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 Table of Contents  
ARTICLE
Year : 2013  |  Volume : 2  |  Issue : 4  |  Page : 214-219

Evaluation of the effectiveness of BACTEC MGIT 960 for the detection of mycobacteria in Bangladesh


1 Department of Microbiology, Stamford University , 51 Siddeswari Road, Dhaka 1217; Mycobacteriology Laboratory, International Centre for Diarrheal Disease Research, (icdddr,b), Mohakhali, Dhaka 1212, Bangladesh
2 Department of Microbiology, Stamford University , 51 Siddeswari Road, Dhaka 1217, Bangladesh
3 Mycobacteriology Laboratory, International Centre for Diarrheal Disease Research, (icdddr,b), Mohakhali, Dhaka 1212, Bangladesh

Date of Web Publication28-Feb-2017

Correspondence Address:
Rashed Noor
Department of Microbiology, Stamford University Bangladesh, 51 Siddeswari Road, Dhaka 1217
Bangladesh
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Source of Support: None, Conflict of Interest: None


DOI: 10.1016/j.ijmyco.2013.09.001

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  Abstract 


Objective: Tuberculosis (TB) caused by Mycobacterium tuberculosis has been identified as a re-emerging infectious disease with public health importance globally. Exploitation of new laboratory techniques for precise identification of mycobacteria in clinical specimens is of great importance to improve the diagnosis as part of the global TB control efforts.
Methods: The current study was conducted for the evaluation of BACTEC MGIT 960 method in comparison with Lowenstein–Jensen (LJ) culture and light emitting diode (LED) fluorescence microscopy for isolation of mycobacteria among TB suspects from Bangladesh. A total of 421 specimens were tested with these methods.
Results: Among the tested samples, 3.6% (n = 15) were LED fluorescence microscopy positive; while 18 (4.2%) and 45 (10.6%) were recovered from LJ and MGIT 960 culture. The relative positivity found through MGIT 960 system were 60% and 66.7% higher than that of LJ culture and LED fluorescence microscopy, respectively. Recovery rate of Mycobacterium tuberculosis complex ([MTC], 21 by MGIT and 16 by LJ culture) and non-tubercular mycobacteria ([NTM], 24 by MGIT and 2 by LJ culture) by MGIT 960 was 24% and 96% greater, respectively than LJ culture. Moreover, MGIT 960 was found to be highly sensitive (100%), specific (93.3%), accurate (93.6%) and a more rapid method in detecting mycobacteria when compared with LJ culture.
Conclusion: Extended recovery of NTM and MTC through MGIT 960 urged frequent application of this method to detect mycobacteria more effectively and rapidly.

Keywords: BACTEC MGIT 960, Lowenstein–Jensen culture, Light emitting diode (LED) fluorescence microscopy, Mycobacteria, Mycobacterium tuberculosis complex (MTM), Non-tubercular mycobacteria (NTM)


How to cite this article:
Hasan M, Munshi SK, Momi SB, Rahman F, Noor R. Evaluation of the effectiveness of BACTEC MGIT 960 for the detection of mycobacteria in Bangladesh. Int J Mycobacteriol 2013;2:214-9

How to cite this URL:
Hasan M, Munshi SK, Momi SB, Rahman F, Noor R. Evaluation of the effectiveness of BACTEC MGIT 960 for the detection of mycobacteria in Bangladesh. Int J Mycobacteriol [serial online] 2013 [cited 2019 Aug 19];2:214-9. Available from: http://www.ijmyco.org/text.asp?2013/2/4/214/201120




  Introduction Top


Mycobacteria, belonging to the family Mycobacteriaceae, represent a great challenge in various regions of the world today. Although diagnostics, chemotherapy and vaccination are available, tuberculosis (TB) is far from being eradicated [1] due to several factors, such as drug resistance, poor or improper detection, complacency in the public health sector and poorly managed TB control programs [2],[3],[4]. According to the World Health Organization (WHO), there were an estimated 8.7 million incident cases of TB in 2011, among which 1.4 million people died. However, the TB mortality rate has decreased 41% since 1990 [5].

The most pathogenic mycobacteria for man and animal have been grouped under Mycobacterium tuberculosis complex (MTC) which till now is comprised of eight members with M. tuberculosis being the most frequent member worldwide [6],[7]. At present, aside from M. tuberculosis, there is also a note of resurgence of non-tubercular mycobacteria (NTM) which was previously considered a commensal, but has now been determined to be pathogenic and causing pulmonary diseases as those caused by M. tuberculosis. Moreover, the frequency of the disease caused by specific MTC members and NTM is not yet known. The latter is not less important, as there has been a significant increase in the pulmonary and non-pulmonary infections due to NTMs during the last two to three decades [8],[9],[10],[11].

In Bangladesh, TB remains a major public health problem as 300,000 new cases arise each year, of which 70,000 result in fatality [2],[3],[4],[12],[13],[14],[15],[16]. The disease is endemic in this country due to the prevailing socio-cultural conditions. After India, China, Indonesia, Nigeria, and South Africa, Bangladesh ranks 6th amongst the TB burden countries [12],[13],[17]. Despite such high prevalence, there is very limited systematic information on the genotypes of MTC strains causing TB in Bangladesh.

Although a presumptive diagnosis of pulmonary TB can be made on the basis of a patient's history, and clinical and radiological findings, the definitive bacteriological diagnosis of TB continues to depend on the microscopic examination of Ziehl–Neelsen (Z–N) stained- and auramine-O stained sputum smears and then cultural confirmation [3],[4],[13]. The mycobacterial growth indicator tube (MGIT) system, introduced around 15 years ago, is part of the new-generation of rapid tests for detection of mycobacteria. The technique is based on fluorescence detection of mycobacterial growth in a tube containing a modified Middlebrook 7H9 medium together with fluorescence quenching-based oxygen sensor [18],[19],[20]. Studies on automated MGIT 960 systems have shown maximum recovery of mycobacteria. The automated MGIT 960 system has the advantage of high throughput (>900 samples can be tested in one instrument), rapidity (4–13 days), and easier interpretation of results [21]. All these aspects would be suitable for the TB detection in high-burden and resource-poor settings. However, the test has been studied only as an indirect assay mainly in developed countries.

Along these lines, the present study attempted to measure the efficacy of the BACTEC MGIT 960 method for its application as a routine diagnostic tool along with microscopic and culture methods in projecting the actual scenario of diseases caused by mycobacteria in Bangladesh.


  Materials and methods Top


Settings

The study was carried out in the Mycobacteriology Laboratory of the International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b), Mohakhali, Dhaka, where suspected TB patients came from different areas of Bangladesh.

Study population

A total of 421 sputum samples from patients suspected of having pulmonary TB based on the finding of any kind of abnormalities in chest X-rays were included in this study regardless of age and sex.

Decontamination and processing of the samples

After collecting samples in sterile 50 ml polypropylene conical tubes, they were diluted with an equal volume of 4% sodium hydroxide (NaOH) and 2.9% sodium citrate containing 0.5% N-acetyl-l-cysteine (NALC) and properly mixed. After 15 min holding at room temperature, phosphate buffer saline (PBS) (pH 6.8) was added to make the final volume of the suspension 45 ml. The samples were then centrifuged at 3000rcf for 15 min and the pellets were collected for further analysis [13].

Detection of mycobacteria through LED fluorescence microscopy

Smears were prepared from each of the concentrated (digested and decontaminated) samples. The smear was left for 15–30 min to air dry and was fixed by placing the slide over the hot plate at 85 °C for about 3–5 min. For auramine O staining, the smear was covered with 0.1% auramine O solution for 15 min and washed off with pure distilled and de-ionized water. Then the smear was decolorized with 0.5% acid-alcohol for 2 min and washed off. Finally, the slide was covered with 0.5% potassium permanganate for 2 min and subjected to drying after being washed. The dried smear was examined under an LED fluorescence microscope at 200 × and 400 × magnifications [2],[13],[22].

Isolation of M. tuberculosis by Lowenstein–Jensen(LJ) culture method

An estimated 0.01 ml of the pellet from the processed samples was taken with a wired loop and carefully inoculated onto LJ media. The inoculated culture bottles were incubated at 37 °C for up to eight weeks in a vertical position which allowed the development of individual colonies. During inoculation, the plates were observed weekly for any growth of mycobacteria. Contaminated cultures (e.g. growth of molds, and also those for which the medium was liquefied or turned a dark green) were discarded [2],[4],[13],[23].

Morphological observation and identification of the isolates

It is important to distinguish NTM from MTC. The LJ slants which showed growth within a week were considered rapid growers, i.e., mostly non-tubercular mycobacteria confirmed by morphological examination of colonies and Z–N staining from culture. L–J slants showing growth after 2–6 weeks were considered to be slow growing mycobacteria which were mostly MTC. Colonial morphology of MTC was non-chromogenic, small, whitish or buff-colored, dry, friable and rough, whether smooth, humid, chromogenic, yellow, orange or cream-colored, colonies were considered NTM. The isolates were subjected to Z–N staining for confirmation of the presence of AFB [4],[13].

Detection of mycobacteria on liquid medium (BACTEC MGIT 960)

0.8 ml from suspension of BACTEC MGIT growth supplement and BBL MGIT PANTA antibiotic mixture was added to the MGIT tube. 0.5 ml of the decontaminated and concentrated specimen suspension was then introduced into the MGIT tube and properly mixed. The tube was placed in the MGIT 960 instrument which automatically tested the sample for the duration of the recommended 42 days testing or until positive cases were detected. The machine was checked every day for positive signal. After appearance of the positive signal, the tubes were taken out from the machine and the cultures were observed under light. The mycobacterial growth assumed to appear as small clumps, grains or cords settling down at the bottom of the tube after shaking like snowfall, and contamination, if present, appeared as uniform turbidity in the entire tube [24]. For the re-confirmation of the presence of mycobacteria from MGIT 960, 500 μl of suspension was centrifuged at 10,000rcf, a smear was prepared for Z–N staining and 5 μl were introduced onto blood agar (BA) from the pellet after discarding the supernatant. Z–N staining positive cases were presumptively detected as mycobacteria.

Identification of Mycobacterium from MGIT 960 by BD MGIT™TBc identification test

The BD MGIT™TBc identification test (TBc ID) is a rapid chromatographic immunoassay for the qualitative detection of M. tuberculosis complex antigen from AFB smear-positive BD MGIT tubes. 0.1 ml of sample from MGIT tube containing AFB smear positive isolates was added into the sample well (as indicated by the teardrops) of the appropriately labeled TBc ID device. A reading was taken after 15 min and results were recorded. Positive results were indicated by the appearance of a pink to red line at the Test “T” position and the control “C” position which denotes the detection of MPT64 antigen in the sample. The sensitivity, specificity, accuracy, positive- and negative-predictive values of the MGIT 960 were calculated to measure the efficacy of the method when compared to the LJ culture [3],[4],[13].


  Results Top


Detection of mycobacteria through different microscopic and culture methods

The presence of acid-fast bacilli (AFB) in a smear reflects disease severity and indicates infectivity of the organism to the patient. Out of 421 samples, 15 (3.6%) samples were AFB positive as revealed by observing yellow or greenish cells under LED fluorescence microscopy ([Table 1]). L–J culture method detected 18 (4.3%) positive cases ([Table 1]). Two L–J slants showed growth within a week and were considered ”rapid growers' which were mostly non-tubercular mycobacteria confirmed by morphological examination of colonies and Z–N staining from culture ([Table 2]). Sixteen L–J slants showed growth after 2–6 weeks, which were slow-growing mycobacteria. These were confirmed as MTC after observing small and whitish or buff-colored, dry, friable and rough colonies on L–J media.
Table 1: Frequency of mycobacteria through different detection methods.

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Table 2: Frequency of M. tuberculosis complex (MTM) and non-tubercular mycobacteria (NTM).

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Among the 421 samples, 45 (10.7%) were MGIT 960 positive which were further confirmed by Z–N staining ([Table 1]). The contamination rate was found to be 9.5% which appeared as uniform turbidity in the entire tube and found negative in Z–N staining. Mycobacterial growth was seen as small clumps, grains or cords settling down at the bottom of the tube after shaking like snowfall when visualized in good light. These were further identified and grouped as Mycobacterium by growth pattern on L–J medium, visualizing morphology after Z–N staining and by TBc identification test. Among the 45 MGIT 960 positive samples, 21 (46.7%) were TBc identification test positive and confirmed as MTC ([Table 2]). The median time of their growth was 10.94 days. The rest of the 24 MGIT 960 positive isolates of the TBc ID test showed a negative result and were confirmed as NTM ([Table 2]). Moreover, 21 among the 24 NTM isolates were detected on blood agar and initially confirmed as AFB by Z–N staining. Isolates from the blood agar were further inoculated on L–J medium and 18 out of the 21 isolates were found to be rapid growers, i.e., NTM.

Higher diagnostic efficiency of MGIT 960 method over LJ culture and LED fluorescence microscopy

The relative positivity of the LED fluorescence microscopy, LJ culture and MGIT 960 was found to be 33.3%, 44% and 100%, consecutively. All the 15 smear positive samples were found to be positive using MGIT 960, while 2 failed to grow on L–J culture ([Table 3]). Three L–J culture and 30 MGIT 960 positive samples were LED fluorescence microscopy negative. However, the diagnostic efficacy of MGIT 960 was found to be far more satisfactory when compared with the LJ culture method ([Table 4]). A total of 27 MGIT 960 positive cases were found to be LJ culture negative. The MGIT 960 method claimed a higher sensitivity (100%), specificity (93.3%) and accuracy (93.6%) when compared with the LJ culture ([Table 4]).
Table 3: Comparative analysis of smear positive cases found through LED fluorescence microscopy with that of mycobacterial culture on L–J medium and MGIT 960.

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Table 4: Diagnostic efficacy of MGIT 960 compared with LJ culture (n = 421).

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  Discussion Top


In recent years, the prevalence of tuberculosis (TB) and non-tubercular mycobacterial (NTM) infections have increased worldwide, mainly as a consequence of several factors, such as the AIDS epidemic [25]. Although there is very little information available on diseases associated with NTM in developing countries, a higher frequency of M. tuberculosis has been recovered in recent years in Bangladesh [2],[3],[4],[12],[13],[14]. Such situations addressed the necessity of implementing a more precise diagnosis of mycobacterial infections to initiate proper management of the patients. Proper diagnosis is still a big challenge in developing countries like Bangladesh. The methods presently used for the diagnosis of mycobacteria are either costly or time-consuming [12],[13]. Therefore, a rather rapid, cost-effective and efficient method is required in developing countries. Considering these, the diagnosis efficacy of BACTEC MGIT 960 was evaluated in the present study.

To date, a number of studies have been conducted worldwide on the diagnostic efficacy of the MGIT 960 system for the recovery of mycobacteria from clinical samples [26],[27],[28],[29],[30]. The present study demonstrated that the MGIT 960 system provided 60% and 66.7% better isolation rate of mycobacteria than the LJ culture and LED fluorescence microscopy, respectively. Several studies reported the recovery rate ranging from 80% to 100% for MGIT 960 and from 59.7% to 87.2% for LJ culture [31],[32],[33]. Interestingly, the MGIT 960 method detected NTM 91.7% more effectively than the LJ culture, although for detecting MTC the difference was 23.8%. Such a large discrepancy between the results of the MGIT and LJ culture, especially in cases of NTM detection, might be due to the contamination of rapid growers in MGIT as this method is relatively susceptible to contamination. Also, LJ culture might fail to detect slow-growing NTM.

Moreover, the mean times for isolation of MTC from L–J culture and MGIT 960 were 17.9 days (10–29 days) and 10.9 days (5–22 days), respectively, while it was less than 7 days and 12.3 days (3–30 days), respectively, for the isolation of NTM. In their study, Fernando et al. [33] reported the mean time for the detection of growth at 13.2 days for MGIT 960 and 22.2 days for L–J culture, while Somoskovi et al. [34] claimed 14.3 days and 35.8 days for MGIT 960 and L–J culture, respectively. In the present study, the contamination rate of MGIT 960 and L–J culture was 9.5% and 1.3%, respectively. The rate was high in the initial two months and decreased gradually.

The results revealed that the MGIT 960 system consistently provided a better recovery of all mycobacterial species (both MTC and NTM) more rapidly and more efficiently than the L–J culture. The sensitivity, specificity and accuracy of the MGIT system was also found to be higher when compared with the L–J culture. The major drawback of the present study was the lack of molecular detection of mycobacteria-specific genes to detect their presence more rapidly and accurately for initiating treatment of the patients as early as possible. As a huge threat associated with TB in Bangladesh has been the development of multidrug resistance by M. tuberculosis isolates [2],[3], detection of such cases by conventional solid and liquid culture techniques and also by molecular means could have an impact on TB treatment and eradication. However, this study focused on the effective detection of TB cases by applying a rather cheaper method than using molecular methods in resource-poor settings like Bangladesh. Elevated positivity along with higher sensitivity, specificity and accuracy compared with the routinely used diagnostic methods led the MGIT 960 to be the most effective technique in detecting MTM as well as NTM which might seek its application more frequently in Bangladesh and other developing countries.


  Disclosure Top


The authors have no potential conflicts of interest. All authors agreed to the content of the article and contributed significantly: Mehedi Hasan and Mst. Sabiha Banu Momi performed the data acquisition and primarily drafted the article; Saurab Kishore Munshi and Farjana Rahman analyzed and interpreted data; Rashed Noor created the concept and design of the study, performed the critical revision of the article and approved it for submission.


  Acknowledgement Top


We thank the Mycobacteriology Laboratory, International Center for Diarrheal Disease Research, Bangladesh (icddr,b) for providing us with the facilities to carry out the experiments. We gratefully acknowledge the donors for their support and commitment to icddr,b's research efforts. Current donors providing unrestricted support include: Australian Agency for International Development (AusAID), Government of the People's Republic of Bangladesh; Canadian International Development Agency (CIDA), Swedish International Development Cooperation Agency (Sida), and the Department for International Development, UK (DFID).



 
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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]


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