RESUMO
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-specific primers were confirmed using known clinical isolates and standard strains. Gradient PCR was performed on each primer against its target bacterial gene to determine its optimal amplification 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 final amplification 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 fluids, respectively. The assays also identified Salmonella spp. and Klebsiella pneumoniae co-infections and extra pathogens (1 E. coli and 2 K. pneumoniae) compared with culture. The sensitivity and specificity 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 efficiently detect low bacterial DNA loads with high sensitivity, implying its value in enhancing efficiency of diagnosis of septicemia.
RESUMO
BACKGROUND: Tick- and louse-borne relapsing fever are highly-neglected, vector-borne diseases caused by diverse Borrelia species. Presently, there are no data available on the endemicity of tick- and louse-borne relapsing fever spirochetes in Kenya. Here, we present data of a retrospective study on the seroprevalence of louse-borne relapsing fever (LBRF) in northern Kenya. METHODS: A novel immunoassay, recently established for the diagnosis of LBRF was utilized to screen 2005 blood samples collected from individuals with fever without a source in Turkana County, Kenya between May 2009 and November 2010 for anti-LBRF antibodies. RESULTS: Out of the 2005 sera analyzed, 287 samples (14.3 %) were considered anti-LBRF IgG positive. Subsequent analyses revealed that 87 out of 152 sera randomly selected from these 2005 samples were tested positive (57.2 %) for anti-LBRF IgM antibodies. Most of the IgG and IgM positive samples were from individuals living in northern regions of Turkana County. CONCLUSION: Our serological finding provides strong evidence for the occurrence of LBRF in Kenya.