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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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Geometric mean parasite density per multiplication cycle for individual volunteers. Panel A shows data from the first study and panel B shows data from the second study.
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Figure 4: Geometric mean parasite density per multiplication cycle for individual volunteers. Panel A shows data from the first study and panel B shows data from the second study.

Mentions: Figure 3 shows parasite multiplication rate (PMR) for the first and second study. The mean PMR for first and second studies were 8.26 (range = 3.5-13.37) and 8.00 (range = 3.98-13.77), respectively. However, parasites in the second study, which were detected sooner and at a lower parasite density, remained low until day 11 (Figure 2). The geometric mean parasite density on day 11 was 22.57 parasites/μL (CI = 15.37-33.15) for the first study compared to 2.02 parasites/μL (CI = 0.37-11.03) for second study. This is reflected by the fact that on day 10 and 11, eight out of 11 (seven on day 11) subjects were found parasite positive by smear for the first study whereas for the second study, subjects did not become smear positive until day 12 (two subjects), day 13 (one subject) and day 14 (two subjects). Table 2 shows the geometric mean parasite density in parasites/μL in the first, second, third, and fourth cycles for first and second study. Figure 4A and B show the geometric mean parasite density in parasites/mL per multiplication cycle for individual subjects. Data clearly indicate that growth rates for the second study were initially slow in the first and second or third cycle but increased rapidly in the third or fourth cycle.


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)

Geometric mean parasite density per multiplication cycle for individual volunteers. Panel A shows data from the first study and panel B shows data from the second study.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Geometric mean parasite density per multiplication cycle for individual volunteers. Panel A shows data from the first study and panel B shows data from the second study.
Mentions: Figure 3 shows parasite multiplication rate (PMR) for the first and second study. The mean PMR for first and second studies were 8.26 (range = 3.5-13.37) and 8.00 (range = 3.98-13.77), respectively. However, parasites in the second study, which were detected sooner and at a lower parasite density, remained low until day 11 (Figure 2). The geometric mean parasite density on day 11 was 22.57 parasites/μL (CI = 15.37-33.15) for the first study compared to 2.02 parasites/μL (CI = 0.37-11.03) for second study. This is reflected by the fact that on day 10 and 11, eight out of 11 (seven on day 11) subjects were found parasite positive by smear for the first study whereas for the second study, subjects did not become smear positive until day 12 (two subjects), day 13 (one subject) and day 14 (two subjects). Table 2 shows the geometric mean parasite density in parasites/μL in the first, second, third, and fourth cycles for first and second study. Figure 4A and B show the geometric mean parasite density in parasites/mL per multiplication cycle for individual subjects. Data clearly indicate that growth rates for the second study were initially slow in the first and second or third cycle but increased rapidly in the third or fourth cycle.

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