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  Table of Contents  
ORIGINAL ARTICLE
Year : 2014  |  Volume : 32  |  Issue : 3  |  Page : 281-284
 

Comparison of manual mycobacteria growth indicator tube and epsilometer test with agar proportion method for susceptibility testing of Mycobacterium tuberculosis


1 Department of Medical Microbiology, Mental Health Hospital, 23000 Elazig, Turkey
2 Department of Medical Microbiology, Sisli Etfal Training and Research Hospital, 34371 Sisli, Istanbul, Turkey
3 Department of Medical Microbiology, Medical Faculty, Firat University, 23119 Elazig, Turkey

Date of Submission31-Oct-2012
Date of Acceptance25-Nov-2013
Date of Web Publication10-Jul-2014

Correspondence Address:
N Karabulut
Department of Medical Microbiology, Mental Health Hospital, 23000 Elazig
Turkey
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.136565

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 ~ Abstract 

Background and Objectives: Antimycobacterial susceptibility tests take weeks, and delayed therapy can lead to spread of Mycobacterium tuberculosis. Therefore, rapid, accurate and cost-effective methods are required for proper therapy selection. In this study, the Mycobacteria growth indicator tube (MGIT) and epsilometer test (Etest) methods were compared to the agar proportion method for susceptibility testing of Mycobacterium tuberculosis. Materials and Methods: The susceptibility tests against isoniazid (INH), rifampin (RIF), streptomycin (STM) and ethambutol (ETM) of 51 M. tuberculosis complex isolates were analyzed by the MGIT, Etest and agar proportion methods. Results: The concordance between MGIT/Etest and agar proportion methods was 98% for INH and 100% for RIF, STM, ETM. There were not statistically significant differences in results of the susceptibility tests between MGIT/Etest and the reference agar proportion method. Conclusion: The results have shown that MGIT and Etest methods can be used instead of the agar proportion method, because these two methods are more rapid and easier than the agar proportion method.


Keywords: Agar proportion method, epsilometer test, mycobacteria growth indicator tube, Mycobacterium tuberculosis


How to cite this article:
Karabulut N, Bayraktar B, Bulut Y. Comparison of manual mycobacteria growth indicator tube and epsilometer test with agar proportion method for susceptibility testing of Mycobacterium tuberculosis. Indian J Med Microbiol 2014;32:281-4

How to cite this URL:
Karabulut N, Bayraktar B, Bulut Y. Comparison of manual mycobacteria growth indicator tube and epsilometer test with agar proportion method for susceptibility testing of Mycobacterium tuberculosis. Indian J Med Microbiol [serial online] 2014 [cited 2018 Dec 10];32:281-4. Available from: http://www.ijmm.org/text.asp?2014/32/3/281/136565



 ~ Introduction Top


The incidence of tuberculosis has increased in developed countries in the last decade and in developing countries, tuberculosis still remains as a serious public health problem. Early diagnosis of tuberculosis and susceptibility testing for effective treatment has vital importance to control the spread of Mycobacterium tuberculosis. Therefore, rapid, accurate and cost-effective methods are needed to control this disease efficiently. [1] Furthermore, rapid identification and susceptibility testing of isolates contribute to prevention of spread of resistant organisms. [2] The clinical and laboratory standards institute (CLSI) subcommittee recommends that laboratories use a quick method of susceptibility testing to achieve the goal of reporting the results of M. tuberculosis complex susceptibility testing within 28 days of receipt of a specimen in the laboratory. [3]

The methods recommended by the CLSI are the agar proportion and BACTEC broth methods. Although the agar proportion method is inexpensive, this method has the disadvantage of requiring 3 weeks of incubation. The BACTEC system can determine mycobacteria isolates within less time, but this method has certain limitations such as requiring expensive instrumentation, being standardised for only a few of the first-line anti tuberculosis drugs. In addition, both methods use a single critical drug concentration for determination of drug resistance. [2],[4],[5],[6]

The mycobacteria growth indicator tube (MGIT) is a non-radiometric approach for the detection of mycobacteria. The MGIT contains modified Middlebrook 7H9 broth and oxygen-quenching fluorescence method to detect the amount of oxygen consumed by growing organisms. [1],[7] The epsilometer test (Etest) method was developed for mycobacterial susceptibility testing by Wanger and Mills. The Etest strip contained an exponential gradient of antimicrobial agent determinates minimum inhibitory concentrations (MIC) for antimicrobial agents. [2],[6],[8] The potential advantages of these methods are relatively easy to perform and provide susceptibility results in about a week. [2],[7],[8]

In this study, MGIT and Etest which were suggested as alternative methods to test susceptibility against isoniazid (INH), rifampin (RIF), streptomycin (STM) and ethambutol (ETM) of M. tuberculosis clinical isolates were compared with agar proportion method.


 ~ Materials and Methods Top


Strains

A total of 51 M. tuberculosis0 complex strains were isolated from 467 tuberculosis suspected patients at Medical Microbiology Laboratory of ͺiΊli Etfal Training and Research Hospital, Istanbul, Turkey. All clinical isolates were grown on Lowenstein-Jensen agar (L-J) slants and MGIT media; identified presumptively as mycobacteria by colony morphology and Erlich-Ziehl-Neelsen and Auromine-Rhodamine staining. Identification was confirmed by using MGIT p-nitro benzoic acid, cord factor, nitrate reductase and niacin accumulation tests.

We included the quality-control strains of M. tuberculosis H37RV, which was susceptible to all first-line drugs; ATCC 35838, which was resistant to rifampin; ATCC 35837, which was resistant to ethambutol; ATCC 35822, which was resistant to INH; ATCC 35820, which was resistant to streptomycin.

MGIT susceptibility testing

A volume of 0.5 ml of oleic acid-albumin-dextrose (OADC) and 0.1 ml of the drug stock solution were aseptically added to each MGIT (Becton Dickinson, USA). The growth control (GC) tube did not contain antibiotics. 0.5 ml of the 1:5 diluted suspension of the test culture was inoculated into each MGIT. The final concentration of drugs in MGIT was 0.8 μg/ml for STM, 0.1 μg/ml for INH, 1.0 μg/ml for RIF and 3.5 μg/ml for ETM. All tubes were incubated at 37°C. MGITs were read daily by placing them on a 365-nm ultraviolet transilluminator. An isolate is considered susceptible if the drug-containing tube is not emitting fluorescence within 2 days after GC tube showed positivity. It is considered resistant if the drug-containing tube showed growth on the day of GC positivity or within the following 2 days. [1],[9]

Etest susceptibility testing

Etest strips, containing gradients of STM (0.064-1024 μg/ml) and INH, RIF, ETM (0.016-256 μg/ml) were provided by AB BIODISK, Solna, Sweden. Freshly prepared Middlebrook 7H10 agar with OADC supplement was used. 3-4 week-old colonies of M. tuberculosis from L-J slants were suspended in 3 ml of Middlebrook 7H9 broth in tubes. The tubes were vortexed vigorously for 3-5 min to homogenise the suspension. The large particles were allowed to settle and the supernatant was adjusted to a turbidity equivalent to a McFarland 3.0. The inoculum was swabbed onto the entire surface of the agar plate. The Etest strips were placed on the agar surface after plates were preincubated at 35°C in 5-10% CO 2 for 24 h. The plates were then incubated under the same conditions until an inhibition ellipse was visible (7-10 days). The MIC was interpreted as the point at which the ellipse of inhibition crosses the Etest strip, as described in the Etest technical guide. [2],[6]

Agar proportion susceptibility testing

Commercially prepared drug-impregnated discs (Sensi-Discs, Becton Dickinson, USA) were used as the source of INH, RIF, ETM, STM drugs. Discs used in agar proportion susceptibility tests and drugs concentrations of discs were INH 0.2 and 1 μg/ml, RIF 1 μg/ml, ETM 5 μg/ml, STM 2 μg/ml. Antibacterial activity was determined by an agar dilution technique using Middlebrooks 7H10 agar with OADC enrichment. Colonies collected from a L-J tube were homogenized in Middlebrooks 7H9 broth for achieving turbidity equal to a McFarland 1.0 standard. The standardised suspensions were diluted 10− 2 and 10− 4 in sterile saline and 0.1 ml were inoculated onto the control quadrant and onto each of the drug-containing quadrants. Plates were incubated at 37°C in an atmosphere of 5% CO 2 for 3 weeks. The colonies on the plates were counted after 3 weeks of incubation. Susceptibility to tested drug was determined by comparing the number of colony forming units growing in the drug containing quadrants to the number in the drug-free control quadrant [Figure 1]. Quadrants showing 1% growth or more compared with growth in the control quadrant were considered resistant to that concentration of tested drug, as described in the NCCLS document. [10]
Figure 1: (a) Control quadrant of agar proportion susceptibility test, (b) Isoniazid and streptomycin in agar proportion susceptibility test (c) Rifampin and ethambutol in agar proportion susceptibility test

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Statistical analyses

Statistical analyses were conducted using the SPSS 16 (SPSS Inc, Chicago, IL). The differences in susceptibility results were evaluated by using the Chi-square test, with a P ≤ 0.05 considered to be significant.


 ~ Results Top


A total of 51 M. tuberculosis complex isolates was tested for susceptibility to STM, INH, RIF and ETM with three different methods. The susceptibility patterns of the strains are listed in [Table 1]. Whereas 47 (92%) of the 51 isolates were sensitive to all four drugs by the agar proportion method, 48 (94%) of them were found sensitive by both MGIT and Etest methods.
Table 1: The susceptibility patterns of M. tuberculosis complex strains by the agar proportion, MGIT and etest methods


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While two (4%) isolates were resistant to INH by the agar proportion method, one of these resistant isolates was found susceptible to INH by both MGIT and Etest methods. MIC of this strain was established as 0,125 μg/ml (indicating susceptibility) for INH by the Etest. The other two (4%) isolates were established as resistant to STM by agar proportion, MGIT and Etest methods. The concordance between MGIT/Etest with agar proportion methods was 98% for INH and 100% for RIF, STM, ETM. The sensitivity of the MGIT and Etest was 50% for INH, 100% for RIF, STM and ETM. The specificity of these methods was 100% for all four drugs. While positive predictive value was 100% for four drugs, negative predictive value was 98% for INH, 100% for RIF, STM and ETM. On the basis of the Chi-square test, the difference in performance between MGIT/Etest methods with the agar proportion method turned out not to be statistically significant.


 ~ Discussion Top


Rapid, accurate and cost-effective susceptibility testing methods are required for proper therapy selection for M. tuberculosis and the prevention of the spread of resistant organisms. MGIT and Etest methods may be considered as they are rapid, accurate and easy to perform.

In this study, an excellent agreement was demonstrated between MGIT/E test with the agar proportion method. The concordance between MGIT/Etest with agar proportion methods was 98% for INH and 100% for RIF, STM, ETM. While no false positive result was reported for all drugs, only one false negative result was reported against INH. There was not a significant difference between MGIT/Etest with agar proportion methods (P > 0.05 for all drugs). Similarly, the study of Jaloba et al. found strong correlations between Etest and agar proportion, which are 100% for STM, RIF, ETM and 98% for INH. In another study, between the Etest and the proportion method for drug susceptibility testing of M. tuberculosis was also found considerable agreement by Varma et al.[2] The authors evaluated Etest to be a more rapid method (10 days) than agar proportion (3 weeks) for testing of M. tuberculosis with INH and RIF.

The correlation between proportion method with MGIT and Etest for the first line antituberculosis drugs was investigated by Birinci et al.[11] The study reported that the Etest has the advantage of being a quick and simple method without the need for any additional equipment. It was also suggested that the MGIT and Etest methods could be routinely used instead of the proportion method. Fegou et al.[12] compared the manual MGIT and Etest to the proportion method. Although they established that manual MGIT showed superior sensitivity at detecting resistance to INH, STM and ETM compared to the Etest, they concluded that the proportion method remains the method of choice. The Etest was compared with the standard proportion method using Lowenstein Jensen medium in testing INH, RIF and STM with M. tuberculosis by Freixo et al. [8] Although they achieved extremely good agreement between the methods, they reported Etest to be less useful for developing countries because of requiring expensive media, Etest strips and the specialised equipment (CO 2 incubator).


 ~ Conclusions Top


The agar proportion method is more inexpensive than other methods, but this method has disadvantages such as requiring 3 weeks of incubation, consisting a single critical drug concentration for determination of drug resistance, requiring technical expertise for the interpretation of results. Although the Etest and MGIT methods compared with agar proportion is expensive, these methods are relatively simple and provide susceptibility results in less than a week may be effective for developing countries. These two methods can beroutinely used as they are rapid and easy to perform instead of the agar proportion method.

 
 ~ References Top

1.Palaci M, Ueki SY, Sato DN, Da Silva Telles MA, Curcio M, Silva EA. Evaluation of mycobacteria growth indicator tube for recovery and drug susceptibility testing of Mycobacterium tuberculosis isolates from respiratory specimens. J Clin Microbiol 1996;34:762-4.  Back to cited text no. 1
    
2.Varma M, Kumar S, Kumar A, Bose M. Comparison of Etest and agar proportion method of testing drug susceptibility of M. tuberculosis. Indian J Tuberc 2002;490:217-20.  Back to cited text no. 2
    
3.Woods GL. Susceptibility testing for mycobacteria. Clin Infect Dis 2000;31:1209-15.  Back to cited text no. 3
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4.Joloba ML, Bajaksouzian S, Jacobs MR. Evaluation of Etest for susceptibility testing of Mycobacterium tuberculosis. J Clin Microbiol 2000;38:3834-6.  Back to cited text no. 4
    
5.National Committee for Clinical Laboratory Standarts. Susceptibility testing of mycobacteria, Nocardiae and other aerobic actinomycetes; Approved standard M24-A. Wayne, PA: NCCLS; 2003.  Back to cited text no. 5
    
6.Wanger A, Mills K. Testing of Mycobacterium tuberculosis susceptibility to ethambutol, isoniazid, rifampin, and streptomycin by using Etest. J Clin Microbiol 1996;34:1672-6.  Back to cited text no. 6
    
7.Adjers-Koskela K, Katila ML. Susceptibility testing with the manual mycobacteria growth indicator tube (MGIT) and the MGIT 960 system provides rapid and reliable verification of multidrug-resistant tuberculosis. J Clin Microbiol 2003;41:1235-9.  Back to cited text no. 7
    
8.Freixo IM, Caldas PC, Martins F, Brito RC, Ferreira RM, Fonseca LS, et al. Evaluation of Etest strips for rapid susceptibility testing of Mycobacterium tuberculosis. J Clin Microbiol 2002;40:2282-4.  Back to cited text no. 8
    
9.Goloubeva V, Lecocq M, Lassowsky P, Matthys F, Portaels F, Bastian I. Evaluation of mycobacteria growth indicator tube for direct and indirect drug susceptibility testing of Mycobacterium tuberculosis from respiratory specimens in a Siberian prison hospital. J Clin Microbiol 2001;39:1501-5.  Back to cited text no. 9
    
10.Della-Latta P. Mycobacteriology and antimycobacterial susceptibility testing. In: Isenberg HD, editor. Clinical Microbiology Procedures Handbook. 2 nd ed., Sec. 7. Washington DC: ASM Press; 2004.  Back to cited text no. 10
    
11.Birinci A, Coban AY, Ekinci B, Durupinar B. Comparison of the proportion method with mycobacteria growth indicator tube and E-test for susceptibility testing of Mycobacterium tuberculosis. Mem Inst Oswaldo Cruz 2002;97:351-2.  Back to cited text no. 11
    
12.Fegou E, Jelastopulu E, Nicolaou S, Sevdali M, Anagnostou S, Kanavaki S, et al. Comparison of the manual mycobacteria growth indicator tube and the Etest with the method of proportion for susceptibility testing of Mycobacterium tuberculosis. Chemotherapy 2006;52:174-7.  Back to cited text no. 12
    


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