Simple Procedure for Drug Susceptibility Testing of Mycobacterium tuberculosis Using a Commercial Colorimetic Assay

 The aim of this study was to evaluate a simple method using a commercial colorimetric assay (Alamar Blue Oxidation-Reduction Indicator; Accumed, USA) in a microtiter format for testing the susceptibility of 94 strains of Mycobacterium tuberculosis to isoniazid, rifampicin, ethambutol and streptomycin. The method makes use of one critical concentration of each drug, and the results are available within 8–10 days. Overall, 97.1% agreement with the proportion method was obtained. Full agreement was obtained for isoniazid and rifampicin. The method is simple to perform, permits visual reading of results, and is practicable for laboratories with limited resources.     

Pyrosequencing for Rapid Detection of Extensively Drug-Resistant Mycobacterium tuberculosis in Clinical Isolates and Clinical Specimens

Treating extensively drug-resistant (XDR) tuberculosis (TB) is a serious challenge. Culture-based drug susceptibility testing (DST) may take 4 weeks or longer from specimen collection to the availability of results. We developed a pyrosequencing (PSQ) assay including eight subassays for the rapid identification of Mycobacterium tuberculosis complex (MTBC) and concurrent detection of mutations associated with resistance to drugs defining XDR TB. The entire procedure, from DNA extraction to the availability of results, was accomplished within 6 h. The assay was validated for testing clinical isolates and clinical specimens, which improves the turnaround time for molecular DST and maximizes the benefit of using molecular testing. A total of 130 clinical isolates and 129 clinical specimens were studied. The correlations between the PSQ results and the phenotypic DST results were 94.3% for isoniazid, 98.7% for rifampin, 97.6% for quinolones (ofloxacin, levofloxacin, or moxifloxacin), 99.2% for amikacin, 99.2% for capreomycin, and 96.4% for kanamycin. For testing clinical specimens, the PSQ assay yielded a 98.4% sensitivity for detecting MTBC and a 95.8% sensitivity for generating complete sequencing results from all subassays. The PSQ assay was able to rapidly and accurately detect drug resistance mutations with the sequence information provided, which allows further study of the association of drug resistance or susceptibility with each mutation and the accumulation of such knowledge for future interpretation of results. Thus, reporting of false resistance for mutations known not to confer resistance can be prevented, which is a significant benefit of the assay over existing molecular diagnostic methods endorsed by the World Health Organization.     

Multicenter Evaluation of Bactec MGIT 960 System for Second-Line Drug Susceptibility Testing of Mycobacterium tuberculosis Complex

The Bactec MGIT 960 system for testing susceptibility to second-line drugs was evaluated with 117 clinical strains in a multicenter study. The four drugs studied were levofloxacin, amikacin, capreomycin, and ethionamide. The critical concentration established for levofloxacin and amikacin was 1.5 μg/ml, that established for capreomycin was 3.0 μg/ml, and that established for ethionamide was 5.0 μg/ml. The overall level of agreement between the agar proportion method and the MGIT 960 system was 96.4%, and the levels of agreement for the individuals drugs were 99.1% for levofloxacin, 100% for amikacin, 97.4% for capreomycin, and 88.9% for ethionamide. The rate of reproducibility of the drug susceptibility testing results between the participating laboratories was 99.5%.The increase in the incidence of multidrug-resistant tuberculosis (MDR TB) and the emergence of extensively drug-resistant tuberculosis present tremendous challenges to the global efforts to combat tuberculosis (1, 5, 16, 21). Rapid methods enabling accurate susceptibility testing of first-line and second-line drugs are critical for the early diagnosis of MDR TB and extensively drug-resistant tuberculosis and the initiation of effective regimens. Various drug susceptibility testing (DST) methods that use solid media, including the agar proportion method (AP) and other methods, have the drawback of prolonged turnaround times (TATs). The World Health Organization and the U.S. Centers for Disease Control and Prevention have recommended the use of liquid culture systems for the diagnosis of tuberculosis and DST to improve TATs (22, 25). The Bactec 460 (Becton Dickinson Diagnostic Systems, Sparks, MD), a radiometric liquid system, provided an excellent alternative for testing of the susceptibilities of Mycobacterium tuberculosis complex (MTBC) isolates to streptomycin, isoniazid, rifampin (rifampicin), and ethambutol (SIRE) and to pyrazinamide (PZA) with improved TATs. The MGIT 960 liquid, nonradiometric SIRE DST (Becton Dickinson Diagnostic Systems), whose performance is comparable to that of the Bactec 460 system, has been commercially available since April 2002 (4, 20, 23). The Microbial Diseases Laboratory (MDL) of the California Department of Public Health implemented SIRE DST with the MGIT 960 system in 2004. With confidence in the SIRE DST with the MGIT 960 system, a study that used the same platform to test the susceptibilities of MTBC isolates to four classes of second-line drugs, levofloxacin (LVX), amikacin (AMK), capreomycin (CAP), and ethionamide (ETH), was initiated in November 2004. The study was conducted at two laboratories: MDL and the TB Reference Laboratory of the Veteran Affairs Medical Center (VA) in West Haven, CT. Here we report the results of the multicenter study, in which the critical concentrations of the test drugs were established, the performance of the MGIT 960 system was compared to that of AP, and the interlaboratory reproducibility of the method was evaluated.(Part of this work was presented at the 46th Interscience Conference on Antimicrobial Agents and Chemotherapy, 2006, San Francisco, CA.)     

Comparative Study on Genotypic and Phenotypic Second-Line Drug Resistance Testing of Mycobacterium tuberculosis Complex Isolates

The mycobacterium growth indicator tube (MGIT960) automated liquid medium testing method is becoming the international gold standard for second-line drug susceptibility testing of multidrug- and extensively drug-resistant Mycobacterium tuberculosis complex isolates. We performed a comparative study of the current gold standard in the Netherlands, the Middlebrook 7H10 agar dilution method, the MGIT960 system, and the GenoType MTBDRsl genotypic method for rapid screening of aminoglycoside and fluoroquinolone resistance. We selected 28 clinical multidrug- and extensively drug-resistant M. tuberculosis complex strains and M. tuberculosis H37Rv. We included amikacin, capreomycin, moxifloxacin, prothionamide, clofazimine, linezolid, and rifabutin in the phenotypic test panels. For prothionamide and moxifloxacin, the various proposed breakpoint concentrations were tested by using the MGIT960 method. The MGIT960 method yielded results 10 days faster than the agar dilution method. For amikacin, capreomycin, linezolid, and rifabutin, results obtained by all methods were fully concordant. Applying a breakpoint of 0.5 μg/ml for moxifloxacin led to results concordant with those of both the agar dilution method and the genotypic method. For prothionamide, concordance was noted only at the lowest and highest MICs. The phenotypic methods yielded largely identical results, except for those for prothionamide. Our study supports the following breakpoints for the MGIT960 method: 1 μg/ml for amikacin, linezolid, and clofazimine, 0.5 μg/ml for moxifloxacin and rifabutin, and 2.5 μg/ml for capreomycin. No breakpoint was previously proposed for clofazimine. For prothionamide, a division into susceptible, intermediate, and resistant seems warranted, although the boundaries require additional study. The genotypic assay proved a reliable and rapid method for predicting aminoglycoside and fluoroquinolone resistance.The emergence of multidrug-resistant tuberculosis (MDR-TB) in the 1990s, and more recently, extensively drug resistant tuberculosis (XDR-TB), has revealed the need for new drugs and alternative, second-line treatment regimens. Many of these second-line drugs are either old drugs that had not been frequently used because of side effects or unproven efficacy or newer drugs intended primarily for treatment of other infections (3). Their use necessitated an evaluation of drug susceptibility testing (DST) and a determination of the critical concentrations of these alternative drugs.A variety of techniques are now available for second-line DST, of which the mycobacterium growth indicator tube automated liquid culture system (MGIT960) is probably the most used and best validated at this moment (13). The latest addition to second-line DST are genotypic methods, which detect mutations in the gyrA gene of Mycobacterium tuberculosis that are associated with fluoroquinolone resistance and mutations in the rrs operon that are associated with resistance to capreomycin and the aminoglycosides (2-5).Despite the arrival of these novel tools, many uncertainties remain. Not all methods have been evaluated in comparative studies. Moreover, the critical concentrations for resistance to several second- and third-line drugs, including moxifloxacin and prothionamide, remain the subject of debate (7, 11, 13).In the Netherlands, the Middlebrook 7H10 agar dilution method has been used for second-line DST for 2 decades (18). In order to comply with international standardization requirements, a switch to the MGIT960 method has been initiated.In this study, we have compared the results of the MGIT960 method and the GenoType MTBDRsl assay, a commercially available genotypic second-line DST method, to our reference method, the Middlebrook 7H10 agar dilution method. For the MGIT960 method, we tested the various critical concentrations published for prothionamide and moxifloxacin but also included clofazimine in our drug panel. For the latter drug, which has become increasingly important in the treatment of MDR and XDR-TB, no in vitro DST data for the MGIT960 method were available.     

Antimicrobial Susceptibility Testing of Bilophila wadsworthia Isolates Submitted for Routine Laboratory Examination

MICs of antibiotics against Bilophila wadsworthia isolates were measured by agar and broth microdilution with pyruvic acid and by Etest. The inoculum size influenced greatly agar dilution. Despite discrepancies in MICs depending on the measurement method used, clindamycin consistently showed potent activity. Broth microdilution and Etest appear to be candidates for laboratory susceptibility testing.     

Integration of Mycobacterium tuberculosis Drug Susceptibility Testing and Genotyping with Epidemiological Data Analysis To Gain Insight into the Epidemiology of Drug-Resistant Tuberculosis in Malatya,Turkey

Drug-resistant tuberculosis (TB) presents a major challenge to global TB control. To gain a better understanding of drug-resistant TB epidemiology in Malatya, Turkey, we conducted the present study using 397 Mycobacterium tuberculosis clinical isolates collected from Malatya, Turkey, in recent years (2000-2007). Resistance to any anti-TB drug was found in 29% (114 of 397) of the study isolates, while the multidrug resistance (MDR) rate was ∼4.5% (18 of 397). Resistances to isoniazid (15.5%) and streptomycin (13.4%) were about twice as high as resistance to rifampin (RMP) (6.3%) and ethambutol (EMB) (6.0%). Importantly, 28% (7 of 25) of the RMP-resistant isolates were non-MDR isolates, as when a significant proportion of RMP-resistant isolates in a population are non-MDR, the predictive value of molecular detection of RMP resistance for MDR can be significantly reduced. Both identical and varied drug resistance patterns were seen in the same genotyping-defined clusters, suggesting that both primary and acquired resistance have contributed to the drug-resistant TB epidemic in Malatya, Turkey. In addition, drug-resistant cases were found to be more likely to be males (odds ratio [95% confidence interval], 1.82 [1.13, 2.94]), suggesting a potential role of gender in the epidemiology of drug-resistant TB in the study population. This study demonstrates that the integration of drug susceptibility testing with genotyping and epidemiological data analysis represents a useful approach to studying the epidemiology of drug-resistant TB.Tuberculosis (TB) remains an important global public health problem, and global TB control is further challenged by the rising epidemics of drug-resistant TB worldwide (29). In 2008, the World Health Organization (WHO) reported that worldwide resistance to any of the anti-TB drugs accounted for 20% of all reported TB cases while an estimated 5.3% of all reported TB cases had multidrug resistance (MDR), defined as resistance to, at least, isoniazid (INH) and rifampin (RMP) (28). Furthermore, globally, only 10% of the roughly 500,000 people who develop MDR TB each year receive treatment, leading to more possible cases of MDR TB (9). Because of the disparities in TB control around the world, a better understanding of the dynamics and driving forces of drug-resistant TB epidemics would contribute to the development of more effective strategies for global TB control.Turkey, with a population of around 70 million, had an annual TB incidence of 27.9 per 100,000 people in 2007 (6). Although the TB incidence in Turkey has decreased by half since 1985, several studies have shown the proportion of drug-resistant TB cases to be higher than the global average (3, 5, 10, 11, 20, 24, 26). In 2005, Surucuoglu and colleagues reported that the rate of resistance to any anti-TB drugs among 355 isolates of Mycobacterium tuberculosis obtained from western Turkey was 21.1%, while another study found that among the 1,513 TB cases diagnosed in Istanbul, Turkey, in 2005, 19% were resistant to at least one drug (20, 25). These rates are similar to the rates of drug resistance found in some of the Eastern European countries that are considered to have the highest drug resistance rates in the world (3). The high drug resistance rate poses a major challenge to the control of TB in Turkey. Previous studies of different populations have found that both host and microbial factors can play a role in drug-resistant TB epidemics. Microbial factors such as specific spoligotype families have been implicated as risk factors. TB genotypes belonging to the Beijing and Latin American and Mediterranean (LAM) families are associated with drug resistance (18, 23).Although previous studies reported high rates of drug-resistant TB infection in different regions of Turkey and several studies reported genotyping results of M. tuberculosis isolates collected from different regions of Turkey (10-12), few assessed associations of microbial and host characteristics with drug-resistant TB cases and attempted to determine the factors driving the epidemic of drug-resistant M. tuberculosis infection in the population. To gain a better understanding of the epidemiology of drug-resistant TB in Malatya, Turkey, we analyzed 397 M. tuberculosis clinical isolates collected from Malatya, Turkey, during the time period between 1 January 2000 and 31 December 2007 and their corresponding epidemiological data. Malatya is the third biggest city in Turkey and has a population of approximately 722,000 and an annual TB incidence of 28.5 per 100,000 (6).     

Quantitative Drug Susceptibility Testing of Mycobacterium tuberculosis by Use of MGIT 960 and EpiCenter Instrumentation

Since numbers of drug-resistant Mycobacterium tuberculosis strains are on the rise, the simple classification into “susceptible” and “resistant” strains based on susceptibility testing at “critical concentrations” has to be reconsidered. While future studies have to address the correlation of phenotypic resistance levels and treatment outcomes, a prerequisite for corresponding investigations is the ability to exactly determine levels of quantitative drug resistance in clinical M. tuberculosis isolates. Here we have established the conditions for quantitative drug susceptibility testing for first- and second-line agents using MGIT 960 instrumentation and EpiCenter software equipped with the TB eXiST module. In-depth comparative analysis of a range of well-characterized susceptible and resistant clinical isolates has allowed us to propose conditions for testing and to develop criteria for interpretation.Tuberculosis (TB) is a leading cause of morbidity and mortality worldwide. Developing countries are the most vulnerable, with more than 95% of the cases (32, 33). The present trend is characterized by an alarming emergence of drug resistance (7, 8, 30). Much attention has focused on the burden of multidrug-resistant (MDR) TB, i.e., resistance to the first-line drugs isoniazid and rifampin (rifampicin) (32, 33), and the emergence of extensively drug resistant TB (6, 26). The rise of drug-resistant TB and the increased susceptibility of the human population to TB due to coinfection with human immunodeficiency virus are driving the worldwide TB pandemic and will worsen the situation in the years ahead, with devastating effects in poor countries, whose economies suffer most from this development (19, 20).In the diagnostic laboratory, testing of mycobacteria for drug susceptibility is substantially different from the general testing procedures used in bacteriology. Rather than determining MICs, a single drug concentration, termed the critical concentration, is usually used to categorize a clinical isolate as susceptible or resistant. This “critical concentration” is more an epidemiological parameter (to distinguish “wild-type” strains from “non-wild-type” strains that are able to grow in the presence of higher drug concentrations [5]) than a clinical cutoff value established to guide treatment decisions (14). With growing knowledge about the mechanisms that underlie drug resistance, it has become evident that drug resistance is multifaceted and that different mutations may lead to different levels of resistance. The acquisition of a resistance mutation leading to a decrease in drug susceptibility should not inevitably exclude an anti-TB drug from a treatment regimen, since low-level resistance does not necessarily imply clinical resistance (3). However, until now, different levels of phenotypic resistance have only rarely been taken into account in the procedures used for in vitro drug susceptibility testing (DST) of mycobacteria (4).“Critical concentration”-based DST of primary and secondary drugs has been established for the radiometric Bactec 460 instrumentation (Becton Dickinson Microbiology Systems, Sparks, MD) and is considered the “gold standard” in the testing of second-line drugs (22, 25). However, the Bactec 460 system has several drawbacks: (i) it involves the use of sharps and radioisotopes with the need for disposal; (ii) it is only semiautomated; and (iii) it needs considerable hands-on time. The nonradiometric MGIT (mycobacterial growth indicator tube) 960 platform (Becton Dickinsion) has been evaluated extensively for DST of first-line drugs (1, 2, 15, 29) and has recently also been evaluated for second-line DST (16, 25). In contrast to the Bactec 460 instrumentation, the MGIT 960 platform is a fully automated system that uses a fluorescence-quenching-based oxygen sensor for growth detection.We have previously characterized quantitative drug resistance levels in clinical strains of drug-susceptible and drug-resistant Mycobacterium tuberculosis using radiometric Bactec 460 measurements (28). However, for widespread implementation of quantitative DST in diagnostic mycobacteriology, the technique chosen should be fully automated, compatible with a computerized expert system for interpretation (so as to avoid individual errors and subjectivity), and safe and reliable (e.g., if possible, the system should not use radioactive material nor needles, such as syringes, for inoculation nor needles inside the instrument). To this end, we have subjected a carefully chosen subset of a previously described and well-characterized collection of clinical M. tuberculosis isolates (28) to quantitative measurements of drug susceptibility using the MGIT 960 platform in conjunction with EpiCenter software equipped with the TB eXiST module.     

Predictive Value of Molecular Drug Resistance Testing of Mycobacterium tuberculosis Isolates in Valle del Cauca,Colombia

Previous evaluations of the molecular GenoType tests have promoted their use to detect resistance to first- and second-line antituberculosis drugs in different geographical regions. However, there are known geographic variations in the mutations associated with drug resistance in Mycobacterium tuberculosis, and especially in South America, there is a paucity of information regarding the frequencies and types of mutations associated with resistance to first- and second-line antituberculosis drugs. We therefore evaluated the performance of the GenoType kits in this region by testing 228 M. tuberculosis isolates in Colombia, including 134 resistant and 94 pansusceptible strains. Overall, the sensitivity and specificity of the GenoType MTBDRplus test ranged from 92 to 96% and 97 to 100%, respectively; the agreement index was optimal (Cohen''s kappa, >0.8). The sensitivity of the GenoType MTBDRsl test ranged from 84 to 100% and the specificity from 88 to 100%. The most common mutations were katG S315T1, rpoB S531L, embB M306V, gyrA D94G, and rrs A1401G. Our results reflect the utility of the GenoType tests in Colombia; however, as some discordance still exists between the conventional and molecular approaches in resistance testing, we adhere to the recommendation that the GenoType tests serve as early guides for therapy, followed by phenotypic drug susceptibility testing for all cases.     

Rifampin Heteroresistance in Mycobacterium tuberculosis Cultures as Detected by Phenotypic and Genotypic Drug Susceptibility Test Methods

Tuberculosis patients may harbor both drug-susceptible and -resistant bacteria, i.e., heteroresistance. We used mixtures of rifampin-resistant and -susceptible Mycobacterium tuberculosis strains to simulate heteroresistance in patient samples. Molecular tests can be used for earlier discovery of multidrug resistance (MDR), but the sensitivity to detect heteroresistance is unknown. Conventional phenotypic drug susceptibility testing was the most sensitive, whereas two line probe assays and sequencing were unable to detect the clinically important 1% resistant bacteria.     

Evaluation of the BD Bactec MGIT 320 System for Detection of Mycobacteria and Drug Susceptibility Testing of Mycobacterium tuberculosis

Two different laboratories evaluated growth and detection of mycobacteria and drug susceptibility testing of Mycobacterium tuberculosis by the BD Bactec MGIT 320 using the BD Bactec MGIT 960 (BD Diagnostics, Sparks, MD) as a reference method. Out of 359 processed sputum specimens for detection of mycobacteria, 99.7% were in agreement between the MGIT 320 and MGIT 960. Streptomycin (STR), isoniazid (INH), rifampin (RIF), ethambutol (EMB) (collectively known as SIRE), and pyrazinamide (PZA) drug susceptibility testing was performed on 89 clinical strains, prepared from both liquid and solid inocula. The results of SIRE and PZA were 100% reproducible between the two instruments tested at both laboratories.     

噬菌体生物扩增技术在结核分枝杆菌药敏检测中的应用

目的探讨噬菌体生物扩增技术（PhaB）在结核分枝杆菌（MTB）耐药研究中的应用价值。方法应用PhaB法同时测定233株结核分枝杆菌分离株对异烟肼（INH）、链霉素（SM）、利福平（RFP）及左旋氧氟沙星（LEV）的耐药性,并用改良罗氏培养法（LJ）做对照,比较两种方法的检测结果。结果PhaB法检测INH、SM、RFP、LEV的药敏结果与改良罗氏培养法药敏结果相比符合率分别为97．4％、96．6％、95．3％、96．9％。同时检出耐多药菌株26株,占11．1％。结论PhaB法能快速进行结核分枝杆菌的药敏检测并与金标法U符合率高,可作为基层结防单位MTB耐药性的快速筛选方法。     

Evaluation of BACTEC Mycobacteria Growth Indicator Tube (MGIT 960) Automated System for Drug Susceptibility Testing of Mycobacterium tuberculosis

The reliability of the BACTEC MGIT 960 system, an automated version of the Mycobacteria Growth Indicator Tube (MGIT), for antimicrobial susceptibility testing of Mycobacterium tuberculosis was evaluated on 78 clinical isolates. Rifampin (RMP), isoniazid (INH), streptomycin (SM), and ethambutol (EMB) were tested at the following concentrations: 1.0 microg/ml for RMP, 0.1 and 0.4 microg/ml for INH, 1.0 and 4.0 microg/ml for SM, and 5.0 and 7.5 microg/ml for EMB. Results were compared with those obtained by the BACTEC 460 TB radiometric system. Initially the reproducibility study showed 99.5% agreement on repeat testing with all the four drugs. With susceptibility testing of clinical isolates, excellent agreement between the two systems was found for all the drugs. A total of nine major errors were observed for only three isolates, resistant according to BACTEC MGIT 960 and susceptible according to BACTEC 460 TB, to SM (4.0 microg/ml), INH (0.1 microg/ml), and EMB (5.0 microg/ml) (one isolate) and to SM (1.0 microg/ml), INH (0.4 microg/ml), and EMB (5.0 microg/ml) (two isolates). When these isolates were tested by using the conventional proportion method on L?wenstein-Jensen medium, agreement with BACTEC MGIT 960 was found for five results and with BACTEC 460 TB for the remainder. The time to report results was 7.9 days by MGIT 960 and 7.3 days by BACTEC 460 TB, which was not found statistically significant (P > 0.05). In conclusion, the performance of BACTEC MGIT 960 was found similar to that of BACTEC 460 TB and this new system can be considered a good alternative to the radiometric method for routine susceptibility testing of M. tuberculosis.     

Laboratory Diagnosis of Tuberculosis: Advances in Technology and Drug Susceptibility Testing

There have been rapid technological advances in the detection of Mycobacterium tuberculosis and its drug susceptibility in clinical samples. These include advances in microscopic examination, in vitro culture and application of molecular techniques. The World Health Organization (WHO) has played a large role in evaluating these technologies for their efficacy and feasibility, especially in the developing countries. Amongst these, the Revised National Tuberculosis Control Programme (RNTCP), through its national network of designated microscopy centres and intermediate reference laboratories, has adopted certain technologies that are currently implemented in India. Advances in microscopy technology include fluorescent microscopy using light-emitting diode source, sodium hypochlorite microscopy and vital fluorescent staining of sputum smears. Automation of in vitro culture has markedly reduced the turnaround time (TAT), even in smear-negative samples, and permits simultaneous detection of resistant mutants. Molecular detection of drug resistance has further reduced the TAT, and the cartridge-based nucleic acid amplification test with its performance convenience and rapid results, appears poised to become the future of tuberculosis (TB) diagnosis in all settings, provided the cost of testing is reduced especially for use in private diagnostic settings. This article reviews technologies currently available for the diagnosis of TB, keeping in mind the WHO recommendations and the RNTCP practices. This is a thematic synthesis of data available on diagnosis in literature, preserving the conclusions of the primary studies.     

Direct Susceptibility Testing of Mycobacterium tuberculosis for Pyrazinamide by Use of the Bactec MGIT 960 System

Pyrazinamide (PZA) is a key antituberculosis drug, yet no rapid susceptibility test is commercially available. PZA drug susceptibility testing (DST) was performed directly on sputum samples from 327 patients and compared with the indirect method by using the Bactec MGIT 960 system in the context of patient screening for participation in a drug trial. Compared to standard indirect PZA DST, direct DST was successful in only 59% of cases, but results obtained were highly accurate and available faster. Agreement between the direct and indirect methods varied from 90 to 100% in each laboratory. The median times for obtaining PZA results from the time when the specimen was collected ranged from 11 to 16 days for the direct test and 18 to 95 days for the indirect test across laboratories. The direct method is accurate and reproducible across laboratories. It can be expected to accelerate results in >50% of cases, but it cannot replace indirect DST for PZA. Phenotypic methods remain the gold standard for DST in drug trials. If future studies can optimize the method to decrease the number of uninterpretable results, direct MGIT DST could be the new phenotypic DST standard for clinical trials, providing more rapid detection of resistance to new drugs in experimental regimens.     

Luciferase Reporter Mycobacteriophages for Detection, Identification, and Antibiotic Susceptibility Testing of Mycobacterium tuberculosis in Mexico

The utility of luciferase reporter mycobacteriophages (LRPs) for detection, identification, and antibiotic susceptibility testing of Mycobacterium tuberculosis was prospectively evaluated in a clinical microbiology laboratory in Mexico City, Mexico. Five hundred twenty-three consecutive sputum samples submitted to the laboratory during a 5-month period were included in this study. These specimens were cultivated in Middlebrook 7H9 (MADC), MGIT, and L?wenstein-Jensen (LJ) media. Of the 71 mycobacterial isolates recovered with any of the three media, 76% were detected with the LRPs, 97% were detected with the MGIT 960 method, and 90% were detected with LJ medium. When contaminated specimens were excluded from the analysis, the LRPs detected 92% (54 of 59) of the cultures. The median time to detection of bacteria was 7 days with both the LRPs and the MGIT 960 method. LRP detection of growth in the presence of p-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP) was used for selective identification of M. tuberculosis complex (MTC) and compared to identification with BACTEC 460. Using the LRP NAP test, 47 (94%) out of 50 isolates were correctly identified as tuberculosis complex. The accuracy and speed of LRP antibiotic susceptibility testing with rifampin, streptomycin, isoniazid, and ethambutol were compared to those of the BACTEC 460 method, and discrepant results were checked by the conventional proportion method. In total, 50 MTC isolates were tested. The overall agreement between the LRP and BACTEC 460 results was 98.5%. The median LRP-based susceptibility turnaround time was 2 days (range, 2 to 4 days) compared to 10.5 days (range, 7 to 16 days) by the BACTEC 460 method. Phage resistance was not detected in any of the 243 MTC isolates tested. Mycobacteriophage-based approaches to tuberculosis diagnostics can be implemented in clinical laboratories with sensitivity, specificity, and rapidity that compare favorably with those of the MGIT 960 and BACTEC 460 methods. The phages currently provide the fastest phenotypic assay for susceptibility testing.     

Testing Susceptibility of Multidrug-Resistant Mycobacterium tuberculosis to Second-Line Drugs by Use of Blood Agar

In this study, the susceptibilities of 35 multidrug-resistant (MDR) Mycobacterium tuberculosis clinical isolates to second-line drugs, including kanamycin (KM), rifabutin (RBU), ofloxacin (OFX), p-aminosalicylic acid (PAS), capreomycin (CAP), clofazimine (CFM), and ethionamide (ETH), were investigated on blood agar according to CLSI recommendations. Compared with the results of the Bactec 460 TB system, agreement was 100, 100, 97, 100, 100, 100, and 86% for KM, RBU, OFX, PAS, CAP, CFM, and ETH, respectively. Compared with the results of the proportion method, agreement was 100, 100, 97, 100, 97, 100, and 77% for KM, RBU, OFX, PAS, CAP, CFM, and ETH, respectively.Multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) are major public health problems, especially in developing countries (13, 18, 21). Rapid susceptibility testing is critical for early diagnosis of MDR- and XDR-TB and the initiation of effective regimens (14). The agar proportion method performed on Middlebrook 7H10 and 7H11 agars is the reference method for susceptibility of Mycobacterium tuberculosis according to CLSI recommendations (16). The Bactec 460 TB system (Becton Dickinson Diagnostic Systems, Sparks, MD), Bactec MGIT 960 (Becton Dickinson Diagnostic Systems, Sparks, MD), and Versa TREK system (formerly known as ESP II; Trek Diagnostic Systems, West Lake, OH) are cleared for used by the U.S. FDA for testing M. tuberculosis susceptibility to first-line drugs (16). It has recently been demonstrated that blood agar can be used for routine culture and testing of M. tuberculosis susceptibility to first-line drugs (1-4, 6, 7, 15).In this study, the susceptibilities of 35 MDR M. tuberculosis clinical isolates against the second-line drugs kanamycin (KM), rifabutin (RBU), ofloxacin (OFX), p-aminosalicylic acid (PAS), capreomycin (CAP), clofazimine (CFM), and ethionamide (ETH) were investigated on blood agar, and results were compared to those obtained using the Bactec 460 TB system and the proportion method on Middlebrook 7H10 agar, performed according to CLSI recommendations.Clinical MDR-TB strains were obtained from the Istanbul Faculty of Medicine, Department of Microbiology and Clinical Microbiology, Istanbul, Turkey. Isolates were identified by the Bactec NAP test as M. tuberculosis complex. Susceptibility to first-line drugs was determined by the radiometric method (Bactec 460 TB system) (19, 20). M. tuberculosis H37Rv was used as a control strain. All drugs were obtained from the manufacturers in a chemically pure form. Stock antibiotic solutions and subsequent dilutions were prepared. All stock solutions except CFM, which was stored in the dark at room temperature, were stored at −70°C in small aliquots.The agar proportion method using Middlebrook 7H10 and blood agar was performed with concentrations of 10, 5, 5, 1, 2, 1, and 2 μg/ml for CAP, ETH, KM, CFM, OFX, RBU, and PAS, respectively (9, 10, 12, 16, 17).Middlebrook 7H12 broth (Bactec 12B; Becton Dickinson Microbiology Systems) was used for radiometric testing (5, 11, 16, 19, 20). Second-line drugs (KM, RBU, PAS, CAP, CFM, OFX, and ETH) were tested by using single critical concentrations. Concentrations of second-line drugs tested in the Bactec 460 TB system were 1.25, 1.25, 5, 0.5, 2, 0.5, and 4 μg/ml for CAP, ETH, KM, CFM, OFX, RBU, and PAS, respectively (9, 10, 12, 16, 17).The inoculum was prepared from freshly grown colonies on Löwenstein-Jensen medium. The supernatant of each isolate was adjusted to a 1 McFarland standard. The agar proportion method was performed on both Middlebrook 7H10 agar and blood agar (infusion agar, Becton Dickinson) separately according to CLSI recommendations (16). The plates were monitored twice a week by the naked eye. Resistance was defined as growth of a colony count of >1% on drug-containing quadrants in comparison to drug-free quadrants (16).The Bactec 460 TB system revealed that all isolates were susceptible to KM and CFM. RBU, OFX, PAS, CAP, and ETH resistance was detected in 27, 1, 3, 2, and 17 isolates, respectively. All isolates were susceptible to KM and CFM on Middlebrook 7H10 agar; resistance was detected in 27, 1, 3, 1, and 14 isolates for RBU, OFX, PAS, CAP, and ETH, respectively. On blood agar, all isolates were susceptible to KM and CFM; resistance was detected in 27, 2, 3, 2, and 22 isolates for RBU, OFX, PAS, CAP, and ETH, respectively.Blood agar results were compared with the results obtained using the Bactec 460 TB system and the proportion method on Middlebrook 7H10 agar. Agreement, sensitivity, specificity, positive predictive value, and negative predictive value are shown in Tables ​Tables11 and ​and2.2. The results of susceptibility testing were obtained on the 21st day of incubation by the proportion method on blood and Middlebrook 7H10 agar as recommended by the CLSI. The results of susceptibility testing by the Bactec 460 TB system were obtained on the 5th day of incubation for 31 isolates and on the 8th day for 4 isolates.

TABLE 1.

Comparison of blood agar results and Bactec 460 TB system results^a