Molecular approaches to the identification of mycobacteria (original) (raw)
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Molecular Diagnosis of Mycobacteria Direct Detection of Mycobacteria from Specimens
Tuberculosis is one of the leading infectious diseases in the world and is responsible for more than 2 million deaths and 8 million new cases annually. Because of the slow growth rate of the causative agent Mycobacterium tuberculosis, isolation, identification, and drug susceptibility testing of this organism and other clinically important mycobacteria can take several weeks or longer. During the past several years, many molecular methods have been developed for direct detection, species identification, and drug susceptibility testing of mycobacteria. These methods can potentially reduce the diagnostic time from weeks to days. Currently, two nucleic acid amplification methods, the Enhanced Mycobacterium tuberculosis Direct Test (Gen-Probe) and the Amplicor Mycobacterium tuberculosis Test (Roche Diagnostic Systems), have been approved by the Food and Drug Administration for direct detection of M. tuberculosis from clinical specimens. PCR-based sequencing has become commonly used to identify many mycobacterial species. DNA probes have been widely used for species determination of the most commonly encountered mycobacteria. High-density oligonucleotide arrays (DNA microarrays) also have been applied to simultaneous species identification and detection of mutations that confer rifampin resistance in mycobacteria.
Diagnostic Microbiology and Infectious Disease, 2003
A novel polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis (PRA) of the hsp65 gene was used for the routine identification of mycobacteria in a high throughput clinical laboratory. A total of 2036 clinical isolates were tested by PRA in conjunction with other methods. The PRA identification of M. tuberculosis complex was 100% sensitive and specific, and 74.5% of nontuberculous mycobacteria (NTM) were correctly identified. It gave highly consistent results for Mycobacterium avium complex (MAC) species and for most isolates of M. fortuitum, M. chelonae, and M. kansasii. It had proven to be highly robust and stable despite usage on such a large-scale and is thus particularly suitable for use in high throughput laboratories in areas with a high incidence of tuberculosis.
Journal of Clinical Microbiology, 2006
We evaluated GenoType Mycobacteria Direct (GTMD), a novel commercial assay based on nucleic acid sequence-based amplification technology, for the detection of Mycobacterium tuberculosis complex, M. avium, M. intracellulare, M. kansasii, and M. malmoense directly from clinical specimens. A total of 134 respiratory and extrapulmonary samples from 65 patients were processed. Sensitivity, specificity, positive predictive, and negative predictive values for GTMD were 92, 100, 100, and 77%, respectively. The GMTD technique is useful, reliable, and rapid when used during the normal routine of a clinical laboratory.
Journal of Clinical Microbiology, 2002
The performance of the Amplified Mycobacterium Tuberculosis Direct (AMTD) test (Gen-Probe Inc., San Diego, Calif.) was assessed in a large tertiary care mycobacteriology laboratory. Both acid-fast smear-positive and smear-negative respiratory and nonrespiratory clinical specimens were analyzed. From February 1998 to 4 October 2001, AMTD assays were performed on 391 respiratory specimens and 164 nonrespiratory specimens. The AMTD assay was compared to the "gold standard" of combined culture and clinical diagnosis. The overall sensitivity for all specimens, including those for which no smear result was available, was 91.2%. The overall sensitivities of the assay, including acid-fast smear-positive and -negative specimens, were 97.8 and 77.3% for respiratory and nonrespiratory specimens, respectively. The corresponding specificities for respiratory and nonrespiratory specimens were 99.1 and 98.5%, respectively. The overall specificity for all specimens was 98.9%. Positive and negative predictive values were 93.9 and 99.7% and 91.7 and 96.4% for respiratory and nonrespiratory specimens, respectively. The time saved by using the AMTD test for making a diagnosis of tuberculosis instead of using culture was 8.99 days. Inhibitors to the AMTD assay were found in 3.1% of respiratory specimens and 3.1% of nonrespiratory specimens. The assay, used in a general mycobacteriology laboratory setting, represents an important advance in improving the speed and accuracy of diagnosis in the management of patients with tuberculosis.
Mycobacteriosis and Tuberculosis: Laboratory Diagnosis
The Open Microbiology Journal
Background: Tuberculosis is one of the most important infectious diseases that has claimed its victims throughout much of known human history. With Koch's discovery of the tubercle bacillus as the etiologic agent of the disease, his sanitary and hygienic measures, which were based on his discovery and the development of a vaccine against tuberculosis by Albert Calmette and Camille Guérin in 1921, an attenuated Mycobacterium bovis strain, bacilli Calmette-Guérin (BCG), and the discovery of the first antibiotic against tuberculosis, streptomycin by Selman Waksman in 1943, soon led to the opinion that appropriate control measures had become available for tuberculosis and it had been assumed that the disease could ultimately be eradicated. The emergence of resistant strains of this bacteria and widespread distribution of the disease in the world, and the emergence of the AIDS epidemic destroyed any possibility of global control of tuberculosis in the foreseeable future. Objectives: ...
Journal of Medical Microbiology, 2007
The aim of this study was to improve the identification of Mycobacterium species in the context of a UK teaching hospital. Real-time PCR assays were established to enable the rapid differentiation between Mycobacterium tuberculosis (MTB) complex and Mycobacterium species other than tuberculosis (MOTT), followed by 16S rRNA gene sequencing for the speciation of MOTT. Real-time PCR assays gave comparable results to those from the reference laboratory. The implementation of these PCR assays using an improved bead extraction method has enhanced the mycobacterial diagnostic service at the Royal Free Hospital by providing a rapid means of differentiating between MTB complex and MOTT, and would be simple to implement in similar laboratories. Sequence analysis successfully identified a range of Mycobacterium spp. representative of those encountered in the clinical setting of the authors, including Mycobacterium avium complex, Mycobacterium fortuitum group, Mycobacterium chelonae-Mycobacterium abscessus group, Mycobacterium xenopi and Mycobacterium gordonae. It provides a useful tool for the identification of MOTT when clinically indicated.
Journal of Clinical Microbiology, 2002
Early diagnosis of tuberculosis and screening of other mycobacteria is required for the appropriate management of patients. We have therefore developed a 5-exonuclease fluorogenic PCR assay in a single-tube balanced heminested format that simultaneously detects Mycobacterium tuberculosis complex (MTC) and members of the Mycobacterium genus (MYC) using the 16S ribosomal DNA target directly on clinical samples. One hundred twenty-seven clinical samples (65 smear negative and 62 smear positive) with a positive culture result from 127 patients were tested, including 40 negative control specimens. The finding of both a positive MTC and probe value and a positive MYC probe value confirmed the presence of MTC or mycobacteria with a 100% positive predictive value. However, a negative value for MTC or MYC did not discount the presence of mycobacteria in the specimen. Interestingly, the addition of the MYC probe allowed the diagnosis of an additional 7% of patients with tuberculosis and rapid screening of nontuberculous mycobacteria (NTM). Thus, over 75% of the patients were diagnosed with mycobacterial disease by PCR. The sensitivity was much higher on smearpositive samples (90.3%) than smear-negative samples (49.2%) and was slightly higher for MTC than NTM samples. With regard to the origin of the sample, MTC pulmonary samples gave better results than others. In conclusion, we believe this test may be useful for the rapid detection of mycobacteria in clinical samples and may be a valuable tool when used together with conventional methods and the clinical data available.
Journal of Clinical Microbiology, 2012
We evaluated a new line probe assay (LiPA) kit to identify Mycobacterium species and to detect mutations related to drug resistance in Mycobacterium tuberculosis. A total of 554 clinical isolates of Mycobacterium tuberculosis (n ؍ 316), Mycobacterium avium (n ؍ 71), Mycobacterium intracellulare (n ؍ 51), Mycobacterium kansasii (n ؍ 54), and other Mycobacterium species (n ؍ 62) were tested with the LiPA kit in six hospitals. The LiPA kit was also used to directly test 163 sputum specimens. The results of LiPA identification of Mycobacterium species in clinical isolates were almost identical to those of conventional methods. Compared with standard drug susceptibility testing results for the clinical isolates, LiPA showed a sensitivity and specificity of 98.9% and 97.3%, respectively, for detecting rifampin (RIF)-resistant clinical isolates; 90.6% and 100%, respectively, for isoniazid (INH) resistance; 89.7% and 96.0%, respectively, for pyrazinamide (PZA) resistance; and 93.0% and 100%, respectively, for levofloxacin (LVX) resistance. The LiPA kit could detect target species directly in sputum specimens, with a sensitivity of 85.6%. Its sensitivity and specificity for detecting RIF-, PZA-, and LVX-resistant isolates in the sputum specimens were both 100%, and those for detecting INH-resistant isolates were 75.0% and 92.9%, respectively. The kit was able to identify mycobacterial bacilli at the species level, as well as drug-resistant phenotypes, with a high sensitivity and specificity.