Gaurav SHARMA, Bharti MALHOTRA, P. J. JOHN, Shipra BHARGAVA

Molecular Methods and Culture in Diagnosis of Tuberculous Meningitis in Children

Objectives: This study aimed to detect the incidence of tuberculous meningitis (TM) in pediatric patients using GenoType MTBDRplus, GenoType MTBDRsl, and Mycobacteria Growth Indicator Tube (MGIT). Additionally, it aimed to evaluate the utility of GeneXpert MTB/RIF assay in the detection of Mycobacterium tuberculosis in comparison to MGIT culture. Methods: Children under or equal to 15 years of age who were clinically suspected of tuberculous meningitis (TBM) were included in the study. Cerebrospinal Fluid (CSF) samples were collected and tested for GeneXpert MTB/RIF assay and MGIT culture. Culture-positive samples were further analyzed using Line Probe Assay (LPA) to detect drug-resistant mycobacteria. Results: Out of 272 CSF samples, 28 (10.3%) samples were positive for MTB, and 5 (17.8%) were MDR-TB. GeneXpert MTB/RIF assay detected 23 (8.4%) MTB cases of which 3 (13%) were rifampicin-resistant. MGIT culture detected the presence of MTB organism in 19 (6.9%) cases, of which 6 (31.6%) were isoniazid-resistant and 2 (10.5%) were rifampicin-resistant by first line LPA. None of the samples had Extensively Drug-Resistant TB (XDR-TB). GeneXpert MTB/RIF assay had 73.6% sensitivity and 96.4% specificity taking MGIT as a gold standard. Conclusion: Total positivity for MTB was seen in 10.3% cases, among which 17.8% were MDR-TB; no XDR-TB was detected in pediatric patients. GeneXpert MTB/RIF assay is a rapid and reliable method for diagnosis of tuberculous meningitis but may miss some cases, so samples should also be cultured in MGIT to enhance yield and for extended sensitivity panel. J Microbiol Infect Dis 2021; 11(3):140-146.

Keywords:

Tuberculous meningitis pediatric, CSF, GeneXpert, MGIT,

PDF

___

1. Ducomble T, Tolksdorf K, Karagiannis I, et al. The burden of extra-pulmonary and meningitis tuberculosis: An investigation of national surveillance data, Germany, 2002 to 2009. Euro Surveill 2013; 18:20436.
2. Merkler AE, Reynolds AS, Gialdini G, et al. Neurological complications after tuberculous meningitis in a multi-state cohort in the United States. J Neurol Sci 2017; 375:460-463.
3. Zignol M, Sigmanidis C, Falzon D, et al. Multidrug-resistant tuberculosis in children: evidence from global surveillance. Eur Respir J 2013; 42:701-707.
4. Solomons RS, Visser DH, Friedrich SO, et al. Improved diagnosis of childhood tuberculous meningitis using more than one nucleic acid amplification test. Int J Tuberc Lung Dis 2015; 19:74–80.
5. WHO. World Health Organization. Automated real-time nucleic acid amplification technology for rapid and simultaneous detection of tuberculosis and rifampicin resistance: Xpert MTB/RIF assay for the diagnosis of pulmonary and extra pulmonary TB in adults and children:policy update 2013; https://apps.who.int/iris/handle/10665/112472
6 Murthy J.M.K. Tuberculous meningitis: the challenges. Neurol India 2010; 58:716–722. 7. Blakemore R, Story E, Helb D, et al. Evaluation of the analytical performance of the Xpert MTB/RIF assay. J Clin Microbiol 2010; 48:2495–2501.
8. Barnard M, Gey van Pittius NC, van Helden PD, Bosman M, Coetzee G, Warren RM. The diagnostic performance of the GenoTypeMTBDRplus Version 2 line probe assay is equivalent to that of the Xpert MTB/RIF assay. J Clin Microbiol 2012; 50:3712–3716.
9. RNTCP. Revised National Tuberculosis Control Programme. Standard operating procedures for Mycobacteriology laboratory. ICMR 2010; http://nirt.res.in/pdf/bact.SOP.pdf.
10. Xpert MTB/RIF kit inserts. http://www.cepheid.com/manageddownloads/xpert-mtb-rif-english-package-insert-301-1404-rev-b-february-2015.pdf.
11. Boehme CC, Nabeta P, Hillemann D, et al. Rapid molecular detection of tuberculosis and rifampin resistance. N Engl J Med 2010; 363:1005–1015.
12. Siddiqi SH, Rüsch-Gerdes S. MGIT procedure manual. Foundation for Innovative New Diagnostics, Geneva, Switzerland 2006 Jul:41-51.
13. Martin A, Bombeeck D, Mulders W, Fissette K, De Rijk P, Palomino JC. Evaluation of the TB Ag MPT64 Rapid test for the identification of Mycobacterium tuberculosis complex. Int J Tuberc Lung Dis 2011; 15:703-705.
14. Kumar VG, Urs TA, Ranganath RR MPT 64 Antigen detection for Rapid confirmation of M. tuberculosis isolates. BMC Research Notes 2011; 4:1-4.
15. GenoTypeMTBDRplus, VER 2.0, Instructions for Use, IFU-304A-02, Hain Lifesciences GmbH, Germany 2012.
16. Gardee Y, Dreyer AW, Koornhof HJ, et al. Evaluation of the Genotype® MTBDRsl® VER 2.0 assay for second-line drug resistance detection of Mycobacterium tuberculosis isolates in South Africa. J Clin Microbiol 2017; 55:791-800.
17. Raizada N, Sachdeva KS, Swaminathan S, et al. Piloting Upfront Xpert MTB/RIF Testing on Various Specimens under Programmatic Conditions for Diagnosis of TB & DR-TB in Paediatric Population. PLoS ONE 2015; 10:e0140375.
18. Bhatia R, Dayal R, Jindal S, Agarwal D, Goyal A. GeneXpert for Diagnosis of Tubercular Meningitis. Indian J Pediatr 2016; 83:1353–1355.
19. Bala S, Goyal S. Study on cartridge based nucleic acid amplification test in children with Neurotuberculosis at RNT Medical College, Udaipur, Rajasthan, India. Int J Contemp Pediatr 2019; 6:1588.
20. Krishnakumariamma K, Ellappan K, Muthuraj M, Tamilarasu K, Kumar SV, Joseph NM. Molecular diagnosis, genetic diversity and drug sensitivity patterns of Mycobacterium tuberculosis strains isolated from tuberculous meningitis patients at a tertiary care hospital in South India. PloS ONE 2020; 15:e0240257.
21. Kohli M, Schiller I, Dendukuri N, et al. Xpert MTB/RIF assay for extra-pulmonary tuberculosis and rifampicin resistance. Cochrane Database Syst Rev 2018; 8:CD012768.
22. Banada PP, Sivasubramani SK, Blakemore R, et al. Containment of bioaerosol infection risk by the Xpert MTB/RIF assay and its applicability to point-of-care settings. J Clin Microbiol 2010; 48:3551-3557.
23. Nhu NT, Heemskerk D, Chau TT, et al. Evaluation of GeneXpert MTB/RIF for diagnosis of tuberculous meningitis. J Clin Microbiol 2014; 52:226-233.
24. Das PK, Ganguly SB, Mandal B. Early diagnosis of smear-negative childhood pulmonary tuberculosis and its substantial yield in gastric lavage/aspirates through cartridge-based nucleic acid amplification test (Xpert Mycobacterium tuberculosis/rifampicin assay). Biomed Biotechnol Res J 2019; 3:258-263.
25. Padayatchi N, Bamber S, Dawood H, Bobat R. Multidrug-resistant tuberculous meningitis in children in Durban, South Africa. Pediatr Infect Dis J 2006; 25:147-150.
26. Santiago-García B, Blázquez-Gamero D, Baquero-Artigao F, et al. Pediatric extrapulmonary tuberculosis. Pediatr Infect Dis J 2016; 35:1175-1181.
27. Shah I, Chilkar S. Clinical profile of drug resistant tuberculosis in children. Indian Pediatr 2012; 49:741-744.
28. Al-Mutairi NM, Ahmad S, Mokaddas E, Eldeen HS, Joseph. Occurrence of disputed rpoB mutations among Mycobacterium tuberculosis isolates phenotypically susceptible to rifampicin in a country with a low incidence of multidrug-resistant tuberculosis. BMC Infect Dis 2019; 19:3.