Experience with the quantitative lytA gene real-time polymerase chain reaction for the detection of Streptococcus pneumoniae from pediatric whole blood in Pakistan

Background: We present our experience with optimization and diagnostic use of quantitative real-time polymerase chain reaction (PCR) targeting the lytA gene of Streptococcus pneumoniae for the detection of S. pneumoniae in whole blood of children <5 years of age. The assay was optimized to detect ≥5 CFU/10 μl or 1 copy of DNA/2 μl of blood. Methods: This assay was applied on 1912 whole blood specimens collected from children <5 years of age with pneumonia, of which 35 specimens were lytA positive. The bacterial loads were determined through categorization of load into five different categories, i.e., very high load, high load, moderate load, low load, and very low load. Results: Of the 35 lytA-positive samples, 9 (25.71%), 4 (11.42%), 1 (2.85%), 13 (37.14%), and 8 (22.85%) were categorized as very high load, high load, moderate load, low load, and very low load, respectively. Extracted samples were also subjected to serotyping by the Centers for Disease Control and Prevention PCR scheme. Positive samples with very high load and high load category were serotyped successfully in all instances. A high proportion of samples with low and very low load (61.53% and 75%, respectively) remained untypeable by the currently proposed schemes. Conclusions: LytA PCR assay in whole blood provides rapid and sensitive results for the diagnosis of invasive S. pneumoniae disease in a resource-limited setting, while also being amenable to quantitation and serotyping.