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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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Related in: MedlinePlus

Effect of increased sample input on the LLOD of the UCP-LF CAA assay. AWA-TCA standard series in urine (CAA concentration indicated in upper panel), analysed in triplicate with the UCP-LF wet assay format. Spiked urine was extracted with 1 volume 4% (v/v) TCA, the resulting TCA-supernatant was either tested directly with the UCAA10 assay or first concentrated using 0.5, 4, and 15 mL Amicon centrifugal filter devices. Arrows in the lower panel (a blow up of the y-axis) indicate the QC cutoff threshold for the different assays; left to right: UCAA10, −250, −2000 and −7500. Error bars indicate 1 standard deviation (n = 3).
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Fig2: Effect of increased sample input on the LLOD of the UCP-LF CAA assay. AWA-TCA standard series in urine (CAA concentration indicated in upper panel), analysed in triplicate with the UCP-LF wet assay format. Spiked urine was extracted with 1 volume 4% (v/v) TCA, the resulting TCA-supernatant was either tested directly with the UCAA10 assay or first concentrated using 0.5, 4, and 15 mL Amicon centrifugal filter devices. Arrows in the lower panel (a blow up of the y-axis) indicate the QC cutoff threshold for the different assays; left to right: UCAA10, −250, −2000 and −7500. Error bars indicate 1 standard deviation (n = 3).

Mentions: The standard UCP-LF CAA assay for urine (UCAA10) allows testing of 20 μL TCA-supernatant (50% v/v urine, 2% w/v TCA) with a pre-determined QC cutoff threshold of 10 or 30 pg CAA per mL, respectively for the wet- and dry-reagent format. To improve the lower limit of detection (LLOD), TCA-supernatant was concentrated using centrifugal filter devices. Assuming a single load protocol (meaning that the Amicon concentration devices were loaded ‘only’ once with the indicated maximum capacity), the assessed 0.5, 4 and 15 mL centrifugal devices theoretically would allow a 25-, 200- and 750-fold concentration, respectively. A standard series of AWA-TCA in NHU was analysed in triplicate with the UCAA10, −250, −2000 and −7500 using a freshly sonicated stock solution of UCP reporter particles (referred to as wet assay format [28]). For this format, the LLOD detection of CAA in urine progressed from 10 pg/mL to 0.03 pg/mL when increasing urine sample input from 10 μL to 7.5 mL; a practical improvement by 50% of the theoretical limit (Figure 2). Similar factors were achieved with the dry-reagent assay (UCP particles supplied as a dry pellet [30]). When using the UCAA7500 assay, 15 mL TCA-supernatant (containing 7.5 mL urine) is first concentrated to 500 μL with a 15 mL centrifugal filtration device and then transferred to a 0.5 mL centrifugal device for further concentration to 20 μL. The approximate centrifugation time required for the different devices is 15, 30 and 60 minutes for the 10 kDa molecular weight cutoff filtration devices used in this study.Figure 2


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)

Effect of increased sample input on the LLOD of the UCP-LF CAA assay. AWA-TCA standard series in urine (CAA concentration indicated in upper panel), analysed in triplicate with the UCP-LF wet assay format. Spiked urine was extracted with 1 volume 4% (v/v) TCA, the resulting TCA-supernatant was either tested directly with the UCAA10 assay or first concentrated using 0.5, 4, and 15 mL Amicon centrifugal filter devices. Arrows in the lower panel (a blow up of the y-axis) indicate the QC cutoff threshold for the different assays; left to right: UCAA10, −250, −2000 and −7500. Error bars indicate 1 standard deviation (n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Effect of increased sample input on the LLOD of the UCP-LF CAA assay. AWA-TCA standard series in urine (CAA concentration indicated in upper panel), analysed in triplicate with the UCP-LF wet assay format. Spiked urine was extracted with 1 volume 4% (v/v) TCA, the resulting TCA-supernatant was either tested directly with the UCAA10 assay or first concentrated using 0.5, 4, and 15 mL Amicon centrifugal filter devices. Arrows in the lower panel (a blow up of the y-axis) indicate the QC cutoff threshold for the different assays; left to right: UCAA10, −250, −2000 and −7500. Error bars indicate 1 standard deviation (n = 3).
Mentions: The standard UCP-LF CAA assay for urine (UCAA10) allows testing of 20 μL TCA-supernatant (50% v/v urine, 2% w/v TCA) with a pre-determined QC cutoff threshold of 10 or 30 pg CAA per mL, respectively for the wet- and dry-reagent format. To improve the lower limit of detection (LLOD), TCA-supernatant was concentrated using centrifugal filter devices. Assuming a single load protocol (meaning that the Amicon concentration devices were loaded ‘only’ once with the indicated maximum capacity), the assessed 0.5, 4 and 15 mL centrifugal devices theoretically would allow a 25-, 200- and 750-fold concentration, respectively. A standard series of AWA-TCA in NHU was analysed in triplicate with the UCAA10, −250, −2000 and −7500 using a freshly sonicated stock solution of UCP reporter particles (referred to as wet assay format [28]). For this format, the LLOD detection of CAA in urine progressed from 10 pg/mL to 0.03 pg/mL when increasing urine sample input from 10 μL to 7.5 mL; a practical improvement by 50% of the theoretical limit (Figure 2). Similar factors were achieved with the dry-reagent assay (UCP particles supplied as a dry pellet [30]). When using the UCAA7500 assay, 15 mL TCA-supernatant (containing 7.5 mL urine) is first concentrated to 500 μL with a 15 mL centrifugal filtration device and then transferred to a 0.5 mL centrifugal device for further concentration to 20 μL. The approximate centrifugation time required for the different devices is 15, 30 and 60 minutes for the 10 kDa molecular weight cutoff filtration devices used in this study.Figure 2

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