Prospective study to determine clinical relevance of detection of pneumococcal DNA in sera of children by PCR

Abstract

We undertook a prospective study to evaluate the accuracy of PCR of serum (aimed at the pneumococcal pneumolysin gene) at detecting pneumococcal infections in infants and children. The assay was positive for all blood and cerebrospinal fluid culture-positive samples and for 38 and 44% of patients with lobar pneumonia and acute otitis media, respectively. It was positive for 17% of healthy controls. There was a marked effect of age on the rate of positivity among healthy controls, with the highest rate (33%) being in 2-year-old children, the age group with the highest rate of nasopharyngeal (NP) carriage; the lowest rate was found among infants <2 months of age (13%) and adults ages 18 to 50 years (0%), age groups with the lowest NP pneumococcal carriage rates. Carriers of pneumococci in the nasopharynges had a higher rate of positivity than noncarriers of pneumococci in the nasopharynges for all groups. Our results suggest that although PCR of serum is a sensitive test for the detection of Streptococcus pneumoniae in sterile fluids, its high rate of positivity for healthy controls, related to NP pneumococcal carriage, might exclude it from being useful in detecting deep-seated pneumococcal infections.

FIG. 1

Sensitivity and specificity of the PCR assay. (A) Specificity of the PCR-based assay. PCR tests were conducted with 25 pg of purified DNA from the organisms. Purified S. pneumoniae DNA was used for the positive control. Lanes: 1, molecular size marker; 2, S. pneumoniae; 3, Staphylococcus aureus; 4, Streptococcus mitis; 5, group A Streptococcus; 6, Escherichia coli; 7, Enterococcus faecalis; 8, Pseudomonas aeruginosa; 9, Salmonella group D; 10, H. influenzae; 11, Moraxella catarrhalis. (B) Agarose gel electrophoresis of PCR-amplified products from a 10-fold dilution of purified S. pneumoniae DNA. Lane 1, molecular size marker; lanes 2 to 8, dilution series from 10 ng to 10 fg of DNA (corresponding to 10⁶ to 1 CFU/ml); lane 9, negative control. (C) Agarose gel electrophoresis of PCR-amplified products from culture-positive CSF or blood before and after antibiotic treatment. Lane 1, molecular size marker; lanes 2 to 5, samples from culture-positive CSF and blood before (lanes 2 and 4, respectively) and after (lanes 3 and 5, respectively) antibiotic treatment for 48 h; lane 6, positive control (S. pneumoniae DNA); lane 7, negative control (H₂O).

FIG. 2

Agarose gel electrophoresis of PCR-amplified products from sera. Lane 1, molecular size marker; lanes 2 to 4 and 6 to 8, samples from healthy controls; lanes 5 and 9, samples from blood culture-positive patients; lane 10, negative control; lane 11, positive control.

FIG. 3

PCR results for serum samples from 202 healthy controls, by age.

FIG. 4

Age relation of the proportion of positive serum samples by PCR in relation to NP pneumococcal carriage rate among healthy infants and children in the community younger than age 30 months. The data for the NP carriage rate in the community were derived from a previous report (23).