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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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Time to smear and qPCR positive. Survival curves showing the percent of subjects negative by both smears and qPCR. (A) first study and (B) second study.
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Figure 1: Time to smear and qPCR positive. Survival curves showing the percent of subjects negative by both smears and qPCR. (A) first study and (B) second study.

Mentions: Data from 16 unvaccinated infectivity control subjects enrolled in two different malaria vaccine trials at the WRAIR were analysed by qPCR. Table 1 shows the mean number of days before detection of parasites in both studies by blood smears and qPCR. The earliest parasites detected were in a subject from the second study where parasites were detected by qPCR 120 hours (5.01 days) after the time of challenge, with a parasite density of 0.174 parasite/μL. Parasites were detected by qPCR in all study subjects within seven days following challenge. In the first study, parasites were detected on average 4.34 days (95% CI = 3.70 to 4.98) before smears, whereas in the second study, parasites were detected on average 6.17 (95% CI = 4.57 to 7.77) days before smears. Figure 1A and B are survival curves showing the per cent of subjects negative by both smears and qPCR for first and 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)

Time to smear and qPCR positive. Survival curves showing the percent of subjects negative by both smears and qPCR. (A) first study and (B) second study.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Time to smear and qPCR positive. Survival curves showing the percent of subjects negative by both smears and qPCR. (A) first study and (B) second study.
Mentions: Data from 16 unvaccinated infectivity control subjects enrolled in two different malaria vaccine trials at the WRAIR were analysed by qPCR. Table 1 shows the mean number of days before detection of parasites in both studies by blood smears and qPCR. The earliest parasites detected were in a subject from the second study where parasites were detected by qPCR 120 hours (5.01 days) after the time of challenge, with a parasite density of 0.174 parasite/μL. Parasites were detected by qPCR in all study subjects within seven days following challenge. In the first study, parasites were detected on average 4.34 days (95% CI = 3.70 to 4.98) before smears, whereas in the second study, parasites were detected on average 6.17 (95% CI = 4.57 to 7.77) days before smears. Figure 1A and B are survival curves showing the per cent of subjects negative by both smears and qPCR for first and 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