Use of ELISA and Dipstick-assay for Detection of Strongyloides. stercoralis Infection in Humans ACCEPTED

Transcription

1 JCM Accepts, published online ahead of print on December 00 J. Clin. Microbiol. doi:./jcm.01-0 Copyright 00, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 1 Use of ELISA and Dipstick-assay for Detection of Strongyloides stercoralis Infection in Humans H. Rogier van Doorn 1, Rob Koelewijn, Henk Hofwegen, Henk Gilis 1, Jose C. F. M Wetsteyn, Pieter J. Wismans, Claudine Sarfati, Tony Vervoort and Tom van Gool 1,* 1 Section of Parasitology, Department of Medical Microbiology, Division of Infectious Diseases, Tropical Medicine and AIDS, Department of Internal Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. The Harbor Hospital and Institute of Tropical Diseases, Rotterdam, The Netherlands. Laboratory of Parasitology and Department of Infectious Diseases, Saint-Louis Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France. Prince Leopold Institute of Tropical Medicine, Central Laboratory of Clinical Biology, Antwerp, Belgium. *Corresponding author: H. Rogier van Doorn Department of Medical Microbiology, Academic Medical Center, Room L1- PO Box 0, 00DD Amsterdam, The Netherlands Phone: , Fax: , H.R.vanDoorn@amc.nl Word count: abstract: 1, text: 1 words. tables, 1 figure. Part of this work was presented as a poster at the annual spring meeting of the Dutch Society for Medical Microbiology (NVMM), april 00, Papendal, The Netherlands. 1

2 Abstract A home-made ELISA (AMC-ELISA) and dipstick-assay (Dipstick) for the detection of anti Strongyloides stercoralis antibodies in serum were developed and evaluated together with two commercially available ELISAs (IVD-ELISA, IVD Research, Inc. and Bordier-ELISA, Bordier Affinity Products SA) for their use in serodiagnosis of imported strongyloidiasis. Both commercially available ELISAs have not been evaluated before. The sensitivity of the assays was evaluated using sera from 0 patients with parasitologically proven intestinal strongyloidiasis, and from patients with clinical larva currens. The sensitivities of the AMC-ELISA, Dipstick, IVD-ELISA and Bordier-ELISA were, 1, and % for intestinal strongyloidiasis, respectively. In all tests / sera from patients with larva currens were positive. The specificity was assessed using a large serum bank of 0 sera from patients with various parasitic, bacterial, viral and fungal infectious diseases, sera containing autoimmune antibodies and sera from healthy blood donors. The specificities of AMC-ELISA, Dipstick, IVD-ELISA and Bordier-ELISA were.0,.,. and.%, respectively. Our data suggest that all four assays are sensitive and specific 1 tests for diagnosis of both intestinal and cutaneous strongyloidiasis.

3 Introduction. Strongyloides stercoralis, the causative agent of strongyloidiasis, is an intestinal nematode, endemic throughout tropical and warm, temperate regions of the world The infection is frequently imported in non-endemic areas by travelers and immigrants. The great majority of patients with strongyloidiasis is asymptomatic, although some patients have a variety of symptoms, mostly localized in the skin (larva currens) or the gastro-intestinal system (1). However, immunocompromised patients may experience accelerated autoinfection: the serious, and often fatal hyperinfection syndrome (). Because of this potential of the parasite, reliable diagnosis of patients at risk is needed for accurate recognition and treatment, before immunosuppressive therapy is installed or in patients with HIV infection. Finding the larvae in stool, in duodenal fluid, or occasionally, in other tissues or fluids, by means of microscopy, makes definitive diagnosis of strongyloidiasis. The Baermann- concentration method is one of the most sensitive tests available for assessment of the presence of larvae in fecal samples (). However, because of low larval densities, single examination with this test is insensitive (1). Multiple examinations increase the sensitivity () but these extensive parasitological investigations of individuals, who for the most part are asymptomatic, is unpractical and not cost effective. An alternative for the Baermann test is culture of stool samples (1). For both tests there is a need for freshly produced stools which, in routine practice, are difficult to obtain. 1 Serological methods to detect strongyloides infection have been studied before (,,,, 0,,, ). The most convenient and widely used method is the Enzyme Linked Immunosorbent Assay (ELISA), which detects serum IgG against a crude extract of infective larvae. However, the ELISA is somewhat cumbersome and labor-

4 intensive, and the test requires a certain level of laboratory infrastructure for performance and interpretation of results: factors that have hampered its applicability in endemic areas (). In contrast to ELISA, dipstick-assays are fast and easy to perform (1, ). Studies with other infections demonstrated a high degree of concordance between the results of ELISA and dipstick-assay (, 1, ). In general, serological assays are easier to perform and require easier obtainable patient material (serum versus fresh stools) than parasitological assays. However, most assays are homemade and require advanced laboratory infrastructure for adequate performance. Therefore, the availability of commercially available serological tests would be advantageous. In this study we evaluated a homemade ELISA (AMC-ELISA) and an easy to perform dipstick-assay (Dipstick), based on S. stercoralis antigen, for serodiagnosis of strongyloidiasis. In addition, two commercial ELISAs (IVD-ELISA and Bordier- ELISA), which were recently introduced, were evaluated.

5 Materials and methods Patients and sera. Sera used in this study were collected from patients in the Academic Medical Center (AMC, Amsterdam, The Netherlands), the Prince Leopold Institute of Tropical Medicine (ITG, Antwerp, Belgium), The Harbor Hospital and Institute of Tropical Diseases (Rotterdam, The Netherlands), and Saint Louis Hospital, Paris, France. A total of sera from patients with strongyloides infection were used: 0 from patients suffering from intestinal S. stercoralis infection, which was parasitologically proven by the Baermann-method and from patients with a clinical diagnosis of larva currens. Sera were collected within 1 month after parasitological diagnosis. Sera were stored at -0 C until used. A total of 0 sera from patients with or without an infectious disease were used as negative controls. (Table 1). Thirty-seven sera from patients suffering from parasitologically proven filariasis were also used (Table ). Antigen preparation. S. stercoralis infective larvae were obtained from a Dutch patient with an urticarial rash around the waist and on the buttocks. The patient, who had an intestinal S. stercoralis infection, had visited an endemic area (Uganda) approximately one year before. Feces with rhabditiform larvae were collected over hours and mixed with (sterile) tap water and commercially available wood shavings, and subsequently incubated for seven days at C. After days, the rhabditiform 1 larvae from the feces had completed one reproductive cycle and entered the filariform stage. Infective larvae were separated from fecal cultures by the Baermann- method (). After separation the infective larvae were washed by sedimentation ten times in phosphate buffered saline ph. (PBS) and sonicated on ice at 0 W for

6 half an hour at 0% duty cycle in a cell disrupter B 1 (Branson Ultrasonics, Danbury, Connecticut, U.S.A.). Finally the disrupted larvae were centrifuged at 1,000 x g for 0 min at C and the supernatant was used as the antigen. The protein level of the antigen was determined by the Lowry method (1). Antigen was stored at -0 C until used. Preparation of dipsticks. The dipsticks were prepared as described previously (1). Two lines were made on a nitrocellulose membrane strip (Schleicher & Schüll Bioscience, Dassel, Germany): one with cultured S. stercoralis antigen and a positive control consisting of human IgG (Nordic Immunological Laboratories, Tilburg, The Netherlands). After incubating the membrane strips for 1 hour at room temperature, the membrane strips were blocked (to prevent non-specific binding of protein) by incubating the strips for 0 min in PBS containing % (w/v) chicken egg white (PBS % Egg [Sigma-Aldrich, Inc., St. Louis, Montana, U.S.A.]). After blocking, the membrane strips were washed three times for min with PBS. The dipsticks were stored under dry conditions at room temperature Use of Dipstick. The dipsticks were incubated for 1 min with serum. After washing three times with TNT buffer (0mM Tris, 0.M NaCl, and 0.% Tween, ph.), the dipsticks were incubated with Horseradish Peroxidase (HRP) conjugated goat anti human IgG (Nordic) 1:00 in TNT 0.% Egg and subsequently washed three times and placed in a substrate solution containing,-diaminobenzidine tetrahydrochlorid and H O (ICN Biomedical, Inc., Aurora, Ohio, U.S.A.) for min. Rinsing with tap water stopped the reaction. A brown line at the position of the positive control indicates successful processing, while a brown line at the position of the antigen indicates the presence of S. stercoralis-specific antibodies in the tested serum.

7 Specific antibody ELISA (AMC-ELISA). Flat bottom polystyrene high bind microplates with small wells (Corning, Inc., Acton, Massachusetts, U.S.A.) were used. Wells were coated overnight at C with S. stercoralis antigen in 0 mm carbonate buffer ph.. Subsequently, the wells were treated with PBS 1% Egg at C for 0 min. After washing times with PBS 0.0% Tween-0, serum samples were added for 1 hour at C. After washing five times, bound antibodies were incubated with a 1:00 dilution of HRP-conjugated goat anti human IgG (Nordic) in PBS 1% Egg for 0 min at C. Subsequently, the wells were washed five times and substrate solution (0.1% -amino salicylic acid in phosphate buffer, ph. with 0.0% H 0 [Merck, Whitehouse Station, New Jersey, U.S.A.]) was added for 1 hour in the dark at room temperature. The reaction volume of each reagent at each step was 0 µl per well. The optical density (OD) was read at nm on a Multiskan ascent reader (Labsystems, Helsinki, Finland). A cut-off value was calculated from the mean OD plus twice the standard deviation of the 0 control sera. Setup of AMC-ELISA and Dipstick. For the AMC-ELISA and Dipstick the optimal concentration of S. stercoralis antigen was determined by making serial dilutions: concentrations of µg/ml and 0 µg/ml gave the best results, respectively. The optimal dilution of test serum for both assays was 1:00 and 1:0, respectively. Testing was done with sera from patients with parasitologically proven S. stercoralis infection and from healthy blood donors. Commercial ELISAs. Two commercially available ELISAs were studied. In the Strongyloides Serology Microwell ELISA Kit (IVD research, Inc., Carlsbad, California, U.S.A.) human IgG antibodies react with S. stercoralis antigen and in the ELISA from Bordier (Strongyloides ratti, Bordier Affinity Products SA, Crissier, Switzerland) specific IgG reacts with Strongyloides ratti somatic larval antigens. Both tests were

8 used according to the instructions of the manufacturers. Briefly, for the IVD-ELISA, serum samples were diluted 1: in Dilution Buffer and incubated for min in Strongyloides antigen coated wells. After washing three times with Wash Buffer, drops of peroxidase conjugated Protein A were added for min. Subsequently the wells were washed again three times, drops of Chromogen (tetramethylbenzidine) were added for min and the reaction was stopped with drops of Stop Solution (1M H PO ). The OD was measured at 0nm with 0-0nm as reference wavelength. An OD of > 0.00 indicated a positive result. For the Bordier-ELISA serum samples were diluted 1:00 in TBS-Tween solution and incubated for 0 min in S. ratti somatic larval antigen coated wells. After washes, bound activity was detected with protein A conjugated to alkaline phosphatase. Four washes, incubation with phosphatase substrate and stopping with K PO completed the reaction. Absorbance was measured at 0nm. An OD higher than that of the Weak Positive Control was considered a positive result.

9 Results Sensitivity and specificity. The specificity of all four tests was assessed with 0 sera from patients suffering from different parasitological, bacterial, and viral infections, from patients with high titers of anti nuclear antibodies (ANA) and rheumatoid factor (RF) in their blood (known to cause aspecific reactions in serological tests), and from healthy blood donors (Table 1). The cut-off value for a positive result of the AMC-ELISA was calculated from the mean plus twice the standard deviation at. With this cut-off the specificity of the AMC-ELISA was.0% (0 negative out of 0). The specificity of the Dipstick was.% (1/0), and of the IVD-ELISA and Bordier-ELISA.% (1/0). The sensitivity was calculated by testing sera from patients with S. stercoralis disease: 0 patients with intestinal infection and patients with larva currens. Sensitivities for the AMC-ELISA, the Dipstick, the IVD-ELISA and Bordier-ELISA for intestinal infection were % (/0), 1% (/0), % (0/0), and % (/0), respectively (Table ). In all tests, sera from larva currens patients gave positive results The combined results of the AMC-ELISA and Dipstick are displayed in figure 1. Because cross-reactivity between filarial and strongyloides antigens is a well-known phenomenon (,, 1-1), a large number of sera from patients with documented parasitologically proven filariasis (positive skin snip test for onchocerciasis, positive 1 blood smears for other filariases) were tested separately. The results are displayed in table. These results were not taken into account in the calculation of sensitivity and specificity of each test.

10 Discussion All four assays for serodiagnosis of strongyloidiasis had a good sensitivity and specificity. The homemade AMC-ELISA with S. stercoralis antigen ranked best, followed by the Dipstick with the same antigen, with sensitivities from 1-% and specificities from -%. The commercial IVD-ELISA and Bordier-ELISA had both high specificities (%) but did, to some extent, lack sensitivity (-%). In the home-made AMC-ELISA and Dipstick antigen of S. stercoralis was used because several dominant immunogenic epitopes are not present in Strongyloides cebus, S. ratti, and Strongyloides venezuelensis (,, ). However, large amounts of S. stercoralis antigen are difficult to obtain: stools of infected patients are needed for collection and culture. We therefore developed an economic ELISA method with microtiter plates with smaller wells (i.e., 0µl as opposed to 00µl), resulting in a tenfold reduction of antigen use (0.1µg vs. 1.µg). Isolation and culure of S. stercoralis from stools from one patient resulted in amounts of antigen sufficient to perform more than 0,000 ELISAs. The high sensitivity of the AMC-ELISA for intestinal strongyloidiasis (%) agreed with the results of earlier studies carried out with standard microtiter-plates in endemic and non-endemic areas (,,,, 0,,, ). Both high and low specificities with ELISA have previously been reported (,,,,, 0,,, ). Because of these variable results, we included a large number of 1 control sera from patients suffering from different parasitological, bacterial, and viral infections, from patients with high titers of anti nuclear antibodies (ANA) and rheumatoid factor (RF) in their blood (these sera are known to cause aspecific reactions in serological tests), and from healthy blood donors to determine the

11 specificity. Despite this variety in control groups, a high specificity was achieved. Low cross-reactivity was observed among sera from patients with echinococcosis (AMC- ELISA and Dipstick) and schistosomiasis (all assays). These false positive reactions could also be caused by occult strongyloidiasis To our knowledge, the Dipstick technique from this study has never been used for detection of antibodies against S. stercoralis. For serodiagnosis of other parasitic infections this Dipstick technique has proved to be highly sensitive and specific (, 1, 1, 0, 1). The Dipstick proved easy to fabricate, easy to use and rapid (results in less than one hour). The absence or presence of a line at the position where the antigen was incubated was unambiguous. Both sensitivity and specificity were high (1% and.%, respectively) and the agreement with the AMC-ELISA was high (κ = 0.). The advantage of a Dipstick is that it is easier to perform for less trained technicians and less equipped laboratories. Drawbacks are the use of more antigen, when compared to the AMC-ELISA (1µg vs. 0.1µg), and the inability to quantify test- results. For this reason it is suggested to use the Dipstick for fast diagnosis of strongyloidiasis, and to use the AMC-ELISA for follow-up after treatment. Eight of nine larva currens cases were positive, both in the AMC-ELISA and the Dipstick. All these cases were negative after three consecutive microscopic stool examinations by the Baermann-method, suggesting that single serological examination is more sensitive for diagnosis of larva currens than the Baermann- method, which is considered one of the most sensitive methods of stool examination (,, ). The sensitivity of the Strongyloides Serology Microwell ELISA Kit from IVD research, Inc. (IVD-ELISA) was slightly lower and the specificity in between the results from the AMC-ELISA and Dipstick (Table ). The agreement between both ELISAs was very

12 good (κ = 0.). The IVD-ELISA was very rapid (less than one hour) and easy to perform. For the production of this kit the soluble fraction of S. stercoralis L filariform larvae is used (IVD, personal communication). However, further details about the source, processing and purification of antigens were not available. In addition, the package inserts of this kit are inconsistent: different cut-off values for the ELISA are recommended for different batches of the kit. For the evaluation in this study we used kits from one batch, with a cut-off value of A kit from a later batch, with a cut- off value of 0.00 was also evaluated: when tested, a slightly higher specificity was found, but the sensitivity was reduced dramatically (Table ). The IVD-ELISA has not been cleared by the FDA. In the Bordier-ELISA (Bordier Affinity Products SA), Strongyloides ratti is used as antigen. The test has a lower sensitivity than the other assays, but had an excellent specificity. The test is also easy to perform but takes longer than the IVD-ELISA ( hours). The agreement between the AMC-ELISA and the Bordier-ELISA and between both commercial ELISAs was very good (κ = 0.1 and 0.0, respectively). The use of somatic Strongyloides ratti larval antigens could explain the slightly lower sensitivity of this test because of the incomplete cross-reactivity of both strongyloides species. This kit is also not FDA cleared. Cross-reactivity between filarial and strongyloides antigen is a well-known phenomenon that has been described previously (,, 1-1). With the four tests evaluated in this study we also observed frequent positive reactions among sera from patients with filarial infections (Table ). This strongly suggests cross-reactivity, although double infections cannot be excluded. For calculation of the cut off and specificity of the assays we excluded results from sera of filarial patients. The antigens of the IVD-ELISA appeared to be less cross-reactive than antigens used in 1

13 the other assays, especially for Wuchereria bancrofti antibodies. It has been reported that problems of cross-reactivity can - partially - be overcome by pre-incubation of sera with Onchocerca gutturosa extract to deplete the sera from anti-filarial antibodies (, 1), or by using recombinant S. stercoralis antigens for coating of the microtiter plates (1). When patients have positive strongyloides serology and originate from or have a history of travel to areas in which filariasis is endemic, it is recommended to run a test for filaria antibodies simultaneously. In our experience, patients with strongyloidiasis have higher antibody levels in ELISAs with strongyloides antigen than with filarial antigen; an observation which was reversed in patients with filariasis (data not shown). In the design of our study parasitologically proven patients were chosen as the gold standard. Although in this design it is not possible to prove superiority of the evaluated tests, two observations from the Harbor Hospital support this suggestion. Firstly, in a group of 0 patients with positive serology, parasitological confirmation was obtained with one, two or three Baermann examinations in 0, and patients, respectively. Secondly, among patients, who were clinically strongly suspected to have intestinal strongyloidiasis Baermann examinations were positive in (%), while serology was positive in (1%). False positivity as an explanation for this is unlikely, since the number of positives is much higher than in our control group. False positivity due to filarial cross-reactivity is also unlikely, since filariasis is most uncommon in our patient population. These findings both suggest that serology might be more sensitive than Baermann examinations. In addition, serology is cheaper and less cumbersome for both the patient and the laboratory. 1

14 In summary, the four assays tested are all highly sensitive and specific and can easily be implemented in routine laboratory diagnostics. Tests with S. stercoralis antigen in general have better sensitivity. Cross-reactivity of antifilarial antibodies should be considered in patients from endemic areas. Both the IVD-ELISA and Bordier-ELISA show promising results in this first-time evaluation. Further evaluations are however needed especially with respect to reproducibility of different batches of the IVD-ELISA. 1

15 Acknowledgements Leny Nieuwendijk-de Bruin, Leonie van Boetzelaer-Wittich and Esther van Schie are gratefully acknowledged for excellent technical assistance. We thank Hans Vetter for development of culture methods for S. stercoralis larvae. 1

16 References 1. Arakaki, T., M. Iwanaga, F. Kinjo, A. Saito, R. Asato, and T. Ikeshiro. 10. Efficacy of agarplate culture in detection of Strongyloides stercoralis infection. J Parasitol :-.. Bailey, J. W. 1. A serological test for the diagnosis of Strongyloides antibodies in ex Far East Prisoners of War. Ann Trop Med Parasitol :1-.. Beaver, P. C., R. C. Jung, and C. F. Craig. 1. Clinical parasitology, th ed. Lea & Febiger, Philadelphia.. Buhrer, S. S., H. L. Smits, G. C. Gussenhoven, C. W. van Ingen, and P. R. Klatser. 1. A simple dipstick assay for the detection of antibodies to phenolic glycolipid-i of Mycobacterium leprae. Am J Trop Med Hyg :1-.. Cahill, K. M., and M. Shevchuk. 1. Fulminant, systemic strongyloidiasis in AIDS. Ann Trop Med Parasitol 0:1-.. Conway, D. J., N. S. Atkins, J. E. Lillywhite, J. W. Bailey, R. D. Robinson, J. F. Lindo, D. A. Bundy, and A. E. Bianco. 1. Immunodiagnosis of Strongyloides stercoralis infection: a method for increasing the specificity of the indirect ELISA. Trans R Soc Trop Med Hyg :1-.. Conway, D. J., J. F. Lindo, R. D. Robinson, D. A. Bundy, and A. E. Bianco. 1. Strongyloides stercoralis: characterization of immunodiagnostic larval antigens. Exp Parasitol :-.. Conway, D. J., J. F. Lindo, R. D. Robinson, and D. A. P. Bundy. 1. Towards effective control of Strongyloides stercoralis. Parasitology Today :0-.. Dreyer, G., E. Fernandes-Silva, S. Alves, A. Rocha, R. Albuquerque, and D. Addiss. 1. Patterns of detection of Strongyloides stercoralis in stool specimens: implications for diagnosis and clinical trials. J Clin Microbiol :-1.. Gam, A. A., F. A. Neva, and W. A. Krotoski. 1. Comparative sensitivity and specificity of ELISA and IHA for serodiagnosis of strongyloidiasis with larval antigens. Am J Trop Med Hyg :1-1.. Genta, R. M. 1. Predictive value of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of strongyloidiasis. Am J Clin Pathol : Grove, D. I. 1. Human strongyloidiasis. Adv Parasitol : Gussenhoven, G. C., M. A. van der Hoorn, M. G. Goris, W. J. Terpstra, R. A. Hartskeerl, B. W. Mol, C. W. van Ingen, and H. L. Smits. 1. LEPTO dipstick, a dipstick assay for detection of Leptospira-specific immunoglobulin M antibodies in human sera. J Clin Microbiol :-. 1. Jelinek, T., M. P. Grobusch, and H. D. Nothdurft Use of dipstick tests for the rapid diagnosis of malaria in nonimmune travelers. J Travel Med : Lindo, J. F., D. J. Conway, N. S. Atkins, A. E. Bianco, R. D. Robinson, and D. A. Bundy. 1. Prospective evaluation of enzyme-linked immunosorbent assay and immunoblot methods for the diagnosis of endemic Strongyloides stercoralis infection. Am J Trop Med Hyg 1: Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 11. Protein measurement with the Folin phenol reagent. J Biol Chem 1:-. 1. Maizels, R. M., I. Sutanto, A. Gomez-Priego, J. Lillywhite, and D. A. Denham. 1. Specificity of surface molecules of adult Brugia parasites: cross-reactivity with antibody from Wuchereria, Onchocerca and other human filarial infections. Trop Med Parasitol :-. 1. Muck, A. E., M. L. Pires, and P. J. Lammie. 00. Influence of infection with non-filarial helminths on the specificity of serological assays for antifilarial immunoglobulin G. Trans R Soc Trop Med Hyg : Neppert, J. 1. Cross-reacting antigens among some Filariae and other nematodes. Tropenmed Parasitol :-. 0. Neva, F. A., A. A. Gam, and J. Burke. 11. Comparison of larval antigens in an enzyme-linked immunosorbent assay for strongyloidiasis in humans. J Infect Dis 1:-. 1. Ramachandran, S., R. W. Thompson, A. A. Gam, and F. A. Neva. 1. Recombinant cdna clones for immunodiagnosis of strongyloidiasis. J Infect Dis 1:1-0.. Sato, Y., F. Inoue, S. Kiyuna, and Y. Shiroma. 10. Immunoblot analysis of three antigen preparations from Strongyloides stercoralis larvae in human strongyloidiasis. Jap J Parasitol :-.. Sato, Y., M. Takara, and M. Otsuru. 1. Detection of antibodies in strongyloidiasis by enzymelinked immunosorbent assay (ELISA). Trans R Soc Trop Med Hyg :1-.

17 . Schaffel, R., M. Nucci, E. Carvalho, M. Braga, L. Almeida, R. Portugal, and W. Pulcheri The value of an immunoenzymatic test (enzyme-linked immunosorbent assay) for the diagnosis of strongyloidiasis in patients immunosuppressed by hematologic malignancies. Am J Trop Med Hyg :-0.. Snowden, K., and M. Hommel. 11. Antigen detection immunoassay using dipsticks and colloidal dyes. J Immunol Methods :-.. Tribouley-Duret, J., J. Tribouley, M. Appriou, and R. N. Megraud. 1. Diagnosis of strongyloidiasis using the E.L.I.S.A. test. Ann Parasitol Hum Comp :1-.. Verburg, G. P., and A. de Geus. 10. Strongyloidiasis in former prisoners of war and internees in Southeast Asia during World War II. Ned Tijdschr Geneeskd 1:-.. Vetter, H., and T. Van Gool. 1. Strongyloides stercoralis: een goedaardige parasiet met enkele kwaadaardige karaktertrekken. Ned Tijdschr Med Microbiol :-.. Weil, G. J., P. J. Lammie, and N. Weiss. 1. The ICT Filariasis Test: A rapid-format antigen test for diagnosis of bancroftian filariasis. Parasitol Today 1: Wever, P. C., E. P. Yzerman, E. J. Kuijper, P. Speelman, and J. Dankert Rapid diagnosis of Legionnaires' disease using an immunochromatographic assay for Legionella pneumophila serogroup 1 antigen in urine during an outbreak in the Netherlands. J Clin Microbiol :-. 1. Zhu, Y., W. He, Y. Liang, M. Xu, C. Yu, W. Hua, and G. Chao. 00. Development of a rapid, simple dipstick dye immunoassay for schistosomiasis diagnosis. J Immunol Methods :1-. 1

18 Figures and Tables Table 1: Patients from whom the sera included in this study were drawn. The number of sera used for the evaluation of all three assays is displayed for each condition. a abbreviations for each condition, used on the x-axis in Figure 1. Table : Results of three different assays when tested on true positive and true negative patient sera. a Enzyme Linked Immuno-Sorbent Assay. b In Vitro Diagnostics. NB: filariasis patients were not included in the calculation of sensitivity and specifity of all tests. Table : Results of three different assays for serodiagnosis of strongyloidiasis when tested on sera from filaria patients. a Enzyme Linked Immuno-Sorbent Assay. b In Vitro Diagnostics. c Positive. d Negative. Figure 1: Results of the AMC-ELISA for detection of anti S. stercoralis antibodies in serum combined with the results of the Dipstick. On the x-axis the patients condition is displayed; abbreviations are explained in table 1. On the y-axis the optical density measured at 0nm is displayed. Each circle represents one serum, tested with both AMC-ELISA and Dipstick. Open circles are Dipstick negative, closed circles are Dipstick positive. The dotted line represents the cut-off value for a positive result in the AMC-ELISA: circles above the dotted line are positive, circles under the line negative. NB: filariasis patients are not included in this table.

19 Number Table 1 Type of patient 0 Intestinal Strongyloides stercoralis infection SSI Larva currens LC Filariasis (Onchocerca volvulus ) Filariasis (Mansonella perstans ) Filariasis (Mansonella ozzardi ) Filariasis (Loa loa ) 1 Filariasis (Wuchereria bancrofti ) Schistosomiasis (Schistosoma mansoni ) SM Schistosomiasis (Schistosoma haematobium ) SH Katayama fever KF Hepatic fascioliasis FAS Trichuriasis TRI Hookworm infection HW Ascariasis ASC Echinococcosis (Enterococcus granulosus ) ECH Amebiasis HIS Malaria tropica MAL Visceral leishmaniasis LEI Toxoplasmosis TOX Syphilis SYP Borreliosis BOR HIV-infection HIV CMV-infection CMV EBV-infection EBV HAV-infection HAV HBV-infection HBV Rubella infection RUB Coxsackie B virus infection CBV Candida sepsis CAN Rheumatoid factor RF Anti-nuclear antibodies ANA 0 Healthy blood donors HBD Total 1

20 Intestinal strongyloidiasis No. of positive samples (Total) Sensitivity larva currens No. of positive samples (Total) Control samples No. of positive samples (Total) Specificity AMC-ELISA a (0) % () (0).0% Dipstick (0) 1% () (0).% IVD b -ELISA a cut-off 00 0(0) % () (0).% IVD b -ELISA a cut-off 00 1(0) % () 1(0).% Bordier-ELISA a (0) % () (0).% Table Pos c Neg d Pos c Neg d Pos c Neg d Pos c Neg d Onchocerca volvulus 1 1 Mansonella perstans Mansonella ozzardi Loa loa 1 Wuchereria bancrofti 1 Table Organism No. of sera AMC-ELISA a Dipstick IVD b -ELISA a 00 Bordier-ELISA a Total

21 OD Strongyloides AMC-ELISA and Dipstick-assay / AMC-ELISA value negative dipstick positive dipstick 00 0 SSI LC SM SH KF FAS TRI HW ASC ECH HIS MAL LEI TOX SYP BOR HIV CMV EBV HAV HBV RUB CBV CAN RF ANAHBD