Limits...
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.

Show MeSH

Related in: MedlinePlus

Time to positivity for microscopy, Oxford qPCR and large volume qRT-PCR in KCS. Kaplan-Meier survival curve representing the rate of conversion from negative to positive NAT results by test method (>=LLQ). Solid line, UW large volume qRT-PCR; heavy dashed line, Oxford qPCR; light dotted line, microscopy diagnosis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Time to positivity for microscopy, Oxford qPCR and large volume qRT-PCR in KCS. Kaplan-Meier survival curve representing the rate of conversion from negative to positive NAT results by test method (>=LLQ). Solid line, UW large volume qRT-PCR; heavy dashed line, Oxford qPCR; light dotted line, microscopy diagnosis.

Mentions: Since the larger volume RT-PCR assay showed increased sensitivity at D7.5 in VAC052, the next step was to test blood from 27 subjects sampled during the first cycle of expected parasitaemia in the KCS trial (D6.5-7.5). All 27 subjects included in the analysis developed positive P. falciparum infections by TBS by the end of the trial [15]. Since 100% of the subjects were positive by the gold standard definition, specificity of the assays could not be compared. The large volume qRT-PCR assay was most sensitive in this dataset, followed by the Oxford qPCR, the standard volume qRT-PCR and the dried blood spot (DBS) qRT-PCR (Table 3 and Figure 1). On D6.5, the large volume qRT-PCR assay detected the highest number of positive specimens (10/27), followed by the Oxford qPCR (7/27) and the UW standard volume qRT-PCR assay (1/27). Amongst samples positive on D6.5, all large volume qRT-PCR (n = 10) and standard volume qRT-PCR assay samples (n = 1) were quantifiable within the limits of the assay (i.e., ≥LLQ), whereas all samples positive by Oxford qPCR (n = 7) were in the ‘low positive’ range (i.e., ≥LLD). By D7.0, most samples were qualitatively positive by all assays (27/27 by large volume qRT-PCR, 26/27 by Oxford qPCR, 19/27 by standard volume qRT-PCR and 14/26 by DBS qRT-PCR), which increased further by D7.5. A large proportion of samples tested by Oxford qPCR were identified as ‘low positives’ (i.e., LLQ > result ≥ LLD) (27/60 positive qPCRs on D6.5-D7.5) whereas this was less common amongst qRT-PCR positive samples (0/61 large volume qRT-PCR, 3/43 standard volume qRT-PCR, 5/35 DBS qRT-PCR). Finally, the large volume qRT-PCR and the Oxford qPCR assays became positive (≥LLD) on average on D7.0 compared to D12.5 by microscopy (Figure 1), a 5.5-day lead time over microscopy.Table 3


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)

Time to positivity for microscopy, Oxford qPCR and large volume qRT-PCR in KCS. Kaplan-Meier survival curve representing the rate of conversion from negative to positive NAT results by test method (>=LLQ). Solid line, UW large volume qRT-PCR; heavy dashed line, Oxford qPCR; light dotted line, microscopy diagnosis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Time to positivity for microscopy, Oxford qPCR and large volume qRT-PCR in KCS. Kaplan-Meier survival curve representing the rate of conversion from negative to positive NAT results by test method (>=LLQ). Solid line, UW large volume qRT-PCR; heavy dashed line, Oxford qPCR; light dotted line, microscopy diagnosis.
Mentions: Since the larger volume RT-PCR assay showed increased sensitivity at D7.5 in VAC052, the next step was to test blood from 27 subjects sampled during the first cycle of expected parasitaemia in the KCS trial (D6.5-7.5). All 27 subjects included in the analysis developed positive P. falciparum infections by TBS by the end of the trial [15]. Since 100% of the subjects were positive by the gold standard definition, specificity of the assays could not be compared. The large volume qRT-PCR assay was most sensitive in this dataset, followed by the Oxford qPCR, the standard volume qRT-PCR and the dried blood spot (DBS) qRT-PCR (Table 3 and Figure 1). On D6.5, the large volume qRT-PCR assay detected the highest number of positive specimens (10/27), followed by the Oxford qPCR (7/27) and the UW standard volume qRT-PCR assay (1/27). Amongst samples positive on D6.5, all large volume qRT-PCR (n = 10) and standard volume qRT-PCR assay samples (n = 1) were quantifiable within the limits of the assay (i.e., ≥LLQ), whereas all samples positive by Oxford qPCR (n = 7) were in the ‘low positive’ range (i.e., ≥LLD). By D7.0, most samples were qualitatively positive by all assays (27/27 by large volume qRT-PCR, 26/27 by Oxford qPCR, 19/27 by standard volume qRT-PCR and 14/26 by DBS qRT-PCR), which increased further by D7.5. A large proportion of samples tested by Oxford qPCR were identified as ‘low positives’ (i.e., LLQ > result ≥ LLD) (27/60 positive qPCRs on D6.5-D7.5) whereas this was less common amongst qRT-PCR positive samples (0/61 large volume qRT-PCR, 3/43 standard volume qRT-PCR, 5/35 DBS qRT-PCR). Finally, the large volume qRT-PCR and the Oxford qPCR assays became positive (≥LLD) on average on D7.0 compared to D12.5 by microscopy (Figure 1), a 5.5-day lead time over microscopy.Table 3

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