Comparison of TrueNat polymerase chain reaction and mycobacterium growth indicator tube culture in the diagnosis of pulmonary and extrapulmonary tuberculosis

Poornima Mankara Valsan; J Sudarasana

doi:10.4103/jacm.jacm_6_22

ORIGINAL ARTICLE

Year : 2022 | Volume : 24 | Issue : 1 | Page : 21-25

Comparison of TrueNat polymerase chain reaction and mycobacterium growth indicator tube culture in the diagnosis of pulmonary and extrapulmonary tuberculosis

Poornima Mankara Valsan, J Sudarasana
Department of Microbiology, Baby Memorial Hospital, Kozhikode, Kerala, India

Date of Submission	19-Apr-2022
Date of Acceptance	02-Jun-2022
Date of Web Publication	11-Jul-2022

Correspondence Address:
Poornima Mankara Valsan
Department of Microbiology, Baby Memorial Hospital, Kozhikode, Kerala
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/jacm.jacm_6_22

Abstract

Many diagnostic tests are available for tuberculosis (TB), from light microscopy, fluorescent microscopy, solid culture, liquid culture (Mycobacterium growth indicator tube [MGIT] culture) to molecular methods. The turnaround time and sensitivity of these tests vary depending on the procedure and principle involved. Based on this background, we compared the results of TrueNat Mycobacterium TB (MTB) Polymerase chain reaction (PCR) and MGIT culture in different clinical samples. A retrospective study was conducted in a tertiary care Hospital Kozhikode, Kerala from December 2018 to October 2021. A total of 431 samples (342 extrapulmonary and 119 pulmonary) were received in the laboratory for both TrueNat MTB PCR and MGIT culture. The samples sent for a single test and/or inadequate volume of samples for both tests were excluded from the study. In extrapulmonary samples, TrueNat MTB PCR and MGIT culture were positive for 31 (9%) samples. MGIT culture was positive in 48 (14%). Seventeen samples (5%) were positive only by MGIT culture. The sensitivity and specificity of PCR in extrapulmonary TB samples are 65% and 70%, respectively. Eighteen pulmonary samples (15%) were positive by both TrueNat PCR & MGIT culture. MGIT culture was positive in 20 (17%) pulmonary samples. Two pulmonary samples were positive by MGIT culture only. The sensitivity and specificity of PCR in pulmonary samples are 90% and 96%, respectively. Accurate diagnosis followed by prompt treatment is vital for the elimination of TB from our country by 2025. PCR is found to be a very rapid method in the early diagnosis of TB. However, culture still remains the gold standard test for the diagnosis.

Keywords: Diagnosis of tuberculosis, mycobacterium growth indicator tube, TrueNat mycobacterium tuberculosis polymerase chain reaction

How to cite this article:
Valsan PM, Sudarasana J. Comparison of TrueNat polymerase chain reaction and mycobacterium growth indicator tube culture in the diagnosis of pulmonary and extrapulmonary tuberculosis. J Acad Clin Microbiol 2022;24:21-5

How to cite this URL:
Valsan PM, Sudarasana J. Comparison of TrueNat polymerase chain reaction and mycobacterium growth indicator tube culture in the diagnosis of pulmonary and extrapulmonary tuberculosis. J Acad Clin Microbiol [serial online] 2022 [cited 2023 Nov 30];24:21-5. Available from: https://www.jacmjournal.org/text.asp?2022/24/1/21/350319

Introduction

Tuberculosis (TB) is an important preventable and treatable cause of morbidity and mortality. It is a major public health problem in developing countries like India^[1] A total of 1.5 million people died from TB in 2020. Worldwide, TB is the 13^th leading cause of death and the second leading infectious killer after COVID-19.^[2] World Health Organisation (WHO) had estimated 16% of extrapulmonary TB (EPTB) among the 7.1 million incident cases notified in 2019.^[1]

Diagnosis of EPTB is challenging due to its atypical presentation often similar to neoplasia or inflammatory disorders. The inaccessible sites, paucibacillary nature and non-uniform distribution of mycobacteria may lead to false-negative results thus decreasing the sensitivity of diagnostic tests.^[3] The rapid and accurate diagnosis of TB infection is essential for starting appropriate treatment and control of infection. Point of care (POC) Polymerase chain reaction (PCR) is emerging as a new diagnostic tool for diagnosis of TB. TrueNat Mycobacterium TB (MTB) PCR (Molbio Diagnostics Pvt. Ltd.) is a chip-based POC real-time PCR test for the detection and diagnosis of MTB in EPTB and Pulmonary specimens. This new diagnostic tool has a turnaround time (TAT) of 2 h, less bio-hazard risk and minimal trained personnel requirement. Apart from routine TB diagnosis, TrueNat also gives Rifampicin sensitivity by melt curve analysis.

The gold standard recommended by the WHO for the diagnosis of TB is the use of culture method. In 2007, the WHO endorsed the use of liquid culture media as a gold standard for TB diagnosis based on the recommendations of international experts.^[4] As the TAT of liquid culture (Mycobacterium growth indicator tube [MGIT]) (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) is 42 days, it still remains a dilemma to choose between PCR and culture as the most dependent and reliable diagnostic test. In this background, we compared the results of MGIT culture and TrueNat MTB PCR in the diagnosis of TB in both EPTB and Pulmonary samples.

Materials and Methods

A retrospective study was conducted from December 2018 to October 2021. A total of 431 samples received in the laboratory for TrueNat PCR and AFB culture were included in the study. Samples with inadequate volume for both tests and samples which were received in the laboratory with request for one test-(either MTB PCR or MGIT culture) were excluded from the study. The data were entered and coded in MS Excel. Appropriate statistical methods like percentage, proportion, sensitivity and specificity were calculated.

Mycobacterium growth indicator tube culture and identification of mycobacterium tuberculosis

Samples were processed in Biosafety cabinet level II. All samples except CSF were decontaminated to inhibit other bacterial growth and concentrated by N-acetyl L-cysteine-sodium hydroxide method.^[5]

MGIT tubes and an LJ slant (HiMedia) were inoculated and incubated at 37° for six weeks. The tubes were read every day using Micro MGIT reader (BD). Cultures were considered positive if the green light of the Micro MGIT reader moves towards the red part (between 14-20) and granular growth appears at the bottom of the liquid medium in the tube. AFB smear was prepared from the granular growth in the tube and if acid fast bacilli are seen, the growth is confirmed by TBc ID card (BD Bactec) which detects the Mp64 antigen of MTB complex.^[5] LJ medium slants are inspected every week for growth.

TrueNat polymerase chain reaction

TrueNat PCR was done as per the SOP provided by the manufactures,^[6] All extrapulmonary samples were processed by MTB Plus PCR and pulmonary samples by MTB PCR. In a positive sample, both the target and the internal positive control curves will take a steep, exponential path when the fluorescence crosses the threshold value. At the end of the cycle, the MTB PCR result screen displays “Detected” with Ct value and the colony-forming units per millilitre (CFU/ml). In MTB PLUS PCR, the result screen would display the MTB load as “High”, “Medium”, “Low” or “Very low” for positive specimens.

Results

During the study period, a total of 342 EPTB samples were received in the laboratory, the details of which are given in [Table 1].

Table 1: Details of various extrapulmonary tuberculosis specimens with results of culture and polymerase chain reaction

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Forty-eight (14%) EPTB samples were positive by MGIT culture and 31 (9%) by both PCR and MGIT culture. Seventeen (5%) samples were positive by MGIT culture only. They are pleural fluid-three (8.3%), followed by lymph node-four (8%), tissue-five (7%) CSF-three (4.6%) and pus-two (2.4%). Comparison of TrueNat MTB PCR and MGIT culture for EPTB samples is given in [Table 2].

Table 2: Comparison of TrueNat mycobacterium tuberculosis polymerase chain reaction and mycobacterium growth indicator tube culture for extrapulmonary tuberculosis samples

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The sensitivity and specificity of PCR in EPTB samples are 65% and 70% respectively. The Positive predictive value and negative predictive value of PCR in EPTB samples are 76% and 94%, respectively.

The 119 pulmonary samples which were considered for the study include 97 bronchoalveolar lavage (BAL), 21 sputum and one gastric lavage. The details of pulmonary samples and the results of PCR and culture are included in [Table 3]. Comparison of TrueNat MTB PCR and MGIT culture of pulmonary samples is given in [Table 4].

Table 3: The details of pulmonary samples with results of polymerase chain reaction and culture

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Table 4: Comparison of TrueNat mycobacterium tuberculosis polymerase chain reaction and mycobacterium growth indicator tube culture of pulmonary samples

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The sensitivity and specificity of PCR in pulmonary samples are 90% and 96%, respectively. The positive predictive value and negative predictive value of PCR in pulmonary samples are 86% and 98%, respectively.

Discussion

“The Clock is ticking” is the theme of world TB Day 2021 which conveys the urgent need to tackle this disease. In this COVID-19 pandemic situation, many centres had started CBNAAT and TrueNat PCR for the diagnosis of COVID-19 and TB. As per the national TB elimination Program 2020, all the drug-resistant TB cases are diagnosed using WHO endorsed rapid diagnostics like Cartridge Based Nucleic Acid Amplification Test (CBNAAT)/Line Probe Assay/TrueNat PCR. When compared with the reference standard of culture, molecular methods have suboptimal sensitivity and specificity.^[7]

MGIT culture is essential for the diagnosis of paucibacillary TB and also to perform drug sensitivity tests other than Rifampicin (if required). However, it has certain drawbacks which includes the requirement of Class II Biosafety laboratory infrastructure, well trained personnel, prolonged hands-on time and TAT. Unfortunately, in resource-constrained countries such as India, the laboratory infrastructure is relatively weak and only few centres have these facilities.

In our study of 342 EPTB samples, the sensitivity and specificity of TrueNat PCR compared to MGIT culture were found to be 65% and 70%, respectively. In a study done by Tortoli et al. at Milan in 2012, the sensitivity and specificity of GeneXpert in EPTB samples were 79% and 97%, respectively.^[8] In 2002, Cheng et al. from Hong Kong had published the sensitivity of PCR in EPTB samples to be 72% using the manual in house PCR.^[9]

Mangayarkarasi et al. in 2019 compared TrueNat MTB PCR with solid culture and found the sensitivity and specificity to be 93.1% and 72.5% in pulmonary TB and 96.77% and 76.4%, in EPTB samples.^[6] Disparity between our results and the results of the above study may be due to the low yield of culture positivity in solid media. The liquid culture is found to have higher rate of mycobacterial isolation and shorter time to detection when compared to the culture of same specimen on solid media.^[10] In 2020 Jose et al. has compared the yield of TrueNat MTB Plus PCR with conventional solid culture in EPTB samples. Their reported 100% sensitivity and 95.1% specificity of PCR may be due to the use of highly sensitive MTB Plus PCR with a moderately sensitive solid culture and the limited sample volume.^[11] The increased recovery of MTB using MGIT culture compared to conventional solid culture from paucibacillary EPTB samples is well established by many authors.^{[12],[13],[14]}

We have observed that 16 (4.6%) EPTB samples and two (1.6%) pulmonary samples were TrueNat PCR negative and MGIT culture positive. Tortoli et al. reported 50 (3.3%) EPTB samples which are CBNAAT negative and MGIT culture positive.^[8] According to our study, pleural fluid followed by lymph nodes has poor sensitivity to PCR. Cheng et al. also reported poor sensitivity of PCR in pleural fluid.^[9]

In theory, nucleic acid amplification tests (NAAT) are capable of amplifying a single copy of the target genomic sequence. However, in practice, the sensitivity of various PCR tests varies according to their limit of detection (LOD). The LOD of various NAATs are as follows: TrueNat MTB-100 CFU/ml, TrueNat MTB Plus PCR-30 CFU/ml, Gene Xpert-131 CFU/ml, Gene Xpert Ultra-16 CFU/ml.^[15] The LOD of open PCR systems may vary from 5–10 CFU/ml.^[15] LOD of MGIT culture is 1–2 CFU/ml.^[16] MGIT culture is the most sensitive test available as of today.

Because the TAT of MGIT culture is six weeks, many clinicians prefer PCR for TB diagnosis. Many cases of EPTB may be missed by carrying out PCR as the only test for diagnosis. If sample volume is not sufficient for both tests, MGIT culture should be preferred as the method of diagnosis over PCR in EPTB.

In the case of pulmonary TB, TrueNat MTB PCR shows a good result nearly matching the results of MGIT culture with sensitivity and specificity of 90% and 96% respectively. Our results are in concordance with Kumar et al., Chhattisgarh in 2019 where the sensitivity and specificity of GeneXpert in sputum samples was 97% and 100% respectively.^[17] A multi centric study by Gomathi et al. in 2019 had compared the results of Sputum MGIT culture and TrueNat MTB. They reported the sensitivity of TrueNat to be 88.3% and specificity of 73.8%.^[18] The difference in sensitivity and specificity of the above compared to our study may be due to the large sample volume.

In the present study, ten EPTB and three pulmonary samples were PCR positive and MGIT culture negative. This may be due to the non-viable bacilli present in latent TB or loss of viability during N-acetyl L-cysteine-NAOH decontamination process before MGIT culture. Clinical dilemma in such cases is whether anti-tuberculous treatment should be maintained or discontinued. In this situation, the PCR result should be correlated with clinical and radiological findings.^[12]

Conclusion

In the diagnosis of pulmonary TB, the TrueNat MTB PCR has good sensitivity and specificity (90% and 96%). Although molecular tests hold promise in rapid diagnosis, they have a modest sensitivity and specificity in EPTB samples (65% and 70%). The diagnosis of TB and especially EPTB is based on a combination of tests. Among them, the role of MGIT culture is once more confirmed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1.	World Health Organization. Global Tuberculosis Report 2020. Geneva: World Health Organization; 2020.
2.	World Health Organization. Global Tuberculosis Report 2021. Geneva: World Health Organization; 2021.
3.	Purohit M, Mustafa T. Laboratory diagnosis of extra-pulmonary tuberculosis (EPTB) in resource-constrained setting: State of the art, challenges and the need. J Clin Diagn Res 2015;9:E01-6.
4.	Campelo TA, Cardoso de Sousa PR, Nogueira LL, Frota CC, Zuquim Antas PR. Revisiting the methods for detecting Mycobacterium tuberculosis: What has the new millennium brought thus far? Access Microbiol 2021;3:000245.
5.	Global Laboratory Initiative Advancing TB Diagnosis. Mycobacteriology Laboratory Manual. 1^st ed. Global Laboratory Initiative a Working Group of the Stop TB Partnership; 2014.
6.	Mangayarkarasi V, Sneka P, Sujith R, Jayaprakash. Ergonomic diagnostic tool based on chip mini RT PCR for diagnosis of pulmonary and extra pulmonary tuberculosis. J Pure Appl Microbiol 2019;13:1185-90.
7.	India TB report 2020, National Tuberculosis Elimination Program Anual report, Central Tb division, Ministry of Health and Family Welfare, New Delhi.
8.	Tortoli E, Russo C, Piersimoni C, Mazzola E, Dal Monte P, Pascarella M, et al. Clinical validation of Xpert MTB/RIF for the diagnosis of extrapulmonary tuberculosis. Eur Respir J 2012;40:442-7.
9.	Cheng VC, Yam WC, Hung IF, Woo PC, Lau SK, Tang BS, et al. Clinical evaluation of the polymerase chain reaction for the rapid diagnosis of tuberculosis. J Clin Pathol 2004;57:281-5.
10.	WHO. The Use of Liquid Medium for Culture and DST. Geneva: World Health Organization; 2007.
11.	Jose RA, Gopal K, Johnson AS, Samuel JA, Abraham SS, Goswami T, et al. Evaluation of TrueNat MTB/RIF test in comparison with microscopy and culture for diagnosis of extrapulmonary tuberculosis in a tertiary care centre. J Clin Diagn Res 2021;15:DC05-9.
12.	Thangavelu K, Jamir I, Ellappan K, Krishnakumariamma K, Gopichand P, Sindhusuta D, et al. Comparison of MGIT 960 with Lowenstein Jensen Media for recovery of mycobacteria from extrapulmonary specimens in Southern India. J Clin Diagn Res 2021;15:DC01-4.
13.	Pfyffer GE, Welscher HM, Kissling P, Cieslak C, Casal MJ, Gutierrez J, et al. Comparison of the mycobacteria growth indicator tube (MGIT) with radiometric and solid culture for recovery of acid-fast bacilli. J Clin Microbiol 1997;35:364-8.
14.	Hillemann D, Richter E, Rüsch-Gerdes S. Use of the BACTEC mycobacteria growth indicator tube 960 automated system for recovery of mycobacteria from 9,558 extrapulmonary specimens, including urine samples. J Clin Microbiol 2006;44:4014-7.
15.	Sastry A, Bhat S. Essential of Medical Microbiology. 2^nd ed. New Delhi: Jaypee Publication; 2018. p. 281.
16.	van Zyl-Smit RN, Binder A, Meldau R, Mishra H, Semple PL, Theron G, et al. Comparison of quantitative techniques including Xpert MTB/RIF to evaluate mycobacterial burden. PLoS One 2011;6:e28815.
17.	Kumar P, Bhardwaj P. Diagnosis of pulmonary tuberculosis with cartridge based nucleic acid amplification test and light emitting diode fluorescent microscopy: A comparative study. Int J Adv Med 2019;6:1580-83.
18.	Gomathi NS, Singh M, Singh UB, Myneedu VP, Chauhan DS, Sarin R, et al. Multicentric validation of indigenous molecular test TrueNat™ MTB for detection of Mycobacterium tuberculosis in sputum samples from presumptive pulmonary tuberculosis patients in comparison with reference standards. Indian J Med Res 2020;152:378-85. [PUBMED] [Full text]