BactecMGIT drug susceptibilitytesting methods. Direct and indi- rect PZA susceptibilitytesting was performed as described previously by Siddiqi et al. and according to the manufacturer’s instructions, respec- tively (15, 17). For the direct method, sputum specimens were processed by using the N-acetyl- L -cysteine–sodium hydroxide (NALC-NaOH) method, using a final concentration of 1 to 1.5% NaOH. The remaining pellet was resuspended in phosphate buffer (pH 6.8) up to a final volume of 2 ml and was used as the inoculum for PZA susceptibilitytesting. The resuspended pellet was diluted 1/10, and 0.5 ml was inoculated into the control tube (also containing polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin [PANTA] and the PZA enrichment supplement), while 0.5 ml of the undiluted resuspended pellet was inoc- ulated into the tube containing 100 g/ml PZA (and also containing PANTA and the PZA enrichment supplement). Tubes were incubated in the Bactec960MGIT instrument, according to the 21-day protocol for PZA susceptibilitytesting (17). Direct DST results from the MGIT instru- ment were recorded as susceptible (S), resistant (R), or uninterpretable (U). Indirect DST results were recorded as susceptible or resistant, since tests with uninterpretable results were repeated until valid results were obtained. If the direct or indirect PZA result was resistant, the PZA tube was checked visually for evidence of contamination, and a Ziehl-Neelsen stain and/or blood agar plate assay was performed to rule out contami- nants. If contaminants were found, the result was reported as uninterpre- table. Uninterpretable results were therefore classified as contaminated (including X400 errors reported by the MGIT instrument), growth failure (X200 errors due to insufficient growth, i.e., that the growth units of the control did not reach 400 within 21 days), or instrument failure.
Preparation of inocula. Prior to inoculating the PZA set tubes, 0.8 ml of BACTECMGIT960 PZA supplement was added to both growth control and PZA tubes and 100 l of PZA solution was added to the PZA tubes with an Eppendorf repeater pipette. Inocula were prepared following the instructions of the manufacturer. (i) MGIT cultures were used for PZA susceptibilitytesting no sooner than the day following instrument positivity (day 1) and no later than 5 days following the day of instrument positivity (ⱖ1 day, ⱕ5 days). On days 1 and 2 following positivity, an undiluted inoculum was used, while on days 3 through 5 suspensions were diluted 1:5 with sterile saline. Half a milliliter was then inoculated into the MGIT PZA tubes by using a GILSON Pipetman with sterile tips and safety plugs. The growth control tube was inoculated with 0.5 ml of a 1:10 dilution of the M. tuberculosis suspension. (ii) Cultures grown on Löwen- stein-Jensen (LJ) medium were used for PZA susceptibilitytesting no later than 14 days after the first appearance of colonies on the slant. Colonies were scraped from the medium with a sterile loop. A suspension adjusted to be equivalent to * Corresponding author. Mailing address: Swiss National Center for
The increase in the incidence of multidrug-resistant tuber- culosis (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 meth- ods enabling accurate susceptibilitytesting of first-line and second-line drugs are critical for the early diagnosis of MDR TB and extensively drug-resistant tuberculosis and the initia- tion of effective regimens. Various drug susceptibilitytesting (DST) methods that use solid media, including the agar pro- portion method (AP) and other methods, have the drawback of prolonged turnaround times (TATs). The World Health Or- ganization and the U.S. Centers for Disease Control and Pre- vention 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 Sys- tems, Sparks, MD), a radiometric liquid system, provided an excellent alternative for testing of the susceptibilities of Myco- bacterium tuberculosis complex (MTBC) isolates to streptomy- cin, isoniazid, rifampin (rifampicin), and ethambutol (SIRE) and to pyrazinamide (PZA) with improved TATs. The MGIT960 liquid, nonradiometric SIRE DST (Becton Dickinson Di- agnostic 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 imple- mented SIRE DST with the MGIT960system in 2004. With confidence in the SIRE DST with the MGIT960system, a study that used the same platform to test the susceptibilities of MTBC isolates to four classes of second-line drugs, levofloxa- cin (LVX), amikacin (AMK), capreomycin (CAP), and ethio- namide (ETH), was initiated in November 2004. The study was conducted at two laboratories: MDL and the TB Reference
Mycobacterial purity checks of suspensions used for SIRE and PZA susceptibility tests are important to ensure that the test is performed with a pure culture of a single mycobacterial species. When the test is performed in liquid medium, if the strain is resistant to any of the drugs tested, a mycobacterial purity check from at least one of the vials showing an unex- pected drug resistance is recommended (6). This approach permits checking for any possible contamination of the broths during the inoculation procedure, avoiding possible false-re- sistant results (ME). We have observed that bacterial contam- ination could occur in only one tube during the inoculation procedure, particularly when working with MGIT tubes, which use screw cups instead of rubber septa as with the B460. When more than one tube in the set showed resistant result, the different turbidities of the broths (contaminated broth and broth with growth of M. tuberculosis) could help to identify a possible contaminated tube, but for safety’s sake, in this study we preferred to check all broths showing drug resistance.
Identification of all strains as M. canettii was confirmed by a PCR-based deletion analysis with two panels of RDs (RD1, RD9, and RD10 and RD4, RD12, and TbD1) and by PCR- restriction analysis of the hsp65 gene, as described earlier (9, 17; L. M. Parsons, J. Dormandy, A. Clobridge, J. R. Driscoll, M. Oxtoby, H. W. Taber, and M. Salfinger, Abstr. National TB Controllers Workshop, 2004). Routine drug susceptibility test- ing was performed with all strains by use of the radiometric BACTEC 460TB system (Becton Dickinson Diagnostic Instru- ment Systems, Sparks, MD) and by the agar proportion method (12, 13); M. canettii AFB 0600720 was tested in the BACTEC 460TB system at the Wadsworth Center. All other M. canettii isolates were tested by the agar proportion method at the Unite ´ de la Tuberculose et des Mycobacte ´ries, Institut Pasteur de Guadeloupe, as described earlier (9). M. canettii Percy 25, 65, and 229 were also tested with the BACTECMGIT960system (Becton Dickinson Diagnostic Instrument Systems) (M. Fabre, personal communication).
Conventional indirect drug susceptibilitytesting of Mycobacteriumtuberculosis with liquid medium is well established and of- fers time-saving and reliable results. This multicenter study was carried out to evaluate if drug susceptibilitytesting (DST) can be successfully carried out directly from processed smear-positive specimens (direct DST) and if this approach could offer substan- tial time savings. Sputum specimens were digested, decontaminated, and concentrated by the laboratory routine procedure and were inoculated in BactecMGIT960 as well as Lowenstein-Jensen (LJ) medium for primary isolation. All the processed speci- mens which were acid-fast bacterium (AFB) smear positive were used for setting up direct DST for isoniazid (INH) and rifampin (RIF). After the antimicrobial mixture of polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin (PANTA) was added, the tubes were entered in the MGIT960 instrument using the 21-day protocol (Bactec960pyrazinamide [PZA] pro- tocol). Results obtained by direct DST were compared with those obtained by indirect DST to establish accuracy and time sav- ings by this approach. Of a total of 360 AFB smear-positive sputum specimens set up for direct DST at four sites in three differ- ent countries, 307 (85%) specimens yielded reportable results. Average reporting time for direct DST was 11 days (range, 10 to 12 days). The average time savings by direct DST compared to indirect DST, which included time to isolate a culture and perform DST, was 8 days (range, 6 to 9 days). When results of direct DST were compared with those of indirect DST, there was 95.1% con- cordance with INH and 96.1% with rifampin. These findings indicate that direct DST with the BactecMGIT960system offers further time savings and is a quick method to reliably detect multidrug resistance (MDR) cases.
simple mouth gargling, taking inhalation for 2 - 3 times to obtain cough deeply from chest and spitting into container directly. For indoor patients with suspected of extra pulmonary tuberculosis, representative samples like pleural fluid, ascetic fluid, cere- brospinal fluid, pus, lymph node, tissue biopsy were collected by treating physician. For all samples, decontamination and concentration was done using 4% NAOH-2.9% tri sodium citrate (Petroff’s method). Smear microscopy was done using ZN stain for all samples both before and after processing of samples. All processed samples were in- oculated in liquid medium-MGIT tubes and also in solid medium-LJ medium to com- pare the growth. MGIT tubes with 7 ml of middle brook 7H9 medium was used into which 0.8 ml of PANTA was added to prevent contamination and to enhance growth and 0.5 ml of processed sample was added. Tubes were then incubated at 37˚C for 42 days. Reading was taken daily in first 3 weeks and then once a week for next 3 weeks using manual MGITsystem. Tubes with granular turbidity or positive by semi-auto- mated reader were inoculated on brain heart infusion broth to check for contamination and also smear is prepared for ZN stain and gram stain to check for acid fast bacilli and contamination respectively. If acid fast bacilli are seen by ZN stain and no contamina- tion is there, then it was confirm by rapid test to differentiate M. Tb and NTM. If con- tamination is present, re-decontamination was done. Tubes which were negative after 42 days were declared negative. For LJ culture, 0.1 - 0.2 ml of processed sample was in- oculated and incubated at 37˚C for 6 - 8 weeks.
of appropriate TB drugs. Understanding drug resistance patterns within a given community is also of enormous epidemiological significance, since it provides indicators of the existence and prev- alence of primary and acquired drug resistance, which can be use- ful in the evaluation of the efficacy of anti-TB treatment (25, 26). For most anti-TB drugs, such as rifampin, isoniazid, ethambutol, and streptomycin, several WHO-recommended methods of as- sessing susceptibilitytesting are available. These include the Low- enstein-Jensen, Middlebrook 7H10, Middlebrook 7H11, Bactec 460, and MGIT960 methods. However, only the Bactec 460 and MGIT960 methods are recommended by the WHO for PZA, one of the most important early oral anti-TB agents (6). The main reason for this is that many of the other methods of testing sus- ceptibility to PZA have certain disadvantages, such as poor or slow growth and difficult standardization, that render them unsuitable for use in clinical mycobacteriology laboratories. Several other methods of testing the susceptibility of M. tuberculosis to PZA have been developed. These include the mDST based on the de- tection of pncA mutations, the PZase activity assay, and the color- imetric methods, which are based on MIC and redox indicators (7, 8, 9, 10, 11, 12). A few studies on the performances of these meth- ods have been reported (7, 8, 9, 10, 11, 12); however, few studies have been done on the use of these methods for testing the suscep- tibility of M. tuberculosis to PZA or comparing several methods synchronously using large quantities of clinically isolated M. tu- berculosis strains.
Existing procedures for DST of mycobacteria are adequate for screening but require complementation with quantitative DST measures, in particular for those drugs where heteroge- neity in phenotypic resistance is present. We have established the conditions for quantitative DST using the MGIT960 sys- tem in combination with EpiCenter software equipped with the TBeXiST module, thus providing a fully automated walk-away system for quantitative DST of M. tuberculosis. This platform allows electronic data management and is compatible with expert systems for interpretation. The MGIT960 platform in conjunction with the EpiCenter software shows high consis- tency with Bactec 460 test results over a wide range of con- centrations tested for first- and second-line anti-TB drugs. While we note that further studies are needed to address the correlation of phenotypic resistance levels and treatment out- come, we have summarized our recommendations for quanti- tative DST of M. tuberculosis in Table 5. Widespread imple- mentation of MGIT960 protocols for quantitative DST should provide standardized data to enable the correlation of results from quantitative DST with clinical outcomes by high-through- put statistical analysis in order to address the issue of pheno- typic drug resistance levels and treatment failure. In addition, data sets obtained by an automated standardized procedure based on agreed guidelines provide optimal input for monitor- ing the epidemiology of resistance at a supranational level.
A prospective study was organized by using a total of 1,585 consecutive clinical specimens to determine whether biomass obtained from positive growth in the MGIT960system could be used directly in AccuProbe DNA hybridization tests, the PCR-based Inno-LiPA Rif.TB (LiPA) assay, and a PCR-based DNA sequencing of the rpoB gene for the rapid identification of the Mycobacteriumtuberculosis complex (MTBC) and other mycobacterial species and for the determination of rifampin (RIF) resistance in MTBC strains. The results were compared to routine culture, identification, and susceptibilitytesting techniques performed on the same samples. The study results revealed that the DNA AccuProbe assay (on the day of growth positivity) readily identified 95.7%, the LiPA assay readily identified 98.6%, and rpoB sequencing readily identified 97.1% of the 70 MTBC isolates from mycobacterial growth indicator tubes (MGIT). In addition, application of the LiPA for the identification and RIF susceptibilitytesting of the MTBC in growth-positive MGIT resulted in a turn- around time of less than 2 weeks after specimen receipt. Although DNA sequencing of rpoB required a slightly longer (16 days) turnaround time, this method was capable of identifying several species of nontuberculous mycobacteria in addition to identifying MTBC and determining RIF susceptibility or resistance. The molecular methods were also found to rapidly identify RIF-susceptible and -resistant MTBC in two of the three mixed mycobacterial cultures weeks earlier than conventional methods. In conclusion, the biomass obtained in MGIT at the time of growth positivity in the 960system is sufficient for use in all three molecular tests, and this approach can reduce the turnaround time for reporting results.
Although a variety of molecular biological methods have been shown to have the potential to provide direct detection of Mycobacteriumtuberculosis complex from clinical specimens within a few hours (3, 5), culture still represents the corner- stone on which a definitive diagnosis of tuberculosis and other mycobacterioses relies. In recent years, the development of rapid, reliable methods for culture detection of acid-fast bacilli has been regarded as worthy of absolute priority (12, 13). Reasons for this renewed concern include the serious public health risk due to the reemergence of tuberculosis, the appear- ance of multidrug-resistant strains of M. tuberculosis, and the high incidence of Mycobacterium avium complex disease in patients with AIDS. Currently, mycobacterial culture can be performed with conventional solid media and by one of the available broth-based methods. Of these, the radiometric semi- automated BACTEC 460TB system (Becton Dickinson, Sparks, Md.), which was the first system to permit the signifi- cantly earlier detection of mycobacteria, is now widely ac- cepted as the “gold standard” (4). It has several drawbacks, however: it involves the use of radioactive material, and read- ing of cultures is labor-intensive and is associated with a po- tential risk of cross-contamination. Furthermore the use of needles for inoculation of the vial involves the risk of stick injury. In recent years, several new nonradiometric technolo-
Members of the Mycobacteriumtuberculosis complex (MTBC) differ in virulence attributes, drug resis- tance patterns, and host preferences. The rapid differentiation of these species to determine zoonotic or human sources of tuberculosis disease or to direct treatment can benefit both public health and patient management. Commercially available assays cannot differentiate these species, and published assays have not been evaluated directly on clinical specimens. A real-time PCR assay for the differentiation of M. tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. microti, and M. canettii was developed. The presence or absence of regions of difference (RD) between the genomes of members of the MTBC allowed for the design of a single-tube five-plex real-time PCR assay to differentiate these species. This assay assesses the presence of RD1, RD4, RD9, RD12, and a region exterior to RD9 which is present in all MTBC members. To evaluate the performance of this assay, 192 clinical specimens positive for MTBC by real-time PCR were tested, resulting in a 94% correlation of the real-time PCR with the identification results obtained with cultured material. Additionally, 727 BactecMGIT960-positive cultures were tested, resulting in a 97% concordance between the methods. This real-time PCR is an inexpensive and rapid (2.5-h) method performed in a closed-format system and requiring minimal hands-on time that can be implemented in a clinical laboratory and used directly on clinical specimens.
Strain selection. Strains were made available by six TB National/Supra- national Reference and partner laboratories within the TB-PANNET Consortium to provide wide coverage for most of the lineages observed for the M. tuberculosis complex. Strains were tested for PZA susceptibility and included in the study regardless of testing for other antitubercular drugs. PZA drug susceptibilitytesting (DST) was performed by using a BactecMGIT960 mycobacterial detection system and MGIT960 PZA kits (BD, Franklin Lakes, NJ, USA) according to the manufacturer’s instruc- tions. A total of 1,950 clinical isolates were incorporated in the database. Whenever available, genotyping information (spoligotyping and/or my- cobacterial interspersed repetitive-unit ⫺ variable-number tandem-repeat [MIRU-VNTR] typing ) were collected. The MIRU-VNTRplus web tool (26, 27) was used to define lineage information (similarity search settings for identification: 0.17; distance measure for MIRU-VNTR: cate- gorical, weighting 1; distance measure for spoligotyping: categorical, weighting 1).
The emergence of multidrug-resistant tuberculosis (MDR- TB) caused by Mycobacteriumtuberculosis and, recently, exten- sively drug-resistant tuberculosis caused by an MDR strain that is also resistant to any fluoroquinolone and at least one of the three injectable second-line drugs (kanamycin [KAN], amika- cin [AK], and/or capreomycin [CM]) is a real threat for TB control programs (28). It is obvious that there is a great ne- cessity for rapid, reliable, and economical methods for testing the susceptibility of M. tuberculosis not only to first-line drugs but also to second-line drugs. Access to drug susceptibilitytesting (DST) is a priority, and TB culture is an essential component of TB management. Using the standardized con- ventional DST methods, it takes a minimum of 3 to 8 weeks to identify resistant or susceptible strains on solid media (6, 7). The introduction of liquid culture media such as the manual mycobacterium growth indicator tube (MGIT) reduces the turnaround time compared to that of solid media, taking an average of 15 days to get results (1, 5, 19, 23, 25). In June 2007, the World Health Organization issued a recommendation for the use of liquid media for culture and DST in middle- and low-income countries to address challenges due to the epi- demic of human immunodeficiency virus-associated TB and drug-resistant TB, especially in resource-limited settings (29). Fully automated commercial systems such as the BACTECMGIT960 (Becton Dickinson) have shown their usefulness for the rapid detection of resistance to second-line drugs (12, 24);
This is the first published evaluation of direct DST using the MGITsystem. Similar trials of direct DST by radiometric BACTEC were performed when that system was introduced in the 1980s (14). Both systems share the advantages of being rapid and of testing the actual mycobacterial population caus- ing the patient’s disease instead of a selected subset that is (preferentially) cultivated in vitro during primary isolation. Fortunately, the directMGIT DST system does not appear to have some of the disadvantages that have limited the wide- spread use of direct radiometric BACTEC DST (8). For ex- ample, unlike the directMGIT DST system which used a different “critical proportion” to define resistance than the indirect radiometric BACTEC DST, the criteria for defining resistance in the indirect MGIT DST also appears appropriate for the directMGIT DST. The manufacturer stipulates that indirect MGIT DST results are only valid if the GC tube becomes positive within 12 days of inoculation. The present study found that discordant results tended to occur more fre- quently with the directMGIT method among specimens incu- bated beyond this time. However, this association did not reach statistical significance, with only nine discordant results. Fur- ther experience with the directMGIT DST method is required
Drug susceptibilitytesting (DST) for both primary and sec- ondary antituberculosis drugs with the broth-based radiomet- ric BACTEC 460 TB system (Becton Dickinson Diagnostic Systems, Sparks, MD) is well established and is considered the “gold standard” (15). However, due to increasing concern about the use and disposal of radioactive material, there is a rapid trend toward using commercially available nonradiomet- ric broth-based culture and susceptibilitytesting methods. BACTECMGIT960 (Becton Dickinson Diagnostic Systems) is a new nonradiometric system which is considered equivalent to the BACTEC 460 in performance. Recovery of mycobacte- ria from clinical specimens as well as DST for first-line drugs has been thoroughly studied for the MGIT960system (3, 4, 5, 7, 8, 10, 11, 12). However, no thorough multicenter study has been carried out establishing DST for second-line and newer drugs currently being used in the treatment of tuberculosis. According to the WHO reports, global drug resistance is an increasing concern (18). Some countries are reporting high resistance even against second-line drugs (1, 17). Therefore, it is important that nonradiometric broth-based systems should also offer DST procedures for drugs other than those consid- ered first-line.
We think it is both intuitive and logical to use a quantitative susceptibility method for MDR-TB detection, such as the Trek MycoTB plate or the phage qPCR method. The clinician may seek to maximize dosing for an isolate with a MIC or quantitative result indicating a borderline susceptible range, as discussed for the fluo- roquinolone, or even continue a medication if the MIC or quan- titative result is at the lower end of the resistance range in the setting of extensively drug-resistant TB or within a limited for- mulary. Both of these methods were readily established in the laboratory, provided information for a broad range of drugs, were relatively rapid, and could be useful for DST surveillance or individualization of multidrug regimens. Of course, interpre- tation of a quantitative range is new territory for the TB field, but so are the issues of complex drug resistance that we are now facing. Some operational aspects of the methods should be men- tioned. MTBDRplus and the Xpert MTB/RIF can of course be performed on sputum samples (preferably smear positive) as a direct DST, which saves time over methods requiring culture. The Xpert MTB/RIF method was the fastest (2 h 40 min) and required the fewest repeats, but it is limited to RIF susceptibilitytesting. The MTBDRplus line probe assay additionally yields INH suscep- tibility information, but it required more repeats due to missing control bands. All other methods (L-J proportion, MGIT960, MycoTB, and phage qPCR), being culture based, required time to obtain an adequate isolate. With the MGIT960 SIRE AST method, when a valid result could be obtained using a seed tube from the primary MGIT culture, the turnaround time was good; however this method required the most repeat testing due to contamina- tion. The MycoTB MIC plate method required the least special- ized equipment of all the methods: an incubator and a multichan- nel pipette. When this test was valid on the first try, the turnaround time was 21 days when we used growth from solid medium. However, contamination or no growth may not be ap- parent for 21 days with this method; thus, when repeat testing is required, the turnaround time doubles to 42 days. For the D29 phage method, contamination was less problematic, due to the short incubation times and specificity of the D29 phage, and this is the easiest method to customize for laboratory-specific drug pan- els. We found that this and other culture-based methods per- formed best when our slow-growing MDR and extremely drug- resistant isolates were subcultured on 7H11 agar.
timely matter. In resource poor settings that have many specimens for DST testing, confirmation of discordant results may be a challenge since new molecular rapid as- says are not easily accessible and costly. In RIF resistant isolates, we observed missense mutations at codons 531, 516 and 526 of the rpoB gene. The S531 L missense mu- tation was most common in the rpoB gene accounting for 55.4% (46/83) of all RIF resistant isolates. This muta- tion pattern is similar to other studies [11–15]. High levels of RIF mono-resistance was confirmed by both MGIT960 (33/97, 34%) and LPA (43/97, 44%) in this study. Increasing levels of RIF mono-resistant has been reported in South Africa at rates ranging from 8 to 13% [16–19]. In South Africa, RIF mono-resistance is in- creasingly observed in HIV-infected people . Unfor- tunately, no data was collected on HIV status, limiting our interpretation of results. Due to the continuous in- crease in RIF mono-resistance in South Africa, RIF re- sistance is no longer a reliable marker for MDR-TB, making DST for the identification of MDR-TB even more important. In countries with high RIF mono- resistance, the use of Xpert MTB/RIF as a base-line rapid screening method for MDR-TB should be revised. This may avoid unnecessary exposure to other toxic TB drugs. We suggest DST using LPA from direct positive smear specimens in high burden countries. The disad- vantage of LPA are longer turnaround times when high volumes of specimens need to be processed since the LPA requires separate DNA extraction before PCR and amplicon detection unlike the Xpert MTB/RIF assay that extracts, amplifies and detects resistance in real time.
All MDR-TB isolates were sub-cultured on Middlebrook medium prior to testing. Drug susceptibilitytesting of MDR-TB isolates using the agar proportion method was done for two first-line (EMB and STR) and two second- line drugs (KAN and OFX). The agar proportion method was performed on Middlebrook 7H11 medium accord- ing to the Clinical and Laboratory Standards Institute (CLSI) procedures and recommended critical concentra- tions (Table 3) . Briefly, six-welled Petri plates of Middlebrook7H11 medium (TB Diagnostic Services, South Africa) was used. Two quadrants in each plate contained drug-free medium, one was used as the pro- portional control and the other was used as a quality control. Fully susceptible M. tuberculosis H37Rv refer- ence strain and a known MDR M. tuberculosis isolate were used as quality controls. The other four quadrants contained the drug concentrations (Table 3). Each quad- rant was inoculated with a standard inoculum of 0.1 ml of mycobacterial suspension and the inoculum was dis- tributed by tilting the plate. An aliquot of 0.1 ml of the 1:100 dilutions was used to inoculate the proportional control. The plates were sealed in a plastic bag and incu- bated at 37 °C. The plates were examined 7, 10, 14 and 21 days of incubation. An isolate was classified as resist- ant when the colonies on the drug-containing quadrant were more than 1% compared to the colonies present on the drug-free control quadrant. An XDR-TB was defined as MDR-TB with additional resistance to KAN and OFX.
In contrast, both EMB and SM testing pointed out some problems in use of the two MGIT methods as well as the BACTEC 460 system. In case of EMB, less than 80% of the results obtained by the MGIT methods agreed with those ob- tained by the BACTEC 460 system. The MGIT results agreed best with the BACTEC 460 results when an EMB concentra- tion of 7.5 g/ml was used in all methods. However, the MGIT results agreed better with the expected results given by the WHO when an EMB concentration of 3.5 g/ml was used. Our results indicated that the EMB concentrations commonly ap- plied are suboptimal for both the MGIT methods and the BACTEC 460 method. In addition, our results indicated that the five isolates tested for heteroresistance to EMB truly con- sisted of resistant and susceptible subpopulations. This prob- ably explains at least partly the discordant results obtained. The heteroresistance also indicates that these isolates are not optimal for use as quality control strains.