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Increased sample volume and use of quantitative reverse-transcription PCR can improve prediction of liver-to-blood inoculum size in controlled human malaria infection studies.

Hodgson SH, Douglas AD, Edwards NJ, Kimani D, Elias SC, Chang M, Daza G, Seilie AM, Magiri C, Muia A, Juma EA, Cole AO, Rampling TW, Anagnostou NA, Gilbert SC, Hoffman SL, Draper SJ, Bejon P, Ogutu B, Marsh K, Hill AV, Murphy SC - Malar. J. (2015)

Bottom Line: Controlled human malaria infection (CHMI) studies increasingly rely on nucleic acid test (NAT) methods to detect and quantify parasites in the blood of infected participants.Large volume qRT-PCR at the University of Washington was the most sensitive test and generated quantifiable data more often than any other NAT methodology.Standard quantitative PCR (qPCR) performed at the University of Oxford and standard volume qRT-PCR performed at the University of Washington were less sensitive than the large volume qRT-PCR, especially at 6.5 days after CHMI.

View Article: PubMed Central - PubMed

Affiliation: The Jenner Institute, University of Oxford, Oxford, UK. susanne.hodgson@ndm.ox.ac.uk.

ABSTRACT

Background: Controlled human malaria infection (CHMI) studies increasingly rely on nucleic acid test (NAT) methods to detect and quantify parasites in the blood of infected participants. The lower limits of detection and quantification vary amongst the assays used throughout the world, which may affect the ability of mathematical models to accurately estimate the liver-to-blood inoculum (LBI) values that are used to judge the efficacy of pre-erythrocytic vaccine and drug candidates.

Methods: Samples were collected around the time of onset of pre-patent parasitaemia from subjects who enrolled in two different CHMI clinical trials. Blood samples were tested for Plasmodium falciparum 18S rRNA and/or rDNA targets by different NAT methods and results were compared. Methods included an ultrasensitive, large volume modification of an established quantitative reverse transcription PCR (qRT-PCR) assay that achieves detection of as little as one parasite/mL of whole blood.

Results: Large volume qRT-PCR at the University of Washington was the most sensitive test and generated quantifiable data more often than any other NAT methodology. Standard quantitative PCR (qPCR) performed at the University of Oxford and standard volume qRT-PCR performed at the University of Washington were less sensitive than the large volume qRT-PCR, especially at 6.5 days after CHMI. In these trials, the proportion of participants for whom LBI could be accurately quantified using parasite density value greater than or equal to the lower limit of quantification was increased. A greater improvement would be expected in trials in which numerous subjects receive a lower LBI or low dose challenge.

Conclusions: Standard qPCR and qRT-PCR methods with analytical sensitivities of ~20 parasites/mL probably suffice for most CHMI purposes, but the newly developed large volume qRT-PCR may be able to answer specific questions when more analytical sensitivity is required.

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Related in: MedlinePlus

Correlation analyses. Data were compiled from all samples in both clinical trials where two methods produced results ≥ LLQ. Paired results were plotted as shown; Spearman rank correlation (R); two-tailed p value. Units for x and y axes are log10 p/mL whole blood as determined for paired samples by the methods listed on each axis.
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Fig2: Correlation analyses. Data were compiled from all samples in both clinical trials where two methods produced results ≥ LLQ. Paired results were plotted as shown; Spearman rank correlation (R); two-tailed p value. Units for x and y axes are log10 p/mL whole blood as determined for paired samples by the methods listed on each axis.

Mentions: Next, an inter-assay comparison of quantitative results was performed. All tests that generated results ≥ LLQ were included. With the exception of the pairing of Oxford qPCR results with DBS qRT-PCR results, all datasets showed statistically significant concentration-dependent correlation with one another (Figure 2). The strongest correlation was between large and standard volume qRT-PCR assays. While Oxford and UW assays showed strong correlation, when comparisons were made using Bland-Altman plots to look for quantitative agreement and bias, a consistent quantitative shift between centers was observed (Figure 3), consistent with a previous report showing qualitative agreement of the Oxford data with that of other CHMI centers including UW, but a consistent quantitative shift at Oxford due to blood filtration and calibration differences [5]. DBS qRT-PCR results also diverged from UW liquid results, and this shift resulted in apparent alignment between Oxford qPCR and UW DBS qRT-PCR results.Figure 2


Increased sample volume and use of quantitative reverse-transcription PCR can improve prediction of liver-to-blood inoculum size in controlled human malaria infection studies.

Hodgson SH, Douglas AD, Edwards NJ, Kimani D, Elias SC, Chang M, Daza G, Seilie AM, Magiri C, Muia A, Juma EA, Cole AO, Rampling TW, Anagnostou NA, Gilbert SC, Hoffman SL, Draper SJ, Bejon P, Ogutu B, Marsh K, Hill AV, Murphy SC - Malar. J. (2015)

Correlation analyses. Data were compiled from all samples in both clinical trials where two methods produced results ≥ LLQ. Paired results were plotted as shown; Spearman rank correlation (R); two-tailed p value. Units for x and y axes are log10 p/mL whole blood as determined for paired samples by the methods listed on each axis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4318195&req=5

Fig2: Correlation analyses. Data were compiled from all samples in both clinical trials where two methods produced results ≥ LLQ. Paired results were plotted as shown; Spearman rank correlation (R); two-tailed p value. Units for x and y axes are log10 p/mL whole blood as determined for paired samples by the methods listed on each axis.
Mentions: Next, an inter-assay comparison of quantitative results was performed. All tests that generated results ≥ LLQ were included. With the exception of the pairing of Oxford qPCR results with DBS qRT-PCR results, all datasets showed statistically significant concentration-dependent correlation with one another (Figure 2). The strongest correlation was between large and standard volume qRT-PCR assays. While Oxford and UW assays showed strong correlation, when comparisons were made using Bland-Altman plots to look for quantitative agreement and bias, a consistent quantitative shift between centers was observed (Figure 3), consistent with a previous report showing qualitative agreement of the Oxford data with that of other CHMI centers including UW, but a consistent quantitative shift at Oxford due to blood filtration and calibration differences [5]. DBS qRT-PCR results also diverged from UW liquid results, and this shift resulted in apparent alignment between Oxford qPCR and UW DBS qRT-PCR results.Figure 2

Bottom Line: Controlled human malaria infection (CHMI) studies increasingly rely on nucleic acid test (NAT) methods to detect and quantify parasites in the blood of infected participants.Large volume qRT-PCR at the University of Washington was the most sensitive test and generated quantifiable data more often than any other NAT methodology.Standard quantitative PCR (qPCR) performed at the University of Oxford and standard volume qRT-PCR performed at the University of Washington were less sensitive than the large volume qRT-PCR, especially at 6.5 days after CHMI.

View Article: PubMed Central - PubMed

Affiliation: The Jenner Institute, University of Oxford, Oxford, UK. susanne.hodgson@ndm.ox.ac.uk.

ABSTRACT

Background: Controlled human malaria infection (CHMI) studies increasingly rely on nucleic acid test (NAT) methods to detect and quantify parasites in the blood of infected participants. The lower limits of detection and quantification vary amongst the assays used throughout the world, which may affect the ability of mathematical models to accurately estimate the liver-to-blood inoculum (LBI) values that are used to judge the efficacy of pre-erythrocytic vaccine and drug candidates.

Methods: Samples were collected around the time of onset of pre-patent parasitaemia from subjects who enrolled in two different CHMI clinical trials. Blood samples were tested for Plasmodium falciparum 18S rRNA and/or rDNA targets by different NAT methods and results were compared. Methods included an ultrasensitive, large volume modification of an established quantitative reverse transcription PCR (qRT-PCR) assay that achieves detection of as little as one parasite/mL of whole blood.

Results: Large volume qRT-PCR at the University of Washington was the most sensitive test and generated quantifiable data more often than any other NAT methodology. Standard quantitative PCR (qPCR) performed at the University of Oxford and standard volume qRT-PCR performed at the University of Washington were less sensitive than the large volume qRT-PCR, especially at 6.5 days after CHMI. In these trials, the proportion of participants for whom LBI could be accurately quantified using parasite density value greater than or equal to the lower limit of quantification was increased. A greater improvement would be expected in trials in which numerous subjects receive a lower LBI or low dose challenge.

Conclusions: Standard qPCR and qRT-PCR methods with analytical sensitivities of ~20 parasites/mL probably suffice for most CHMI purposes, but the newly developed large volume qRT-PCR may be able to answer specific questions when more analytical sensitivity is required.

Show MeSH
Related in: MedlinePlus