Repetitive Unit-variable Number Tandem Repeat Research Articles

Spoligotyping analysis of Mycobacterium tuberculosis in Khyber Pakhtunkhwa area, Pakistan.

Background: Spoligotyping is a reproducible, reverse hybridization approach for genotyping of Mycobacterium tuberculosis complex (MTBC). Molecular typing of MTBC is helpful for understanding and controlling tuberculosis epidemics.Methods: Spoligotyping was performed on 166 clinical isolates of Mycobacterium tuberculosis (MTB) collected from 25 districts of Khyber Pakhtunkhwa, Pakistan. Results were compared to SITVIT2, an online database developed by the Institut Pasteur de la Guadeloupe, France.Results: Spoligotyping results showed that 145 strains (88%) displayed known patterns while 21 (12%) were new. Lineage 3/Central Asian strain (L3/CAS) was the predominant family (73%, χ2=19.9, P=0.001), followed by L2/Beijing (5.4%) and L4 (4.2%). L3/CAS1-Delhi was the major sublineage (82%) among the L3/CAS family (χ2=664, P=0.0001). Analysis showed that the majority of the clinical isolates with an unknown pattern had an evolutionary link with the L3/CAS strain, and nine (5.4%) of the unknown strains were epidemiologically linked and were tentatively named L3/CAS-KP (Khyber Pakhtunkhwa).Conclusion: The present study demonstrated that L3/CAS is the predominant lineage of MTB, widely distributed in different areas of the Khyber Pakhtunkhwa province of Pakistan. Spoligotyping patterns of some clinical isolates could not be matched to other reported patterns in an international database. Other tools, such as mycobacterial interspersed repetitive unit–variable number tandem repeat (MIRU-VNTR), will be helpful in future investigations into the epidemiological characteristics of clinical isolates in the Khyber Pakhtunkhwa province.

Infectivity of Mycobacterium tuberculosis Genotypes and Outcome of Contact Investigation in Classroom in Guangxi, China.

Objective To evaluate the infectivity of Mycobacterium tuberculosis (M.tb) genotypes of index cases in the classroom of adolescent schools in Guangxi, China. Methods Adolescent school tuberculosis (TB) contact investigations were conducted for all reported index TB cases from November 2016 to December 2017 in Guangxi, China. Genotypes of index cases and contact cases were identified by 15-loci mycobacterial interspersed repetitive units–variable number tandem repeat and spoligotyping. Outcome variable was 5 levels' order of tuberculin skin test (TST) results to new active TB [0-5 mm, 6-9 mm, 10-14 mm, ≥ 15 mm (without TB), and ≥15 mm (with TB)]. Multivariate ordered logistic regression analysis was performed to evaluate the independent effect of genotypes of index case on contact screening outcome. Results Beijing genotype occurred more commonly in female index patients. One genotypic cluster of two index cases and one cluster of two contact cases were detected. The association between infectivity of Beijing genotype of index cases and outcome of contact investigation was statistically significant in univariate analysis but no so after adjustment for characteristics of contacts and sex of index cases (P value=0.057). Female index cases increased the chance for TB infection/being active TB among contacts (ordinal odds ratio = 1.39, 95% confidence interval: 1.21, 1.60). Contacts who studied in the middle school, who with non-Han ethnicity and who without BCG scar had increased risk for TB infection/being active TB. Conclusion There was not enough evidence from our data to support that Beijing strains were more infective than non-Beijing strains in TB transmission in school setting.

A first insight into the application of high discriminatory MIRU-VNTR typing using QIAxcel technology for genotyping Mycobacterium bovis isolated from the Delta area in Egypt

Mycobacterium bovis is a notorious infectious agent leading to serious economic losses for cattle farms worldwide. Analysis of the widely spreading genotypes is vital for tracing infections, understanding transmission dynamics, and controlling the cluster growth. This study aimed to evaluate the discrimination ability of 15 mycobacterial interspersed repetitive unit-variable number tandem repeats (MIRU-VNTR) loci and to assess the extremely efficient loci subset for molecular epidemiological investigations of M.bovis from farms in the Delta area of Egypt. The discriminating ability of MIRU-VNTR genotyping using 15 loci {2 exact tandem repeat (ETR) loci, 6 MIRU loci, 4 Mtub loci, and 3 Queen's University of Belfast (QUB) group loci)} were evaluated on 61 M.bovis isolates from cattle (Holstein Frisian) and buffaloes. The results indicate that there are 48 genotypes: 3 unique genotypes and 45 genotypes with shared similarities. Using the MIRU-VNTRplus database, M.bovis ID 7540/01 and ID 5346/02 were the nearest lineages to both groups. Six loci (MIRU10, QUB11b, QUB26, ETRA, Mtub30, and Mtub39) were highly discriminating, seven other loci (Mtub21, MIRU26, QUB4156, MIRU04 (ETRD), MIRU16, MIRU 40, and ETRC) gave moderate discriminatory power, and the last two loci (Mtub04 and MIRU31) were poorly discriminative. MIRU-VNTR typing generally proved efficacy and high discriminatory power, with a collective allele's diversification of 0.9641. Both the six highly discriminating (DI = 0.9492) and the seven moderately discriminating loci (DI = 0.9269) evidenced to be suitable for M.bovis first-step initial genotyping from cattle herds in Egypt. MIRU-VNTR is rapid and effective in the genotyping of M.bovis from cattle and buffaloes in Egypt.

Transmission dynamics study of tuberculosis isolates with whole genome sequencing in southern Sweden

Epidemiological contact tracing complemented with genotyping of clinical Mycobacterium tuberculosis isolates is important for understanding disease transmission. In Sweden, tuberculosis (TB) is mostly reported in migrant and homeless where epidemiologic contact tracing could pose a problem. This study compared epidemiologic linking with genotyping in a low burden country. Mycobacterium tuberculosis isolates (n = 93) collected at Scania University Hospital in Southern Sweden were analysed with the standard genotyping method mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTR) and the results were compared with whole genome sequencing (WGS). Using a maximum of twelve single nucleotide polymorphisms (SNPs) as the upper threshold of genomic relatedness noted among hosts, we identified 18 clusters with WGS comprising 52 patients with overall pairwise genetic maximum distances ranging from zero to nine SNPs. MIRU-VNTR and WGS clustered the same isolates, although the distribution differed depending on MIRU-VNTR limitations. Both genotyping techniques identified clusters where epidemiologic linking was insufficient, although WGS had higher correlation with epidemiologic data. To summarize, WGS provided better resolution of transmission than MIRU-VNTR in a setting with low TB incidence. WGS predicted epidemiologic links better which could consolidate and correct the epidemiologically linked cases, avoiding thus false clustering.

Prevalence, Drug Resistance, and Genotypic Diversity of the Mycobacterium tuberculosis Beijing Family in Ecuador.

The Beijing family, the most successful Mycobacterium tuberculosis lineage, is considered hypervirulent, associated with clustering and has a strong association with multidrug-resistant tuberculosis. The Beijing strains have spread worldwide and also to Latin America. Genotyping of a countrywide collection of 380 M. tuberculosis strains from Ecuador, with 24-loci mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTR), revealed only six Beijing strains, but four of these were MDR-TB. There was no clustering as all six strains had very distinct MIRU-VNTR profiles that have not been reported in the rest of Latin America. Although active transmission for Beijing has been described for the neighboring countries Peru and Colombia, there is no evidence that Beijing strains in Ecuador are more frequently transmitted than other strains. Moreover, the low prevalence (1.6%) of the Beijing sublineage in Ecuador challenges the concept of hyperadaptability and transmissibility of the Beijing strains in our country.

Simplified Model to Survey Tuberculosis Transmission in Countries Without Systematic Molecular Epidemiology Programs.

Systematic molecular/genomic epidemiology studies for tuberculosis surveillance cannot be implemented in many countries. We selected Panama as a model for an alternative strategy. Mycobacterial interspersed repetitive unit–variable-number tandem-repeat (MIRU-VNTR) analysis revealed a high proportion (50%) of Mycobacterium tuberculosis isolates included in 6 clusters (A–F) in 2 provinces (Panama and Colon). Cluster A corresponded to the Beijing sublineage. Whole-genome sequencing (WGS) differentiated clusters due to active recent transmission, with low single-nucleotide polymorphism–based diversity (cluster C), from clusters involving long-term prevalent strains with higher diversity (clusters A, B). Prospective application in Panama of 3 tailored strain–specific PCRs targeting marker single-nucleotide polymorphisms identified from WGS data revealed that 31.4% of incident cases involved strains A–C and that the Beijing strain was highly represented and restricted mainly to Colon. Rational integration of MIRU-VNTR, WGS, and tailored strain–specific PCRs could be a new model for tuberculosis surveillance in countries without molecular/genomic epidemiology programs.

Genetic diversity of Mycobacterium tuberculosis complex strains isolated from livestock workers and cattle in Nigeria

Molecular typing techniques are useful in understanding tuberculosis epidemiology; yet, they have been under-utilised at the human-animal interface in Nigeria. Sixty-four Mycobacterium tuberculosis complex (MTBC) isolates including 42 M. tuberculosis, 13 M. bovis and nine M. africanum obtained from livestock workers (LW, n = 47) and their cattle (n = 17) in three geographical zones of Nigeria were genotyped to identify and evaluate the genetic diversity of the circulating MTBC using spoligotyping. Distribution into clades of M. tuberculosis revealed; 45.3% Uganda I- [SIT46- cattle: 1; LW: 28], 14.1% Latin American Mediterranean- [SIT61, cattle: 1; LW: 8], and 1.6% T- [SIT53—LW: 1]. The M. bovis strains were 6.3% SB0944 [cattle: 4] and 1.6% each of SB0300, SB1026, SB1027 and SB1439 [cattle: 4]. Seventeen MTBC isolates [cattle: 7; LW: 10] yielded 14 new spoligotype patterns including three M. tuberculosis strains (three isolates), five M. bovis strains (five isolates) and six M. africanum strains (nine isolates), two of which belonged to MAF1. Only few families namely, the not previously described Uganda I-, LAM and SB0944 are predominant among the LW and cattle, with other types in lower prevalences. The strain population structure indicates an intriguing diversity and possible zoonotic linkage with consequences for TB control in the country. The need to employ newer molecular techniques such as Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeats and whole genome sequence to decipher circulating MTBC strains in Nigeria is advocated.

Evaluation of the 15 and 24-loci MIRU-VNTR genotyping tools with spoligotyping in the identification of Mycobacterium tuberculosis strains and their genetic diversity in molecular epidemiology studies

Background: The transmission dynamics of Mycobacterium tuberculosis (Mtb) using various genotyping tools has been studied globally and a particular tool for genotyping Mtb is the mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTR). Tuberculosis (TB) remains an important public health problem worldwide and Sri Lanka being a country of tourist destination; because of major development projects undergoing, it has a high proportion of tourists and immigrants from Asia and Europe that are characterized with highest TB incidences and drug-resistant clinical isolates. Hence, in order to address the question of Mtb genetic diversity, we investigated the discriminatory power of both MIRU-VNTR typing of 15 and 24 loci with spoligotyping to differentiate Mtb isolates.Method: Acid-fast bacilli positive sputum samples (n = 150) from first visit patients were collected. Decontamination of sputum and extraction of genomic DNA were carried out using standard techniques. The isolates were characterized by MIRU-VNTR for both the 15 and 24 loci and spoligotyping.Results: In our study population, MIRU-VNTR 15 and 24 loci did not show a significant difference among the identified M. tuberculosis strains. However, MIRU 24 loci yielded an additional strain LAM, which is of T1 origin. 15 loci strain grouping had more clusters of strains grouped together while 24 loci differentiated the same cluster of strains into distinct strain types.Conclusion: We conclude that the use of 15-locus MIRU-VNTR typing is sufficient for a first-line epidemiological study to genotype M. tuberculosis, but the additional discriminatory power of 24 loci MIRU-VNTR has been able to differentiate samples within highly homologous groups.

Whole genome sequencing-based analysis of tuberculosis (TB) in migrants: rapid tools for cross-border surveillance and to distinguish between recent transmission in the host country and new importations.

BackgroundThe analysis of transmission of tuberculosis (TB) is challenging in areas with a large migrant population. Standard genotyping may fail to differentiate transmission within the host country from new importations, which is key from an epidemiological perspective.AimTo propose a new strategy to simplify and optimise cross-border surveillance of tuberculosis and to distinguish between recent transmission in the host country and new importationsMethodsWe selected 10 clusters, defined by 24-locus mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR), from a population in Spain rich in migrants from eastern Europe, north Africa and west Africa and reanalysed 66 isolates by whole-genome sequencing (WGS). A multiplex-allele-specific PCR was designed to target strain-specific marker single nucleotide polymorphisms (SNPs), identified from WGS data, to optimise the surveillance of the most complex cluster.ResultsIn five of 10 clusters not all isolates showed the short genetic distances expected for recent transmission and revealed a higher number of SNPs, thus suggesting independent importations of prevalent strains in the country of origin. In the most complex cluster, rich in Moroccan cases, a multiplex allele-specific oligonucleotide-PCR (ASO-PCR) targeting the marker SNPs for the transmission subcluster enabled us to prospectively identify new secondary cases. The ASO-PCR-based strategy was transferred and applied in Morocco, demonstrating that the strain was prevalent in the country.ConclusionWe provide a new model for optimising the analysis of cross-border surveillance of TB transmission in the scenario of global migration.

Mycobacterium tuberculosis transmission in an ethnically-diverse high incidence region in England, 2007\u201311

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

Genotyping of swine Mycobacterium avium subsp. hominissuis isolates from Kyushu, Japan.

The incidence of diseases caused by nontuberculous mycobacteria (NTM) is increasing annually worldwide, including Japan. Mycobacterium avium subsp. hoiminissuis (MAH) is one of the most common NTM species responsible for chronic lung diseases in animals and humans. In the current study, mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing was employed to characterize the genetic diversity of swine MAH isolates from Kyushu, Japan. In total, 309 isolates were obtained from the lymph nodes of 107 pigs not displaying any clinical signs of disease, of which 307 were identified as MAH, comprising 173 strains. Based on eight established MIRU-VNTR loci, the MAH strains represented 50 genotypes constituting three lineages, and 29 had not been described in the Mac French National Institute for Agricultural Research Nouzilly MIRU-VNTR (Mac-INMV) database. MAH was the dominant M. avium complex (MAC) in pigs from Kyushu, and there was high genetic diversity among genotype profiles of MAH from Kyushu. We identified three predominant genotype profiles in the tested area sharing high relatedness with genotype profiles of strains isolated in European countries. MAH was the most common NTM in pigs from Kyushu and exhibited high diversity, with new strain-derived genotypes.

Genotypic analysis of Mycobacterium tuberculosis strains collected from He′nan Province by 24-locus variable-number tandem-repeat typing and spoligotyping

Objective To investigate the genotype of M. tuberculosis in He′nan Province. Methods A total of 668 M. tuberculosis clinical strains collected in difference regions of He′nan Province during 2015 were genotyped by two standard methods, including classical 24-locus mycobacterium interspersed repetitive unit variable-number tandem-repeat (MIRU-VNTR) typing and spoligotyping. Results The 668 isolates were divided into 11 clusters and 35 patterns by spoligotyping. Among the 558 Beijing strains, 546 were typical Beijing strains and the other 12 were atypical Beijing strains. Among the 110 non-Beijing strains, eight were new strains and the remaining 102 non-Beijing strains were divided into 10 families. There were 76 isolates belonging to T family, including 59 of T1 families, 7 of T2 families, and 10 of T3 families. The 668 strains were divided into 550 gene patterns by standard 24-locus VNTR, including 508 un-clustered patterns and 160 clustered into 42 clusters. The largest cluster contained 21 strains, the other clusters contained 2-20 strains. Conclusion Beijing strain is still the most prevalent M. tuberculosis in He′nan Province. Key words: Mycobacterium tuberculosis; 24-locus mycobacterium interspersed repetitive unit-variable number tandem repeat; Spoligotyping

Genomic sequencing is required for identification of tuberculosis transmission in Hawaii

BackgroundTuberculosis (TB) caused an estimated 1.4 million deaths and 10.4 million new cases globally in 2015. TB rates in the United States continue to steadily decline, yet rates in the State of Hawaii are perennially among the highest in the nation due to a continuous influx of immigrants from the Western Pacific and Asia. TB in Hawaii is composed of a unique distribution of genetic lineages, with the Beijing and Manila families of Mycobacterium tuberculosis (Mtb) comprising over two-thirds of TB cases. Standard fingerprinting methods (spoligotyping plus 24-loci Mycobacterial Interspersed Repetitive Units-Variable Number Tandem Repeats [MIRU-VNTR] fingerprinting) perform poorly when used to identify actual transmission clusters composed of isolates from these two families. Those typing methods typically group isolates from these families into large clusters of non-linked isolates with identical fingerprints. Next-generation whole-genome sequencing (WGS) provides a new tool for molecular epidemiology that can resolve clusters of isolates with identical spoligotyping and MIRU-VNTR fingerprints.MethodsWe performed WGS and SNP analysis and evaluated epidemiological data to investigate 19 apparent TB transmission clusters in Hawaii from 2003 to 2017 in order to assess WGS’ ability to resolve putative Mtb clusters from the Beijing and Manila families. This project additionally investigated MIRU-VNTR allele prevalence to determine why standard Mtb fingerprinting fails to usefully distinguish actual transmission clusters from these two Mtb families.ResultsWGS excluded transmission events in seven of these putative clusters, confirmed transmission in eight, and identified both transmission-linked and non-linked isolates in four. For epidemiologically identified clusters, while the sensitivity of MIRU-VNTR fingerprinting for identifying actual transmission clusters was found to be 100%, its specificity was only 28.6% relative to WGS. We identified that the Beijing and Manila families’ significantly lower Shannon evenness of MIRU-VNTR allele distributions than lineage 4 was the cause of standard fingerprinting’s poor performance when identifying transmission in Beijing and Manila family clusters.ConclusionsThis study demonstrated that WGS is necessary for epidemiological investigation of TB in Hawaii and the Pacific.

Genetic diversity of drug resistant Mycobacterium Tuberculosis in local area of Southwest China: a retrospective study

BackgroundBy 2014 although tuberculosis (TB) incidence had fallen by an average of 1.5% per year since 2000 and was 18% lower than the level of in 2000, 1.5 million people died for TB in that year. One of reason was that drug resistant Mycobacterium tuberculosis (DRTB) spread. This study aims to determine drug resistant characteristics and genotype of DRTB that isolated from patients in a tuberculosis referral hospital of southwest China.MethodsFive hundred thirty-eight drug resistant tuberculosis samples were collected from July 2013 to March 2015. All the isolates were identified by genomic deletions in region of difference 105 (RD105) and genotyped by mycobacterial interspersed repetitive unit-variable number tandem repeat typing (MIRU-VNTR). Polymorphism and cluster analysis of each locus was carried out using Bionumerics Version 3.0 and phyloviz software.ResultsFive hundred thirty-eight TB strains included 503 Mycobacterium tuberculosis (MTB) isolates and 35 non Mycobacterium tuberculosis (NMTB) isolates. Of 503 isolates Beijing family type was 447 (88.9%, 447/503) and non-Beijing family type was 56 (11.1%, 56/503). Five hundred three DRTB isolates were divided into 345 genotypes, of which 265 isolates were single genotype and the remaining 238 strains were classified into 80 clusters with cluster rate of 47.3% and cluster ratio of 31.4%. Sixty-nine clusters belonged to Beijing family with cluster rate was 48.3% and clustering ratio was 32.9%. The non - Beijing family had 11 clusters with a cluster rate of 39.3% and the clustering ratio of 19.6%. Beijing genotype had a significant correlation with the age (P < 0.05), the retreatment patients (P < 0.05) and the city of Chongqing (P < 0.05), not with gender (P > 0.05). In the 9 Beijing genotype clusters each cluster contained some patients who lived in the same region.ConclusionsBeijing genotype was the predominant in the patients with DRTB in our hospital. In Chongqing retreatment patients with Beijing genotype MTB may be patient with DRTB. Drug resistance test (DST), regular medication and strict follow-up are very important for patients with Beijing genotype MTB. In Chongqing control and treatment of DRTB should be paid attention. Transmission and relations of patients with DRTB need to be further research.

Creating a web-based electronic tool to aid tuberculosis (TB) cluster investigation: data integration in TB surveillance activities in the United Kingdom, 2013 to 2016.

Molecular technology to identify relatedness between Mycobacterium tuberculosis complex isolates, representative of possible tuberculosis (TB) transmission between individuals, continues to evolve. At the same time, tools to utilise this information for public health action to improve TB control should also be implemented. Public Health England developed the Strain Typing Module (STM) as an integral part of the web-based surveillance system used in the United Kingdom following the roll-out of prospective 24 loci mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) strain typing. The creation of such a system required data integration and linkage, bringing together laboratory results and patient notification information. The STM facilitated widespread access to patient strain typing and clustering results for the public health community working in TB control. In addition, the system provided a log of cluster review and investigation decision making and results. Automated real-time data linkage between laboratory and notification data are essential to allow routine use of genotyping results in TB surveillance and control. Outputs must be accessible by those working in TB control at a local level to have any impact in ongoing public health activity.

Set-up and validation of mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) analysis of Mycobacterium tuberculosis using BioNumerics software.

The objective was to describe and validate a new and alternative software procedure for 24-locus mycobacterial interspersed repetitive unit-variable number-tandem repeat (MIRU-VNTR) typing of Mycobacterium tuberculosis (Mtb) based on the multipurpose BioNumerics software. DNA from randomly selected isolates of Mtb from two European laboratories, including external control samples for MIRU-VNTR typing, were analysed. Samples were genotyped using the commercial 24-locus VNTR typing kit from GenoScreen. The PCR amplified fragments were separated by capillary electrophoresis. For the subsequent analyses, the currently used software GeneMapper was compared with BioNumerics. The endpoint was the level of concordance when comparing genotyping results obtained from BioNumerics with results obtained from GeneMapper and the ECDC proficiency study reference results. Also, the number of necessary manual standard size corrections and allele assignments in the two different software methods were compared. In total, 272 DNA samples, including the ECDC proficiency panel, were analysed. For all samples, there were 100% concordance of results. For a randomly selected set of 96 samples the numbers of manual corrections needed for size standards were 199 with GeneMapper versus zero for BioNumerics. The numbers of manual corrections for allele assignments were 122 with GeneMapper versus 16 with BioNumerics. In conclusion, we have validated the multipurpose software BioNumerics for standard 24-locus MIRU-VNTR typing and the software shows promising benefits in terms of simplification and minimization of hand-on time.

Recent transmission of pulmonary tuberculosis and its influencing factors in Jing'an district, Shanghai, 2010-2015

Objective: To understand the recent transmission of Mycobacterium tuberculosis (MTB), and to identify the influencing factors of recent transmission among pulmonary tuberculosis (TB) patients in Jing'an district, Shanghai. Methods: The genotypes and drug resistances of MTB isolated from TB patients registered in the TB designated hospitals in Jing'an district during 2010-2015 were analyzed through 12-loci Mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR)(QUB11b, QUB18, Mtub21, Miru26, QUB26, Mtub04, Miru31, Miru40, VNTR2372, VNTR3820, 3232, 4120), and tested for drug susceptibility as well. With the results of field epidemiological investigation, univariate and multivariate analyses were performed to analyze the distribution of the clusters and influencing factors on recent transmission. Results: This study enrolled 80 TB patients, 23 (28.75%) had a resistance to at least one anti-TB drug, and the prevalence of multidrug-resistant tuberculosis (MDR-TB) was 16.25%. A total of 65 genotypes were identified with 58 (72.50%, 58/80) being unique and 7 clusters with 2-10 isolated in each cluster. The proportion of clustering was 27.50% (22/80). Results from the multivariate analysis revealed that multidrug- resistance (OR=35.799, 95%CI: 4.239-302.346) and having comorbidity with TB (OR=7.695, 95%CI: 1.421-41.658) were independently associated with the clustering, which suggesting a recent transmission. The field investigation to the clustered cases proved that the patients in two clusters had epidemiological links, one was between family members, and the other contained 10 MDR-TB patients with 9 knowing each other which have a definite connection and 1 having the possible connection with them. Conclusion: Recent transmission of tuberculosis happened among TB patients in Jing'an district, with high risks among the MDR-TB patients.

A Quantitative Evaluation of MIRU-VNTR Typing Against Whole-Genome Sequencing for Identifying Mycobacterium tuberculosis Transmission: A Prospective Observational Cohort Study.

BackgroundMycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR) typing is widely used in high-income countries to determine Mycobacterium tuberculosis relatedness. Whole-genome sequencing (WGS) is known to deliver greater specificity, but no quantitative prospective comparison has yet been undertaken.MethodsWe studied isolates from the English Midlands, sampled consecutively between 1 January 2012 and 31 December 2015. In addition to routinely performed MIRU-VNTR typing, DNA was extracted from liquid cultures and sequenced using Illumina technology. Demographic and epidemiological data for the relevant patients were extracted from the Enhanced Tuberculosis Surveillance system run by Public Health England. Closely related samples, defined using a threshold of five single nucleotide variants (SNVs), were compared to samples with identical MIRU-VNTR profiles, to samples from individuals with shared epidemiological risk factors, and to those with both characteristics.Findings1999 patients were identified for whom at least one M. tuberculosis isolate had been MIRU-VNTR typed and sequenced. Comparing epidemiological risk factors with close genetic relatedness, only co-residence had a positive predictive value of over 5%. Excluding co-resident individuals, 18.6% of patients with identical MIRU-VNTR profiles were within 5 SNVs. Where patients also shared social risk factors and ethnic group, this rose to 48%. Only 8% of MIRU-VNTR linked pairs in lineage 1 were within 5 SNV, compared to 31% in lineage 4.InterpretationIn the setting studied, this molecular epidemiological study shows MIRU-VNTR typing and epidemiological risk factors are poorly predictive of close genomic relatedness, assessed by SNV. MIRU-VNTR performance varies markedly by lineage.FundingPublic Health England, Health Innovation Challenge Fund, NIHR Health Protection Research Unit Oxford, NIHR Oxford Biomedical Research Centre.

Genomic Investigation of a Mycobacterium tuberculosis Outbreak Involving Prison and Community Cases in Florida, United States.

.We used whole-genome sequencing to investigate a tuberculosis outbreak involving U.S.-born persons in the prison system and both U.S.- and foreign-born persons in the community in Florida over a 7-year period (2009–2015). Genotyping by spacer oligonucleotide typing and 24-locus mycobacterial interspersed repetitive unit-variable number tandem repeat suggested that the outbreak might be clonal in origin. However, contact tracing could not link the two populations. Through a multidisciplinary approach, we showed that the cluster involved distinct bacterial transmission networks segregated by country of birth. The source strain is of foreign origin and circulated in the local Florida community for more than 20 years before introduction into the prison system. We also identified novel transmission links involving foreign and U.S.-born cases not discovered during contact investigation. Our data highlight the potential for spread of strains originating from outside the United States into U.S. “high-risk” populations, such as prisoners, with subsequent movement back to the general community.

Towards better prediction of Mycobacterium tuberculosis lineages from MIRU-VNTR data

The determination of lineages from strain-based molecular genotyping information is an important problem in tuberculosis. Mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing is a commonly used molecular genotyping approach that uses counts of the number of times pre-specified loci repeat in a strain. There are three main approaches for determining lineage based on MIRU-VNTR data - one based on a direct comparison to the strains in a curated database, and two others, on machine learning algorithms trained on a large collection of labeled data.All existing methods have limitations. The direct approach imposes an arbitrary threshold on how much a database strain can differ from a given one to be informative. On the other hand, the machine learning-based approaches require a substantial amount of labeled data. Notably, all three methods exhibit suboptimal classification accuracy without additional data.We explore several computational approaches to address these limitations. First, we show that eliminating the arbitrary threshold improves the performance of the direct approach. Second, we introduce RuleTB, an alternative direct method that proposes a concise set of rules for determining lineages. Lastly, we propose StackTB, a machine learning approach that requires only a fraction of the training data to outperform the accuracy of both existing machine learning methods.Our approaches demonstrate superior performance on a training dataset collected in New York City over 10 years, and the improvement in performance translates to a held-out testing set. We conclude that our methods provide opportunities for improving the determination of pathogenic lineages based on MIRU-VNTR data.