Rapid and Accurate Diagnosis of Acute Pyogenic Meningitis Due to Streptococcus Pneumoniae, Haemophilus influenzae Type b and Neisseria meningitidis Using A Multiplex PCR Assay

Abstract

Introduction: Acute bacterial meningitis is one of the major causes of morbidity and mortality in children and geriatric population, especially in developing countries. Methods of identification are standard culture and other phenotypic tests in many resource poor settings.

Aim: To use molecular methods for the improvement of aetiological diagnosis of acute pyogenic meningitis in patients.

Materials and methods: CSF samples of 125 patients were included for the study. Gram staining and culture were performed according to standard procedures. Antigen was detected using commercial latex agglutination test kit. Multiplex PCR was performed using previously published primers and protocols. Fischer's exact test was used for finding association between presence of the disease and clinical/biochemical parameters, considering two tailed p<0.05 as statistically significant. Sensitivity, specificity, positive and negative predictive values were calculated using Graphpad QuicCalc software.

Results: A total of 39 cases (31.2%) were confirmed to be of acute pyogenic meningitis based on biochemical methods. Only 10/39 was positive for the three organisms tested. Multiplex PCR was able to detect one additional isolate each of Streptococcus pneumoniae and Haemophilus influenzae type b. When compared with multiplex PCR as the gold standard, culture and latex agglutination tests had same sensitivity (80%), specificity (100%), PPV (100%) and NPV (97.8%), whereas Gram stain had poor sensitivity (40%) and good specificity (95.6%). Detection rates were higher in multiplex PCR for the two organisms Streptococcus pneumoniae and Haemophilus influenzae type b.

Conclusion: Multiplex PCR was more sensitive than culture or antigen detection, and employing this assay can significantly increase the speed and accuracy of identification of the pathogen.

Keywords: Bacterial meningitis; Culture; Gram stain; Latex agglutination test; Multiplex polymerase chain reaction.

[Table/Fig-2]:

Clinical manifestations of the patients with confirmed bacterial meningitis (n=39). Percentage of the patients showing each clinical sign was represented.

[Table/Fig-3]:

Gel electrophoresis of the samples after polymerase chain reaction; Lane 1- 50 bp molecular weight marker, Lane 2- 10 bp molecular weight marker, Lane 3 – Negative control, Lane 4- Positive controls {S. pneumoniae (80 bp), H. influenzae (180 bp) and N. meningitidis (110 bp)}, Lane 5- 12- Samples positive for Streptococcus pneumoniae, Lane 13 – Sample positive for Haemophilus influenzae, Lane 14 –Sample positive for Neisseria meningitidis, Lane 15- Sample negative for all three targeted organisms, Lane 16- 50 bp molecular weight marker.