Utility of high-performance liquid chromatography analysis of mycolic acids and partial 16S rRNA gene sequencing for routine identification of Mycobacterium spp. in a national reference laboratory

Toney, Nadege C.; Toney, Sean; Butler, W. Ray

doi:10.1016/j.diagmicrobio.2010.02.011

Cited by 18 publications

(8 citation statements)

References 51 publications

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“…The obtained results did not significantly extend the specificity or sensitivity relative to the standard AFB staining method. Novel TB diagnosis methods involve genetic analysis of DNA or RNA (Daley et al, 2008;Toney et al, 2010). Xpert MTB/RIF assay is an example of a sensitive, specific and rapid PCR-based method (Chang et al, 2012;Lawn et al, 2011).…”

Section: Discussionmentioning

confidence: 99%

Rapid method for Mycobacterium tuberculosis identification using electrospray ionization tandem mass spectrometry analysis of mycolic acids

Szewczyk

Kowalski

Janiszewska-Drobińska³

et al. 2013

Diagnostic Microbiology and Infectious Disease

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Mycolic acids, which play a crucial role in the architecture of mycobacterial cell walls, were analyzed using electrospray ionization tandem mass spectrometry (ESI-MS/MS). A targeted analysis based on the 10 most abundant and characteristic multiple reaction monitoring (MRM) pairs was used to profile the crude fatty acid mixtures from Mtb and several nontuberculous mycobacterial strains. Comparative analysis yielded unique profiles for mycolic acids, enabling the reliable identification of mycobacterial species. In a case-control study of TB and non-TB Polish patients, we demonstrated the potential diagnostic utility of our approach for the rapid diagnosis of active TB with sensitivity and specificity surpassing those of existing methods. This robust method allows the identification of TB-positive patients after 2 h of sample preparation in the case of direct sputum analysis or 10 days of culturing, both of which are followed by one minute of LC-MS/MS analysis.

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Section: Discussionmentioning

confidence: 99%

Rapid method for Mycobacterium tuberculosis identification using electrospray ionization tandem mass spectrometry analysis of mycolic acids

Szewczyk

Kowalski

Janiszewska-Drobińska³

et al. 2013

Diagnostic Microbiology and Infectious Disease

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“…Standard laboratory practice for NTM speciation entails either high-performance liquid chromatography (HPLC) or molecular methods, which are generally preferred [20, 48•, 50, 51]. Molecular NTM genotyping options include commercially available DNA probes, PCR product-restriction enzyme analysis (PRA), or 16S ribosomal RNA sequencing [20, 48•].…”

Section: Ntm Diagnosismentioning

confidence: 99%

Nontuberculous Mycobacteria in Cystic Fibrosis

Skolnik

Kirkpatrick

Quon

2016

Curr Treat Options Infect Dis

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Opinion statementNontuberculous mycobacteria (NTM) are found in approximately 10 % of cystic fibrosis (CF) patients, but only a portion will develop NTM disease. The management of CF lung disease should be optimized, including antibiotic therapy targeted to the individual’s usual airway bacteria, prior to considering treatment for NTM lung disease. Those who meet criteria for NTM lung disease may not necessarily require treatment and could be monitored expectantly if symptoms and radiographic findings are minimal. However, the presence of Mycobacterium abscessus complex (MABSC), severe lung disease, and/or anticipated lung transplant should prompt NTM therapy initiation. For CF patients with Mycobacterium avium complex (MAC), recommended treatment includes triple antibiotic therapy with a macrolide, rifampin, and ethambutol. Azithromycin is generally our preferred macrolide in CF as it is better tolerated and has fewer drug-drug interactions. MABSC treatment is more complex and requires an induction phase (oral macrolide and two IV agents including amikacin) as well as a maintenance phase (nebulized amikacin and two to three oral antibiotics including a macrolide). The induction phase may range from one to three months (depending on infection severity, treatment response, and medication tolerability). For both MAC and MABSC, treatment duration is extended 1-year post-culture conversion. However, in patients who do not achieve culture negative status but tolerate therapy, we consider ongoing treatment for mycobacterial suppression and prevention of disease progression.

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“…These include DNA probe hybridization-and nucleic acid amplification-based assays with or without sequencing (1-4) and high-performance liquid chromatography (HPLC) to evaluate the mycolic acid composition (1,2). More recently, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been shown to be a reliable and rapid means for the identification of bacterial species, including mycobacteria through the recognition of specific proteins (5), PCR amplification profiles (6), or protein spectral profiles (7,8).…”

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confidence: 99%

Rapid Inactivation of Mycobacterium and Nocardia Species before Identification Using Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

et al. 2014

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The identification of mycobacteria outside biocontainment facilities requires that the organisms first be rendered inactive. Exposure to 70% ethanol (EtOH) either before or after mechanical disruption was evaluated in order to establish a safe, effective, and rapid inactivation protocol that is compatible with identification of Mycobacterium and Nocardia species using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). A combination of 5 min of bead beating in 70% EtOH followed by a 10-min room temperature incubation period was found to be rapidly bactericidal and provided high-quality spectra compared to spectra obtained directly from growth on solid media. The age of the culture, the stability of the refrigerated or frozen lysates, and freeze-thaw cycles did not adversely impact the quality of the spectra or the identification obtained. Different approaches have been developed to hasten the identification of clinically significant mycobacteria compared to traditional biochemical phenotyping. These include DNA probe hybridization-and nucleic acid amplification-based assays with or without sequencing (1-4) and high-performance liquid chromatography (HPLC) to evaluate the mycolic acid composition (1, 2). More recently, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been shown to be a reliable and rapid means for the identification of bacterial species, including mycobacteria through the recognition of specific proteins (5), PCR amplification profiles (6), or protein spectral profiles (7,8). Advanced databases for use with the latter approach are currently being expanded. Regardless of which option is used, it is essential that cultures are rendered nonviable prior to processing outside appropriate biocontainment facilities for obvious safety reasons. Previous reports have identified a variety of ways in which mycobacteria, including Mycobacterium tuberculosis can be successfully inactivated prior to manipulation, including heat treatment (9), exposure to lethal irradiation with or without photosensitizing chemicals (10-12), alcohol exposure (13), or a combination of treatments (14-16). While most were successful, temperatures and/or exposure times varied in order to achieve uniform cell death. Treatment with disinfecting agents such as 2% glutaraldehyde was not considered an option as it can result in extensive cross-linking of the nucleic acids, proteins, and other cellular components that are necessary for the MALDI-TOF MS identification process. Within the past year, two publications described the successful adaptation of a mycobacterial inactivation/ extraction protocol for use with MALDI-TOF MS-based identification (17, 18). Herein, we report a detailed description of an even more convenient process that was first presented in part in 2013 (19). MATERIALS AND METHODSAll studies involving mycobacteria were performed in a biological safety level 3 (BSL-3) laboratory. For various aspects of this study, 28 strains encompass...

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Utility of high-performance liquid chromatography analysis of mycolic acids and partial 16S rRNA gene sequencing for routine identification of Mycobacterium spp. in a national reference laboratory

Cited by 18 publications

References 51 publications

Rapid method for Mycobacterium tuberculosis identification using electrospray ionization tandem mass spectrometry analysis of mycolic acids

Rapid method for Mycobacterium tuberculosis identification using electrospray ionization tandem mass spectrometry analysis of mycolic acids

Nontuberculous Mycobacteria in Cystic Fibrosis

Rapid Inactivation of Mycobacterium and Nocardia Species before Identification Using Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

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