Susceptibility Testing Of Mycobacterium Tuberculosis Research Articles

Evaluation of Drug Susceptibility Testing of Mycobacterium tuberculosis Isolates Against First Line Antituberculosis Drugs With a New Agar Medium

Tüberküloz (TB), dünya genelinde enfeksiyon kaynaklı hastalıklar arasında ölümün önde gelen nedenlerinden biri olan bulaşıcı bir hastalıktır. TB’nin epidemiyolojik dağılımına göre, yüksek oranda TB basili ile enfekte hastaların bulunduğu ülkelerin büyük bir bölümü orta ve düşük gelirli ülkelerdir. Bu nedenle çalışmada düşük gelirli ülkelerde ve laboratuvarlarda TB ve çok ilaca dirençli ( TB izolatlarının ilaç duyarlılık testleri için kolayca kullanılabilecek yeni bir besiyeri olan AYC.2.2 agar besiyerinin klinik izolatlarla etkinliğinin değerlendirilmesi amaçlanmıştır. Çalışmada Mycobacterium tuberculosis ATCC 27294 (H37Rv, tüm ilaçlara duyarlı), ATCC 35822 (izoniazid dirençli), ATCC 35838 (rifampisin dirençli), ATCC 35820 (streptomisin dirençli) ve ATCC 35837 (etambutol dirençli) referans suşlar ve 34 klinik izolat test edilmiştir. Çalışmadaki tüm anti TB ilaçlar ve izolatlar için kullanılan referans yöntem ve AYC.2.2 agar kullanılarak yapılan agar proporsiyon metodu arasında duyarlılık, özgüllük, pozitif prediktif değer (PPD), negatif prediktif değer (NPD) ve uyum hesaplanmıştır. İzoniazid ve rifampisin için tüm değerler %100 bulunmuştur. Etambutol için duyarlılık %100, özgüllük %88.23, PPD %89.47, NPD %100, uyum %94 olarak hesaplanmıştır. Streptomisin için duyarlılık %100, özgüllük %77.77, PPD %80, NPD %100, uyum %88 bulunmuştur. M. tuberculosis ATCC suşları klinik izolatların değerlendirilmesine dahil edilmemiş ve bunlardaki uyum %100 bulunmuştur. Elde edilen sonuçlara göre, sıvı otomatize sistemlere oranla üreme süresi dezavantaj olmasına rağmen, AYC.2.2. agar duyarlılık sonuçlarının belirlenmesinde otomatik sistemler kadar etkili bir şekilde kullanılabilmiştir. Maliyetinin düşük olması ve hazırlanmasının kolay olması en büyük avantajlarındandır. Ancak, rutin kullanıma geçmeden önce çok merkezli çalışmalara ihtiyaç vardır.

Evaluation of Xpert MTB/XDR test for susceptibility testing of Mycobacterium tuberculosis to first and second-line drugs in Uganda.

Drug-Resistant Tuberculosis (DR-TB) is one of the major challenges to TB control. This was a blinded, laboratory-based cross-sectional study using sputum samples or culture isolates. Samples were from patients with rifampicin-resistant-TB and/or with high risk for isoniazid (INH) resistance and/or 2nd line fluoroquinolones (FQ) and injectable agents (IAs). The diagnostic accuracy of the Xpert® MTB/XDR test was compared to MGIT960 and the Hain Genotype® MTBDRplus and MDRsl assays (LPA) as reference DST methods. Factors for laboratory uptake of the Xpert® MTB/XDR test were also evaluated. Of the 100 stored sputum samples included in this study, 65/99 (65.6%) were resistant to INH, 5/100 (5.0%) were resistant to FQ and none were resistant to IAs using MGIT960. The sensitivity and specificity, n (%; 95% Confidence Interval, CI) of Xpert® MTB/XDR test for; INH was 58 (89.2; 79.1-95.5) and 30 (88.2; 72.5-96.6) and for FQ; 4 (80.0; 28.3-99.4) and 95 (100; 96.2-100), respectively. Using LPA as a reference standard, a total of 52/98 (53.1%) were resistant to INH, 3/100 (3.0%) to FQ, and none to IA. The sensitivity and specificity, n (%; 95%CI) of Xpert® MTB/XDR test compared to LPA for; INH was 50 (96.1; 86.7-99.5) and 34 (74.0; 58.8-85.7) for FQ 3 (100; 29.2-100) and 96 (99.0; 94.3-99.9) respectively. The factors for laboratory uptake and roll-out of the Xpert® MTB/XDR test included: no training needed for technicians with, and one day for those without, previous Xpert-ultra experience, recording and reporting needs were not different from those of Xpert-ultra, the error rate was 4/100 (4%), one (1%) indeterminate rate and test turn-around-time were 1hr/45 minutes. There is high sensitivity and specificity of Xpert® MTB/XDR test for isoniazid and fluoroquinolones. There are acceptable Xpert® MTB/XDR test attributes for the test uptake and roll-out.

The Effect of Inoculum Size on Antimicrobial Susceptibility Testing of Mycobacterium tuberculosis.

Phenotypic drug susceptibility testing (DST) requires a standardized amount of inoculum to produce reproducible susceptibility results. The most critical step in the application of DST in Mycobacterium tuberculosis isolates is the preparation of the bacterial inoculum. In this study, the effect of bacterial inoculum prepared in various McFarland turbidities on primary antituberculosis drug susceptibility of M. tuberculosis strains was investigated. Five standard ATCC strains (ATCC 27294 [H37Rv], ATCC 35822 [izoniazid-resistant], ATCC 35838 [rifampicin-resistant], ATCC 35820 [streptomycin-resistant], ATCC 35837 [ethambutol-resistant]) were tested. Inoculums of McFarland standard of 0.5, 1, 2, 3, and 1:100 dilutions of 1 McFarland standard of each strain were used. The effect of inoculum size on DST results was determined by the proportion method in Lowenstein-Jensen (LJ) medium and nitrate reductase assay (NRA) in the LJ medium. In both test methods, the increase in inoculum size did not affect the DST results of the strains. On the contrary, DST results were obtained more rapidly as a result of the use of dense inoculum. DST results obtained in all McFarland turbidities were found to be 100% compatible with the recommended amount of inoculum, 1:100 dilution of 1 McFarland standard (inoculum size of gold standard method). In conclusion, the use of a high amount of inoculum did not change the drug susceptibility profile of tuberculosis bacilli. Minimizing manipulations during the inoculum preparation phase of susceptibility testing, this outcome will decrease the need for equipment and make the test application easier, particularly in developing countries. IMPORTANCE During DST application, it can be challenging to evenly homogenize TB cell clumps with lipid-rich cell walls. These experiments must be carried out under Biosafety Level-3 (BSL-3) laboratory conditions with personal protective equipment and taking safety precautions because the procedures applied at this stage cause the formation of bacillus-laden aerosols and carry a serious risk of transmission. Considering this situation, this stage is important given that it is not possible to establish a BSL-3 laboratory in poor and developing countries. Reducing the manipulations to be applied during the preparation of bacterial turbidity will minimize the risk of aerosol formation. Perhaps there will be no need to do these steps for susceptibility tests in these countries or even in developed countries.

Determination of critical concentration for drug susceptibility testing of Mycobacterium tuberculosis against para-aminosalicylic acid with clinical isolates with thyA, folC and dfrA mutations

Background & ObjectivesAccurate determination of antimicrobial resistance profiles is of great importance to formulate optimal regimens against multidrug-resistant tuberculosis (MDR-TB). Although para-aminosalicylic acid (PAS) has been widely used clinically, the reliable testing methods for PAS susceptibility were not established. Herein, we aimed to establish critical test concentration for PAS on the Mycobacterial Growth Indicator Tube (MGIT) 960 in our laboratory settings.MethodsA total of 102 clinical isolates were included in this study, including 82 wild-type and 20 resistotype isolates. Minimum inhibitory concentration (MIC) was determined by MGIT 960. Whole-genome sequencing was used to identify the mutation patterns potentially conferring PAS resistance. Sequence alignment and structure modelling were carried out to analyze potential drug-resistant mechanism of folC mutant.ResultsOverall, the Minimum inhibitory concentration (MIC) distribution demonstrated excellent separation between wild-type and resistotype isolates. The wild-type population were all at least 1 dilution below 4 μg/ml, and the resistotype population were no lower than 4 μg/ml, indicating that 4 μg/ml was appropriate critical concentration to separate these two populations. Of 20 mutant isolates, 12 (60.0%) harbored thyA mutations, 2 (10%) had a mutation on upstream of dfrA, and the remaining isolates had folC mutations. Overall, thyA and folC mutations were scattered throughout the whole gene without any one mutation predominating. All mutations within thyA resulted in high-level resistance to PAS (MIC > 32 μg/ml); whereas the MICs of isolates with folC mutations exhibited great diversity, ranged from 4 to > 32 μg/ml, and sequence and structure analysis partially provided the possible reasons for this diversity.ConclusionsWe propose 4 μg/ml as tentative critical concentration for MGIT 960. The major mechanism of PAS resistance is mutations within thyA and folC in MTB isolations. The whole-gene deletion of thyA locus confers high-level resistance to PAS. The diversity of many distinct mutations scattered throughout the full-length folC gene challenges the PCR-based mutation analysis for PAS susceptibility.

Evaluation of the broth microdilution plate methodology for susceptibility testing of Mycobacterium tuberculosis in Peru

BackgroundTuberculosis (TB) is a communicable, preventable and curable disease caused by the bacterium Mycobacterium tuberculosis (MTB). Peru is amongst the 30 countries with the highest burden of multidrug-resistant tuberculosis (MDR-TB) worldwide. In the fight against drug-resistant tuberculosis, the UKMYC6 microdilution plate was developed and validated by the CRyPTIC project. The objective of the study was to evaluate the use of the broth microdilution (BMD) plate methodology for susceptibility testing of drug-resistant MTB strains in Peru.MethodsMTB strains isolated between 2015 and 2018 in Peru were used. 496 nationally-representative strains determined as drug-resistant by the routine 7H10 Agar Proportion Method (APM) were included in the present study. The Minimum Inhibitory Concentration (MIC) of 13 antituberculosis drugs were determined for each strain using the UKMYC6 microdilution plates. Diagnostic agreement between APM and BMD plate methodology was determined for rifampicin, isoniazid, ethambutol, ethionamide, kanamycin and levofloxacin. Phenotypes were set using binary (or ternary) classification based on Epidemiological cut-off values (ECOFF/ECV) proposed by the CRyPTIC project. Whole Genome Sequencing (WGS) was performed on strains with discrepant results between both methods.ResultsMIC distributions were determined for 13 first- and second-line anti-TB drugs, including new (bedaquiline, delamanid) and repurposed (clofazimine, linezolid) agents. MIC results were available for 80% (397/496) of the strains at 14 days and the remainder at 21 days. The comparative analysis determined a good agreement (0.64 ≤ k ≤ 0.79) for the drugs rifampicin, ethambutol, ethionamide and kanamycin, and the best agreement (k > 0.8) for isoniazid and levofloxacin. Overall, 12% of MIC values were above the UKMYC6 plate dilution ranges, most notably for the drugs rifampicin and rifabutin. No strain presented MICs higher than the ECOFF/ECV values for the new or repurposed drugs. Discrepant analysis using genotypic susceptibility testing by WGS supported half of the results obtained by APM (52%, 93/179) and half of those obtained by BMD plate methodology (48%, 86/179).ConclusionsThe BMD methodology using the UKMYC6 plate allows the complete susceptibility characterization, through the determination of MICs, of drug-resistant MTB strains in Peru. This methodology shows good diagnostic performances for rifampicin, isoniazid, ethambutol, ethionamide, kanamycin and levofloxacin. It also allows for the characterization of MICs for other drugs used in previous years against tuberculosis, as well as for new and repurposed drugs recently introduced worldwide.

Second-Line Drug Susceptibility Testing of Mycobacterium tuberculosis Isolates from Patients in Bayelsa State, Nigeria

Aim: To determine the second-line drug susceptibility testing of Mycobacterium tuberculosis isolates from patients in Bayelsa State, Nigeria.
 Study Design: A cross sectional study was carried out in this research.
 Place and Duration of Study: Directly Observed Treatment Short-course (DOTS) Centers across Bayelsa State, between March 2020 and November 2021.
 Methodology: Ethical approval was obtained from Bayelsa State Ministry of Health, Yenagoa, Nigeria. Informations such as age, sex and residential address was obtained with the help of a questionnaire. A total of 100 sputum sample was collected from 100 patients across all the Local Government Areas. Sputum sample decontamination and homogenization was done using the Sodium Hydroxide/N- Acetyl -L- Cysteine Citrate Solution. Sputum samples were cultured on solid Lowenstein Jensen (LJ) Media. All growths on LJ media were confirmed with Ziehl Neelsen staining and Standard Bioline antigen test. Drug susceptibility test was carried out after bacterial DNA was extracted and amplified using Line Probe Assay (MTBDRplus assay ver 2).
 Results: Out of 100 patients, 15 had confirmed growth of Mycobacterium tuberculosis. All isolate had no form of mutation on gyr gene, meaning 100% of isolates were susceptibility to flouroquinolones. There were also no mutation detected on rrs gene therefore all strains are also susceptible to Kanamycin, Amikasin and capreomycin. Out of the 15 isolates 14 had no mutation on the eis gene while 1% had mutation of WT2 and MUT1.
 Conclusion: A good percentage of the isolates are susceptible to second line drugs, therefore cases of extensive drug resistance is not common in Bayelsa State, Nigeria.

Comparison of Different Methods for Drug Susceptibility Testing of Mycobacterium tuberculosis to Rifampicin

The objective: to compare results of drug susceptibility testing to rifampicin by molecular genetic methods and phenotypic tests of Mycobacterium tuberculosis isolates obtained from clinical specimens of tuberculosis patients.Subjects and Methods. 915 samples of M. tuberculosis DNA and 426 cultures were used in this study. Genotypic tests (TB-TEST (BIOCIP-IMB, Russia), GenoType MTBDRplusV2) and phenotypic technologies (absolute concentration method, Bactec MGIT 960 system, Sensititre Myco TB kit) were used.Results. A high percentage (98.7%; CI 97.7-99.7%) of confirmation of the results of the molecular genetic test (TB-TEST) by the phenotypic test (absolute concentration method) was demonstrated. In some cases, the Bactec MGIT 960 system as well as the absolute concentration method were shown to produce false negative results of rifampicin resistance in some cases.

Single-cell raman spectroscopy: a powerful diagnostic tool for identification and drug susceptibility testing of Mycobacterium tuberculosis

Close to one quarter of the world’s population is infected with Mycobacterium tuberculosis (Mtb). Tuberculosis (TB) is hard to treat and causes 3,800 deaths each day. Multidrug-resistant TB (MDR-TB) is a global public health threat. Rapid identification and drug susceptibility testing (DST) of Mtb is key to prevent the spread of MDR-TB. We studied whether single-cell Raman spectroscopy is suitable to provide reliable species identification (ID) and DST results while significantly accelerating the time-to-result (TTR) compared to the standard of care (SoC), that is PCR-based species ID within one day and phenotypic DST results at 7-10 days of culture-positivity.

Drug susceptibility testing of Mycobacterium tuberculosis using next generation sequencing and Mykrobe software

Introduction. Mycobacterium tuberculosis is the causative agent of tuberculosis. Drug susceptibility testing is performed by phenotypic and molecular tests. Commonly used for phenotypic drug susceptibility testing is the automated BACTEC system in a liquid culture medium. Drug susceptibility by line probe molecular tests was introduced almost 15 years ago. Recently whole genome sequencing (WGS) analysis of M. tuberculosis strains demonstrated that genotyping of drug-resistance could be accurately performed. Several software tools were developed.Our study aimed to perform whole-genome sequencing on phenotypically confirmed multi-drug resistant (MDR) M. tuberculosis strains, to identify drug-resistant mutations and to compare whole-genome sequencing profiles with line probe assay and phenotypic results.Materials and methods. We performed analysis on 34 MDR M. tuberculosis Bulgarian strains. Phenotypic drug susceptibility testing was performed on the BACTEC system. For molecular testing of drug susceptibility to first- and second-line tuberculostatics, we applied line probe assay Geno Type MTBDR plus v.1.0 и Geno Type MTBDR sl v.1.0. Sequencing was performed on MiSeq. Generated FASTQ files were analyzed for known drugresistant mutations with the software platform Mykrobe v.0.8.1.Results. All three methods — phenotypic analysis using the BACTEC system, genetic analysis of strains applying the Geno Type test and Mykrobe software gave comparable sensitivity/resistance results for the studied strains. All phenotypically proven rifampicin and isoniazid-resistant strains were 100% confirmed using Mykrobe software. The C-15T mutation is a marker for isoniazid resistance in strains of the SIT41 spoligotype. We observed a 75% (21/28) agreement between BACTEC and Mykrobe for ethambutol resistance. Phenotypically, 87% (n = 27) of the strains are resistant to streptomycin, but only 59% (n = 19) are proven by Mykrobe software. Comparing phenotypic and genotypic resistance to ofloxacin, amikacin and kanamycin, we observed 100% coincidence of results.Conclusions. Whole-genome sequencing approach is relatively expensive and laborious but useful for detailed analysis such as epidemiological genotyping and molecular drug susceptibility testing.

Validation of a novel medium for drug susceptibility testing of Mycobacterium Tuberculosis against First- and Second-Line drugs: AYC.2.2 Agar and AYC.2.1 Broth.

The aim of this study was the validation of AYC.2.2 agar and AYC.2.1 broth for the breakpoint values of first- and second-line drugs for Mycobacterium tuberculosis. A total of 12 isolates including 5 reference strains and 7 well-defined clinical isolates were tested for their antituberculosis susceptibilities. Inhibitory effects of first- and second-line antituberculous drugs including isoniazid, rifampicin, streptomycin, ethambutol, amikacin, capreomycin, kanamycin, para-aminosalicylic acid, ethionamide, rifabutin, ofloxacin, levofloxacin, and moxifloxacin were tested. According to the minimal inhibitory concentration values obtained in 7H10 agar, 7H9-S broth, AYC.2.2 agar, and AYC.2.1 broth, category agreement is 100%, and very major discrepancy (MAD), MAD, and minor discrepancy ratios were determined as 0 for all drugs. It was concluded that breakpoint values by CLSI recommendation for 7H10 agar can be also used for AYC.2.2 agar and AYC.2.1 broth. In addition, further multicenter studies are needed to use the new medium in routine mycobacteriology laboratories.

A comparative study between microscopy and culture in detection of M.tb among smear negative pulmonary and extra pulmonary tuberculosis

A comparative study between microscopy and culture in detection of M.tb among smear negative pulmonary and extra pulmonary tuberculosis - IJMR- Print ISSN No: - 2394-546X Online ISSN No:- 2394-5478 Article DOI No:- 10.18231/2394-5478.2018.0066, Indian Journal of Microbiology Research-Indian J Microbiol Res

Resazurin assay for rapid drug susceptibility testing of Mycobacterium tuberculosis

Introduction: Tuberculosis, a disease of antiquity continues to cause high morbidity and mortality. In Sustainable Development Goals and End Tb Strategy proposed by WHO includes Drug Susceptibility Testing as major component. Conventional methods are time consuming and molecular methods are rapid but expensive and demanding. Resazurin assay is a rapid colorimetric redox indicator method of Drug Susceptibility Testing. Materials and Methods: 67 strains of M. tuberculosis isolated from pulmonary tuberculosis patients were tested using indirect absolute concentration method by resazurin assay and compared with drug incorporated Lowenstein – Jensen media for Isoniazid and Rifamycin. Results: There was 91.5 % and 91.7% concordance between both methods for INH and Rifamycin respectively. Resazurin assay for INH was 100% sensitive and 61.54% specific. Sensitivity and specificity for RMP was 98.04% and 62.5% respectively. The duration required for INH was 7.0 ± 2.07 and for RMP 6.58 ± 1.58 days. Conclusions: Resazurin assay is sensitive, rapid, cheap, technically simple drug susceptibility method. Keywords: M. tuberculosis, resazurin assay, drug susceptibility.

Genotyping and drug susceptibility testing of Mycobacterium tuberculosis in Iran: a multi-centre study.

Tuberculosis (TB) is a deadly infection and caused 1.4 million deaths in 2018. Assessing the geographic distribution of major lineages of Mycobacterium tuberculosis can contribute greatly to TB control. Mycobacterial interspersed repetitive unit variable number tandem repeat (MIRU-VNTR) typing is commonly used to differentiate various lineages of M. tuberculosis. A total of 2747 clinical specimens were collected consecutively from October 2018 through June 2019. Clinical isolates were identified as M. tuberculosis using standard biochemical tests. The standard 15-locus MIRU-VNTR typing was used for the genotyping of clinical isolates. Drug susceptibility testing was performed using the conventional proportion method. From the collected specimens, 100 were culture positive for M. tuberculosis. Using MIRU-VNTR, 99 different patterns were detected among the 100 isolates. They were distributed in one cluster comprising two strains and 98 unique patterns. Most of our isolates were similar to New-1 and Delhi/CAS strains. Of the M. tuberculosis isolates, 83 (83.0%) were pan-susceptible and 17 (17.0%) were resistant to at least one drug. Our study showed that MIRU-VNTR is a useful method for studying the genetic diversity of M. tuberculosis isolates in different regional settings and will help the health authorities to construct a preventive programme for TB.

Whole-genome sequencing in drug susceptibility testing of Mycobacterium tuberculosis in routine practice in Lyon, France

Rapid and correct determination of Mycobacterium tuberculosis (MTB) drug susceptibility is a challenge for tuberculosis (TB) management. Phenotypic drug susceptibility testing (DST) remains the reference method but is time consuming. In this study, genotypic prediction of the first-line drug susceptibility profile obtained by whole-genome sequencing (WGS) was compared with that obtained by phenotypic DST and the line probe assay (LPA). All MTB strains isolated from patients during routine practice at the mycobacteria laboratory of Lyon University Hospital, France, between November 2016 and July 2019 were included (n=274). Isolates were tested for the first-line drugs using phenotypic DST (Mycobacteria Growth Indicator Tube) and for genotypic prediction of the susceptibility profile with LPA and WGS. Considering phenotypic DST as the reference, WGS predicted resistance to rifampicin, isoniazid, ethambutol and pyrazinamide with sensitivities of 100%, 100%, 100% and 93.8%, respectively, and susceptibility to these drugs with specificities of 99.6%, 100%, 98.5% and 100%, respectively. Performance of the LPA was poorer, with sensitivity of 83.3% for rifampicin and 85.7% for isoniazid resistance. Five isolates were classified as susceptible according to phenotypic DST (1 for rifampicin, 4 for ethambutol) while WGS detected resistance mutations in rpoB and embB genes. WGS, used under appropriate quality-control conditions, has good performance to predict the resistance profile for the four first-line drugs and can correct phenotypic DST results. This study highlights the need for future guidelines recommending WGS as the initial tool in routine practice in areas where the prevalences of TB and drug-resistant MTB are low.

Genotypic characterization of ‘inferred’ rifampin mutations in GenoType MTBDRplus assay and its association with phenotypic susceptibility testing of Mycobacterium tuberculosis.

In GenoType MTBDRplus assay [line probe assay (LPA)], when Mycobacterium tuberculosis (M. tuberculosis) sample DNA fails to hybridize to at least 1 rpoB wild-type probe and any mutation probe, it is inferred as rifampin (RIF)-resistant. In this study, we sought to identify such ‘inferred’ mutations in M. tuberculosis isolates (n = 203) by rpoB gene sequencing and determined their association with phenotypic resistance. D516Y, H526N, L511P mutations were associated with both phenotypically sensitive (59%, n = 38/64) and resistant (23.7%, n = 33/139) antimicrobial susceptibility testing (AST) results, whereas S531W mutation was associated with only RIF-resistant isolates (33%, n = 46/139). These results demonstrated that, at standard drug concentrations, some ‘inferred’ mutations may be missed by RIF-AST (phenotypically sensitive). The use of LPA permits identification of these RIF-resistant isolates, and incorporation of additional mutation probes (e.g., S531W) could further increase LPA specificity. Further studies are needed to establish the significance of the type of ‘inferred’ mutation with clinical/treatment outcomes.

MDR/XDR-TB Colour Test for drug susceptibility testing of Mycobacterium tuberculosis, Northwest Ethiopia

BackgroundAppropriate technology tests are needed for Mycobacterium tuberculosis drug-susceptibility testing (DST) in resource-constrained settings. This study was performed to evaluate the MDR/XDR-TB Colour Test (a colour platethin-layer agar test; TB-CX) for M. tuberculosis DST by directly testing sputum at University of Gondar Hospital. MethodsSputum samples were each divided into two aliquots. One aliquot was mixed with disinfectant and applied directly to the TB-CX quadrant petri-plate containing culture medium with and without isoniazid, rifampicin, or ciprofloxacin. Concurrently, the other aliquot was decontaminated with sodium hydroxide, centrifuged, and cultured on Lӧwenstein–Jensen medium; the stored M. tuberculosis isolates were then sub-cultured in BACTEC Mycobacteria Growth Indicator Tube (MGIT) 960 for reference DST. ResultsThe TB-CX test yielded DST results for 94% (123/131) of positive samples. For paired DST results, the median number of days from sputum processing to DST was 12 for TB-CX versus 35 for LJ-MGIT (p<0.001). Compared with LJ-MGIT for isoniazid, rifampicin, and multidrug-resistant tuberculosis, TB-CX had 59%, 96%, and 95% sensitivity; 96%, 94%, and 98% specificity; and 85%, 94%, and 98% agreement, respectively. All ciprofloxacin DST results were susceptible by both methods. ConclusionThe TB-CX test was simple and rapid for M. tuberculosis DST. Discordant DST results may have resulted from sub-optimal storage and different isoniazid concentrations used in TB-CX versus the reference standard test.

Revisiting problems and solutions to decrease Mycobacterium tuberculosis pyrazinamide false resistance when using the Bactec MGIT 960 system.

Pyrazinamide (PZA) is a first-line key drug used in combination with other agents for the treatment of tuberculosis (TB). Phenotypic and molecular assays for testing susceptibility of Mycobacterium tuberculosis (Mtb) to PZA have been developed, with the assay in liquid medium at acidic pH in the Bactec MGIT 960 (M960) system being routinely used in the mycobacteriology laboratories. However, false resistance to PZA by this method was reported to occur by several investigators, mostly due to high Mtb inoculum, which may impair drug activity by increasing the pH of the medium. In this study, a revision of the literature on the issue of false resistance in the M960 PZA assay was performed. In the reports examined, all improvements of the M960 test proposed to decrease false resistant results were based on the use of reduced inoculum densities of Mtb cells, to be easily translated into laboratory practice.

Evaluation of nitrate reductase assay on Lowenstein–Jensen medium for drug susceptibility testing of Mycobacterium tuberculosis

Introduction: Multidrug-resistant tuberculosis is of increasing public health concern and a rapid and inexpensive drug susceptibility test is needed in resource limited settings. Aim: This study aims to compare the efficacy of drug susceptibility testing (DST) of Mycobacterium tuberculosis using nitrate reductase assay (NRA) on solid Lowenstein–Jensen (LJ) medium with proportion method on LJ medium as reference standard. Materials and Methods: From November 2011 to July 2012, 100 isolates of M. tuberculosis were tested for susceptibility to first-line drugs on LJ medium by proportion method and NRA using LJ with potassium nitrate. Results: For various drugs, the sensitivity and specificity by NRA on solid medium were as follows: streptomycin 96.6% and 98.5%; isoniazid 96.87% and 97%; rifampicin 96.3% and 96%; and ethambutol 78.9% and 91.4%, respectively. The concordance for rifampicin and isoniazid between NRA method and LJ proportion method was found to be >95%. Conclusion: NRA was found to be a rapid and sensitive method for DST of M. tuberculosis.

Automated detection of bacterial growth on 96-well plates for high-throughput drug susceptibility testing of Mycobacterium tuberculosis

M. tuberculosis grows slowly and is challenging to work with experimentally compared with many other bacteria. Although microtitre plates have the potential to enable high-throughput phenotypic testing of M. tuberculosis, they can be difficult to read and interpret. Here we present a software package, the Automated Mycobacterial Growth Detection Algorithm (AMyGDA), that measures how much M. tuberculosis is growing in each well of a 96-well microtitre plate. The plate used here has serial dilutions of 14 anti-tuberculosis drugs, thereby permitting the MICs to be elucidated. The three participating laboratories each inoculated 38 96-well plates with 15 known M. tuberculosis strains (including the standard H37Rv reference strain) and, after 2 weeks' incubation, measured the MICs for all 14 drugs on each plate and took a photograph. By analysing the images, we demonstrate that AMyGDA is reproducible, and that the MICs measured are comparable to those measured by a laboratory scientist. The AMyGDA software will be used by the Comprehensive Resistance Prediction for Tuberculosis: an International Consortium (CRyPTIC) to measure the drug susceptibility profile of a large number (>30000) of samples of M. tuberculosis from patients over the next few years.

Corrigendum: Automated detection of bacterial growth on 96-well plates for high-throughput drug susceptibility testing of Mycobacterium tuberculosis.

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