Likelihood Of Cross-contamination Research Articles

A highly sensitive 1-tube nested real-time RT-PCR assay using LNA-modified primers for detection of respiratory syncytial virus

Respiratory syncytial virus (RSV) causes serious respiratory tract infection worldwide. The relatively low RSV load makes it difficult to detect in frail, elderly, and severely immune-compromised patients. In the present study, we developed a locked nucleic acid–-based 1-tube nested real-time RT-PCR (OTNRT-PCR) assay with the advantages of extremely high sensitivity, facile operability, and less likelihood of cross-contamination. The sensitivity, specificity, and clinical performance of the OTNRT-PCR assay were compared in parallel with a conventional TaqMan probe-based real-time PCR (qRT-PCR) assay and a traditional 2-step nested RT-PCR assay. The limit of detection of the OTNRT-PCR assay was 1.02 × 10−1 TCID50/mL, equivalent to the traditional 2-step nested RT-PCR assay and 25-fold lower than the qRT-PCR assay. Of 616 nasopharyngeal aspirates tested, 143 RSV-negative samples by qRT-PCR were confirmed as positive by sequencing the OTNRT-PCR products. We therefore conclude that OTNRT-PCR is more sensitive than qRT-PCR for detection of RSV in clinical samples.

THE SPREAD OF FOOT INFECTION AND ITS IMPACT ON THE OUTCOMES OF MAJOR AMPUTATIONS OF LOWER EXTREMITIES IN DIABETIC PATIENTS

Aim To assess the spread of foot infection and its impact on the outcomes of major amputations of lower extremities in diabetic patients. Method In a multicentre retrospective and prospective cohort study, we included adult diabetic patients (≥ 18 years) who underwent a major amputation of a lower limb in 5 hospitals between 2000 and 2009, 2012 and 2014. A total of 51 patients were included (of which 27 (52.94%) were men and 24 (47.06%) were women) with the mean age of 65.51 years (SD=16.99). Concomitant section9s osseous slice biopsy (BA) and percutaneous bone biopsy of the distal site (BD) were performed during limb amputation. A new surgical set-up and new instruments were used to try and reduce the likelihood of cross-contamination during surgery. A positive culture was defined as the identification of at least 1 species of bacteria not belonging to the skin flora or at least 2 bacteria belonging to the skin flora (CoNS (coagulase negative staphylococci), Corynebacterium spp, Propionibacterium acnes ) with the same antibiotic susceptibility profiles. A doubtful culture was defined as the identification of 1 species of bacteria belonging to the skin flora. The patients were followed-up for 1 year. Stump outcomes were assessed on the delay of complete healing, equipment, need of re-intervention and antibiotics. Results In total, 51 BA were performed during major lower limb amputations (17 above the knee and 34 below the knee) in diabetic patients. Nine (17.65%) bacterial culture results from BA specimens were positive, 7 (13.73%) doubtful and 35 (68.63%) sterile. Before amputation, 23 patients (45.1%) had not received any antibiotics, including 16 (31.37%) with an antibiotic-free interval of 15 days or more. Microorganisms identified in BA were also cultured from the distal site in 33.33% of the cases. Positive BA was associated with prolonged complete stump healing, delay of complete healing (more than 6 months), re-amputation and the need of antibiotics Conclusions The microorganisms identified from BA play a role in stump healing in diabetic patients. BA is useful during major limb amputation due to infectious complications and antibiotic therapy could be corrected on the basis of the BA culture results.

Microbiological quality of typical foods served in Thai restaurants in Vienna, Austria

The microbiological quality, pH and serving size of typical meals served in 18 Thai restaurants in Vienna were studied. During the summer and winter season, one ready-to-eat food portion was sampled per restaurant and per food category (A: raw or insufficient heat treatment; B: heat treated main dish garnished/mixed with raw side-dishes; C: thorough heat treatment, served alone; total number = 108). In addition, ― where possible ― some typical side dishes were sampled (n = 25). The average weight of main dishes varied dependent on category from 360―510 g. The lowest pH was observed in group A (average 4.5), most probably because of the addition of acids via lime juice. Expectedly, group A had significantly higher total aerobic counts (6.1 ± 0.8 log cfu/g) than group B (4.5 ± 1.7 log cfu/g) and C (2.6 ± 0.9 log cfu/g). In side-dishes, average total aerobic counts were higher in raw vegetables (4.9 log cfu/g) than in rice (2.6 log cfu/g). With respect to hygiene indicator bacteria, there was a clear association between presumably insufficient heat treatment and/or the likelihood of cross-contamination (groups A B) on the one hand and the presence of E. coli and Enterobacteriaceae on the other. All 133 samples tested negative for Salmonella spp., Listeria monocytogenes and Campylobacter spp. (25 g aliquots). In groups A and B the warning limits for total aerobic count and Enterobacteriaceae established by the German Society for Microbiology and Hygiene were exceeded in 30-60 % of the samples. For food classification we used an empirical scheme taking into account heat treatment and the likelihood of cross-contamination, assuming that these two factors would be associated with the risk of presence of pathogenic bacteria in the ready-to-eat food portion. This empirical classification was well reflected in total aerobic counts. As regards pathogens, the usefulness of such a classification scheme could not be evaluated due to the low frequency of pathogens. This indicates that on this issue further studies should be conducted.

An outbreak of Campylobacter jejuni associated with consumption of chicken, Copenhagen, 2005.

In May/June 2005 an outbreak of diarrhoeal illness occurred among company employees in Copenhagen. Cases were reported from seven of eight companies that received food from the same catering kitchen. Stool specimens from three patients from two companies were positive for Campylobacter jejuni. We performed a retrospective cohort study among employees exposed to canteen food in the three largest companies to identify the source of the outbreak and to prevent further spread. Using self-administered questionnaires we collected information on disease, days of canteen food eaten and food items consumed. The catering kitchen was inspected and food samples were taken. Questionnaires were returned by 295/348 (85%) employees. Of 247 employees who ate canteen food, 79 were cases, and the attack rate (AR) was 32%. Consuming canteen food on 25 May was associated with illness (AR 75/204, RR=3.2, 95%CI 1.3-8.2). Consumption of chicken salad on this day, but not other types of food, was associated with illness (AR=43/97, RR=2.3, 95%CI 1.3-4.1). Interviews with kitchen staff indicated the likelihood of cross-contamination from raw chicken to the chicken salad during storage. This is the first recognised major Campylobacter outbreak associated with contaminated chicken documented in Denmark. It is plausible that food handling practices contributed to transmission, and awareness of safe food handling and storage has since been raised among kitchen staff. The low number of positive specimens accrued in this outbreak suggests a general underascertainment of adult cases in the laboratory reporting system by a factor of 20.

Ground-Applied Insecticide Rescue Treatments in Whorl-Stage Corn to Control First Generation European Corn Borer Larvae, 1995

Abstract An irrigated corn field near Delmont, SD, was chosen to evaluate granular and liquid insecticide treatments for management of first-generation ECB, larvae. Preapplication counts indicated that the site had a severe larval infestation (shotholing damage was observed in >90% of surveyed plants). Stadial composition of the population was 18, 39, 32, and 11 % 1st-, 2nd-, 3rd-, and 4th-instar larvae, respectively. Two untreated control plots were used to compare 6 granular insecticide formulations and 9 liquid treatments for efficacy in preventing larval tunneling damage. The study was arranged in a RCBD with 4 replications. Individual treatment plots were single, 30.5-m rows spaced 76 cm apart. All treatment rows were buffered on either side by an untreated row to minimize the likelihood of cross-contamination between plots due to insecticide drift. Treatments were applied on 6 Jul at whorl (approximately 1 wk prior to tasseling) stage of development. Granules were directed into plant whorls using a pneumatic applicator powered by a 3.5 hp engine mounted on a Highboy. Noble metering units attached to a modified chain-driven apparatus regulated the amount of granules released into the applicator. Liquid treatments were applied to whorls using a single-nozzle (TeeJet 8003 flat fan) CO2-propelled sprayer system mounted on the Highboy. Approximately 5 wk following insecticide applications (15 Aug), 20 corn plants were randomly chosen from each plot. Each plant was split vertically and examined for ECB tunneling damage. Cavity number and length data were analyzed with SAS’s General Linear Models procedure and means were compared using DMRT.

Solid phase capture method for the specific amplification of microbial nucleic acids—avoidance of false-positive and false-negative reactions

DNA amplification assays such as the polymerase chain reaction are being developed for the amplification of small quantities of microbial nucleic acids. These assays offer the potential for a great deal of sensitivity. However, the high level of sensitivity increases the likelihood of cross-contamination of amplified products and the generation of false-positive reactions. In addition, substances in body fluids can inhibit the efficient performance of the amplification reactions. We have developed an assay format in which microbial nucleic acids are specifically bound to a solid phase surface. Contaminating DNA and enzyme inhibitors present in the sample are removed by washing prior to the performance of the amplification reaction. We could use this system to amplify and detect small amounts of HIV DNA diluted in whole blood. The assay system could distinguish target DNA from contaminating DNA fragments generated by prior amplification reactions.