Evaluation of Nested PCR for Rapid Diagnosis of Pediatric Tuberculous Meningitis

Abstract

Introduction: Pediatric Tuberculous Meningitis (TBM) is a highly morbid, often fatal disease and its prompt diagnosis is fundamental to the proper management. Cerebrospinal fluid (CSF) is paucibacillary in such cases and conventional bacteriological methods for detecting M.tuberculosis has inadequate sensitivity, which limits their utility as diagnostic method. Molecular techniques, detecting DNA of M.tuberculosis in CSF, have better sensitivity and thus could be a potent tool for rapid diagnosis of TBM. Aim: To evaluate Nested PCR protocol targeting 38 kDa gene for rapid detection of M.tuberculosis complex in clinically suspected cases of pediatric TBM. Materials and Methods: In this prospective case control study was total of 109 subjects, age ranging from between 6 months and 12 years were included. Case group comprised of 75 clinically suspected cases and control group comprised of 34 children with non tubercular CNS infection or some non infectious CNS disorders. 1-3 ml of CSF was aseptically collected and assessed for Mycobacterium tuberculosis by centrifugation of CSF. Smear was prepared ZN staining, inoculation of LJ medium & BacT/alert bottles from the deposit was done on same day. A portion of deposit was kept at –20°C for DNA extraction and nested PCR was performed later. Result: Among case group, there were 33 (44%) males and 42 (56%) females, with maximum number (33/75) of cases belonging to 6 months-4-year group. Nested PCR showed sensitivity and specificity of 89.3% and 97and respectively, for detection of M.tuberculosis. Sensitivity and specificity of BacT/ Alert 3D, LJ Culture and ZN staining were 28%,8%, 2.67%and 100%,100%, 100% respectively. Conclusion: Nested PCR targeting 38kDa gene (protein antigen b) proved to be highly sensitive and specific for the detection of M.tuberculosis in CSF in respect with the current gold standard, LJ culture. Nested PCR has the potential as an effective tool for early diagnosis of pediatric TBM.

Keywords

• automated liquid culture
• meningeal tuberculosis
• polymerase chain reaction