All in-text references underlined in blue are linked to publications on ResearchGate, letting you... more All in-text references underlined in blue are linked to publications on ResearchGate, letting you access and read them immediately.
This chapter includes a description of nonradioactive broth culture systems and rapid molecular s... more This chapter includes a description of nonradioactive broth culture systems and rapid molecular systems for detection of drug resistance, as well as the standard agar proportion method. The antimicrobial agents that are used in the treatment of mycobacterial infections are discussed in the chapter for the most commonly encountered species. The Clinical and Laboratory Standards Institute (CLSI) document on drug susceptibility testing of mycobacteria currently recommends that first-line testing include ethambutol (EMB), RMP, INH, and pyrazinamide (PZA). The chapter describes drug susceptibility testing of M. tuberculosis complex. Broth microdilution is the method recommended by the CLSI for susceptibility testing of nontuberculous mycobacteria (NTM). The CLSI provides guidelines for testing M. avium complex (MAC), M. kansasii, M. marinum, and the rapidly growing mycobacteria. General recommendations regarding the broth microdilution method and QC that apply to all NTM are discussed in the chapter. The chapter describes specific details related to MAC, M. kansasii, M. marinum, and the rapidly growing mycobacteria. It also talks about incubation temperature and time for each species or group. The chapter explains that Nocardia spp. and other aerobic actinomycetes (Actinomadura, Rhodococcus equi, Gordonia, Tsukamurella, and rarely Streptomyces spp.) can cause serious disease in immunocompromised and occasionally even healthy hosts. The recommended method for testing Nocardia and other aerobic actinomycetes is broth microdilution.
The international journal of tuberculosis and lung disease : the official journal of the International Union against Tuberculosis and Lung Disease, 2000
Healthy People 2010, an initiative from the federal government, calls for action from tuberculosi... more Healthy People 2010, an initiative from the federal government, calls for action from tuberculosis controllers and tuberculosis laboratories in the fight to eliminate tuberculosis. Many patients, such as immunocompromised patients and those infected with multidrug-resistant tuberculosis strains, pose a challenge for care and diagnosis. Fortunately, many changes have occurred in the last decade to facilitate more rapid and accurate testing to assist with the care of these patients. California, Florida, New York and Texas have almost 50% of the tuberculosis cases in the United States, and their public health laboratories utilize different approaches to meet the same goal of rapid and accurate testing of specimens. With the targets of Healthy People 2010 (e.g., to reduce the average time for a laboratory to confirm and report tuberculosis cases to 2 days for 75% of cases) already looming on the horizon, innovative methods for achieving these goals should be evaluated. Using these publi...
Polyphasic taxonomic methods were employed to characterize a new species of slowly growing, nonpi... more Polyphasic taxonomic methods were employed to characterize a new species of slowly growing, nonpigmented mycobacteria. We propose the name Mycobacterium triplex sp. nov. for this new taxon. Conventional identification testing demonstrated a group of similar organisms that were geographically widespread in the United States. Commercially available nucleic-acid probes specific for the Mycobacterium avium complex were unreactive for these strains. High-performance liquid chromatography analysis of the mycolic acids revealed mycolate profiles that closely resembled Mycobacterium simiae. Comparative 16S rRNA sequence data confirmed the phylogenetic relationship of the strains with the slowly growing mycobacteria. Representative-type strains have been deposited in the American Type Culture Collection as strain ATCC 700071 [corrected].
Microscopic examination of respiratory specimens for acid-fast bacilli (AFB) plays a key role in ... more Microscopic examination of respiratory specimens for acid-fast bacilli (AFB) plays a key role in the initial diagnosis of tuberculosis, monitoring of treatment, and determination of eligibility for release from isolation. The objective of this study was to compare the sensitivity obtained with smears for detection of AFB (AFB smears) made directly from respiratory specimens (direct AFB smears) to that obtained with parallel smears made from concentrates of the specimens (concentrated AFB smears). A total of 2,693 specimens were evaluated; 1,806 were from the University of California Irvine Medical Center Medical Microbiology Laboratory (UCIMC), which serves a tertiary-care hospital with outpatient clinics, and 887 were from the Microbial Disease Laboratory at the California Department of Public Health (MDL), which receives specimens from outpatient facilities and clinics on Pacific islands. Of the 353 AFB culture-positive specimens at UCIMC, there was a statistically significant dif...
We explored sharing nucleic acid amplification testing (NAAT) for detection of Mycobacterium tube... more We explored sharing nucleic acid amplification testing (NAAT) for detection of Mycobacterium tuberculosis complex (MTBC) and molecular and phenotypic drug susceptibility testing between two state public health tuberculosis (TB) laboratories, and evaluated turnaround times and cost-effectiveness. From September 1, 2012, to May 30, 2013, the Wisconsin State Laboratory of Hygiene (Wisconsin Lab) submitted specimens to the Microbial Diseases Laboratory of the California Department of Public Health (California Lab) for NAAT and molecular drug susceptibility testing (MDST) by pyrosequencing, and culture-based TB drug susceptibility testing by the BACTEC(TM) MGIT(TM) 960 system. A total of 182 specimens were referred to the California Lab, and 47 TB cases and 12 drug-resistant cases were identified. The average time for specimen transport was two days, which included one day for processing and packaging in the submitting laboratory. The average turnaround time for NAAT was 0.3 days at the ...
The international journal of tuberculosis and lung disease : the official journal of the International Union against Tuberculosis and Lung Disease, 2015
Rifampin (RMP) resistant Mycobacterium tuberculosis is usually assumed to be resistant to all rif... more Rifampin (RMP) resistant Mycobacterium tuberculosis is usually assumed to be resistant to all rifamycins. Increasing evidence indicates, however, that some rpoB mutations, detectable by rapid molecular diagnostics, confer resistance to RMP but not to rifabutin (RBT), suggesting that RBT may be effective for the treatment of M. tuberculosis with these mutations. To determine if specific rpoB mutations reliably predict differential phenotypic resistance to RMP and RBT. We selected 60 clinical M. tuberculosis isolates from a repository of multinational multidrug-resistant tuberculosis isolates and stratified them into two groups: 1) those with rpoB mutations suspected to confer differential resistance to RMP and RBT, and 2) those expected to be cross-resistant to RMP and RBT. These assumptions were tested by comparing the phenotypic susceptibilities of RMP/RBT with those predicted by mutations in the rpoB gene. Of 20 suspected RMP-resistant/RBT-susceptible isolates, 15 were RMP-resista...
The international journal of tuberculosis and lung disease : the official journal of the International Union against Tuberculosis and Lung Disease, 2009
Drug-resistant strains of Mycobacterium tuberculosis are increasing worldwide and pose a major th... more Drug-resistant strains of Mycobacterium tuberculosis are increasing worldwide and pose a major threat to global health. However, it remains unsettled whether drug-resistant mutants are fixed in the bacterial population or if they would revert in the absence of drug pressure. To document the occurrence of isoniazid (INH) reversion in a patient with multidrug-resistant tuberculosis (TB) and investigate its association with fitness cost. Genotypic and phenotypic assays were used to characterize the reversion of INH resistance in isolates from a patient with pulmonary TB. The pre-reversion katG mutation was reconstructed in a pan-susceptible laboratory strain (H37Rv DeltakatG::katG W300G) and tested for susceptibility to INH and oxidative stress. Genotyping and drug susceptibility testing showed that an isogenic strain of M. tuberculosis reverted from an INH-resistant to a susceptible phenotype in the absence of INH therapy. The genotypic basis of this reversion was mapped to the katG c...
Reliable molecular diagnostics, which detect specific mutations associated with drug resistance, ... more 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 IS 6110 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-...
The Bactec MGIT 960 system for testing susceptibility to second-line drugs was evaluated with 117... more The Bactec MGIT 960 system for testing susceptibility to second-line drugs was evaluated with 117 clinical strains in a multicenter study. The four drugs studied were levofloxacin, amikacin, capreomycin, and ethionamide. The critical concentration established for levofloxacin and amikacin was 1.5 μg/ml, that established for capreomycin was 3.0 μg/ml, and that established for ethionamide was 5.0 μg/ml. The overall level of agreement between the agar proportion method and the MGIT 960 system was 96.4%, and the levels of agreement for the individuals drugs were 99.1% for levofloxacin, 100% for amikacin, 97.4% for capreomycin, and 88.9% for ethionamide. The rate of reproducibility of the drug susceptibility testing results between the participating laboratories was 99.5%.
Prompt laboratory reporting of tuberculosis (TB) test results is necessary for TB control. To und... more Prompt laboratory reporting of tuberculosis (TB) test results is necessary for TB control. To understand the extent of and factors contributing to laboratory reporting delays and the impact of reporting delays on initiation of treatment of TB patients, we analyzed data from 300 consecutive culture-positive TB cases reported in four California counties in 1998. Laboratory reporting to the specimen submitter was delayed for 26.9% of smear-positive patients and 46.8% of smear-negative patients. Delays were associated with the type of laboratory that performed the testing and with delayed transport of specimens. Referral laboratories (public health and commercial) had longer median reporting time frames than hospital and health maintenance organization laboratories. Among patients whose treatment was not started until specimens were collected, those with delayed laboratory reporting were more likely to have delayed treatment than patients with no laboratory reporting delays (odds ratio ...
To assess the clinical impact of a molecular beacon (MB) assay that detects multidrug-resistant t... more To assess the clinical impact of a molecular beacon (MB) assay that detects multidrug-resistant tuberculosis (MDR TB), we retrospectively reviewed records of 127 MDR TB patients with and without MB testing between 2004 and 2007. Use of the MB assay reduced the time to detection and treatment of MDR TB.
All in-text references underlined in blue are linked to publications on ResearchGate, letting you... more All in-text references underlined in blue are linked to publications on ResearchGate, letting you access and read them immediately.
This chapter includes a description of nonradioactive broth culture systems and rapid molecular s... more This chapter includes a description of nonradioactive broth culture systems and rapid molecular systems for detection of drug resistance, as well as the standard agar proportion method. The antimicrobial agents that are used in the treatment of mycobacterial infections are discussed in the chapter for the most commonly encountered species. The Clinical and Laboratory Standards Institute (CLSI) document on drug susceptibility testing of mycobacteria currently recommends that first-line testing include ethambutol (EMB), RMP, INH, and pyrazinamide (PZA). The chapter describes drug susceptibility testing of M. tuberculosis complex. Broth microdilution is the method recommended by the CLSI for susceptibility testing of nontuberculous mycobacteria (NTM). The CLSI provides guidelines for testing M. avium complex (MAC), M. kansasii, M. marinum, and the rapidly growing mycobacteria. General recommendations regarding the broth microdilution method and QC that apply to all NTM are discussed in the chapter. The chapter describes specific details related to MAC, M. kansasii, M. marinum, and the rapidly growing mycobacteria. It also talks about incubation temperature and time for each species or group. The chapter explains that Nocardia spp. and other aerobic actinomycetes (Actinomadura, Rhodococcus equi, Gordonia, Tsukamurella, and rarely Streptomyces spp.) can cause serious disease in immunocompromised and occasionally even healthy hosts. The recommended method for testing Nocardia and other aerobic actinomycetes is broth microdilution.
The international journal of tuberculosis and lung disease : the official journal of the International Union against Tuberculosis and Lung Disease, 2000
Healthy People 2010, an initiative from the federal government, calls for action from tuberculosi... more Healthy People 2010, an initiative from the federal government, calls for action from tuberculosis controllers and tuberculosis laboratories in the fight to eliminate tuberculosis. Many patients, such as immunocompromised patients and those infected with multidrug-resistant tuberculosis strains, pose a challenge for care and diagnosis. Fortunately, many changes have occurred in the last decade to facilitate more rapid and accurate testing to assist with the care of these patients. California, Florida, New York and Texas have almost 50% of the tuberculosis cases in the United States, and their public health laboratories utilize different approaches to meet the same goal of rapid and accurate testing of specimens. With the targets of Healthy People 2010 (e.g., to reduce the average time for a laboratory to confirm and report tuberculosis cases to 2 days for 75% of cases) already looming on the horizon, innovative methods for achieving these goals should be evaluated. Using these publi...
Polyphasic taxonomic methods were employed to characterize a new species of slowly growing, nonpi... more Polyphasic taxonomic methods were employed to characterize a new species of slowly growing, nonpigmented mycobacteria. We propose the name Mycobacterium triplex sp. nov. for this new taxon. Conventional identification testing demonstrated a group of similar organisms that were geographically widespread in the United States. Commercially available nucleic-acid probes specific for the Mycobacterium avium complex were unreactive for these strains. High-performance liquid chromatography analysis of the mycolic acids revealed mycolate profiles that closely resembled Mycobacterium simiae. Comparative 16S rRNA sequence data confirmed the phylogenetic relationship of the strains with the slowly growing mycobacteria. Representative-type strains have been deposited in the American Type Culture Collection as strain ATCC 700071 [corrected].
Microscopic examination of respiratory specimens for acid-fast bacilli (AFB) plays a key role in ... more Microscopic examination of respiratory specimens for acid-fast bacilli (AFB) plays a key role in the initial diagnosis of tuberculosis, monitoring of treatment, and determination of eligibility for release from isolation. The objective of this study was to compare the sensitivity obtained with smears for detection of AFB (AFB smears) made directly from respiratory specimens (direct AFB smears) to that obtained with parallel smears made from concentrates of the specimens (concentrated AFB smears). A total of 2,693 specimens were evaluated; 1,806 were from the University of California Irvine Medical Center Medical Microbiology Laboratory (UCIMC), which serves a tertiary-care hospital with outpatient clinics, and 887 were from the Microbial Disease Laboratory at the California Department of Public Health (MDL), which receives specimens from outpatient facilities and clinics on Pacific islands. Of the 353 AFB culture-positive specimens at UCIMC, there was a statistically significant dif...
We explored sharing nucleic acid amplification testing (NAAT) for detection of Mycobacterium tube... more We explored sharing nucleic acid amplification testing (NAAT) for detection of Mycobacterium tuberculosis complex (MTBC) and molecular and phenotypic drug susceptibility testing between two state public health tuberculosis (TB) laboratories, and evaluated turnaround times and cost-effectiveness. From September 1, 2012, to May 30, 2013, the Wisconsin State Laboratory of Hygiene (Wisconsin Lab) submitted specimens to the Microbial Diseases Laboratory of the California Department of Public Health (California Lab) for NAAT and molecular drug susceptibility testing (MDST) by pyrosequencing, and culture-based TB drug susceptibility testing by the BACTEC(TM) MGIT(TM) 960 system. A total of 182 specimens were referred to the California Lab, and 47 TB cases and 12 drug-resistant cases were identified. The average time for specimen transport was two days, which included one day for processing and packaging in the submitting laboratory. The average turnaround time for NAAT was 0.3 days at the ...
The international journal of tuberculosis and lung disease : the official journal of the International Union against Tuberculosis and Lung Disease, 2015
Rifampin (RMP) resistant Mycobacterium tuberculosis is usually assumed to be resistant to all rif... more Rifampin (RMP) resistant Mycobacterium tuberculosis is usually assumed to be resistant to all rifamycins. Increasing evidence indicates, however, that some rpoB mutations, detectable by rapid molecular diagnostics, confer resistance to RMP but not to rifabutin (RBT), suggesting that RBT may be effective for the treatment of M. tuberculosis with these mutations. To determine if specific rpoB mutations reliably predict differential phenotypic resistance to RMP and RBT. We selected 60 clinical M. tuberculosis isolates from a repository of multinational multidrug-resistant tuberculosis isolates and stratified them into two groups: 1) those with rpoB mutations suspected to confer differential resistance to RMP and RBT, and 2) those expected to be cross-resistant to RMP and RBT. These assumptions were tested by comparing the phenotypic susceptibilities of RMP/RBT with those predicted by mutations in the rpoB gene. Of 20 suspected RMP-resistant/RBT-susceptible isolates, 15 were RMP-resista...
The international journal of tuberculosis and lung disease : the official journal of the International Union against Tuberculosis and Lung Disease, 2009
Drug-resistant strains of Mycobacterium tuberculosis are increasing worldwide and pose a major th... more Drug-resistant strains of Mycobacterium tuberculosis are increasing worldwide and pose a major threat to global health. However, it remains unsettled whether drug-resistant mutants are fixed in the bacterial population or if they would revert in the absence of drug pressure. To document the occurrence of isoniazid (INH) reversion in a patient with multidrug-resistant tuberculosis (TB) and investigate its association with fitness cost. Genotypic and phenotypic assays were used to characterize the reversion of INH resistance in isolates from a patient with pulmonary TB. The pre-reversion katG mutation was reconstructed in a pan-susceptible laboratory strain (H37Rv DeltakatG::katG W300G) and tested for susceptibility to INH and oxidative stress. Genotyping and drug susceptibility testing showed that an isogenic strain of M. tuberculosis reverted from an INH-resistant to a susceptible phenotype in the absence of INH therapy. The genotypic basis of this reversion was mapped to the katG c...
Reliable molecular diagnostics, which detect specific mutations associated with drug resistance, ... more 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 IS 6110 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-...
The Bactec MGIT 960 system for testing susceptibility to second-line drugs was evaluated with 117... more The Bactec MGIT 960 system for testing susceptibility to second-line drugs was evaluated with 117 clinical strains in a multicenter study. The four drugs studied were levofloxacin, amikacin, capreomycin, and ethionamide. The critical concentration established for levofloxacin and amikacin was 1.5 μg/ml, that established for capreomycin was 3.0 μg/ml, and that established for ethionamide was 5.0 μg/ml. The overall level of agreement between the agar proportion method and the MGIT 960 system was 96.4%, and the levels of agreement for the individuals drugs were 99.1% for levofloxacin, 100% for amikacin, 97.4% for capreomycin, and 88.9% for ethionamide. The rate of reproducibility of the drug susceptibility testing results between the participating laboratories was 99.5%.
Prompt laboratory reporting of tuberculosis (TB) test results is necessary for TB control. To und... more Prompt laboratory reporting of tuberculosis (TB) test results is necessary for TB control. To understand the extent of and factors contributing to laboratory reporting delays and the impact of reporting delays on initiation of treatment of TB patients, we analyzed data from 300 consecutive culture-positive TB cases reported in four California counties in 1998. Laboratory reporting to the specimen submitter was delayed for 26.9% of smear-positive patients and 46.8% of smear-negative patients. Delays were associated with the type of laboratory that performed the testing and with delayed transport of specimens. Referral laboratories (public health and commercial) had longer median reporting time frames than hospital and health maintenance organization laboratories. Among patients whose treatment was not started until specimens were collected, those with delayed laboratory reporting were more likely to have delayed treatment than patients with no laboratory reporting delays (odds ratio ...
To assess the clinical impact of a molecular beacon (MB) assay that detects multidrug-resistant t... more To assess the clinical impact of a molecular beacon (MB) assay that detects multidrug-resistant tuberculosis (MDR TB), we retrospectively reviewed records of 127 MDR TB patients with and without MB testing between 2004 and 2007. Use of the MB assay reduced the time to detection and treatment of MDR TB.
Uploads