Pneumococcal DNA is not detectable in the blood of healthy carrier children by real-time PCR targeting the lytA gene

Abstract

The diagnosis of invasive pneumococcal disease (IPD) is currently based on culture methods, which lack sensitivity, especially after antibiotic therapy. Molecular methods have improved sensitivity and do not require viable bacteria; however, their use is complicated by reports of low specificity with some assays. The present study investigated the specificity of a real-time PCR targeting lytA for the detection of IPD. A group of 147 healthy children, aged 6 months to 16 years (mean 6.4 years, median 4.9 years, interquartile range 6.4 years), who were in hospital for routine examinations, were tested for pneumococcal carrier status and for the presence of detectable pneumococcal DNA in their blood by real-time PCR targeting the pneumococcal lytA gene. In addition, 35 culture-positive biological samples were analysed. Urine was examined for the presence of pneumococcal DNA and C-polysaccharide antigen. Carriage was detected in 77 of the 147 subjects (52.4 %); however, regardless of carrier status, none of the subjects had a positive result from blood. Analysis of the culture-positive biological samples yielded positive results in 100 % (15/15) of cerebrospinal fluid samples and 95 % (19/20) of blood samples. All urine samples from healthy carriers were negative for DNA, whilst antigenuria was detected in 44/77 carriers (57.1 %). In conclusion, real-time PCR is both sensitive and specific and can be a useful tool in the routine diagnosis of IPD. Its sensitivity, which surpasses that of other methods for this purpose, does not come at the cost of reduced specificity.

Detection of Pneumococcal DNA in Blood by Polymerase Chain Reaction for Diagnosing Pneumococcal Pneumonia in Young Children From Low- and Middle-Income Countries

“…Controls in those studies were not a random selection from the community but were attending hospital for allergies or celiac disease [4] or had previously been vaccinated with PCV-9 in a clinical trial [10], were older (mean age of approximately 5 years), had lower prevalence (50%) [4, 10] and density [4] of nasopharyngeal pneumococcal carriage, and possibly had higher socioeconomic status. The South African study also used serum rather than whole blood [10].…”

Association between pneumococcal load and disease severity in adults with pneumonia

“…Qualitative PCR has been useful for detection of pneumococcal DNA in blood specimens, especially from children (Azzari et al, 2011;Marchese et al, 2011), but detection in respiratory specimens has been difficult to interpret because of the inability to differentiate colonization from infection (Dagan et al, 1998;Murdoch et al, 2003). The choice of PCR assay can have a noticeable A. M. Werno, T. P. Anderson and D. R. Murdoch effect on results.…”

Burden of Pneumococcal Disease in Northern Togo before the Introduction of Pneumococcal Conjugate Vaccine

“…Real-time PCR on whole blood allowed identification of additional possible Sp cases: in young children, culture and PCR together detected 8 times more cases than culture alone. Recent data from the PERCH study suggest that lytA PCR may have limited specificity (personal communication, MD Knoll): in several PERCH sites, DNA-emia was seen in healthy controls, which could be explained by the transfer of genetic material from the nasopharynx to the blood in colonized children; however, these results run counter to earlier analyses showing high specificity for blood PCR [39,40]. Elsewhere, we reported findings from latent class analysis models combining laboratory and radiological data from our patients (Blake et al, accepted for publication): we showed that standardized CXR interpretation was the best approach to diagnosing Sp pneumonia and estimated that one third of pneumonia cases were attributable to pneumococcus, a proportion that increased with age from 20 to 50% and was consistent with clinical trials and observational data [5].…”