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Evaluation Of Multiplex Polymerase Chain Reaction Using Two Primers And Its Comparison With Conventional Isolation Techniques For The Diagnosis Of...

602 words - 3 pages

Patients and samples
This study was approved by the Institutional Ethics Committee. After taking written informed consent of the subjects, total of 115 CSF samples were collected from patients more than 12 years of age irrespective of HIV status admitted to Nehru Hospital, Post Graduate Institute of Medical Education and Research (PGIMER). All the samples were subjected to Ziehl Neelsen (ZN) staining, conventional Lowenstein Jensen (LJ) culture, BACTEC Mycobacterial growth indicator tube (MGIT) liquid culture and multiplex PCR using IS6110 and MPB64 primers. The 55 TBM patients were divided into ‘confirmed’ TBM, ‘highly probable’ TBM, ‘probable’ TBM , ‘possible’ TBM groups and non-TBM groups included 60 patients of bacterial, viral or fungal causes of meningitis and non infectious causes of CNS symptoms based on criteria by Ahuja et al15. The ‘highly probable’, ‘probable’ and ‘possible’ TBM groups were grouped together as ‘suspected’ TBM for analysis (Table 1 & 2).

Table 1. Classification criteria15:
Clinical criteria Supporting evidence
Mandatory: Fever and headache >2 weeks
1) CSF: Cells >20/cmm, lymphocytes >60%, proteins 100 mg%, sugar <60% of corresponding blood sugar, negative Gram stain, India stain and VDRL where relevant.

Optional: Vomiting, neck stiffness, altered sensorium, seizures or focal neurologic deficit
2) Active extraneural TB: as evidenced by appropriate mycobacterial tests, radiology, histopathological examination.
3) Clinical response to ATT and relief of symptoms

Table 2. Study groups:
Smear / culture Supporting evidence
Confirmed TBM (9) Positive +/-
Highly probable (4) Negative 3
Probable (39) Negative 2
Possible (3) Negative 1

Specimen processing and DNA isolation:
All CSF samples were processed in a biosafety cabinet. Approximately 2 ml of CSF sample was collected and was divided...

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