Browsing by Author "Odoyo, Erick"

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    Development of Two Multiplex PCR Assays for Rapid Detection of Eleven Gram-Negative Bacteria in Children with Septicemia
    (Tropical Medicine and Health, 2024) Miringu, Gabriel; Musyoki, Abednego; Muriithi, Betty; Wandera, Ernest; Waithiru, Dan; Odoyo, Erick; Shoji, Hisashi; Menza, Nelson; Ichinose, Yoshio
    Aim This study aimed to develop a multiplex PCR assay for simultaneous detection of major Gram-negative etiologies of septicemia and evaluate its performance. Methods Multiplex PCR (mPCR) assays were developed targeting 11 bacterial strains. Species-specifc primers were confrmed using known clinical isolates and standard strains. Gradient PCR was performed on each primer against its target bacterial gene to determine its optimal amplifcation condition. The minimum detectable DNA concentration of the two assays was evaluated by adjusting bacterial DNA concentration to 100 ng/μL and, tenfold serially diluting it up to 10 pg/μL with DNAse-free water. The diagnostic accuracy of mPCR assays was established by subjecting the assays to 60 clinical blood samples. Results Two mPCR assays were developed. Optimal primer annealing temperature of 55 °C was established and utilized in the fnal amplifcation conditions. The assays detected all targeted bacteria, with a 100 pg minimum detectable DNA concentration. Pathogens were not detected directly from whole blood, but after 4 h and 8 h of incubation, 41% (5/12) and 100% (12/12) of the bacteria were detected in culture fuids, respectively. The assays also identifed Salmonella spp. and Klebsiella pneumoniae co-infections and extra pathogens (1 E. coli and 2 K. pneumoniae) compared with culture. The sensitivity and specifcity of the mPCR were 100.0% (71.7–100.0) and 98.0% (90.7–99.0), respectively. The area under the ROC curve was 1.00 (1.00–1.00). Conclusions The mPCR assays demonstrated substantial potential as a rapid tool for septicemia diagnosis alongside the traditional blood culture method. Notably, it was able to identify additional isolates, detect co-infections, and efciently detect low bacterial DNA loads with high sensitivity, implying its value in enhancing efciency of diagnosis of septicemia