Molecular epidemiology of Mycobacterium tuberculosis in Baja California,Mexico: A result of human migration?

The State of Baja California (BC) exhibits the highest incidence and prevalence rates of tuberculosis (TB), and multidrug-resistant TB (MDR-TB) in Mexico. However information about the circulation of M. tuberculosis lineages in BC and Mexico as a whole is limited. Here, we describe the genetic relationship and genetic diversity among M. tuberculosis clinical isolates (n = 140) collected in BC between October 2009 and April 2011 with other regions of Mexico, the United States, and Latin America. All specimens were genotyped based on 24 mycobacterial interspersed repetitive units (MIRU)-variable number of tandem repeats (VNTR) loci. Population structure and minimum spanning tree (MST) analyses were used to assess the genetic diversity and distribution of BC isolates in comparison to USA and South America strains. Among the nine lineages observed, LAM, Haarlem and S were the most frequent identified in BC. Population structure analysis clustered most BC isolates (41%) into three distinctive groups that included strains from San Diego and South America, whereas other BC strains (22%) clustered with other Mexican strains. A subset of isolates (12%) seemed to be autochthonous of BC, while 25% were cosmopolitan and grouped into multiple clusters. It is highly likely that the TB genetic structure observed in BC is due to human migration.Additional studies are required to determine the mechanism involved in the phylogeographic distribution of M. tuberculosis in Mexico. Implementation of domestic molecular TB surveillance programs is required to better understand the molecular epidemiology of TB not only in the region but at the national level.     

Molecular epidemiology and genetic diversity of Mycobacterium tuberculosis complex in the Cross River State,Nigeria

This study provides with a first insight on Mycobacterium tuberculosis complex epidemiology and genetic diversity in the Cross River State, Nigeria. Starting with 137 smear positive patients recruited over a period of 12 months (June 2008 to May 2009), we obtained 97 pure mycobacterial isolates out of which 81 (83.5%) were identified as M. tuberculosis complex. Genotyping revealed a total of 27 spoligotypes patterns with 10 clusters (n = 64% or 79% of clustered isolates, 2–32 isolates/cluster), with patients in the age group range 25–34 years being significantly associated with shared-type pattern SIT61 (p = 0.019). Comparison with SITVIT2 database showed that with the exception of a single cluster (SIT727/H1), all other clusters observed were representative of West Africa; the two main lineages involved were LAM10-CAM (n = 42/81% or 51.8%) of M. tuberculosis and AFRI_2 sublineage of Mycobacterium africanum (n = 27/81% or 33.3%). Subsequent 12-loci MIRU typing resulted in a total of 13 SIT/MIT clusters (n = 52 isolates, 2–9 isolates per cluster), with a resulting recent n ? 1 transmission rate of 48.1%. Available drug-susceptibility testing (DST) results for 58/81 clinical isolates revealed 6/58% or 10.4% cases of multiple drug-resistance (MDR); 5/6 MDR cases were caused by strains belonging to LAM10-CAM lineage (a specific cluster SIT61/MIT266 in 4/6 cases, and an orphan spoligotype pattern in 1/6 case). Additionally, MIT266 was associated with streptomycin resistance (p = 0.016). All the six MDRTB isolates were concomitantly resistance to streptomycin and ethambutol; however, 4/6 MDR strains with identical MIRU patterns were characterized by consecutive strain numbers hence the possibility of laboratory cross contamination could not be excluded in 3/4 serial cases. The present preliminary study underlines the usefulness of spoligotyping and 12-loci MIRU–VNTRs to establish a baseline of circulating genotypic lineages of M. tuberculosis complex in Nigeria.     

Efficient Differentiation of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> Strains of the W-Beijing Family from Russia using Highly Polymorphic VNTR Loci

The W-Beijing family is a widespread Mycobacterium tuberculosis clonal lineage that frequently causes epidemic outbreaks. This family is genetically homogeneous and conserved, so ETR-VNTR (exact tandem repeat-variable number of tandem repeats) typing is insufficient for strain differentiation, due to a common ETR-A to E profile (42435). This leads to the false clustering in molecular epidemiological studies, especially in the regions of predominance of the W-Beijing family. In this study, we searched for VNTR loci with a high evolutionary rate of polymorphism in the W-Beijing genome. Here we further evaluated VNTR typing on a set of 99 Mycobacterium tuberculosis clinical isolates and reference strains. These isolates were characterized and classified into several genotype families based on three ETR loci (A, C, E) and eight additional loci [previously described as QUB (Queen’s University Belfast) or MIRU (Mycobacterial Interspersed Repetitive Units) or Mtubs]. Ninety-nine strains were divided into 74 VNTR-types, 51 isolates of the W-Beijing family identified by IS6110 RFLP-typing (the restriction fragment length polymorphism-typing) and/or spoligotyping were subdivided into 30 VNTR-types. HGDI (the Hunter–Gaston discriminatory index) for all studied loci was close to that of IS6110 RFLP typing, a “gold standard” method for subtyping M. tuberculosis complex strains. The QUB 26 and QUB 18 loci located in the PPE genes were highly polymorphic and more discriminative than other loci (HGDI is 0.8). Statistically significant increase of tandem repeats number in loci ETR-A, -E, QUB 26, QUB 18, QUB 11B, Mtub21 was revealed in the W-Beijing group compared to genetically divergent non-W-Beijing strains. Thirty-six isolates were subjected to IS6110 RFLP typing. The congruence between results of the IS6110 RFLP typing and 11-loci VNTR typing was estimated on 23 isolates of the W-Beijing family. These isolates were subdivided into 9 IS6110-RFLP types and 13 VNTR types. The poor profiles correlation (0.767) reflects the differences in the rate and type of evolution between genome regions targeted by IS6110-RFLP and VNTR typing. VNTR typing in proposed format is powerful tool for discrimination of M. tuberculosis strains with different level of genetic relationship.     

Concordance of variable-number tandem repeat (VNTR) and large sequence polymorphism (LSP) analyses of Mycobacterium tuberculosis strains

Variable-number tandem repeat (VNTR) and large sequence polymorphism (LSP) analyses were compared to determine whether VNTR analysis was effective for population genetic analysis of Mycobacterium tuberculosis strains. A total of 682 strains, 510 Beijing genotype and 172 non-Beijing genotype strains, were studied.The number of repeats was investigated for 24 VNTR loci: the 15 loci of “optimized miru”, the 8 loci of “Beijing option”, and 1 locus for “JATA12”. Six loci (miru31, Mtub4, QUB4156c, QUB3232, VNTR3820, and VNTR4120) showed significantly different median numbers of repeats in strains belonging to different lineages defined by LSP (P < 0.01, Mann–Whitney U test). When a minimum-spanning tree (MST) was reconstructed using these 6 loci, most strains clustered in the expected branches in the MST branches. However, topology of the MST was not congruent with the evolutional hypothesis of M. tuberculosis, indicating that MST analysis using VNTR data should not use for phylogeny of the organism.When the standardized index of association (sI_A) was calculated using data for the 6 VNTR loci, the value of sI_A was significantly different from zero (Monte Carlo simulation with 10,000 resamplings) in every lineage, indicating the linkage disequilibrium in different lineage strains of M. tuberculosis. These results were consistent with the hypothesis that clonal evolution of lineages of the organism has occurred.Therefore, the 6 loci identified in this study would be effective for M. tuberculosis population genetic analysis due to their significantly different median numbers of repeat and linkage disequilibrium though VNTR data was not effective for phylogeny of the organism.     

Penitentiary population of Mycobacterium tuberculosis in Kyrgyzstan: Exceptionally high prevalence of the Beijing genotype and its Russia-specific subtype

Here, we present results of the first study of the Mycobacterium tuberculosis genotypes circulating in Kyrgyzstan. We focused on the incarcerated population known to be at high-risk for tuberculosis (TB) and with a significant impact on TB incidence in the general population. Beijing genotype was detected in 42 of 56 M. tuberculosis sputum-extracted DNA samples from newly-diagnosed adult pulmonary TB patients. RIF and INH resistance was genotypically detected in 28% and 55% samples; 13 of 15 MDR strains belonged to Beijing genotype. 12-locus MIRU-VNTR typing showed 8 of 56 samples to be mixed cases; 7 of them contained a Beijing strain. MIRU analysis demonstrated a high homogeneity of the studied collection (HGI = 0.66) while 28 of 56 strains had a profile 223325153533 corresponding to Beijing/M2 subtype highly prevalent in different Russian settings. Three hypervariable loci, QUB-3232, VNTR-3820 and VNTR-4120, permitted to further subdivide 28 Beijing/M2 strains into 11 subtypes shared by 1 to 9 strains. To conclude, all markers taken together, the penitentiary population of M. tuberculosis in Kyrgyzstan exhibited a strong genetic affinity to Russia and a weak relatedness to East Asia.     

A first insight on the population structure of Mycobacterium tuberculosis complex as studied by spoligotyping and MIRU-VNTRs in Bogotá, Colombia

With an incidence of 25.6/100,000 in 2008, tuberculosis (TB) remains an important public health problem in Colombia. In this study, a total of 152 Mycobacterium tuberculosis complex strains isolated in Bogotá, Colombia between years 1995 and 2007 were genotyped by spoligotyping and 12-loci MIRU-VNTRs. The various spoligotyping-based genotypic lineages in our sample were: Latin American & Mediterranean (LAM) n = 75, 49.34%; Haarlem, n = 38, 25.0%; ill-defined T group, n = 21, 13.82%; S family, n = 5, 3.29%; X clade, n = 2, 1.32%; Beijing, n = 1, 0.65%, while strains with unknown signatures (n = 10) represented 6.58% of isolates. Using spoligotyping as a first molecular marker and MIRU-VNTRs as second marker, we obtained 102 single patterns and 14 clustered patterns (n = 52 strains from 49 patients, 2–8 strains per cluster). The MIRU-VNTRs patterns corresponded to 50 MITs for 109 strains and 43 orphan patterns. The most frequent patterns were MIT190 (n = 12), MIT45 (n = 10), and MIT25 (n = 9). The Hunter & Gaston discriminatory index (HGDI) of both methodologies used together showed a value of 0.992. In our setting, the HGDI of five loci subset (MIRU10, 16, 23, 26 and 40) contributed most to the discriminatory power of 12-loci format used (HGDI = 0.977). The lineage distribution of M. tuberculosis showed that more than 3/4 of strains in Bogotá are commonly found in Latin America, Caribbean, and Europe. This observation might reflect the shared post-Columbus history of Colombia and its Latin-American neighbors as well as strains brought in by 20th century immigrants from Europe. We also demonstrate the usefulness of MIRU-VNTR to detect suspected links among patients and polyclonal infections.     

Molecular characterization of Mycobacterium bovis isolates from patients with tuberculosis in Baja California,Mexico

The incidence of tuberculosis (TB) from Mycobacterium bovis in humans is likely to be underestimated and in some cases even ignored in most developing countries. This may be due to the difficulty of differentiating TB caused by either Mycobacterium tuberculosis or M. bovis.Our objectives were to determine the prevalence of M. bovis human disease among the patients referred for study to the Tuberculosis Laboratory of the Tijuana General Hospital in Baja California, Mexico and to characterize molecularly the clinical isolates using 8 loci of MIRU-VNTR.A cross-sectional analysis of all culture-proven cases of tuberculosis was conducted during the period from January 1, 2011 through June 30, 2013. Clinical isolates that exhibited resistance to pyrazinamide (Z) were submitted for molecular analysis.A total of 2699 clinical samples were cultured during the study period and 600 (22%) that tested positive were processed for drug susceptibility for first line drugs. Sixty-four (10.7%) of the tested isolates tested were resistant to Z, and 27 (4.5%) of those were subsequently identified molecularly as M. bovis. Three of the M. bovis isolates were polyresistant to Z, isoniazid (H), ethambutol (E) and rifampicin (R) (Z + H + E, Z + E and Z + R); the rest were only resistant only to Z. VNTR typing, based on the 8 VNTR loci commonly tested for M. bovis, detected 12 allelic profiles (genotypes).The real burden of M. bovis cases among the total reported human tuberculosis cases can only be known from especially designed studies in which, during a specific period, all specimens submitted to tuberculosis diagnosis in one or more laboratories are cultured on the appropriate media and the isolated mycobacteria are analyzed to differentiate M. bovis from M. tuberculosis and other Mycobacterium species.     

Mycobacterium tuberculosis Beijing family: Analysis of the epidemiological and clinical factors associated with an emerging lineage in the urban area of Milan

The Mycobacterium tuberculosis Beijing genotype raises major concern because of global spreading, hyper-virulence and association with multi-drug resistance (MDR). The aims of the study were to evaluate role of Beijing family in the epidemiological setting of Milan and to identify predictors associated with the spreading of this lineage. Overall 3830 TB cases were included. Beijing family accounted for 100 isolates (2.6%). Prevalence grew from 1.7% to 5.4% in the period 1996–2009. Foreign origin increased significantly the risk of having a Beijing strain: the greatest risk was observed among patients coming either from China [AOR = 57.7, 95%CI (26.3–126.8)] or from Former Soviet countries [AOR = 33.9, 95%CI (12.8–99.6)]. Also MDR was independently associated with Beijing family [AOR = 2.7, 95%CI (1.3–5.8)], whereas male gender and younger age only approximated the statistical significance [p 0.051 and p 0.099, respectively].However, the percentage of cases attributable to MDR strains decreased over time, both in the Beijing group and in the non-Beijing group.97 isolates were grouped in 37 sub-lineages: MT11, MT33 were predominant. Beijing family is an emerging lineage in Milan. Origin from countries like China and Ukraine and MDR are significantly associated with Beijing. The broad range of the sub-lineages reflects the recent dynamics of the migration flows to our area. This scenario can prelude to a constant increase in the spreading of Beijing strains in the near future.     

Mutation rates of spoligotypes and variable numbers of tandem repeat loci in Mycobacterium tuberculosis

Variable numbers of tandem repeat (VNTR) loci and spoligotypes are molecular markers used to study genetic diversity of bacteria such as Mycobacterium tuberculosis. Knowledge about the rate at which molecular fingerprints change, or the mutation rate, is important for interpreting molecular epidemiological data and for studying quantitative models of the evolution and epidemiology of bacteria. In this paper we estimate the mutation rates of spoligotypes and VNTR loci of M. tuberculosis using published data sets from epidemiological studies. Our method makes use of a compound parameter: twice the product of the effective population size and the mutation rate. We use a standard procedure to estimate this population genetic parameter which describes genetic diversity. The ratio of estimated diversity parameters for different markers can be used to generate new estimates for markers with unknown mutation rates. We apply this method to generate new estimates along with confidence intervals. We found mutation rates for VNTR loci to be around 7 × 10^?4 to 1.5 × 10^?2 per locus per year, and for spoligotypes to be around 0.02–0.09 per year. The spoligotype rate is similar to previous estimates while the VNTR rates are at least an order of magnitude higher than previously reported. These findings confirm the high level of discrimination observed using multilocus VNTR typing, and suggest that caution should be taken not to underestimate the extent of recent transmission when using this marker.     

Distinct clinical and epidemiological features of tuberculosis in New York City caused by the RDRio Mycobacterium tuberculosis sublineage

BackgroundGenetic tracking of Mycobacterium tuberculosis is a cornerstone of tuberculosis (TB) control programs. The RD^Rio M. tuberculosis sublineage was previously associated with TB in Brazil. We investigated 3847 M. tuberculosis isolates and registry data from New York City (NYC) (2001–2005) to: (1) affirm the position of RD^Rio strains within the M. tuberculosis phylogenetic structure, (2) determine its prevalence, and (3) define transmission, demographic, and clinical characteristics associated with RD^Rio TB.MethodsIsolates classified as RD^Rio or non-RD^Rio M. tuberculosis by multiplex PCR were further classified as clustered (?2 isolates) or unique based primarily upon IS6110-RFLP patterns and lineage-specific cluster proportions were calculated. The secondary case rate of RD^Rio was compared with other prevalent M. tuberculosis lineages. Genotype data were merged with the data from the NYC TB Registry to assess demographic and clinical characteristics.ResultsRD^Rio strains were found to: (1) be restricted to the Latin American–Mediterranean family, (2) cause approximately 8% of TB cases in NYC, and (3) be associated with heightened transmission as shown by: (i) a higher cluster proportion compared to other prevalent lineages, (ii) a higher secondary case rate, and (iii) cases in children. Furthermore, RD^Rio strains were significantly associated with US-born Black or Hispanic race, birth in Latin American and Caribbean countries, and isoniazid resistance.ConclusionsThe RD^Rio genotype is a single M. tuberculosis strain population that is emerging in NYC. The findings suggest that expanded RD^Rio case and exposure identification could be of benefit due to its association with heightened transmission.     

不同可变数目串联重复序列组合对中国流行结核分枝杆菌分辨力的评价研究

目的 在规范化的结核分枝杆菌（Mycobacterium tuberculosis,MTB）可变数目串联重复序列（variable number of tandem repeats,VNTR）基因分型的基础上,构建我国31个省（自治区、直辖市）VNTR数据库,每个省优化一套VNTR位点组合,为我国结核病预防控制策略的制定提供科学依据。方法 对2007－2008年全国结核病耐药性基线调查的4 116株MTB15位点VNTR（15-VNTR）基因分型。汉高指数（Hunter-Gaston Index,HGI）分析每个位点的分辨率。依据谱系流行特征,以分辨率高和稳定性强为原则,为各省设计一套VNTR优化组合（12-VNTR、10-VNTR、8-VNTR和5-VNTR）,采用HGI和成簇率进行评价。结果 完成了涵盖率为96.36%（3 966/4 116）MTB完整15-VNTR图谱。发现QUB11b、MIRU26等7个高分辨率位点;QUB26、MIRU16、Mtub21、QUB11b在部分地区遗传稳定性差。内蒙古自治区、重庆市、黑龙江省的最优组合为10-VNTR,其他各省的最佳组合为8-VNTR。结论 VNTR数据库的建立将推动全国范围MTB传染源的追踪;各省优化VNTR组合的推出有助于当地结核病疫情的监测和群体遗传学的研究。     

A novel liposome adjuvant DPC mediates Mycobacterium tuberculosis subunit vaccine well to induce cell-mediated immunity and high protective efficacy in mice

Tuberculosis (TB) is a serious disease around the world, and protein based subunit vaccine is supposed to be a kind of promising novel vaccine against it. However, there is no effective adjuvant available in clinic to activate cell-mediated immune responses which is required for TB subunit vaccine. Therefore, it is imperative to develop new adjuvant. Here we reported an adjuvant composed of dimethyl dioctadecylammonium (DDA), Poly I:C and cholesterol (DPC for short). DDA can form a kind of cationic liposome with the ability to deliver and present antigen and can induce Th1 type cell-mediated immune response. Poly I:C, a ligand of TLR3 receptor, could attenuate the pathologic reaction induced by following Mycobacterium tuberculosis challenge. Cholesterol, which could enhance rigidity of lipid bilayer, is added to DDA and Poly I:C to improve the stability of the adjuvant. The particle size and Zeta-potential of DPC were analyzed in vitro. Furthermore, DPC was mixed with a TB fusion protein ESAT6-Ag85B-MPT64(190-198)-Mtb8.4-Rv2626c (LT70) to construct a subunit vaccine. The subunit vaccine-induced immune responses and protective efficacy against M. tuberculosis H37Rv infection in C57BL/6 mice were investigated. The results showed that the DPC adjuvant with particle size of 400 nm and zeta potential of 40 mV was in good stability. LT70 in the adjuvant of DPC generated strong antigen-specific humoral and cell-mediated immunity, and induced long-term higher protective efficacy against M. tuberculosis infection (5.41 ± 0.38 log₁₀ CFU) than traditional vaccine Bacillus Calmette–Guerin (BCG) (6.01 ± 0.33 log₁₀ CFU) and PBS control (6.53 ± 0.26 log₁₀ CFU) at 30 weeks post-vaccination. In conclusion, DPC would be a promising vaccine adjuvant with the ability to stimulate Th1 type cell-mediated immunity, and could be used in TB subunit vaccine.     

Phylogeographical and molecular characterization of an emerging Mycobacterium tuberculosis clone in Trinidad and Tobago

We report on a fine molecular and phylogenetical characterization of circulating Mycobacterium tuberculosis strains isolated from patients during a 1-year period in Trinidad and Tobago (T&T). The spoligotyping data coupled to minisatellite typing and available epidemiological data showed that a single major clone of “evolutionary modern” tubercle bacilli (SIT566) was responsible for more than half of the tuberculosis (TB) cases. It preferentially infected younger age groups (mean 39.1 years versus 47.7 years for other genotypes, p < 0.0005), and was overrepresented in Port-of-Spain (1 out of 3 patients). A comparison of genotyping results to data gathered for 6 Caribbean countries (n = 2653 clinical isolates) showed that the overall lineage distribution in T&T was completely different from its neighbors, e.g., T&T was the only country harboring a unique sublineage of the Latin American & Mediterranean (LAM) family, designated LAM-10CAM with phylogeographical specificity for Cameroon and neighboring countries in West Africa; interestingly 3/4 of the patients within this group in T&T were African descendants. Similarly, strains belonging to East African Indian (EAI) lineage with phylogeographical specificity for the Indian subcontinent, were found in T&T (13% of all strains), but were absent among the neighboring countries. Although the predominant SIT566 was not yet detected elsewhere in the Caribbean, available information underlined that this genotype was already present in the United States as imported cases of disease among T&T-born patients. Characterization of SIT566 strains using 12-, 15- and 24-loci MIRU typing, and comparison of results to international databases showed that these isolates were characterized by a common 12-loci MIRU pattern 224315153324 corresponding to MIRU International Type—MIT633 in 21/25 strains tested, as well as its 4 variants; an orphan pattern

, MIT27—

, MIT117—

, and MIT1158—

. Extended 24-loci MIRU typing led to a predominant pattern 224315153324323483334323 in a total of 16/21 MIT633 isolates, as well as identification of 3 supplemental patterns. Comparison of 24-loci MIRU data with the international database MIRU-VNTRplus showed the unique nature of the patterns obtained in T&T. Further analysis using the Levenshtein algorithm showed that the first 2 closest matches with the SIT566/MIT633 clone belonged to the X lineage strains in MIRU-VNTRplus. This observation corroborates our preliminary spoligotyping-based analysis using minimum spanning and neighbor-joining trees, which suggested a phylogenetical relatedness of the SIT566 clone with SIT119, which represents X1 lineage prototype in SpolDB4 database. We hypothesize that the predominant SIT566 clone might have evolved from a pool of X lineage M. tuberculosis strains with phylogeographical affinity for Anglo-Saxon descendants.     

Molecular epidemiology of Mycobacterium tuberculosis isolates from Kerala,India using IS6110-RFLP,spoligotyping and MIRU-VNTRs

Tuberculosis (TB) continues to be a major health problem in India, and there is very little information about the prevalent genotypes of tubercle bacilli that cause TB in India, especially in Kerala. Our aim was to study the different circulating strains of Mycobacterium tuberculosis (MTB) that are prevalent in Kerala, India. We analyzed 168 MTB isolates from as many pulmonary TB patients using IS6110-RFLP, spoligotyping and MIRU-VNTRs. The results of IS6110-RFLP revealed that majority of isolates had null copy (10.89%) or single copy (44.87%) of IS6110 insertion. Low copy (<6) isolates accounted for 71.5% in the isolates studied. Genotypic clade designations were done by comparing with the SITVIT2 database which showed 68 patterns; of which 51 corresponded to different shared types whereas 17 patterns were orphans. Among the 51 SITs recorded, 42 SITs matched a preexisting SIT in the SITVIT2 database, whereas 9 SITs were newly-created. Majority of the isolates (64.28%) belonged to the ancestral East-African Indian (EAI) lineage. MIRU-40 and 31 (HGDI = 0.6555 and 0.6524) showed highest discrimination, while MIRU-2 and 20 (HGDI = 0.0354 and 0.0696) had the least discriminatory power. ETR-A and B (HGDI 0.7382 and 0.6743) discriminated better as compared to other MIRU loci. The overall HGDI for MIRU-VNTRs at 0.9735 (calculated for 166 isolates) showed a better discriminatory power than spoligotyping used alone. This study of MTB genotypic diversity was useful by providing a first snapshot of circulating MTB genotypic clones in Kerala.     

Multi-locus variable-number tandem repeat analysis (MLVA) reveals heterogeneity of Mycobacterium bovis strains and multiple genotype infections of cattle in Ethiopia

Bovine tuberculosis (BTB) remains a major threat to animal and human health, and obstructs international and inter-regional livestock trade in Ethiopia. Many aspects of epidemiology of BTB and its causative agent, Mycobacterium bovis (M. bovis) are not well known. Aims of the study were to elucidate molecular characteristics of M. bovis strains using MLVA typing method. Further aim was to determine infection pressure associated with occurrence of multiple genotypes in individual infected cattle. Data and samples were collected in the period July 2006–January 2007 in cattle slaughtered at five representative abattoirs across the country. Molecular investigation of the isolates was carried out using multilocus variable-number tandem-repeat analysis (MLVA) of 28 variable numbers of tandem repeat (VNTR) loci, and the results were compared to spoligotyping. This study is believed to contribute to the knowledge of molecular genetics and epidemiology of M. bovis in Ethiopia and elsewhere with similar BTB epidemic situation and livestock production settings. Four-hundred and six tissue samples from 337 carcasses revealing gross pathologic lesions compatible with BTB were collected from five abattoirs. Fifty-eight isolates obtained from cultured samples were subjected to region of difference (RD) analysis and MLVA typing. RD confirmed all isolates as being M. bovis. MLVA revealed a high heterogeneity of M. bovis (19 genotypes) and the discriminatory power of MLVA was higher than for spoligotyping (Hunter–Gaston Diversity Index (HGDI) 0.92 vs. 0.82). Adoption of the nine VNTR loci with ⩾3 alleles provided good differentiation between the isolates. However, differentiation was optimized when MLVA was combined with spoligotyping (HGDI = 0.99). MLVA confirmed infections with multiple genotypes in 38.5% (10/26) of individual animals. In conclusion, the usefulness of MLVA for genotyping M. bovis in high prevalence settings was demonstrated. BTB in Ethiopia is caused by heterogeneous populations of M. bovis and individual carcasses were often infected with different genotypes, indicating a high infection pressure perhaps related to the absence of protective immunity conferred by infection.     

Factors of loss to follow-up during tuberculosis treatment in a low-incidence region

ObjectiveThe proportion of successfully treated tuberculosis (TB) patients remains below the WHO target in France, because of a high proportion of loss to follow-up. We aimed to identify factors associated with loss to follow-up in northern France, a low-incidence area.MethodsBetween 1997 and 2017, all consecutive patients diagnosed with TB at the Tourcoing Hospital, except those infected with multidrug-resistant or extensively drug-resistant strains, were included in a retrospective cohort study. A logistic regression analysis was performed to determine factors associated with loss to follow-up.ResultsOne hundred and ninety patients were included. Previous TB treatment was reported in 32 patients (17%), extrapulmonary TB in 107 (56%), and HIV infection in 44 (23%). The proportion of loss to follow-up was 15%. In multivariate analysis, the risk of loss to follow-up decreased in case of first TB treatment (OR 0.36; 95% CI: 0.14–0.92, P = 0.03) and increased in non-HIV-infected patients (OR 7.67; 95% CI: 1.00–59.0, p = 0.05). Support for compliance was more frequent in HIV-infected patients (23% vs. 7%, p = 0.005).ConclusionThe proportion of loss to follow-up was high. HIV infection was associated with a lower risk of loss to follow-up, likely to be due to more frequent support for compliance.     

Genetic variants at 18q11.2 and 8q24 identified by genome-wide association studies were not associated with pulmonary tuberculosis risk in Chinese population

ObjectiveGenome-wide association study (GWAS) recently identified several susceptibility loci in ASAP1 gene on chromosome 8q24 for tuberculosis (TB) in a Russian population, but no relevant studies have been performed to validate these findings. In addition, previous GWAS in Ghana and Gambia found that the variant rs4331426 at 18q11.2 was a susceptibility locus for TB. However, the follow-up studies reported conflicting results. Herein, we investigated the contribution of genetic variants at 8q24 and 18q11.2 to TB in Chinese population.MethodsWe genotyped four genetic variants at 8q24 (rs10956514 and rs11774633) and 18q11.2 (rs4331426 and rs6507226) in a case–control study with 355 newly bacteriologically confirmed pulmonary TB cases and 395 healthy controls using TaqMan allelic discrimination assay. Subsequently, we conducted a meta-analysis including 4 reported studies in Chinese populations and our case–control study with a total of 3118 cases and 3226 controls to further evaluate the relationship between rs4331426 at 18q11.2 and TB risk.ResultsWe did not find significant association between genetic variants at 8q24 and risk of TB (rs10956514: OR = 0.89, 95%CI: 0.72–1.09, P = 0.253; rs11774633: OR = 0.86, 95%CI: 0.69–1.08, P = 0.206). We did not observe significant association for genetic variants at 18q11.2 (rs4331426: OR = 0.62, 95%CI: 0.34–1.14, P = 0.122; and rs6507226: OR = 0.98, 95%CI: 0.80–1.20, P = 0.853). Moreover, the pooled results from the Meta-analysis further supported that rs4331426 at 18q11.2 was not associated with TB risk in Chinese population (OR = 0.90, 95% CI: 0.63–1.29).ConclusionsOur findings indicate that TB risk-associated loci at 8q24 and 18q11.2 identified by GWAS from the other populations may not contribute to TB susceptibility in Chinese population.     

Allopatric tuberculosis host–pathogen relationships are associated with greater pulmonary impairment

BackgroundHost pathogen relationships can be classified as allopatric, when the pathogens originated from separate, non-overlapping geographic areas from the host; or sympatric, when host and pathogen shared a common ancestral geographic location. It remains unclear if host–pathogen relationships, as defined by phylogenetic lineage, influence clinical outcome. We sought to examine the association between allopatric and sympatric phylogenetic Mycobacterium tuberculosis lineages and pulmonary impairment after tuberculosis (PIAT).MethodsPulmonary function tests were performed on patients 16 years of age and older who had received ⩾20 weeks of treatment for culture-confirmed M. tuberculosis complex. Forced Expiratory Volume in 1 min (FEV1) ⩾80%, Forced Vital Capacity (FVC) ⩾80% and FEV1/FVC >70% of predicted were considered normal. Other results defined pulmonary impairment. Spoligotype and 12-locus mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) were used to assign phylogenetic lineage. PIAT severity was compared between host–pathogen relationships which were defined by geography and ethnic population. We used multivariate logistic regression modeling to calculate adjusted odds ratios (aOR) between phylogenetic lineage and PIAT.ResultsSelf-reported continental ancestry was correlated with Mycobacterium. tuberculosis lineage (p < 0.001). In multivariate analyses adjusting for phylogenetic lineage, age and smoking, the overall aOR for subjects with allopatric host–pathogen relationships and PIAT was 1.8 (95% confidence interval [CI]: 1.1, 2.9) compared to sympatric relationships. Smoking >30 pack-years was also associated with PIAT (aOR: 3.2; 95% CI: 1.5, 7.2) relative to smoking <1 pack-years.ConclusionsPIAT frequency and severity varies by host–pathogen relationship and heavy cigarette consumption, but not phylogenetic lineage alone. Patients who had disease resulting from allopatric–host–pathogen relationship were more likely to have PIAT than patients with disease from sympatric–host–pathogen relationship infection. Further study of this association may identify ways that treatment and preventive efforts can be tailored to specific lineages and racial/ethnic populations.     

青海省结核分枝杆菌临床分离株可变数目串联重复序列基因多态性研究

目的 初步了解青海省结核分枝杆菌临床分离株基因多态性和基因分型特征。方法 2009-2012年收集青海省疾病预防控制中心分离的结核分枝杆菌临床分离株,提取DNA,对15个可变数目串联重复序列(VNTR)位点进行PCR扩增和产物电泳分析,使用BioNumerics软件对菌株进行聚类分析。结果 共检测251株结核分枝杆菌临床分离株的15个VNTR位点,显示这些菌株有明显的基因多态性,15个VNTR位点中Hunter-Gaston指数>0.6的VNTR位点有6个,位点分辨能力最高的是MIRU26,经聚类分析,可分为4个基因群,238个基因型。4个基因群分别占4.9%、91.9%、1.6%和1.6%。结论 青海省流行的结核分枝杆菌菌株存在明显的VNTR基因多态性。