Detection ofHaemophilus influenzaeandStreptococcus pneumoniae DNA in Blood Culture by a Single PCR Assay

An Haemophilus influenzae type b conjugate vaccine trial was recently concluded in The Gambia. One of the main end- points of the trial was the incidence of H. influenzae type b pneumonia in children enrolled in the study. Children were assessed for pneumonia by using the World Health Organiza- tion criteria: cough or difficult breathing and a respiration rate of 50 breaths per minute or more in children ages 2 to 11 months or 40 breaths per minute or more in children ages 12 to59months.However,thechildrenenrolledinthestudywere seen by physicians whenever they were ill, and blood was cul- tured for conditions other than pneumonia. Therefore, the blood cultures tested in the present study were not exclusively for suspected cases of pneumonia. PCR was introduced into the study as a sensitive means of detection of H. influenzae type b. It was envisaged that the technique would be able to detect a few more cases of H. influenzaetype b infection than culture, thereby increasing the power of the detection method in the trial for the pneumonia endpoint. Previous studies in The Gambia have shown thatH. influenzae type b and Streptococcus pneumoniae are the two organismsmostlikelytobeisolatedfromclinicalsamplesfrom children with suspected pneumoniae (2, 3, 5). It was therefore decided to screen blood cultures from the trial for both H. influenzae type b and S. pneumoniae. In this way it was rea- soned that the vaccination trial would provide a forum for testing not only the sensitivity of PCR but also the robustness of the technique in this environment. It was initially intended that buffy coat DNA obtained from EDTA-treated samples would be used for PCR analysis in the vaccinationtrialstudy.Thefeasibilityofthisapproachwasfirst investigated by comparing the PCR results obtained by using DNA extracted from EDTA-treated blood and blood culture samples obtained from 8 patients whose culture results were positive for H. influenzae type b and from 15 patients whose culture results were positive forS. pneumoniae. Prior to DNA extraction, erythrocytes were removed from samples by dex- tran sedimentation to eliminate a possible inhibitory effect by heme compounds on the PCR. Pellets obtained from superna- tants devoid of erythrocytes were used for DNA extraction (4). Separate PCR assays were used to detectH. influenzaetype b