Journal of Microbiology

Research Support, Non-U.S. Gov't

Reliability of Non-Culturable Virus Monitoring by PCR-Based Detection Methods in Environmental Waters Containing Various Concentrations of Target RNA: Eung Seo Koo , Chang-Hoon Yoo , Youjin Na , Soo Young Park , Hey Rhyoung Lyoo , Yong Seok Jeong; J. Microbiol. 2012;50(5):726-734. Published online November 4, 2012; DOI: https://doi.org/10.1007/s12275-012-2279-y

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Owing to the lack of practical cell culture system for human noroviruses (HuNoV), various detection methods based on conventional reverse transcription-PCR (RT-PCR) and the quantitative real-time PCR have been major tools for monitoring environmental water safety. In this study, we showed that the proportion of water sample concentrates used for one-step RT-PCR significantly influences false-negative findings of the non-culturable viruses. In total, 59 archived samples of previously analyzed water concentrates were reexamined for HuNoV RNA by the one-step RT-PCR and semi-nested PCR. Using new aliquots for RNA extraction for every trial, up to 20 PCR trials were performed for each archive to determine whether the crosscheck results supported the previous determinations. We reconfirmed that 27.6% (8/29) of the samples were HuNoV-positive samples: 6.7% (1/15) from groundwater, 33.3% (3/9) from river water, and 80% (4/5) from treated sewage effluent (TSE). These results corresponded to the ratio of previously negative HuNoV samples now identified as positive (8/30): 6.7% (1/15) from groundwater, 20% (1/5) from river water, and 60% (6/10) from TSE. To elucidate the cause of these results, 16 different concentrations of murine norovirus (MNV) RNA (from 2×102 to 8×103 copies, divided into 10 tubes for each concentration) were subjected to one-step RT-PCR. The detection frequency and reproducibility decreased sharply when the number of MNV RNA copies fell below threshold levels. These observations suggest that the proportion of water concentrate used for PCR-based detection should be considered carefully when deciding viral presence in certain types of environmental water, particularly in regard with legal controls.

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Article

Evaluation of Negative Results of BacT/Alert 3D Automated Blood Culture System: M. Esra Kocoglu , Aysen Bayram , Iclal Balcl; J. Microbiol. 2005;43(3):257-259.; DOI: https://doi.org/2216 [pii]

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Abstract PDF: Although automated continuous-monitoring blood culture systems are both rapid and sensitive, false-positive and false-negative results still occur. The objective of this study, then, was to evaluate negative results occurring with BacT/Alert 3D blood culture systems. A total of 1032 samples were cultured with the BacT/Alert 3D automated blood culture system, using both aerobic (BPA) and anaerobic (BPN) media, and 128 of these samples yielded positive results. A total of 904 negative blood samples were then subcultured in 5% sheep blood agar, eosin methylene blue, chocolate agar, and sabouraud-dextrose agar. Organisms growing on these subcultures were subsequently identified using both Vitek32 (bioMerieux, Durham, NC) and conventional methods. Twenty four (2.6%) of the 904 subcultures grew on the subculture media. The majority (83.3%) of these were determined to be gram-positive microorganisms. Fourteen (58.3%) were coagulase-negative staphylococci, two (8.3%) were Bacillus spp., one (4.2%) was Staphylococcus aureus, and one (4.2%) was identified as Enterococcus faecium. Streptococcus pneumoniae and Neisseria spp. were isolated together in two (8.3%) vials. Gram-negative microorganisms comprised 12.5% of the subcultures, of which two (8.3%) were found to be Pseudomonas aeruginosa, and one (4.2%) was Pseudomonas fluorescens. The other isolate (4.2%) was identified as Candida albicans. We conclude that the subculture of negative results is valuable in the BacT/Alert 3D system, especially in situations in which only one set of blood cultures is taken.

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