Detection of Haemophilus influenzae in cerebrospinal fluids by polymerase chain reaction DNA amplification.
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.
van Ketel
R J
RJ
Department of Medical Microbiology, Academic Medical Center, Amsterdam, The Netherlands.
de Wever
B
B
van Alphen
L
L
D001483:Base Sequence; D004269:DNA, Bacterial / Q000134:cerebrospinal fluid*; D006193:Haemophilus influenzae / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008583:Meningitis, Haemophilus / Q000134:cerebrospinal fluid / Q000175:diagnosis* / Q000382:microbiology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D004269:DNA, Bacterial