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Measurement of parasitological data by quantitative real-time PCR from controlled human malaria infection trials at the Walter Reed Army Institute of Research.

Kamau E, Alemayehu S, Feghali KC, Komisar J, Regules J, Cowden J, Ockenhouse CF - Malar. J. (2014)

Bottom Line: Data showed that at low parasite densities, a combination of sequestration and stochastic effects of low copy number DNA may impact qPCR detection and the parasite detection limit.Smear positive is an endpoint which antimalarial rescue is imperative whereas early detection of parasitological data by qPCR can allow for better anticipation of the endpoint.This would allow for early treatment to reduce clinical illness and risk for study participants.

View Article: PubMed Central - HTML - PubMed

Affiliation: Military Malaria Research Program, Malaria Vaccine Branch, Walter Reed Army Institute of Research, 503 Robert Grant Ave, Silver Spring, Maryland, USA. edwin.kamau@us.army.mil.

ABSTRACT

Background: The use of quantitative real-time PCR (qPCR) has allowed for precise quantification of parasites in the prepatent period and greatly improved the reproducibility and statistical power of controlled human malaria infection (CHMI) trials. Parasitological data presented here are from non-immunized, control-challenged subjects who participated in two CHMI trials conducted at the Walter Reed Army Institute of Research (WRAIR).

Methods: Standardized sporozoite challenge was achieved through the bite of five Anopheles stephensi mosquitoes infected with the 3D7clone of the NF54 strain of Plasmodium falciparum. Blood smears were scored positive when two unambiguous parasites were found. Analysis of parasitological PCR data was performed on log-transformed data using an independent sample t-test when comparing the two studies. The multiplication rate of blood-stage parasites was estimated using the linear model.

Results: On average, parasites were detected 4.91 days (95% CI = 4.190 to 5.627) before smears. The earliest parasites were detected within 120 hours (5.01 days) after challenge. Parasite densities showed consistent cyclic patterns of blood-stage parasite growth in all volunteers. The parasite multiplication rates for both studies was 8.18 (95% CI = 6.162 to 10.20). Data showed that at low parasite densities, a combination of sequestration and stochastic effects of low copy number DNA may impact qPCR detection and the parasite detection limit.

Conclusion: Smear positive is an endpoint which antimalarial rescue is imperative whereas early detection of parasitological data by qPCR can allow for better anticipation of the endpoint. This would allow for early treatment to reduce clinical illness and risk for study participants. To use qPCR as the primary endpoint in CHMI trials, an algorithm of two positives by qPCR where one of the positives must have parasite density of at least 2 parasites/μL is proposed.

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Parasite density as measured by qPCR. The geometric mean is shown from both studies with 95% confidence intervals, from the day which qPCR was initiated (day 5 after challenge) until the day the subjects became smear positive and treatment was initiated.
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Figure 2: Parasite density as measured by qPCR. The geometric mean is shown from both studies with 95% confidence intervals, from the day which qPCR was initiated (day 5 after challenge) until the day the subjects became smear positive and treatment was initiated.

Mentions: Parasite densities showed consistent cyclic patterns of blood-stage parasite growth in all volunteers from both studies. Figure 2 shows the geometric mean of parasite density from both studies. Parasites in the second study were detected sooner by qPCR compared to the first study and at a lower density. Parasite geometric mean density first cycle was 0.444 parasites/μL (95% CI = 0.274 to 0.718) for the first study and 0.202 parasites/μL (95% CI = 0.107 to 0.383) for the second study. Geometric mean parasite density by smears for the first study was 3.29 parasites/μL (95% CI 2.00 to 5.42) and 1.68 (95% CI = 0.97 to 2.92) for the second study. Geometric mean parasite density by qPCR analysis on the first day of qPCR positivity for the first study was 0.394 parasites/μL (95% CI = 0.272 to 0.572) and 0.174 parasites/μL (95% CI = 0.079 to 0.383) for second study. Interestingly, the geometric mean parasite density by qPCR at the day of smear positive for first study was 23.92 parasites/μL (95% CI = 16.43 to 34.80) and 35.74 parasites/μL (95% CI = 25.43 to 50.23) for second study.


Measurement of parasitological data by quantitative real-time PCR from controlled human malaria infection trials at the Walter Reed Army Institute of Research.

Kamau E, Alemayehu S, Feghali KC, Komisar J, Regules J, Cowden J, Ockenhouse CF - Malar. J. (2014)

Parasite density as measured by qPCR. The geometric mean is shown from both studies with 95% confidence intervals, from the day which qPCR was initiated (day 5 after challenge) until the day the subjects became smear positive and treatment was initiated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4128310&req=5

Figure 2: Parasite density as measured by qPCR. The geometric mean is shown from both studies with 95% confidence intervals, from the day which qPCR was initiated (day 5 after challenge) until the day the subjects became smear positive and treatment was initiated.
Mentions: Parasite densities showed consistent cyclic patterns of blood-stage parasite growth in all volunteers from both studies. Figure 2 shows the geometric mean of parasite density from both studies. Parasites in the second study were detected sooner by qPCR compared to the first study and at a lower density. Parasite geometric mean density first cycle was 0.444 parasites/μL (95% CI = 0.274 to 0.718) for the first study and 0.202 parasites/μL (95% CI = 0.107 to 0.383) for the second study. Geometric mean parasite density by smears for the first study was 3.29 parasites/μL (95% CI 2.00 to 5.42) and 1.68 (95% CI = 0.97 to 2.92) for the second study. Geometric mean parasite density by qPCR analysis on the first day of qPCR positivity for the first study was 0.394 parasites/μL (95% CI = 0.272 to 0.572) and 0.174 parasites/μL (95% CI = 0.079 to 0.383) for second study. Interestingly, the geometric mean parasite density by qPCR at the day of smear positive for first study was 23.92 parasites/μL (95% CI = 16.43 to 34.80) and 35.74 parasites/μL (95% CI = 25.43 to 50.23) for second study.

Bottom Line: Data showed that at low parasite densities, a combination of sequestration and stochastic effects of low copy number DNA may impact qPCR detection and the parasite detection limit.Smear positive is an endpoint which antimalarial rescue is imperative whereas early detection of parasitological data by qPCR can allow for better anticipation of the endpoint.This would allow for early treatment to reduce clinical illness and risk for study participants.

View Article: PubMed Central - HTML - PubMed

Affiliation: Military Malaria Research Program, Malaria Vaccine Branch, Walter Reed Army Institute of Research, 503 Robert Grant Ave, Silver Spring, Maryland, USA. edwin.kamau@us.army.mil.

ABSTRACT

Background: The use of quantitative real-time PCR (qPCR) has allowed for precise quantification of parasites in the prepatent period and greatly improved the reproducibility and statistical power of controlled human malaria infection (CHMI) trials. Parasitological data presented here are from non-immunized, control-challenged subjects who participated in two CHMI trials conducted at the Walter Reed Army Institute of Research (WRAIR).

Methods: Standardized sporozoite challenge was achieved through the bite of five Anopheles stephensi mosquitoes infected with the 3D7clone of the NF54 strain of Plasmodium falciparum. Blood smears were scored positive when two unambiguous parasites were found. Analysis of parasitological PCR data was performed on log-transformed data using an independent sample t-test when comparing the two studies. The multiplication rate of blood-stage parasites was estimated using the linear model.

Results: On average, parasites were detected 4.91 days (95% CI = 4.190 to 5.627) before smears. The earliest parasites were detected within 120 hours (5.01 days) after challenge. Parasite densities showed consistent cyclic patterns of blood-stage parasite growth in all volunteers. The parasite multiplication rates for both studies was 8.18 (95% CI = 6.162 to 10.20). Data showed that at low parasite densities, a combination of sequestration and stochastic effects of low copy number DNA may impact qPCR detection and the parasite detection limit.

Conclusion: Smear positive is an endpoint which antimalarial rescue is imperative whereas early detection of parasitological data by qPCR can allow for better anticipation of the endpoint. This would allow for early treatment to reduce clinical illness and risk for study participants. To use qPCR as the primary endpoint in CHMI trials, an algorithm of two positives by qPCR where one of the positives must have parasite density of at least 2 parasites/μL is proposed.

Show MeSH
Related in: MedlinePlus