A robust dry reagent lateral flow assay for diagnosis of active schistosomiasis by detection of Schistosoma circulating anodic antigen

https://doi.org/10.1016/j.exppara.2013.06.017Get rights and content

Highlights

  • Improved lateral flow strip assay to diagnose active Schistosoma infection.

  • Assay materials can be stored and shipped worldwide, both at ambient temperature.

  • Assay materials shipped from the Netherlands, evaluated in Africa by local staff.

  • The lateral flow strip assay performed as least as good as the ELISA.

  • Validation of lightweight reader for analysis of the test strips.

Abstract

An earlier reported laboratory assay, performed in The Netherlands, to diagnose Schistosoma infections by detection of the parasite antigen CAA in serum was converted to a more user-friendly format with dry reagents. The improved assay requires less equipment and allows storage and worldwide shipping at ambient temperature. Evaluation of the new assay format was carried out by local staff at Ampath Laboratories, South Africa. The lateral flow (LF) based assay utilized fluorescent ultrasensitive up-converting phosphor (UCP) reporter particles, to be read by a portable reader (UPlink) that was also provided to the laboratory. Over a period of 18 months, about 2000 clinical samples were analyzed prospectively in parallel with a routinely carried out CAA–ELISA. LF test results and ELISA data correlated very well at CAA concentrations above 300 pg/mL serum. At lower concentrations the UCP–LF test indicates a better performance than the ELISA. The UCP–LF strips can be stored as a permanent record as the UCP label does not fade. At the end of the 18 months testing period, LF strips were shipped back to The Netherlands where scan results obtained in South Africa were validated with different UCP scanning equipment including a novel, custom developed, small lightweight UCP strip reader (UCP-Quant), well suited for testing in low resource settings.

Conclusion

The dry format UCP–LF assay was shown to provide a robust and easy to use format for rapid testing of CAA antigen in serum. It performed at least as good as the ELISA with respect to sensitivity and specificity, and was found to be superior with respect to speed and simplicity of use. Worldwide shipping at ambient temperature of the assay reagents, and the availability of small scanners to analyze the CAA UCP–LF strip, are two major steps towards point-of-care (POC) applications in remote and resource poor environments to accurately identify low (30 pg CAA/mL serum; equivalent to about 10 worm pairs) to heavy Schistosoma infections.

Introduction

Schistosomiasis control has recently gained increasing interest by the commitment of the 65th World Health Assembly (World Health Organization (WHO), 2012) to support interruption of transmission and even elimination is considered feasible in a number of countries. For these efforts, the availability of highly accurate diagnostics becomes a key issue and alternatives for the current standard of microscopical counting of parasite eggs in urine or stool to diagnose active schistosome infections are urgently needed. The detection of parasite-derived circulating antigens in various diagnostic assays with high sensitivity and specificity has been described extensively (Gabriel et al., 2012, Mendoza et al., 2009, Utzinger et al., 2011, van Lieshout et al., 2000, Wilson et al., 2006). Two well described circulating antigens for Schistosoma, both applied to diagnose active infection, are the circulating cathodic antigen (CCA) and the circulating anodic antigen (CAA).

The circulating cathodic antigen CCA can be detected in untreated urine of individuals with active Schistosoma mansoni infection. After initial development of monoclonal antibody based ELISA’s (de Jonge et al., 1990), a rapid point-of-care (POC) test for detection of CCA in urine has been described (van Dam et al., 2004). Several versions of this test were developed before eventually outsourcing it to Rapid Medical Diagnostics (Pretoria, South-Africa); the device has now been evaluated in various studies (Coulibaly et al., 2011, Legesse and Erko, 2007, Midzi et al., 2009, Shane et al., 2011, Standley et al., 2010). The POC–CCA test provides a rapid visual result based on a carbon or gold label but is developed for urine testing only. In the various evaluations it showed sufficiently high sensitivity and specificity to be taken up as an alternative to egg microscopy in mapping studies and field surveys. The test is particularly well-suited to accurately demonstrate moderate to heavy S. mansoni infections and can be considered as a useful method for S. mansoni diagnosis in peripheral health centers and schistosomiasis control programs (Coulibaly et al., 2011). Unfortunately, the accuracy of the POC–CCA test in Schistosoma haematobium infections is variable and needs to be further evaluated (Midzi et al., 2009, Obeng et al., 2008, Stothard et al., 2009).

Also for the second well-described schistosomal circulating antigen, circulating anodic antigen CAA, highly sensitive and specific monoclonal antibody based ELISA’s were developed and applied in numerous epidemiological and laboratory studies (Agnew et al., 1995, Deelder et al., 1989, Leutscher et al., 2008, van Dam et al., 1996a, van Lieshout et al., 1995). CAA is a genus-specific antigen with a unique carbohydrate structure (Bergwerff et al., 1994), present in serum and urine of hosts infected with various species of Schistosoma, including species infecting cattle (de Bont et al., 1996, Flowers et al., 2002, Gabriel et al., 2002). The test requires a trichloroacetic acid (TCA)-precipitation step after which CAA can be detected in the supernatant. Because of this sample pretreatment procedure and the fact that only an ELISA is available for testing, detection of CAA – despite showing a much larger potential for very sensitive detection of active schistosomiasis – for many years remained a laboratory-based assay. When the test is implemented in the daily routine, with several built-in controls and samples tested in duplicate, the ELISA may detect CAA in serum at levels as low as 40 pg/mL (Leutscher et al., 2008). When the assay is performed on a less regular basis and samples are tested only once, this sensitivity may not always be reached. ELISA standard series indicate an exponential increase in signal from around 300 to 10,000 pg/mL (Corstjens et al., 2008); a concentration of 300 pg/mL seems a “safe” level to prevent unacceptable high numbers of false positives when testing under non-optimal conditions.

Schistosoma worm pairs excrete a steady amount of CAA in the bloodstream upon feeding and the day-to-day variation of CAA in serum is fairly constant implying that the time of day is irrelevant for sample collection (Polman et al., 1998). Studies on in vitro incubated worms as well as studies with experimentally infected animals have indicated that a single worm pair would excrete a daily amount of CAA in the order of 40 ng, corresponding to 1–10 pg/mL blood (van Dam et al., 1996a, Wilson et al., 2006). In contrast to CCA which shares Lewis-X epitopes with various host components (van Dam et al., 1996b), the CAA carbohydrate structure (repeating GalNAC and GlcA disaccharides) is completely unique and no biological equivalent has so far been described. The use of CAA specific monoclonal antibodies in combination with ultra-sensitive detection platforms could thus be expected to result in further sensitivity improvements without compromising specificity.

In order to pursue the above, and also to further improve the robustness of the CAA assay and make it more applicable for future POC applications, we recently introduced a lateral flow based platform in combination with an ultrasensitive reporter technology. The resulting LF assay demonstrated an analytical sensitivity down to 1 pg/mL, about 10-fold better than the CAA–ELISA (Corstjens et al., 2008). However, the applied format was not yet optimal for distribution because of a limited batch size and due to the fact that some of the reagents needed refrigeration. Furthermore, the requirement of a sonication step added to the complexity of the assay. Here we describe a further advance towards a field applicable test through the introduction of dry reagents. The improved field-applicable assay was tested in a routine diagnostic setting in South Africa by local staff, with dry assay materials that were shipped at ambient temperature from The Netherlands. In parallel, a custom designed lightweight reader to analyze the UCP–LF strips was tested successfully.

Section snippets

Patient population and sample treatment

During a period of 18 months, 2599 serum samples were routinely analyzed for schistosomiasis by CAA–ELISA using the standard operating procedures of the Department of Serology of Ampath Laboratories (ISO 15189 and GLP/GPC certified, under supervision of Dr. L.H. van Rooyen, Dr. du Buisson, Kramer, Swart, Bouwer Inc., Centurion, South-Africa). These sera were additionally evaluated using the CAA UCP–LF strip, and after quality control, full data records of 1979 samples were obtained. The samples

Development of a robust and user friendly platform

Fig. 1 shows an overview of the dry reagent UCP–LF assay format. Drying of UCP reporter materials into a specific reporter release pad was described by Niedbala et al. (Niedbala et al., 2001). The same protocol with minor modifications (e.g. sucrose instead of trehalose) was used to dry the reporter in flat-bottom polypropylene tubes or polystyrene 8-wells strips. The reporter solution dried homogenously, sticking as a ring to the wall at the bottom of the tube/well; rehydration occurred

Discussion

The need for accurate diagnosis of active schistosome infections has recently been emphasized in several review papers (Bergquist et al., 2009, Ekpo et al., 2012, Utzinger et al., 2011). Routine diagnosis in field settings is still based on stool and urine microscopy with obvious limitations and shortcomings as low sensitivity, high variability, laborious and unhygienic procedures. Particularly, infants and preschool-aged children are difficult to diagnose by stool- and urine-based

Conclusion

The second version of the UCP–LF assay for CAA detection in serum samples involves only dried reagents and therefore allows storage and worldwide shipping of the assay at ambient temperature. The omission of a sonication step reduced complexity of the assay thereby allowing the assay to be performed by third parties after minimal training. The sensitivity of the dry assay format has been shown to be at least as good as that of the more complex and lab-based CAA–ELISA, but the variability in

Acknowledgments

Ms. D. Kornelis at LUMC and Mrs. Gloria Molaba at Ampath Laboratories are acknowledged for performing the UCP–LF assays, strip analysis and data processing. The UPlink™ reader and UCP particles were previously provided by OraSure Technologies Inc. Part of this development work was supported with financial support from US National Institutes of Health Grant UO1DE017855 and the University of Georgia Research Foundation, Inc. (SCORE project, funded by the Bill and Melinda Gates Foundation).

References (49)

  • J.R. Stothard et al.

    An evaluation of urine-CCA strip test and fingerprick blood SEA-ELISA for detection of urinary schistosomiasis in schoolchildren in Zanzibar

    Acta Trop.

    (2009)
  • J. Utzinger et al.

    From innovation to application: social-ecological context, diagnostics, drugs and integrated control of schistosomiasis

    Acta Trop.

    (2011)
  • G.J. van Dam et al.

    Schistosoma mansoni excretory circulating cathodic antigen shares Lewis-x epitopes with a human granulocyte surface antigen and evokes host antibodies mediating complement-dependent lysis of granulocytes

    Blood

    (1996)
  • L. van Lieshout et al.

    Immunodiagnosis of schistosomiasis by determination of the circulating antigens CAA and CCA, in particular in individuals with recent or light infections

    Acta Trop.

    (2000)
  • Z.Q. Yan et al.

    Rapid quantitative detection of Yersinia pestis by lateral-flow immunoassay and up-converting phosphor technology-based biosensor

    Sens. Actuators B

    (2006)
  • H.J. Zijlmans et al.

    Detection of cell and tissue surface antigens using up-converting phosphors: a new reporter technology

    Anal. Biochem.

    (1999)
  • A. Agnew et al.

    The relationship between worm burden and levels of a circulating antigen (CAA) of five species of Schistosoma in mice

    Parasitology

    (1995)
  • D.G. Colley et al.

    A five-country evaluation of a point-of-care circulating cathodic antigen urine assay for the prevalence of Schistosoma mansoni

    Am. J. Trop. Med. Hyg.

    (2013)
  • P. Corstjens et al.

    Use of up-converting phosphor reporters in lateral-flow assays to detect specific nucleic acid sequences: a rapid, sensitive DNA test to identify human papillomavirus type 16 infection

    Clin. Chem.

    (2001)
  • P.L. Corstjens et al.

    Infrared up-converting phosphors for bioassays

    IEE Proc. Nanobiotechnol.

    (2005)
  • P.L. Corstjens et al.

    Up-converting phosphor technology-based lateral flow assay for detection of Schistosoma circulating anodic antigen in serum

    J. Clin. Microbiol.

    (2008)
  • J.T. Coulibaly et al.

    Accuracy of urine circulating cathodic antigen (CCA) test for Schistosoma mansoni diagnosis in different settings of Cote d’Ivoire

    PLoS Negl. Trop. Dis.

    (2011)
  • J. de Bont et al.

    Circulating antigen levels in serum of cattle naturally infected with Schistosoma mattheei

    Parasitology

    (1996)
  • A.M. Deelder et al.

    Sensitive determination of circulating anodic antigen in Schistosoma mansoni infected individuals by an enzyme-linked immunosorbent assay using monoclonal antibodies

    Am. J. Trop. Med. Hyg.

    (1989)
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