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1.  Mycobacterium tuberculosis Pyrazinamide Resistance Determinants: a Multicenter Study 
mBio  2014;5(5):e01819-14.
ABSTRACT
Pyrazinamide (PZA) is a prodrug that is converted to pyrazinoic acid by the enzyme pyrazinamidase, encoded by the pncA gene in Mycobacterium tuberculosis. Molecular identification of mutations in pncA offers the potential for rapid detection of pyrazinamide resistance (PZAr). However, the genetic variants are highly variable and scattered over the full length of pncA, complicating the development of a molecular test. We performed a large multicenter study assessing pncA sequence variations in 1,950 clinical isolates, including 1,142 multidrug-resistant (MDR) strains and 483 fully susceptible strains. The results of pncA sequencing were correlated with phenotype, enzymatic activity, and structural and phylogenetic data. We identified 280 genetic variants which were divided into four classes: (i) very high confidence resistance mutations that were found only in PZAr strains (85%), (ii) high-confidence resistance mutations found in more than 70% of PZAr strains, (iii) mutations with an unclear role found in less than 70% of PZAr strains, and (iv) mutations not associated with phenotypic resistance (10%). Any future molecular diagnostic assay should be able to target and identify at least the very high and high-confidence genetic variant markers of PZAr; the diagnostic accuracy of such an assay would be in the range of 89.5 to 98.8%.
IMPORTANCE
Conventional phenotypic testing for pyrazinamide resistance in Mycobacterium tuberculosis is technically challenging and often unreliable. The development of a molecular assay for detecting pyrazinamide resistance would be a breakthrough, directly overcoming both the limitations of conventional testing and its related biosafety issues. Although the main mechanism of pyrazinamide resistance involves mutations inactivating the pncA enzyme, the highly diverse genetic variants scattered over the full length of the pncA gene and the lack of a reliable phenotypic gold standard hamper the development of molecular diagnostic assays. By analyzing a large number of strains collected worldwide, we have classified the different genetic variants based on their predictive value for resistance which should lead to more rapid diagnostic tests. This would assist clinicians in improving treatment regimens for patients.
doi:10.1128/mBio.01819-14
PMCID: PMC4212837  PMID: 25336456
2.  High-dose vitamin D3 during intensive phase treatment of pulmonary tuberculosis: a double-blind randomised controlled trial 
Lancet  2011;377(9761):242-250.
Background
Vitamin D was used to treat tuberculosis in the pre-antibiotic era, and its metabolites induce antimycobacterial immunity in vitro. Clinical trials investigating the effect of adjunctive vitamin D on sputum culture conversion are lacking.
Methods
We conducted a multi-centre randomised controlled trial of adjunctive vitamin D in adults with sputum smear-positive pulmonary tuberculosis in London, UK. 146 participants were allocated to receive 2.5 mg vitamin D3 or placebo at baseline and at 2, 4 and 6 weeks after starting standard tuberculosis treatment. The primary endpoint of the trial was time from initiation of antimicrobial therapy to sputum culture conversion. Participants were genotyped for TaqI and FokI polymorphisms of the vitamin D receptor (VDR), and interaction analyses were conducted to determine the influence of VDR genotype on response to vitamin D. This trial is registered with ClinicalTrials.gov (NCT00419068).
Findings
126 participants were included in the primary efficacy analysis (62 allocated to intervention, 64 allocated to placebo). Median time to sputum culture conversion was 36.0 days in the intervention arm and 43.5 days in the placebo arm (adjusted HR 1.39; 95% CI 0.90-2.16, P=0.14). TaqI genotype modified the effect of vitamin D supplementation on time to sputum culture conversion (Pinteraction=0.03), with enhanced response seen only in participants with the tt genotype (HR 8.09, 95% CI 1.36-48.01, P=0.02). FokI genotype did not modify the effect of vitamin D supplementation (Pinteraction=0.85). Mean serum 25-hydroxyvitamin D at 8 weeks was 101.4 nmol/L vs. 22.8 nmol/L in intervention vs. placebo arms (95% CI for difference 68.6-88.2 nmol/L, P<0.001).
Interpretation
Administration of four doses of 2.5 mg vitamin D3 elevated serum 25-hydroxyvitamin D concentrations in patients receiving intensive phase treatment for pulmonary tuberculosis and reduced time to sputum culture conversion in participants with the tt genotype of the TaqI VDR polymorphism.
doi:10.1016/S0140-6736(10)61889-2
PMCID: PMC4176755  PMID: 21215445
3.  Evolution and transmission of drug resistant tuberculosis in a Russian population 
Nature genetics  2014;46(3):279-286.
The molecular mechanisms determining transmissibility and prevalence of drug-resistant tuberculosis in a population were investigated through whole genome sequencing of 1,000 prospectively-obtained patient isolates from Russia. Two-thirds belonged to the Beijing lineage, which was dominated by two homogeneous clades. MDR genotypes were found in 48% of isolates overall and 87% of the major clades. The most common rifampicin-resistance rpoB mutation was associated with fitness-compensatory mutations in rpoA or rpoC, and a novel intragenic compensatory substitution was identified. The proportion of MDR cases with XDR-tuberculosis was 16% overall with 65% of MDR isolates harboring eis mutations, selected by kanamycin therapy, which may drive the expansion of strains with enhanced virulence. The combination of drug resistance and compensatory mutations displayed by the major clades confer clinical resistance without compromising fitness and transmissibility, revealing a biological contribution to the tuberculosis program weaknesses driving the persistence and spread of M/XDR-tuberculosis in Russia and beyond.
doi:10.1038/ng.2878
PMCID: PMC3939361  PMID: 24464101
4.  Proposal of a Consensus Set of Hypervariable Mycobacterial Interspersed Repetitive-Unit–Variable-Number Tandem-Repeat Loci for Subtyping of Mycobacterium tuberculosis Beijing Isolates 
Journal of Clinical Microbiology  2014;52(1):164-172.
Mycobacterium tuberculosis Beijing strains represent targets of special importance for molecular surveillance of tuberculosis (TB), especially because they are associated with spread of multidrug resistance in some world regions. Standard 24-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) typing lacks resolution power for accurately discriminating closely related clones that often compose Beijing strain populations. Therefore, we evaluated a set of 7 additional, hypervariable MIRU-VNTR loci for better resolution and tracing of such strains, using a collection of 535 Beijing isolates from six world regions where these strains are known to be prevalent. The typeability and interlaboratory reproducibility of these hypervariable loci were lower than those of the 24 standard loci. Three loci (2163a, 3155, and 3336) were excluded because of their redundant variability and/or more frequent noninterpretable results compared to the 4 other markers. The use of the remaining 4-locus set (1982, 3232, 3820, and 4120) increased the number of types by 52% (from 223 to 340) and reduced the clustering rate from 58.3 to 36.6%, when combined with the use of the standard 24-locus set. Known major clonal complexes/24-locus-based clusters were all subdivided, although the degree of subdivision varied depending on the complex. Only five single-locus variations were detected among the hypervariable loci of an additional panel of 92 isolates, representing 15 years of clonal spread of a single Beijing strain in a geographically restricted setting. On this calibrated basis, we propose this 4-locus set as a consensus for subtyping Beijing clonal complexes and clusters, after standard typing.
doi:10.1128/JCM.02519-13
PMCID: PMC3911419  PMID: 24172154
5.  PolyTB: A genomic variation map for Mycobacterium tuberculosis 
Summary
Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is the second major cause of death from an infectious disease worldwide. Recent advances in DNA sequencing are leading to the ability to generate whole genome information in clinical isolates of M. tuberculosis complex (MTBC). The identification of informative genetic variants such as phylogenetic markers and those associated with drug resistance or virulence will help barcode Mtb in the context of epidemiological, diagnostic and clinical studies. Mtb genomic datasets are increasingly available as raw sequences, which are potentially difficult and computer intensive to process, and compare across studies. Here we have processed the raw sequence data (>1500 isolates, eight studies) to compile a catalogue of SNPs (n = 74,039, 63% non-synonymous, 51.1% in more than one isolate, i.e. non-private), small indels (n = 4810) and larger structural variants (n = 800). We have developed the PolyTB web-based tool (http://pathogenseq.lshtm.ac.uk/polytb) to visualise the resulting variation and important meta-data (e.g. in silico inferred strain-types, location) within geographical map and phylogenetic views. This resource will allow researchers to identify polymorphisms within candidate genes of interest, as well as examine the genomic diversity and distribution of strains. PolyTB source code is freely available to researchers wishing to develop similar tools for their pathogen of interest.
doi:10.1016/j.tube.2014.02.005
PMCID: PMC4066953  PMID: 24637013
Mycobacterium tuberculosis; Database; Genomics; Software; Molecular epidemiology; Whole-genome sequencing
6.  First Evaluation after Implementation of a Quality Control System for the Second Line Drug Susceptibility Testing of Mycobacterium tuberculosis Joint Efforts in Low and High Incidence Countries 
PLoS ONE  2013;8(10):e76765.
Three networks/projects involving 27 European countries were established to investigate the quality of second-line drug (SLD) susceptibility testing with conventional and molecular methods. 1. The “Baltic-Nordic TB-Laboratory Network” comprised 11 reference laboratories in the Baltic-Nordic States. They performed SLD testing in the first phase with a panel of 20 Mycobacterium tuberculosis strains. After several laboratories made technical changes a second panel of 10 strains with a higher proportion of resistant strains were tested. Although the concordance for Ofloxacin, Kanamycin, and Capreomycin was consistently high, the largest improvements in performance were achieved for the analysis of Ofloxacin resistant (from 88.9 to 95.0%), and Capreomycin resistant (from 71.0 to 88.9%) strains. 2. Within the FP7 TB PAN-NET project (EU Grant agreement 223681) a quality control panel to standardize the EQA (External Quality Assurance) for first-line drugs (FLD) and SLD testing for phenotypic and molecular methods was established. The strains were characterized by their robustness, unambiguous results when tested, and low proportion of secondary drug resistances. 3. The (European Reference Laboratory Network-TB) ERLN-TB network analyzed four different panels for drug resistance testing using phenotypic and molecular methods; in two rounds in 2010 the 31 participating laboratories began with 5 strains, followed by 10 strains and 6 additional crude DNA extracts in 2011 and 2012 were examined by conventional DST and molecular methods. Overall, we demonstrated the importance of developing inter-laboratory networks to establish quality assurance and improvement of SLD testing of M. tuberculosis.
doi:10.1371/journal.pone.0076765
PMCID: PMC3795631  PMID: 24146924
7.  Rapid diagnostics of tuberculosis and drug resistance in the industrialized world: clinical and public health benefits and barriers to implementation 
BMC Medicine  2013;11:190.
In this article, we give an overview of new technologies for the diagnosis of tuberculosis (TB) and drug resistance, consider their advantages over existing methodologies, broad issues of cost, cost-effectiveness and programmatic implementation, and their clinical as well as public health impact, focusing on the industrialized world. Molecular nucleic-acid amplification diagnostic systems have high specificity for TB diagnosis (and rifampicin resistance) but sensitivity for TB detection is more variable. Nevertheless, it is possible to diagnose TB and rifampicin resistance within a day and commercial automated systems make this possible with minimal training. Although studies are limited, these systems appear to be cost-effective. Most of these tools are of value clinically and for public health use. For example, whole genome sequencing of Mycobacterium tuberculosis offers a powerful new approach to the identification of drug resistance and to map transmission at a community and population level.
doi:10.1186/1741-7015-11-190
PMCID: PMC3765611  PMID: 23987891
Diagnosis; Drug resistance; Tuberculosis; Public health; Whole genome sequencing
8.  Ethnic Variation in Inflammatory Profile in Tuberculosis 
PLoS Pathogens  2013;9(7):e1003468.
Distinct phylogenetic lineages of Mycobacterium tuberculosis (MTB) cause disease in patients of particular genetic ancestry, and elicit different patterns of cytokine and chemokine secretion when cultured with human macrophages in vitro. Circulating and antigen-stimulated concentrations of these inflammatory mediators might therefore be expected to vary significantly between tuberculosis patients of different ethnic origin. Studies to characterise such variation, and to determine whether it relates to host or bacillary factors, have not been conducted. We therefore compared circulating and antigen-stimulated concentrations of 43 inflammatory mediators and 14 haematological parameters (inflammatory profile) in 45 pulmonary tuberculosis patients of African ancestry vs. 83 patients of Eurasian ancestry in London, UK, and investigated the influence of bacillary and host genotype on these profiles. Despite having similar demographic and clinical characteristics, patients of differing ancestry exhibited distinct inflammatory profiles at presentation: those of African ancestry had lower neutrophil counts, lower serum concentrations of CCL2, CCL11 and vitamin D binding protein (DBP) but higher serum CCL5 concentrations and higher antigen-stimulated IL-1 receptor antagonist and IL-12 secretion. These differences associated with ethnic variation in host DBP genotype, but not with ethnic variation in MTB strain. Ethnic differences in inflammatory profile became more marked following initiation of antimicrobial therapy, and immunological correlates of speed of elimination of MTB from the sputum differed between patients of African vs. Eurasian ancestry. Our study demonstrates a hitherto unappreciated degree of ethnic heterogeneity in inflammatory profile in tuberculosis patients that associates primarily with ethnic variation in host, rather than bacillary, genotype. Candidate immunodiagnostics and immunological biomarkers of response to antimicrobial therapy should be derived and validated in tuberculosis patients of different ethnic origin.
Author Summary
Mycobacterium tuberculosis (MTB) is the causative agent of tuberculosis. Genetically distinct strains of MTB cause disease in particular ethnic groups, and these strains vary in their ability to elicit inflammatory responses from antigen-presenting cells in vitro. Circulating and antigen-stimulated concentrations of inflammatory mediators (‘inflammatory profile’) might therefore be expected to differ between tuberculosis patients of different ethnic origin; however, this question has not previously been addressed. We therefore conducted a study to characterise ethnic variation in inflammatory profiles in a cohort of 128 newly-diagnosed tuberculosis patients in London, UK. Patients of African vs. Eurasian ancestry had distinct inflammatory profiles at presentation; differences did not relate to MTB strain variation between groups, but they did associate with ethnic variation in host genotype. Moreover, immunological correlates of the rate of MTB clearance from sputum differed between patients of African vs. Eurasian ancestry. Our findings provide insight into the mechanisms underlying ethnic variation in inflammatory profile in tuberculosis patients, and indicate that candidate immunodiagnostics and immunological biomarkers of response to tuberculosis therapy should be derived and validated in tuberculosis patients of different ethnic origin.
doi:10.1371/journal.ppat.1003468
PMCID: PMC3701709  PMID: 23853590
9.  Comparative Drug Resistance of Mycobacterium abscessus and M. chelonae Isolates from Patients with and without Cystic Fibrosis in the United Kingdom 
Journal of Clinical Microbiology  2013;51(1):217-223.
The isolation of rapidly growing mycobacteria (RGM), particularly Mycobacterium abscessus, from individuals with cystic fibrosis (CF) is associated with poor clinical outcome due to broad drug resistance and the difficulty of eradicating the organisms. Susceptibility testing is recommended to guide therapy. A disc diffusion method is used in the United Kingdom, whereas in the United States, the CLSI (Clinical and Laboratory Standards Institute) recommends the broth dilution method. The purpose of this study was to investigate whether the two methods produced comparable drug resistance profiles and to test the hypotheses that the disc diffusion method overscores resistance and that isolates of M. abscessus/M. chelonae from CF patients are more likely than those from non-CF patients to show drug resistance, as a result of CF patients' greater exposure to antibiotic therapy. A total of 82 isolates (58 M. abscessus and 24 M. chelonae isolates) were tested blindly against 15 antimicrobials by broth dilution and the disc diffusion method. Isolates tested by the broth microdilution showed high levels of resistance; susceptibility to amikacin, clarithromycin, tobramycin (only in M. chelonae), and cefoxitin (only in M. abscessus) was shown. Tigecycline results varied widely depending on which breakpoint was used. Agreement between methods for a few drugs (e.g., cefoxitin and amikacin) was poor. Although there were drug resistance differences between CF and non-CF isolates, these did not reach statistical significance. The CLSI method provided more robust breakpoints, standardization, and reproducibility. An analysis of the implementation of the CLSI method demonstrated ease of use and similar drug resistance findings for the two species.
doi:10.1128/JCM.02260-12
PMCID: PMC3536196  PMID: 23135941
10.  Diagnostic Accuracy of the GenoType MTBDRsl Assay for Rapid Diagnosis of Extensively Drug-Resistant Tuberculosis in HIV-Coinfected Patients 
Journal of Clinical Microbiology  2013;51(1):243-248.
The Russian Federation is a high-tuberculosis (TB)-burden country with high rates of Mycobacterium tuberculosis multidrug resistance (MDR) and extensive drug resistance (XDR), especially in HIV-coinfected patients. Rapid and reliable diagnosis for detection of resistance to second-line drugs is vital for adequate patient management. We evaluated the performance of the GenoType MTBDRsl (Hain Lifescience GmbH, Nehren, Germany) assay on smear-positive sputum specimens obtained from 90 HIV-infected MDR TB patients from Russia. Test interpretability was over 98%. Specificity was over 86% for all drugs, while sensitivity varied, being the highest (71.4%) for capreomycin and lowest (9.4%) for kanamycin, probably due to the presence of mutations in the eis gene. The sensitivity of detection of XDR TB was 13.6%, increasing to 42.9% if kanamycin (not commonly used in Western Europe) was excluded. The assay is a highly specific screening tool for XDR detection in direct specimens from HIV-coinfected TB patients but cannot be used to rule out XDR TB.
doi:10.1128/JCM.02513-12
PMCID: PMC3536261  PMID: 23152552
11.  Prevalence of Nontuberculous Mycobacteria in Cystic Fibrosis Clinics, United Kingdom, 2009 
Emerging Infectious Diseases  2013;19(7):1128-1130.
Incidence of pulmonary infection with nontuberculous mycobacteria (NTM) is increasing among persons with cystic fibrosis (CF). We assessed prevalence and management in CF centers in the United Kingdom and found 5.0% of 3,805 adults and 3.3% of 3,317 children had recently been diagnosed with NTM. Of those, 44% of adults and 47% of children received treatment.
doi:10.3201/eid1907.120615
PMCID: PMC3713964  PMID: 23764198
nontuberculous mycobacteria; cystic fibrosis; Mycobacterium avium complex; Mycobacterium absessus complex; bacteria; United Kingdom; tuberculosis and other mycobacteria
12.  Evaluation of Tuberculosis Diagnostics in Children: 2. Methodological Issues for Conducting and Reporting Research Evaluations of Tuberculosis Diagnostics for Intrathoracic Tuberculosis in Children. Consensus From an Expert Panela 
The Journal of Infectious Diseases  2012;205(Suppl 2):S209-S215.
Confirming the diagnosis of childhood tuberculosis is a major challenge. However, research on childhood tuberculosis as it relates to better diagnostics is often neglected because of technical difficulties, such as the slow growth in culture, the difficulty of obtaining specimens, and the diverse and relatively nonspecific clinical presentation of tuberculosis in this age group. Researchers often use individually designed criteria for enrollment, diagnostic classifications, and reference standards, thereby hindering the interpretation and comparability of their findings. The development of standardized research approaches and definitions is therefore needed to strengthen the evaluation of new diagnostics for detection and confirmation of tuberculosis in children.
In this article we present consensus statements on methodological issues for conducting research of Tuberculosis diagnostics among children, with a focus on intrathoracic tuberculosis. The statements are complementary to a clinical research case definition presented in an accompanying publication and suggest a phased approach to diagnostics evaluation; entry criteria for enrollment; methods for classification of disease certainty, including the rational use of culture within the case definition; age categories and comorbidities for reporting results; and the need to use standard operating procedures. Special consideration is given to the performance of microbiological culture in children and we also recommend for alternative methodological approaches to report findings in a standardized manner to overcome these limitations are made. This consensus statement is an important step toward ensuring greater rigor and comparability of pediatric tuberculosis diagnostic research, with the aim of realizing the full potential of better tests for children.
doi:10.1093/infdis/jir879
PMCID: PMC3334504  PMID: 22476719
13.  Control of (Multi)Drug Resistance and Tuberculosis Incidence over 23 Years in the Context of a Well-Supported Tuberculosis Programme in Rural Malawi 
PLoS ONE  2013;8(3):e58192.
Background
The rise in tuberculosis (TB) incidence following generalized HIV epidemics can overwhelm TB control programmes in resource-limited settings, sometimes accompanied by rising rates of drug resistance. This has led to claims that DOTS-based TB control has failed in such settings. However, few studies have described the effect of a sustained and well-supported DOTS programme on TB incidence and drug resistance over a long period. We present long-term trends in incidence and drug resistance in rural Malawi.
Methods
Karonga District in northern Malawi has an adult HIV prevalence of ∼10%. A research group, the Karonga Prevention Study, collaborates with the National Tuberculosis Programme to support core TB control activities. Bacteriological, demographic and clinical (including HIV status) information from all patients starting TB treatment in the District have been recorded since 1988. During that period isolates from each culture-positive TB patient were exported for drug sensitivity testing. Antiretroviral therapy (ART) has been widely available since 2005.
Results
Incidence of new smear-positive adult TB peaked at 124/100,000/year in the mid-90s, but has since fallen to 87/100,000/year. Drug sensitivity information was available for 95% (3132/3307) of all culture-positive cases. Initial resistance to isoniazid was around 6% with no evidence of an increase. Fewer than 1% of episodes involved a multi-drug resistant strain.
Discussion
In this setting with a generalised HIV epidemic and medium TB burden, a well-supported DOTS programme enhanced by routine culture and drug sensitivity testing may well have reduced TB incidence and maintained drug resistance at low levels.
doi:10.1371/journal.pone.0058192
PMCID: PMC3590148  PMID: 23483994
14.  Detection of Resistance to Second-Line Antituberculosis Drugs by Use of the Genotype MTBDRsl Assay: a Multicenter Evaluation and Feasibility Study 
Journal of Clinical Microbiology  2012;50(5):1593-1597.
The rate of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) has been steadily increasing in countries of the former USSR. The availability of rapid and reliable methods for the detection of drug resistance to second-line drugs is vital for adequate patient management. We evaluated the performance of the Genotype MTBDRsl assay compared to that of phenotypic drug susceptibility testing (Becton Dickinson Bactec MGIT 960 system) with a test panel of 200 Mycobacterium tuberculosis isolates at four sites in Eastern Europe. The interpretability of the Genotype MTBDRsl assay was over 95%. The sensitivity for the detection of resistance to fluoroquinolones, ethambutol, amikacin, and capreomycin varied between 77.3% and 92.3%; however, it was much lower for kanamycin (42.7%). The sensitivity for the detection of XDR TB was 22.6%. The test specificity was over 82% for all drugs. The assay presents a good screening tool for the rapid detection of resistance to individual second-line drugs and can be recommended for use in countries with a high burden of MDR/XDR TB. The sensitivity for the detection of kanamycin resistance needs improvement.
doi:10.1128/JCM.00039-12
PMCID: PMC3347138  PMID: 22378910
15.  Common variants at 11p13 are associated with susceptibility to tuberculosis 
Nature genetics  2012;44(3):257-259.
After imputation of data of the 1000 Genomes Project into a genome-wide data set of Ghanaian tuberculosis cases and controls, we identified a resistance locus on chromosome 11p13, downstream of the Wilms' tumour 1 gene. The strongest signal was obtained at SNP rs2057178 (P = 2.63 × 10−9). Replication in Gambian, Indonesian and Russian TB case-control study groups increased the significance level to P = 2.57 × 10−11.
doi:10.1038/ng.1080
PMCID: PMC3427019  PMID: 22306650
16.  A genome wide association study of pulmonary tuberculosis susceptibility in Indonesians 
BMC Medical Genetics  2012;13:5.
Background
There is reason to expect strong genetic influences on the risk of developing active pulmonary tuberculosis (TB) among latently infected individuals. Many of the genome wide linkage and association studies (GWAS) to date have been conducted on African populations. In order to identify additional targets in genetically dissimilar populations, and to enhance our understanding of this disease, we performed a multi-stage GWAS in a Southeast Asian cohort from Indonesia.
Methods
In stage 1, we used the Affymetrix 100 K SNP GeneChip marker set to genotype 259 Indonesian samples. After quality control filtering, 108 cases and 115 controls were analyzed for association of 95,207 SNPs. In stage 2, we attempted validation of 2,453 SNPs with promising associations from the first stage, in 1,189 individuals from the same Indonesian cohort, and finally in stage 3 we selected 251 SNPs from this stage to test TB association in an independent Caucasian cohort (n = 3,760) from Russia.
Results
Our study suggests evidence of association (P = 0.0004-0.0067) for 8 independent loci (nominal significance P < 0.05), which are located within or near the following genes involved in immune signaling: JAG1, DYNLRB2, EBF1, TMEFF2, CCL17, HAUS6, PENK and TXNDC4.
Conclusions
Mechanisms of immune defense suggested by some of the identified genes exhibit biological plausibility and may suggest novel pathways involved in the host containment of infection with TB.
doi:10.1186/1471-2350-13-5
PMCID: PMC3287960  PMID: 22239941
17.  Survival of drug resistant tuberculosis patients in Lithuania: retrospective national cohort study 
BMJ Open  2011;1(2):e000351.
Objective
To establish risk factors influencing survival of patients with multidrug-resistant and extensively drug-resistant tuberculosis (MDR/XDRTB).
Design
All MDR/XDRTB cases (n=1809) reported from 2002 to 2008 in Lithuania with a known outcome were included in the survival analysis.
Results
Median survival for MDRTB and XDRTB patients was 4.1 (95% CI 3.7 to 4.4) and 2.9 (95% CI 2.2 to 3.9) years. In a multivariable analysis adjusting for other patient characteristics, the difference in survival between MDRTB and XDRTB patients was not significant (HR=1.29 (0.91 to 1.81)). Older age (HR=4.80 (3.16 to 7.29)) for 60+ vs <30 years, rural living (HR=1.20 (1.02 to 1.40)), alcohol use (HR=1.49 (1.13 to 1.96)) for alcoholic versus moderate use, unemployment (HR=1.79 (1.31 to 2.46)), lower education levels (HR=1.50 (1.08 to 2.07)) for primary level versus tertiary level, cavitary disease (HR=1.54 (1.29 to 1.83)) and being smear positive at the time of MDR/XDRTB diagnosis (HR=1.47 (1.19 to 1.82)) were associated with poorer survival. HIV positivity significantly affected survival (HR=3.44 (1.92 to 6.19)) for HIV positive versus HIV negative; HR=1.60 (1.28 to 2.01) for HIV not tested versus HIV negative). There was no difference in survival of patients who acquired MDR/XDRTB during treatment compared with patients with primary MDR/XDRTB (HR=1.01 (0.85 to 1.19)). Treatment with a second-line drug improved survival (HR=0.40 (0.34 to 0.47)). In a subgroup with genotyped TB strains, a Beijing family of strains was associated with poorer survival (HR=1.71 (1.19 to 2.47)).
Conclusions
Social factors, rural living, HIV infection and Beijing strain family impact on survival. Survival of MDR/XDRTB patients is short. Rapid drug resistance identification, early administration of appropriate treatment and achieving high cure rates, expansion of HIV testing and antiretroviral treatment are necessary for optimal management of MDR/XDRTB.
Article summary
Article focus
The Baltic States have consistently had one of the world's highest rates of drug resistant tuberculosis (TB) including multidrug- and extensively drug-resistant tuberculosis (MDR/XDRTB) despite an extensive tuberculosis control programmes.
Patients with TB in Lithuania show a relatively high mortality for unknown reasons.
The study analyses factors influencing survival of patients with MDR/XDRTB in Lithuania.
Key messages
Patients with MDR/XDRTB show a poor survival regardless of HIV status, but there was no significant difference between MDRTB and XDRTB.
Patients infected with Beijing TB family strains and co-infected with HIV with underlying social problems have worse survival. However, only few TB patients are tested for HIV.
Addressing accompanying social and health problems (eg, alcohol dependency), access to care in rural settings and expansion of HIV testing and antiretroviral treatment are necessary to improve survival.
Strength and limitations of the study
This is the first national study to analyse 7 years of national surveillance data covering a large cohort of MDR/XDRTB patients.
The study demonstrates the value of long-term survival cohorts, as well as pointing to the absence of comparable UK data.
Limitations: limited data on HIV status (due to the low HIV testing coverage in the early years of the TB programme) and limited genotyping data for the XDRTB isolates.
doi:10.1136/bmjopen-2011-000351
PMCID: PMC3225583  PMID: 22123922
18.  Investigating Transmission of Mycobacterium bovis in the United Kingdom in 2005 to 2008▿ 
Journal of Clinical Microbiology  2011;49(5):1943-1950.
Due to an increase in bovine tuberculosis in cattle in the United Kingdom, we investigated the characteristics of Mycobacterium bovis infection in humans and assessed whether extensive transmission of M. bovis between humans has occurred. A cross-sectional study linking demographic, clinical, and DNA fingerprinting (using 15-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat [MIRU-VNTR] typing) data on cases reported between 2005 and 2008 was undertaken. A total of 129 cases of M. bovis infection in humans were reported over the period, with a decrease in annual incidence from 0.065 to 0.047 cases per 100,000 persons. Most patients were born pre-1960, before widespread pasteurization was introduced (73%), were of white ethnicity (83%), and were born in the United Kingdom (76%). A total of 102 patients (79%) had MIRU-VNTR typing data. A total of 31 of 69 complete MIRU-VNTR profiles formed eight distinct clusters. The overall clustering proportion determined using the n − 1 method was 33%. The largest cluster, comprising 12 cases, was indistinguishable from a previously reported West Midlands outbreak strain cluster and included those cases. This cluster was heterogeneous, having characteristics supporting recent zoonotic and human-to-human transmission as well as reactivation of latent disease. Seven other, smaller clusters identified had demographics supporting recrudescence rather than recent infection. A total of 33 patients had incomplete MIRU-VNTR profiles, of which 11 may have yielded 2 to 6 further small clusters if typed to completion. The incidence of M. bovis in humans in the United Kingdom remains low, and the epidemiology is predominantly that of reactivated disease.
doi:10.1128/JCM.02299-10
PMCID: PMC3122646  PMID: 21430093
19.  Analysis of undiagnosed tuberculosis-related deaths identified at post-mortem among HIV-infected patients in Russia: a descriptive study 
BMC Infectious Diseases  2011;11:276.
Background
Tuberculosis remains a serious public health threat and economic burden in Russia with escalating rates of drug resistance against a background of growing HIV-epidemic. Samara Oblast is one of the regions of the Russian Federation where more than 1% of the population is affected by the HIV-epidemic; almost half of the cases are concentrated in the largely-industrial city of Togliatti with a population of 800 000.
Methods
We conducted a retrospective analysis of errors leading to death of HIV-positive patients in general health care hospitals in Togliatti, Russia, in 2008. All (n = 29) cases when tuberculosis was established at autopsy as a cause of death were included.
Results
Median length of hospital stay was 20 days; in 11 cases the death occurred within the first 24 hours of admission. All cases were known to be HIV-positive prior to admission, however HAART was not initiated for any case, and no relevant tests to assess severity of immunosupression were performed despite their availability. No appropriate diagnostic algorithms were applied to confirm tuberculosis. Major gaps were identified in the work of hospital and consulting physicians including insufficient records keeping. In almost all patients earlier regular HIV-relevant tests were not performed due to poor compliance of patients, many of whom abused alcohol and drugs.
Conclusions
We conclude that introduction of prompt and accurate diagnostics tests, adequate treatment protocols and intensive training of physicians in management of AIDS and TB is vital. This should include reviewing standards of care for HIV-positive individuals with accompanying social problems.
doi:10.1186/1471-2334-11-276
PMCID: PMC3215671  PMID: 22008481
20.  The Role of the Interferon Gamma Release Assay in Assessing Recent Tuberculosis Transmission in a Hospital Incident 
PLoS ONE  2011;6(6):e20770.
In 2007, an extensive contact screening investigation into onward transmission of tuberculosis was instigated at a hospital in Northern Ireland following diagnosis of pulmonary multi-drug resistant TB in a healthcare worker. Interferon gamma release assays (IGRAs) were used to test 333 patients and 98 staff. We investigated for evidence of onward transmission and recent infection based on analysis of clinical, demographic and IGRA data. We also described within-patient variability of IGRA results. Among patients and staff, increasing age of patients was the only factor associated with IGRA positivity. Greatest within-subject variability of IU/mL in serially-tested patients/staff was seen in those with a positive IGRA test and this did not correlate with increased exposure to the index case. IGRA positivity being largely explained by increasing age in patients and previous TB contact in staff lends weight to the conclusion that IGRA positivity reflected previous infection rather than recent transmission.
doi:10.1371/journal.pone.0020770
PMCID: PMC3113857  PMID: 21695149
21.  Survival of Civilian and Prisoner Drug-Sensitive, Multi- and Extensive Drug- Resistant Tuberculosis Cohorts Prospectively Followed in Russia 
PLoS ONE  2011;6(6):e20531.
Objective and Methods
A long-term observational study was conducted in Samara, Russia to assess the survival and risk factors for death of a cohort of non-multidrug resistant tuberculosis (non-MDRTB) and multidrug resistant tuberculosis (MDRTB) civilian and prison patients and a civilian extensive drug-resistant tuberculosis (XDRTB) cohort.
Results
MDRTB and XDRTB rates of 54.8% and 11.1% were identified in the region. Half (50%) of MDRTB patients and the majority of non-MDRTB patients (71%) were still alive at 5 years. Over half (58%) of the patients died within two years of establishing a diagnosis of XDRTB. In the multivariate analysis, retreatment (HR = 1.61, 95%CI 1.04, 2.49) and MDRTB (HR = 1.67, 95%CI 1.17, 2.39) were significantly associated with death within the non-MDR/MDRTB cohort. The effect of age on survival was relatively small (HR = 1.01, 95%CI 1.00, 1.02). No specific factor affected survival of XDRTB patients although median survival time for HIV-infected versus HIV-negative patients from this group was shorter (185 versus 496 days). The majority of MDRTB and XDRTB strains (84% and 92% respectively) strains belonged to the Beijing family. Mutations in the rpoB (codon 531 in 81/92; 88.8%), katG (mutation S315T in 91/92, 98.9%) and inhA genes accounted for most rifampin and isoniazid resistance respectively, mutations in the QRDR region of gyrA for most fluroquinolone resistance (68/92; 73.5%).
Conclusions
Alarmingly high rates of XDRTB exist. Previous TB treatment cycles and MDR were significant risk factors for mortality. XDRTB patients' survival is short especially for HIV-infected patients. Beijing family strains comprise the majority of drug-resistant strains.
doi:10.1371/journal.pone.0020531
PMCID: PMC3112205  PMID: 21695213
22.  Molecular Epidemiology of Mycobacterium tuberculosis, Buenos Aires, Argentina 
Emerging Infectious Diseases  2011;17(3):528-531.
To analyze the molecular epidemiology of Mycobacterium tuberculosis strains at a hospital in Buenos Aires, Argentina, and mutations related to multidrug-resistant and extensively drug-resistant tuberculosis, we conducted a prospective case–control study. Our findings reinforce the value of incorporating already standardized molecular methods for rapidly detecting resistance.
doi:10.3201/eid1703100394
PMCID: PMC3165991  PMID: 21392451
Tuberculosis; tuberculosis and other mycobacteria; molecular epidemiology; drug resistance; Argentina; Mycobacterium tuberculosis; dispatch
23.  Association analysis of the LTA4H gene polymorphisms and pulmonary tuberculosis in 9115 subjects 
Summary
Immunoregulatory eicosanoids have been implicated in protection from mycobacterial infection in cell and animal models. Recently, a study of the zebrafish embryo demonstrated that mutants of the lta4h gene, which encodes the leukotriene A4 hydrolase (LTA4H) enzyme of the eicosanoid pathway, have hypersusceptibility to Mycobacterium marinum infection. It also reported that heterozygosity at the two single nucleotide polymorphisms rs1978331 and rs2660898 located in introns of the LTA4H gene, a human homologue of lta4h, is associated with protection from pulmonary tuberculosis. To replicate this association we genotyped six LTA4H gene polymorphisms in samples from 3703 pulmonary tuberculosis patients and 5412 healthy controls collected in Russia. We found no evidence of the protective effect of heterozygosity at the polymorphisms rs1978331 and rs2660898 (P = 0.29 and 0.49) and no association of the alleles of any of the six polymorphisms (P = 0.13–0.81). These results suggest that common polymorphisms in the LTA4H gene do not play any major role in susceptibility to clinical pulmonary tuberculosis.
doi:10.1016/j.tube.2010.11.001
PMCID: PMC3040790  PMID: 21112816
Tuberculosis; Gene; SNP; Association; Heterozygosity
24.  Increasing reports of non-tuberculous mycobacteria in England, Wales and Northern Ireland, 1995-2006 
BMC Public Health  2010;10:612.
Background
Non-tuberculous mycobacteria have long been identified as capable of causing human disease and the number at risk, due to immune-suppression, is rising. Several reports have suggested incidence to be increasing, yet routine surveillance-based evidence is lacking. We investigated recent trends in, and the epidemiology of, non-tuberculous mycobacterial infections in England, Wales and Northern Ireland, 1995-2006.
Methods
Hospital laboratories voluntarily report non-tuberculous mycobacterial infections to the Health Protection Agency Centre for Infections. Details reported include age and sex of the patient, species, specimen type and source laboratory. All reports were analysed.
Results
The rate of non-tuberculous mycobacteria reports rose from 0.9 per 100,000 population in 1995 to 2.9 per 100,000 in 2006 (1608 reports). Increases were mainly in pulmonary specimens and people aged 60+ years. The most commonly reported species was Mycobacterium avium-intracellulare (43%); M. malmoense and M. kansasii were also commonly reported. M. gordonae showed the biggest increase over the study period rising from one report in 1995 to 153 in 2006. Clinical information was rarely reported.
Conclusions
The number and rate of reports increased considerably between 1995 and 2006, primarily in older age groups and pulmonary specimens. Increases in some species are likely to be artefacts but real changes in more pathogenic species, some of which will require clinical care, should not be excluded. Enhanced surveillance is needed to understand the true epidemiology of these infections and their impact on human health.
doi:10.1186/1471-2458-10-612
PMCID: PMC2964631  PMID: 20950421
25.  Associations between Mycobacterium tuberculosis Strains and Phenotypes 
Emerging Infectious Diseases  2010;16(2):272-280.
This population-based study was used to investigate strong associations between phenotypes and genotypes.
To inform development of tuberculosis (TB) control strategies, we characterized a total of 2,261 Mycobacterium tuberculosis complex isolates by using multiple phenotypic and molecular markers, including polymorphisms in repetitive sequences (spoligotyping and variable-number tandem repeats [VNTRs]) and large sequence and single-nucleotide polymorphisms. The Beijing family was strongly associated with multidrug resistance (p = 0.0001), and VNTR allelic variants showed strong associations with spoligotyping families: >5 copies at exact tandem repeat (ETR) A, >2 at mycobacterial interspersed repetitive unit 24, and >3 at ETR-B associated with the East African–Indian and M. bovis strains. All M. tuberculosis isolates were differentiated into 4 major lineages, and a maximum parsimony tree was constructed suggesting a more complex phylogeny for M. africanum. These findings can be used as a model of pathogen global diversity.
doi:10.3201/eid1602.091032
PMCID: PMC2958017  PMID: 20113558
Tuberculosis and other mycobacteria; microbial evolution; genetic polymorphisms; drug resistance; phenotypes; bacteria; research

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