Angela M. Starks

Molecular Detection of Mutations Associated with First- and Second-Line Drug Resistance Compared with Conventional Drug Susceptibility Testing of Mycobacterium tuberculosis

ABSTRACT The emergence of multi- and extensively drug-resistant tuberculosis is a significant impediment to the control of this disease because treatment becomes more complex and costly. Reliable and timely drug susceptibility testing is critical to ensure that patients receive effective treatment and become noninfectious. Molecular methods can provide accurate and rapid drug susceptibility results. We used DNA sequencing to detect resistance to the first-line antituberculosis drugs isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol (EMB) and the second-line drugs amikacin (AMK), capreomycin (CAP), kanamycin (KAN), ciprofloxacin (CIP), and ofloxacin (OFX). Nine loci were sequenced: rpoB (for resistance to RIF), katG and inhA (INH), pncA (PZA), embB (EMB), gyrA (CIP and OFX), and rrs, eis, and tlyA (KAN, AMK, and CAP). A total of 314 clinical Mycobacterium tuberculosis complex isolates representing a variety of antibiotic resistance patterns, genotypes, and geographical origins were analyzed. The molecular data were compared to the phenotypic data and the accuracy values were calculated. Sensitivity and specificity values for the first-line drug loci were 97.1% and 93.6% for rpoB, 85.4% and 100% for katG, 16.5% and 100% for inhA, 90.6% and 100% for katG and inhA together, 84.6% and 85.8% for pncA, and 78.6% and 93.1% for embB. The values for the second-line drugs were also calculated. The size and scope of this study, in numbers of loci and isolates examined, and the phenotypic diversity of those isolates support the use of DNA sequencing to detect drug resistance in the M. tuberculosis complex. Further, the results can be used to design diagnostic tests utilizing other mutation detection technologies.

Pathogenesis of Infection by Clinical and Environmental Strains of Vibrio vulnificus in Iron-Dextran-Treated Mice

ABSTRACT Vibrio vulnificus is an opportunistic pathogen that contaminates oysters harvested from the Gulf of Mexico. In humans with compromising conditions, especially excess levels of iron in plasma and tissues, consumption of contaminated seafood or exposure of wounds to contaminated water can lead to systemic infection and disfiguring skin infection with extremely high mortality. V. vulnificus-associated diseases are noted for the rapid replication of the bacteria in host tissues, with extensive tissue damage. In this study we examined the virulence attributes of three virulent clinical strains and three attenuated oyster or seawater isolates in mouse models of systemic disease. All six V. vulnificus strains caused identical skin lesions in subcutaneously (s.c.) inoculated iron dextran-treated mice in terms of numbers of recovered CFU and histopathology; however, the inocula required for identical frequency and magnitude of infection were at least 350-fold higher for the environmental strains. At lethal doses, all strains caused s.c. skin lesions with extensive edema, necrosis of proximate host cells, vasodilation, and as many as 108CFU/g, especially in perivascular regions. These data suggest that the differences between these clinical and environmental strains may be related to growth in the host or susceptibility to host defenses. In non-iron dextran-treated mice, strains required 105-fold-higher inocula to cause an identical disease process as with iron dextran treatment. These results demonstrate that s.c. inoculation of iron dextran-treated mice is a useful model for studying systemic disease caused by V. vulnificus.

Construction and Phenotypic Evaluation of a Vibrio vulnificus vvpE Mutant for Elastolytic Protease

ABSTRACT Vibrio vulnificus is an opportunistic gram-negative pathogen that commonly contaminates oysters. Predisposed individuals who consume raw oysters can die within days from sepsis, and even otherwise healthy people are susceptible to serious wound infection after contact with contaminated seafood or seawater. Numerous secreted and cell-associated virulence factors have been proposed to account for the fulminating and destructive nature of V. vulnificusinfections. Among the putative virulence factors is an elastolytic metalloprotease. We cloned and sequenced the vvpE gene encoding an elastase of V. vulnificus ATCC 29307. The functions of the elastase were assessed by constructingvvpE insertional knockout mutants and evaluating phenotypic changes in vitro and in mice. Although other types of protease activity were still observed in vvpE mutants, elastase activity was completely absent in the mutants and was restored by reintroducing the recombinant vvpE gene. In contrast to previous characterization of elastase as a potential virulence factor, which was demonstrated by injecting the purified protein into animals, inactivation of the V. vulnificus vvpE gene did not affect the ability of the bacteria to infect mice and cause damage, either locally in subcutaneous tissues or systemically in the liver, in both iron-treated and normal mice. Furthermore, a vvpE mutant was not affected with regard to cytolytic activity toward INT407 epithelial cells or detachment of INT407 cells from culture dishes in vitro. Therefore, it appears that elastase is less important in the pathogenesis of V. vulnificus than would have been predicted by examining the effects of administering purified proteins to animals. However, V. vulnificus utilizes a variety of virulence factors; hence, the effects of inactivation of elastase alone could be masked by other compensatory virulence factors.

Mutations at embB Codon 306 Are an Important Molecular Indicator of Ethambutol Resistance in Mycobacterium tuberculosis

ABSTRACT Ethambutol resistance in clinical Mycobacterium tuberculosis isolates is associated primarily with missense mutations in the embB gene. However, recent reports have described the presence of embB mutations, especially those at embB codon 306, in isolates susceptible to ethambutol. To clarify the role of embB mutations in ethambutol resistance, we sequenced the ethambutol resistance-determining region in spontaneous ethambutol-resistant mutants. In our study, 66% of spontaneous mutants contained a single point mutation in embB, with 55% of these occurring at embB 306. The MIC of ethambutol for spontaneous mutants was increased two- to eightfold relative to the pansusceptible M. tuberculosis strains from which the mutants were generated. To further characterize the role of embB 306 mutations, we directly introduced mutant alleles, embB(M306V) or embB(M306I), into pansusceptible M. tuberculosis strains and conversely reverted mutant alleles in spontaneous ethambutol-resistant mutants back to those of the wild type via allelic exchange using specialized linkage transduction. We determined that the MIC of ethambutol was reduced fourfold for three of the four spontaneous ethambutol-resistant embB 306 mutants when the mutant allele was replaced with the wild-type embB allele. The MIC for one of the spontaneous mutants genetically reverted to wild-type embB was reduced by only twofold. When the wild-type embB allele was converted to the mutant allele embB(M306V), the ethambutol MIC was increased fourfold, and when the allele was changed to M306I, the ethambutol MIC increased twofold. Our data indicate that embB 306 mutations are sufficient to confer ethambutol resistance, and detection of these mutations should be considered in the development of rapid molecular tests.

Diagnostic performance and costs of Capilia TB for Mycobacterium tuberculosis complex identification from broth-based culture in Bangkok, Thailand

Objectives  Broth‐based culture (BBC) systems are increasingly being used to detect Mycobacterium tuberculosis complex (MTBC) in resource‐limited. We evaluated the performance, time to detection and cost of the Capilia TB identification test from broth cultures positive for acid‐fast bacilli (AFB) in Thailand.

A standardised method for interpreting the association between mutations and phenotypic drug resistance in Mycobacterium tuberculosis

A clear understanding of the genetic basis of antibiotic resistance in Mycobacterium tuberculosis is required to accelerate the development of rapid drug susceptibility testing methods based on genetic sequence. Raw genotype–phenotype correlation data were extracted as part of a comprehensive systematic review to develop a standardised analytical approach for interpreting resistance associated mutations for rifampicin, isoniazid, ofloxacin/levofloxacin, moxifloxacin, amikacin, kanamycin, capreomycin, streptomycin, ethionamide/prothionamide and pyrazinamide. Mutation frequencies in resistant and susceptible isolates were calculated, together with novel statistical measures to classify mutations as high, moderate, minimal or indeterminate confidence for predicting resistance. We identified 286 confidence-graded mutations associated with resistance. Compared to phenotypic methods, sensitivity (95% CI) for rifampicin was 90.3% (89.6–90.9%), while for isoniazid it was 78.2% (77.4–79.0%) and their specificities were 96.3% (95.7–96.8%) and 94.4% (93.1–95.5%), respectively. For second-line drugs, sensitivity varied from 67.4% (64.1–70.6%) for capreomycin to 88.2% (85.1–90.9%) for moxifloxacin, with specificity ranging from 90.0% (87.1–92.5%) for moxifloxacin to 99.5% (99.0–99.8%) for amikacin. This study provides a standardised and comprehensive approach for the interpretation of mutations as predictors of M. tuberculosis drug-resistant phenotypes. These data have implications for the clinical interpretation of molecular diagnostics and next-generation sequencing as well as efficient individualised therapy for patients with drug-resistant tuberculosis. A comprehensive basis for interpreting mutations to predict antibiotic resistance in tuberculosis http://ow.ly/hhwJ30g9jCY

Collaborative Effort for a Centralized Worldwide Tuberculosis Relational Sequencing Data Platform

Continued progress in addressing challenges associated with detection and management of tuberculosis requires new diagnostic tools. These tools must be able to provide rapid and accurate information for detecting resistance to guide selection of the treatment regimen for each patient. To achieve this goal, globally representative genotypic, phenotypic, and clinical data are needed in a standardized and curated data platform. A global partnership of academic institutions, public health agencies, and nongovernmental organizations has been established to develop a tuberculosis relational sequencing data platform (ReSeqTB) that seeks to increase understanding of the genetic basis of resistance by correlating molecular data with results from drug susceptibility testing and, optimally, associated patient outcomes. These data will inform development of new diagnostics, facilitate clinical decision making, and improve surveillance for drug resistance. ReSeqTB offers an opportunity for collaboration to achieve improved patient outcomes and to advance efforts to prevent and control this devastating disease.

Integration of Published Information Into a Resistance-Associated Mutation Database for Mycobacterium tuberculosis

Tuberculosis remains a major global public health challenge. Although incidence is decreasing, the proportion of drug-resistant cases is increasing. Technical and operational complexities prevent Mycobacterium tuberculosis drug susceptibility phenotyping in the vast majority of new and retreatment cases. The advent of molecular technologies provides an opportunity to obtain results rapidly as compared to phenotypic culture. However, correlations between genetic mutations and resistance to multiple drugs have not been systematically evaluated. Molecular testing of M. tuberculosis sampled from a typical patient continues to provide a partial picture of drug resistance. A database of phenotypic and genotypic testing results, especially where prospectively collected, could document statistically significant associations and may reveal new, predictive molecular patterns. We examine the feasibility of integrating existing molecular and phenotypic drug susceptibility data to identify associations observed across multiple studies and demonstrate potential for well-integrated M. tuberculosis mutation data to reveal actionable findings.

Use of a marker plasmid to examine differential rates of growth and death between clinical and environmental strains of Vibrio vulnificus in experimentally infected mice

Vibrio vulnificus is Gram‐negative bacterium that contaminates oysters, causing highly lethal sepsis after consumption of raw oysters and wound infection. We previously described two sets of V. vulnificus strains with different levels of virulence in subcutaneously inoculated iron dextran‐treated mice. Both virulent, clinical strains and attenuated, environmental strains could be recovered in high numbers from skin lesions and livers; however, the attenuated environmental strains required significantly higher numbers of colony‐forming units (cfu) in the inoculum to produce lethal infection. Using some of these strains and an additional clinical strain, we presently asked if the different abilities to cause infection between the clinical and environmental strains were due to differences in rates of growth or death of the bacteria in the mouse host. We therefore constructed a marker plasmid, pGTR902, that functions as a replicon only in the presence of arabinose, which is not present in significant levels in animal tissues. V. vulnificus strains containing pGTR902 were inoculated into iron dextran‐treated and untreated mice. Measuring the proportion of bacteria that had maintained the marker plasmid recovered from mice enabled us to monitor the number of in vivo divisions, hence growth rate; whereas measuring the number of marker plasmid‐containing bacteria recovered enabled the measurement of death of the vibrios in the mice. The numbers of bacterial divisions in vivo for all of the strains over a 12–15 h infection period were not significantly different in iron dextran‐treated mice; however, the rate of death of one environmental strain was significantly higher compared with the clinical strains. Infection of non‐iron dextran‐treated mice with clinical strains demonstrated that the greatest effect of iron dextran‐treatment was increased growth rate, while one clinical strain also experienced increased death in untreated mice. V. vulnificus inoculated into iron dextran‐treated mice replicated extremely rapidly over the first 4 h of infection with doubling times of approximately 15–28 min. In contrast, one of the environmental strains exhibited a reduced early growth rate. These results demonstrate that differences in virulence among naturally occurring V. vulnificus can be explained by diverse abilities to replicate rapidly in or resist defences of the host. The marker plasmid pGTR902 should be useful for examining virulence of bacteria in terms of differentiating growth verses death in animal hosts for most Gram‐negative bacteria.

Unexpected decline in tuberculosis cases coincident with economic recession - United States, 2009

BackgroundSince 1953, through the cooperation of state and local health departments, the U.S. Centers for Disease Control and Prevention (CDC) has collected information on incident cases of tuberculosis (TB) disease in the United States. In 2009, TB case rates declined -11.4%, compared to an average annual -3.8% decline since 2000. The unexpectedly large decline raised concerns that TB cases may have gone unreported. To address the unexpected decline, we examined trends from multiple sources on TB treatment initiation, medication sales, and laboratory and genotyping data on culture-positive TB.MethodsWe analyzed 142,174 incident TB cases reported to the U. S. National Tuberculosis Surveillance System (NTSS) during January 1, 2000-December 31, 2009; TB control program data from 59 public health reporting areas; self-reported data from 50 CDC-funded public health laboratories; monthly electronic prescription claims for new TB therapy prescriptions; and complete genotyping results available for NTSS cases. Accounting for prior trends using regression and time-series analyses, we calculated the deviation between observed and expected TB cases in 2009 according to patient and clinical characteristics, and assessed at what point in time the deviation occurred.ResultsThe overall deviation in TB cases in 2009 was -7.9%, with -994 fewer cases reported than expected (P < .001). We ruled out evidence of surveillance underreporting since declines were seen in states that used new software for case reporting in 2009 as well as states that did not, and we found no cases unreported to CDC in our examination of over 5400 individual line-listed reports in 11 areas. TB cases decreased substantially among both foreign-born and U.S.-born persons. The unexpected decline began in late 2008 or early 2009, and may have begun to reverse in late 2009. The decline was greater in terms of case counts among foreign-born than U.S.-born persons; among the foreign-born, the declines were greatest in terms of percentage deviation from expected among persons who had been in the United States less than 2 years. Among U.S.-born persons, the declines in percentage deviation from expected were greatest among homeless persons and substance users. Independent information systems (NTSS, TB prescription claims, and public health laboratories) reported similar patterns of declines. Genotyping data did not suggest sudden decreases in recent transmission.ConclusionsOur assessments show that the decline in reported TB was not an artifact of changes in surveillance methods; rather, similar declines were found through multiple data sources. While the steady decline of TB cases before 2009 suggests ongoing improvement in TB control, we were not able to identify any substantial change in TB control activities or TB transmission that would account for the abrupt decline in 2009. It is possible that other multiple causes coincident with economic recession in the United States, including decreased immigration and delayed access to medical care, could be related to TB declines. Our findings underscore important needs in addressing health disparities as we move towards TB elimination in the United States.