An Integrated Approach to Detection of Mycobacterium Tuberculosis Using BD MGIT 320 System and other Diagnostic Modalities

Background: Application of automated liquid culture system like Becton, Dickinson and Company (BD) MGIT 320 can shorten the recovery time of Mycobacteria in clinical diagnostic laboratories as compared to Lowenstein Jensen (LJ) medium. The present study aimed to compare the results of BD MGIT 320 culture system with that of LJ medium and also to evaluate the usefulness of molecular methods as an adjunct. Methods: Specimen from clinically suspected cases of tuberculosis were processed as per standard guidelines for smear microscopy, solid culture in LJ media, liquid culture in BD MGIT 320 system and Line probe assay. Polymerase chain reaction was performed on extracted DNA from MGIT positive specimen to detect IS6110 and MPB64 gene targets. Result: Out of total 83 specimens, a statistically significant higher isolation rate (25.3%, n=21) was shown by MGIT 320 system in comparison to LJ medium (12.04%, n=10). Line Probe Assay was positive in 37.3% (n=31) and direct microscopy was positive in only 13.25% (n=11) specimen. The average duration of detection was found to be more rapid by MGIT320 than LJ culture (18 days vs 24.1 days). Among the 21 MGIT positive specimens 7 were found positive for both IS6110 and MPB64 gene while 7 specimens were found positive only for IS6110. Conclusion: The automated liquid culture systems like BD MGIT 320 have a higher isolation rate and are more rapid as compared to solid media. But adoption of Line probe assay combined with MGIT culture will definitely yield rapid and efficient detection of Mycobacteria.


Introduction
The increasing incidence of tuberculosis (TB) and emergence of multidrug resistant tuberculosis (MDRTB) has made it essential for health care laboratories to detect Mycobacteria rapidly along with drug susceptibility pattern. In 2015, the world had an estimated 10.4 million new TB cases; India recorded an incidence of 2.84 million with 0.13 million drug resistant TB cases. [1] As conventional culture methods are cumbersome and need longer incubation, adoption of automated culture system like BD (Becton, Dickinson and Company) MGIT (Mycobacterium Growth Indicator Tube) 320 liquid culture system can shorten the recovery time of Mycobacteria. [2] The MGIT TBc identification test based on detection of MPT64, a protein secreted by Mycobacterium tuberculosis (MTb) complex can detect MTb complex in 15 minutes from positive MGIT cultures. Liquid culture shortens the time of detection in comparison to solid culture but it is still not a rapid test as labor intensive biochemical methods are required for species identification and it takes longer incubation for smear-negative specimens. [3,4,5] In recent times, laboratories are adopting nucleic acid amplification (NAA) diagnostic technologies owing to their rapidity and sensitivity. Methods like Line probe assay (LPA) or Polymerase Chain Reaction (PCR) assay as adjunct to detect MTb complex from clinical specimen can overcome the limitations of liquid culture.
The present study aimed to compare the results of BD MGIT 320 culture system with that of Lowenstein-Jensen (LJ) medium for recovery rates and the mean time required to detect Mycobacteria from clinical specimens. Our study also aimed to evaluate the usefulness of molecular methods as an adjunct for rapid detection.

Materials and Methods
The study involves specimens from clinically suspected cases of tuberculosis (both pulmonary and extrapulmonary) received in Mycobacteriology laboratory for detection of Mycobacteria during a period of 18 months from September, 2015 to February, 2017. Inoculation of MGIT medium: After labeling MGIT tube with specimen number, 0.8 mL of Growth Supplement/ MGIT PANTA (polymyxin, amphotericin B, nalidixic acid, trimethoprim, and azlocillin) antibiotic mixture was added just prior to inoculation of 0.5 mL of the suspension into a BD MGIT 320 tube. After proper mixing, tubes were entered into the instrument and allowed for automatic testing for the duration of the recommended 42 days. Positive MGIT tubes were processed for the MGIT TBc identification test; positive result of which confirmed growth of Mtb complex.

Line Probe Assay (LPA):
The GenoType MTBDRplus VER 2.0 (Hain Lifesciences GmBH, Nehren, Germany) is a commercially available line probe assay based on multiplex PCR combined with reverse hybridization on nitrocellulose strips, targeting common mutations and was performed according to the manufacturer's instructions [9]. This assay identifies Rifampicin (RIF) and Isoniazid (INH) resistance by detecting the most common mutations of the rpoB gene and the katG and inhA genes, respectively, and can be used on direct specimens as well as culture isolates. Mycobacterial DNA was extracted in BSL-3 laboratory according to manufacturer's instructions. [9] Conventional PCR : Multiplex PCR assay was performed for identification of M. tuberculosis using specific pair of primers designed to amplify IS6110 and MPB64 gene in the Mtb complex and the expected band size was 123bp for IS6110 and 240 bp for MPB64. [10] The sequences of primer used were IS6110F: 5'-CCTGCGAGCGTAGGCGT-3', IS6110R: 5'-CTCGTCCAGCGCCGCTTCGG-3', MPB64F: 5'-TCCGCTGCCAGTCGTCTTCC-3', MPB64R: 5'-GTCCTCGCGAGTCTAGGCCA-3' . [10] Briefly, following components were added to a sterile eppendorf tube (for 20 µL reaction volume)-10 µL universal PCR mix 2X (Promega), 2 µL of each of the forward and reverse primers (10pm/ µL), 1 µL of template and rest nuclease free water. The positive control included was the DNA of M. tuberculosis H37Rv strain and negative control was the PCR grade water. DNA amplification was performed for 40 cycles following an initial denaturation step at 95 o C for 5 minutes, denaturation at 94 o C for 1 minute, annealing at 65 o C for 1.5 minutes, extension at 72 o C for 1.5 minutes and final extension at 72 o C for 10 minutes followed by storage at 4 o Celsius until detection. The amplified products were then run on 1.5% agarose gel electrophoresis stained with ethidium bromide using 100bp ladder followed by detection of target band using gel documentation system (BIORAD) (Fig 1).
Quality control of all the methods was performed using M. tuberculosis, H37Rv strain.
All MGIT positive specimens (except one pleural fluid) were initially found positive for MTb complex by LPA, of which only 8 specimens were positive on direct microscopy. The average duration of MGIT positivity was http://www.pacificejournals.com/aabs found to be 18 days while LJ media showed an average duration of 24.1 days for positive growth (Table 2). In case of pulmonary specimens, the mean recovery time was 17.3 days whereas in extra-pulmonary specimens it was found to be 18.9 days by the BD MGIT 320 TB system in the present study.
Multiplex PCR of all MGIT positive specimens showed positive result for both IS6110 and MPB64 gene in 7 numbers, negative for both the targets in another 7, while 7 specimens were found positive only for IS6110 (Table 2).
Drug Susceptibility: Results of LPA positive specimens showed that 12 were susceptible for both Rifampicin (RIF) and Isoniazid (INH), 3 were indeterminate and 3 were resistant to both the drugs while one specimen was susceptible to Rifampicin and indeterminate to Isoniazid; yet another was Isoniazid monoresistant.

Discussion
The increased incidence of tuberculosis worldwide has made rapid and efficient culture strategies very essential which can easily be adopted in a clinical Mycobacteriology laboratory. Solid egg based medium like LJ medium is the current "gold standard" culture method for detection of Mycobacteria [6,11]. One of the culture methods, MGIT liquid culture system is easy to handle, non-radiometric, and at present does not need costly instrumentation. The BD MGIT 320 culture system contains only one drawer, holds 320 MGIT tubes and can be placed on a bench top. It is designed for laboratories with limited space and workload. MGIT 320 can be used by a laboratory that cultures up to 40 Mycobacteriology specimens per week, with an incubation period of 6 weeks, and performs up to five drug susceptibility tests per week. [2] Our study compared BD MGIT 320 system with LJ culture method for Mycobacteria and evaluated following important parameters -the rate of recovery and mean time to detection. Combined use of liquid and solid media is essential in good laboratory practice for successful isolation of mycobacterium which we followed in our study. The present study showed higher isolation rate in BD MGIT 320 system than LJ culture (25.3% vs 12.04%) and was found  [14,16,17] One smear positive pus specimen took 34 days to detection by MGIT system while another smear negative pus specimen flagged positive on day 30. Both the isolates were found to be MDRTB by LPA.
Thus, in this study MGIT 320 was found to be more rapid and efficient method than the conventional LJ media. Though culture contamination is often encountered in most of the laboratories, meticulous adherence to the manufacturer's manual and protocols, and rapid transport and/or refrigeration of samples could overcome any such problem in our study.
NAA based diagnostic techniques have the potential to increase the sensitivity as well as to considerably reduce the detection time. [18] Most studies have used IS6110 as a target for PCR-based diagnosis of tuberculosis with variable degrees of success. [19] However, this insertion element is absent in a proportion of Mtb isolates which limits its use as a sole target for the detection of tuberculosis in India. [20,21] Use of two or more gene targets for amplification has been demonstrated to increase the diagnostic yield of MTB infection in some earlier studies. [22,23] We evaluated the role of PCR using two different targets IS6110 and MPB64 specific for MTb complex in MGIT positive cases and found presence of IS6110 in 66.6% isolates while both IS6110 and MPB64 in 33.3% and none of the targets amplified in the rest. http://www.pacificejournals.com/aabs

Limitation of The Study
Though we detected the susceptibility to Rifampicin and Isoniazid using LPA in all cases, we performed MGIT DST only in those isolates which were found resistant/ indeterminate by LPA. Comparison of DST result by both the methods would have been more useful which we intend to follow in future studies. Also due to limited number of cases in the present study it is too early to comment on the usefulness of PCR assay using multiple targets.

Conclusion
Hence, we conclude that the automated culture systems like BD MGIT 320 have a higher isolation rate as compared to solid media (LJ). But adoption of Line probe assay combined with MGIT culture will definitely yield rapid and efficient detection of Mycobacterium as well as multidrug resistant TB cases.