Comparison of next-generation droplet digital PCR with quantitative PCR for enumeration of Naegleria fowleri in environmental water and clinical samples.

Naegleria fowleri in recreational waters is a serious health threat. A rapid and accurate method to determine this pathogen in water is vital to develop effective control strategies. In this study, we compared two molecular methods: droplet digital polymerase chain reaction (ddPCR) and quantitative PCR (qPCR) assays in identifying N. fowleri from clinical and environmental samples. Strong agreement between ddPCR and qPCR methods over clinical DNA samples was observed. The limit of detection (LOD) for ddPCR and qPCR assays were 2·5 and 25 N. fowleri per reaction respectively. In the comparative analysis using N. fowleri genomic DNA, quantitative results obtained from ddPCR and qPCR assays showed no significant difference. The assay specificity for ddPCR and qPCR assays were 100 and 86% respectively. Results from both PCR assays indicated N. fowleri was present in surface water samples from Lake Pontchartrain during our study period. In general, the ddPCR performance demonstrated in this study on clinical and environmental samples lead to greater confidence of ddPCR technology on field application. For precise quantification using qPCR, we recommend using ddPCR to quantify the standard materials before qPCR application. SIGNIFICANCE AND IMPACT OF THE STUDY: This study explored the application of ddPCR and qPCR methods in identifying Naegleria fowleri from both clinical and environmental water samples. Strong agreement between ddPCR and qPCR methods over clinical DNA samples was observed. Naegleria fowleri was present in surface water samples from Lake Pontchartrain during our study period. The ability of N. fowleri to survive in brackish water is therefore a potential risk factor for people who engage in water-related recreational activities. The ddPCR performance demonstrated in this study on clinical and environmental samples lead to greater confidence of ddPCR technology on field application.

Impact of disinfectant wipes on the risk of Campylobacter jejuni infection during raw chicken preparation in domestic kitchens.

AIMS: In the present study, we conducted a quantitative microbial risk assessment forecasting the exposure to Campylobacter jejuni contaminated surfaces during preparation of chicken fillets and how using a disinfectant-wipe intervention to clean a contaminated work area decreases the risk of infection following the preparation of raw chicken fillet in a domestic kitchen. METHODS AND RESULTS: Using a Monte Carlo simulation of the risk of transferring Camp. jejuni strain A3249, from various surfaces to hands and subsequently transferring it to the mouth was forecasted. The use of a disinfectant-wipe intervention to disinfect contaminated surface area was also assessed. Several assumptions were used as input parameters in the classical Beta-Poisson model to determine the risk of infection. The disinfectant-wipe intervention reduced the risk of Camp. jejuni infection by 2-3 orders on all fomites. CONCLUSIONS: The use of disinfectant wipes after the preparation of raw chicken meat reduces the risk of Camp. jejuni infections. SIGNIFICANCE AND IMPACT OF THE STUDY: This risk assessment shows that the use of disinfectant wipes to decontaminate surface areas after chicken preparation reduces the annual risk of Camp. jejuni infections up to 99·2%, reducing the risk from 2 : 10 to 2 : 1000.

The presence of atrazine and atrazine-degrading bacteria in the residential, cattle farming, forested and golf course regions of Lake Oconee.

AIMS: To assess the concentration of atrazine in Lake Oconee and develop a qPCR assay as a potential marker for the presence of atrazine-degrading bacteria indicating atrazine contamination. METHODS AND RESULTS: Water and sediment samples were collected from the Oconee Lake at four golf course sites, two residential sites, one cattle farming site and a forested site. Atrazine concentration at the study sites was determined using an ELISA kit and indicated the presence of atrazine from 0·72 ppb at the forested sites to 1·84 ppb at the golf course sites. QPCR results indicate the presence of atzA gene (atrazine chlorohydrolase) from 1·51 × 10(2) gene copies at the residential sites to 3·31 × 10(5) gene copies per 100 ml of water at the golf course regions of the lake and correlated (r = 0·64) with atrazine concentration. Sediment samples had higher atzA gene copies compared with the water samples (P < 0·05). CONCLUSIONS: Atrazine concentration and the highest quantity of atzA gene were detected in the golf course regions of the lake. Overall, atrazine concentration monitored in Lake Oconee was below the Environment Protection Agency (EPA) regulatory standards. SIGNIFICANCE AND IMPACT OF THE STUDY: Quantitative PCR is an efficient technique for assessing the presence of atrazine catabolism gene as a functional marker for atrazine-degrading bacteria and the presence of atrazine contamination.