Nested PCR Assay for Eight Pathogens: A Rapid Tool for Diagnosis of Bacterial Meningitis.

PURPOSE: Bacterial meningitis is a dreadful infectious disease with a high mortality and morbidity if remained undiagnosed. Traditional diagnostic methods for bacterial meningitis pose a challenge in accurate identification of pathogen, making prognosis difficult. The present study is therefore aimed to design and evaluate a specific and sensitive nested 16S rDNA genus-based polymerase chain reaction (PCR) assay using clinical cerebrospinal fluid (CSF) for rapid diagnosis of eight pathogens causing the disease. METHODS: The present work was dedicated to development of an in-house genus specific 16S rDNA nested PCR covering pathogens of eight genera responsible for causing bacterial meningitis using newly designed as well as literature based primers for respective genus. A total 150 suspected meningitis CSF obtained from the patients admitted to Central India Institute of Medical Sciences (CIIMS), India during the period from August 2011 to May 2014, were used to evaluate clinical sensitivity and clinical specificity of optimized PCR assays. RESULTS: The analytical sensitivity and specificity of our newly designed genus-specific 16S rDNA PCR were found to be ≥92%. With such a high sensitivity and specificity, our in-house nested PCR was able to give 100% sensitivity in clinically confirmed positive cases and 100% specificity in clinically confirmed negative cases indicating its applicability in clinical diagnosis. CONCLUSIONS: Our in-house nested PCR system therefore can diagnose the accurate pathogen causing bacterial meningitis and therefore be useful in selecting a specific treatment line to minimize morbidity. Results are obtained within 24 h and high sensitivity makes this nested PCR assay a rapid and accurate diagnostic tool compared to traditional culture-based methods.

Diagnosis of tuberculosis in an Indian population by an indirect ELISA protocol based on detection of Antigen 85 complex: a prospective cohort study.

BACKGROUND: Diagnosis of tuberculosis (TB) remains problematic despite many new advanced diagnostic methods. A reliable and rapid diagnostic test, which could be performed in any standard pathology laboratory, would help to obtain definitive early diagnoses of TB. In the present study we describe a prospective evaluation for demonstrating Antigen (Ag) 85 complex in the sera from TB patients. METHODS: Indirect ELISA, employing monoclonal antibodies (mAb) against the purified Ag 85 complex, was used to demonstrate Ag 85 complex in sera from TB patients. Serum samples were obtained from 197 different groups of patients: confirmed TB {n = 24}, clinically diagnosed TB {n = 104}, disease controls {n = 49} and healthy controls {n = 20}. Receiver operating curve (ROC) was used to calculate the cut off value and comparison between TB and non-TB groups were done by the chi-square test. RESULTS: The indirect ELISA method, using an mAb against Ag 85 complex, yielded 82% sensitivity (95% confidence interval [CI] 67 to 93%) and 86% specificity (95% CI, 57 to 98%) for the diagnosis of TB. The serum positivities for Ag 85 complex in cases of confirmed and clinically diagnosed TB patients were 96% (23/24) and 79% (82/104) respectively, while the positivity for patients in the non-tuberculosis group was 14% (10/69). CONCLUSION: The detection of Ag 85 complex in sera from TB patients by indirect ELISA using mAb against purified Ag 85 complex gives a reliable diagnosis and can be used to develop an immunodiagnostic assay with increased sensitivity and specificity.