Alexander Sloutsky

Comprehensive Treatment of Extensively Drug-Resistant Tuberculosis

BACKGROUND Extensively drug-resistant tuberculosis has been reported in 45 countries, including countries with limited resources and a high burden of tuberculosis. We describe the management of extensively drug-resistant tuberculosis and treatment outcomes among patients who were referred for individualized outpatient therapy in Peru. METHODS A total of 810 patients were referred for free individualized therapy, including drug treatment, resective surgery, adverse-event management, and nutritional and psychosocial support. We tested isolates from 651 patients for extensively drug-resistant tuberculosis and developed regimens that included five or more drugs to which the infecting isolate was not resistant. RESULTS Of the 651 patients tested, 48 (7.4%) had extensively drug-resistant tuberculosis; the remaining 603 patients had multidrug-resistant tuberculosis. The patients with extensively drug-resistant tuberculosis had undergone more treatment than the other patients (mean [+/-SD] number of regimens, 4.2+/-1.9 vs. 3.2+/-1.6; P<0.001) and had isolates that were resistant to more drugs (number of drugs, 8.4+/-1.1 vs. 5.3+/-1.5; P<0.001). None of the patients with extensively drug-resistant tuberculosis were coinfected with the human immunodeficiency virus (HIV). Patients with extensively drug-resistant tuberculosis received daily, supervised therapy with an average of 5.3+/-1.3 drugs, including cycloserine, an injectable drug, and a fluoroquinolone. Twenty-nine of these patients (60.4%) completed treatment or were cured, as compared with 400 patients (66.3%) with multidrug-resistant tuberculosis (P=0.36). CONCLUSIONS Extensively drug-resistant tuberculosis can be cured in HIV-negative patients through outpatient treatment, even in those who have received multiple prior courses of therapy for tuberculosis.

Genomic analysis identifies targets of convergent positive selection in drug-resistant Mycobacterium tuberculosis.

M. tuberculosis is evolving antibiotic resistance, threatening attempts at tuberculosis epidemic control. Mechanisms of resistance, including genetic changes favored by selection in resistant isolates, are incompletely understood. Using 116 newly sequenced and 7 previously sequenced M. tuberculosis whole genomes, we identified genome-wide signatures of positive selection specific to the 47 drug-resistant strains. By searching for convergent evolution—the independent fixation of mutations in the same nucleotide position or gene—we recovered 100% of a set of known resistance markers. We also found evidence of positive selection in an additional 39 genomic regions in resistant isolates. These regions encode components in cell wall biosynthesis, transcriptional regulation and DNA repair pathways. Mutations in these regions could directly confer resistance or compensate for fitness costs associated with resistance. Functional genetic analysis of mutations in one gene, ponA1, demonstrated an in vitro growth advantage in the presence of the drug rifampicin.

The folate pathway is a target for resistance to the drug para‐aminosalicylic acid (PAS) in mycobacteria

The increasing rate of multidrug‐resistant tuberculosis has led to more use of second‐line antibiotics such as para‐aminosalicylic acid (PAS). The mode of action of PAS remains unclear, and mechanisms of resistance to this drug are undefined. We have isolated PAS‐resistant transposon mutants of Mycobacterium bovis BCG with insertions in the thymidylate synthase (thyA) gene, a critical determinant of intracellular folate levels. BCG thyA mutants have reduced thymidylate synthase activity and are resistant to known inhibitors of the folate pathway. We also find that mutations in thyA are associated with clinical PAS resistance. We have identified PAS‐resistant Mycobacterium tuberculosis isolates from infected patients, which harbour mutations in thyA and show reduced activity of the encoded enzyme. Thus, PAS acts in the folate pathway, and thyA mutations probably represent a mechanism of developing resistance not only to PAS but also to other drugs that target folate metabolism.

First Demonstration of Combined kV/MV Image-Guided Real-Time Dynamic Multileaf-Collimator Target Tracking

PURPOSE For intrafraction motion management, a real-time tracking system was developed by combining fiducial marker-based tracking via simultaneous kilovoltage (kV) and megavoltage (MV) imaging and a dynamic multileaf collimator (DMLC) beam-tracking system. METHODS AND MATERIALS The integrated tracking system employed a Varian Trilogy system equipped with kV/MV imaging systems and a Millennium 120-leaf MLC. A gold marker in elliptical motion (2-cm superior-inferior, 1-cm left-right, 10 cycles/min) was simultaneously imaged by the kV and MV imagers at 6.7 Hz and segmented in real time. With these two-dimensional projections, the tracking software triangulated the three-dimensional marker position and repositioned the MLC leaves to follow the motion. Phantom studies were performed to evaluate time delay from image acquisition to MLC adjustment, tracking error, and dosimetric impact of target motion with and without tracking. RESULTS The time delay of the integrated tracking system was approximately 450 ms. The tracking error using a prediction algorithm was 0.9 +/- 0.5 mm for the elliptical motion. The dose distribution with tracking showed better target coverage and less dose to surrounding region over no tracking. The failure rate of the gamma test (3%/3-mm criteria) was 22.5% without tracking but was reduced to 0.2% with tracking. CONCLUSION For the first time, a complete tracking system combining kV/MV image-guided target tracking and DMLC beam tracking was demonstrated. The average geometric error was less than 1 mm, and the dosimetric error was negligible. This system is a promising method for intrafraction motion management.

Multicenter Evaluation of Ethambutol Susceptibility Testing of Mycobacterium tuberculosis by Agar Proportion and Radiometric Methods

ABSTRACT Reproducibility of ethambutol (EMB) susceptibility test results for Mycobacterium tuberculosis has always been difficult for a variety of reasons, including the narrow range between the critical breakpoint for EMB resistance and the MIC for susceptible strains, borderline results obtained with the BACTEC 460TB method, the presence of microcolonies determined using the agar proportion (AP) method, and a lack of agreement between these two testing methods. To assess the frequency of these problems, M. tuberculosis drug susceptibility data were collected in a multicenter study involving four laboratories. Resistant, borderline, and susceptible isolates were shared among the laboratories to measure interlaboratory test agreement. Half of isolates determined by BACTEC 460TB to be resistant were determined to be susceptible by the AP method. Isolates determined to be resistant to EMB by both BACTEC 460TB and AP methods were almost always resistant to isoniazid. Results from isolates tested by the BACTEC 460TB method with an EMB concentration of 3.75 μg/ml in addition to the standard 2.5 μg/ml did not show improved agreement by the AP method. While these results do not indicate that the AP method is more accurate than the BACTEC 460TB method, laboratories should not report EMB monoresistance based on BACTEC 460TB results alone. Monoresistance to EMB should only be reported following confirmation by the AP method. Microcolonies could not be confirmed as resistant by the BACTEC 460TB method or by repeat testing with the AP method and do not appear to be indicative of resistance.

Dominant role of the sst1 locus in pathogenesis of necrotizing lung granulomas during chronic tuberculosis infection and reactivation in genetically resistant hosts.

Significant host heterogeneity in susceptibility to tuberculosis exists both between and within mammalian species. Using a mouse model of infection with virulent Mycobacterium tuberculosis (Mtb), we identified the genetic locus sst1 that controls the progression of pulmonary tuberculosis in immunocompetent hosts. In this study, we demonstrate that within the complex, multigenic architecture of tuberculosis susceptibility, sst1 functions to control necrosis within tuberculosis lesions in the lungs; this lung-specific sst1 effect is independent of both the route of infection and genetic background of the host. Moreover, sst1-dependent necrosis was observed at low bacterial loads in the lungs during reactivation of the disease after termination of anti-tuberculosis drug therapy. We demonstrate that in sst1-susceptible hosts, nonlinked host resistance loci control both lung inflammation and production of inflammatory mediators by Mtb-infected macrophages. Although interactions of the sst1-susceptible allele with genetic modifiers determine the type of the pulmonary disease progression, other resistance loci do not abolish lung necrosis, which is, therefore, the core sst1-dependent phenotype. Sst1-susceptible mice from tuberculosis-resistant and -susceptible genetic backgrounds reproduce a clinical spectrum of pulmonary tuberculosis and may be used to more accurately predict the efficacy of anti-tuberculosis interventions in genetically heterogeneous human populations.

Aggressive Regimens for Multidrug-Resistant Tuberculosis Decrease All-Cause Mortality

Rationale A better understanding of the composition of optimal treatment regimens for multidrug-resistant tuberculosis (MDR-TB) is essential for expanding universal access to effective treatment and for developing new therapies for MDR-TB. Analysis of observational data may inform the definition of an optimized regimen. Objectives This study assessed the impact of an aggressive regimen–one containing at least five likely effective drugs, including a fluoroquinolone and injectable–on treatment outcomes in a large MDR-TB patient cohort. Methods This was a retrospective cohort study of patients treated in a national outpatient program in Peru between 1999 and 2002. We examined the association between receiving an aggressive regimen and the rate of death. Measurements and Main Results In total, 669 patients were treated with individualized regimens for laboratory-confirmed MDR-TB. Isolates were resistant to a mean of 5.4 (SD 1.7) drugs. Cure or completion was achieved in 66.1% (442) of patients; death occurred in 20.8% (139). Patients who received an aggressive regimen were less likely to die (crude hazard ratio [HR]: 0.62; 95% CI: 0.44,0.89), compared to those who did not receive such a regimen. This association held in analyses adjusted for comorbidities and indicators of severity (adjusted HR: 0.63; 95% CI: 0.43,0.93). Conclusions The aggressive regimen is a robust predictor of MDR-TB treatment outcome. TB policy makers and program directors should consider this standard as they design and implement regimens for patients with drug-resistant disease. Furthermore, the aggressive regimen should be considered the standard background regimen when designing randomized trials of treatment for drug-resistant TB.

RNA signatures allow rapid identification of pathogens and antibiotic susceptibilities

With rising rates of drug-resistant infections, there is a need for diagnostic methods that rapidly can detect the presence of pathogens and reveal their susceptibility to antibiotics. Here we propose an approach to diagnosing the presence and drug-susceptibility of infectious diseases based on direct detection of RNA from clinical samples. We demonstrate that species-specific RNA signatures can be used to identify a broad spectrum of infectious agents, including bacteria, viruses, yeast, and parasites. Moreover, we show that the behavior of a small set of bacterial transcripts after a brief antibiotic pulse can rapidly differentiate drug-susceptible and -resistant organisms and that these measurements can be made directly from clinical materials. Thus, transcriptional signatures could form the basis of a uniform diagnostic platform applicable across a broad range of infectious agents.

Genetic Determinants of Drug Resistance in Mycobacterium tuberculosis and Their Diagnostic Value

RATIONALE The development of molecular diagnostics that detect both the presence of Mycobacterium tuberculosis in clinical samples and drug resistance-conferring mutations promises to revolutionize patient care and interrupt transmission by ensuring early diagnosis. However, these tools require the identification of genetic determinants of resistance to the full range of antituberculosis drugs. OBJECTIVES To determine the optimal molecular approach needed, we sought to create a comprehensive catalog of resistance mutations and assess their sensitivity and specificity in diagnosing drug resistance. METHODS We developed and validated molecular inversion probes for DNA capture and deep sequencing of 28 drug-resistance loci in M. tuberculosis. We used the probes for targeted sequencing of a geographically diverse set of 1,397 clinical M. tuberculosis isolates with known drug resistance phenotypes. We identified a minimal set of mutations to predict resistance to first- and second-line antituberculosis drugs and validated our predictions in an independent dataset. We constructed and piloted a web-based database that provides public access to the sequence data and prediction tool. MEASUREMENTS AND MAIN RESULTS The predicted resistance to rifampicin and isoniazid exceeded 90% sensitivity and specificity but was lower for other drugs. The number of mutations needed to diagnose resistance is large, and for the 13 drugs studied it was 238 across 18 genetic loci. CONCLUSIONS These data suggest that a comprehensive M. tuberculosis drug resistance diagnostic will need to allow for a high dimension of mutation detection. They also support the hypothesis that currently unknown genetic determinants, potentially discoverable by whole-genome sequencing, encode resistance to second-line tuberculosis drugs.

Practical Strategies for Performance Optimization of the Enhanced Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test

ABSTRACT The enhanced Gen-Probe Amplified Mycobacterium Tuberculosis Direct (MTD) test was evaluated using a combined set of 338 acid-fast smear-positive and smear-negative, respiratory and nonrespiratory clinical specimens received by the Massachusetts State Tuberculosis Laboratory from September 1999 through March 2002. Microbiological culture was used as the reference method; therefore, the sensitivity and specificity of the MTD test were calculated for culture-positive specimens only. The initial assessment indicated that the overall sensitivity, specificity, and positive and negative predictive values of the MTD test for all specimens grouped together were 62, 98, 99, and 68%, respectively. A detailed discrepancy analysis revealed that two major factors causing negative MTD results in specimens that were culture positive for M. tuberculosis complex were patient treatment with antituberculosis drugs prior to testing and the presence of inhibitory substances in the specimen. Based on these findings, a protocol for optimizing MTD test performance in this setting is proposed in which (i) specimens from patients taking antituberculosis medications are excluded from testing and (ii) all initially MTD-negative or MTD-equivocal specimens are subjected to testing for inhibitors. If this strategy was followed, the MTD test sensitivity would be at least 91%, a significant improvement over the initial sensitivity of 62%. Accordingly, the negative predictive value would increase from 68 to 91%.