3.168; p < 0.001) and LAM sublineage isolates (aOR 1.023, 95% CI 1.023–2.333; p = 0.039) were associated with the presence of cavitations. Resistance to at least one drug (aOR 25.763, 95% CI 7.096–93.543; p < 0.001) and having isolates other than Haarlem and LAM sublineages (aOR 6.740, 95% CI 1.704–26.661; p = 0.007) were associated with treatment failure. In a second model, multidrug resistance was associated with treatment failure (aOR 31.497, 95% CI 5.119–193.815; p < 0.001). Having more than 6 years of formal education was not associated with treatment failure.ConclusionsKnowing M. tuberculosis genetic diversity plays an essential role in disease development and outcomes, and could have important implications for guiding treatment and improving tuberculosis control.

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.

A first insight into genetic diversity of Mycobacterium bovis isolated from extrapulmonary tuberculosis patients in South Tunisia assessed by spoligotyping and MIRU VNTR.

IntroductionIn Tunisia, almost 77% of clinically and bacteriologically diagnosed cases of extrapulmonary tuberculosis (EPTB) are zoonotic TB, caused by M. bovis. Although several studies have analyzed bovine TB in cattle in Tunisia, no study has evaluated the risk of transmission to humans in such an endemic country. We aimed to study the genetic diversity of M. bovis human isolates, to ascertain the causes of human EPTB infection by M. bovis and to investigate the distribution and population structure of this species in Tunisia.Materials and methodsA total of 110 M. bovis isolates taken from patients with confirmed EPTB were characterized by spoligotyping and MIRU-VNTR typing methods.ResultsAmong the 15 spoligotypes detected in our study, 6 (SB0120, SB0121, SB2025, SB1200, SB1003 and SB0134) were the most prevalent (83.5%) of which SB0120, SB0121 and SB2025 were the most prevailing. MIRU-VNTR typing method showed a high genotypic and genetic diversity. The genetic differentiation based on MIRU-VNTR was significant between populations from South East (Tataouine, Medenine) and Central West (Gafsa, Sidi Bouzid, Kasserine) regions. Of note, 13/15 (86.7%) spoligotypes detected in our study were previously identified in cattle in Tunisia with different frequencies suggesting a peculiar ability of some genotypes to infect humans. Using combined spoligotyping and MIRU-VNTR method, a high clustering rate of 43.9% was obtained. Our results underlined that human EPTB due to M. bovis was more commonly found in female gender and in young patients. Most of our patients, 66.4% (73/110) were raw milk or derivatives consumers, whereas 30.9% (34/110) patients would have contracted EPTB through contact with livestock. The findings suggest that the transmission of Zoonotic TB caused by M. bovis to humans mainly occurred by oral route through raw milk or derivatives.ConclusionOur study showed the urgent need of a better veterinary control with the implementation of effective and comprehensive strategies in order to reach a good protection of animals as well as human health.

Surveillance of tuberculosis (TB) cases attributable to relapse or reinfection in London, 2002-2015.

Recurrence of TB in an individual can occur due to relapse of the same strain or reinfection by a different strain. The contribution of reinfection and relapse to TB incidence, and the factors associated with each are unknown. We aimed to quantify and describe cases attributable to relapse or reinfection, and identify associated risk factors in order to reduce recurrence. We categorised recurrent TB cases from notifications in London (2002–2015) as relapse or reinfection using molecular (MIRU VNTR strain type) and epidemiological information (hierarchical approach using time since notification, site of disease and method of case finding). Factors associated with each outcome were determined using logistic regression in Stata Version 13.1 (2009–2015 only). Of 43,465 TB cases, 1.4% (618) were classified as relapse and 3.8% (1,637) as reinfection. The proportion with relapse decreased from 2002 (2.3%) to 2015 (1.3%), while the proportion of reinfection remained around 4%. Relapse was more common among recent migrants (<1 year, odds ratio (OR) = 1.99, p = 0.005), those with a social risk factor (OR = 1.51, p = 0.033) and those with central nervous system, spinal, miliary or disseminated TB (OR = 1.75, p = 0.001). Reinfection was more common among long term migrants (>11 years, OR = 1.67, p = <0.001), those with a social risk factor (OR = 1.96, p = <0.001) and within specific areas in London. Patients with social risk factors were at increased risk of both relapse and reinfection. Characterising those with relapsed disease highlights patients at risk and factors associated with reinfection suggest groups where transmission is occurring. This will inform TB control programs to target appropriate treatment and interventions in order to reduce the risk of recurrence.

A molecular epidemiological analysis of tuberculosis trends in South Korea

Molecular epidemiological data are needed to assess tuberculosis (TB)-management policy outcomes in South Korea. IS6110 restriction fragment-length polymorphism (IS6110-RFLP) and mycobacterial interspersed repetitive unit–variable-number tandem repeat (MIRU–VNTR) analyses are major molecular epidemiological tools for investigating the transmission or reactivation of active TB. Here, we determined trends in the clustering rate (i.e., the prevalence of Mycobacterium tuberculosis isolates with identical genotype patterns) of active TB and related differences between the 1990s and 2000s in Korea. M. tuberculosis isolates (1,007) of nationwide origins were analyzed by IS6110-RFLP and 24-locus standardized MIRU-VNTR genotyping. The clustering rate was measured by IS6110-RFLP, 24-locus MIRU-VNTR, and both analytical methods in combination. IS6110-RFLP, 24-locus MIRU-VNTR typing, and the combined method revealed 882, 754, and 983 distinct profiles; 809, 651, and 961 unique isolates; and 198, 356, and 46 clustered isolates grouped into 73, 103, and 22 clusters, respectively. In addition, we confirmed that the clustering rates in the 2000s decreased by 11.2%, 2.1%, and 3.1% relative to that in the 1990s using the three methods, respectively. Furthermore, in multivariate analysis, the younger-age group (<30) clustered more frequently than the older-age group (>50), based on all the three methods. Our study is the first report to provide nationwide molecular epidemiological information on TB in Korea.

Genetic diversity & drug sensitivity profiles of Mycobacterium tuberculosis isolates from two slums of Jaipur city, Rajasthan, India.

Background & objectives:Slums are considered as hotspots of tuberculosis (TB). The study of genetic diversity and drug susceptibility profile of Mycobacterium tuberculosis (MTB) will help understand the transmission dynamics and can be used for better prevention and control of the disease. The aim of this study was to determine the drug susceptibility profiles and genetic diversity using the random amplified polymorphic DNA (RAPD) and mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU VNTR) of MTB isolates from sputum samples of pulmonary TB patients residing in the two slums of Jaipur city in Rajasthan, India.Methods:Sputum samples collected from pulmonary TB patients, their contacts and suspects during 2010-2012 were processed for microscopy and mycobacterial culture. Drug susceptibility testing was done by one per cent indirect proportion method on Lowenstein–Jensen medium for first-line anti-TB drugs rifampicin, isoniazid, ethambutol and streptomycin. MTB DNA was extracted by physicochemical method, and DNA fingerprinting was done by RAPD and MIRU VNTR analysis.Results:Among 175 sputum samples collected, 75 were positive (43.8%) for acid-fast bacilli, 83 for MTB culture and four were contaminated. Fifty two isolates (62.7%) were fully sensitive to four drugs, and five (6%) were multidrug resistant (MDR). RAPD analysis of 81 isolates revealed six clusters containing 23 (28.4%) isolates, and 58 (71.6%) were unique. MIRU VNTR analysis clustered 20 (24.7%) isolates, and 61 (75.3%) were unique.Interpretation & conclusions:About 62.7 per cent isolates from the sputum samples from slum areas were sensitive to four drugs; six per cent of isolates were MDR. Poly-resistance other than MDR was high (16%). About one-fourth isolates were clustered by either method. RAPD was rapid, less expensive but had low reproducibility. MIRU VNTR analysis could identify to greater extent the epidemiological link in the population studied.

A quantitative and efficient approach to select MIRU–VNTR loci based on accumulation of the percentage differences of strains for discriminating divergent Mycobacterium tuberculosis sublineages

Although several optimal mycobacterial interspersed repetitive units–variable number tandem repeat (MIRU–VNTR) loci have been suggested for genotyping homogenous Mycobacterium tuberculosis, including the Beijing genotype, a more efficient and convenient selection strategy for identifying optimal VNTR loci is needed. Here 281 M. tuberculosis isolates were analyzed. Beijing genotype and non-Beijing genotypes were identified, as well as Beijing sublineages, according to single nucleotide polymorphisms. A total of 22 MIRU–VNTR loci were used for genotyping. To efficiently select optimal MIRU–VNTR loci, we established accumulations of percentage differences (APDs) between the strains among the different genotypes. In addition, we constructed a minimum spanning tree for clustering analysis of the VNTR profiles. Our findings showed that eight MIRU–VNTR loci displayed disparities in h values of ≥0.2 between the Beijing genotype and non-Beijing genotype isolates. To efficiently discriminate Beijing and non-Beijing genotypes, an optimal VNTR set was established by adding loci with APDs ranging from 87.2% to 58.8%, resulting in the construction of a nine-locus set. We also found that QUB11a is a powerful locus for separating ST10s (including ST10, STF and STCH1) and ST22s (including ST22 and ST8) strains, whereas a combination of QUB11a, QUB4156, QUB18, Mtub21 and QUB26 could efficiently discriminate Beijing sublineages. Our findings suggested that two nine-locus sets were not only efficient for distinguishing the Beijing genotype from non-Beijing genotype strains, but were also suitable for sublineage genotyping with different discriminatory powers. These results indicate that APD represents a quantitative and efficient approach for selecting MIRU–VNTR loci to discriminate between divergent M. tuberculosis sublineages.Emerging Microbes & Infections (2017) 6, e68; doi:10.1038/emi.2017.58; published online 26 July 2017

Высокая распространённость генотипа B0/W148 MycobacteriuM tuberculosisу больных вич-инфекцией, сочетанной с туберкулёзом, в пермском крае и иркутской области

The population with HIV-infection plays significant role in ongoing tuberculosis pandemic. Immunosup-pression due to HIV-infection is one of the causes of TB disseminated forms in this group of people. Having low immune status is also often associated with a polyclonal M.tuberculosis infection. Aim of the research: comparative assessment of epidemic genotypes of M.tuberculosis prevalence and mixed genotypes identification within HIV-TB co-infected patients in two Russian regions.Materials and methods. The DNAs of 78 clinical isolates from Irkutsk Region (IR) and 64strains from Perm Krai (PK) have been genotyped by MIRU VNTR 24 and RD105/RD207. Strains were obtained from patients who did not have significant age and sex differences. In the PK age of the patients was 34.5±0.9, in IR – 34.4±1.5 years. The samples were obtained from 67.2 and 65.4% of men, respectively.Result. The study of the M.tuberculosis indicates significant predominance of Beijing genotype strains in patients with TB-HIV of PK (92.2%) compared to the IR (59.5%) (χ2=18.0; p&amp;#60;0.01). The prevalence of MDR pathogens in TB-HIV patients exceeded 50%. The mixed genotype detection in the PK and IR was high (14.1 and 12.7% respectively). The level of virulent strains B0/W148 was 34.4% in PK patients and 25.3% in IR ones. Analysis of the results suggests the epidemic spread of MDR-TB in the immunocompromised individuals.Conclusions: The identified trends may indicate that Perm Kray have a process of active dissemination of transmissible strains of M.tuberculosis within HIV-infected population.

Comment on: Characterization of the embB gene in Mycobacterium tuberculosis isolates from Barcelona and rapid detection of main mutations related to ethambutol resistance using a low-density DNA array

Sir, We agree with Moure et al.1 that fast genotypic methods will play an increasingly prominent role in drug susceptibility testing for the Mycobacterium tuberculosis complex (MTBC).2,3 We would, however, like to point out that the embB (Rv3795) Glu378Ala polymorphism, which is detected by probe 3 of their newly developed low-density DNA array, is not a marker for ethambutol resistance.4–7 Instead, Ala represents the ancestral amino acid at this codon (Figure 1), whereas Glu is present in all modern MTBC (lineages 2, 3 and 4).6–9 The MIRU–VNTR data of the 51 ethambutol-resistant isolates from the study by Moure et al.1 are largely congruent with this finding. All 49 phylogenetically modern MTBC isolates had the embB 378 Glu variant. Isolate 5765 was a representative of Mycobacterium bovis, which is consistent with the fact that it harboured the Ala variant and was pyrazinamide resistant. By contrast, it was unclear why isolate 233R, which appeared to be M. bovis based on its MIRU–VNTR signature, had the Glu variant (experimental error or a homoplastic event might account for this discrepancy). Figure 1. Whole-genome phylogeny of 219 isolates representative of all major MTBC lineages.9 Glu at codon 378 is a marker for modern MTBC, which all share the TbD1 deletion and include the lineage 4 M. tuberculosis H37Rv laboratory strain that is used as the reference/wild-type ... In light of these data, the results of probe 3 would be predicted to lead to systematic false-positive reports, which calls into question the validity of this probe. This underlines that the entire MTBC diversity has to be considered when designing and validating genotypic drug susceptibility testing assays.7,10

Estimates of Genetic Variability of &amp;lt;i&amp;gt;Mycobacterium tuberculosis&amp;lt;/i&amp;gt; Complex and Its Association with Drug Resistance in Cameroon

The present study investigates the genetic diversity among Mycobacterium tuberculosis complex circulating in the Centre region of Cameroon and analyzes the relationship between genotypes and drug resistance patterns. Spoligotyping was performed by PCR-amplification followed by the reverse hybridization of 298 cultured specimens. Spoligotypes patterns were identified by comparison to reference strains in SPolDB4 database via the MIRU VNTR plus web application. About 97.65% of all tuberculosis (TB) cases were attributed to M. tuberculosis. A total of 65 different profiles were identified. Of these, 40 were represented as Shared Types (ST) while the others were orphans. LAM10_CAM and Haarlem families were the most prevalent genetic families with 51.01% and 14.09% respectively. ST 61, a member of the LAM10_ CAM family formed the largest cluster with 128 (42.95%) isolates. No association was found between genotypes with regard to drug resistance and HIV sero-status. However, there was a significant association between genotypes and age groups. Patients belonging to 15 - 24 and 35 - 44 age groups were more likely infected by LAM10_CAM strains compared to others. The population structure of Mycobacterium tuberculosis complex strains from the Centre region was found to be diverse and the spoligotype 61 of the LAM10_CAM family was highly predominant. Isolates of the LAM10_CAM seem to be not associated with drug resistance.

MIRU–VNTR profiles of three major Mycobacterium tuberculosis spoligotypes found in western India

We performed 12 loci MIRU-VNTR on 327 Mycobacterium tuberculosis (Mtb) isolates belonging to three major spoligotypes MANU1, CAS1_Delhi and Beijing from Mumbai, western India and two proximal rural locations. Complete allele and drug susceptibility data was available for 232 isolates. These included 143 MANU1 (ST100), 65 CAS1_Delhi (ST26) and 24 Beijing (ST1) isolates. Of the 232 isolates, 26 were rural consisting 6 CAS1_Delhi and 20 MANU1 isolates. Using eBURST multi-locus sequence typing (MLST), cluster analyses was performed for each of the spoligotypes and drug susceptibility profiles. MANU1 MLST consisted of 90 related isolates (clustered and grouped) and 53 singletons; CAS1_Delhi MLST consisted of 44 related isolates and 21 singletons; Beijing MLST consisted of 10 related isolates and 14 singletons. Although the number of related isolates were different in MANU1 (63%), CAS1_Delhi (68%) and Beijing (42%) clusters, it was not statistically significant. Furthermore, it was observed that while MANU1 and CAS1_Delhi singletons (n=74) had only 12 (16%) MDR isolates, the Beijing MLST had 8/14 (57%) MDR singleton isolates. Phylogenetic ananlysis using minimum spanning tree (MST) and a UPGMA radial tree revealed MANU1 had the largest number of nodes as compared to the CAS1_Delhi and Beijing spoligotypes. Additionally the CAS isolates were more homogeneous than the MANU1 isolates. The 12 loci MIRU-VNTR was used to provide greater discrimination than spoligotyping, but 6 of the 12 loci provided less than 50% discriminatory power. The highest discrimination was achieved using locus 26 (80%). Our results concur with recent reports that the most discriminatory MIRU-VNTR combination varied across different lineages. The results also highlight the need for more robust genetic markers for studying the transmission of Mtb in endemic regions like India.

Implication of the RDRioMycobacterium tuberculosis sublineage in multidrug resistant tuberculosis in Portugal

Multidrug and extensively drug resistant Mycobacterium tuberculosis are a threat to tuberculosis control programs. Genotyping methods, such as spoligotyping and MIRU–VNTR typing (Mycobacterial Interspersed Repetitive Units), are useful in monitoring potentially epidemic strains and estimating strain phylogenetic lineages and/or genotypic families. M. tuberculosis Latin American Mediterranean (LAM) family is a major worldwide contributor to tuberculosis (TB). LAM specific molecular markers, Ag85C103 single nucleotide polymorphism (SNP) and RDRio long-sequence polymorphism (LSP), were used to characterize spoligotype signatures from 859 patient isolates from Portugal. LAM strains were found responsible for 57.7% of all tuberculosis cases. Strains with the RDRio deletion (referred to as RDRio) were estimated to represent 1/3 of all the strains and over 60% of the multidrug resistant (MDR) strains. The major spoligotype signature SIT20 belonging to the LAM1 RDRio sublineage, represented close to 1/5th of all the strains, over 20% of which were MDR. Analysis of published datasets according to stipulated 12loci MIRU–VNTR RDRio signatures revealed that 96.3% (129/134) of MDR and extensively drug resistant (XDR) clusters were RDRio. This is the first report associating the LAM RDRio sublineage with MDR. These results are an important contribution to the monitoring of these strains with heightened transmission for future endeavors to arrest MDR–TB and XDR–TB.

Outbreak of reproductive disorders and mycobacteriosis in swine associated with a single strain of Mycobacterium avium subspecies hominissuis

In a breeding and fattening pig farm an increasing number of cases of abortion and generalized mycobacteriosis at slaughter occurred. Pathological findings compatible with mycobacteriosis, acid-fast organisms in tissues, and isolation of mycobacteria from tissue samples including fetuses, lungs and reproductive organs from sows, genital swabs, mesenteric lymph nodes, and from a sperm sample revealed the cause of the disease. Bacterial cultures were identified as Mycobacterium avium subsp. hominissuis using IS901-/IS1245-specific PCR. Genotyping of selected isolates from animals as well as from their environment by MIRU–VNTR analysis showed that the herd was infected with one single outbreak strain. The same genotype was also isolated from pigs of two other farms which showed comparable symptoms and were in direct contact with the index farm as well as from their environment. Immunological host responses detected by tuberculin skin test and ELISA gave positive results at herd level only. Despite the detection of other potential pathogens mycobacteria were regarded as the causative agent of the reproductive disorders.To our knowledge this is the first report of an epidemic mycobacterial infection in a pig holding associated with reproductive disorders, which could be attributed to one single virulent strain, and the first report of detection of M. avium subsp. hominissuis in pig sperm.

A novel method based on high resolution melting (HRM) analysis for MIRU–VNTR genotyping of Mycobacterium tuberculosis

The mycobacterial interspersed repetitive unit–variable number tandem repeat (MIRU–VNTR) method is one of the most important methods that have been used in recent years for genotyping Mycobacterium tuberculosis. Agarose gel electrophoresis and capillary electrophoresis have been used to determine the size of amplicons, however, both of these methods have shortcomings. Here, we develop and evaluate a novel method for MIRU–VNTR typing based on high resolution melting (HRM) analysis. The MIRU40 locus was selected to evaluate different real-time PCR machines and the accuracy of our method; the Roche LightCycler 480 provided greatest consistency between the T m value and repeat number and was used in subsequent evaluations. Our method gives greater accuracy in comparison with conventional agarose gel electrophoresis (98.9% vs. 90.9%, p = 0.017), and, with the help of fitting formulae, can be used to obtain the number of MIRU tandem repeats from the T m value. To validate our method we analyzed 12 classical MIRU loci to genotype 88 clinical isolates. The number of MIRU tandem repeats was determined accurately, quickly and conveniently.

Molecular characterization of Mycobacterium avium subsp. paratuberculosis using multi-locus short sequence repeat (MLSSR) and mycobacterial interspersed repetitive units–variable number tandem repeat (MIRU–VNTR) typing methods

The aim of this study was to characterize 38 bovine strains of Mycobacterium avium subsp. paratuberculosis isolated in Ireland using 11 multi locus short sequence repeat (MLSSR) loci and 8 mycobacterial interspersed repetitive units–variable number of tandem repeat (MIRU–VNTR) loci. The discriminatory power of these two typing methods alone and combined was evaluated, as was the epidemiology of the isolates and the genotypes obtained. Using the MIRU–VNTR typing method (8 loci analysed), only 3 subtypes were detected with a discrimination index (DI) of 0.54, but one MIRU–VNTR type has not been identified in other studies. In contrast the MLSSR method (using 11 loci) differentiated the 38 MAP isolates into 18 types with DI of 0.92. Among these 18 types 6 have not been recorded previously. The combination of the 2 methods (MIRU–VNTR and MLSSR) produced 22 distinct genotypes giving a maximal DI of 0.94. According to the allelic diversity, some markers are more polymorphic than others and must be applied in priority for the differentiation of MAP bovine isolates. This is the first report of genotyping data for MAP on the island of Ireland and will be very useful for analysing its epidemiology, transmission and virulence.

MIRU–VNTR typing of Mycobacterium avium in animals and humans: Heterogeneity of Mycobacterium avium subsp. hominissuis versus homogeneity of Mycobacterium avium subsp. avium strains

Epidemiological studies on Mycobacterium avium are requisite for revealing infection sources and disease transmission. They are based upon genotyping methods like RFLP and MIRU–VNTR. In our study, MIRU–VNTR typing was applied to 121 previously RFLP typed M. avium field isolates to compare the discriminatory power of both methods. The applicability of MIRU–VNTR typing was studied for isolates from a limited geographic area, namely 41 M. avium subsp. avium and 80 M. avium subsp. hominissuis isolates. Among the former, exhibiting 12 IS 901 RFLP types, five MIRU–VNTR types were found with discriminatory index ( DI) of 0.716. Among the latter, exhibiting 56 IS 1245 RFLP types, 18 MIRU–VNTR types were found with DI of 0.866. Concomitant use of both methods increased DI to 0.981 and 0.995, respectively. MIRU–VNTR typing employing the selected markers provided discernible discrimination among M. avium subsp. hominissuis isolates, but more discriminative markers are needed for M. avium subsp. avium isolates.

Molecular characterization of Mycobacterium avium subspecies paratuberculosis Types II and III isolates by a combination of MIRU–VNTR loci

Mycobacterial interspersed repetitive units and variable number tandem repeats typing (MIRU–VNTR) is a useful technique that has been recently applied to characterize members of the Mycobacterium avium complex (MAC). The aim of this study was to examine the genetic variability among a collection of Spanish M. avium subspecies paratuberculosis ( M. a. paratuberculosis) isolates with a combination of MIRU–VNTR loci. For this purpose we tested six MIRU–VNTR loci (MIRU-2, MIRU-3, VNTR-25, VNTR-32, VNTR-292 and VNTR-259) in 70 M. a. paratuberculosis isolates of Types II and III that were recovered from 22 Spanish localities during a nine-year period (1998–2007). The combination of five loci (MIRU-2, MIRU-3, VNTR-25, VNTR-32 and VNTR-259) enabled the differentiation of 12 allelic profiles, with a resulting Hunter and Gaston discriminatory index (HGDI) of 0.84. Moreover, we obtained MIRU–VNTR patterns that were unique for each of the M. a. paratuberculosis types analyzed (II and III); other patterns were host-related or restricted to geographic areas. Therefore, this MIRU–VNTR approach could be a useful sub-typing molecular tool in order to get a better sense of the epidemiology of Johne's disease.

Transmission of Mycobacterium tuberculosis in a Family Proved by Genotyping

Molecular genetic methods have been applied in various epidemiologic studies including investigations of disease acquisition by contact. This report describes the use of various molecular genetic methods in tracing possible household transmission of tuberculosis by contact. Four Mycobacterium tuberculosis strains, each from four members of a family, were first isolated and identified in the clinical laboratory of the Chest Hospital and were submitted to the National Reference Laboratory of Mycobacteriology for further confirmation and genotyping. In this study, IS6110 restriction fragment length polymorphism (RFLP), spacer oligonucleotide typing (spoligotyping) and mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTR), and rpoB gene sequencing were used for genotyping. All four strains were found to have identical spoligotypes, MIRU-VNTR patterns, and similar IS6110 RFLP profiles. The results of the drug susceptibility test and of rpoB sequencing showed that all four strains were rifampicin resistant. Household transmission through close contact was thus proved by genotyping. We conclude that all four family members were infected with the same lineage of M. tuberculosis.