Multicenter evaluation of the fully automated Bactec MGIT 960 system for susceptibility testing of Mycobacterium tuberculosis to pyrazinamide: comparison with the radiometric Bactec 460TB system

One hundred and fifty clinical isolates of Mycobacterium tuberculosis were tested for susceptibility to pyrazinamide using the fully automated Bactec MGIT 960 system and the radiometric Bactec 460TB system. The overall concordance rate between MGIT 960 and radiometric system was 100% and the mean turnaround times to report the susceptibility test results were almost identical (6.37 and 6.8 days, respectively).     

Combined use of the fully automated Bactec MGIT 960 system and a PCR-restriction fragment length polymorphism analysis for routine detection and identification of mycobacteria from clinical samples

We developed a method to identify the insertion sites of transposons in the chromosome of Salmonella using one step only. In this method, the Salmonella's genomic DNA is directly sequenced using a transposon internal primer. Reliable direct sequencing was achieved using high purity genomic DNA and an improved protocol for automated sequence machine. This note is intended to promote the use of direct sequencing, which we found to be reliable, efficient and inexpensive as compared to the other currently used methods.     

Evaluation of BACTEC MGIT 960 System for Testing Susceptibility of Mycobacterium tuberculosis to First-Line Drugs in China

Background

The purpose of this study was to evaluate the performance of the BACTEC MGIT 960 (M960) system compared with the proportion method (PM) on Löwenstein-Jensen (L-J) medium in a peripheral laboratory in China for the testing of Mycobacterium tuberculosis (MTB) susceptibility to streptomycin (SM), isoniazid (INH) rifampicin (RIF) and ethambutol (EMB) a combination known as SIRE.

Methods

The susceptibility of 205 clinical isolates of MTB to SM, INH, RIF and EMB was performed with the M960 system. The drugs were tested at the following concentrations: 1.0 µg/ml for SM, 0.1 µg/ml for INH, 1.0 µg/ml for RIF, and 5.0 µg/ml for EMB. The results were compared with those obtained by the L-J PM. The L-J PM at an arbiter site was used to resolve any discordant results.

Results

The overall consistency was 96.6% and concordance values were 95.6% for SM, 97.6% for INH, 98.0% for RIF and 95.1% for EMB. The overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the M960 system for PM (the standard method) was 95.6%, 97.3%, 96.2% and 96.9% respectively, and the sensitivity were 93.3% for SM, 96.9% for INH, 97.4% for RIF and 94.6% for EMB, the specificity were 96.9% for SM, 98.2% for INH, 98.4% for RIF and 95.5% for EMB, the PPV were 94.6% for SM, 97.9% for INH, 97.4% for RIF and 94.6% for EMB, the NPV were 96.2% for SM, 97.3% for INH, 98.4% for RIF and 95.5% for EMB. The turnaround time with the M960 system (median 8.0 days, ranged from 5 to 14 days) was significantly shorter than that with the PM (28 days or 42 days).

Conclusion

There was a substantial degree of agreement between the two methods. The M960 system was a reliable and rapid method for SIRE susceptibility testing of tuberculosis in China.     

Comparative evaluation under routine conditions of the nitrate reduction assay, the proportion assay and the MGIT 960 assay for drug susceptibility testing of clinical isolates of Mycobacterium tuberculosis

The performance of the nitrate reductase assay (NRA) was compared with the proportion method (PM) on Lowenstein-Jensen medium and the BACTEC MGIT960 assay under routine conditions using 160 clinical isolates of Mycobacterium tuberculosis with a high proportion of resistant strains. The mean time to obtain results was 8.8 days and the overall agreements between NRA and PM and NRA and M960 were 95% and 94%, respectively. NRA was easy to perform and represents a useful tool for the rapid screening of drug-resistant M. tuberculosis strains in low-resource countries.     

Evaluation of indirect susceptibility testing of Mycobacterium tuberculosis to the first- and second-line, and alternative drugs by the newer MB/BacT system

In order to evaluate the Organon Teknika MB/BacT system used for testing indirect susceptibility to the alternative drugs ofloxacin (OFLO), amikacin (AMI), and rifabutin (RIF), and to the usual drugs of standard treatment regimes such as rifampin (RMP), isoniazid (INH), pyrazinamide (PZA), streptomycin (SM), ethambutol (EMB), and ethionamide (ETH), cultures of clinical specimens from 117 patients with pulmonary tuberculosis under multidrug-resistant investigation, admitted sequentially for examination from 2001 to 2002, were studied. Fifty of the Mycobacterium tuberculosis cultures were inoculated into the gold-standard BACTEC 460 TB (Becton Dickinson) for studying resistance to AMI, RIF, and OFLO, and the remaining 67 were inoculated into Lowenstein Jensen (LJ) medium (the gold standard currently used in Brazil) for studying resistance to RMP, INH, PZA, SM, EMB, and ETH. We observed 100% sensitivity for AMI (80.8-100), RIF (80.8-100), and OFLO (78.1-100); and 100% specificity for AMI (85.4-100), RIF (85.4-100), and OFLO (86.7-100) compared to the BACTEC system. Comparing the results obtained in LJ we observed 100% sensitivity for RMP (80-100), followed by INH-95% (81.8-99.1), EMB-94.7% (71.9-99.7), and 100% specificity for all drugs tested except for PZA-98.3 (89.5-99.9) at 95% confidence interval. The results showed a high level of accuracy and demonstrated that the fully automated, non-radiometric MB/BacT system is indicated for routine use in susceptibility testing in public health laboratories.     

Microbial sensor for drug susceptibility testing of Mycobacterium tuberculosis

Aims

Drug susceptibility testing (DST) of clinical isolates of Mycobacterium tuberculosis is critical in treating tuberculosis. We demonstrate the possibility of using a microbial sensor to perform DST of M. tuberculosis and shorten the time required for DST.

Methods and Results

The sensor is made of an oxygen electrode with M. tuberculosis cells attached to its surface. This sensor monitors the residual oxygen consumption of M. tuberculosis cells after treatment with anti‐TB drugs with glycerine as a carbon source. In principle, after drug pretreatment for 4–5 days, the response differences between the sensors made of drug‐sensitive isolates are distinguishable from the sensors made of drug‐resistant isolates. The susceptibility of the M. tuberculosis H37Ra strain, its mutants and 35 clinical isolates to six common anti‐TB drugs: rifampicin, isoniazid, streptomycin, ethambutol, levofloxacin and para‐aminosalicylic acid were tested using the proposed method. The results agreed well with the gold standard method (LJ) and were determined in significantly less time. The whole procedure takes approximately 11 days and therefore has the potential to inform clinical decisions.

Conclusions

To our knowledge, this is the first study that demonstrates the possible application of a dissolved oxygen electrode‐based microbial sensor in M. tuberculosis drug resistance testing. This study used the microbial sensor to perform DST of M. tuberculosis and shorten the time required for DST.

Significance and Impact of the Study

The overall detection result of the microbial sensor agreed well with that of the conventional LJ proportion method and takes less time than the existing phenotypic methods. In future studies, we will build an O₂ electrode array microbial sensor reactor to enable a high‐throughput drug resistance analysis.     

Evaluation of rapid alternative methods for drug susceptibility testing in clinical isolates of Mycobacterium tuberculosis

A study was carried out to compare the performance of a commercial method (MGIT) and four inexpensive drug susceptibility methods: nitrate reductase assay (NRA), microscopic observation drug susceptibility (MODS) assay, MTT test, and broth microdilution method (BMM). A total of 64 clinical isolates of Mycobacterium tuberculosis were studied. The Lowenstein-Jensen proportion method (PM) was used as gold standard. MGIT NRA, MODS, and MTT results were available on an average of less than 10 days, whereas BMM results could be reported in about 20 days. Most of the evaluated tests showed excellent performance for isoniazid and rifampicin, with sensitivity and specificity values > 90%. With most of the assays, sensitivity for ethambutol was low (62-87%) whereas for streptomycin, sensitivity values ranged from 84 to 100%; NRA-discrepancies were associated with cultures with a low proportion of EMB-resistant organisms while most discrepancies with quantitative tests (MMT and BMM) were seen with isolates whose minimal inhibitory concentrations fell close the cutoff MGIT is reliable but still expensive. NRA is the most inexpensive and easiest method to perform without changing the organization of the routine PM laboratory performance. While MODS, MTT, and BMM, have the disadvantage from the point of view of biosafety, they offer the possibility of detecting partial resistant strains. This study shows a very good level of agreement of the four low-cost methods compared to the PM for rapid detection of isoniazid, rifampicin and streptomycin resistance (Kappa values > 0.8); more standardization is needed for ethambutol.     

Evaluation of susceptibility of Mycobacterium bovis to antituberculous drugs by radiometric BACTEC 460TB system

Susceptibility of Mycobacterium bovis strains to antituberculous drugs (isoniazid and rifampin) was detected by radiometric BACTEC 460TB system. M.bovis strains were isolated from tissue samples showing tuberculous lesions collected at an abbattoir from cattle belonging to 47 tuberculosis outbreaks occurring in Northern Italy in 1995-1999. Forty-six out of 61 strains (75.4%) resulted susceptible to both isoniazid and rifampin. Thirteen strains (21.3%) were resistant to isoniazid only. No strains showed resistance to rifampin only. Two strains (3.3%) resulted resistant to both drugs, showing antituberculous multidrug-resistance. Given the compulsory eradication program of bovine tuberculosis by elimination of infected animals and the ban on antituberculous drug treatments in animals, detection of resistant M. bovis strains appears of great interest.     

Simple and rapid method for detection of nitrate reductase activity of Mycobacterium tuberculosis and Mycobacterium canettii grown in the Bactec MGIT960 system

Mycobacterium tuberculosis reduces nitrate very strongly as compared to Mycobacterium bovis and M. bovis BCG. Nitrate reductase, in conjunction with niacin accumulation, constitutes one of the major biochemical tests used in clinical microbiology laboratories to differentiate M. tuberculosis from other members of the M. tuberculosis complex, as well as nontuberculous Mycobacteria. Determination of nitrate reductase activity is currently performed using cultures grown on solid media with a slow detection time and the need for large quantities of bacilli, as otherwise the test is not reliable. Hereby, we propose a nitrate reduction test coupled to Bactec MGIT960 system as a simple, rapid and economic method with a total gain of time of about 3 to 4 weeks over the conventional solid medium. In our study, almost all the M. tuberculosis and Mycobacterium canettii strains gave a strongly positive nitrate reductase result within 1 day of positive detection by the MGIT960 system. In contrast, M. bovis, M. bovis BCG and M. africanum strains remained negative even after 14 days of incubation. The possibility to detect nitrate reductase within 1 to 3 days of a positive culture using MGIT960 opens new perspectives with the possibility of confirming M. tuberculosis — starting directly from pathological specimens.     

结核分枝杆菌吡嗪酰胺耐药性检测方法的研究进展

吡嗪酰胺(pyrazinamide,PZA)是重要的一线抗结核药物,与异烟肼、利福平和乙胺丁醇构成治疗方案的核心。因其疗效较好,被广泛应用于结核病的治疗过程。然而,近年来随着耐多药结核病的出现,PZA耐药导致部分患者治疗失败,因此常规开展PZA药物敏感性试验对于减少耐药性的发生显得极为重要。由于PZA在酸性条件下才能发挥作用,而结核分枝杆菌在酸性环境下生长不良,故PZA耐药性检测一直是临床中的难题。本文结合国内外最新研究进展,就结核分枝杆菌PZA耐药性检测方法的研究进行阐述,期望能为更有效地诊治结核病提供新思路。     

Mycobacterium tuberculosis: a model system for structural genomics

Over the past five years, genomics has had a major impact on Mycobacterium tuberculosis research. With the publication of the sequences of two virulent strains (H37Rv and CDC1551) and three closely related sequences, M. tuberculosis is becoming a model system for proteomics and structural genomics initiatives. Together with the promise of structures of proteins with novel folds, high-resolution structures of drug targets are providing the basis for rational inhibitor design, with the goal of the development of novel anti-tuberculars. In addition, this work is aiding scientists in the quest for an effective vaccine against this persistent pathogen.     

Flow cytometry as a tool to identify Mycobacterium tuberculosis interaction with the immune system and drug susceptibility

Flow cytometric analysis is a useful and widely employed tool to identify immunological alterations caused by different microorganisms, including Mycobacterium tuberculosis. However, this tool can be used for several others analysis. We will discuss some applications for flow cytometry to the study of M. tuberculosis, mainly on cell surface antigens, mycobacterial secreted proteins, their interaction with the immune system using inflammatory cells recovered from peripheral blood, alveolar and pleura spaces and the influence of M. tuberculosis on apoptosis, and finally the rapid determination of drug susceptibility. All of these examples highlight the usefulness of flow cytometry in the study of M. tuberculosis infection.     

Comparison of the performance of two mycobacteriophage D29-based protocols for fluoroquinolone susceptibility testing in Mycobacterium tuberculosis

We tested a mycobacteriophage D29-based method for fluoroquinolone susceptibility assessment in clinical isolates of Mycobacterium tuberculosis. The method was incapable of identifying susceptible strains as such, although a slightly different published protocol successfully identified resistant and susceptible strains. Thus, caution is necessary when choosing an “in-house” D29-based protocol for testing of drug resistance.     

Comparison of the proportion method with mycobacteria growth indicator tube and E-test for susceptibility testing of Mycobacterium tuberculosis

The aim of this study was to investigate the correlation between proportion method with mycobacteria growth indicator tube (MGIT) and E-test for Mycobacterium tuberculosis. Forty clinical isolates were tested. MGIT and E-test with the first line antituberculous drugs correlated with the proportion method. Our results suggested that MGIT and E-test methods can be routinely used instead of the proportion method.     

Evaluation of the GenoCard as a tool for transport and storage of samples for tuberculosis molecular drug susceptibility testing

Early identification and monitoring of the spread of resistant M. tuberculosis strains is essential to control tuberculosis. The paper-based transport device GenoCard enables the safe shipment of inactivated biological material and strains to be used for molecular detection of drug resistance.     

Evaluation of the BD MGIT TBc Identification Test (TBc ID), a rapid chromatographic immunoassay for the detection of Mycobacterium tuberculosis complex from liquid culture

The BACTEC MGIT 960 system is increasingly used to culture Mycobacterium tuberculosis. We evaluated the performance of the new immunochromatographic assay BD MGIT TBc Identification Test (TBc ID) for the rapid identification of M. tuberculosis complex in clinical samples when performed directly from BACTEC MGIT 960 culture positive for acid-fast bacilli (AFB).Of 92 cultures evaluated, the sensitivity and specificity of the TBc ID test was 98.5% and 100%, respectively compared to sequencing of the 16S rRNA gene. One culture that was TBc ID test negative but that was identified as M. tuberculosis by 16S rRNA sequencing was confirmed to have a mutation in the mpt64 gene.The TBc ID test is an easy and sensitive method for the identification of M. tuberculosis complex in liquid culture medium, does not require a high level of skills, neither any additional specific equipment and gives results in 15 min, which provide a good alternative for the rapid identification of M. tuberculosis complex in liquid medium.     

Preclinical testing of new drugs for tuberculosis: current challenges

The continuing global epidemic of tuberculosis, the increasing rate of multidrug resistant (MDR) tuberculosis and the more recent emergence of extensively drug resistant (XDR) tuberculosis are great causes for concern. A major international effort is currently underway to optimize current drug therapies and to discover new drugs that are active against these organisms. This effort has created a pipeline of new candidate drugs at various stages of preclinical and early clinical evaluations. Major challenges still exist, however, varying from the standardization and application of current animal models and their application to drug discovery and characterization to the fact that our knowledge about the basic biology of the MDR and XDR strains of Mycobacterium tuberculosis is minimal at best.     

Drug susceptibility testing of Mycobacterium tuberculosis by the broth microdilution method with 7H9 broth

In this study, we have evaluated the broth microdilution method (BMM) for susceptibility testing of Mycobacterium tuberculosis. A total of 43 clinical isolates of M. tuberculosis and H37Rv as a control strain were studied. All isolates were tested by the proportion method and the BMM for isoniazid (INH), rifampicin (RIF), streptomycin (STR), and ethambutol (ETM). The proportion method was carried out according to the National Committee for Clinical Laboratory Standards (NCCLS) on L?wenstein-Jensen (LJ) medium. The BMM was carried out using 7H9 broth with 96 well-plates. All strains were tested at 3.2-0.05 micro g/ml, 16-0.25 micro g/ml, 32-0.5 micro g/ml, and 32-0.5 micro g/ml concentrations for INH, RIF, STR, and ETM, respectively. When the BMM was compared with the proportion method, sensitivity was 100, 100, 96.9, and 90.2%, while specificity was 100, 85.7, 90.9, and 100% for INH, RIF, STR, and ETM, respectively. The plates were examined 7, 10, 14, and 21 days after incubation. The majority of the result were obtained at 14th days after incubation, while the proportion method result were ended in 21-28 days. According to our results, it may be suggested that the BMM is suitable for early determining of multidrug-resistance-M. tuberculosis strains in developed or developing countries.