A real-time PCR assay for detection of isoniazid resistance in Mycobacterium tuberculosis clinical isolates

AimsMycobacterium tuberculosis and non-tuberculous mycobacteria (NTM) are clinically different, and the rapid detection and differentiation of M. tuberculosis complex (MTBC) and NTM is crucial for patient management and infection control. Given the slow growth of most pathogenic mycobacteria, nucleic acid amplification assays are excellent tools for direct identification of mycobacteria in clinical specimens. Recently, a multiplex real-time PCR assay was developed that can directly detect 20 mycobacterial species in clinical specimens. Here, we evaluated the diagnostic performance of the assay for diagnosing mycobacterial disease under routine laboratory conditions.MethodsA total of 3334 specimens collected from 1437 patients suspected of tuberculosis infection were subjected to acid-fast bacilli staining, conventional culture and the multiplex real-time PCR assay. To evaluate the sensitivity and specificity of the assay, the overall diagnosis of tuberculosis was defined by positive culture plus medical history, and the 2007 American Thoracic Society and Infectious Disease Society of America diagnostic criteria for NTM disease were applied.ResultsThe sensitivity, specificity, positive predictive value and negative predictive value were 87.5%, 99.6%, 96.1% and 98.5%, respectively, for the detection of MTBC isolates and 53.3%, 99.9%, 95.2%, and 98.9%, respectively, for detecting NTM isolates.ConclusionsThus, the assay can correctly differentiate between MTBC and NTM isolates in clinical specimens and would be a useful tool for the rapid differentiation of tuberculosis and NTM disease, despite its limited sensitivity for the diagnosis of NTM disease.

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Real-Time PCR Assay for Detection of blaZ Genes in Staphylococcus aureus Clinical Isolates

Journal of Clinical Microbiology ◽

10.1128/jcm.03413-13 ◽

2014 ◽

Vol 52 (4) ◽

pp. 1259-1261 ◽

Cited By ~ 15

Author(s):

L. A. Pereira ◽

G. B. Harnett ◽

M. M. Hodge ◽

J. A. Cattell ◽

D. J. Speers

Keyword(s):

Staphylococcus Aureus ◽

Real Time ◽

Real Time Pcr ◽

Clinical Isolates ◽

Pcr Assay

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Detection of the plasmid-mediated colistin-resistance genemcr-1in clinical isolates and stool specimens obtained from hospitalized patients using a newly developed real-time PCR assay: Table 1.

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkw192 ◽

2016 ◽

Vol 71 (8) ◽

pp. 2344-2346 ◽

Cited By ~ 25

Author(s):

R. H. T. Nijhuis ◽

K. T. Veldman ◽

J. Schelfaut ◽

A. Van Essen-Zandbergen ◽

E. Wessels ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Hospitalized Patients ◽

Clinical Isolates ◽

Colistin Resistance ◽

Pcr Assay ◽

Stool Specimens

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Rapid Detection of Fluoroquinolone-Resistant and Heteroresistant Mycobacterium tuberculosis by Use of Sloppy Molecular Beacons and Dual Melting-Temperature Codes in a Real-Time PCR Assay

Journal of Clinical Microbiology ◽

10.1128/jcm.02271-10 ◽

2010 ◽

Vol 49 (3) ◽

pp. 932-940 ◽

Cited By ~ 37

Author(s):

S. Chakravorty ◽

B. Aladegbami ◽

K. Thoms ◽

J. S. Lee ◽

E. G. Lee ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Real Time ◽

Melting Temperature ◽

Real Time Pcr ◽

Rapid Detection ◽

Molecular Beacons ◽

Pcr Assay

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Use of Real-Time PCR and Fluorimetry for Rapid Detection of Rifampin and Isoniazid Resistance-Associated Mutations in Mycobacterium tuberculosis

Journal of Clinical Microbiology ◽

10.1128/jcm.38.9.3194-3199.2000 ◽

2000 ◽

Vol 38 (9) ◽

pp. 3194-3199 ◽

Cited By ~ 69

Author(s):

Maria J. Torres ◽

Antonio Criado ◽

Jose C. Palomares ◽

Javier Aznar

Keyword(s):

Mycobacterium Tuberculosis ◽

Real Time ◽

Real Time Pcr ◽

Antimicrobial Agents ◽

Dna Amplification ◽

Single Strand Conformation Polymorphism ◽

Clinical Isolates ◽

Polymorphism Analysis ◽

Antibiotic Resistant ◽

Hybridization Probes

Very fast amplification of DNA in small volumes can be continuously monitored with a rapid cycler that incorporates fluorimetric detection. Primers were designed to amplify a 157-bp fragment of therpoB gene spanning codons 526 and 531 and a 209-bp fragment of the katG gene spanning codon 315 of Mycobacterium tuberculosis. Most mutations associated with resistance to rifampin (RMP) and isoniazid (INH) in clinical isolates occur in these codons. Two pairs of hybridization probes were synthesized; one in each pair was 3′ labeled with fluorescein and hybridized upstream of the codon with the mutation; the other two probes were 5′ labeled with LightCycler-Red 640. Each pair of probes recognized adjacent sequences in the amplicon. After DNA amplification was finished by using a LightCycler, the temperature at which the Red 640 probe melted from the product was determined in a 3-min melt program. Twenty M. tuberculosis clinical isolates susceptible to streptomycin, INH, RMP, and ethambutol and 36 antibiotic-resistant clinical M. tuberculosis isolates (16 resistant to RMP, 16 to INH, and 4 to both antimicrobial agents) were amplified, and the presence of mutations was determined using single-strand conformation polymorphism analysis, the LiQor automated sequencer, and the LightCycler system. Concordant results were obtained in all cases. Within 30 min, the LightCycler method correctly genotyped all the strains without the need of any post-PCR sample manipulation. Overall, this pilot study demonstrated that real-time PCR coupled to fluorescence detection is the fastest available method for the detection of RMP and INH resistance-associated mutations in M. tuberculosis clinical isolates.

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