Analysis of UCP-LF test result

For standardization and quantitative expression of the results UCP-LF assay data are presented as ratio values (Corstjens et al. Reference Corstjens, Zuiderwijk, Brink, Li, Feindt, Neidbala and Tanke2001), i.e. the UCP signal measured at the Test (T) line divided by the UCP signal measured at the Flow Control (FC) line (Fig. 1). The cut-off threshold value is then defined as the ratio T/FC value above which a sample is designated reactive or positive. These threshold values may be influenced by technical factors such as batch-to-batch variations, as well as (immuno-)epidemiological settings (e.g. co-infections, age and geography). Table 1 shows the minimal level of variation that is observed in a cut-off threshold when testing different populations with UCP-CAA LF strip batches prepared from the same set of material following a strict quality control protocol. In practice, when studying prevalence and clinical sensitivity, a low- and high-sensitivity cut-off threshold are applied. These values are usually defined, respectively, as the average and the highest value both plus two s.d. of a series of samples from a set of confirmed negative controls. If proper control samples are not available, cut-off thresholds are determined from a standard series of AWA-TCA (containing approximately 3% w/v of CAA and CCA) spiked in NHS or negative urine. Ratio values can also be adjusted by varying the amount of capture antibodies on the T and FC line. In general the composition of the T line is optimized for analytical sensitivity, such that the optimal load of capture antibody still results in a detectable specific T line signal with the lowest concentration of target antigen. The FC line can be adapted to give the desired ratio either at the low or high end of the antigen concentration curve. These conditions are directly linked to the sensitivity of the UCP reader used. CAA or CCA concentrations are determined by fitting ratio T/FC values to the standard curve obtained with the AWA-TCA series (Corstjens et al. Reference Corstjens, van Lieshout, Zuiderwijk, Kornelis, Tanke, Deelder and van Dam2008).

Table 1. Variation in the UCP-LF CAA cut-off threshold when testing different populations

^a Average, average ratio of the negatives; Stdev, s.d.; Max, largest ratio value determined in the selection; Low and High represent low- and high-specificity cut-off threshold. In contrast to the high specificity threshold, the low specificity threshold may not lead to 100% clinical specificity. Results obtained utilizing the 20 μL assay for CAA detection in serum (SCAA20).

^b the Netherlands, Burundi, Senegal: sera were also used in previous studies developing the CAA- and CCA-ELISA (Krijger et al. Reference Krijger, van Lieshout and Deelder1994; Polman et al. Reference Polman, Diakhate, Engels, Nahimana, van Dam, Ferreira, Deelder and Gryseels2000). South Africa: as described in Van Dam et al. (Reference van Dam, de Dood, Lewis, Deelder, van Lieshout, Tanke, van Rooyen and Corstjens2013). the Netherlands, as described in Corstjens et al. (Reference Corstjens, van Lieshout, Zuiderwijk, Kornelis, Tanke, Deelder and van Dam2008). Digits indicate the number of sera tested.

^c Different batches of LF strips and UCP conjugate may influence the UCP-LF ratio value. Batches are identified by production year and two letters indicating the LF strip and UCP batch, respectively.

^d Results from Burundi, the Netherlands and Senegal group for the UCP-CAA LF assay (unpublished) were reported to SCORE for the 2010 annual progress report.

Antigen – towards single worm detection with the UCP-LF CAA assay

The UCP-LF format for antigen testing was extensively studied for the detection of CAA. Previous studies indicated the potential of the UCP-LF assay to detect low-grade infections (Corstjens et al. Reference Corstjens, van Lieshout, Zuiderwijk, Kornelis, Tanke, Deelder and van Dam2008) and showed the development and application of a convenient and robust dry reagent format (Fig. 2). In the dry format a sonication step of the UCP conjugate could be omitted and further adjustments allowed worldwide shipping and storage at ambient temperature and improved shelf life (van Dam et al. Reference van Dam, de Dood, Lewis, Deelder, van Lieshout, Tanke, van Rooyen and Corstjens2013). The dry reagent format requires basic laboratory equipment (a microcentrifuge and a thermoshaker) for the TCA sample pre-treatment step and the 37 °C incubation step. The assay was developed for use with serum or plasma samples and requires an input of 20  μL TCA extract; in practice for efficient testing of a larger series, a 50 μL serum/plasma sample is requested as it enables fast and easy recovery of sufficient clear TCA supernatant (Table 2) and leaves enough material for a duplicate result if required. The lower limit of detection (LLOD) for the wet and dry reagent can be batch-dependent (batches, with a maximum of 2000 LF strips, are manually produced). The maintained quality control (QC) standard demands a LLOD of 10 and 30 pg CAA per ml for the standard 50  μL serum test (SCAA20) in the wet and dry format, respectively (Table 3). The clinical specificity of the assay is 100% based on sets of negative controls both from endemic and non-endemic countries. Recently the assay was also successfully applied for detection of CAA levels in urine. Moreover, the introduction of a concentration step after TCA extraction utilizing disposable centrifugal concentration devices provided further improvement of the sensitivity. Depending on the amount of sample and capacity of the applied concentration device, either 0·5, 4 or even 15 mL, theoretical improvements of the LLOD with a factor of 25, 200 or even 750 can be achieved. In practice the gain will be less, but development of a robust reproducible assay that can achieve levels well below 1 pg/mL (Fig. 3) is certainly feasible. Note that generally the sample volume will be restricted when testing clinical serum samples; therefore, centrifugal devices larger than 0·5 mL are not likely to be applied in blood-based assays. The 1 pg/mL level in serum is expected to allow identification of single worm infections, as in vitro worm culture studies and studies with experimentally infected baboons indicate steady state serum CAA levels of ~5 pg/mL (van Dam et al. Reference van Dam, Bogitsh, van Zeyl, Rotmans and Deelder1996a; Wilson et al. Reference Wilson, van Dam, Kariuki, Farah, Deelder and Coulson2006). For serum analysis, the sample volume is restricted, therefore limiting the increase in sensitivity that can be reached with concentrating, but for urine samples large volume applications using 4 or even 15 mL concentration devices will not cause a significant problem. Obviously it would be useful if the filtrate from urine filtrations performed for egg microscopy could be used in the CAA concentration assay. This was tested recently using freshly collected urine (experiments performed at Public Health Laboratory-Ivo de Carneri (PHL-IdC), Pemba, Tanzania, courtesy of Dr Marco Albonico and Dr Beatrice Barda) as well as stored frozen urine samples (Kahama et al. Reference Kahama, Kremsner, van Dam and Deelder1998). CAA levels in the filtrates were equal to these in unfiltered urine (results not shown). The use of concentration devices usually resulted in a reduction of the background signal of the negative controls, which maintained or improved the accuracy of the assay. Figure 3 shows the results of a typical assay employing concentration of a TCA-extracted dilution series of CAA in buffer. Filter devices with a 10 kDa cut-off were used and only for samples containing high CAA levels a minor portion was detected in the flow-through (ultrafiltrate) after concentration. As the molecular mass of CAA in AWA-TCA preparations may be different from CAA material present in serum or urine, the optimal pore size of the filtration membrane for testing clinical samples (either serum or urine) still needs to be determined.

Fig. 2. UCP-LF antigen detection assay format (CAA and CCA). The UCP-LF antigen test is available as a ‘wet reagent assay’ and a ‘dry reagent assay’. The ‘wet’ format is performed in well-equipped laboratories by qualified staff and has a 3-fold better analytical sensitivity than the ‘dry’ format assay. The ‘dry’ format allows convenient shipping and storage at ambient temperature and is less demanding in equipment and training. Sample preparation is identical for both formats and requires a TCA extraction. The TCA supernatant can be concentrated to increase sensitivity.

Fig. 3. Concentration devices to improve LLOD. Analysis of an AWA-TCA (containing 3% w/w CAA) standard series and comparison of the 20 μL assay and the 500 μL concentration assay. The standard series was prepared in 1×PBS with 0·5% Tween-20, and extracted with an equal volume of 4% (w/v) TCA; 20 μL of TCA supernatant was analysed with the UCP-LF assay without concentration, and 500 μL TCA-sup was analysed after 25-fold concentration with the Amicon 0·5 mL centrifugal device (10 kDa molecular weight cut-off membrane).

Table 2. Required clinical sample size for the various UCP-LF assay formats

^a Serum and urine collection is standardized. Saliva collection, however, is quite variable; different saliva collectors are available (Malamud et al. Reference Malamud, Bau, Niedbala and Corstjens2005; Corstjens and Malamud, Reference Corstjens, van Lieshout, Zuiderwijk, Kornelis, Tanke, Deelder and van Dam2008) and also saliva can be collected from different parts of the mouth. Currently assays are evaluated using either whole mouth-stimulated saliva (clarified by centrifugation) or saliva collected using Salivette collectors (SARSTEDT AG & Co.).

^b The required sample amount is the volume needed to ensure maximum sample input. The minimum requested sample volume is 50 μL; this allows convenient sample handling (transport and storage) and extraction of the sample with TCA.

^c The maximum sample input translates to the maximum volume of the biological matrix (relating to the CAA concentration) that is actually used in the assay. Lower amounts are feasible but will affect the lower limit of detection (note: high infection grades may require 10- or 100-fold dilution of the sample). Higher amounts are possible when using the 500, 4000 and 15 000 concentration devices; this requires reloading of the devices which can be done multiple times. Note that for urine the actual sample input is only half that of serum; a consequence of the fact that TCA extraction of serum samples generates a large pellet (about 50% of the total volume), whereas urine commonly generates only a small pellet. The composition of saliva is quite variable and TCA extractions in general generate a larger pellet than observed for urine samples, but significantly less than the serum samples.

^d In the foreseen combined testing approach, supplying 50 μL for the antigen assays would also allow testing for antibodies.

^e For antibody testing the sample input is flexible, however the volume and dilution will affect the cut-off threshold. Cut-off levels are best determined from a group representing the same ethnicity and geography.

Table 3. Quality control and lower limit of detection levels of the different UCP-LF CAA assay formats

^a Results achieved in the saliva-based matrix are not included. The saliva-based assay is not fully explored and determined levels are still considered tentative; clinical saliva-based samples from Schistosoma infected individuals were not yet tested.

^b The dry assay format is developed for use by third parties (allows convenient transport of dry reagents without a cold chain). The wet assay format is used at LUMC to achieve maximum sensitivity.

^c The QC (quality control) values present the detection values that need to be achieved when testing normal human sera or urine spiked with AWA-TCA (containing 3% w/w CAA). The CAA-20 level is the most stringent; UCP conjugates that do not pass the CAA-20 QC will not be used for preparation of dry reagents.

^d The LLOD (lower limit of detection) is the level achievable when testing under ideal laboratory conditions or when performing multiple (triplicate or more) experiments.

Antigen – development of the UCP-LF CCA assay

A UCP-LF assay for detection of CCA is being explored following similar protocols as utilized for CAA (Fig. 2). In the assay, CCA first binds to the UCP reporter (liquid phase) and the resulting UCP-CCA complex is then captured on the T line of the LF strip (solid matrix phase). The T line contains the same CCA-specific antibody as present on the UCP reporter particles, and thereby differs from the antibodies as used in the POC-CCA (van Dam et al. Reference van Dam, Wichers, Ferreira, Ghati, van Amerongen and Deelder2004). It recognizes the repeated structure of multiple Lewis x fragments from CCA (van Dam et al. Reference van Dam, Bergwerff, Thomas-Oates, Rotmans, Kamerling, Vliegenthart and Deelder1994). Because the Lewis x determinant and its repeats are also found in host-derived glycans that could be present in serum and urine (van Dam et al. Reference van Dam, Claas, Yazdanbakhsh, Kruize, van Keulen, Ferreira, Rotmans and Deelder1996b), these compounds may generate a biological background level below which a CCA-specific signal cannot be distinguished. Because of the observed natural background signals it was previously decided to maintain cut-off levels of, respectively, 2460 and 1140 pg CCA per ml urine and serum (with TCA extraction), in order to obtain a specificity of 98% (Polman et al. Reference Polman, Diakhate, Engels, Nahimana, van Dam, Ferreira, Deelder and Gryseels2000). Note that for CAA, this is completely different because of the unique polysaccharide structure, consisting of repeating disaccharide units of GlcA and GalNAc (Bergwerff et al. Reference Bergwerff, van Dam, Rotmans, Deelder, Kamerling and Vliegenthart1994), which up to now has not shown any homology to other polysaccharide structures found in nature. Initial experiments with a standard series of CCA (AWA-TCA) in buffer, urine and serum (from non-endemic controls) indicate the feasibility of the UCP-LF CCA assay (Fig. 4). Proper assay cut-off thresholds need to be determined with larger sets of urine and serum samples. The current LLOD in urine and serum is approximately 300 pg mL⁻¹, with a somewhat higher background signal for the serum assay, but further optimization of the T and FC line of the LF strip and/or assay buffer conditions will probably increase the analytical sensitivity (performance of the assay without biological matrix) to the same level as the UCP-LF CAA assay. Filtration devices with varying molecular weight limits are being explored for removal of the above-mentioned natural background. The goal is to find a protocol that allows removal of the background to such an extent that CCA levels may be used to provide additional information. It is known from past ELISA results that the different schistosome species excrete CAA and CCA in varying quantities and that the antigens may exhibit different clearance patterns from the circulation (de Jonge et al. Reference de Jonge, Fillie, Hilberath, Krijger, Lengeler, de Savigny, van Vliet and Deelder1989; Agnew et al. Reference Agnew, Fulford, de Jonge, Krijger, Rodriguezchacon, Gutsmann and Deelder1995). For this reason, development of a multiplex assay allowing simultaneous and quantitative detection of both antigens on a single LF strip will be started. This assay will also contribute to the clarification of the clearance mechanism of the two antigens from the body, which is relevant for determination of treatment efficacy and the need for (rapid) retreatment. We suggest that such an approach would also be able to indicate species specificity (of S. mansoni and S. haematobium as well as other species, including the veterinarian species) and potentially allow identification of mixed infections.

Fig. 4. Performance of the UCP-CCA assay. In the UCP-CCA assay the anti-CCA antibody #54-4C2 is immobilized at 200 ng per 4 mm on the T line of the LF strip (capture) and covalently coupled to the UCP reporter particle 25 μg per mg UCP reporter (detection). (Panel A) Ratio values determined with the UCP-CCA assay; analysis is of an AWA-TCA (3% w/w CCA) standard series in buffer, urine and serum. (Panel B) The UCP-CCA ratio value of 29 urine reference samples (various origins) compared with the visually recorded semi-quantitative POC-CCA test. The POC-CCA (RMD) scores are semi-quantified as: 0, trace, 1, 2, 3 or 4; 1 indicates the assay threshold below which samples are classified as no response (0) or a trace signal (TS), 1–4 indicate a low, medium, medium-high and high response, respectively.

Applications – quantitative genus-specific assays to measure response vs. infection intensity

Detection of CAA in serum or urine identifies infections by (probably) all known Schistosoma species. In addition to the major human species, CAA detection has also been demonstrated in Schistosoma intercalatum, Schistosoma matthei, Schistosoma bovis, Schistosoma curassoni, Heterobilharzia americana (Kremsner et al. Reference Kremsner, De, Simarro, Muhlschlegel, Mir, Sima, Feldmeier, Bienzle and Deelder1993; Agnew et al. Reference Agnew, Fulford, de Jonge, Krijger, Rodriguezchacon, Gutsmann and Deelder1995; Flowers et al. Reference Flowers, Hammerberg, Wood, Malarkey, van Dam, Levy and McLawhorn2002; Gabriel et al. Reference Gabriel, de Bont, Phiri, Masuku, Riveau, Schacht, Deelder, van Dam and Vercruysse2002) as well as Schistosoma mekongi, Schistosoma nasale, Schistosoma spindale and Schistosoma indicum (unpublished). Microscopy for schistosome eggs is generally only performed on either stool or urine depending on the expected Schistosoma species. It has been reported that in endemic areas with mixed infections, only one will be identified (usually S. haematobium as urine microhaematuria dipsticks allow easier detection of infections than stool microscopy necessary for S. mansoni) and large groups of infected patients could go undetected and untreated (Gutman et al. Reference Gutman, Fagbemi, Alphonsus, Eigege, Miri and Richards2008; Tchuem Tchuenté et al. Reference Tchuem Tchuenté, Momo, Stothard and Rollinson2013). With the recent demonstration of schistosomes being capable of forming hybrids between species that allows infection of both humans and ruminants, there will be a greater need for control programmes to identify these hybrid schistosome infections (Huyse et al. Reference Huyse, Webster, Geldof, Stothard, Diaw, Polman and Rollinson2009; Webster et al. Reference Webster, Diaw, Seye, Webster and Rollinson2013). Moreover, only standardized microscopy techniques for counting schistosome eggs provide a measure for the intensity of infection being linked with morbidity (King et al. Reference King, Dickman and Tisch2005). Obviously for these reasons a quantitative and genus-specific assay is preferred over a qualitative species-specific assay. It is also more useful for evaluating drug efficacy and studying (community) levels of associated morbidity, transmission pressure, anti-fecundity effects, as well as validation of elimination approaches. Previously CAA and CCA ELISAs have been shown to indicate infection intensities by significant correlations with egg counts as well as worm burdens in experimental infections (Deelder et al. Reference Deelder, Qian, Kremsner, Acosta, Rabello, Enyong, Simarro, Vanetten, Krijger, Rotmans, Fillie, de Jonge, Agnew and van Lieshout1994; Agnew et al. Reference Agnew, Fulford, de Jonge, Krijger, Rodriguezchacon, Gutsmann and Deelder1995; Wilson et al. Reference Wilson, van Dam, Kariuki, Farah, Deelder and Coulson2006). They were applied to, for example, analysis of worm burden variations (van Lieshout et al. Reference van Lieshout, Polderman, de Vlas, de Caluwe, Krijger, Gryseels and Deelder1995), determining different schemes of treatment (van Lieshout et al. Reference Deelder, Qian, Kremsner, Acosta, Rabello, Enyong, Simarro, Vanetten, Krijger, Rotmans, Fillie, de Jonge, Agnew and van Lieshout1994) or studying relations with different clinical appearances of the disease (de Jonge et al. Reference de Jonge, Rabello, Krijger, Kremsner, Rocha, Katz and Deelder1991).

Association between CAA levels, egg and worm counts

Using the more recently developed UCP-LF assays, previous ELISA data were confirmed with an improved resolution in the lower infection range (i.e. low worm burden). Figure 5 shows the relationship between CAA levels, worm counts and stool egg counts for a series of eight samples from previous vaccinated baboon studies (Kariuki et al. Reference Kariuki, Farah, Yole, Mwenda, van Dam, Deelder, Wilson and Coulson2004, Reference Kariuki, Farah, Wilson and Coulson2008; Wilson et al. Reference Wilson, van Dam, Kariuki, Farah, Deelder and Coulson2006), and four samples from a recent graded infection experiment (IPR, Nairobi, Dr Thomas Kariuki and Dr Philip LoVerde). Serum CAA levels (UCP-LF ratio value, Fig. 5A) and worm counts correlate well, similarly to (or slightly better than) egg and worm counts (Fig. 5B) or egg counts and CAA levels (Fig. 5C). This shows that the UCP-LF CAA assay can adequately estimate worm burdens especially at low egg counts; the UCP-LF CAA assay identified the baboon that had no eggs but 74 worms upon perfusion. Moreover, the fact that the UCP-LF CAA assay performed on a set of stored serum samples from almost 15 years ago still showed an excellent correlation with the previously determined CAA-ELISA (Fig. 5D) not only indicates the high stability of the polysaccharide analyte (CAA), but also the robustness of the immunodiagnostic assay even though testing was performed with a different mouse monoclonal anti-CAA antibody preparation and two different detection platforms.

Fig. 5. CAA serum levels versus egg and worm count. (Panel A–C) The relation between the number of eggs per gram stool, serum CAA levels expressed as UCP-LF ratio values and the total number of worms counted after perfusion of 8 baboons from previous vaccination studies (diamonds) and 4 baboons from a graded infection experiment (squares, Kariuki and LoVerde, unpublished). (Panel D) Comparison of ELISA CAA concentrations as determined in baboon serum samples from previous vaccinations studies (Kariuki et al. Reference Kariuki, Farah, Yole, Mwenda, van Dam, Deelder, Wilson and Coulson2004, Reference Kariuki, van Dam, Deelder, Farah, Yole, Wilson and Coulson2006) vs. UCP-LF CAA concentrations determined during a visit in 2012 to IPR (Nairobi, Kenya) using the same stored serum samples (n = 24). The dotted lines indicate the assay value above which an exponential increase in signal is measured for both assays (van Dam et al. Reference van Dam, de Dood, Lewis, Deelder, van Lieshout, Tanke, van Rooyen and Corstjens2013).

Evaluating drug efficiency by changes in the CAA levels

Analysis of the effect of drug administration by quantitation of CAA levels rather than monitoring egg production may provide more accurate results. Drug administration may only temporarily affect egg production and recapture later: not all worms may die, some worms may for a period of time suffer from the drug and not produce eggs, but eventually recover and again produce eggs. Determination of CAA levels immediately before and shortly after drug administration may be a better indicator for monitoring drug efficiency; CAA production will persist as long as there are live worms around, but probably at a lower level as compared with healthy worms. Factors regarding the antigen turnover time (clearance from the body; what is the optimal time point after drug administration to determine CAA levels?) and potential variation in concentration when analysing for example urine rather than blood, are still questions to be solved. Figure 6 shows the result of a study with a stored collection of urine samples from individuals with S. haematobium infections (Kahama et al. Reference Kahama, Kremsner, van Dam and Deelder1998); the set comprised four individuals without eggs in the urine and 16 with egg counts varying from 17 to 2380 eggs. CAA levels were determined before and 2 months after administration of the drug praziquantel. Panel A indicates that in all egg-positives the level of CAA decreases; however, the CAA concentration of seven individuals remains well above the cut-off threshold indicating a persistent active infection. Egg counts at 2 months post-drug treatment indicate three individuals with only 1 egg and one individual with 10 eggs. The decrease in urine CAA level indicates the effectiveness of the drug, but it also indicates that especially individuals with high egg counts may require a larger or multiple drug dose(s). Note that the UCP-LF CAA assay easily identified individuals with a low egg count.

Fig. 6. CAA levels before and after drug treatment. A set of urine samples from 20 individuals before and 2 months after praziquantel treatment (Kahama et al. Reference Kahama, Kremsner, van Dam and Deelder1998) were analysed with the 20 μL urine UCP-LF CAA assay (UCAA10, wet reagents). (Panel A) Scatter plot of the UCP-LF ratio values measured in the UCAA10 before and after treatment with praziquantel. Dashed lines indicate the UCP-LF cut-off threshold (0·0571) determined for this particular set of samples. The solid line indicates the ‘no change in CAA concentration’ position; samples with values below this line indicate a decrease of the CAA concentration 2 months after treatment. (Panel B and C) The decrease in CAA concentration (UCP-LF ratio value) and number of eggs (10 mL urine filtrate) showing the respective values ‘before’ and 2 months ‘after’ treatment with praziquantel. Dashed lines indicate cut-off thresholds for the UCP-LF ratio value (0·0571, panel A) and the egg count (arbitrarily set at 0·5 eggs per 10 mL, 1 egg is the minimum number counted).

Antibody – UCP-LF assay platform for antibody detection

The presence of antibodies against Schistosoma is a clear marker of (past) exposure to the pathogen and as such is most useful for diagnosis, for example, of people originating from non-endemic regions (Bergquist, Reference Bergquist1992). Although a limited correlation with infection intensity is sometimes observed (van Dam et al. Reference van Dam, Stelma, Gryseels, Falcao Ferreira, Talla, Niang, Rotmans and Deelder1996c), the presence of specific antibodies cannot be considered as an accurate marker for active Schistosoma infections. The absence of specific antibodies in most cases would indicate that no exposure has taken place in the past, although this may also be a consequence of chronic infection and/or indicate an immunocompromised individual or other unknown issue. It was shown (Whitty et al. Reference Whitty, Mabey, Armstrong, Wright and Chiodini2000) that about 30% of egg-positive cases had negative SEA serology, a phenomenon which was confirmed by others (unpublished).

Results obtained with an in-house ELISA (Deelder and Kornelis, Reference Deelder and Kornelis1981) detecting IgG antibodies against Schistosoma SEA were used to evaluate two new UCP-LF based antibody assays. The assays, both developed by applying the consecutive flow (CF) format, use a generic Ig-specific protein-coated UCP label (Fig. 7). The CF consists of three rapid sequential flow steps that allow unrestricted enrichment of the targeted antibodies at their respective test lines before interaction with the generic Ig specific reporter. A set of samples from a previous WHO serum bank was analysed with two types of LF strips, one containing a T line comprised of SEA antigen and one with a T line of SCAP. Figure 8 (panel A) shows the excellent correlation of the SEA antibody ELISA and UCP-CF assays. The results (Fig. 8B) further indicate that SCAP extract provides a potential alternative for the more difficult to isolate SEA; SCAP is a crude antigen preparation from freshly shed cercariae whereas SEA is prepared from eggs recovered from the liver. Further optimization and evaluation of this assay is required to obtain the same level of specificity and sensitivity as the UCP SEA antibody assay. The UCP-LF antibody assays (UCP-CF) will also be analysed for applications using non-invasive samples such as urine and saliva: the presence of antibodies in urine was recently demonstrated for S. mansoni and S. haematobium (Elhag et al. Reference Elhag, Abdelkareem, Yousif, Frah and Mohamed2011), earlier studies mentioned detection of anti-S. mansoni (Santos et al. Reference Santos, Garcia, Orsini, Disch, Katz and Rabello2000); and anti-S. japonicum antibodies (Wang et al. Reference Wang, Xue, Lou, Zhang, Zhang, Wu and Shen2002) in saliva.

Fig. 7. UCP-LF antibody detection format (anti-SEA and anti-SCAP). UCP-LF assay utilizing three sequential flow steps, referred to as consecutive flow (CF). The assay utilizes an Ig-specific label (UCP particles coated with protein A); disease/pathogen specificity is only determined by the T-line (capture zone) on the LF strip. Multiple T-lines can be used as shown in Fig. 1.

Fig. 8. Performance of two UCP-CF antibody assays. Comparison of the UCP-CF antibody assays with an in-house ELISA to detect anti-SEA antibodies using a set of sera from a previous WHO serum bank, comprised of 28 antibody negatives and 34 antibody positives. (Panel A) The ELISA and UCP-CF performed with SEA. (Panel B) The UCP-CF assays performed with SEA and SCAP. The high and low specificity cut-off thresholds for this experiment are determined according to the protocol described for the antigen assay. The dotted line indicates a threshold for the UCP-CF SEA antibody assay such that the UCP-CF test results match the SEA antibody ELISA. The indicated ELISA cut-off is defined as the average value of the negatives plus 3 s.d.

Implications and outlook

The UCP-LF based antigen assays for human (as well as veterinary) schistosomiasis approach ultimate sensitivity for detection of active infections and are presented as robust low complexity (user-friendly) assay formats for screening and detection of low-grade infection in endemic areas. In combination with the (multiplex) rapid UCP-LF based antibody assays to indicate exposure and/or previous infections with Schistosoma as well as other pathogens, a complete set of assays using a similar platform is available. The assays can be performed on serum and plasma, as well as non-invasive sample fluids such as urine or possibly saliva; further research is required to clarify whether urine or saliva are full alternatives to blood-based testing.