Use of blood agar along with Lowenstein-Jensen media for rapid isolation of Mycobacterium tuberculosis for early drug susceptibility testing

ABSTRACTFour out of 143 phenotypically isoniazid-resistant but rifampin-susceptibleMycobacterium tuberculosisstrains that were isolated from patients in Germany in 2011 had mutations in the rifampin resistance-determining region ofrpoB. After performing drug susceptibility testing (DST) with two methods, the proportion method on Löwenstein-Jensen medium and using the Bactec 960 Mycobacteria Growth Indicator Tube system, we conclude that the two methods are equally reliable for phenotypic DST and MIC determination.

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Thin layer agar-based direct phenotypic drug-susceptibility testing on sputum in Eswatini rapidly detects Mycobacterium tuberculosis growth and rifampicin resistance, otherwise missed by WHO endorsed diagnostic tests.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02263-20 ◽

2021 ◽

Author(s):

E. Ardizzoni ◽

E. Ariza ◽

D. Mulengwa ◽

Q. Mpala ◽

R. de La Tour ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Thin Layer ◽

Susceptibility Testing ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Field Conditions ◽

Reference Laboratory ◽

Solid Media ◽

Specific Setting ◽

Lowenstein Jensen

BACKGROUND: Xpert®MTB/RIF rapidly detects resistance to rifampicin (RR), however this test misses the I491F-RR conferring rpoB mutation, common in Southern Africa. In addition, Xpert®MTB/RIF does not distinguish between viable and dead Mycobacterium tuberculosis (MTB). OBJECTIVE: To investigate the ability of thin layer agar (TLA) direct drug-susceptibility testing (DST) to detect MTB and its drug-resistance profiles in field conditions in Eswatini. DESIGN: Consecutive samples were tested in parallel with Xpert®MTB/RIF and TLA for rifampicin (1.0 μg/ml) and ofloxacin (2.0 μg/ml). TLA results were compared at the Reference Laboratory in Antwerp with indirect DST on Löwenstein-Jensen or 7H11 solid media and additional phenotypic and genotypic testing to resolve discordance. RESULTS: TLA showed a positivity rate for MTB detection of 7.1% versus 10.0% for Xpert®MTB/RIF. Of a total of 4547 samples included in the study, 200 isolates were available for comparison to the composite reference. Within a median of 18.4 days, TLA detected RR with 93.0% sensitivity (CI-77.4-98.0) and 99.4% specificity (CI 96.7-99.9), versus 62.5% (CI 42.7-78.8) and 99.3% (CI 96.2-99.9) for Xpert®MTB/RIF. Eight isolates, 28.6% of all RR confirmed isolates, carried the I491F mutation, all detected by TLA. TLA also correctly identified 183 of the 184 ofloxacin-S isolates (99.5% specificity, CI 97.0-99.9). CONCLUSIONS: In field conditions, TLA rapidly detects RR, and in this specific setting contributed to detection of additional RR patients over Xpert®MTB/RIF, mainly but not exclusively due to I491F. TLA also accurately excluded fluoroquinolones resistance.

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Comparison of Proportion and Resistance Ratio Methods for Drug Susceptibility Testing of Mycobacterium tuberculosis Isolated from Patients Attending National Tuberculosis Centre, Nepal

SAARC Journal of Tuberculosis Lung Diseases and HIV/AIDS ◽

10.3126/saarctb.v5i1.3078 ◽

2010 ◽

Vol 5 (1) ◽

pp. 13-20

Author(s):

S Acharya ◽

P Ghimire ◽

DK Khadka ◽

S Nepali

Keyword(s):

Mycobacterium Tuberculosis ◽

Fluorescence Microscopy ◽

Susceptibility Testing ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Resistance Ratio ◽

National Tuberculosis ◽

Smear Negative ◽

Culture Positive ◽

Good Agreement

Background: Tuberculosis (TB) is among the most serious infectious cause of global morbidity and mortality. Emergence of Multi-drug resistant tuberculosis (MDR-TB) is posing an increased threat to TB control programs. Drug susceptibility testing (DST) of Mycobacterium tuberculosis (M. tuberculosis) isolates is important for tackling such problems. Setting: National Tuberculosis Centre (NTC), Thimi, Bhaktapur, Nepal. Objectives: Comparative evaluation of two in vitro DST methods in determining susceptibility of M. tuberculosis isolates from patients attending NTC, to front-line anti-TB drugs: (Isoniazid-INH, Rifampicin-RFP, Streptomycin-SM, and Ethambutol-EMB). Methodology: This study was conducted from Sep 2006-Jun 2007. A total of 862 sputum samples (diagnosis or follow up cases) collected from patients (type of patients or their categories was not differentiated in this study) attending NTC bacteriology lab for sputum direct smear microscopy were analyzed using fluorescence microscopy. All smear positive samples, smear negative samples requested for culture were cultured. All culture positive samples confirmed as M. tuberculosis by biochemical tests were processed for DST by both proportion (PR) and resistance ratio (RR) methods. Results: Out of 862 sputum samples analyzed, 226 (26.2%) samples were positive for Acid Fast Bacilli (AFB) by fluorescence microscopy. Among 323 samples 226 smear positive samples and 97 smear negative samples requested for culture), 221 (68.4%) were culture positive, 92 (28.5%) were culture negative and 10 (3.1%) were contaminated. Out of 221 isolates of M. tuberculosis, 57.5% were resistant to one or more drugs by the PR method and 56.6% by the RR method. Similarly, MDR isolates were 29.9% and 29% by PR and RR methods respectively. On correlation analysis using Mc Nemar Chi-square test, no significant difference between the two tests were observed (p>0.05). The results showed high agreement between both methods and agreement rates to INH, RFP, SM and EMB were 93.2%, 93.7%, 93.2% and 94.1% respectively. Similarly, the agreement rates between both methods using kappa analysis showed kappa (k) value of 0.86, 0.85, 0.86 and 0.84 for INH, RFP, SM and EMB respectively, which is believed to be good agreement between both methods (k=0.80 to 1.00: Very good agreement). Conclusion: In conclusion, this study showed that both the Proportion and Resistance ratio methods are equally good for determining drug susceptibility of M. tuberculosis. Keywords: Mycobacterium tuberculosis; Drug Susceptibility Testing; Proportion Method; Resistance Ratio Method. DOI: 10.3126/saarctb.v5i1.3078 SAARC J. Tuber. Lung Dis. HIV/AIDS 2008 Vol.5(1) 13-20

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Comparison of the Performances of Two In-House Rapid Methods for Antitubercular Drug Susceptibility Testing

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00960-08 ◽

2008 ◽

Vol 53 (2) ◽

pp. 808-810 ◽

Cited By ~ 6

Author(s):

Agustina I. de la Iglesia ◽

Emma J. Stella ◽

Héctor R. Morbidoni

Keyword(s):

Drug Resistance ◽

Mycobacterium Tuberculosis ◽

Sensitivity And Specificity ◽

Susceptibility Testing ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Antitubercular Drug ◽

Rapid Methods ◽

Resistance Assessment

ABSTRACT Resistance to rifampin (rifampicin), isoniazid, and streptomycin of 69 Mycobacterium tuberculosis isolates was analyzed by an in-house method based on mycobacteriophage D29 and a colorimetric micromethod. Both methods showed sensitivity and specificity values ranging from 93% to 100%. These simple methods offer an option for drug resistance assessment of M. tuberculosis.

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