Search

Abstract 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.

Background Pseudomonads are gram negative bacteria and readily form biofilms in the environment, allowing long-term colonization and persistence in sinks, water systems. They pose a risk of life-threatening opportunistic infections in immune-compromised individuals. MDR strains, make treatment increasingly difficult. Environmentally persistent MDR strains are typically problematic within healthcare facilities, however, data on MDR pseudomonad reservoirs in settings with community-acquired infections to inform preventive interventions, in resource-constrained settings is scarce. Here, we determined reservoirs and antibiotic susceptibility of Pseudomonas species in water sources in Kisumu County, Kenya with reported high levels of community acquired pseudomonad infections. Methods We adopted a cross-sectional design, randomly collecting 297 samples from tap heads, sinks, tanks, vendor and household storage containers in six selected sub-locations and one hospital (KCRH) . Standard microbiological procedures were used for identification and AST of the isolates. Results We isolated Pseudomonads from 14.1% of the samples collected, predominantly from the community 10.4%. Seven different pseudomonads were identified, with Pseudomonas aeruginosa predominating 6.7% overall, in the community samples 5.7%, and among isolates from water tanks 21.4%. Pseudomonad isolates were 62% non-susceptible to piperacillin, 57% to tigecycline, 24% meropenem, 21% cefepime, 19% levofloxacin and 14% colistin. Carbapenem resistance was mainly detected in P. aeruginosa 80% (8/10) from Milimani sub-location 75% (6/8). 45% of the isolates recovered were MDR, mainly community-associated carbapenem-resistant P. aeruginosa (CRPA) 42%, strains susceptible to colistin. The MDR pseudomonads exhibited high multiple antibiotic resistance indices, ranging from 0.43 to 1. Conclusion This study reveals a higher prevalence of MDR pseudomonads, including CRPA strains in community water sources. These potential conduits of drug resistance present a critical public health threat, especially among immunocompromised. Regular cleaning of water storage facilities, water treatment and implementation of antimicrobial stewardship programs, are required to prevent a rise in AMR and eliminate the environmental reservoirs that put the vulnerable populations at risk.