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Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction
Published on: November 1, 2018
Real-time qPCR improves meningitis pathogen detection in invasive bacterial-vaccine preventable disease surveillance
Eileen M Dunne1, Silivia Mantanitobua2, Shalini P Singh2
1Murdoch Childrens Research Institute, Infection and Immunity, Parkville, 3052, Australia.
Abstract:
As part of the World Health Organization Invasive Bacterial-Vaccine Preventable Diseases (IB-VPD) surveillance in Suva, Fiji, cerebrospinal fluid (CSF) samples from suspected meningitis patients of all ages were examined by traditional methods (culture, Gram stain, and latex agglutination for bacterial antigen) and qPCR for Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae. Of 266 samples tested, pathogens were identified in 47 (17.7%). S. pneumoniae was the most common pathogen detected (n = 17) followed by N. meningitidis (n = 13). The use of qPCR significantly increased detection of IB-VPD pathogens (P = 0.0001): of 35 samples that were qPCR positive for S. pneumoniae, N. meningitidis, and H. influenzae, only 10 were culture positive. This was particularly relevant for N. meningitidis, as only 1/13 cases was culture positive. Molecular serotyping by microarray was used to determine pneumococcal serotypes from 9 of 16 (56%) of samples using DNA directly extracted from CSF specimens. Results indicate that qPCR significantly increases detection of S. pneumoniae, N. meningitidis, and H. influenzae in CSF, and that application of molecular diagnostics is a feasible way to enhance local and global surveillance for IB-VPD.
Insights
Quantitative PCR (qPCR) significantly enhances the detection of invasive bacterial-vaccine preventable diseases (IB-VPD) pathogens like Streptococcus pneumoniae and Neisseria meningitidis in cerebrospinal fluid samples. This molecular method improves surveillance for meningitis in Fiji.
Area of Science:
- Microbiology
- Infectious Diseases
- Molecular Diagnostics
Background:
- The World Health Organization (WHO) conducts Invasive Bacterial-Vaccine Preventable Diseases (IB-VPD) surveillance.
- Meningitis surveillance is crucial for public health, particularly in resource-limited settings.
Purpose of the Study:
- To evaluate the utility of quantitative PCR (qPCR) in detecting key IB-VPD pathogens in cerebrospinal fluid (CSF) samples.
- To compare the diagnostic yield of qPCR with traditional methods for meningitis diagnosis.
Main Methods:
- Cerebrospinal fluid samples from suspected meningitis patients were analyzed using culture, Gram stain, latex agglutination, and qPCR.
- qPCR targeted Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae.
- Molecular serotyping by microarray was employed for pneumococcal serotypes.
Main Results:
- Pathogens were identified in 47 (17.7%) of 266 CSF samples.
- Streptococcus pneumoniae (n=17) and Neisseria meningitidis (n=13) were the most frequently detected pathogens.
- qPCR significantly increased pathogen detection (P=0.0001), identifying 35 positive samples where only 10 were culture-positive. Notably, only 1 of 13 N. meningitidis cases was culture-positive.
Conclusions:
- Quantitative PCR substantially enhances the detection of S. pneumoniae, N. meningitidis, and H. influenzae in CSF samples.
- Molecular diagnostics, including qPCR and microarray-based serotyping, offer a feasible approach to improve local and global IB-VPD surveillance.
- The findings highlight the limitations of traditional culture methods for certain bacterial meningitis pathogens.
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