Comparative analysis of a hup B gene based diagnostic PCR with other conventional techniques for pediatric tuberculous meningitis

Abstract

Tuberculous meningitis, an extrapulmonary manifestation of tuberculosis, is associated with poor prognosis and high mortality in the pediatric age group with delayed treatment. The main causative agent in humans is Mycobacterium tuberculosis but other members of the M. tuberculosis complex especially Mycobacterium bovis, have also been implicated as potential human pathogens. Our study utilizes the hup B gene (Rv2986c in M. tuberculosis and Mb3010c in M. bovis) as a target for a two-step 'in house' nested polymerase chain reaction (PCR) in cerebrospinal fluid samples and can differentiate between M. tuberculosis and M. bovis. The products obtained for M. tuberculosis and M. bovis are 116 and 89 base pair respectively due to a 27 base pair deletion at the C-terminal of hup B gene of M. bovis. Fifty cases, suggestive of tuberculous meningitis, and fifty controls (admitted for other causes) were chosen as per Modified Ahuja's criteria and correlated with response to therapy. They were regrouped retrospectively as definitive tuberculous meningitis [25] and non-tuberculous meningitis [75]. Of the non-tuberculous meningitis category, 45 patients were diagnosed as pyogenic meningitis while the rest were diagnosed as febrile seizures [16], hypocalcemic seizures [11] and enteric encephalopathy [3]. Mycobacterial DNA extracted from cerebrospinal fluid samples were subjected to a two-step nested PCR. A sensitivity of 92% and specificity of 98.7% was obtained. M. tuberculosis and M. bovis infection was seen in 56% and 35% of the PCR positive cases respectively while 9% showed co-infection with both species. Thus this novel gene target shows promising potential as a rapid molecular diagnostic aid in pediatric tuberculous meningitis where rapid institution of antituberculosis therapy is essential.