Comparison of the Mycobacterium Growth Indicator Tube Method and the Method of Proportion for Drug Susceptibility Testing of Mycobacterium Tuberculosis Mycobacterium Tuberculosis

Objective: Tuberculosis is an important public health problem in developed and, especially, developing countries. The incidence of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) has increased in recent years. Mycobacterial culture and susceptibility testing must be rapidly concluded for effective treatment and control of the disease. The present study evaluated the reliability of the Mycobacterium Growth Indicator Tube (MGIT) method for testing the susceptibility of M. tuberculosis to four first-line antimicrobial drugs by comparing MGIT results to those obtained by the method of proportion (MOP), which served as the reference method. Materials and Methods: A total of 60 clinical isolates (28 sputum, 7 bronchoalveolar lavage, 7 cerebrospinal fluid, 3 gastric aspirates, 5 urine, 4 pleural fluid and 6 other specimens) of M. tuberculosis were tested for susceptibility to streptomycin (SM), isoniazid (INH), ethambutol (EMB) and rifampin (RIF). MOP was carried out according to National Committe for Clinical Laboratory Standards (NCCLS) on Löwenstein-Jensen medium. MGIT susceptibility testing was performed according to the protocol provided by the manufacturer. Results: Resistance was detected in 18.3% and 16.7% of the isolates for INH, 13.3% and 10.0% for RIF, 16.7% and 11.7% for SM and 6.7% and 8.3% for EMB by MOP and MGIT, respectively. Conclusion: MOP remains the method of choice, however, the correlation between MOP and MGIT suggested that MGIT can also be used routinely and that it is a reliable method for testing susceptibility of M. tuberculosis strains to first-line anti-tuberculosis drugs.


Introduction
An ancient disease, tuberculosis remains one of the major causes of disability and death worldwide.The incidence of tuberculosis has been increasing dramatically throughout the world in the last decade.Control of the disease requires rapid identification of patients and prompt implementation of drug therapy.Furthermore, treatment and control of tuberculosis has been increasingly complicated by the emergence of drug resistance [1][2][3].
Drug-resistant Mycobacterium tuberculosis strains represent a serious public health problem.Resistance to the four primary drugs-INH, SM, EMB and RIF-makes tuberculosis difficult to treat.The emergence of MDR-TB within the last decade has posed an exceptionally severe threat and underscores the significance of rapid antimicrobial susceptibility testing of MDR-TB strains to effectively treat patients [4][5][6].
Among the methods that are used for drug susceptibility testing, MOP is universally accepted as the "gold standard" (NCCLS, 2000) [7].However, it generally requires 3 to 4 weeks to obtain a final result.The performance standard of a new system should be comparable with that of the BACTEC 460 TB technology, in addition to carrying a low risk of needle punctures and disposal of radioactive waste.Recently, the nonradiometric, fully automated Bactec MGIT 960 system (MGIT) (Becton Dickinson, Sparks, MD) technology has provided a new step forward, as it allows continuous monitoring of positive fluorescence, which is based on bacterial growth.This MGIT system has been proposed as a method for determining susceptibility of M. tuberculosis strains to the first-line antituberculosis drugs: INH, RIF, EMB and SM [4][5][6][8][9][10].
In this study, we evaluated, under the routine conditions of a clinical microbiology laboratory, the reliability of the MGIT system for susceptibility testing of 60 clinical M. tuberculosis isolates to four first-line anti-tuberculosis agents: INH, RIF, EMB and SM.The results were compared to those obtained by the conventional MOP using Löwenstein-Jensen (LJ) medium, which is proposed as the "gold standard".

Strains and Specimens
Sixty strains of M. tuberculosis were isolated from various clinical specimens.All specimens were processed following the standard NALC-NaOH method for digestion, decontamination, and concentration.The concentrated sediment was suspended in approximately 2 to 3 mL phosphate buffer (pH 6.8) and mixed thoroughly.A smear was prepared for acid-fast staining, and culture media were inoculated according to the laboratory standard procedure for primary isolation.All clinical isolates were grown on LJ medium slants and identified as M. tuberculosis according to growth rates, pigmentation, properties of colonies, the presence of acidfast bacilli, nitrate reduction, niacin accumulation and heat stable catalase tests.Sixty isolates were tested by the MOP and MGIT system to determine susceptibility to four first-line anti-tuberculosis agents: INH, RIF, EMB and SM.All procedures in the experimental protocol were approved by the Ethics Committee of Medical Faculty.

Method of Proportion
Each strain was tested for susceptibility to INH, RIF, EMB and SM using MOP on LJ medium.LJ tubes containing an anti-tuberculosis agent and a control (anti-tuberculosis agent-free) were provided by Salubris AŞ, Istanbul.Each of two dilutions (10 -3 and 10 -5 of a 10 7 -10 8 cfu/mL suspension) was inoculated into three control LJ tubes and into one tube containing either 4 µg/L SM, 0.2 or 1 µg/L INH, 40 µg/L RIF, or 2 µg/L EMB.Colonies were counted after 21, 28 and 42 days of incubation at 37 0 C. The criterion for resistance was 1% or more colonies in the drug-containing medium when compared to the number of colonies developing in the drug-free medium [1,3,[7][8][9]11].
Mycobacterial Growth Indicator Tube-Antibiotic Susceptibility Test (MGIT-AST): MGIT-AST (Becton Dickinson, Sparks, MD, ABD) was performed as recommended by the manufacturer.MGIT tubes were prepared by adding 0.8 mL of oleic acid-albumin-dextrose-catalase in each of the five tubes that together constituted an MGIT-AST kit: growth control, INH, SM, EMB and RIF.Antibiotics were added at a volume of 100 ml to stock solutions.Final concentrations were 1.0 µg/L for SM, 0.1 µg/L for INH, 1 µg/L for RIF, and 5.0 µg/L for EMB.A 1:5 sterile saline concentration of McFarland 0.5 standard suspension was inoculated into each tube at a volume of 0.5 mL.Drug susceptibility testing sets were entered into the MGIT instrument and continuously monitored until a susceptible or resistant result was obtained.The drug susceptibility testing set results was reported by the instrument (determined by the software algorithms, once the GC becomes positive) [2,[4][5][6].Total 60

Statistical Analysis
The χ 2 test (Spearman's correlation test) was used for comparing susceptibility results of MGIT with MOP.

Results
Sixty strains of M. tuberculosis were isolated from various clinical specimens (Table 1).
Sixty strains of M. tuberculosis were studied for susceptibility to all four anti-tuberculosis agents.Using MOP on LJ medium, we found that eleven strains were resistant to at least INH, 8 strains were resistant to RIF, 10 strains were resistant to SM, 4 strains were resistant to EMB, and 6 strains were MDR-TB (resistant to INH and RIF).Using MGIT, we found that ten strains were resistant to at least INH, 6 strains were resistant to RIF, 7 strains were resistant to SM, 5 strains were resistant to EMB, and 5 strains were MDR-TB (Table 2).
When compared, the results of susceptibility testing in this study detected resistance in 18.3% and 16.7% of the isolates for INH, 13.3% and 10.0% for RIF, 16.7% and 11.7% for SM and 6.7% and 8.3% for EMB by MOP and MGIT, respectively (Table 3).The x 2 test (Spearman's correlation test), which was used for comparing susceptibility results of the BACTEC MGIT 960 with MOP, found no statistically significant difference between the means (x 2 =0.568, p>0.05).

Discussion
The importance of rapid availability of M. tuberculosis drug resistance results is universally acknowledged.Detection of resistance at the genetic level by the presence of mutations in certain loci, although promising, is still far from finding its place among the techniques of diagnostic mycobacteriology.Furthermore, this approach is hindered by the presence of multiple resistance mechanisms for the majority of antimycobacterial drugs [3].
Phenotypic susceptibility testing, therefore, remains the method of choice.Standard methods for determining the drug susceptibility of M. tuberculosis, such as MOP on Middlebrook 7H10 agar and LJ, require 3 to 4 weeks to complete.Meanwhile, patients with resistant organisms receive treatment with an ineffective drug regimen.This delay may lead to additional drug resistance and failure to control the disease.Given the increasing rate of TB, there is a need to develop more rapid and efficient methods for mycobacterial testing (diagnosis, susceptibility testing).Of the commonly used methods, only the BACTEC 460 system can provide results within 30 days.Unfortunately, both the cost and the management of radioactive waste limit the use of the BACTEC system.MGIT, a broth-based non-radiometric system is an alternative to the radiometric system.By adding M. tuberculosis suspension to a drug-containing MGIT tubes, an anti-mycobacterial susceptibility test (AST) can be performed.MGIT-AST is a new test that is based on critical concentrations and developed in liquid media.MGIT-AST results can be obtained in less than 14 days.Furthermore, the handling of MGIT-AST is easy and secure [1-6, 8, 9, 11-14].In this study, we evaluated the reliability of MGIT in comparison with the reference method (MOP on LJ medium) using 60 strains of M. tuberculosis.
When compared, the results of susceptibility testing in this study detected resistance in 18.3% and 16.7% of the isolates for INH, 13.3% and 10.0% for RIF, 16.7% and 11.7% for SM and 6.7% and 8.3% for EMB by MOP and MGIT, respectively.The χ 2 test (Spearman's correlation test), which was used for comparing susceptibility results of the BACTEC MGIT 960 with MOP, found no statistically significant difference between the means χ 2 =0,568, p>0.05).
The performances (sensitivity, specificity, rapidity) of MGIT and MOP seemed comparable in several studies conducted recently.Birinci et al. [15] found a better correlation between MGIT and MOP on LJ medium.Said et al. [16] reported that the MGIT 960 system performed well for SM and EMB when compared to MOP (sensitivity in detecting resistance 95% and 92%, respectively).Safwat et al. [11] reported that the total efficiency of MGIT was 82% for susceptibility testing to SM, while it was 96% for INH, 90% for RIF and 80% for EMB.Safwat et al. [11] suggested that the MGIT system is not yet sufficiently accurate to warrant the elimination of LJ media for susceptibility testing to first line anti-tuberculosis drugs.Cambau et al. [1] found that the concordance between the two methods was 95.3-100% when considering the results for each drug/strain combination separately.Kontos et al. [9] reported that the overall agreement between MGIT and MOP was 99.1%.High sensitivity (the ability to detect true resistance) and specificity (the ability to detect true susceptibility) values of the MGIT system were demonstrated by this study for susceptibility testing of M. tuberculosis.Aktas et al. [17] reported the concordance between the MGIT and MOP susceptibility tests for first-line drugs as100% and 90%; 98% and 90% for INH; 100% and 90% for RIF; and 98% and 94% for EMB, respectively.Another study found INH and RIF with 97.1% and 100% concordance, and SM and EMB with 91.4% and 94.2% concordance between the two methods, respectively [14].
The emergence of MDR-TB and, more recently, of extensively drug-resistant (XDR-TB) is a real threat to TB control and elimination.Over 400,000 new cases of MDR-TB occur each year, and although the true incidence is currently unknown, XDR cases are recognized in every setting where there has been the capacity to detect them.The long-term vision for the full control of MDR-TB requires the scaling-up of culture and drug-susceptibility testing capacity, which is very limited in disease-endemic countries, and the expanded use of hightechnology assays for rapid determination of resistance [18].
In our study, 60 strains of M. tuberculosis were evaluated for susceptibility to all four anti-tuberculosis agents.Using MOP on LJ medium, we found that eleven strains were resistant to at least INH, 8 strains were resistant to RIF, 10 strains were resistant to SM, 4 strains were resistant to EMB, and 6 strains were MDR-TB.Using MGIT, we found that ten strains were resistant to at least INH, 6 strains were resistant to RIF, 7 strains were resistant to SM, 5 strains were resistant to EMB, and 5 strains were MDR-TB.
Surveillance of drug-resistance has not been performed at the national level in Turkey, however, local studies have reported various ranges of MDR-TB.Gonlugur et al. [19] reported resistance rates of 17.7% for INH, 11.4% for SM, 4.4% for RIF, 5.1% for EMB and 3.8% for MDR-TB.Ucar et al. [20] reported resistance rates in seven different regions of Turkey between 2003 and 2006 as 12.3%, 10.1%, 6% and 15.8% for INH, RIF, EMB and SM, respectively.The MDR-TB rate was 10% for this 4-year period in the same study.
In our study, resistance to INH was detected in 18.3% and 16.7% of the isolates for INH, 13.3% and 10.0% for RIF, 16.7% and 11.7% for SM and 6.7% and 8.3% for EMB by MOP and MGIT, respectively.The MDR-TB rates detected in our study were 10% (6 strains) using MOP and 8.3% (5 strains) using MGIT.
In conclusion, our results showed that there were no statistically significant differences in susceptibility testing results between MGIT and MOP.MGIT-AST is a reliable method that produced results congruent with those obtained by the proportion method on LJ medium for routine drug susceptibility testing of M. tuberculosis.