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Detection of Haemophilus influenzae in cerebrospinal fluids by polymerase chain reaction DNA amplification
R J van Ketel1, B de Wever, L van Alphen
1Department of Medical Microbiology, Academic Medical Center, Amsterdam, The Netherlands.
Abstract:
Two primer sets were chosen for the detection of Haemophilus influenzae in cerebrospinal fluid by polymerase chain reaction (PCR) DNA amplification. One primer set was selected from sequences encoding a capsulation-associated protein and reacted with target DNA from all 15 capsulate H. influenzae strains (all serotypes) examined. The other primer set was selected from the DNA sequence of a gene encoding for outer-membrane protein P6 and reacted with the 15 capsulate and 10 non-capsulate strains of H. influenzae tested. This primer set also reacted with the closely related species H. haemolyticus and H. aegyptius, and with two of nine H. parainfluenzae strains. In reconstruction experiments, PCR DNA amplification was able to detect as few as five H. influenzae cells when 40 cycles of amplification were used. Two hundred cerebrospinal fluid (CSF) samples collected consecutively from patients suffering from meningitis were investigated by PCR; 40 were culture-positive for H. influenzae and 39 of these were also clearly positive in the PCR test with both primer sets. Contamination occurred to some extent with 40 cycles of amplification but was completely eliminated when the number of cycles was reduced to 35. We conclude that the two primer sets are appropriate for the detection of H. influenzae by PCR, each having its own specificity. When these two primer sets are used, PCR is a technique of equivalent sensitivity to culture for the detection of H. influenzae in CSF.
Insights
Polymerase chain reaction (PCR) effectively detects Haemophilus influenzae in cerebrospinal fluid. Two primer sets demonstrated high sensitivity and specificity, comparable to traditional culture methods for diagnosing meningitis.
Area of Science:
- Microbiology
- Molecular Biology
- Clinical Diagnostics
Background:
- Haemophilus influenzae is a significant cause of bacterial meningitis, particularly in children.
- Accurate and rapid diagnostic methods are crucial for timely treatment and improved patient outcomes.
Purpose of the Study:
- To evaluate the efficacy of two distinct primer sets for detecting Haemophilus influenzae in cerebrospinal fluid using polymerase chain reaction (PCR).
- To compare the sensitivity and specificity of PCR with conventional culture methods for H. influenzae detection.
Main Methods:
- Development and testing of two primer sets targeting specific H. influenzae genes (capsulation-associated protein and outer-membrane protein P6).
- Application of PCR for DNA amplification in cerebrospinal fluid samples from meningitis patients.
- Reconstruction experiments to determine the limit of detection for PCR.
- Comparison of PCR results with bacterial culture results.
Main Results:
- One primer set detected all 15 capsulate H. influenzae strains, while the other detected both capsulate and non-capsulate strains, with some cross-reactivity with related species.
- PCR could detect as few as five H. influenzae cells per sample.
- PCR correctly identified 39 out of 40 culture-positive cerebrospinal fluid samples.
- Optimized PCR protocols (35 cycles) eliminated contamination issues observed at 40 cycles.
Conclusions:
- The evaluated primer sets are suitable for detecting H. influenzae in cerebrospinal fluid via PCR, offering distinct specificity profiles.
- PCR demonstrates sensitivity equivalent to culture for H. influenzae detection in cerebrospinal fluid samples.
- PCR offers a rapid and sensitive alternative for diagnosing H. influenzae meningitis.