|Year : 2012 | Volume
| Issue : 2 | Page : 218-221
Presumptive identification of Mycobacterium tuberculosis complex based on cord formation in BACTEC MGIT 960 medium
R Singhal, J Arora, M Bhalla, P Lal, S Reza, D Behera, VP Myneedu
Department of Microbiology, Lala Ram Swarup Institute of Tuberculosis and Respiratory Diseases, New Delhi 110 030, India
|Date of Submission||26-Dec-2011|
|Date of Acceptance||15-Mar-2012|
|Date of Web Publication||28-May-2012|
V P Myneedu
Department of Microbiology, Lala Ram Swarup Institute of Tuberculosis and Respiratory Diseases, New Delhi 110 030
Source of Support: None, Conflict of Interest: None
We considered samples received for culture of mycobacteria using BACTEC MGIT 960 system over a period of 1 year. Tubes flagged positive by MGIT were evaluated for presence of serpentine cording. The cord formation was compared with isolates identified as Mycobacterium tuberculosis complex (MTC) based on p-nitrobenzoic acid (PNB) test. Cords were found in 591 isolates of which 584 (98.8%) were confirmed as MTC. The sensitivity and specificity of cord formation were found to be 99.7% and 89.9%, respectively.
Keywords: Cord, mycobacterium tuberculosis, p-nitrobenzoic acid
|How to cite this article:|
Singhal R, Arora J, Bhalla M, Lal P, Reza S, Behera D, Myneedu V P. Presumptive identification of Mycobacterium tuberculosis complex based on cord formation in BACTEC MGIT 960 medium. Indian J Med Microbiol 2012;30:218-21
|How to cite this URL:|
Singhal R, Arora J, Bhalla M, Lal P, Reza S, Behera D, Myneedu V P. Presumptive identification of Mycobacterium tuberculosis complex based on cord formation in BACTEC MGIT 960 medium. Indian J Med Microbiol [serial online] 2012 [cited 2020 May 28];30:218-21. Available from: http://www.ijmm.org/text.asp?2012/30/2/218/96697
| ~ Introduction|| |
Mycobacteriology laboratory plays a pivotal role in the tuberculosis control by timely detection and identification of mycobacteria. In resource-limited settings, use of rapid and cost-effective procedure is of paramount importance. Most laboratories perform solid culture-based biochemical tests for identification of mycobacteria which could take 4-6 weeks.  Molecular techniques produce faster results but require funds, infrastructure, and technical expertise. Other rapid techniques such as CAPILIA are expensive and are not easily available in India. 
In liquid cultures, Mycobacterium tuberculosis complex (MTC) displays characteristic serpentine cord formation. Cord effect is caused by the major mycolic acid containing molecules such as trehalose-6, 6-dimycolate (TDM), a component of the mycobacterium cell wall, implicated in major immune-modular mechanism that is responsible for rendering MTC virulent.  Non- tuberculous mycobacteria (NTM) are scattered or dispersed without any orientation. 
The EXPAND-TB (Expanding Access to New Diagnostics for Tuberculosis) project is collaboration between the World Health Organization (WHO), Global Laboratory Initiative (GLI), Foundation for Innovative New Diagnostics (FIND), and the Stop TB Partnership Global Drug Facility (GDF). In India, EXPAND TB has established liquid culture systems in Intermediate Reference Laboratories (IRLs) and National Reference Laboratories (NRLs) to improve timely case detection and management of TB and multidrug resistant (MDR) TB in National TB Control Programme.
Many of these laboratories use liquid culture-based p-nitrobenzoic acid (PNB) test for identification which takes 8-12 days. The PNB inhibits MTC at a concentration of 500 μg/ml, whereas NTM are resistant to this concentration.  In such settings, identification of mycobacteria could be done by cord formation on microscopy of acid-fast bacilli (AFB). Yet few studies have evaluated its precision against established identification systems. ,, We designed this study to determine the reliability of the cord formation in MGIT 960 medium as a presumptive method for rapid identification of MTC or NTM in our setup.
| ~ Materials and Methods|| |
Study design: The study was conducted in the Department of Microbiology which is a NRL for Tuberculosis (TB). Our institute is a tertiary care referral TB and respiratory diseases hospital and DOTS-Plus site for the treatment of MDR patients. Drug susceptibility testing (DST) is performed on request by clinicians for deciding regimens for management of TB patients. Most DST requisitions are for previously treated patients (either relapse or failure cases). Our MGIT 960 liquid culture system is accredited for culture and susceptibility testing for first-line and second-line drugs by Supra National Reference Laboratory, SNRL, Antewerp, Belgium.
All pulmonary and extrapulmonary samples received for culture and drug susceptibility testing of mycobacteria from June 2010 to July 2011 were considered. A total of 1049 samples were received for isolation of mycobacteria by MGIT 960 system.
Sample processing and culture
The samples were digested and decontaminated by standard NALC-NaOH method. , Final suspension of samples was prepared in phosphate buffer saline (PBS) of which 500 μl was inoculated into 7 ml MGIT tube and a loop-full inoculated on the Lowenstein-Jensen (LJ) medium. The inoculated MGIT tubes were incubated at 37°C in MGIT 960 system till positive or for 42 days. The instrument automatically monitors for an hourly increase of fluorescence and alerts when tube is positive. The AFB smear was prepared from positive tubes and presence or absence of serpentine cording was noted on microscopy. Serpentine cording was defined as tight rope like aggregates of AFB. Morphologically, smear not meeting the said criterion was considered as negative for cording. , All slides were screened for cords by a microbiologist. For internal quality control, slides of M. tuberculosis H37Rv and M. gordonae cultures were prepared and examined with each lot of slides. All tubes tagged negative at the end of 42 days by MGIT were confirmed using AFB smear. The LJ slopes were examined weekly for appearance of mycobacteria till maximum of 8 weeks.
Identification of positive cultures
For identification, PNB test was performed by inoculation of positive culture in MGIT tubes with PNB and incubated in the MGIT 960 instrument. Another tube was inoculated as growth control (GC), which flagged positive by the MGIT system when growth unit of 400 was obtained. Cultures sensitive to PNB with cut off values of less than or equal to 100 were identified as MTC. A value more than 100 was indicative of NTM.  All NTM obtained by PNB were confirmed by conventional biochemical tests, i.e., niacin, nitrate reductase, and heat stable catalase performed with growth on LJ slopes.  The PNB and biochemicals were put by two different technicians, who were blinded to microscopy results. An independent observer correlated the microscopy and PNB results.
The sensitivity, specificity, positive predictive value, and negative predictive value for presence/absence of cord formation among identified MTC and NTM isolates were calculated using a two by two table.
| ~ Result|| |
A total of 1049 samples were received for culture and DST, of which 954 (90.9%) were pulmonary, all being sputum samples. Of the 95 (9.1%) extrapulmonary specimens, lymph node aspirates were most common 34 (35.8%), followed by 23 (24.2%) FNAC, 20 (21.1%) pus, 8 (8.4%) pleural fluids, 2 (2.1%) ascitic fluids, 2 (2.1%) tissues, 2 (2.1%) pott spine abscess, 2 (2.1%) cerebrospinal fluid, 2 (2.1%) gastric aspirate, 1 (1.1%) urine, and 1 (1.1%) thyroid abscess.
Of 1049 samples, 716 samples were indicated as positive by MGIT 960 system. Remaining 333 samples were incubated in MGIT 960 till 42 days and confirmed as culture negative by ZN smears prepared from tube. Of the positive tubes, 560 (78.3%) were from smear-positive samples with AFB grades varying from 1+ to 3+. All 716 MGIT positive tubes were screened for presence of AFB along with identification of cords. Of these, 655 (91.5%) tubes showed AFB on smear microscopy. Cords were found in 591 (90.2%) isolates, and atypical morphology with scattered short bacilli or in loose clumps was seen in 64 (9.8%) isolates [Figure 1] and [Figure 2]; [Table 1]. Contaminants were grown in 61 (5.8%) tubes on microscopy and/or blood agar and were excluded from analysis.
|Figure 1: Cord formation in Mycobacterium tuberculosis complex. Ziehl– Neelsen staining; ×1000 or ×100; Tight rope like aggregates of Mycobacterium tuberculosis|
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|Figure 2: Atypical morphology of nontubercular mycobacteri. Ziehl– Neelsen staining; ×100´; nonoriented, dispersed, and small clumps of short nontuberculous mycobacteria|
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Among 655 positives, 586 (89.5%) and 69 (10.5%) were identified as MTC and NTM, respectively by PNB method [Table 1]. All NTM identified by PNB method were confirmed as NTM by conventional biochemical identification. Of 591 cords, 584 were confirmed as MTC (98.8%) and seven as NTM. Among atypical morphology isolates, 62 were identified as NTM (96.9%). Overall concordance (98.6%) was found between cord formation and MTC identification [Table 2].
Average time required for MTC for positive signal in culture was found to be 7.4 days, whereas average time required for NTM was 4.1 days. For PNB test, the median time to obtain presumptive identification of MTC was 6.8 days and for NTM was 3.2 days. Identification by conventional biochemicals for NTM took an average of 3-6 weeks from inoculation.
| ~ Discussion|| |
Mycobacteriology laboratory plays a central role in tuberculosis control by providing accurate isolation and identification of Mycobacterium spp. Use of rapid techniques for identification have been advocated by CDC, in order to reduce transmission. These rapid methods are expensive and require elaborate infrastructure and skill set.  In liquid cultures, positive tubes are screened for AFB and cording.  Some studies have found cord formation as a simple, rapid, and economical test for presumptive identification of MTC. , However, validity of this method must be firmly established against standard techniques in different settings. In this study, we compared the cord formation with PNB test in liquid culture.
A positivity rate of 716 (68.3%) in MGIT was obtained in our study. Of the indicated positives, 5.8% were found to be contaminated which is comparable to other studies.  Gram-positive bacteria were found to be commonest contaminants as also found in other studies.  The Middlebrook 7H9 is highly enriched media, and organisms especially bacteria can easily grow and utilize the oxygen, hereby giving false-positive indication. 
We found sensitivity, specificity, positive predictive value, and negative predictive value of the cord formation for MTC to be 99.7%, 89.9%, 98.8%, and 96.9%, respectively. The sensitivity of MTC identification in our study is similar to other studies; Simeao et al. (95.4%), Kadam et al. (99.6%), McCarter et al. (89.2%), Yagupsky et al. (88.3%), Zuhre Badak et al. (90.6%), Tu et al. (92.9%), and Koksalan et al. (92.7%). ,,,,,, Attori et al. found low sensitivity of 63.5%.  Our samples were mostly smear-positive pulmonary samples in which MTC form cords. Paucity of AFB on culture may be misread as absence of cording.  More paucibacillary samples, especially extrapulmonary samples, need to be considered for outcome in such cases. Outcome of cording is also dependent on handling of specimen, expertise in microscopy, and strain variation. 
Most studies have reported good specificity of cord formation for MTC ranging from 92.9% to 99.6%. ,,,, However, the Indian study by Kadam et al. found 46% of NTM showing cording.  We found the specificity to be 89.9%. Incomplete cords produced by NTM may be read as true cords by inexperienced microscopist.
We found the median time for MTC and NTM identification by PNB inhibition test in the MGIT 960 system as 6.8 and 3.2 days, respectively. Confirmation of NTM by conventional biochemicals took an average of 3-6 weeks from inoculation. Identification based on cords gave the result on day 0 thereby yielding the fastest result.
In resource-limited countries, cost is the major determining factor in feasibility of any test. PNB inoculation in MGIT costs approximately Rs. 300 per isolate. The cost for identification of MTC using LJ is approximately Rs 70 per isolate. For identification based on cords, no additional cost is incurred for conducting the test.
To conclude, we obtained good positive and negative predictive values for preliminary identification of MTC based on cord formation in MGIT tubes. This method provides results on same day at no additional cost. However, thoroughly trained and experienced microscopist with regular rechecks and quality control is imperative for obtaining precise results for presumptive identification of MTC.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]
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