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Improved sensitivity of the urine CAA lateral-flow assay for diagnosing active Schistosoma infections by using larger sample volumes.

Corstjens PL, Nyakundi RK, de Dood CJ, Kariuki TM, Ochola EA, Karanja DM, Mwinzi PN, van Dam GJ - Parasit Vectors (2015)

Bottom Line: Only a single 2 mL urine sample is needed, but analysing larger sample volumes could still increase test accuracy.The UCAA2000 test is an appropriate candidate for accurate identification of all infected individuals in low-endemic regions.Assay materials do not require refrigeration and collected urine samples may be stored and transported to central test laboratories without the need to be frozen.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Einthovenweg 20, P.O. Box 9600, 2300 RC, Leiden, the Netherlands. p.corstjens@lumc.nl.

ABSTRACT

Background: Accurate determination of Schistosoma infection rates in low endemic regions to examine progress towards interruption of transmission and elimination requires highly sensitive diagnostic tools. An existing lateral flow (LF) based test demonstrating ongoing infections through detection of worm circulating anodic antigen (CAA), was improved for sensitivity through implementation of a protocol allowing increased sample input. Urine is the preferred sample as collection is non-invasive and sample volume is generally not a restriction.

Methods: Centrifugal filtration devices provided a method to concentrate supernatant of urine samples extracted with trichloroacetic acid (TCA). For field trials a practical sample volume of 2 mL urine allowed detection of CAA down to 0.3 pg/mL. The method was evaluated on a set of urine samples (n = 113) from an S. mansoni endemic region (Kisumu, Kenya) and compared to stool microscopy (Kato Katz, KK). In this analysis true positivity was defined as a sample with either a positive KK or UCAA test.

Results: Implementation of the concentration method increased clinical sensitivity (Sn) from 44 to 98% when moving from the standard 10 μL (UCAA10 assay) to 2000 μL (UCAA2000 assay) urine sample input. Sn for KK varied between 23 and 35% for a duplicate KK (single stool, two slides) to 52% for a six-fold KK (three consecutive day stools, two slides). The UCAA2000 assay indicated 47 positive samples with CAA concentration above 0.3 pg/mL. The six-fold KK detected 25 egg positives; 1 sample with 2 eggs detected in the 6-fold KK was not identified with the UCAA2000 assay.

Conclusions: Larger sample input increased Sn of the UCAA assay to a level indicating 'true' infection. Only a single 2 mL urine sample is needed, but analysing larger sample volumes could still increase test accuracy. The UCAA2000 test is an appropriate candidate for accurate identification of all infected individuals in low-endemic regions. Assay materials do not require refrigeration and collected urine samples may be stored and transported to central test laboratories without the need to be frozen.

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CAA standard series in urine analysed with the UCP-LF concentration assays. The amount of urine analysed per strip increased from 10, 250 to 2000 μL, respectively for the UCAA10, −250 and −2000 assay. The UCAA250 and UCA2000 require concentration of the TCA-supernatant using Amicon Centrifugal Filter Devices Ultra-0.5 and Ultra-4, respectively.
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Fig3: CAA standard series in urine analysed with the UCP-LF concentration assays. The amount of urine analysed per strip increased from 10, 250 to 2000 μL, respectively for the UCAA10, −250 and −2000 assay. The UCAA250 and UCA2000 require concentration of the TCA-supernatant using Amicon Centrifugal Filter Devices Ultra-0.5 and Ultra-4, respectively.

Mentions: The urine samples collected in Kisumu were tested at IPR (Nairobi) with the UCP-LF dry-reagent assay. These assay materials were transported from the Netherlands to Kenya without cooling, and on arrival stored at ambient temperature. Not needing a cold chain for shipping was an important feature that reduced cost and logistic issues. All 113 samples were tested (in singlet) with the UCAA10 and −250 assay. However, material restrictions only allowed testing of 73 samples with the UCAA2000 assay. Scanning of the LF strips and analysis of the result was also performed at IPR utilizing a lightweight reader with appropriate software as described for other studies [35]. CAA concentrations were determined from standard curves obtained with NHU spiked with AWA-TCA tested with the UCAA10, −250 and −2000 assay (Figure 3) using a 4 parameter curve fit method. Samples were classified as positive, indecisive or negative according the predetermined QC cutoff thresholds. The number of CAA positives increased from 21 (identified with the UCAA10 assay) to 30 when using the concentration based assays UCAA250. In the subset of 73 samples that were tested with all 3 UCAA assays, the number of CAA positives increased from 15 in the UCAA10, to 20 in the UCAA250, to 31 in the UCAA2000, respectively. Increased sample volume obviously did result in an increase of the number of identified CAA positives, raising the percentage positives in the (non-random) subset from 19%, to 27%, to 42% for the respective assays (Table 1). Moreover, positives identified by the UCAA10 assay were all confirmed with the UCAA250 assay, and similarly from the UCAA250 assay to the UCAA2000 assay. Out of the 6 UCAA10 indecisive samples 50% was a true positive as demonstrated with the larger sample volume assay (UCAA250). The single sample classified indecisive with the UCAA250 did not test positive with the UCAA2000 assay. We speculate that the percentage of indecisive samples that actually turn out positive may vary per setting. The pre-determined QC cutoff thresholds firmly maintain 100% specificity. Depending on the objective of the study, when targeting 100% sensitivity the indecisive group should be included in the positive group.Figure 3


Improved sensitivity of the urine CAA lateral-flow assay for diagnosing active Schistosoma infections by using larger sample volumes.

Corstjens PL, Nyakundi RK, de Dood CJ, Kariuki TM, Ochola EA, Karanja DM, Mwinzi PN, van Dam GJ - Parasit Vectors (2015)

CAA standard series in urine analysed with the UCP-LF concentration assays. The amount of urine analysed per strip increased from 10, 250 to 2000 μL, respectively for the UCAA10, −250 and −2000 assay. The UCAA250 and UCA2000 require concentration of the TCA-supernatant using Amicon Centrifugal Filter Devices Ultra-0.5 and Ultra-4, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4418045&req=5

Fig3: CAA standard series in urine analysed with the UCP-LF concentration assays. The amount of urine analysed per strip increased from 10, 250 to 2000 μL, respectively for the UCAA10, −250 and −2000 assay. The UCAA250 and UCA2000 require concentration of the TCA-supernatant using Amicon Centrifugal Filter Devices Ultra-0.5 and Ultra-4, respectively.
Mentions: The urine samples collected in Kisumu were tested at IPR (Nairobi) with the UCP-LF dry-reagent assay. These assay materials were transported from the Netherlands to Kenya without cooling, and on arrival stored at ambient temperature. Not needing a cold chain for shipping was an important feature that reduced cost and logistic issues. All 113 samples were tested (in singlet) with the UCAA10 and −250 assay. However, material restrictions only allowed testing of 73 samples with the UCAA2000 assay. Scanning of the LF strips and analysis of the result was also performed at IPR utilizing a lightweight reader with appropriate software as described for other studies [35]. CAA concentrations were determined from standard curves obtained with NHU spiked with AWA-TCA tested with the UCAA10, −250 and −2000 assay (Figure 3) using a 4 parameter curve fit method. Samples were classified as positive, indecisive or negative according the predetermined QC cutoff thresholds. The number of CAA positives increased from 21 (identified with the UCAA10 assay) to 30 when using the concentration based assays UCAA250. In the subset of 73 samples that were tested with all 3 UCAA assays, the number of CAA positives increased from 15 in the UCAA10, to 20 in the UCAA250, to 31 in the UCAA2000, respectively. Increased sample volume obviously did result in an increase of the number of identified CAA positives, raising the percentage positives in the (non-random) subset from 19%, to 27%, to 42% for the respective assays (Table 1). Moreover, positives identified by the UCAA10 assay were all confirmed with the UCAA250 assay, and similarly from the UCAA250 assay to the UCAA2000 assay. Out of the 6 UCAA10 indecisive samples 50% was a true positive as demonstrated with the larger sample volume assay (UCAA250). The single sample classified indecisive with the UCAA250 did not test positive with the UCAA2000 assay. We speculate that the percentage of indecisive samples that actually turn out positive may vary per setting. The pre-determined QC cutoff thresholds firmly maintain 100% specificity. Depending on the objective of the study, when targeting 100% sensitivity the indecisive group should be included in the positive group.Figure 3

Bottom Line: Only a single 2 mL urine sample is needed, but analysing larger sample volumes could still increase test accuracy.The UCAA2000 test is an appropriate candidate for accurate identification of all infected individuals in low-endemic regions.Assay materials do not require refrigeration and collected urine samples may be stored and transported to central test laboratories without the need to be frozen.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Einthovenweg 20, P.O. Box 9600, 2300 RC, Leiden, the Netherlands. p.corstjens@lumc.nl.

ABSTRACT

Background: Accurate determination of Schistosoma infection rates in low endemic regions to examine progress towards interruption of transmission and elimination requires highly sensitive diagnostic tools. An existing lateral flow (LF) based test demonstrating ongoing infections through detection of worm circulating anodic antigen (CAA), was improved for sensitivity through implementation of a protocol allowing increased sample input. Urine is the preferred sample as collection is non-invasive and sample volume is generally not a restriction.

Methods: Centrifugal filtration devices provided a method to concentrate supernatant of urine samples extracted with trichloroacetic acid (TCA). For field trials a practical sample volume of 2 mL urine allowed detection of CAA down to 0.3 pg/mL. The method was evaluated on a set of urine samples (n = 113) from an S. mansoni endemic region (Kisumu, Kenya) and compared to stool microscopy (Kato Katz, KK). In this analysis true positivity was defined as a sample with either a positive KK or UCAA test.

Results: Implementation of the concentration method increased clinical sensitivity (Sn) from 44 to 98% when moving from the standard 10 μL (UCAA10 assay) to 2000 μL (UCAA2000 assay) urine sample input. Sn for KK varied between 23 and 35% for a duplicate KK (single stool, two slides) to 52% for a six-fold KK (three consecutive day stools, two slides). The UCAA2000 assay indicated 47 positive samples with CAA concentration above 0.3 pg/mL. The six-fold KK detected 25 egg positives; 1 sample with 2 eggs detected in the 6-fold KK was not identified with the UCAA2000 assay.

Conclusions: Larger sample input increased Sn of the UCAA assay to a level indicating 'true' infection. Only a single 2 mL urine sample is needed, but analysing larger sample volumes could still increase test accuracy. The UCAA2000 test is an appropriate candidate for accurate identification of all infected individuals in low-endemic regions. Assay materials do not require refrigeration and collected urine samples may be stored and transported to central test laboratories without the need to be frozen.

Show MeSH
Related in: MedlinePlus