Evolutionary history and global spread of the Mycobacterium tuberculosis Beijing lineage.

Mycobacterium tuberculosis strains of the Beijing lineage are globally distributed and are associated with the massive spread of multidrug-resistant (MDR) tuberculosis in Eurasia. Here we reconstructed the biogeographical structure and evolutionary history of this lineage by genetic analysis of 4,987 isolates from 99 countries and whole-genome sequencing of 110 representative isolates. We show that this lineage initially originated in the Far East, from where it radiated worldwide in several waves. We detected successive increases in population size for this pathogen over the last 200 years, practically coinciding with the Industrial Revolution, the First World War and HIV epidemics. Two MDR clones of this lineage started to spread throughout central Asia and Russia concomitantly with the collapse of the public health system in the former Soviet Union. Mutations identified in genes putatively under positive selection and associated with virulence might have favored the expansion of the most successful branches of the lineage.

Tuberculosis Mortality and Living Conditions in Bern, Switzerland, 1856-1950.

BACKGROUND Tuberculosis (TB) is a poverty-related disease that is associated with poor living conditions. We studied TB mortality and living conditions in Bern between 1856 and 1950. METHODS We analysed cause-specific mortality based on mortality registers certified by autopsies, and public health reports 1856 to 1950 from the city council of Bern. RESULTS TB mortality was higher in the Black Quarter (550 per 100,000) and in the city centre (327 per 100,000), compared to the outskirts (209 per 100,000 in 1911-1915). TB mortality correlated positively with the number of persons per room (r = 0.69, p = 0.026), the percentage of rooms without sunlight (r = 0.72, p = 0.020), and negatively with the number of windows per apartment (r = -0.79, p = 0.007). TB mortality decreased 10-fold from 330 per 100,000 in 1856 to 33 per 100,000 in 1950, as housing conditions improved, indoor crowding decreased, and open-air schools, sanatoria, systematic tuberculin skin testing of school children and chest radiography screening were introduced. CONCLUSIONS Improved living conditions and public health measures may have contributed to the massive decline of the TB epidemic in the city of Bern even before effective antibiotic treatment became finally available in the 1950s.

Tuberculosis-related mortality in people living with HIV in Europe and Latin America: an international cohort study

BACKGROUND Management of tuberculosis in patients with HIV in eastern Europe is complicated by the high prevalence of multidrug-resistant tuberculosis, low rates of drug susceptibility testing, and poor access to antiretroviral therapy (ART). We report 1 year mortality estimates from a multiregional (eastern Europe, western Europe, and Latin America) prospective cohort study: the TB:HIV study. METHODS Consecutive HIV-positive patients aged 16 years or older with a diagnosis of tuberculosis between Jan 1, 2011, and Dec 31, 2013, were enrolled from 62 HIV and tuberculosis clinics in 19 countries in eastern Europe, western Europe, and Latin America. The primary endpoint was death within 12 months after starting tuberculosis treatment; all deaths were classified according to whether or not they were tuberculosis related. Follow-up was either until death, the final visit, or 12 months after baseline, whichever occurred first. Risk factors for all-cause and tuberculosis-related deaths were assessed using Kaplan-Meier estimates and Cox models. FINDINGS Of 1406 patients (834 in eastern Europe, 317 in western Europe, and 255 in Latin America), 264 (19%) died within 12 months. 188 (71%) of these deaths were tuberculosis related. The probability of all-cause death was 29% (95% CI 26-32) in eastern Europe, 4% (3-7) in western Europe, and 11% (8-16) in Latin America (p<0·0001) and the corresponding probabilities of tuberculosis-related death were 23% (20-26), 1% (0-3), and 4% (2-8), respectively (p<0·0001). Patients receiving care outside eastern Europe had a 77% decreased risk of death: adjusted hazard ratio (aHR) 0·23 (95% CI 0·16-0·31). In eastern Europe, compared with patients who started a regimen with at least three active antituberculosis drugs, those who started fewer than three active antituberculosis drugs were at a higher risk of tuberculosis-related death (aHR 3·17; 95% CI 1·83-5·49) as were those who did not have baseline drug-susceptibility tests (2·24; 1·31-3·83). Other prognostic factors for increased tuberculosis-related mortality were disseminated tuberculosis and a low CD4 cell count. 18% of patients were receiving ART at tuberculosis diagnosis in eastern Europe compared with 44% in western Europe and 39% in Latin America (p<0·0001); 12 months later the proportions were 67% in eastern Europe, 92% in western Europe, and 85% in Latin America (p<0·0001). INTERPRETATION Patients with HIV and tuberculosis in eastern Europe have a risk of death nearly four-times higher than that in patients from western Europe and Latin America. This increased mortality rate is associated with modifiable risk factors such as lack of drug susceptibility testing and suboptimal initial antituberculosis treatment in settings with a high prevalence of drug resistance. Urgent action is needed to improve tuberculosis care for patients living with HIV in eastern Europe. FUNDING EU Seventh Framework Programme.

Red deer as maintenance host for bovine tuberculosis, Alpine region.

To estimate the prevalence of bovine tuberculosis in the Alpine region, we studied the epidemiology of Mycobacterium caprae in wildlife during the 2009-2012 hunting seasons. Free-ranging red deer (Cervus elaphus) were a maintenance host in a hot-spot area, mainly located in Austria.

Tuberculosis in Cape Town: An age-structured transmission model.

BACKGROUND Tuberculosis (TB) is the leading cause of death in South Africa. The burden of disease varies by age, with peaks in TB notification rates in the HIV-negative population at ages 0-5, 20-24, and 45-49 years. There is little variation between age groups in the rates in the HIV-positive population. The drivers of this age pattern remain unknown. METHODS We developed an age-structured simulation model of Mycobacterium tuberculosis (Mtb) transmission in Cape Town, South Africa. We considered five states of TB progression: susceptible, infected (latent TB), active TB, treated TB, and treatment default. Latently infected individuals could be re-infected; a previous Mtb infection slowed progression to active disease. We further considered three states of HIV progression: HIV negative, HIV positive, on antiretroviral therapy. To parameterize the model, we analysed treatment outcomes from the Cape Town electronic TB register, social mixing patterns from a Cape Town community and used literature estimates for other parameters. To investigate the main drivers behind the age patterns, we conducted sensitivity analyses on all parameters related to the age structure. RESULTS The model replicated the age patterns in HIV-negative TB notification rates of Cape Town in 2009. Simulated TB notification rate in HIV-negative patients was 1000/100,000 person-years (pyrs) in children aged <5 years and decreased to 51/100,000 in children 5-15 years. The peak in early adulthood occurred at 25-29 years (463/100,000 pyrs). After a subsequent decline, simulated TB notification rates gradually increased from the age of 30 years. Sensitivity analyses showed that the dip after the early adult peak was due to the protective effect of latent TB and that retreatment TB was mainly responsible for the rise in TB notification rates from the age of 30 years. CONCLUSION The protective effect of a first latent infection on subsequent infections and the faster progression in previously treated patients are the key determinants of the age-structure of TB notification rates in Cape Town.

Standard genotyping overestimates transmission of Mycobacterium tuberculosis among immigrants in a low incidence country.

Immigrants from high tuberculosis (TB) incidence regions are a risk group for TB in low-incidence countries such as Switzerland. In a previous analysis of a nationwide collection of 520 Mycobacterium tuberculosis isolates from 2000-2008, we identified 35 clusters comprising 90 patients based on standard genotyping (24-loci MIRU-VNTR and spoligotyping). Here, we used whole genome sequencing (WGS) to revisit these transmission clusters. Genome-based transmission clusters were defined as isolate pairs separated by ≤12 single nucleotide polymorphisms (SNPs). WGS confirmed 17/35 (49%) MIRU-VNTR clusters; the other 18 clusters contained pairs separated by >12 SNPs. Most transmission clusters (3/4) of Swiss-born patients were confirmed by WGS, as opposed to 25% (4/16) of clusters involving only foreign-born patients. The overall clustering proportion using standard genotyping was 17% (90 patients, 95% confidence interval [CI]: 14-21%), but only 8% (43 patients, 95% CI: 6-11%) using WGS. The clustering proportion was 17% (67/401, 95% CI: 13-21%) using standard genotyping and 7% (26/401, 95% CI: 4-9%) using WGS among foreign-born patients, and 19% (23/119, 95% CI: 13-28%) and 14% (17/119, 95% CI: 9-22%), respectively, among Swiss-born patients. Using weighted logistic regression, we found weak evidence for an association between birth origin and transmission (aOR 2.2, 95% CI: 0.9-5.5, comparing Swiss-born patients to others). In conclusion, standard genotyping overestimated recent TB transmission in Switzerland when compared to WGS, particularly among immigrants from high TB incidence regions, where genetically closely related strains often predominate. We recommend the use of WGS to identify transmission clusters in low TB incidence settings.

Role of FbpA and FbpB in the pathogenesis and mycolyltransferase activity of Mycobacterium tuberculosis

Mycobacterium tuberculosis infects more people worldwide each year than any other single organism. The Antigen 85 Complex, a family of fibronectin-binding proteins (Fbps) found in several species of mycobacteria and possibly involved in host interaction, is considered among the putative virulence factors of M. tuberculosis. These proteins are implicated in the production of trehalose dimycolate (TDM) and arabinogalactan-mycolate (AG-M), two prominent components of the mycobacterium cell wall and potent modulators of the immune system during infection. For these reasons, the principal members of the complex, FbpA and FbpB, were the focus of these studies. The genes encoding these proteins, fbpA and fbpB, were each disrupted by insertion of a kanamycin resistance cassette in a pathogenic strain of M. tuberculosis, H37Rv. Neither mutation affected growth in routine broth culture. Thin layer chromatography analysis of TDM and AG-M showed no difference in content between the parent strain H37Rv and the FbpA- and FbpB-deficient mutants grown under two different culture conditions. However, metabolic radiolabeling of the strains showed that the production of TDM (but not its precursor TMM) was delayed in the FbpA- and FbpB-deficient mutants compared to the parent H37Rv. During this same labeling period, FbpA-deficient mutant LAa1 failed to produce AG-M and in the FpbB-deficient mutant LAb1 production was decreased. In macrophage tissue culture assay, LAa1 failed to multiply when bacteria in early log phase were used to infect monolayers while LAb1 grew like the parent strain. The growth deficiency of LAa1 as well as the deficiencies in TDM and AG-M production were restored by complementing LAa1 with a functional fbpA gene. These results suggest that the FbpA and FbpB proteins are involved in synthesis of TDM (but not its precursor TMM) as well as AG-M. Other members of the complex appear to compensate for defects in synthesis caused by mutation of single genes in the complex over time. Mutation of the FbpA gene causes greater in vivo effect than mutation of the FbpB gene despite very similar deficiencies in the rate of production of mycolate containing molecules on the cell surface. ^

Factors associated with diabetes in tuberculosis patients in Harris County, Texas 1995--2004

Objective. To determine the prevalence and factors associated with diabetes in tuberculosis patients in Harris County, Texas. ^ Background. Tuberculosis and diabetes mellitus are two diseases of immense public health significance. Various epidemiologic studies have established an association between the two conditions. While many studies have identified factors associated with the conditions individually, few have looked at factors associated with their co-occurrence particularly in the United States. Furthermore, most of those studies are hospital-based and may not be representative of the population. The aim of this study was to determine the prevalence and distribution of diabetes among tuberculosis patients in Harris County, Texas and to identify the factors associated with diabetes in tuberculosis. ^ Methods. A population-based case control study was performed using secondary data from the Houston Tuberculosis Initiative (HTI) collected from October 1995 to September 2004. Socio-demographic characteristics and clinical variables were compared between tuberculosis patients with diabetes and non-diabetic tuberculosis patients. Logistic regression analysis was performed to identify associations. Survival at 180 days post tuberculosis diagnosis was assessed by Cox regression. ^ Results. The prevalence of diabetes among the tuberculosis (TB) population was 14.4%. The diabetics (cases) with a mean age 53 ± 13.3 years were older than the non-diabetics (controls) with a mean age of 39 ± 18.5 years (p<0.001). Socio-demographic variables that were independently associated with the risk of diabetes were age (OR 1.04, p<0.001) and Hispanic ethnicity (OR 2.04, p<0.001). Diabetes was associated with an increased risk of pulmonary tuberculosis disease (OR 1.33, p<0.028). Among individuals with pulmonary TB, diabetes was associated with positive sputum acid-fast bacilli (AFB) smear (OR 1.47, p<0.005) and culture (OR 1.83, p<0.018). Diabetics were more likely to have cavitary lung disease than non-diabetics (OR 1.50, p<0.002). After adjustment for age and HIV status, the risk of dying within 180 days of TB diagnosis was significantly increased in the diabetics (HR 1.51, p<0.002). ^ Conclusion. Diabetes mellitus was more prevalent in our tuberculosis patients than in the general population. The tuberculous diabetic may be more infectious and has a higher risk of death. It is therefore imperative to screen diabetics for TB and TB patients for diabetes. ^

Outcomes of tuberculosis positive non-injection drug users in Harris County from October 1995 through September 2004

Background. The population-based Houston Tuberculosis Initiative (HTI) study has enrolled and gathered demographic, social, behavioral, and disease related data on more than 80% of all reported Mycobacterium Tuberculosis (MTB) cases and 90% of all culture positive patients in Houston/Harris County over a 9 year period (from October 1995-September 2004). During this time period 33% (n=1210) of HTI MTB cases have reported a history of drug use. Of those MTB cases reporting a history of drug use, a majority of them (73.6%), are non-injection drug users (NIDUs). ^ Other than HIV, drug use is the single most important risk factor for progression from latent to infectious tuberculosis (TB). In addition, drug use is associated with increased transmission of active TB, as seen by the increased number of clonally related strains or clusters (see definition on page 30) found in this population. The deregulatory effects of drug use on immune function are well documented. Associations between drug use and increased morbidity have been reported since the late 1970's. However, limited research focused on the immunological consequence of non-injection drug use and its relation to tuberculosis infection among TB patients is available. ^ Methods. TB transmission patterns, symptoms, and prevalence of co-morbidities were a focus of this project. Smoking is known to suppress Nitric Oxide (NO) production and interfere with immune function. In order to limit any possible confounding due to smoking two separate analyses were done. Non-injection drug user smokers (NIDU-S) were compared to non-drug user smokers (NDU-S) and non-injection drug user non-smokers (NIDU-NS) were compared to non-drug user non-smokers (NDU-NS) individually. Specifically proportions, chi-square p-values, and (where appropriate) odds ratios with 95% confidence intervals were calculated to assess characteristics and potential associations of co-morbidities and symptoms of TB among NIDUs HTI TB cases. ^ Results. Significant differences in demographic characteristics and risk factors were found. In addition drug users were found to have a decreased risk for cancer, diabetes mellitus, and chronic pulmonary disease. They were at increased risk of having HIV/AIDS diagnosis, liver disease, and trauma related morbidities. Drug users were more likely to have pulmonary TB disease, and a significantly increased amount of clonally related strains of TB or "clusters" were seen in both smokers and non-smoker drug users when compared to their non-drug user counterparts. Drug users are more likely to belong to print groups (clonally related TB strains with matching spoligotypes) including print one and print three and the Beijing family group, s1. Drug users were found to be no more likely to experience drug resistance to TB therapy and were likely to be cured of disease upon completion of therapy. ^ Conclusion. Drug users demographic and behavioral risk factors put them at an increased risk contracting and spreading TB disease throughout the community. Their increased levels of clustering are evidence of recent transmission and the significance of certain print groups among this population indicate the transmission is from within the social family. For these reasons a focus on this "at risk population" is critical to the success of future public health interventions. Successful completion of directly observed therapy (DOT), the tracking of TB outbreaks and incidence through molecular characterization, and increased diagnostic strategies have led to the stabilization of TB incidence in Houston, Harris County over the past 9 years and proven that the Houston Tuberculosis Initiative has played a critical role in the control and prevention of TB transmission. ^

Analysis of Mycobacterium tuberculosis in the state of Texas for rifampin resistance using molecular beacons

Mycobacterium tuberculosis, a bacillus known to cause disease in humans since ancient times, is the etiological agent of tuberculosis (TB). The infection is primarily pulmonary, although other organs may also be affected. The prevalence of pulmonary TB disease in the US is highest along the US-Mexico border, and of the four US states bordering Mexico, Texas had the second highest percentage of cases of TB disease among Mexico-born individuals in 1999 (CDC, 2001). Between the years of 1993 and 1998, the prevalence of drug-resistant (DR) TB was 9.1% among Mexican-born individuals and 4.4% among US-born individuals (CDC, 2001). In the same time period, the prevalence of multi-drug resistant (MDR) TB was 1.4% among Mexican-born individuals and 0.6% among US-born individuals (CDC, 2001). There is a renewed urgency in the quest for faster and more effective screening, diagnosis, and treatment methods for TB due to the resurgence of tuberculosis in the US during the mid-1980s and early 1990s (CDC, 2007a), and the emergence of drug-resistant, multidrug-resistant, and extremely drug-resistant tuberculosis worldwide. Failure to identify DR and MDR-TB quickly leads to poorer treatment outcomes (CDC, 2007b). The recent rise in TB/HIV comorbidity further complicates TB control efforts. The gold standard for identification of DR-TB requires mycobacterial growth in culture, a technique taking up to three weeks, during which time DR/MDR-TB individuals harboring resistant organisms may be receiving inappropriate treatment. The goal of this study was to determine the sensitivity and specificity of real-time quantitative polymerase chain reaction (qPCR) using molecular beacons in the Texas population. qPCR using molecular beacons is a novel approach to detect mycobacterial mutations conferring drug resistance. This technique is time-efficient and has been shown to have high sensitivity and specificity in several populations worldwide. Rifampin (RIF) susceptibility was chosen as the test parameter because strains of M. tuberculosis which are resistant to RIF are likely to also be MDR. Due to its status as a point of entry for many immigrants into the US, control efforts against TB and drug-resistant TB in Texas is a vital component of prevention efforts in the US as a whole. We show that qPCR using molecular beacons has high sensitivity and specificity when compared with culture (94% and 87%, respectively) and DNA sequencing (90% and 96%, respectively). We also used receiver operator curve analysis to calculate cutoff values for the objective determination of results obtained by qPCR using molecular beacons. ^

Role of T cell response during vaccine immunity to tuberculosis

Tuberculosis remains one of the leading causes of death in man due to a single infectious agent. An estimated one-third of the world's population is infected with the causative agent, Mycobacterium tuberculosis (Mtb), despite the availability of the widely used vaccine, BCG. BCG has significantly varying protection rates with the lowest level of protection seen with the most common form of TB, adult pulmonary TB. Thus, numerous studies are being conducted to develop a more efficient vaccine. The ideal candidate vaccine would possess the ability to induce a solid and strong Th1 response, as this is the subset of T cells primarily involved in clearance of the infection. A novel vaccine should also induce such a response that may be recalled and expanded upon subsequent infection. Our group has introduced a mutant of a virulent strain of Mtb which lacks a component of the immunogenic antigen 85 complex (Ag85). Our vaccine, ΔfbpA, does not secrete the fibronectin binding protein Ag85A, and this has shown to lead to its attenuation in both murine macrophages and mice. Previous studies have also proven that ΔfbpA is more protective in mice than BCG against virulent aerosol challenge with Mtb. This study addresses the mechanisms of protection observed with ΔfbpA by phenotyping responding T cells. We first evaluated the ability of dendritic cells to present the mycobacteria to naïve T cells, an in vitro mock of primary immunization. We also measured the response of primed T cells to macrophage-presented mycobacteria to interpret the possible response of a vaccinated host to a boost. We concluded that ΔfbpA can elicit a stronger Th1 response compared to BCG in vitro, and further observed that this enhanced response is at least partly due to the presence of proteins encoded by a region of the genome absent in all strains of BCG. Finally, we observed this heightened Th1 response in the mouse model after primary vaccination and a virulent aerosol challenge. The cytolytic T cell response was also measured after virulent challenge and was found to be superior in the ΔfbpA-treated group when compared to the BCG group. ^

Trends in anti-tuberculosis drug resistance from 2003--2007 at Pham Ngoc Thach tuberculosis and lung disease hospital, Ho Chi Minh City, Vietnam

Vietnam is one of the countries with the highest prevalence and incidence of tuberculosis (TB) in the world (1). Although Vietnam has had many successes in TB control, it still faces the challenge of drug resistant and multidrug-resistant tuberculosis (MDR-TB). MDR-TB appears to be relatively stable, but data on MDR-TB continues to be scarce and routine testing of all isolates for drug susceptibility is not performed under Vietnam's National Tuberculosis Program (6). Pham Ngoc Thach Hospital (PNT), the leading tuberculosis and lung disease hospital in Ho Chi Minh City, serves as a reference hospital and laboratory for both Ho Chi Minh City and the Southern Vietnam region. This study is an unmatched, nested case-control study consisting of a secondary analysis of a previously created dataset composed of drug susceptibility and basic demographic data from a cohort of patients diagnosed with tuberculosis at PNT from 2003 through 2007 in order to calculate the prevalence of resistance among acid-fast bacilli smear-positive patients. The susceptibility records for the years 2003-2004 were not representative of the entire population, but over the years 2005-2007 the investigator found a decrease in resistance to all primary TB drugs on which records were available, as well as MDR-TB. Overall, females showed a higher proportion of resistance to TB drugs than males, and females had a greater likelihood of presenting with MDR-TB than males (OR=1.77). Persons 35-54 had greater likelihood of having MDR-TB than younger and older age groups. Among the population with HIV data, HIV-positivity was associated with greater likelihood of MDR-TB (OR=1.70, 95% CI=0.97-3.11). This study shows that rates of TB drug resistance are high, but declining, in one of Vietnam's largest TB hospitals, and that females and HIV-positive individuals are possible high-risk groups in this population.^