The Effectiveness of HEPA Filters on DNA

Held, Kara F.; Rundell, Clark; Thibeault, Robert; Ghidoni, Daniel; Magoon, Daniel; Nguyen, Trinh Duy; Fisher, Alyson; Parker, Bretna; Spenlinhauer, Tania; Gordon, Joan; Nesbitt, S. W.

doi:10.1177/1535676018766080

Cited by 3 publications

(1 citation statement)

References 2 publications

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“…As with the experimental insectary-reared flies, other potential sources of contamination, such as carry-over contamination during the DNA extraction or amplification stages, were ruled out by use of controls (negative extraction controls and negative template controls respectively). In addition, pre-amplification setup was performed within a dedicated PCR workstation with HEPA-filtered airflow, which is known to reduce aerosolised DNA contamination [47]. Using the quantitative DNA approach, a two-step Cq cut-off protocol revealed a more accurate true positive sample population infection prevalence of 0.47% (95% CI [0.36%, 0.73%]).…”

Section: Discussionmentioning

confidence: 99%

Caught in a trap: DNA contamination in tsetse xenomonitoring can lead to over-estimates ofTrypanosoma bruceiinfection

Saldanha,

Lea,

Manangwa

et al. 2024

Preprint

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Background: Tsetse flies (Glossina sp.) are vectors ofTrypanosoma bruceisubspecies that cause human African trypanosomiasis (HAT). Capturing and screening tsetse is critical for HAT surveillance. Classically, tsetse have been microscopically analysed to identify trypanosomes, but this is increasingly replaced with molecular xenomonitoring. Nonetheless, sensitiveT. brucei-detection assays, such as TBR-PCR, are vulnerable to DNA cross-contamination. This may occur at capture, when often multiple live tsetse are retained temporarily in the cage of a trap. This study set out to determine whether infected tsetse can contaminate naive tsetse withT. bruceiDNA via faeces when co-housed. Methodology/Principle Findings: Insectary-reared teneralG. morsitans morsitanswere fed an infectiousT. b. brucei-spiked bloodmeal. At 19 days post-infection, infected and naive tsetse were caged together in the following ratios: (T1) 9:3, (T2) 6:6 (T3) 1:11 and a control (C0) 0:12 in triplicate. Following 24-hour incubation, DNA was extracted from each fly and screened for parasite DNA presence using PCR and qPCR. All insectary-reared infected flies were positive forT. bruceiDNA using TBR-qPCR. However, naive tsetse also tested positive. Even at a ratio of 1 infected to 11 naive flies, 91% of naive tsetse gave positive TBR-qPCR results. Furthermore, the quantity ofT. bruceiDNA detected in naive tsetse was significantly correlated with cage infection ratio. With evidence of cross-contamination, field-caught tsetse from Tanzania were then assessed using the same screening protocol. End-point TBR-PCR predicted a sample population prevalence of 24.8%. Using qPCR and Cq cut-offs optimised on insectary-reared flies, we estimated that prevalence was 0.5% (95% confidence interval [0.36, 0.73]). Conclusions/Significance: Our results show that infected tsetse can contaminate naive flies withT. bruceiDNA when co-caged, and that the level of contamination can be extensive. Whilst simple PCR may overestimate infection prevalence, quantitative PCR offers a means of eliminating false positives.

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Section: Discussionmentioning

confidence: 99%

Caught in a trap: DNA contamination in tsetse xenomonitoring can lead to over-estimates ofTrypanosoma bruceiinfection

Saldanha,

Lea,

Manangwa

et al. 2024

Preprint

View full text Add to dashboard Cite

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Capability of the Airstream Helmet for Protecting Mine Workers from Diesel Particulate Matter

Noll

Lee

Vanderslice

et al. 2021

Mining, Metallurgy & Exploration

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Caught in a trap: DNA contamination in tsetse xenomonitoring can lead to over-estimates of Trypanosoma brucei infection

Saldanha,

Lea,

Manangwa

et al. 2024

PLoS Negl Trop Dis

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Background Tsetse flies (Glossina sp.) are vectors of Trypanosoma brucei subspecies that cause human African trypanosomiasis (HAT). Capturing and screening tsetse is critical for HAT surveillance. Classically, tsetse have been microscopically analysed to identify trypanosomes, but this is increasingly replaced with molecular xenomonitoring. Nonetheless, sensitive T. brucei-detection assays, such as TBR-PCR, are vulnerable to DNA cross-contamination. This may occur at capture, when often multiple live tsetse are retained temporarily in the cage of a trap. This study set out to determine whether infected tsetse can contaminate naïve tsetse with T. brucei DNA via faeces when co-housed. Methodology/Principle findings Insectary-reared teneral G. morsitans morsitans were fed an infectious T. b. brucei-spiked bloodmeal. At 19 days post-infection, infected and naïve tsetse were caged together in the following ratios: (T1) 9:3, (T2) 6:6 (T3) 1:11 and a control (C0) 0:12 in triplicate. Following 24-hour incubation, DNA was extracted from each fly and screened for parasite DNA presence using PCR and qPCR. All insectary-reared infected flies were positive for T. brucei DNA using TBR-qPCR. However, naïve tsetse also tested positive. Even at a ratio of 1 infected to 11 naïve flies, 91% of naïve tsetse gave positive TBR-qPCR results. Furthermore, the quantity of T. brucei DNA detected in naïve tsetse was significantly correlated with cage infection ratio. With evidence of cross-contamination, field-caught tsetse from Tanzania were then assessed using the same screening protocol. End-point TBR-PCR predicted a sample population prevalence of 24.8%. Using qPCR and Cq cut-offs optimised on insectary-reared flies, we estimated that prevalence was 0.5% (95% confidence interval [0.36, 0.73]). Conclusions/Significance Our results show that infected tsetse can contaminate naïve flies with T. brucei DNA when co-caged, and that the level of contamination can be extensive. Whilst simple PCR may overestimate infection prevalence, quantitative PCR offers a means of eliminating false positives.

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The Effectiveness of HEPA Filters on DNA

Cited by 3 publications

References 2 publications

Caught in a trap: DNA contamination in tsetse xenomonitoring can lead to over-estimates ofTrypanosoma bruceiinfection

Caught in a trap: DNA contamination in tsetse xenomonitoring can lead to over-estimates ofTrypanosoma bruceiinfection

Capability of the Airstream Helmet for Protecting Mine Workers from Diesel Particulate Matter

Caught in a trap: DNA contamination in tsetse xenomonitoring can lead to over-estimates of Trypanosoma brucei infection

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