Donald G. Catanzaro

Evaluation of Genetic Mutations Associated with Mycobacterium tuberculosis Resistance to Amikacin, Kanamycin and Capreomycin: A Systematic Review

Background Rapid molecular diagnostics for detecting multidrug-resistant and extensively drug-resistant tuberculosis (M/XDR-TB) primarily identify mutations in Mycobacterium tuberculosis (Mtb) genes associated with drug resistance. Their accuracy, however, is dependent largely on the strength of the association between a specific mutation and the phenotypic resistance of the isolate with that mutation, which is not always 100%. While this relationship is well established and reliable for first-line anti-TB drugs, rifampin and isoniazid, it is less well-studied and understood for second-line, injectable drugs, amikacin (AMK), kanamycin (KAN) and capreomycin (CAP). Methodology/Principal Findings We conducted a systematic review of all published studies evaluating Mtb mutations associated with resistance to AMK, KAN, CAP in order to characterize the diversity and frequency of mutations as well as describe the strength of the association between specific mutations and phenotypic resistance in global populations. Our objective was to determine the potential utility and reliability of these mutations as diagnostic markers for detecting AMK, KAN and CAP resistance. Mutation data was reviewed for 1,585 unique clinical isolates from four continents and over 18 countries. Mutations in the rrs, tlyA, eis promoter and gidB genes were associated with AMK, KAN and/or CAP resistance. Conclusions/Significance The rrs A1401G mutation was present in the majority of AMK, KAN and CAP resistant Mtb strains reviewed, but was also found in 7% of CAP susceptible strains. The 1401 mutation alone, however, was not found with sufficient frequency to detect more than 70–80% of global Mtb strains resistant to AMK and CAP, and 60% of strains resistant to KAN. Additional mutations in the rrs, eis promoter, tlyA and gidB genes appear to be associated with resistance and could improve sensitivity and specificity of future diagnostics.

Predicting Extensively Drug-Resistant Mycobacterium tuberculosis Phenotypes with Genetic Mutations

ABSTRACT Molecular diagnostic methods based on the detection of mutations conferring drug resistance are promising technologies for rapidly detecting multidrug-/extensively drug-resistant tuberculosis (M/XDR TB), but large studies of mutations as markers of resistance are rare. The Global Consortium for Drug-Resistant TB Diagnostics analyzed 417 Mycobacterium tuberculosis isolates from multinational sites with a high prevalence of drug resistance to determine the sensitivities and specificities of mutations associated with M/XDR TB to inform the development of rapid diagnostic methods. We collected M/XDR TB isolates from regions of high TB burden in India, Moldova, the Philippines, and South Africa. The isolates underwent standardized phenotypic drug susceptibility testing (DST) to isoniazid (INH), rifampin (RIF), moxifloxacin (MOX), ofloxacin (OFX), amikacin (AMK), kanamycin (KAN), and capreomycin (CAP) using MGIT 960 and WHO-recommended critical concentrations. Eight genes (katG, inhA, rpoB, gyrA, gyrB, rrs, eis, and tlyA) were sequenced using Sanger sequencing. Three hundred seventy isolates were INHr, 356 were RIFr, 292 were MOXr/OFXr, 230 were AMKr, 219 were CAPr, and 286 were KANr. Four single nucleotide polymorphisms (SNPs) in katG/inhA had a combined sensitivity of 96% and specificities of 97 to 100% for the detection of INHr. Eleven SNPs in rpoB had a combined sensitivity of 98% for RIFr. Eight SNPs in gyrA codons 88 to 94 had sensitivities of 90% for MOXr/OFXr. The rrs 1401/1484 SNPs had 89 to 90% sensitivity for detecting AMKr/CAPr but 71% sensitivity for KANr. Adding eis promoter SNPs increased the sensitivity to 93% for detecting AMKr and to 91% for detecting KANr. Approximately 30 SNPs in six genes predicted clinically relevant XDR-TB phenotypes with 90 to 98% sensitivity and almost 100% specificity.

Genetic Mutations Associated with Isoniazid Resistance in Mycobacterium tuberculosis: A Systematic Review

Background Tuberculosis (TB) incidence and mortality are declining worldwide; however, poor detection of drug-resistant disease threatens to reverse current progress toward global TB control. Multiple, rapid molecular diagnostic tests have recently been developed to detect genetic mutations in Mycobacterium tuberculosis (Mtb) genes known to confer first-line drug resistance. Their utility, though, depends on the frequency and distribution of the resistance associated mutations in the pathogen population. Mutations associated with rifampicin resistance, one of the two first-line drugs, are well understood and appear to occur in a single gene region in >95% of phenotypically resistant isolates. Mutations associated with isoniazid, the other first-line drug, are more complex and occur in multiple Mtb genes. Objectives/Methodology A systematic review of all published studies from January 2000 through August 2013 was conducted to quantify the frequency of the most common mutations associated with isoniazid resistance, to describe the frequency at which these mutations co-occur, and to identify the regional differences in the distribution of these mutations. Mutation data from 118 publications were extracted and analyzed for 11,411 Mtb isolates from 49 countries. Principal Findings/Conclusions Globally, 64% of all observed phenotypic isoniazid resistance was associated with the katG315 mutation. The second most frequently observed mutation, inhA-15, was reported among 19% of phenotypically resistant isolates. These two mutations, katG315 and inhA-15, combined with ten of the most commonly occurring mutations in the inhA promoter and the ahpC-oxyR intergenic region explain 84% of global phenotypic isoniazid resistance. Regional variation in the frequency of individual mutations may limit the sensitivity of molecular diagnostic tests. Well-designed systematic surveys and whole genome sequencing are needed to identify mutation frequencies in geographic regions where rapid molecular tests are currently being deployed, providing a context for interpretation of test results and the opportunity for improving the next generation of diagnostics.

Frequency and Geographic Distribution of gyrA and gyrB Mutations Associated with Fluoroquinolone Resistance in Clinical Mycobacterium Tuberculosis Isolates: A Systematic Review

Background The detection of mutations in the gyrA and gyrB genes in the Mycobacterium tuberculosis genome that have been demonstrated to confer phenotypic resistance to fluoroquinolones is the most promising technology for rapid diagnosis of fluoroquinolone resistance. Methods In order to characterize the diversity and frequency of gyrA and gyrB mutations and to describe the global distribution of these mutations, we conducted a systematic review, from May 1996 to April 2013, of all published studies evaluating Mycobacterium tuberculosis mutations associated with resistance to fluoroquinolones. The overall goal of the study was to determine the potential utility and reliability of these mutations as diagnostic markers to detect phenotypic fluoroquinolone resistance in Mycobacterium tuberculosis and to describe their geographic distribution. Results Forty-six studies, covering four continents and 18 countries, provided mutation data for 3,846 unique clinical isolates with phenotypic resistance profiles to fluoroquinolones. The gyrA mutations occurring most frequently in fluoroquinolone-resistant isolates, ranged from 21–32% for D94G and 13–20% for A90V, by drug. Eighty seven percent of all strains that were phenotypically resistant to moxifloxacin and 83% of ofloxacin resistant isolates contained mutations in gyrA. Additionally we found that 83% and 80% of moxifloxacin and ofloxacin resistant strains respectively, were observed to have mutations in the gyrA codons interrogated by the existing MTBDRsl line probe assay. In China and Russia, 83% and 84% of fluoroquinolone resistant strains respectively, were observed to have gyrA mutations in the gene regions covered by the MTBDRsl assay. Conclusions Molecular diagnostics, specifically the Genotype MTBDRsl assay, focusing on codons 88–94 should have moderate to high sensitivity in most countries. While we did observe geographic differences in the frequencies of single gyrA mutations across countries, molecular diagnostics based on detection of all gyrA mutations demonstrated to confer resistance should have broad and global utility.

Novel katG mutations causing isoniazid resistance in clinical M. tuberculosis isolates.

We report the discovery and confirmation of 23 novel mutations with previously undocumented role in isoniazid (INH) drug resistance, in catalase-peroxidase (katG) gene of Mycobacterium tuberculosis (Mtb) isolates. With these mutations, a synonymous mutation in fabG1g609a, and two canonical mutations, we were able to explain 98% of the phenotypic resistance observed in 366 clinical Mtb isolates collected from four high tuberculosis (TB)-burden countries: India, Moldova, Philippines, and South Africa. We conducted overlapping targeted and whole-genome sequencing for variant discovery in all clinical isolates with a variety of INH-resistant phenotypes. Our analysis showed that just two canonical mutations (katG 315AGC-ACC and inhA promoter-15C-T) identified 89.5% of resistance phenotypes in our collection. Inclusion of the 23 novel mutations reported here, and the previously documented point mutation in fabG1, increased the sensitivity of these mutations as markers of INH resistance to 98%. Only six (2%) of the 332 resistant isolates in our collection did not harbor one or more of these mutations. The third most prevalent substitution, at inhA promoter position -8, present in 39 resistant isolates, was of no diagnostic significance since it always co-occurred with katG 315. 79% of our isolates harboring novel mutations belong to genetic group 1 indicating a higher tendency for this group to go down an uncommon evolutionary path and evade molecular diagnostics. The results of this study contribute to our understanding of the mechanisms of INH resistance in Mtb isolates that lack the canonical mutations and could improve the sensitivity of next generation molecular diagnostics.

Detection of Low-Level Mixed-Population Drug Resistance in Mycobacterium tuberculosis Using High Fidelity Amplicon Sequencing.

Undetected and untreated, low-levels of drug resistant (DR) subpopulations in clinical Mycobacterium tuberculosis (Mtb) infections may lead to development of DR-tuberculosis, potentially resulting in treatment failure. Current phenotypic DR susceptibility testing has a theoretical potential for 1% sensitivity, is not quantitative, and requires several weeks to complete. The use of “single molecule-overlapping reads” (SMOR) analysis with next generation DNA sequencing for determination of ultra-rare target alleles in complex mixtures provides increased sensitivity over standard DNA sequencing. Ligation free amplicon sequencing with SMOR analysis enables the detection of resistant allele subpopulations at ≥0.1% of the total Mtb population in near real-time analysis. We describe the method using standardized mixtures of DNA from resistant and susceptible Mtb isolates and the assay’s performance for detecting ultra-rare DR subpopulations in DNA extracted directly from clinical sputum samples. SMOR analysis enables rapid near real-time detection and tracking of previously undetectable DR sub-populations in clinical samples allowing for the evaluation of the clinical relevance of low-level DR subpopulations. This will provide insights into interventions aimed at suppressing minor DR subpopulations before they become clinically significant.

Performance Comparison of Three Rapid Tests for the Diagnosis of Drug-Resistant Tuberculosis.

Background The aim of this study was to compare the performance of several recently developed assays for the detection of multi- and extensively drug-resistant tuberculosis (M/XDR-TB) in a large, multinational field trial. Methods Samples from 1,128 M/XDR-TB suspects were examined by Line Probe Assay (LPA), Pyrosequencing (PSQ), and Microscopic Observation of Drug Susceptibility (MODS) and compared to the BACTEC MGIT960 reference standard to detect M/XDR-TB directly from patient sputum samples collected at TB clinics in India, Moldova, and South Africa. Results Specificity for all three assays was excellent: 97–100% for isoniazid (INH), rifampin (RIF), moxifloxacin (MOX) and ofloxacin (OFX) and 99–100% for amikacin (AMK), capreomycin (CAP) and kanamycin (KAN) resistance. Sensitivities were lower, but still very good: 94–100% for INH, RIF, MOX and OFX, and 84–90% for AMK and CAP, but only 48–62% for KAN. In terms of agreement, statistically significant differences were only found for detection of RIF (MODS outperformed PSQ) and KAN (MODS outperformed LPA and PSQ) resistance. Mean time-to-result was 1.1 days for LPA and PSQ, 14.3 days for MODS, and 24.7 days for MGIT. Conclusions All three rapid assays evaluated provide clinicians with timely detection of resistance to the drugs tested; with molecular results available one day following laboratory receipt of samples. In particular, the very high specificity seen for detection of drug resistance means that clinicians can use the results of these rapid tests to avoid the use of toxic drugs to which the infecting organism is resistant and develop treatment regiments that have a higher likelihood of yielding a successful outcome.

The Global Consortium for Drug-resistant Tuberculosis Diagnostics (GCDD): design of a multi-site, head-to-head study of three rapid tests to detect extensively drug-resistant tuberculosis

BackgroundDrug-resistant tuberculosis (DR-TB) remains a threat to global public health, owing to the complexity and delay of diagnosis and treatment. The Global Consortium for Drug-resistant Tuberculosis Diagnostics (GCDD) was formed to develop and evaluate assays designed to rapidly detect DR-TB, so that appropriate treatment might begin more quickly. This paper describes the methodology employed in a prospective cohort study for head-to-head assessment of three different rapid diagnostic tools.MethodsSubjects at risk of DR-TB were enrolled from three countries. Data were gathered from a combination of patient interviews, chart reviews, and laboratory testing from each site’s reference laboratory. The primary outcome of interest was reduction in time from specimen arrival in the laboratory to results of rapid drug susceptibility tests, as compared with current standard mycobacterial growth indicator tube (MGIT) drug susceptibility tests.ResultsSuccessful implementation of the trial in diverse multinational populations is explained, in addition to challenges encountered and recommendations for future studies with similar aims or populations.ConclusionsThe GCDD study was a head-to-head study of multiple rapid diagnostic assays aimed at improving accuracy and precision of diagnostics and reducing overall time to detection of DR-TB. By conducting a large prospective study, which captured epidemiological, clinical, and biological data, we have produced a high-quality unique dataset, which will be beneficial for analyzing study aims as well as answering future DR-TB research questions. Reduction in detection time for XDR-TB would be a major public health success as it would allow for improved treatment and more successful patient outcomes. Executing successful trials is critical in assessment of these reductions in highly variable populations.Trial registrationClinicalTrials.gov NCT02170441.

Evaluation of pyrosequencing for detecting extensively drug-resistant Mycobacterium tuberculosis among clinical isolates from four high-burden countries.

ABSTRACT Reliable molecular diagnostics, which detect specific mutations associated with drug resistance, are promising technologies for the rapid identification and monitoring of drug resistance in Mycobacterium tuberculosis isolates. Pyrosequencing (PSQ) has the ability to detect mutations associated with first- and second-line anti-tuberculosis (TB) drugs, with the additional advantage of being rapidly adaptable for the identification of new mutations. The aim of this project was to evaluate the performance of PSQ in predicting phenotypic drug resistance in multidrug- and extensively drug-resistant tuberculosis (M/XDR-TB) clinical isolates from India, South Africa, Moldova, and the Philippines. A total of 187 archived isolates were run through a PSQ assay in order to identify M. tuberculosis (via the IS6110 marker), and to detect mutations associated with M/XDR-TB within small stretches of nucleotides in selected loci. The molecular targets included katG, the inhA promoter and the ahpC-oxyR intergenic region for isoniazid (INH) resistance; the rpoB core region for rifampin (RIF) resistance; gyrA for fluoroquinolone (FQ) resistance; and rrs for amikacin (AMK), capreomycin (CAP), and kanamycin (KAN) resistance. PSQ data were compared to phenotypic mycobacterial growth indicator tube (MGIT) 960 drug susceptibility testing results for performance analysis. The PSQ assay illustrated good sensitivity for the detection of resistance to INH (94%), RIF (96%), FQ (93%), AMK (84%), CAP (88%), and KAN (68%). The specificities of the assay were 96% for INH, 100% for RIF, FQ, AMK, and KAN, and 97% for CAP. PSQ is a highly efficient diagnostic tool that reveals specific nucleotide changes associated with resistance to the first- and second-line anti-TB drug medications. This methodology has the potential to be linked to mutation-specific clinical interpretation algorithms for rapid treatment decisions.

Rapid Drug Susceptibility Testing of Drug-Resistant Mycobacterium tuberculosis Isolates Directly from Clinical Samples by Use of Amplicon Sequencing: a Proof-of-Concept Study

ABSTRACT Increasingly complex drug-resistant tuberculosis (DR-TB) is a major global health concern and one of the primary reasons why TB is now the leading infectious cause of death worldwide. Rapid characterization of a DR-TB patients complete drug resistance profile would facilitate individualized treatment in place of empirical treatment, improve treatment outcomes, prevent amplification of resistance, and reduce the transmission of DR-TB. The use of targeted next-generation sequencing (NGS) to obtain drug resistance profiles directly from patient sputum samples has the potential to enable comprehensive evidence-based treatment plans to be implemented quickly, rather than in weeks to months, which is currently needed for phenotypic drug susceptibility testing (DST) results. In this pilot study, we evaluated the performance of amplicon sequencing of Mycobacterium tuberculosis DNA from patient sputum samples using a tabletop NGS technology and automated data analysis to provide a rapid DST solution (the Next Gen-RDST assay). One hundred sixty-six out of 176 (94.3%) sputum samples from the Republic of Moldova yielded complete Next Gen-RDST assay profiles for 7 drugs of interest. We found a high level of concordance of our Next Gen-RDST assay results with phenotypic DST (97.0%) and pyrosequencing (97.8%) results from the same clinical samples. Our Next Gen-RDST assay was also able to estimate the proportion of resistant-to-wild-type alleles down to mixtures of ≤1%, which demonstrates the ability to detect very low levels of resistant variants not detected by pyrosequencing and possibly below the threshold for phenotypic growth methods. The assay as described here could be used as a clinical or surveillance tool.