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1.  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.
PMCID: PMC3795631  PMID: 24146924
2.  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.
PMCID: PMC3765611  PMID: 23987891
Diagnosis; Drug resistance; Tuberculosis; Public health; Whole genome sequencing
3.  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.
PMCID: PMC3701709  PMID: 23853590
4.  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.
PMCID: PMC3536196  PMID: 23135941
5.  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.
PMCID: PMC3536261  PMID: 23152552
6.  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.
PMCID: PMC3713964  PMID: 23764198
nontuberculous mycobacteria; cystic fibrosis; Mycobacterium avium complex; Mycobacterium absessus complex; bacteria; United Kingdom; tuberculosis and other mycobacteria
7.  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.
PMCID: PMC3334504  PMID: 22476719
8.  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.
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.
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.
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.
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.
PMCID: PMC3590148  PMID: 23483994
9.  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.
PMCID: PMC3347138  PMID: 22378910
10.  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.
PMCID: PMC3427019  PMID: 22306650
11.  A genome wide association study of pulmonary tuberculosis susceptibility in Indonesians 
BMC Medical Genetics  2012;13:5.
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.
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.
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.
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.
PMCID: PMC3287960  PMID: 22239941
12.  Survival of drug resistant tuberculosis patients in Lithuania: retrospective national cohort study 
BMJ Open  2011;1(2):e000351.
To establish risk factors influencing survival of patients with multidrug-resistant and extensively drug-resistant tuberculosis (MDR/XDRTB).
All MDR/XDRTB cases (n=1809) reported from 2002 to 2008 in Lithuania with a known outcome were included in the survival analysis.
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)).
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.
PMCID: PMC3225583  PMID: 22123922
13.  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.
PMCID: PMC3122646  PMID: 21430093
14.  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.
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.
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.
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.
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.
PMCID: PMC3215671  PMID: 22008481
15.  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.
PMCID: PMC3113857  PMID: 21695149
16.  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.
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%).
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.
PMCID: PMC3112205  PMID: 21695213
17.  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.
PMCID: PMC3165991  PMID: 21392451
Tuberculosis; tuberculosis and other mycobacteria; molecular epidemiology; drug resistance; Argentina; Mycobacterium tuberculosis; dispatch
18.  Association analysis of the LTA4H gene polymorphisms and pulmonary tuberculosis in 9115 subjects 
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.
PMCID: PMC3040790  PMID: 21112816
Tuberculosis; Gene; SNP; Association; Heterozygosity
19.  Increasing reports of non-tuberculous mycobacteria in England, Wales and Northern Ireland, 1995-2006 
BMC Public Health  2010;10:612.
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.
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.
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.
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.
PMCID: PMC2964631  PMID: 20950421
20.  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.
PMCID: PMC2958017  PMID: 20113558
Tuberculosis and other mycobacteria; microbial evolution; genetic polymorphisms; drug resistance; phenotypes; bacteria; research
21.  Discriminatory Ability of Hypervariable Variable Number Tandem Repeat Loci in Population-based Analysis of Mycobacterium tuberculosis Strains, London, UK 
Emerging Infectious Diseases  2009;15(10):1609-1616.
Hypervariable loci should be included in standardized panels because they can provide consistent comparable results in multiple settings.
To address conflicting results about the stability of variable number tandem repeat (VNTR) loci and their value in prospective molecular epidemiology of Mycobacterium tuberculosis, we conducted a large prospective population-based analysis of all M. tuberculosis strains in a metropolitan setting. Optimal and reproducible conditions for reliable PCR and fragment analysis, comprising enzymes, denaturing conditions, and capillary temperature, were identified for a panel of hypervariable loci, including 3232, 2163a, 1982, and 4052. A total of 2,261 individual M. tuberculosis isolates and 265 sets of serial isolates were analyzed by using a standardized 15-loci VNTR panel, then an optimized hypervariable loci panel. The discriminative ability of loci varied substantially; locus VNTR 3232 varied the most, with 19 allelic variants and Hunter-Gaston index value of 0.909 unNN. Hypervariable loci should be included in standardized panels because they can provide consistent comparable results at multiple settings, provided the proposed conditions are adhered to.
PMCID: PMC2866407  PMID: 19861054
Tuberculosis; tuberculosis and other mycobacteria; metropolitan area; bacterial genetics; genotyping; reproducibility; bacteria; London; research
22.  An Integrated Approach to Rapid Diagnosis of Tuberculosis and Multidrug Resistance Using Liquid Culture and Molecular Methods in Russia 
PLoS ONE  2009;4(9):e7129.
To analyse the feasibility, cost and performance of rapid tuberculosis (TB) molecular and culture systems, in a high multidrug-resistant TB (MDR TB) middle-income region (Samara, Russia) and provide evidence for WHO policy change.
Performance and cost evaluation was conducted to compare the BACTEC™ MGIT™ 960 system for culture and drug susceptibility testing (DST) and molecular systems for TB diagnosis, resistance to isoniazid and rifampin, and MDR TB identification compared to conventional Lowenstein-Jensen culture assays.
698 consecutive patients (2487 sputum samples) with risk factors for drug-resistant tuberculosis were recruited. Overall M. tuberculosis complex culture positivity rates were 31.6% (787/2487) in MGIT and 27.1% (675/2487) in LJ (90.5% and 83.2% for smear-positive specimens). In total, 809 cultures of M. tuberculosis complex were isolated by any method. Median time to detection was 14 days for MGIT and 36 days for LJ (10 and 33 days for smear positive specimens) and indirect DST in MGIT took 9 days compared to 21 days on LJ. There was good concordance between DST on LJ and MGIT (96.8% for rifampin and 95.6% for isoniazid). Both molecular hybridization assay results correlated well with MGIT DST results, although molecular assays generally yielded higher rates of resistance (by approximately 3% for both isoniazid and rifampin).
With effective planning and logistics, the MGIT 960 and molecular based methodologies can be successfully introduced into a reference laboratory setting in a middle incidence country. High rates of MDR TB in the Russian Federation make the introduction of such assays particularly useful.
PMCID: PMC2744930  PMID: 19774085
23.  Clinical and ultrasonographic features of abdominal tuberculosis in HIV positive adults in Zambia 
The diagnosis of abdominal tuberculosis (TB) is difficult, especially so in health care facilities in developing countries where laparoscopy and colonoscopy are rarely available. There is little information on abdominal TB in HIV infection. We estimated the prevalence and clinical features of abdominal (excluding genitourinary) TB in HIV infected adults attending the University Teaching Hospital, Zambia.
We screened 5,609 medical inpatients, and those with fever, weight loss, and clinical features suggestive of abdominal pathology were evaluated further. A clinical algorithm was used to specify definitive investigations including laparoscopy or colonoscopy, with culture of biopsies and other samples.
Of 140 HIV seropositive patients with these features, 31 patients underwent full evaluation and 22 (71%) had definite or probable abdominal TB. The commonest presenting abdominal features were ascites and persistent tenderness. The commonest ultrasound findings were ascites, para-aortic lymphadenopathy (over 1 cm in size), and hepatomegaly. Abdominal TB was associated with CD4 cell counts over a wide range though 76% had CD4 counts <100 cells/μL.
The clinical manifestations of abdominal TB in our HIV-infected patients resembled the well-established pattern in HIV-uninfected adults. Patients with fever, weight loss, abdominal tenderness, abdominal lymphadenopathy, ascites and/or hepatomegaly in Zambia have a high probability of abdominal TB, irrespective of CD4 cell count.
PMCID: PMC2678139  PMID: 19374757
24.  Performance of the Genotype® MTBDRPlus assay in the diagnosis of tuberculosis and drug resistance in Samara, Russian Federation 
Russia is a high tuberculosis (TB) burden country with a high prevalence of multidrug resistant tuberculosis (MDRTB). Molecular assays for detection of MDRTB on clinical specimens are not widely available in Russia.
We performed an evaluation of the GenoType® MTBDRplus assay (HAIN Lifescience GmbH, Germany) on a total of 168 sputum specimens from individual patients at a public health laboratory in Central Russia, as a model of a middle income site in a region with high levels of drug resistance. Phenotypic drug resistance tests (DST) were performed on cultures derived from the same sputum specimens using the BACTEC 960 liquid media system.
Interpretable GenoType® MTBDRplus results were obtained for 154(91.7%) specimens with readability rates significantly higher in sputum specimens graded 2+ and 3+ compared to 1+ (RR = 1.17 95%CI 1.04–1.32). The sensitivity and specificity of the assay for the detection of rifampicin (RIF) and isoniazid (INH) resistance and MDR was 96.2%, 97.4%, 97.1% and 90.7%, 83.3%, 88.9% respectively. Mutations in codon 531 of the rpoB gene and codon 315 of the katG gene dominated in RIF and INH resistant strains respectively. Disagreements between phenotypical and molecular tests results (12 samples) could be explained by the presence of rare mutations in strains circulating in Russia and simultaneous presence of resistant and sensitive bacilli in sputum specimens (heteroresistance).
High sensitivity, short turnaround times and the potential for screening large numbers of specimens rapidly, make the GenoType® MTBDRplus assay suitable as a first-line screening assay for drug resistant TB.
PMCID: PMC2660363  PMID: 19284561
25.  Mycobacterium bovis Strains Causing Smear-Positive Human Tuberculosis, Southwest Ireland 
Emerging Infectious Diseases  2008;14(12):1931-1934.
Mycobacterium bovis caused 3% of human tuberculosis cases in southwest Ireland during 1998–2006. Of 11 M. bovis strains genotyped, 9 belonged to common animal spoligotypes. Seven strains were from sputum and potential sources of human-centered disease transmission. Ten-locus variable-number tandem repeat typing gave unique strain profiles and would detect disease outbreaks.
PMCID: PMC2634610  PMID: 19046525
Mycobacterium bovis; tuberculosis; molecular epidemiology; respiratory tract infections; dispatch

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