Determining the 95% limit of detection for waterborne pathogen analyses from primary concentration to qPCR

Stokdyk, Joel P.; Firnstahl, Aaron D.; Spencer, Susan K.; Burch, Tucker R.; Borchardt, Mark A.

doi:10.1016/j.watres.2016.03.026

Cited by 47 publications

(42 citation statements)

References 36 publications

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“…36 As an alternative supporting analysis, the concentration of L. pneumophila was estimated at the theoretical 95 % detection limit of the Legiolert assay (3 most probable number (MPN) in the total volume of water assayed). 37 Because MAC was never detected while flushing showers in the St. Paul building, an upper limit was estimated based on the theoretical 95 % detection limit of the qPCR assay (i.e., 3 copies in the total volume of water analyzed by qPCR). 37 Exponential dose-response models were used for both Legionella health endpoints based on a guinea pig model reported by Armstrong and Haas 38 , which has been validated against human outbreak data 39 and with "uncertain parameter" values as reported in Hamilton et al 32 For MAC health endpoints, exponential dose-response models were used for pulmonary infection and cervical lymphadenitis (i.e., Tomioka model and Jorgensen 1 model, respectively), and an approximate beta-Poisson model for systemic infection (i.e., Yangco model).…”

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confidence: 99%

Flushing of stagnant premise water systems after the COVID-19 shutdown can reduce infection risk by Legionella and Mycobacterium spp

Hozalski

LaPara

Zhao

et al. 2020

Preprint

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The unprecedented widespread closing of buildings due to the COVID-19 pandemic has allowed water to stagnate in premise plumbing systems, creating conditions that may facilitate the growth of opportunistic pathogens. In this study, we flushed and collected samples from showers in buildings that had been unoccupied for approximately two months and quantified Legionella pneumophila using a commercial cultivation-based assay. In addition, all bacteria, Legionella spp., L. pneumophila, L. pneumophila serogroup 1, non-tuberculous mycobacteria (NTM), and Mycobacterium avium complex (MAC) were analyzed using quantitative PCR (qPCR). Despite low or negligible total chlorine in the stagnant pre-flush water samples, L. pneumophila were not detected by either method; Legionella spp., NTM, and MAC, however, were widespread. Using quantitative microbial risk assessment (QMRA), estimated risks of clinical illness from exposure to legionella and MAC via showering were generally low, but the risk of subclinical infection via Legionella spp. could exceed a 10-7 daily risk threshold if just a small fraction (≥0.1 %) of those legionellae detected by qPCR are highly infectious. Flushing cold and hot water lines rapidly restored a total chlorine (as chloramine) residual and decreased all bacterial gene targets to building inlet water levels within 30 min. Following flushing, the chlorine residual rapidly dissipated and bacterial gene targets rebounded, approaching pre-flush concentrations after 6 to 7 days of stagnation. These results suggest that stagnant water in premise plumbing may contain elevated levels of opportunistic pathogens; flushing, however, can rapidly improve water quality and reduce the health risk but the improvement will be short-lived if building disuse persists.

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confidence: 99%

Flushing of stagnant premise water systems after the COVID-19 shutdown can reduce infection risk by Legionella and Mycobacterium spp

Hozalski

LaPara

Zhao

et al. 2020

Preprint

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“…Focusing on DNA-dependent detection methods (e.g., PCR), let us assume that every copy, C, of the target DNA sequence in a sample has a small, fixed probability of causing a positive result in a reaction, φ. The probability of a positive test result can then be described by a "single-hit" model 82,83 :…”

Section: Discussionmentioning

confidence: 99%

Pooled samples and eDNA-based detection can facilitate the “clean trade” of aquatic animals

Brunner

2020

Sci Rep

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the regional and international trade of live animals facilitates the movement, spillover, and emergence of zoonotic and epizootic pathogens around the world. Detecting pathogens in trade is critical for preventing their continued movement and introduction, but screening a sufficient fraction to ensure rare infections are detected is simply infeasible for many taxa and settings because of the vast numbers of animals involved-hundreds of millions of live animals are imported into the U.S.A. alone every year. Batch processing pools of individual samples or using environmental DnA (eDnA)-the genetic material shed into an organism's environment-collected from whole consignments of animals may substantially reduce the time and cost associated with pathogen surveillance. Both approaches, however, lack a framework with which to determine sampling requirements and interpret results. Here I present formulae for pooled individual samples (e.g,. swabs) and eDNA samples collected from finite populations and discuss key assumptions and considerations for their use with a focus on detecting Batrachochytrium salamandrivorans, an emerging pathogen that threatens global salamander diversity. While empirical validation is key, these formulae illustrate the potential for eDnA-based detection in particular to reduce sample sizes and help bring clean trade into reach for a greater number of taxa, places, and contexts.

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“…Utilizing serially diluted purified ISAV, a precise cut‐off Ct value (Ct LoD ) was identified where at least 95% of biological and technical replicates of a low concentration ISAV dilution could be detected by RT‐qPCR (Table ). A 95% LoD was chosen as this has been common practice in past qPCR studies (Bustin et al., ; Forootan et al., ; Nutz et al., ; Stokdyk, Firnstahl, Spencer, Burch, & Borchardt, ). A standard deviation calculated from the average Ct values of all biological replicates of this low‐titre dilution then enabled the calculation of another limit of detection, representing a significant shift in ISAV Ct values between time points (∆Ct LoD ).…”

Section: Discussionmentioning

confidence: 99%

Accelerated ISAV replication detection by cell culture methods combined with time‐monitoring RT‐qPCR

Arseneau

Gautreau

Boston

et al. 2018

Journal of Fish Diseases

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Infectious salmon anaemia (ISA) is a viral disease that affects farmed Atlantic salmon (Salmo salar L.), often leading to mass mortalities. A quick detection of the ISA virus (ISAV) is crucial for decision‐making and can prevent the occurrence of future outbreaks. Screening done by Canada's National Aquatic Animal Health Laboratory System (NAAHLS) uses quantitative reverse transcription PCR (RT‐qPCR) followed by sequencing of PCR amplicons. As neither technique provides information regarding the infectivity of the virus, suspected virulent strains are subsequently tested using viral isolation. However, this stepwise process can require significant time to deliver results. To speed up this delivery, we have improved on these pre‐existing techniques by combining the use of cell culture with RT‐qPCR to detect replicative virus in as little as 5 days. Preliminary assays enabled the establishment of a minimal shift in Ct values over time, which is representative of viral replication in cultured cells. Subsequent blind panel analyses allowed the establishment of the optimal sampling days, as well as diagnostic sensitivity (DSe) and specificity (DSp) estimates. This method could be adopted not only by laboratories conducting diagnostic analyses for ISAV, but also for other slow‐replicating viral agents that replicate through a budding mechanism.

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Determining the 95% limit of detection for waterborne pathogen analyses from primary concentration to qPCR

Cited by 47 publications

References 36 publications

Flushing of stagnant premise water systems after the COVID-19 shutdown can reduce infection risk by Legionella and Mycobacterium spp

Flushing of stagnant premise water systems after the COVID-19 shutdown can reduce infection risk by Legionella and Mycobacterium spp

Pooled samples and eDNA-based detection can facilitate the “clean trade” of aquatic animals

Accelerated ISAV replication detection by cell culture methods combined with time‐monitoring RT‐qPCR

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