Spoligotyping Methods Research Articles

Epidemiological Analysis of Human and Animal Originated Mycobacterium bovis Strains by Spoligotyping and MIRU-VNTR Methods

This study aims to investigate the genotypic similarities between human and animal-originated isolates by spoligotyping and 24 loci MIRU-VNTR for molecular epidemiological analysis of M. bovis isolates. In this study, isolates were obtained between 2019-2022 from 58 humans and 50 bovines. All isolates were initially identified with the GenoType MTBC kit and genotyped with spoligotyping and 24 loci MIRU-VNTR methods and their lineage relationships were shown in the dendrogram. When the human and animal-originated isolates were tested by the spoligotyping method, eight different clusters and 29 different genotypes were observed. Among these genotypes, the most common ones were found to be SIT1118/SB0989 (19.23%), SIT482/SB0120 (16.35%), SIT685/SB0288 (12.5%) and they were detected in isolates of both human and animal-originated. SB1593 (12.5%) was detected only in isolates of animal-originated. Other genotypes were found as SIT3529/SB0920, SIT1185/SB0897, SIT3710/SB1595, SIT688/SB0129, SIT3687/SB1625, SB0419, SB2466, SB1231, and SB2510. Nine different clusters and 55 different genotypes were obtained with MIRU-VNTR. ETR-C, QUB2163b, QUB26, and Mtub04 had the most allelic diversity. It was observed that MIRU02, MIRU20, MIRU24, MIRU27, and MIRU39 did not indicate allelic diversity. In conclusion, it was seen that the spoligotyping was easier to implement and evaluate compared to MIRU-VNTR. It was concluded that both tests can be used safely. Similar genotypes in humans and animals indicate the importance of zoonotic transmission of bovine tuberculosis.

Genotyping of Mycobacterium tuberculosis Complex Isolates from Patients with Tuberculosis by MIRU-VNTR and Spoligotyping Methods

Genotyping of Mycobacterium tuberculosis Complex Isolates from Patients with Tuberculosis by MIRU-VNTR and Spoligotyping Methods

Determination of genetic diversity of multidrug-resistant Mycobacterium tuberculosis strains in Turkey using 15 locus MIRU-VNTR and spoligotyping methods

ABSTRACT Tuberculosis (TB) remains the leading cause of deaths from infectious disease worldwide. Nowadays, the tendency of Mycobacterium tuberculosis complex (MTBC) to spread between continents due to uncontrolled migration movements shows that TB is a global health problem. The number of studies for the detection of MTBC strains’ epidemiological features in areas with TB spread risk using molecular-based methods such as spoligotyping and Mycobacterial Interspersed Repetitive Unit (MIRU) Variable Number Tandem Repeats (VNTR) at the clonal level is insufficient. In this study, it was aimed to determine the phylogenetic relationships of MTBC strains at the species level by spoligotyping and 15 locus MIRU-VNTR (MIRU-VNTR15) molecular methods of 96 multidrug-resistant (MDR) MTBC strains isolated from sputum samples of patients with a preliminary diagnosis of pulmonary TB or suspected contact history those sent to National Tuberculosis Reference Laboratory from the centers that are members of the Tuberculosis Laboratory Surveillance Network. The phylogenetic relationship between 96 MDR-TB strains was investigated with the combination of bead-based spoligotyping and MIRU-VNTR15 methods on the MAGPIX® Milliplex Map device. In this study, it was determined that the T1 family is more common in our country and LAM7-TUR family is less common than the Beijing family unlike other studies. It was determined that the strains in the same cluster had different locus profiles, and there was no transmission from the same clone in the clonal typing we performed with spoligotyping and MIRU-VNTR15.

The evaluation of second line drug susceptibilities and molecular epidemiological profiles of multidrug resistance Mycobacterium tuberculosis isolates isoleted from different region of Turkey

Introduction: This study was planned to determine the second line drug resistance and molecular epidemiological profiles of multidrug resistant Mycobacterium tuberculosis isolates isolated from different geographical regions of Turkey.
 Material and Method: In our study, 63 MDR M. tuberculosis isolates were evaluated for the drug susceptibility sent from different tuberculosis laboratories of Turkey. Secondary antituberculosis drugs resistance was evaluated by indirect proportion method. Epidemiological origins were evaluated by using IS6110-RFLP and spoligotyping methods. 
 Results: Cycloserine, ethionamide, capreomycin, thiacetozone, ofloxacin, kanamsin and paraaminosalicylic acid resistance rates were 15.87%, 19.04%, 7.93%, 6.34%, 11.11%, 12.69% and 6.34.%, respectively. According to spoligotyping results, 11 different patterns were obtained, including 52 isolates consisting of 5 clusters and 11 patterns consisting of a single isolate. When we compared our results with the spoligotype database in the world; 42 of 52 isolates forming 5 clusters were identified as predefined spoligotypes (LAM7-TUR, LAM9, T clade). 10 isolates showed the characteristics of the U spoligotype family. Of the 11 isolates that produced 11 different patterns, 8 were Haarlem and T spoligotypes. It was found that 2 isolates had the characteristics of Orphan and 1 isolate had the characteristics of BOV family. 
 Conclusion: In our study, LAM7-TUR, LAM9, T clade spoligotype families are common in our country and in the world were determined.

Molecular characterization of Mycobacterium bovis strains isolated from cattle and humans by spoligotyping and 24-locus MIRU-VNTR, and prevalence of positive IGRA in slaughterhouse workers in Southern Turkey.

Mycobacterium bovis is a zoonotic member of the Mycobacterium tuberculosis complex with a wide range of hosts, mainly cattle. Molecular epidemiological studies should be conducted to determine the transmission route, zoonotic risk factors, and phylogenetic relationships of M. bovis strains. Aims: This study aimed to characterize bovine and human M. bovis isolates by molecular methods. Molecular characterization and clonal relationship of strains isolated from tissue and organ samples of 76 cattle with positive tuberculin tests were collected from a slaughterhouse, and four M. bovis strains isolated from clinical materials of patients with suspected pulmonary TB isolates were analyzed using 24-locus MIRU-VNTR and spoligotyping methods. QuantiFERON-TB Gold Plus (QFT-Plus; Qiagen) was used to determine the prevalence of latent TB infection among 21 slaughterhouse personnel including 7 veterinarians, 12 butchers, 1 caretaker, and 1 veterinary technician. SB0288/SIT685 type was detected in both cattle and humans by the spoligotyping method. When evaluating MIRU-VNTR, the presence of a 100% compatible pattern between human and bovine isolates was not detected, but some human samples were found to be 91.6% similar to a bovine sample. In addition, 21 slaughterhouse workers were screened with the interferon gamma-released assay (IGRA) and a 23.8% positivity was detected. Clonal similarity was determined between the bovine and human isolates using the MIRU-VNTR and spoligotyping methods and IGRA positivity in the occupational group suggested that M. bovis might be associated with pulmonary tuberculosis in humans.

Spoligotyping and polymerase chain reaction based mycobacterium bovis strains typing with methods (enterobacterial repetitive intergenic consensus-polymerase chain reaction, randomly amplified polymorphic dnas-polymerase chain reaction and out polymerase chain reaction).

In this study, it was aimed to investigate Mycobacterium bovis strains isolated from lungs and lymph nodes of slaughtered animals on clonal level by using different methods such as spoligotyping, enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR), randomly amplified polymorphic DNAs (RAPD-PCR) and OUT-PCR. Comparative evaluation of these methods was further conducted. A total of 38 M. bovis isolates were evaluated in the study. DNA isolation of all M. bovis strains isolated from pruvat free Löwenstein Jensen medium was done by boiling method for ERIC-PCR, RAPD-PCR, and OUT PCR. Mickle device was used for DNA isolation for spoligotyping method. In 38 M. bovis isolates examined in our study, 4 different groups were determined by spoligotyping and RAPD-PCR test methods, and 5 different groups were detected in ERIC-PCR tests. In the OUT-PCR tests, the band which provides sufficient type separation was not observed. ERIC-PCR, RAPD-PCR, and OUT-PCR methods are easily applicable, simple, and relatively inexpensive methods for evaluating the differences between origins in the typing of M. bovis. The tests need to be evaluated in more detail with extensive studies.

Genotypic diversity of multi- and pre-extremely drug-resistant Mycobacterium tuberculosis isolates from Morocco.

In Morocco, the prevalence of multidrug resistant tuberculosis (MDR-TB) continues to increase especially within previously treated cases; these MDR cases may evolve to extensively drug resistant tuberculosis (XDR-TB) raising major concern to TB control programs. From an epidemiological window, scarce informations are available about the genetic diversity of Mycobacterium tuberculosis (MTB) strains fueling these forms of resistance. The aim of this study was to assess to genetic diversity of MDR-MTB strains. Hence, this prospective study was conducted on patients diagnosed with MDR-TB at Pasteur Institute of Casablanca from 2010 to 2013. A total of 70 MDR-MTB isolates were genotyped by spoligotyping and 15-loci MIRU-VNTR methods. Spoligotyping generated four orphan patterns, five unique profiles whereas 61 strains were grouped in nine clusters (2 to 25 strains per cluster), the clustering rates being 87.1%. Subtyping by 15 loci MIRU-VNTR splitted all clusters already established by spoligotyping and generated 70 unique profiles not recognized in SITVIT2 database; clustering rate was equal to zero. HGDI analysis of 15 loci MIRU demonstrated that eight out of 15 loci were highly discriminant. Of note, all pre-XDR strains belongs to many clades, meaning that there no association between gyrA mutants and particular clade. Overall, the data generated by this study (i) describe the population structure of MDR MTBC in Morocco which is highly homogenous, (ii) confirm that TB in Morocco is almost exclusively transmitted by modern and evolutionary lineages with high level of biodiversity seen by MIRU, and (iii) validate the use of optimized 15-loci MIRU-VNTR format for future investigations in Morocco.

Molecular Characteristics and Drug Resistance of Mycobacterium tuberculosis Isolate Circulating in Shaanxi Province, Northwestern China.

Objective: Shaanxi is the most highly populated province with high burdens of tuberculosis in northwestern China. The aim of this study was to investigate the molecular characteristics and drug resistance of Mycobacterium tuberculosis isolates from Shaanxi province of China in 2018. Methods: Phenotypic drug susceptibility testing and spoligotyping methods were performed on 518 M. tuberculosis isolates; drug-resistant isolates were sequenced in 11 drug loci, including katG, inhA, oxyR-ahpC, rpoB, embB, rpsL, rrs1 (nucleotides 388-1084), gyrA, gyrB, rrs2 (nucleotides 1158-1674), and eis. Results: The prevalences of isoniazid, rifampicin, ethambutol, streptomycin, ofloxacin, and kanamycin resistance were 22.0%, 19.3%, 7.9%, 23.8%, 10.4%, and 3.3%, respectively. The Beijing family (82.8%) was the predominant genotype, followed by the T (9.3%), H (0.6%), CAS (0.4%), LAM (0.4%), and U (0.4%) families. The percentage of Beijing genotype in a central area (88.1%) was higher than in the south (77.3%) and the north area (80.1%) (p < 0.05), while the sex, age, and treatment history between Beijing and non-Beijing family were not statistically different. Mutation analysis found that the most prevalent mutations were katG315, rpoB531, embB306, rpsL43, gyrA94, and rrs1401; the Beijing family exhibited a high rate of isoniazid-resistant isolates carrying katG315 mutations (p < 0.05). Furthermore, compared with the phenotypic data, the sensitivities of isoniazid, rifampicin, ethambutol, streptomycin, ofloxacin, and kanamycin resistance by sequencing base on 11 loci were 85.1%, 94.0%, 53.7%, 74.8%, 77.8%, and 64.7%, respectively. Conclusions: Shaanxi has a serious epidemic of drug-resistant tuberculosis, Beijing family is the predominant genotype, and the distribution showed geographic diversity. The prevalence of Beijing genotypes has a tendency to promote the transmission of high-level isoniazid-resistant M. tuberculosis. Besides, the hot spot regions localized in the embB, rrs2, and eis gene appear not to serve as excellent biomarkers for predicting ethambutol and kanamycin resistance in Shaanxi.

Genetic evaluation of Mycobacteriumbovis isolates with MIRU-VNTR and spoligotyping

Background/aimDetermining the epidemiological characteristics of M. bovis strains isolated from human and animal tuberculosis cases will assist in taking more appropriate and effective control measures in controlling tuberculosis originating from animals. Materials and methodsIn this study, 32 M. bovis isolates of animal origin and 10 of human origin were isolated and identified in the Çukurova region between March 2011 and June 2012. The 12-locus MIRU-VNTR and spoligotyping methods were used. ResultsSix different patterns were determined by spoligotyping and 10 by MIRU-VNTR. When both methods were used together, the number of patterns was found to be 28; MIRU4, MIRU26, MIRU31, and MIRU40 had the highest locus discrimination powers by MIRU-VNTR. The isolates concentrated in the SB0120 pattern at the rate of 42.85% in spoligotyping. By the same method, it was seen that 7 isolates were M bovis ssp. caprae pattern and 2 human isolates were M. bovis BCG pattern. Nevertheless, spoligotyping and MIRU-VNTR patterns showed that 5 M. bovis isolates of human origin were 100% compatible with isolates originating from cattle. ConclusionIn this study, we determined that the use of spoligotyping and MIRU-VNTR methods together was found to be more sensitive in the epidemiological analysis of M. bovis isolates.

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.

Detection of Beijing strains of MDR M. tuberculosis and their association with drug resistance mutations in katG, rpoB, and embB genes

BackgroundMolecular epidemiological studies of Mycobacterium tuberculosis (MTB) are the core of current research to find out the association of the M. tuberculosis genotypes with its outbreak and transmission. The high prevalence of the Beijing genotype strain among multidrug resistance (MDR) TB has already been reported in various studies around India. The overall objective of this study was to detect the prevalence of Beijing genotype strains of MDR M. tuberculosis and their association with the clinical characteristics of TB patients.MethodsIn this study 381 M. tuberculosis clinical isolates were obtained from sputum samples from 2008 to 2014. The multiplex-PCR and Spoligotyping (n = 131) methods were used to investigate the prevalence of the Beijing genotype strain by targeting the Rv2820 gene and their association with drug resistance and clinical characteristics of TB patients. The drug susceptibility testing of first-line anti-TB drugs was performed by using the proportion method and MGIT960. A collection of isolates having Beijing and non-Beijing strains were also characterized to see if Beijing genotype strains had a higher rate of mutations at codons 516, 526 and 531 of the 81-bp region of the rpoB gene, codon 315 of the katG gene, and codon 306 of the embB gene.ResultsThe sensitivities and specificities of multiplex-PCR assay compared to that of standard Spoligotyping was detected to be 100%. Further, we observe that the multi drug-resistance was significantly associated with Beijing genotype strains (p = 0.03) and a strong correlation between Beijing genotype strains and specific resistance mutations at the katG315, rpoB531, and embB306 codons (p = < 0.0001, < 0.0001 & 0.0014 respectively) was also found.ConclusionsThis rapid, simple, and cost-effective multiplex PCR assay can effectively be used for monitoring the prevalence of Beijing genotype strains in low resource settings. Findings of this study may provide a scientific basis for the development of new diagnostic tools for detection and effective management of DR-TB in countries with a higher incidence rate of Beijing genotype strains.

A tuberculosis school outbreak in China, 2018: reaching an often overlooked adolescent population.

Adolescents have been largely neglected from tuberculosis control efforts. In low- to medium burden settings much of the tuberculosis burden in this age group occurs from school outbreaks. We report on a large tuberculosis outbreak in adolescents from a boarding high school in Jiangsu Province, China. From March to June 2018, a tuberculosis outbreak occurred in a boarding high school. We conducted an outbreak investigation involving clinical diagnostic tests and molecular analysis to determine the outbreak origin. Cases were detected through symptom screening, tuberculin skin testing (TST), chest radiography, sputum smear, solid sputum culture and GeneXpert MTB/RIF. Mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) genotyping and spoligotyping methods were performed on Mycobacterium tuberculosis (M. tuberculosis) isolates to identify the outbreak origin. A total of 845 students and 131 teachers/staff attended a TST screening for tuberculosis infection. The prevalence of elevated tuberculin reactions at ≥5, ≥10 and ≥15 mm was 12.19% (119/976), 6.35% (62/976) and 3.28% (32/976), respectively. Radiographic abnormalities were present in 5.73% (56 of 976) individuals, 40 students and 16 teachers/staff. Of these, 12 students were diagnosed with confirmed tuberculosis. In total, 14 students (two index cases and 12 confirmed cases) were diagnosed and reported in the tuberculosis outbreak, an attack rate of 1.7% (14/847) among students (two index cases and 845 screened students). Results from MIRU-VNTR typing and spoligotyping analyses demonstrated that three M. tuberculosis strains belong to the Beijing family with corresponding MIRU-VNTR alleles. This school-based tuberculosis outbreak among adolescents demonstrates that transmission among individuals in this age group is common and must be prioritised. It suggests that identifying and timely diagnosis of smear-positive cases, especially in the early phase of outbreaks, is the key to preventing further spread among close contacts.

Genetic diversity of Mycobacterium tuberculosis isolates causing pulmonary and extrapulmonary tuberculosis in the capital of Iran

ObjectivesEvaluation of the genetic diversity of Mycobacterium tuberculosis (M.tb) and determining if the association between a specific genotype and the site of infection is crucial. Accordingly, the current study aimed at comparing predominant M.tb genotypes in pulmonary (PTB) and extrapulmonary tuberculosis (EPTB) isolates circulating in the capital of Iran. MethodsThe genetic diversity of culture-confirmed PTB and EPTB isolates were evaluated by Spoligotyping and MIRU-VNTR (mycobacterial interspersed repetitive-unit–variable-number tandem-repeat) typing methods. Genotyping data were analyzed with SITVIT, MIRU-VNTRplus, and TBminer databases. To assess adjusted associations, chi-square/the Fisher exact test and multiple logistic regression model were applied. ResultsURAL2 (NEW-1) (28/88; 31.8%) and CAS1-DELHI (25/84; 29.8%) genotypes were predominant in EPTB and PTB strains, respectively. Based on MIRU-VNTR typing, 158 different MIRU-VNTR patterns were identified. Clustering rate and minimum estimate of the proportion of TB caused by recent transmission was 4.1% and 8.1%, respectively. ConclusionsThe current study provided new insight into circulating genotypes of M.tb in PTB and EPTB patients in Tehran, Iran. This low percentage of TB transmission rate, demonstrated that mode of TB transmission was mainly associated with reactivation of latent TB rather than recently transmitted infection in this region. There was no significant difference in the association between the genotypes of M.tb strains and the site of the disease.

Molecular Characterization of Mycobacterium tuberculosis Strains with TB-SPRINT.

We evaluated Tuberculosis-Spoligo-Rifampicin-Isoniazid Typing (TB-SPRINT), a microbead-based method for spoligotyping and detection of rifampicin and isoniazid resistance in Mycobacterium tuberculosis. For that, 67 M. tuberculosis complex strains were retrospectively selected. Membrane-based spoligotyping, restriction fragment length polymorphism, DNA sequencing/pyrosequencing of rpoB, katG, and inhA promoter, TB-SPRINT, and SNP typing were performed. Concordance between spoligotyping methods was 99.6% (2,785/2,795 spoligotype data points). For most of the discordant cases, the same lineage was assigned with both methods. Concordance between phenotypic drug susceptibility testing and TB-SPRINT for detecting rifampicin and isoniazid resistance was 98.4% (63/64) and 93.8% (60/64), respectively. Concordance between DNA sequencing/pyrosequencing and TB-SPRINT for detecting mutations in rpoB, katG, and inhA were 98.4% (60/61), 100% (64/64), and 96.9% (62/64), respectively. In conclusion, TB-SPRINT is a rapid and easy-to-perform assay for genotyping and detecting drug resistance in a single tube; therefore, it may be a useful tool to improve epidemiological surveillance.

"Comparision of ERIC-PCR, OUT- PCR and Spoligotyping Methods to Diagnose of Cross- Contamination with Mycobacterium tuberculosis"

"Comparision of ERIC-PCR, OUT- PCR and Spoligotyping Methods to Diagnose of Cross- Contamination with Mycobacterium tuberculosis"

Molecular Typing of Mycobacterium bovis from Cattle Reared in Midwest Brazil.

Mycobacterium bovis is the causative agent of bovine tuberculosis (BTB), the pathogen responsible for serious economic impact on the livestock sector. In order to obtain data on isolated M. bovis strains and assist in the control and eradication program for BTB, a cross sectional descriptive molecular epidemiology study in the Brazilian Midwest was conducted. Through spoligotyping and 24-loci MIRU-VNTR methods, 37 clinical isolates of M. bovis circulating in the region were analyzed, 10 isolated from the state of Mato Grosso, 12 from the state of Mato Grosso do Sul and 15 from the state of Goiás. The spoligotyping analysis identified 10 distinct M. bovis profiles (SB0121 n = 14, SB0295 n = 6, SB0140 n = 6, SB0881 n = 3, SB1144 n = 2, SB1145 n = 2, SB0134 n = 1, SB1050 n = 1, SB1055 n = 1, SB1136 n = 1) grouped in six clusters and four orphan patterns. The MIRU-VNTR 24-loci grouped the same isolates in six clusters and 22 unique orphan patterns, showing higher discriminatory power than spoligotyping. When associating the results of both techniques, the isolates were grouped in five clusters and 24 unique M. bovis profiles. Among the 24-loci MIRU-VNTR evaluated, two, ETR-A and QUB 11b loci, showed high discriminatory ability (h = ≥ 0.50), while MIRU 16, MIRU 27, ETR-B, ETR-C, Mtub21 and QUB 26 loci showed moderate ability (h = 0.33 or h = 0.49) and were the most effective in evaluating the genotypic similarities among the clinical M. bovis isolate samples. Herein, the 29 patterns found amongst the 37 isolates of M. bovis circulating in the Brazilian Midwest can be due to the animal movement between regions, municipalities and farms, thus causing the spread of various M. bovis strains in herds from Midwest Brazil.

Determination of genotypic varieties and genotyping of multiple drug-resistant tuberculosis by the RFLP and spoligotyping methods.

The purpose of the present study was to determine the distribution and epidemiological features of mycobacteria with molecular methods. Fifty-five culture-positive samples were analyzed by polymerase chain reaction-restriction enzyme length polymorphism (PCR-RFLP) at species level, and their molecular typing was performed with spoligotyping. The IS6110 region and the locus of gene coding for Hsp65 were amplified. RFLP profiles were obtained by cutting the Hsp65 region with the Hae III and BstE II (Eco91I) enzymes. Spoligotyping was carried out by commercial kit. The H37Rv strain was used as the control. All samples showed the same cutting pattern with the H37Rv strain. The RFLP profiles of 9 strains identified as "mycobacteria other than tuberculosis" were compatible with the M. tuberculosis complex. Spoligotyping of 55 isolates detected 13 different genetic profiles. The Beijing genotype was not detected. One isolate was described as an orphan strain according to the SpolDB4 database. The most frequently detected family was T1 with 32 strains (64%), followed by 9 isolates (18%) belonging to the LAM7 TUR family. PCR-RFLP is a specific, rapid, and effective method in routine diagnosis of mycobacteria. Spoligotyping is an ideal method in the determination of genotypic varieties of mycobacteria.

Molecular Typing Characteristic and Drug Susceptibility Analysis of Mycobacterium tuberculosis Isolates from Zigong, China.

China is one of the 22 countries with high TB burden worldwide, and Sichuan contained the second-largest number of TB cases among all of the Chinese provinces. But the characteristics of Mycobacterium tuberculosis circulated in Zigong, Sichuan, were still unknown. To investigate the character and drug resistance profile, 265 clinical isolates were cultured from tuberculosis patient's sputum samples in the year of 2010, of which the genetic profile was determined by using Spoligotyping and MIRU-VNTR typing methods, and the drug sensibility testing to the four first-line and four second-line antituberculosis (anti-TB) drugs was performed by using proportion method on Lowenstein-Jensen (L-J) media. The major Spoligotype was Beijing family (143/265, 53.96%), followed by T (80/265, 30.19%) and H (9/265, 3.40%) genotypes; the total Hunter-Gaston discrimination index (HGDI) of the 24 loci MIRU-VNTR was 0.9995. About 27.17% (72/265) of the isolates were resistant to at least one of the eight tested anti-TB drugs, and for Beijing and non-Beijing family isolates the proportion of drug resistance was 28.47% (41/144) and 25.62% (31/121), respectively. That is, the most prevalent genotype here was Beijing family, and the 24 loci VNTR analysis could supply a high resolution for genotyping, and Beijing and non-Beijing isolates had no difference (p > 0.05) for drug resistance.

Mixed Infections and Rifampin Heteroresistance among Mycobacterium tuberculosis Clinical Isolates.

Mixed infections and heteroresistance of Mycobacterium tuberculosis contribute to the difficulty of diagnosis, treatment, and control of tuberculosis. However, there is still no proper solution for these issues. This study aimed to investigate the potential relationship between mixed infections and heteroresistance and to determine the high-risk groups related to these factors. A total of 499 resistant and susceptible isolates were subjected to spoligotyping and 24-locus variable-number tandem repeat methods to analyze their genotypic lineages and the occurrence of mixed infections. Two hundred ninety-two randomly selected isolates were sequenced on their rpoB gene to examine mutations and heteroresistance. The results showed that 12 patients had mixed infections, and the corresponding isolates belonged to Manu2 (n = 8), Beijing (n = 2), T (n = 1), and unknown (n = 1) lineages. Manu2 was found to be significantly associated with mixed infections (odds ratio, 47.72; confidence interval, 9.68 to 235.23; P < 0.01). Four isolates (1.37%) were confirmed to be heteroresistant, which was caused by mixed infections in three (75%) isolates; these belonged to Manu2. Additionally, 3.8% of the rifampin-resistant isolates showing no mutation in the rpoB gene were significantly associated with mixed infections (χ(2), 56.78; P < 0.01). This study revealed for the first time that Manu2 was the predominant group in the cases of mixed infections, and this might be the main reason for heteroresistance and a possible mechanism for isolates without any mutation in the rpoB gene to become rifampin resistant. Further studies should focus on this lineage to clarify its relevance to mixed infections.

Study of Mycobacterium bovis genotypes in human and bovine isolates using spoligotyping, MIRUVNTR and RFLP-PCR

Aims and objectivesThe aim of this study is to investigate and detect the prevalence of Mycobacterium bovis subtypes (Mycobacterium bovis subtype bovis and Mycobacterium bovis subtype caprae) in humans and compare the genetic diversity of Mycobacterium bovis in humans and cattle with spoligotyping methods, as well as pyrazinamide susceptibility study of subtypes. MethodsExamining these subtypes with molecular epidemiology techniques is particularly important due to different treatment of M. bovis diverse subtypes in humans. Culture tests were performed on clinical samples that were isolated from Lowenstein-Jensen culture medium. Identification tests were performed to differentiate Mycobacterium bovis from Mycobacterium tuberculosis. DNA was extracted and spoligotyping (spacer oligonucleotide typing) was performed using the DRb and DRa primers. ResultsThe results were analyzed with the SPOLDB4 site. PCR-RFLP method of pncA gene was used to evaluate the resistance to pyrazinamide and pncA gene polymorphism. ConclusionsMycobacterium bovis subtype bovis in the Iranian population was reported with a frequency of 0.6% which was below the average of the previous reviews. In this study, all the strains were M. bovis subtype bovis resistant to pyrazinamide. No Mycobacterium bovis subtype caprae was detected. The only shared ST between humans and cattle was ST694. Mycobacterium bovis subtype bovis with ST 595 was reported as human bovis index.