Evaluation of PCR Using TRC ₄ and IS 6110 Primers in Detection of Tuberculous Meningitis

Abstract

We have evaluated a new set of primers (TRC 4 ) in comparison with the IS6110 primers commonly used in PCR to detect tuberculous meningitis among children. The levels of concordance between the results of IS6110 PCR and TRC 4 PCR with cerebrospinal fluid specimens from patients with clinically confirmed tuberculous meningitis were 80 and 86%, respectively. Results with the two primer sets were concordant for 55 positive and 22 negative specimens (n ‫؍‬ 98). We conclude that the sensitivity of PCR can be increased by using both IS6110 and TRC 4 primers.Tuberculosis (TB) is the major cause of death worldwide and is due to a single pathogen (16). Childhood TB in general and TB meningitis in particular owe their existence to unsuspected, undiagnosed, or incompletely treated adults in the community. Among children, mortality due to TB occurs mainly due to the neural form of TB, namely, TB meningitis. About 3% of the pediatric admissions to hospitals in India are due to TB meningitis, with reported mortality ranging from 17 to 71% (2, 14). Prompt diagnosis is critical for initiating appropriate therapy and facilitating measures to prevent dissemination of this highly contagious disease. The prevalence of childhood TB meningitis remains largely underestimated because clinical manifestations are nonspecific in early stages of the disease and bacteriologic confirmation is available only for a small proportion of patients. Also, clinical diagnosis of childhood TB meningitis is difficult due to its varied clinical presentations. Further, routinely used tests employed for clinical diagnosis of TB are inadequate to detect extrapulmonary forms of TB like TB meningitis.PCR is currently the most sensitive and rapid method to detect extrapulmonary Mycobacterium tuberculosis (1,5,8,11,15). We used as a new target TRC 4 , which was cloned and characterized previously in our laboratory (10). TRC 4 is a conserved repetitive element with specificity for M. tuberculosis complex. The aim of this paper was to compare the efficiency of a PCR with a target chosen from this cloned fragment with that of a PCR with the widely used IS6110 sequence in detecting M. tuberculosis in cerebrospinal fluid (CSF) samples from children with meningitis.CSF specimens from children suffering from meningitis, aged to 12 years, were included in the study if at least four of the following seven indicators of disease were in evidence: first, the presence of clinical features, such as gradual loss of playful activity, irritability, clouding of consciousness, convulsions, neck stiffness, and cranial nerve or motor defects; second, a CSF lymphocyte count at admission greater than 10 ϫ 10 6 / liter; third, a CSF protein level at admission greater than 80 mg/dl; fourth, a CSF glucose/blood glucose ratio at admission of less than 0.5; fifth, contact with an intrafamilial adult positive for pulmonary TB; sixth, induration of 10 mm or more on tuberculin testing with 1 TU of purified protein derivative; and seventh, positive radiological features of primary complex in...

Central Nervous System Tuberculosis: Pathogenesis and Clinical Aspects

“…Experimentally, PCR assays are able to detect M. tuberculosis DNA down to an estimated 2-CFU/ml concentration (10). Many of these assays utilize primers directed at the insertion sequence IS6110 (30,77,102,137,166,167,180,240), which is a repetitive element exclusively found in the genome of the M. tuberculosis complex (230), although isolates with low copy numbers and even no copy numbers have been identified (230). A recent example of this method includes the use of an IS6110 uniplex PCR assay that had 98% sensitivity (NPV, 99%) and a specificity of 100% (PPV ϭ 100%) against the "gold standard" of culture (167) and an overall sensitivity of 76.4% (NPV, 59.9%) and specificity of 89.2% (PPV, 94.7%) when clinical TBM was included (166).…”

Advances in Molecular Diagnosis of Tuberculosis

“…Different investigators have used separate gene targets like MPB 64 [10], repetitive sequences [13], GC repeats [14], dev R [15], 38kD [17], TRC 4 [18], IS 1081 [19] and a patented system from CDRI Lucknow. IS-1081 based system has been further modified and a new nested PCR target of this gene has been developed at CDFD, Hyderabad.…”

“…PCR has been used to confirm the diagnosis in case of lymphadenopathy. Positivity rates varying from 40-90% have been reported by PCR in case of tubercular lymphadenitis [29][30][31][32].…”

“…PCR has been extensively tried for the detection of M tuberculosis specific sequences in the CSF as well as biopsy specimens. As compared to 5-20% positivity with demonstration of AFB and mycobacterial cultures, PCR has been found to be positive in 50-70% of specimens from cases having cardinal features as well as biochemical/cytological evidence of neurotuberculosis [10,18,25].…”

“…targeting insertion and repetitive elements, various protein encoding genes and ribosomal RNA. Developments in this area have been very rapid and a large number of PCR assays targeting different gene stretches of M tuberculosis have been described [8][9][10][11][12][13][14][15][16][17][18][19][20]. It would be difficult to comment on their relative merits as limited comparative application data is available.…”