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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 8  |  Issue : 2  |  Page : 153-156

Evaluation of thin-layered agar for Mycobacterium tuberculosis isolation and drug susceptibility testing


Department of Microbiology, Sri Ramachandra Medical College and Research Institute, Chennai, Tamil Nadu, India

Date of Web Publication14-Jun-2019

Correspondence Address:
Sridharan Sathyamoorthy Kopula
Department of Microbiology, Sri Ramachandra Medical College and Research Institute, Chennai, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmy.ijmy_45_19

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  Abstract 


Background: Tuberculosis (TB) is India's major public health problem. According to the WHO, India harbors the largest number of cases, and TB control remains a challenge in diagnosis, drug resistance, and treatment. We undertook this study to compare the isolation rates of Mycobacterium tuberculosis (MTB) in agar, egg-based media, incidence of multidrug-resistant (MDR), and extended drug resistance (XDR) in MTB. This study aimed to compare and evaluate thin-layered agar (TLA) for the cultivation and drug susceptibility pattern of MTB with the conventional egg-based Lowenstein–Jensen's (LJ) medium and to differentiate atypical Mycobacterium by incorporating para-nitrobenzoic acid (PNB) in the TLA medium. This cross-sectional study was conducted in Sri Ramachandra Medical College and Research Institute, Porur, Chennai. Methods: A total of 68 smear-positive samples were inoculated into TLA (Middle Brook 7H 11) and LJ media with and without antibiotics (rifampicin, isoniazid, and ofloxacin) simultaneously after decontamination by the modified Petroff's method. TLA with PNB was also used to differentiate the growth of nontuberculous mycobacterium (NTM). Incubation was done at 37°C, and reading was taken every 3rd day for 6 weeks in case of TLA and for 8 weeks in case of LJ medium. Results: Out of the 68 samples, 64 (94.1%) grew in LJ, and the growth observed at the end of the 1st, 2nd, 3rd, 4th, 5th, and 6–10th weeks was 0, 12 (18.8%), 10 (15.6%), 14 (21.9%), 15 (23.4%), and 13 (20.3%), respectively. Similarly, in TLA, 65 (95.5%) samples were grown, among which 22 (33.8%) grew in the 1st week and the rest (43 [66.2%]) in the 2nd week. MDR and XDR were observed in 4 (5.8%) and 3 (4.4%) samples, respectively. Seven of them were NTM. Conclusions: TLA is a better medium, with time to positivity ranging from 1 to 2 weeks with drug susceptibility and the pattern is also comparable with LJ medium. Incorporation of PNB in TLA helps in differentiating NTM.

Keywords: Extended drug resistance tuberculosis, multidrug resistance tuberculosis, nontuberculous mycobacterium, thin-layered agar


How to cite this article:
Tharmalingam D, Kopula SS, Palraj KK. Evaluation of thin-layered agar for Mycobacterium tuberculosis isolation and drug susceptibility testing. Int J Mycobacteriol 2019;8:153-6

How to cite this URL:
Tharmalingam D, Kopula SS, Palraj KK. Evaluation of thin-layered agar for Mycobacterium tuberculosis isolation and drug susceptibility testing. Int J Mycobacteriol [serial online] 2019 [cited 2019 Jul 22];8:153-6. Available from: http://www.ijmyco.org/text.asp?2019/8/2/153/260379




  Introduction Top


Resistance in Mycobacterium tuberculosis (MTB) is a universal health issue, due to its restricted treatment alternatives, poor clinical outcome, and intensified local epidemics.[1] Multidrug-resistant (MDR) tuberculosis (TB) which is defined as resistance to isoniazid and rifampicin is being increasingly reported in newly diagnosed TB (2.84%) and in previously treated cases (11.60%) nowadays.[2] As per the Global Report for Tuberculosis 2017, the incidence of tuberculosis (TB) in India accounts for about one-fourth of the world's TB cases.[2] Reasons backing for the current epidemic and uninterrupted spread of MDR-TB comprise the uprising of HIV infections and delay in laboratory identification and susceptibility testing of MTB isolates. Universally followed methods for the drug susceptibility testing (DST) of MTB are the resistance ratio method, absolute concentration method, and proportionate method, which involves observation of sluggishly growing colonies (up to 6 weeks).[3] Hence, there is a delay in the initiation of drug therapy, especially in case of extended drug resistance or MDR cases. Even though molecular methods are available, not all the laboratories have the facility to perform and are also not cost-effective. This necessitates a quick and nominal susceptibility testing method other than molecular assays for the early identification of MDR cases in a laboratory with minimal facilities, to control and manage the spread of such epidemics. Henceforth, this study was undertaken in our center to evaluate thin-layered agar (TLA) for cultivation and drug susceptibility of MTB for quicker isolation and susceptibility results.

Aims of the study

  • Primary: To compare and evaluate TLA for the cultivation and drug susceptibility pattern of MTB with the conventional egg-based Lowenstein–Jensen's (LJ) medium
  • Secondary: To differentiate atypical mycobacterium by incorporating para-nitrobenzoic acid (PNB) in the TLA medium.



  Methods Top


A cross-sectional study was conducted at Sri Ramachandra Medical College and Research Institute, Porur, Chennai, after obtaining approval from the Institutional Ethics Committee (Ref: CSP-MED/14/OCT/19/185). Samples that were sent for acid-fast staining to the central laboratory were enrolled in the study. A total of 2915 respiratory samples during the study period (November 2014–October 2015) meant for acid-fast staining were screened, of which 107 showed acid-fast bacilli (AFB) positivity. All the samples were processed in biosafety cabinet Level 3 as per recommendations. The samples which were positive irrespective of the grade were included in the study for further culture and susceptibility testing. All samples were decontaminated and concentrated by modified Petroff's method.[4] The decontaminated samples were inoculated into the following culture medias procured commercially, and reading was taken on every 3rd day for 6 weeks for TLA and 8 weeks for LJ medium. ([Figure 1] and [Figure 2] showing growth of the samples in Plain LJ and TLA medium).
Figure 1: Growth in Lowenstein–Jensen medium

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Figure 2: Growth in thin-layered agar medium

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  1. LJ medium, plain
  2. LJ medium with antitubercular drugs (rifampicin 40 μg, isoniazid 0.2 μg, and ofloxacin 2 μg) to detect drug susceptibility
  3. TLA (Middlebrook 7H11), plain
  4. TLA (Middlebrook 7H11) with PNB
  5. TLA (Middlebrook 7H11) with antitubercular drugs (rifampicin 2 μg, isoniazid 0.2 μg, and ofloxacin 2 μg).[5]


Each lot of the medium prepared was tested for its quality with the standard H37RV strain. All the inoculated cultures (plain and with drugs) were read every 3rd day. The TLA tubes were incubated for 6 weeks and LJ tubes were incubated for 8 weeks before declared as “no growth.” The cultures showing growth were subjected to acid-fast staining (Kinyoun's method), for the confirmation of Mycobacterium. The time of growth was recorded. Simultaneously, susceptibility testing for rifampicin, isoniazid, and ofloxacin was done, as per the recommended concentration. The isolates which were showing growth in the presence of drug were reported “resistant” after the specified periods.


  Results Top


Of the 2915 respiratory samples screened by acid-fast staining method (Kinyoun's method), 107 (3.7%) were AFB positive and taken up for cultivation and antibiotic susceptibility testing. The drugs tested were rifampicin, isoniazid, and ofloxacin by LJ method and TLA method. Among the 107 AFB-positive samples, 80 (74.7%) were of male and 27 (25.3%) were of female. The male: female ratio was 2.9:1. The age-wise distributions of AFB smear-positive (n = 107) samples are shown in [Table 1].
Table 1: Age-wise distribution of the participants who were acid-fast bacilli smear positive

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The breakup of the clinical samples included in the study is shown in [Figure 3].
Figure 3: Breakup of clinical samples

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Time of growth

Lowenstein–Jensen medium

Among the 107 samples, 106 (99.1%) grew. The shortest time of growth was 14 days; 12 (11.3%) samples grew at the end of the 2nd week. The turnaround time for the rest of the isolates in days was as follows: 20 (18.9%), 31 (29.2%), and 34 (32.1%). Growth of all the samples in weeks is shown in [Figure 4].
Figure 4: Growth of samples in Lowenstein–Jensen medium in weeks

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Thin-layered agar

Of the 107 samples processed, growth occurred in 106 and all grew within the 2nd week. Most of the growth (51, 48.1%) occurred by the end of the 1st week and the rest 55 (51.9%) grew at the end of the 2nd week. Four isolates grew in the presence of PNB and hence, they are atypical mycobacteria, which were not characterized further. The time of growth of all the samples in TLA in weeks is shown in [Figure 5]. Comparison of the growth of samples in LJ medium and TLA medium is shown in [Figure 6].
Figure 5: Growth in thin-layered agar

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Figure 6: Comparison of growth in thin-layered agar medium and Lowenstein–Jensen medium

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  Discussion Top


TB being a chronic granulomatous disease having significant morbidity and mortality rates, an early microbiological diagnosis by culture and susceptibility helps clinicians to start treatment early, thereby limiting the spread of bacilli into the community. The results of this study showed the advantages of using TLA for the isolation of MTB and DST, as the growth occurred early with the information regarding susceptibility to rifampicin, isoniazid, and ofloxacin. Additionally, the presence of PNB in TLA helps in identifying the isolates as atypical mycobacteria without doing further testing procedures. In our study, >94% of the smear-positive samples originated from the respiratory system such as sputum, lavages, and pleural fluid, comparable with the global prevalence which reports that >80% are pulmonary TB.

In this study of 2915 samples which were received for smear testing on clinical suspicion or to rule out TB, 107 samples were positive with different grading, which means 3.7% were smear positive, i.e., open TB, which were included in this project for cultivation and DST. In the present study, we used the conventional egg-based LJ medium and agar-based TLA medium for the cultivation of TB bacilli and DST. In the conventional LJ medium, the earliest growth was seen after 2 weeks and the maximum number of samples grew at the 5th week, i.e., 34 (32.1%). However, TLA showed early growth, i.e., 55 (51.9%) grew in the 1st week itself. This finding correlates with a study done by Battagloli et al.[6] who had reported the mean time of detection being 12 days for TLA medium and 44 days for LJ medium. Robledo et al.[7] in 2008 also reported shorter detection time in TLA medium and mycobacteria growth indicator tube than LJ medium. They have reported more contamination in LJ medium than in TLA medium but in contrast to our study which showed more contamination with Aspergillus and aerobic spore bearers in TLA than LJ medium. The rapidity of the growth observed in TLA medium may probably be due to more surface area for growth and hence more aeration, which also explains more contamination rate in TLA medium. When comparing LJ medium is in McCartney's and hence less contamination comparatively. We incorporated PNB-500 μg/ml in TLA, which will help in the detection of atypical mycobacteria and mycobacteria at the same time. MTB is susceptible to PNB and atypical mycobacteria are resistant to PNB and so the presence or absence of growth in the TLA medium with PNB helps to differentiate MTB from atypical mycobacteria. In the present study, out of 107 samples' growth observed in TLA, 4 (3.7%) were atypical mycobacteria.

Drug susceptibility testing

Mycobacterium DST was done in the present study by incorporating rifampicin, isoniazid, and ofloxacin, with the concentration being rifampicin 40 μg, isoniazid 0.2 μg, and ofloxacin 2 μg in LJ medium and in TLA medium, the concentration of rifampicin is less, i.e. 2 μg, We detected 3 (2.8%) of our study isolates as MDR. Among the 3 isolates 2 (1.9%) of them was resistant to all the 3 drugs tested and 1(0.9%) isolate resistance to rifampicin and isoniazid. Study published by Sharma et al.[8] in 2010, with 218 sputum positive samples reported two (1.1%) of their test isolates as MDR.


  Conclusions Top


The results of the present study clearly show that agar-based medium is effective for cultivation and DST of MTB as it shows early growth. It also has an advantage of differentiating between MTB and nontuberculous mycobacterium by incorporating PNB. As this method is neither expensive nor technically demanding, it can be adopted for the cultivation and DST of mycobacterium, especially in resource-poor settings.

Acknowledgment

We are intended to extend our heartfelt thanks to the management, Sri Ramachandra Medical College and Research Institute, SRIHER, for providing excellent infrastructures and facilities to carry out this project and complete it successfully.

Financial support and sponsorship

This article is a self-funded project.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Coeck N, de Jong BC, Diels M, de Rijk P, Ardizzoni E, Van Deun A, et al. Correlation of different phenotypic drug susceptibility testing methods for four fluoroquinolones in Mycobacterium tuberculosis. J Antimicrob Chemother 2016;71:1233-40.  Back to cited text no. 1
    
2.
India TB Report 2018. Revised National TB Control Programme, Annual Status report. Ch. 4; 2018. p. 28. Availaber from: http://www.tbcindia.gov.in. [Last accessed on 2018 Dec].  Back to cited text no. 2
    
3.
Kohli A, Bashir G, Fatima A, Jan A, Wani NU, Ahmad J. Rapid drug-susceptibility testing of mycobacterium tuberculosis clinical isolates to first-line antitubercular drugs by nitrate reductase assay: A comparison with proportion method. Int J Mycobacteriol 2016;5:469-74.  Back to cited text no. 3
  [Full text]  
4.
Burdz TV, Wolfe J, Kabani A. Evaluation of sputum decontamination methods for Mycobacterium tuberculosis using viable colony counts and flow cytometry. Diagn Microbiol Infect Dis 2003;47:503-9.  Back to cited text no. 4
    
5.
Minion J, Leung E, Menzies D, Pai M. Microscopic-observation drug susceptibility and thin layer agar assays for the detection of drug resistant tuberculosis: A systematic review and meta-analysis. Lancet Infect Dis 2010;10:688-98.  Back to cited text no. 5
    
6.
Battaglioli T, Rintiswati N, Martin A, Palupi KR, Bernaerts G, Dwihardiani B, et al. Comparative performance of thin layer agar and Löwenstein–Jensen culture for diagnosis of tuberculosis. Clin Microbiol Infect 2013;19:E502-8.  Back to cited text no. 6
    
7.
Robledo J, Mejia GI, Paniagua L, Martin A, Guzmán A. Rapid detection of rifampicin and isoniazid resistance in Mycobacterium tuberculosis by the direct thin-layer agar method. Int J Tuberc Lung Dis 2008;12:1482-4.  Back to cited text no. 7
    
8.
Sharma SK, Singla R, Sarda P, Mohan A, Makharia G, Jayaswal A, et al. Safety of 3 different reintroduction regimens of antituberculosis drugs after development of antituberculosis treatment-induced hepatotoxicity. Clin Infect Dis 2010;50:833-9.  Back to cited text no. 8
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
    Tables

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