Hunter-Gaston Discriminatory Index Research Articles

Optimization of MLVA loci combination using metaheuristic methods

Multilocus variable number tandem repeat analysis (MLVA) utilizes short DNA repeat polymorphism in genomes, which is termed variable number tandem repeat (VNTR), to differentiate closely related organisms. One research challenge is to find an optimal set of VNTR to distinguish different members accurately. An intuitive method is to use an exhaustive search method. However, this method is not an efficient way to find optimal solutions from a dataset comprising many attributes (loci) due to the curse of dimensionality. In this study, metaheuristic methods are proposed to find an optimal set of loci combination. Basic genetic algorithm (BGA) and modified genetic algorithm (MGA) were proposed in our previous work for this purpose. However, they require prior knowledge from an experienced user to specify the minimum number of loci for achieving good results. To impose no such expertise requirement for parameter setting, a GA with Duplicates (GAD), which allows the inclusion of duplicated loci in a chromosome (potential solution) during the search process, is developed. The study also investigates the search performance of a hybrid metaheuristic method, namely quantum-inspired differential evolution (QDE). Hunter-Gaston Discriminatory Index (HGDI) is used to indicate the discriminatory power of a loci combination. Two Mycobacterium tuberculosis MLVA datasets obtained from a public portal and a local laboratory respectively, are used. The results obtained by using exhaustive search and metaheuristic methods are first compared, followed by a performance comparison among BGA, MGA, GAD, and QDE by a statistical approach. The best-performing GA method (i.e., GAD) and QDE are selected for a performance comparison with several recent metaheuristic methods using both MLVA datasets by a statistical approach. The statistical results show that both GAD and QDE could achieve higher HGDI than the recent methods using a small but informative set of loci combination.

The mutation rate of rpoB gene showed an upward trend with the increase of MIRU10, MIRU39 and QUB4156 repetitive number

BackgroundMycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) is a frequently used typing method for identifying the Beijing genotype of Mycobacterium tuberculosis (Mtb), which is easily transformed into rifampicin (RIF) resistance. The RIF resistance of Mtb is considered to be highly related with the mutation of rpoB gene. Therefore, this study aimed to analyze the relationship between the repetitive number of MIRU loci and the mutation of rpoB gene.MethodsAn open-source whole-genome sequencing data of Mtb was used to detect the mutation of rpoB gene and the repetitive number of MIRU loci by bioinformatics methods. Cochran-Armitage analysis was performed to analyze the trend of the rpoB gene mutation rate and the repetitive number of MIRU loci.ResultsAmong 357 rifampicin-resistant tuberculosis (RR-TB), 304 strains with mutated rpoB genes were detected, and 6 of 67 rifampicin susceptible strains were detected mutations. The rpoB gene mutational rate showed an upward trend with the increase of MIRU10, MIRU39, QUB4156 and MIRU16 repetitive number, but only the repetitive number of MIRU10, MRIU39 and QUB4156 were risk factors for rpoB gene mutation. The Hunter-Gaston discriminatory index (HGDI) of MIRU10 (0.65) and QUB4156 (0.62) was high in the overall sample, while MIRU39 (0.39) and MIRU16 (0.43) showed a moderate discriminatory Power.ConclusionThe mutation rate of rpoB gene increases with the addition of repetitive numbers of MIRU10, QUB4156 and MIRU39 loci.

Fingerprinting of Mycobacterium tuberculosis isolates by MIRU-VNTR genotyping and detection of isoniazid resistance by real-time PCR.

Introduction. Tuberculosis (TB) is a great public health problem in developing countries such as Egypt. Genotyping of Mycobacterium tuberculosis isolates has a prominent role in the field of TB prevention.Aim. This study aimed to evaluate real-time PCR using Minor Groove Binder (MGB) probes and to identify circulating lineages/sub-lineages of M. tuberculosis and their transmission patterns.Hypothesis. We hypothesize that MIRU-VNTR technique is efficient in identifying circulating M. tuberculosis lineages in Egypt.Methodology. Fifty sputum specimens positive for acid-fast bacilli were included. Isoniazid (INH) resistance was detected using the 1 % proportion method. Real-time PCR using MGB-probes was used for simultaneous detection of TB infection and INH resistance. Partial sequencing of the katG gene was used to confirm INH resistance results. A standard 15 Mycobacterial Interspersed Repetitive Unit Variable Number Tandem Repeat (15-MIRU-VNTR) approach was used for genotyping through the MIRU-VNTRplus online platform.Results. Only seven specimens showed phenotypic resistance to INH. M. tuberculosis was detected in all samples, while a mutation in the katG gene codon 315 was detected only in five samples, which were also phenotypically INH-resistant. Sequencing of the katG gene showed codon 315 mutation genotypically and phenotypically in the five INH-resistant isolates. Molecular genotyping of M. tuberculosis isolates revealed that the majority of isolates (26/50, 52 %) belonged to the S family of lineage_4. A low clustering rate (2 %) was observed among our isolates. According to the Hunter-Gaston Discriminatory Index (HGDI), 11 MIRU-VNTR loci were highly or moderately discriminative, while four loci were less polymorphic.Conclusion. MIRU-VNTR genotyping revealed a low clustering rate with a low recent transmission rate of M. tuberculosis strains in Alexandria, Egypt.

The shortcut of mycobacterial interspersed repetitive unit-variable number tandem repeat typing for Mycobacterium tuberculosis differentiation.

The 24-loci mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) genotyping has been used as an international standard method for Mycobacterium tuberculosis (Mtb) genotyping. However, different optimized VNTR loci sets for improving the discrimination of specific Mtb genotypes have been proposed. In this regard, we investigated the efficacy of accumulation of the percentage differences (APDs) compared with the least absolute shrinkage and selection operator (LASSO) regression strategy to identify a customized genotype-specific VNTR loci set which provides a resolution comparable to 24-loci MIRU-VNTR in divergent Mtb populations. We utilized Spoligotyping and 24-loci MIRU-VNTR typing for genotyping 306 Mtb isolates. The APD and LASSO regression approaches were used to identify a customized VNTR set in our studied isolates. Besides, the Hunter-Gaston discriminatory index (HGDI), sensitivity, and specificity of each selected loci set were calculated based on both strategies. The selected loci based on LASSO regression compared with APD-based loci showed a better discriminatory power for identifying all studied genotypes except for T genotype, which APD-based loci showed promising discriminative power. Our findings suggested the LASSO regression rather than the APD approach is more effective in the determination of possible discriminative VNTR loci set to precise discrimination of our studied Mtb population and may be beneficial to be used in finding reduced number loci sets in other Mtb genotypes or sublineages. Moreover, we proposed customized genotype-specific MIRU-VNTR loci sets based on the LASSO regression and APD approaches for precise Mtb strains identification. As the proposed VNTR sets offered a comparable discriminatory power to the standard 24 MIRU-VNTR loci set could be promising alternatives to the standard genotyping for using in resource-limited settings.

Prevalence and antimicrobial-resistant characterization of Bacillus cereus isolated from ready-to-eat rice products in Eastern China.

Bacillus cereus is a major food-borne bacterial pathogen in the world, which can cause diarrhea and emetic syndrome. This study aimed to reveal the quantitative prevalence of B. cereus in ready-to-eat (RTE) rice products in Eastern China and to gain essential information on the characteristics of B. cereus isolates. A total of 91 out of the 1071 samples were positive for B. cereus. The contamination level of B. cereus in 0.5 % of RTE rice product samples outnumbered 103 CFU/g. The number of B. cereus attained 105−106 CFU/g in one sample. The distribution patterns of virulence genes in B. cereus isolates were identified. 84.6% of the B. cereus isolates had at least one enterotoxin or emetic toxin gene. The predominant pattern was XXV. 9.9% of isolates belonged to it and possessed one enterotoxin gene entFM. The occurrence rate of hblACD and nheABC was 36.3% and 47.3%, respectively. Antimicrobial susceptibility tests revealed a high resistance rate toward penicillin, and 23.1% of the isolates were multi-drug resistant. B. cereus isolates were genotyped by using ERIC-PCR. 89 genotypes were determined. The Hunter Gaston Discriminatory Index (HGDI) attained 0.9995. Relationships analysis revealed that Group A B. cereus isolates tended to carry hblA, hblC, hblD, nheA, nheB, and show resistance to penicillin/trimethoprim/sulfamethoxazole. This study was useful for updating the knowledge of the contamination status of B. cereus in RTE rice products in China.

Developing customized stepwise MIRU-VNTR typing for tuberculosis surveillance in Georgia.

IntroductionMycobacterial Interspersed Repetitive Units–Variable Tandem Repeats (MIRU-VNTR) typing has been widely used for molecular epidemiological studies of tuberculosis (TB). However, genotyping tools for Mycobacterium tuberculosis (Mtb) may be limiting in some settings due to high cost and workload. In this study developed a customized stepwise MIRU-VNTR typing that prioritizes high discriminatory loci and validated this method using penitentiary system cohort in the country of Georgia.MethodsWe used a previously generated MIRU-VNTR dataset from recurrent TB cases (32 cases) in Georgia and a new dataset of TB cases from the penitentiary system (102 cases) recruited from 2014 to 2015. A Hunter-Gaston Discriminatory Index (HGDI) was calculated utilizing a 24 standard loci panel, to select high discriminatory power loci, subsequently defined as the customized Georgia-specific set of loci for initial typing. The remaining loci were scored and hierarchically grouped for second and third step typing of the cohort. We then compared the processing time and costs of the customized stepwise method to the standard 24-loci method.ResultsFor the customized Georgia-specific set that was used for initial typing, 10 loci were selected with a minimum value of 0.32 to the highest HGDI score locus. Customized 10 loci (step 1) typing of 102 Mtb patient isolates revealed 35.7% clustered cases. This proportion was reduced to 19.5% after hierarchical application of 2nd and 3rd step typing with the corresponding groups of loci. Our customized stepwise MIRU-VNTR genotyping approach reduced the quantity of samples to be typed and therefore overall processing time and costs by 42.6% each.ConclusionOur study shows that our customized stepwise MIRU-VNTR typing approach is a valid alternative of standard MIRI-VNTR typing panels for molecular epidemiological investigation in Georgia that saves time, workload and costs. Similar approaches could be developed for other settings.

Assessment of genotyping markers in the molecular characterization of a population of clinical isolates of Fusarium in Colombia.

:Introduction:Fusarium is a very heterogeneous group of fungi, difficult to classify, with a wide range of living styles, acting as saprophytes, parasites of plants, or pathogens for humans and animals. Prevalence of clinical fusariosis and lack of effective treatments have increased the interest in the precise diagnosis, which implies a molecular characterization of Fusarium populations.Objective: We compared different genotyping markers in their assessment of the genetic variability and molecular identification of clinical isolates of Fusarium.Materials and methods: We evaluated the performance of the fingerprinting produced by two random primers: M13, which amplifies a minisatellite sequence, and (GACA)4, which corresponds to a simple repetitive DNA sequence. Using the Flunter Gaston Discriminatory Index (HGDI), an analysis of molecular variance (AMOVA), and a Mantel test, the resolution of these markers was compared to the reference sequencing-based and PCR genotyping methods.Results: The highest HGDI value was associated with the M13 marker followed by (GACA)4. AMOVA and the Mantel tests supported a strong correlation between the M13 classification and the reference method given by the partial sequencing of the transcription elongation factor 1-alpha (TEF1-α) and rDNA 28S.Conclusion: The strong correlation between the M13 classification and the sequencing- based reference together with its higher resolution demonstrates its adequacy for the characterization of Fusarium populations.

Highly Discriminative Genotyping of Mycobacterium abscessus Complex Using a Set of Variable Number Tandem Repeats in China.

In this study, our aims were to comparatively analyze the power of variable number tandem repeat (VNTR) typing to discriminate isolates within subspecies and to identify a potential genetic marker for better molecular typing of Mycobacterium abscessus complex (MABC) strains. A total of 103 clinical MABC isolates were collected from a nationwide cross-sectional study in China. Eighteen VNTR loci were chosen to genotype the MABC isolates. Of the 103 clinical MABC isolates, there were 76 (73.8%) M. abscessus subsp. abscessus (MAA) and 27 (26.2%) M. abscessus subsp. massiliense (MAM) isolates. Among the patients with MAA lung diseases, the percentage of patients older than 45 years (67.1%) was significantly higher than that of patients with MAM lung diseases [33.3%, adjusted odds ratio (aOR) = 0.36, 95% CI = 0.13–0.98, p = 0.046]. Fifteen VNTR loci were designated as being “highly discriminant” in our sample, except for TR109. The total of 103 MABC isolates were fully discriminated into 103 unique patterns by an 18-locus VNTR set [Hunter–Gaston Discriminatory Index (HGDI) = 1.000], of which the inclusion of the top 12 loci yielded a comparative HGDI value (HGDI = 0.9998). Remarkably, the order of the diversity of the VNTR loci showed significant difference between the MAA and MAM isolates. TR137 and TR2, two loci with high diversity indices for the MAA isolates, only yielded poor discriminatory power for the MAM isolates; the allelic diversity (h) values were 0.0000 and 0.2621, respectively. A detailed analysis of TR137 in combination with the other 17 VNTR loci showed that the combination of TR137–TR2 could fully distinguish MAA from MAM isolates. In conclusion, our data revealed that MAA is more prone to affect elderly patients. Additionally, the population structure of the MABC isolates circulating in China has high diversity. The combined use of the TR137 and TR2 loci provides a simple criterion for the precise identification of MABC to the subspecies level.

Evaluation of the discriminatory power of spoligotyping and 19-locus mycobacterial interspersed repetitive unit-variable number of tandem repeat analysis (MIRU-VNTR) of Mycobacterium bovis strains isolated from cattle in Algeria.

Bovine tuberculosis (bTB) caused by Mycobacterium (M.) bovis and M. caprae is a transmissible disease of livestock, notifiable to the World Organization for Animal Health (OIE). BTB particularly affects cattle and small ruminants and can be transmitted to humans thereby posing a significant threat to veterinary and public health worldwide. M. bovis is the principal cause of bTB in Algeria. In order to better understand the route of spreading and elaborate an eradication program, isolation and characterization of mycobacteria from Algerian cattle was performed. Sixty strains belonging to the M. tuberculosis complex were analyzed by spoligotyping, thereof 42 by 19-locus-MIRU-VNTR-typing. Spoligotyping revealed 16 distinguishable patterns (Hunter-Gaston discriminatory index [HGDI] of 0.8294), with types SB0120 (n = 20) and SB0121 (n = 13) being the most frequent patterns, representing 55% of the strains. Analyses based on 19-locus-MIRU-VNTR yielded 32 different profiles, five clusters and one orphan pattern, showing higher discriminatory power (HGDI = 0.9779) than spoligotyping. Seven VNTR-loci [VNTR 577 (alias ETR C), 2163b (QU11b), 2165 (ETR A), 2461 (ETR B), 3007 (MIRU 27), 2163a (QUB11a) and 3232 (QUB 3232)] were the most discriminative loci (HGDI ˃ 0.50). In conclusion, 19-locus-MIRU-VNTR yielded more information than spoligotyping concerning molecular differentiation of strains and better supports the elucidation of transmission routes of M. bovis between Algerian cattle herds.

A Prospective Cohort Study on the Prevalent and Recurrent Tuberculosis Isolates Using the MIRU-VNTR Typing.

The study aims to describe the clustering characteristics of Mycobacterium tuberculosis (M.tb) strains circulating in eastern China and determine the ratio of relapse and reinfection in recurrent patients. We recruited sputum smear-positive pulmonary tuberculosis cases from five cities of Jiangsu Province, China, during August 2013 and December 2015. Patients were followed for the treatment outcomes and recurrence based on a cohort design. M.tb strains were isolated and genotyped using the 12-locus MIRU-VNTR. The Beijing family was identified by the extended Region of Difference (RD) analysis. The Hunter-Gaston Discriminatory Index (HGDI) was used to judge the resolution ability of MIRU-VNTR. The odds ratio (OR) together with 95% confidence interval (CI) were used to estimate the strength of association. We performed a cluster analysis on 2098 M.tb isolates and classified them into 545 genotypes and five categories (I, 0.19%; II, 0.43%; III, 3.34%; IV, 77.46%; V, 18.59%). After adjusting for potential confounders, the Beijing family genotype (OR = 118.63, 95% CI: 79.61–176.79, P = 0.001) was significantly related to the dominant strain infections. Patients infected with non-dominant strains had a higher risk of the pulmonary cavity (OR = 1.39, 95% CI: 1.01–1.91, P = 0.046). Among 37 paired recurrent cases, 22 (59.46%) were determined as endogenous reactivation, and 15 (40.54%) were exogenous reinfection. The type of M.tb strains prevalent in Jiangsu Province is relatively single. Beijing family strains infection is dominant in local tuberculosis cases. Endogenous reactivation appears to be a major cause of recurrent tuberculosis in Eastern China. This finding emphasizes the importance of case follow-up and monitoring after the completion of antituberculosis treatment.

Multispacer Sequence Typing for Mycobacterium bovis Genotyping.

The molecular typing of Mycobacterium bovis, which causes bovine tuberculosis, can be accomplished by combining different polymorphic markers, contributing to its epidemiological investigation. Multispacer sequence typing (MST) is a sequencing-based method that employs intergenic regions susceptible to higher mutation rates given the low selection pressure. It has been applied to M. tuberculosis, but not to M. bovis. The aim of this study was to evaluate a MST for M. bovis. A total of 58 strains isolated from tissues with lesions suggestive of bovine tuberculosis, coming from cattle herds in six Brazilian states and four standard samples of M. bovis were typified employing the MST technique. Fourteen intergenic regions were used, and four types of genetic events were reported: single nucleotide mutation (SNP), insertion, deletion, and tandem repeat (TR). Seven loci were chosen for typing. Twenty-eight type sequences (ST) were identified, indicating type sequences (ST) were identified, indicating a 92.9% HGDI (Hunter Gaston Discriminatory Index). The data were used to analyze the evolutionary patterns of these isolates and correlate them to phylogeographic lineages based on the formation of clonal complexes generated from eBURST software. Later, we associated the MST with spoligotyping technique, currently considered the gold standard for classification of M. bovis. The results support the MST as an alternative method for genotyping of M. bovis. The method has the advantage of sequencing and the availability of sequences analyzed in public databases, which can be used by professionals around the world as a tool for further analysis. This was the first study to identify the variability of isolates of M. bovis by the MST method.

Genetic diversity of Mycobacterium bovis evaluated by spoligotyping and MIRU‐VNTR in an intensive dairy cattle breeding area in Mexico

Bovine tuberculosis (bTB) is mainly caused by Mycobacterium bovis. In Mexico, dairy cattle play an important role in the persistence and spread of the bacillus. In order to describe M.bovis genetic diversity, we genotyped a total of 132 strains isolated from slaughtered cattle with bTB suggestive lesions between 2009 and 2010 in Hidalgo, Mexico, using a panel of 9-loci mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTR) and spoligotyping. We found 21 spoligotypes, and 124 isolates were grouped in 13 clusters. The most frequent spoligotypes were SB0121 (49, 37.1%) and SB0673 (27, 20.5%); three new spoligotypes were reported SB02703, SB02704 and SB02705. We observed 37 MIRU-VNTR patterns, 107 isolates were grouped in 12 clusters and 25 isolates were unique. Spoligotypes SB0121, SB0673, SB0140, SB0145 and SB0120 showed marked subdivision applying MIRU-VNTR method; meanwhile, spoligotypes SB0971 and SB0327 showed single MIRU-VNTR profiles. The Hunter-Gaston discriminatory index (HGDI) was 0.88, 0.78 and 0.90 for 9-loci MIRU-VNTR, spoligotyping and both methods, respectively. Additionally, allelic diversity (h) analysis showed high diversity for QUB3232, QUB26 and QUB11b with h=0.79, 0.66 and 0.63, respectively. Overall, high genetic variability was observed among M.bovis isolates. Thus, the use of 9-loci MIRU-VNTR panel is enough to describe genetic diversity, evolution and distribution of M.bovis. This study supports the use of these tools for subsequent epidemiological studies in high incidence areas.

Molecular Epidemiology of Xanthomonas euvesicatoria Strains from the Balkan Peninsula Revealed by a New Multiple-Locus Variable-Number Tandem-Repeat Analysis Scheme.

Bacterial spot of pepper and tomato is caused by at least three species of Xanthomonas, among them two pathovars of Xanthomonas euvesicatoria, which are responsible for significant yield losses on all continents. In order to trace back the spread of bacterial spot pathogens within and among countries, we developed the first multilocus variable number of tandem repeat analyses (MLVA) scheme for pepper- and tomato-pathogenic strains of X. euvesicatoria. In this work, we assessed the repeat numbers by DNA sequencing of 16 tandem repeat loci and applied this new tool to analyse a representative set of 88 X. euvesicatoria pepper strains from Bulgaria and North Macedonia. The MLVA-16 scheme resulted in a Hunter–Gaston Discriminatory Index (HGDI) score of 0.944 and allowed to resolve 36 MLVA haplotypes (MTs), thus demonstrating its suitability for high-resolution molecular typing. Strains from the different regions of Bulgaria and North Macedonia were found to be widespread in genetically distant clonal complexes or singletons. Sequence types of the variable number of tandem repeats (VNTR) amplicons revealed cases of size homoplasy and suggested the coexistence of different populations and different introduction events. The large geographical distribution of MTs and the existence of epidemiologically closely related strains in different regions and countries suggest long dispersal of strains on pepper in this area.

Genotypic diversity of Mycobacterium tuberculosis isolates and its association with drug-resistance status in Xinjiang, China

Xinjiang is a unique region of Central Asian part of China. It is widely noted for high tuberculosis burden and particularly for growing prevalence of drug resistance. Understanding genotypic distribution of Mycobacterium tuberculosis could help clarify unknown causes for the spread of drug-resistant strains. We analyzed 986 M. tuberculosis isolates collected from Xinjiang. Two genotyping schemes, i.e., spoligotyping and multiple-locus variable number tandem repeats (VNTR), were used to determine the phylogenetic lineages and their association with drug-resistances. The M. tuberculosis isolates studied displayed wide distribution of spoligotypic lineages, including Beijing, T, CAS, Ural, LAM, MANU, H, X, EAI, S, Microti, and BOV. The dominant Beijing lineage showed statistical difference from non-Beijing lineages in patients ages (P < 0.001), ethnic groups (P < 0.001) and resistance of three or more drugs (P = 0.008). Further analysis of the year of 2017 subset (n = 257) using VNTR scheme revealed an extremely high discrimination power (Hunter-Gaston discriminatory index = 0.9994). Cluster analysis showed a much lower recent transmission index (7.93%), indicating that the high drug-resistant tuberculosis in this region was mainly caused by reactivation or inappropriate therapy rather than by recent transmission. These data would be valuable for making and implementing policies for improving tuberculosis treatment and care in Xinjiang.

Molecular and genetic analysis of &lt;i&gt;Mycobacterium tuberculosis&lt;/i&gt; population in the Vologda Region with low tuberculosis incidence

The Vologda Region is characterized by a relatively calm epidemic situation for tuberculosis in Russia: the incidence rate in 2010—2018 is decreased from 45.2 to 15.8 per 100 thousand of the population (44.4 in Russia). However, the proportion of patients with multiple drug resistance (MDR) of the pathogen increased from 12.1% in 2016 to 23.7% in 2018. The aim of the study was to characterize the genetic structure of the M. tuberculosis population and identify the main genotypes associated with the primary multidrug resistance of the pathogen in the Vologda Region. A total of 82 strains of M. tuberculosis isolated in 2018 from newly diagnosed tuberculosis patients were studied. Drug susceptibility testing was performed using the standard method of absolute concentration and BACTEC MGIT 960 kit. M. tuberculosis strains were assigned to the Beijing genotype and its main subtypes based on the analysis of specific markers. The Beijing strains were subtyped by the MIRU-VNTR method (24 standard loci), calculating the Hunter-Gaston Discriminatory Index (HGDI). Other strains of the non-Beijing group were spoligotyped. The majority of the strains were of the Beijing genotype (62.2%; 51 of 82). The most numerous cluster was Central Asian/Russian (41.5%; 34 of 82 strains). The shares of the Central Asia Outbreak (CAO) subtype and cluster B0/W148 amounted to 8.5% and 7.3%, respectively. The non-Beijing strains belonged to the genetic families T (11%; 9 of 82), LAM (11%), Haarlem (6.1%), and Ural (4.9%). Among 82 M. tuberculosis isolates, 33 (40.2%) MDR strains were identified, counting 27 of the Beijing genotype, including those of the Central Asian/Russian — 18 (66.7%), B0/W148 and CAO — 4 each (14.8%) clusters. MIRU-VNTR typing of 51 Beijing strains revealed 22 profiles (HGDI = 0.852); the largest clusters were 94-32 (35.3%) and 95-32 (15.7%), which included strains Central Asian/Russian and CAO. Four strains of genotype B0/W148 belonged to cluster 100-32. The loci QUB26 (HGDI = 0.493) and MIRU26 (HGDI = 0.388) had the highest polymorphism. For the first time, a molecular genetic study carried out in the Vologda region revealed the heterogeneity of the M. tuberculosis population with strains of the Beijing genotype dominated. At the same time, the share of the associated with MDR, epidemiologically and clinically significant cluster Beijing B0/W148, well defined in Russia and abroad, was only 7.3%, which is significantly less than in other regions of the Northwestern Federal District of the Russian Federation (~19%). Concurrent, representatives of the Central Asian/Russian cluster of the Beijing genotype prevailed in the structure of genotypes and among MDR M. tuberculosis strains.

Molecular Epidemiologic Analysis of Mycobacterium tuberculosis among Prison Inmates in Selected Prisons in the Philippines

Tuberculosis (TB) remains an important public health problem in developing countries like the Philippines. The success of the National TB Control Program depends on a clear understanding of the dynamics of transmission and spread of TB in high-risk populations in the community. We conducted a molecular epidemiologic analysis of M tuberculosis isolates collected from inmates with pulmonary TB in selected prisons in the Philippines. A total of 25 isolates were characterized and genotyped using Spoligotyping and 15-loci MIRU-VNTR (mycobacterial interspersed repetitive units–variable number of tandem repeats) typing. The majority of the patients were male (84%) and aged 30–49 yr old (68%). Eighteen (72%) of the culture-positive patients had severe pulmonary TB, 13 (52%) were smear-positive, and seven (28%) were classified as having a high bacillary load. Twenty isolates (80%) were susceptible to all the first-line drugs. Two (8%) were multidrug- resistant (MDR) and isolated from patients in the same prison, one of which was resistant to all first-line drugs. Three isolates (12%) were streptomycin-monoresistant. There were nine identified Spoligo-International Types (SITs), with SIT19 as the predominant (40%). One isolate (4%) did not match any SIT in the SpoIDB4 database, while three were not assessed due to inadequate DNA for analysis. The distribution of strains according to major M tuberculosis clades were as follows: EAI2_Manilla (48%) &gt; LAM2 (20%) &gt; LAM6 (8%) = LAM9 (8%). Spoligotyping identified two clusters and 13 genotypes (four unique strains) with a Hunter-Gaston discriminatory index (HGDI) of 0.83. MIRU-VNTR typing identified two clusters and 23 genotypes (HGDI = 0.993). Combined Spoligotyping and MIRU-VNTR typing also identified two clusters and 23 genotypes (HGDI = 0.993). There were no significant associations shown among host demographic factors, severity of the disease, drug resistance, and M tuberculosis strain. We conclude that our patient population was infected predominantly by M tuberculosis belonging to the EAI2_Manila clade

Genotypes of Mycobacterium tuberculosis isolates circulating in Shaanxi Province, China.

ObjectivesThe prevalence of drug-resistant TB in Shaanxi Province is higher than other areas. This study was aimed to investigate the genetic diversity and epidemiology of Mycobacterium tuberculosis clinical strains in Shaanxi Province, China.MethodsFrom January to December 2016, a total of 298 Mycobacterium tuberculosis clinical isolates from smear-positive pulmonary tuberculosis patients were genotyped by Mcspoligotyping and 15-locus VNTR.ResultsWe found that the Beijing family strains was the most prominent family(81.54%, 243/298). Other family strains included T family(9.06%, 27/298), U family(0.67%, 2/298), LAM9 family(0.34%, 1/298) and Manu family(0.34%, 1/298). The rates of multidrug-resistant (MDR) M.Tuberculosis, age, type of case and education between Beijing and non-Beijing family strains were not statistically different, while the distribution in the three different regions among these was statistically significant.VNTR results showed that strains were classified into 280 genotypes, and 33 (11.07%) strains could be grouped into 14 clusters. 11 of the 15-VNTR loci were highly or moderately discriminative according to the Hunter-Gaston discriminatory index.ConclusionsWe concluded that the Beijing family genotype was the most prevalent genotype and 15-locus VNTR typing might be suitable for genotyping of M. tuberculosis in Shaanxi Province. There was less association between Beijing family genotypes and drug resistance in our study area.

Typing of ΦSP–3 lytic Salmonella bacteriophages obtained from various fecal sources

Although several reports are available on both ΦSP–1 and ΦSP–3 lytic Salmonella bacteriophages obtained from poultry, further research is required to study the effectiveness of ΦSP–3 type on serovars isolated from other sources. In the present study, we aimed to isolate bacteriophages from 8 serovars previously obtained from 869 fecal samples (calf, dairy cow, buffalo, and camel), genotype the bacteriophages, and detect the cross-lytic activities of the bacteriophages on Salmonella enterica subsp. enterica serovar Kentucky, S.Anatum, and S.Muenchen. A total of 16 bacteriophages were detected as ΦSP–3 type via PCR. The Hunter-Gaston Discriminatory Index of SDS-PAGE was calculated to be 0.825. Determination of multiplicity of infection (MOI) values were different for each bacteriophage according to the cross-lytic activity assessment. The MOI of the most effective S. Kentucky bacteriophage was 79.11 μg/mL for 2.5×104 cells, whereas that of the most ineffective S.Muenchen bacteriophage was 1.142 μg/μL for 2.5×104 cells. In conclusion, it was assumed that owing to the high and cross-lytic activity of the S. Kentucky bacteriophage, it has a larger host range, which differs in the lytic activities of each bacteriophage, despite being the same serovar, and that calf feces is the most important source for obtaining Salmonella bacteriophages.

Comparative Evaluation of Band-Based Genotyping Methods for Mycobacterium intracellulare and Its Application for Epidemiological Analysis.

Mycobacterium intracellulare is a leading cause of nontuberculous mycobacterial pulmonary disease, with a rapidly increasing prevalence worldwide. This bacterium, commonly distributed in soil and water, is known to be transmitted through the environment rather than between people. Therefore, it is imperative to establish distinguishable genotyping methods to understand the clinical outcome, disease relapses, and epidemiology. Therefore, in this study, representative band-based genotyping methods were performed using M. intracellualre clinical isolates, and their Hunter–Gaston discriminatory index (HGDI) was 0.947, 0.994, and 1 for variable number tandem repetition (VNTR), VNTR-mycobacterial interspersed repetitive units, pulsed field gel electrophoresis, and repetitive sequence based-PCR, respectively. Although VNTR showed relatively low HGDI, co-infection with other M. intracellualre strains could be determined by loci showing allele diversity from 0 to 0.69. Additionally, genetic distance of clinical isolates from Gyeongnam/Korea, and other regions/countries were visualized by minimum spanning tree (MST) using the globally available VNTR profiles. The results of MST revealed that M. intracellulare isolated from patients in Gyeongnam/Korea had specific VNTR genotypes, which may be evidence of the geographic distribution of M. intracellulare specific genotypes. The comparative results of genotyping techniques and geographical characteristics in this study may provide fundamental information for the epidemiology of M. intracellulare.

Enhancing the differentiation of specific genotypes in Mycobacterium tuberculosis population

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