Treatment of tuberculosis in a region with high drug resistance: outcomes, drug resistance amplification and re-infection

Introduction

Emerging antituberculosis drug resistance is a serious threat for tuberculosis (TB) control, especially in Eastern European countries.

Methods

We combined drug susceptibility results and molecular strain typing data with treatment outcome reports to assess the influence of drug resistance on TB treatment outcomes in a prospective cohort of patients from Abkhazia (Georgia). Patients received individualized treatment regimens based on drug susceptibility testing (DST) results. Definitions for antituberculosis drug resistance and treatment outcomes were in line with current WHO recommendations. First and second line DST, and molecular typing were performed in a supranational laboratory for Mycobacterium tuberculosis (MTB) strains from consecutive sputum smear-positive TB patients at baseline and during treatment.

Results

At baseline, MTB strains were fully drug-susceptible in 189/326 (58.0%) of patients. Resistance to at least H or R (PDR-TB) and multidrug-resistance (MDR-TB) were found in 69/326 (21.2%) and 68/326 (20.9%) of strains, respectively. Three MDR-TB strains were also extensively resistant (XDR-TB). During treatment, 3/189 (1.6%) fully susceptible patients at baseline were re-infected with a MDR-TB strain and 2/58 (3.4%) PDR-TB patients became MDR-TB due to resistance amplification. 5/47 (10.6%) MDR- patients became XDR-TB during treatment. Treatment success was observed in 161/189 (85.2%), 54/69 (78.3%) and 22/68 (32.3%) of patients with fully drug susceptible, PDR- and MDR-TB, respectively. Development of ofloxacin resistance was significantly associated with a negative treatment outcome.

Conclusion

In Abkhazia, a region with high prevalence of drug resistant TB, the use of individualized MDR-TB treatment regimens resulted in poor treatment outcomes and XDR-TB amplification. Nosocomial transmission of MDR-TB emphasizes the importance of infection control in hospitals.     

Spread of extensively drug-resistant tuberculosis in KwaZulu-Natal province, South Africa

Background

In 2005 a cluster of 53 HIV-infected patients with extensively drug-resistant tuberculosis (XDR-TB) was detected in the Msinga sub-district, the catchment area for the Church of Scotland Hospital (CoSH) in Tugela Ferry, in KwaZulu-Natal province (KZN), South Africa. KZN is divided into 11 healthcare districts. We sought to determine the distribution of XDR TB cases in the province in relation to population density.

Methods

In this cross-sectional study, the KZN tuberculosis laboratory database was analysed. Results of all patients with a sputum culture positive for Mycobacterium tuberculosis from January 2006 to June 2007 were included. Drug-susceptibility test results for isoniazid, rifampicin, ethambutol, streptomycin, kanamycin and ofloxacin were available for all patients as well as the location of the hospital where their clinical diagnosis was made.

Findings

In total, 20858 patients attending one of 73 hospitals or their adjacent clinics had cultures positive for M. tuberculosis. Of these, 4170 (20%) were MDR-TB cases. Four hundred and forty three (11%) of the MDR tuberculosis cases displayed the XDR tuberculosis susceptibility profile. Only 1429 (34%) of the MDR-TB patients were seen at the provincial referral hospital for treatment. The proportion of XDR-TB amongst culture-confirmed cases was highest in the Msinga sub-district (19.6%), followed by the remaining part of the Umzinyati district (5.9%) and the other 10 districts (1.1%). The number of hospitals with at least one XDR-TB case increased from 18 (25%) to 58 (80%) during the study period.

Interpretation

XDR-TB is present throughout KZN. More than 65% of all diagnosed MDR-TB cases, including XDR-TB patients, were left untreated and likely remained in the community as a source of infection.     

Characterization of the Genetic Diversity of Extensively-Drug Resistant Mycobacterium tuberculosis Clinical Isolates from Pulmonary Tuberculosis Patients in Peru

Background

Peru holds the fourth highest burden of tuberculosis in the Americas. Despite an apparently well-functioning DOTS control program, the prevalence of multidrug resistant tuberculosis (MDR-TB) continues to increase. To worsen this situation, cases of extensively drug resistance tuberculosis (XDR-TB) have been detected. Little information exists about the genetic diversity of drug-susceptible vs. MDR-TB and XDR-TB.

Methods

Cryopreserved samples of XDR strains from 2007 to 2009 (second semester), were identified and collected. Starting from 227 frozen samples, a total of 142 XDR-TB strains of Mycobacterium tuberculosis complex (MTBC; 1 isolate per patient) were retained for this study. Each strain DNA was analyzed by spoligotyping and the 15-loci Mycobacterial Interspersed Repetitive Unit (MIRU-15).

Results

Among the 142 isolates analyzed, only 2 samples (1.41%) could not be matched to any lineage. The most prevalent sublineage was Haarlem (43.66%), followed by T (27.46%), LAM (16.2%), Beijing (9.15%), and X clade (1.41%). Spoligotype analysis identified clustering for 128/142 (90.1%) isolates vs. 49/142 (34.5%) with MIRUs. Of the samples, 90.85% belonged to retreated patients. The drug resistant profile demonstrated that 62.67% showed resistance to injectable drugs capreomycin (CAP) and kanamycin (KAN) vs. 15.5% to CAP alone and 21.8% to KAN alone. The SIT219/T1 and SIT50/H3 were the most prevalent patterns in our study. The spoligoforest analysis showed that SIT53/T1 was at the origin of many of the T lineage strains as well as a big proportion of Haarlem lineage strains (SIT50/H3, followed by SIT47/H1, SIT49/H3, and SIT2375/H1), as opposed to the SIT1/Beijing strains that did not appear to evolve into minor Beijing sublineages among the XDR-TB strains.

Conclusion

In contrast with other Latin-American countries where LAM sublineage is the most predominant, we found the Haarlem to be the most common followed by T sublineage among the XDR-TB strains.     

Programmatically Selected Multidrug-Resistant Strains Drive the Emergence of Extensively Drug-Resistant Tuberculosis in South Africa

Background

South Africa shows one of the highest global burdens of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB). Since 2002, MDR-TB in South Africa has been treated by a standardized combination therapy, which until 2010 included ofloxacin, kanamycin, ethionamide, ethambutol and pyrazinamide. Since 2010, ethambutol has been replaced by cycloserine or terizidone. The effect of standardized treatment on the acquisition of XDR-TB is not currently known.

Methods

We genetically characterized a random sample of 4,667 patient isolates of drug-sensitive, MDR and XDR-TB cases collected from three South African provinces, namely, the Western Cape, Eastern Cape and KwaZulu-Natal. Drug resistance patterns of a subset of isolates were analyzed for the presence of commonly observed resistance mutations.

Results

Our analyses revealed a strong association between distinct strain genotypes and the emergence of XDR-TB in three neighbouring provinces of South Africa. Strains predominant in XDR-TB increased in proportion by more than 20-fold from drug-sensitive to XDR-TB and accounted for up to 95% of the XDR-TB cases. A high degree of clustering for drug resistance mutation patterns was detected. For example, the largest cluster of XDR-TB associated strains in the Eastern Cape, affecting more than 40% of all MDR patients in this province, harboured identical mutations concurrently conferring resistance to isoniazid, rifampicin, pyrazinamide, ethambutol, streptomycin, ethionamide, kanamycin, amikacin and capreomycin.

Conclusions

XDR-TB associated genotypes in South Africa probably were programmatically selected as a result of the standard treatment regimen being ineffective in preventing their transmission. Our findings call for an immediate adaptation of standard treatment regimens for M/XDR-TB in South Africa.     

Fitness of Mycobacterium tuberculosis Strains of the W-Beijing and Non-W-Beijing Genotype

Background

Multidrug resistant tuberculosis (MDR-TB) is a major threat for global tuberculosis control. The W-Beijing Mycobacterium tuberculosis genotype has been associated with drug resistance. Elucidation of the mechanisms underlying this epidemiological finding may have an important role in the control of MDR-TB. The aim of this study was to evaluate the fitness of drug-susceptible and MDR M. tuberculosis strains of the W-Beijing genotype compared with that of Non-W-Beijing strains.

Methodology/Principal Findings

Fitness of M. tuberculosis strains was determined by evaluating the difference in the growth curves obtained in the MGIT960 automated system and assessing the competitive growth capacity between W-Beijing and non-W-Beijing strains. The W-Beijing MDR strains had a significant longer lag phase duration compared to the other strains but did not present a significant fitness cost. When grown in competition they had an advantage only in medium containing 0.1% Tween 80.

Conclusions/Significance

It was not possible to confirm a selective advantage of W-Beijing strains to grow, except for differences in their resistance to Tween 80. Further studies are needed to elucidate the putative advantage of W-Beijing strains compared to other genotypes.     

Multidrug-resistant tuberculosis treatment outcomes in Karakalpakstan, Uzbekistan: treatment complexity and XDR-TB among treatment failures

Background

A pilot programme to treat multidrug-resistant TB (MDR-TB) was implemented in Karakalpakstan, Uzbekistan in 2003. This region has particularly high levels of MDR-TB, with 13% and 40% among new and previously treated cases, respectively.

Methodology

This study describes the treatment process and outcomes for the first cohort of patients enrolled in the programme, between October 2003 and January 2005. Confirmed MDR-TB cases were treated with an individualised, second-line drug regimen based on drug susceptibility test results, while suspected MDR-TB cases were treated with a standardised regimen pending susceptibility results.

Principal Findings

Of 108 MDR-TB patients, 87 were started on treatment during the study period. Of these, 33 (38%) were infected with strains resistant to at least one second-line drug at baseline, but none had initial ofloxacin resistance. Treatment was successful for 54 (62%) patients, with 13 (15%) dying during treatment, 12 (14%) defaulting and 8 (8%) failing treatment. Poor clinical condition and baseline second-line resistance contributed to treatment failure or death. Treatment regimens were changed in 71 (82%) patients due to severe adverse events or drug resistance. Adverse events were most commonly attributed to cycloserine, ethionamide and p-aminosalicylic acid. Extensively drug resistant TB (XDR-TB) was found among 4 of the 6 patients who failed treatment and were still alive in November 2006.

Conclusions

While acceptable treatment success was achieved, the complexity of treatment and the development of XDR-TB among treatment failures are important issues to be addressed when considering scaling up MDR-TB treatment.     

Management of extensively drug-resistant tuberculosis in Peru: cure is possible

Aim

To describe the incidence of extensive drug-resistant tuberculosis (XDR-TB) reported in the Peruvian National multidrug-resistant tuberculosis (MDR-TB) registry over a period of more than ten years and present the treatment outcomes for a cohort of these patients.

Methods

From the Peruvian MDR-TB registry we extracted all entries that were approved for second-line anti-TB treatment between January 1997 and June of 2007 and that had Drug Susceptibility Test (DST) results indicating resistance to both rifampicin and isoniazid (i.e. MDR-TB) in addition to results for at least one fluoroquinolone and one second-line injectable (amikacin, capreomycin and kanamycin).

Results

Of 1,989 confirmed MDR-TB cases with second-line DSTs, 119(6.0%) XDR-TB cases were detected between January 1997 and June of 2007. Lima and its metropolitan area account for 91% of cases, a distribution statistically similar to that of MDR-TB. A total of 43 XDR-TB cases were included in the cohort analysis, 37 of them received ITR. Of these, 17(46%) were cured, 8(22%) died and 11(30%) either failed or defaulted treatment. Of the 14 XDR-TB patients diagnosed as such before ITR treatment initiation, 10 (71%) were cured and the median conversion time was 2 months.

Conclusion

In the Peruvian context, with long experience in treating MDR-TB and low HIV burden, although the overall cure rate was poor, a large proportion of XDR-TB patients can be cured if DST to second-line drugs is performed early and treatment is delivered according to the WHO Guidelines.     

The Beijing genotype and drug resistant tuberculosis in the Aral Sea region of Central Asia

Background

After the collapse of the Soviet Union, dramatically increasing rates of tuberculosis and multidrug-resistant tuberculosis (MDR-TB) have been reported from several countries. This development has been mainly attributed to the widespread breakdown of TB control systems and declining socio-economic status. However, recent studies have raised concern that the Beijing genotype of Mycobacterium tuberculosis might be contributing to the epidemic through its widespread presence and potentially enhanced ability to acquire resistance.

Methods

A total of 397 M. tuberculosis strains from a cross sectional survey performed in the Aral Sea region in Uzbekistan and Turkmenistan have been analysed by drug susceptibility testing, IS6110 fingerprinting, and spoligotyping.

Results

Fifteen isolates showed mixed banding patterns indicating simultaneous infection with 2 strains. Among the remaining 382 strains, 152 (40%) were grouped in 42 clusters with identical fingerprint and spoligotype patterns. Overall, 50% of all isolates were Beijing genotype, with 55% of these strains appearing in clusters compared to 25% of non-Beijing strains. The percentage of Beijing strains increased with increasing drug resistance among both new and previously treated patients; 38% of fully-susceptible isolates were Beijing genotype, while 75% of MDR-TB strains were of the Beijing type.

Conclusion

The Beijing genotype is a major cause of tuberculosis in this region, it is strongly associated with drug resistance, independent of previous tuberculosis treatment and may be strongly contributing to the transmission of MDR-TB. Further investigation around the consequences of Beijing genotype infection for both tuberculosis transmission and outcomes of standard short course chemotherapy are urgently needed.     

Comparative genomic analysis of Mycobacterium tuberculosis clinical isolates

Background

Due to excessive antibiotic use, drug-resistant Mycobacterium tuberculosis has become a serious public health threat and a major obstacle to disease control in many countries. To better understand the evolution of drug-resistant M. tuberculosis strains, we performed whole genome sequencing for 7 M. tuberculosis clinical isolates with different antibiotic resistance profiles and conducted comparative genomic analysis of gene variations among them.

Results

We observed that all 7 M. tuberculosis clinical isolates with different levels of drug resistance harbored similar numbers of SNPs, ranging from 1409–1464. The numbers of insertion/deletions (Indels) identified in the 7 isolates were also similar, ranging from 56 to 101. A total of 39 types of mutations were identified in drug resistance-associated loci, including 14 previously reported ones and 25 newly identified ones. Sixteen of the identified large Indels spanned PE-PPE-PGRS genes, which represents a major source of antigenic variability. Aside from SNPs and Indels, a CRISPR locus with varied spacers was observed in all 7 clinical isolates, suggesting that they might play an important role in plasticity of the M. tuberculosis genome. The nucleotide diversity (Л value) and selection intensity (dN/dS value) of the whole genome sequences of the 7 isolates were similar. The dN/dS values were less than 1 for all 7 isolates (range from 0.608885 to 0.637365), supporting the notion that M. tuberculosis genomes undergo purifying selection. The Л values and dN/dS values were comparable between drug-susceptible and drug-resistant strains.

Conclusions

In this study, we show that clinical M. tuberculosis isolates exhibit distinct variations in terms of the distribution of SNP, Indels, CRISPR-cas locus, as well as the nucleotide diversity and selection intensity, but there are no generalizable differences between drug-susceptible and drug-resistant isolates on the genomic scale. Our study provides evidence strengthening the notion that the evolution of drug resistance among clinical M. tuberculosis isolates is clearly a complex and diversified process.

Electronic supplementary material

The online version of this article (doi: 10.1186/1471-2164-15-469) contains supplementary material, which is available to authorized users.     

High Frequency of Resistance,Lack of Clinical Benefit,and Poor Outcomes in Capreomycin Treated South African Patients with Extensively Drug-Resistant Tuberculosis

Background

There are limited data about the epidemiology and treatment-related outcomes associated with capreomycin resistance in patients with XDR-TB. Capreomycin achieves high serum concentrations relative to MIC but whether capreomycin has therapeutic benefit despite microbiological resistance remains unclear.

Methods

We reviewed the susceptibility profiles and outcomes associated with capreomycin usage in patients diagnosed with XDR-TB between August 2002 and October 2012 in two provinces of South Africa. Patients whose isolates were genotypically tested for capreomycin resistance were included in the analysis.

Results

Of 178 XDR-TB patients 41% were HIV-infected. 87% (154/178) isolates contained a capreomycin resistance-conferring mutation [80% (143/178) rrs A1401G and 6% (11/178) were heteroresistant (containing both the rrs A1401G mutation and wild-type sequences)]. Previous MDR-TB treatment, prior usage of kanamycin, or strain type was not associated with capreomycin resistance. 92% (163/178) of XDR-TB patients were empirically treated with capreomycin. Capreomycin resistance decreased the odds of sputum culture conversion. In capreomycin sensitive and resistant persons combined weight at diagnosis was the only independent predictor for survival (p=<0.001). By contrast, HIV status and use of co-amoxicillin/clavulanic acid were independent predictors of mortality (p=<0.05). Capreomycin usage was not associated with survival or culture conversion when the analysis was restricted to those whose isolates were resistant to capreomycin.

Conclusion

In South Africa the frequency of capreomycin conferring mutations was extremely high in XDR-TB isolates. In those with capreomycin resistance there appeared to be no therapeutic benefit of using capreomycin. These data inform susceptibility testing and the design of treatment regimens for XDR-TB in TB endemic settings.     

Is HIV Infection a Risk Factor for Multi-Drug Resistant Tuberculosis? A Systematic Review

Background

Tuberculosis (TB) is an important cause of human suffering and death. Human immunodeficiency virus (HIV), multi-drug resistant TB (MDR-TB), and extensive drug resistant tuberculosis (XDR-TB) have emerged as threats to TB control. The association between MDR-TB and HIV infection has not yet been fully investigated. We conducted a systematic review and meta-analysis to summarize the evidence on the association between HIV infection and MDR-TB.

Methods and Results

Original studies providing Mycobacterium tuberculosis resistance data stratified by HIV status were identified using MEDLINE and ISI Web of Science. Crude MDR-TB prevalence ratios were calculated and analyzed by type of TB (primary or acquired), region and study period. Heterogeneity across studies was assessed, and pooled prevalence ratios were generated if appropriate. No clear association was found between MDR-TB and HIV infection across time and geographic locations. MDR-TB prevalence ratios in the 32 eligible studies, comparing MDR-TB prevalence by HIV status, ranged from 0.21 to 41.45. Assessment by geographical region or study period did not reveal noticeable patterns. The summary prevalence ratios for acquired and primary MDR-TB were 1.17 (95% CI 0.86, 1.6) and 2.72 (95% CI 2.03, 3.66), respectively. Studies eligible for review were few considering the size of the epidemics. Most studies were not adjusted for confounders and the heterogeneity across studies precluded the calculation of a meaningful overall summary measure.

Conclusions

We could not demonstrate an overall association between MDR-TB and HIV or acquired MDR-TB and HIV, but our results suggest that HIV infection is associated with primary MDR-TB. Future well-designed studies and surveillance in all regions of the world are needed to better clarify the relationship between HIV infection and MDR-TB.     

Optimizing multiplex SNP-based data analysis for genotyping of Mycobacterium tuberculosis isolates

Background

Multiplex ligation-dependent probe amplification (MLPA) is a powerful tool to identify genomic polymorphisms. We have previously developed a single nucleotide polymorphism (SNP) and large sequence polymorphisms (LSP)-based MLPA assay using a read out on a liquid bead array to screen for 47 genetic markers in the Mycobacterium tuberculosis genome. In our assay we obtain information regarding the Mycobacterium tuberculosis lineage and drug resistance simultaneously. Previously we called the presence or absence of a genotypic marker based on a threshold signal level. Here we present a more elaborate data analysis method to standardize and streamline the interpretation of data generated by MLPA. The new data analysis method also identifies intermediate signals in addition to classification of signals as positive and negative. Intermediate calls can be informative with respect to identifying the simultaneous presence of sensitive and resistant alleles or infection with multiple different Mycobacterium tuberculosis strains.

Results

To validate our analysis method 100 DNA isolates of Mycobacterium tuberculosis extracted from cultured patient material collected at the National TB Reference Laboratory of the National Center for Tuberculosis and Lung Diseases in Tbilisi, Republic of Georgia were tested by MLPA. The data generated were interpreted blindly and then compared to results obtained by reference methods. MLPA profiles containing intermediate calls are flagged for expert review whereas the majority of profiles, not containing intermediate calls, were called automatically. No intermediate signals were identified in 74/100 isolates and in the remaining 26 isolates at least one genetic marker produced an intermediate signal.

Conclusion

Based on excellent agreement with the reference methods we conclude that the new data analysis method performed well. The streamlined data processing and standardized data interpretation allows the comparison of the Mycobacterium tuberculosis MLPA results between different experiments. All together this will facilitate the implementation of the MLPA assay in different settings.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-572) contains supplementary material, which is available to authorized users.     

Mycobacterium tuberculosis Strains Potentially Involved in the TB Epidemic in Sweden a Century Ago

A hundred years ago the prevalence of tuberculosis (TB) in Sweden was one of the highest in the world. In this study we conducted a population-based search for distinct strains of Mycobacterium tuberculosis complex isolated from patients born in Sweden before 1945. Many of these isolates represent the M. tuberculosis complex population that fueled the TB epidemic in Sweden during the first half of the 20^th century.

Methods

Genetic relationships between strains that caused the epidemic and present day strains were studied by spoligotyping and restriction fragment length polymorphism.

Results

The majority of the isolates from the elderly population were evolutionary recent Principal Genetic Group (PGG)2/3 strains (363/409 or 88.8%), and only a low proportion were ancient PGG1 strains (24/409 or 5.9%). Twenty-two were undefined. The isolates demonstrated a population where the Euro-American superlineage dominated; in particular with Haarlem (41.1%) and T (37.7%) spoligotypes and only 21.2% belonged to other spoligotype families. Isolates from the elderly population clustered much less frequently than did isolates from a young control group population.

Conclusions

A closely knit pool of PGG2/3 strains restricted to Sweden and its immediate neighbours appears to have played a role in the epidemic, while PGG1 strains are usually linked to migrants in todaýs Sweden. Further studies of these outbreak strains may give indications of why the epidemic waned.     

Serious Treatment Related Adverse Drug Reactions amongst Anti-Retroviral Na?ve MDR-TB Patients

Background

Globally treatment outcomes for multidrug-resistant Mycobacterium tuberculosis (MDR-TB) remain poor and this is compounded by high drug toxicity. Little is known about the influence of adverse drug reactions (ADRs) on treatment outcomes in South Africa.

Methods

We evaluated the impact of severe ADRs among a prospective cohort of MDR-TB patients in South Africa (2000–2004). The HIV-infected study participants were anti-retroviral naïve.

Results

Of 2,079 patients enrolled, 1,390 (66.8%) were included in this analysis based on known HIV test results (39.1% HIV-infected). At least one severe ADR was reported in 83 (6.9%) patients with ototoxicity being the most frequent ADR experienced (38.9%).

Conclusions

We found that being HIV-infected but antiretroviral naïve did not increase occurrence of SADRs in patients on second-line anti-tuberculosis drugs. Early screening and proactive management of ADRs in this patient population is essential, especially given the rollout of decentralized care and the potential for overlapping toxicity of concomitant MDR-TB and HIV treatment.     

High Prevalence of inhA Promoter Mutations among Patients with Drug-Resistant Tuberculosis in KwaZulu-Natal,South Africa

Background

Drug-resistant tuberculosis (TB) remains extremely difficult to treat because there are often few remaining active medications and limited diagnostic options to detect resistance. Resistance to isoniazid is typically caused by mutations in either katG or the inhA promoter. inhA mutations confer low-level resistance to isoniazid and cross-resistance to ethionamide while katG mutations confer high-level isoniazid resistance and no cross-resistance. Line Probe Assays (LPAs) that detect mutations in katG and inhA are currently performed on all positive TB cultures in KwaZulu-Natal province, South Africa, but the frequency of inhA mutations in drug-resistant TB patients has not been examined.

Methods

We sought to determine the proportion of patients who could potentially benefit from high-dose isoniazid and who may be resistant to ethionamide. We reviewed 994 LPA (Hain MTBDRplus) results at the TB reference laboratory in KwaZulu-Natal to determine the frequency of mutations in either katG or the inhA promoter. We stratified these results by drug-resistance category (i.e., MDR-TB, pre-XDR-TB, and XDR-TB) as determined by phenotypic drug-susceptibility testing.

Results

Among MDR- and XDR-TB isolates, the prevalence of inhA mutations without a concurrent katG mutation was 14.8% and 10.3% respectively. The prevalence of inhA mutations with OR without a katG mutation was 30.3% and 82.8%, respectively.

Conclusion

More than 10% of patients with MDR- and XDR-TB may benefit from high-dose isoniazid. Although ethionamide is empirically included in all MDR- and XDR-TB regimens, nearly a third of MDR-TB patients and a majority of XDR-TB patients likely have resistance to ethionamide. Laboratories performing line probe assays should report specific band patterns so that clinicians may adjust treatment regimens accordingly.     

Genetic diversity of Mycobacterium tuberculosis isolates from Tibetans in Tibet, China

Background

Tuberculosis (TB) is a serious health problem in Tibet where Tibetans are the major ethnic group. Although genotyping of Mycobacterium tuberculosis (M. tuberculosis) isolates is a valuable tool for TB control, our knowledge of population structure of M. tuberculosis circulating in Tibet is limited.

Methodology/Principal Findings

In our study, a total of 576 M. tuberculosis isolates from Tibetans in Tibet, China, were analyzed via spoligotyping and 24-locus MIRU-VNTR. The Beijing genotype was the most prevalent family (90.63%, n = 522). Shared-type (ST) 1 was the most dominant genotype (88.89%, n = 512). We found that there was no association between the Beijing genotype and sex, age and treatment status. In this sample collection, 7 of the 24 MIRU-VNTR loci were highly or moderately discriminative according to their Hunter-Gaston discriminatory index. An informative set of 12 loci had similar discriminatory power with 24 loci set.

Conclusions/Significance

The population structure of M. tuberculosis isolates in Tibetans is homogeneous and dominated by Beijing genotype. The analysis of 24-locus MIRU-VNTR data might be useful to select appropriate VNTR loci for the genotyping of M. tuberculosis.     

Alarming Levels of Drug-Resistant Tuberculosis in HIV-Infected Patients in Metropolitan Mumbai,India

Background

Drug-resistant tuberculosis (DR-TB) is a looming threat to tuberculosis control in India. However, no countrywide prevalence data are available. The burden of DR-TB in HIV-co-infected patients is likewise unknown. Undiagnosed and untreated DR-TB among HIV-infected patients is a major cause of mortality and morbidity. We aimed to assess the prevalence of DR-TB (defined as resistance to any anti-TB drug) in patients attending public antiretroviral treatment (ART) centers in greater metropolitan Mumbai, India.

Methods

A cross-sectional survey was conducted among adults and children ART-center attendees. Smear microscopy, culture and drug-susceptibility-testing (DST) against all first and second-line TB-drugs using phenotypic liquid culture (MGIT) were conducted on all presumptive tuberculosis patients. Analyses were performed to determine DR-TB prevalence and resistance patterns separately for new and previously treated, culture-positive TB-cases.

Results

Between March 2013 and January 2014, ART-center attendees were screened during 14135 visits, of whom 1724 had presumptive TB. Of 1724 attendees, 72 (4%) were smear-positive and 202 (12%) had a positive culture for Mycobacterium tuberculosis. Overall DR-TB was diagnosed in 68 (34%, 95% CI: 27%–40%) TB-patients. The proportions of DR-TB were 25% (29/114) and 44% (39/88) among new and previously treated cases respectively. The patterns of DR-TB were: 21% mono-resistant, 12% poly-resistant, 38% multidrug-resistant (MDR-TB), 21% pre-extensively-drug-resistant (MDR-TB plus resistance to either a fluoroquinolone or second-line injectable), 6% extensively drug-resistant (XDR-TB) and 2% extremely drug-resistant TB (XDR-TB plus resistance to any group-IV/V drug). Only previous history of TB was significantly associated with the diagnosis of DR-TB in multivariate models.

Conclusion

The burden of DR-TB among HIV-infected patients attending public ART-centers in Mumbai was alarmingly high, likely representing ongoing transmission in the community and health facilities. These data highlight the need to promptly diagnose drug-resistance among all HIV-infected patients by systematically offering access to first and second-line DST to all patients with ‘presumptive TB’ rather than ‘presumptive DR-TB’ and tailor the treatment regimen based on the resistance patterns.     

The Balance of Apoptotic and Necrotic Cell Death in Mycobacterium tuberculosis Infected Macrophages Is Not Dependent on Bacterial Virulence

Background

An important mechanism of Mycobacterium tuberculosis pathogenesis is the ability to control cell death pathways in infected macrophages: apoptotic cell death is bactericidal, whereas necrotic cell death may facilitate bacterial dissemination and transmission.

Methods

We examine M.tuberculosis control of spontaneous and chemically induced macrophage cell death using automated confocal fluorescence microscopy, image analysis, flow cytometry, plate-reader based vitality assays, and M.tuberculosis strains including H37Rv, and isogenic virulent and avirulent strains of the Beijing lineage isolate GC1237.

Results

We show that bacterial virulence influences the dynamics of caspase activation and the total level of cytotoxicity. We show that the powerful ability of M.tuberculosis to inhibit exogenously stimulated apoptosis is abrogated by loss of virulence. However, loss of virulence did not influence the balance of macrophage apoptosis and necrosis – both virulent and avirulent isogenic strains of GC1237 induced predominantly necrotic cell death compared to H37Rv which induced a higher relative level of apoptosis.

Conclusions

This reveals that macrophage necrosis and apoptosis are independently regulated during M. tuberculosis infection of macrophages. Virulence affects the level of host cell death and ability to inhibit apoptosis but other strain-specific characteristics influence the ultimate mode of host cell death and alter the balance of apoptosis and necrosis.     

Spoligotyping and drug resistance analysis of Mycobacterium tuberculosis strains from national survey in China

Background

Tuberculosis (TB), caused by Mycobacterium tuberculosis complex (MTBC), is one of the major causes of death in the world today. Although China has the second largest global case rate of tuberculosis, a systematic study of TB prevalence in China has not been completed. From 2006 to 2007, the base line surveillance of tuberculosis was carried out by Ministry of Health, and more than 4000 representative strains were selected from 31 provinces in China.

Methodology/Principal Findings

The aim of the present research was to survey the genotypes of representative Mycobacterium tuberculosis (M. tuberculosis) strains from China using spacer oligonucleotide typing (spoligotyping), and to analyze the relationship between genotype and drug resistance for the first time. A total of 4017 clinical isolates were collected from 2007 to 2008 throughout China. Among those M. tuberculosis isolates, 2500 (62.2%) isolates were Beijing genotypes. The percentage of Beijing genotypes in northern China was higher than in southern China (76.5% vs. 53.2%). Additionally, the frequencies of rifampin-resistant, ofloxacin-resistant and multidrug-resistant isolates were significantly higher in Beijing genotype strains than non-Beijing strains. Furthermore, a novel genotype named “China Southern genotype (CS)” was only isolated from Fujian and Guangdong provinces. Hence, it is very practical to uncover the reason for prevalence of the CS type in southern China.

Conclusions/Significance

In conclusion, Beijing family genotypes were still the predominant genotype throughout China, which exhibited a greater correlation with rifampin-resistance, ofloxacin-resistance and MDR phenotypes than other TB spoligotypes, and some regions of China showed several unique characters in the distribution of M. tuberculosis genotypes. Our research represents an important contribution for the TB control and research community, which completes broad pictures on drug resistance levels and distribution of M. tuberculosis strain types over China.     

The Impact of Mouse Passaging of Mycobacterium tuberculosis Strains prior to Virulence Testing in the Mouse and Guinea Pig Aerosol Models

Background

It has been hypothesized that the virulence of lab-passaged Mycobacterium tuberculosis and recombinant M. tuberculosis mutants might be reduced due to multiple in vitro passages, and that virulence might be augmented by passage of these strains through mice before quantitative virulence testing in the mouse or guinea pig aerosol models.

Methodology/Principal Findings

By testing three M. tuberculosis H37Rv samples, one deletion mutant, and one recent clinical isolate for survival by the quantitative organ CFU counting method in mouse or guinea pig aerosol or intravenous infection models, we could discern no increase in bacterial fitness as a result of passaging of M. tuberculosis strains in mice prior to quantitative virulence testing in two animal models. Surface lipid expression as assessed by neutral red staining and thin-layer chromatography for PDIM analysis also failed to identify virulence correlates.

Conclusions/Significance

These results indicate that animal passaging of M. tuberculosis strains prior to quantitative virulence testing in mouse or guinea pig models does not enhance or restore potency to strains that may have lost virulence due to in vitro passaging. It is critical to verify virulence of parental strains before genetic manipulations are undertaken and comparisons are made.