WO2012145398A1 - Diagnostic test for schistosoma haematobium - Google Patents

Abstract

Provided is a diagnostic test for S. haematobium, using a differential filter enrichment method on a urine sample and a colorimetric assay to detect the patient's own anti- schistosome antibodies on the surface of S. haematobium eggs. Also provided is a kit for performing the diagnostic test.

Description

DIAGNOSTIC TEST FOR SCHISTOSOMA HAEMATOBIUM

[0001] This application claims the benefit of priority of U.S. Provisional Application Serial No. 61/476,630, filed April 18, 2011, the disclosure of which is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] Schistosomes are parasitic trematodes, which are endemic in 53 countries. About 85% of people with schistosomiasis live in Africa. It is estimated that 652 million people are at risk for schistosomiasis, 200 million are infected with the parasite, and 120 million have evidence of clinical infection. Twenty million of those who are infected with S.

haematobium have serious morbidity. Among human parasitic diseases, schistosomiasis is second only to malaria in terms of socioeconomic and public health consequences. (Gryseels et al. 2006.)

[0003] Schistosoma haematobium is one of the most prevalent of the several Schistosoma species that infect humans. The other common Schistosoma species— S. mansoni, S.

japonicum, S. guineensis, and S. intercalatum— generally cause gastrointestinal pathology, whereas S. haematobium causes genitourinary disease. Risk factors for being infected with S. haematobium include poverty, lack of potable water, lack of sanitary facilities, poor housing, and close contact with fresh water. Water development projects and man-made lakes and irrigation ditches have contributed to the spread of the disease. Children show much higher prevalence of infection in endemic areas compared with adults. Primary infection does not confer immunity against further infection, and people can develop heavy worm burdens over time.

[0004] The life cycle of S. haematobium begins with free-swimming larval cercaria that are released from infected fresh- water Bulinus snails and burrow into human skin when in close contact. The cercaria transform into schistosomula and travel in the bloodstream to the lungs and liver, where, after about three weeks, they mature into adult flukes. The parasite then migrates to the terminal venules in the wall of the bladder, pelvic venous plexus, and genitourinary system, where they can live for approximately three to thirty years. (Gryseels et al. 2006.) After copulation, each pregnant female produces about 20-200 eggs per day, mostly between the hours of 10 AM and 2 PM, as a result of circadian rhythms. (Gryseels et al. 2006; Kahama et al. 1998.) Most eggs are excreted in the bladder and voided in the urine. Eggs that are exposed to fresh water may hatch and release miracidia, which can then infect fresh water snails. (Gryseels et al. 2006.)

[0005] Adult worms largely escape the human immue response by coating themselves with human serum proteins. S. haematobium eggs are highly immunogenic and are responsible for the majority of the morbidity and mortality. The eggs contribute to hematuria, calcified bladder, obstructive uropathy, fibrosis of the neck of the bladder, hydroureter,

hydronephrosis, and bladder cancer. The eggs can be found in both the human male and female reproductive organs. (Gryseels et al. 2006.) People with mild worm burdens can have minimal or no symptoms. In areas heavily endemic for S. haematobium, painless hematuria in adolescence is nearly universal and is indicative of S. haematobium infection.

[0006] S. haematobium eggs are excreted into the urine covered in human

immunoglobulins. (Koech et al. 1984.) Fluorescent antibody tests showed both IgM and IgG, while immunodiffusion revealed IgG, IgM, IgA and C3. It was found that protein precipitates in human urine from people infected with S. haematobium had human IgG, IgM, IgA, IgE and complement C3 (Bosompem et al. 1996). Humans generate a IgM immune response followed by IgA, IgG, and IgE to adult worms and eggs and that IgG and IgE levels correlate with egg burden (Osada et al. 2003; Woolhouse et al. 2000; Mutapi et al. 1998). IgG levels rise dramatically after children are reinfected with S. haematobium (Mutapi et al. 1998).

[0007] Children living in S. haematobium endemic areas receive praziquantel for the treatment of schistosomiasis as part of organized mass drug administration campaigns. An inexpensive, easy-to-use, rapid diagnostic test with sufficient sensititivity for diagnosing S. haematobium infection would be useful for targeting drug treatment toward only infected individuals, monitoring S. haematobium resistance to praziquantel, improving

epidemiological mapping and monitoring of disease prevalence, and helping to guide public health intervention and prevention strategies. Current successful S. haematobium

epidemiological mapping strategies include school-based questionnaires about self-reported hematuria, as well as teacher-initiated testing of student's urine for blood. The WHO currently advocates repeated stool and/or urine examinations of at least 50 people within a defined ecological zone to quantify the severity and type of infecting Schistosoma species (Brooker et al. 2009). [0008] The inclusive societal costs and benefits to presumptively treating at-risk people for possible schistosomiasis has been compared with treatment based on the results of a potential rapid diagnostic testing. It was found that a rapid diagnostic test costing less than $3 would provide financial incentive to make treatment decisions based on the results of the rapid diagnostic test as well as allow for mass screenings for the disease to improve targeted interventions. A rapid diagnostic test costing $5 would be cost neutral. (Stothard 2009.)

[0009] The performance of a diagnostic assay can be measured by its sensitivity— the ability of the test to identify positive results in people having a disease— and by its specificity— the ability of the test to identify negative results in undiseased people. For example, a test that is not sensitive will miss some people who are infected with S.

haematobium, while a test that is not specific will return positive results in people who are not infected with S. haematobium.

[0010] The gold- standard test for detecting S. haematobium is seeing the eggs in a urine specimen under the microscope (Gryseels et al. 2006). Typically this is done by centrifuging or filtering the urine to concentrate the eggs onto filter paper that can then be evaluated microscopically. While urine microscopy is highly specific if eggs are identified, the sensitivity can suffer due to intermittent egg shedding and when a low number of eggs are present in the urine. Urine microscopy usually involves filtering two different lOmL samples from the each person and evaluating both samples under the microscope and averaging the number of eggs and reported as # eggs/lOmL. Urine microscopy requires a microscope, a trained technician, and the time and resources to read the slides. These limitations make urine microscopy difficult to perform, especially in the field, and during mass screenings or epidemiological surveys.

[0011] Hematuria, and to a lesser extent, proteinuria, are associated with S. haematobium infection. Urine dipsticks for microscopic hematuria and proteinuria are being used to guide treatment and public health interventions for S. haematobium. Urine dipsticks are inexpensive, and hematauria is highly specific for S. haematobium infections in endemic areas. However, hematuria has low sensitivity for diagnosing light infections. (Houmsou et al. 2011; Fatiregun et al. 2009; Sousa-Figueiredo et al. 2009; Ugbomoiko et al. 2009; Nduka et al. 2008; French et al. 2007.) [0012] Antibody-based assays can be used detect serum IgG, IgM, or IgE against soluble worm antigen or crude egg antigen using enzyme immunoassay, indirect haemagglutination, or immunofluorescence. These techniques are very sensitive, but not very specific in that they can cross-react with parasites other than S. haematobium. In addition, seroconversion does not occur until 4-8 weeks after the initial infection, and antibodies can still be detected in individuals two or more years after the infection has been cured. Moreover, antibody- based assays are expensive and difficult to carry out under field conditions in most endemic areas. (Gryseels et al. 2006.)

[0013] An ELISA urine dipstick test that detects schistosome circulating cathodic antigen (CCA) has been developed and is commercially supplied by Rapid Medical Diagnostics (South Africa). CCA is a group of proteins regurgitated by the worm into the host circulation, and is then excreted in the host urine at concentrations that can be detected by ELISA. CCA is not genus specific, meaning that infection with any schistosome will be detected. A further disadvantage of the CCA dipstick test is that it costs between $2.60 and $4.60 per test. Additionally, CCA has low sensitivity for detecting S. haematobium infection.

[0014] PCR and oligonucleotide probes can also be used to detect parasite DNA in urine, but these methods are technically challenging, expensive, and have limited field application. (Ibironke et al. 2011)

[0015] Several different methods of diagnosing S. haematobium have been evaluated using latent class analysis (LCA), including urine antigen detection of S. haematobium complexed to C3 (98% sensitive and 35% specific), detection of anti-schistosome IgG from dried blood spots (48% sensitive and 57% specific), ultrasound examination (74% sensitive and 65%o specific), and urine microscopy (93% sensitive and 98% specific) (Koukounari et al. 2009). There have been conflicting reports on the sensitivity and specificity of schistosome CCA, but it is thought to be relatively insensitive for detecting S. haematobium (Stothard 2009).

[0016] There is no commercially available diagnostic test for S. haematobium that is quick, inexpensive, easy to perform, and has a high sensitivity and specificity. In resource-poor countries with endemic S. haematobium, a rapid diagnostic test is urgently needed to direct treatment and resources, to conduct epidemiological mapping, and to monitor the

effectiveness of drug treatment. Developed countries, such as the United States, have large numbers of returning travelers and immigrants from S. haematobium endemic areas, and could also benefit from an easily performed test for S. haematobium.

SUMMARY OF THE INVENTION

[0017] The present invention provides a rapid diagnostic test for S. haematobium (RDT- Sh), using a differential filter enrichment method on a urine sample and a colorimetric assay to detect the patient's own anti-schistosome antibodies on the surface of S. haematobium eggs. The method of the invention is rapid, inexpensive, and does not require a highly trained technician to perform the assay or read the results.

[0018] In one aspect, the invention provides a method for detecting a Schistosoma haematobium infection in a human subject by passing a sample through a filter, wherein the sample comprises urine from the human subject and an anti-human immunoglobulin antibody linked to an enzyme; and applying to the filter a substrate for the enzyme, wherein reaction of the enzyme with the substrate produces a detectable signal; and whereby detection of the signal indicates a Schistosoma haematobium infection in the subject. This method describes the steps of the RDT-Sh of the invention.

[0019] In another aspect, the invention provides a method for detecting Schistosoma haematobium eggs in a urine sample by passing a sample through a filter, wherein the sample comprises urine from a human subject and an anti-human immunoglobulin antibody linked to an enzyme; and applying to the filter a substrate for the enzyme, wherein reaction of the enzyme with the substrate produces a detectable signal; and whereby detection of the signal indicates the presence of Schistosoma haematobium eggs in the urine sample. This method describes the steps of the RDT-Sh of the invention.

[0020] In a further aspect, the invention provides a kit containing a syringe, a filter on or in a housing that is capable of being connected to the syringe, a first reagent comprising an anti- human immunoglobulin antibody linked to an enzyme, and a second reagent comprising a substrate for the enzyme. This kit can be used to carry out the RDT-Sh of the invention. The kit can include components sufficient in number for carrying out one test or multiple tests.

[0021] In one embodiment of the methods of the invention, the sample is passed through the filter using a syringe. In one embodiment, the filter used in the methods and kit of the invention has a pore size of at least about 10 microns. In another embodiment, the filter has a pore size of about 40 microns or less. [0022] The sample preferably comprises at least about 10 mL of urine. The anti-human immunoglobulin antibody used in the methods or kit of the invention is preferably an anti- human IgG antibody. In one embodiment, the enzyme coupled to the antibody is horseradish peroxidase, and the substrate is 3,3'5,5'-tetramethylbenzidine base (TMB). In the methods of the invention, the signal is preferably detected within twenty minutes of applying the substrate to the filter.

[0023] The methods of the invention can include an optional step, wherein the sample is passed through a first filter having a pore size of about 200 microns to about 1 ,000 microns prior to passing the sample through the filter that will collect the eggs. In this optional embodiment, the first filter preferably has a pore size of about 400 microns. The first filter can be optionally included in the kit of the invention.

[0024] Another optional step that can be included in the methods of the invention is passing a wash solution through the filter after filtering the sample, but before applying the substrate for the enzyme to the filter.

[0025] In an additional aspect, the invention provides an algorithm for diagnosing

Schistosoma haematobium infection. In the first step, urine samples are visually inspected, and all grossly bloody samples (i.e., having gross hematuria) are categorized as positive for S. haematobium infection. In the second step, all urine samples that are negative for gross hematuria are evaluated for microscopic hematuria by dipstick analysis, wherein samples that are positive for microscopic hematuria are categorized as positive for S. haematobium infection. In the third step, all urine samples that are negative for microscopic hematuria are evaluated using RDT-Sh, wherein samples that are positive by RDT-Sh are categorized as positive for S. haematobium infection, and wherein samples that are negative by RDT-Sh are categorized as negative for S. haematobium infection. Performing the RDT-Sh on only those samples without hematuria, as provided by the algorithm of the invention, improves the sensitivity and specificity of the diagnostic test.

[0026] All described aspects of the present invention can employ any of the transitional phrases "comprising," "consisting essentially of," and "consisting of to define what unrecited additional components or steps, if any, are included in or excluded from the scope of a claim directed to the invention. [0027] The term "about" as used in this disclosure means "±10%" when modifying a quanity and "approximately" when modifying a percentage.

[0028] The individual embodiments of the invention disclosed herein can be used in any combination with one another.

[0029] All publications cited in this application are hereby incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The drawings illustrate particular embodiments of the invention and are not intended to be limiting. Other embodiments are described throughout the specification.

[0031] Figure 1 shows an embodiment of the invention using a syringe (1) to pass a sample (2) through a filter contained within a housing (3).

[0032] Figure 2 shows a cross-section of the embodiment shown in Figure 1. The filter (4) is contained in a housing (3), which is attached to a syringe (1) through which the sample (2) is passed.

[0033] Figure 3 shows an embodiment of the invention wherein a filter (3) can be attached to a female-female luer lock coupling device (2), which attaches to a syringe (1) through which a sample can be passed.

[0034] Figure 4 shows an embodiment of the invention wherein a first filter (3) is contained within a housing (2) that is attached on one side to a syringe (1) and on the other side to a second filter (5), contained within a housing (4).

[0035] Figure 5 shows an algorithm to rapidly identify individuals with S. haematobium infection.

DETAILED DESCRIPTION

[0036] Human immunoglobulins coat S. haematobium eggs in the urine (Koech et al.

1984). These immune complexes have complicated the use of ELISA tests for detection of egg antigens (Uga et al. 1989; Koech et al. 1984). The RDT-Sh of the invention works by identifying these immune complexes on the S. haematobium eggs. Anti-human

immunoglobulin antibodies conjugated to enzymes, and their substrates, are inexpensive and readily available. [0037] According to the European Association of Urology (EAU), a light S. haematobium infection is < 100 eggs/1 OmL urine and >400 eggs/1 OmL is a severe infection (Bichlet et al. 2006). The World Health Organization (WHO) classifies a light infection as <50 eggs/ 1 OmL urine and a heavy infection as >50 eggs/ 1 OmL (WHO Meeting Report 2010).

[0038] The mapping and treatment of neglected tropical diseases is being facilitated by the use of accurate rapid diagnostic tests that exist for malaria, onchocerciasis, visceral leishmaniasis, and lymphatic filariasis (Brooker et al. 2009). The urine antibody test to detect CCA has good sensitivity and specificity for S. mansoni, but not for S. haematobium.

Concurrently using CCA and the RDT-Sh of the invention on a single urine sample could allow for non-invasive, accurate, and rapid mapping of schistosomiasis throughout Africa, where S. mansoni and S. haematobium frequently co-exist. Additionally, after successful treatment with praziquantel, both the CCA and RDT-Sh should both become negative. This will allow for monitoring of praziquantel resistance, an increasing public health concern, especially since there are few acceptable treatment alternatives for schistosomiasis.

[0039] The invention provides a method for detecting a Schistosoma haematobium infection in a human subject by passing a sample through a filter, wherein the sample comprises urine from the human subject and an anti-human immunoglobulin antibody linked to an enzyme; and applying to the filter a substrate for the enzyme, wherein reaction of the enzyme with the substrate produces a detectable signal; and whereby detection of the signal indicates a Schistosoma haematobium infection in the subject. This method describes the steps of the RDT-Sh of the invention. The invention also provides a method for detecting S. haematobium eggs in a urine sample using the RDT-Sh of the invention.

[0040] The invention further provides a kit containing a syringe, a filter on or in a housing that is capable of being connected to the syringe, a first reagent comprising an anti-human immunoglobulin antibody linked to an enzyme, and a second reagent comprising a substrate for the enzyme. The kit can be used to carry out the RDT-Sh of the invention.

[0041] The sample can be passed through the filter using any means that will entrap S. haematobium eggs on the filter. For example, the sample can be passed through the filter by any type of pump device, including a positive displacement pump, a gravity pump, or a centrifugal pump. A syringe is an example of a positive displacement pump. In a preferred embodiment of the invention, the sample is drawn up into a syringe and passed through a filter coupled to the syringe (Fig. 1). Alternatively, the sample can be passed through the filter by gravity, using, for example, a conical funnel with the filter located at one end of its stem, or a Buchner funnel. In another alternative, the sample can be passed through the filter by use of a vacuum, for example, using a bottle-top filtering unit.

[0042] The filter used in the RDT-Sh can be made of any suitable material, including but not limited to cellulose, polyethersulfone, polycarbonate, polypropylene,

polytetrafluoroethylene, nylon, and the like. S. haematobium eggs are approximately 110- 170 microns x 40-70 microns in size. Urine can contain components such as ammonia, salts, water, proteins, carbohydrates, red blood cells (6-8 microns), white blood cells (10 microns), trichomoniasis (10 microns), crystals, yeast (10-20 microns), and bacteria (< 3 microns). Therefore, the filter preferably has pores that are small enough to retain S. haematobium eggs, but large enough to pass other debris in the urine. Preferably, the filter has a pore size of at least about 10 microns, 20 microns, or 30 microns. Preferably, the filter has a pore size of less than about 70, 60, 50, or 40 microns. Preferably, the pore size is between about 10 and about 40 microns, most preferably between about 20 and about 30 microns.

[0043] The filter can be connected to the funnel, syringe, other pumping device, etc., by any means that ensures that the sample will pass through the filter. For example, where a Buchner funnel is used, the filter is connected to the funnel by resting on the bottom of the bowl of the funnel, on top of the exit holes through which the sample passes out of the funnel. Where a syringe is used, the filter is preferably connected to the syringe by a housing having a luer lock coupling device. For example, the filter can be affixed to one side of a female- female adaptor (Fig. 3), for example, using glue. Alternatively, the filter can be contained in a housing (Fig. 2, Fig. 4). It is preferable that the housing is configured such that the filter can be removed after the RDT-Sh is performed, which will allow for microscopic analysis of the filter to quantify the number of eggs, if desired.

[0044] The sample preferably comprises between about 1 mL and about 1000 mL urine, more preferably at least about 10 mL urine, even more preferably at least about 60 mL urine, and most preferably at least about 100, 200, 300, 400, 500, 600, 700, 800, or 900 mL urine. When the sample passed through the filter has a larger volume, the incidence of false negative results is lower. Utilizing an entire urine sample for the RDT-Sh will add as many eggs as possible onto the filter and should provide improved sensitivity, especially in light infections. [0045] The anti-human immunoglobulin antibody linked to an enzyme is added to the urine prior to filtering in a concentration sufficient to bind to S. haematobium eggs in the sample. Preferably, the antibody is added to the urine sample at a final concentration in the sample of between about 0.1 ng/mL and about 1000 ng/mL. Preferably, the final concentration of antibody in the sample is between about 1 ng/mL and about 5 ng/mL, most preferably about 2 ng/mL. More or less antibody may be required depending upon the extent of infection, with lighter infections requiring a higher concentration of antibody for detection. Antibody concentrations that are too high might result in increased incidence of false positives. One of ordinary skill in the art is able to determine the concentration of antibody required under the particular conditions.

[0046] The anti-human immunoglobulin antibody can be an antibody against human IgA, IgE, IgG, or IgM. In a preferred embodiment, the antibody is an anti-human IgG antibody.

[0047] The antibody can be conjugated to any enzyme that can be reacted with a substrate to yield a detectable signal. The reaction between the enzyme and the substrate can be a spectrophotometric, f uorimetric, chemiluminescent, or radio assay. Preferably, the reaction between the enzyme and the substrate produces a signal that can be observed by the naked eye or by the use of equipment that detects absorbance or emission of light, including UV, visible, and IR wavelengths. Preferably, the reaction between the enzyme and the substrate is a colorimetric reaction. The reaction can be a direct reaction or a coupled reaction. One example of a coupled reaction uses the hexokinase enzyme, which can be assayed by coupling its production of glucose-6-phosphate to NADPH production, using glucoses- phosphate dehydrogenase.

[0048] Non-limiting examples of enzyme-substrate pairs suitable for use in the invention include:

• horseradish peroxidase as the enzyme and 3,3'5,5'-tetramethylbenzidine base (TMB); 2,2'-azino-di-(3-ethylbenzthiazolinesulfonic acid), sodium salt (ABTS); 3-amino-9-ethyl carbazole (AEC); 4-chloro-l-naphthol; 3,3'-diaminobenzidine (DAB); 3,3'-diaminobenzidine, tetrahydrochloride salt; o-phenylenediamine (OPD); o-phenylenediamine, tetrahydrochloride salt; 4-aminoantipyrine; 4- aminoantipyrine hydrochloride; 5 -aminosalicylic acid; 2,4,-dichlorophenol; N,N- diethyl-m-toluidine; Ν,Ν-dimethylaniline; 3-methyl-2- benzothiazolinonehydrazone hydrochloride hydrate; or 2,4,6-tribromo-3- hydroxybenzoic acid as the substrate;

alkaline phosphatase as the enzyme and 5-bromo-4-chloro-3-indolyl phosphate, p- tol (X-Phos, BCIP); 5-bromo-4-chloro-3-indolyl phosphate, disodium salt (X- Phos, diNa salt); 6-chloro-3-indolyl phosphate, p-tol (Red-Phos®); 3-indoxyl phosphate, disodium salt; naphthol AS-MX phosphate; naphthol AS-TR phosphate, disodium salt; naphthol AS phosphate, disodium salt; a-naphthyl acid phosphate, sodium salt; p-nitrophenyl phosphate, disodium salt (p-NPP); 3-0- methylfluorescein phosphate, MCHA salt; 4-methylumbelliferyl phosphate; blue tetrazolium; disodium 1-naphthyl phosphate hydrate; disodium 4- nitrophenylphosphate hexahydrate; fast red B salt, 1,5-naphthalenedisulfonate; 2- (4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride; monosodium 1- naphthyl phosphate monohydrate; nitro blue tetrazolium; 4-nitrophenylphosphoric acid bis[tris(hydroxymethyl)aminomethane] salt hydrate; tetranitro blue tetrazolium; or variamine blue B diazonium salt, as the substrate;

β-galactosidase as the enzyme and 5-bromo-4-chloro-indolyl-P-D- galactopyranoside (X-Gal); 5-bromo-6-chloro-3-indolyl-P-D-galactopyranoside; 5-bromo-3-indolyl-P-D-galactopyranoside; 6-chloro-3-indolyl-P-D-galactoside (Red Gal®); 5-chloro-3-indolyl-P-D-galactoside; 3-indolyl-P-D-galactoside; 6- bromo-2-naphthyl-P-D-galactoside; 5-carboxy-2-nitrophenyl-P-D-galactoside; o- nitrophenyl-P-D-galactoside (ONPG); p-nitrophenyl-P-D-galactoside; 2- nitrophenyl-P-D-galactopyranoside; 4-nitrophenyl-P-D-galactopyranoside;

fluorescein di-P-D-galactoside; 4-methylumbelliferyl-P-D-galactoside; or resorufin-P-D-galactopyranoside as the substrate;

β-glucuronidase and 5-bromo-4-chloro-3-indolyl-P-D-glucuronic acid, sodium salt (X-Gluc, Na salt); 5-bromo-4-chloro-3-indolyl-P-D-glucuronic acid, MCHA salt (X-Gluc); 6-chloro-3-indolyl-P-D-glucuronide (Red-Gluc®); 6-bromo-2-naphthyl- β-D-glucopyranoside; naphthol AS-BI-P-D-glucuronide; or 4-methylumbelliferyl- β-D-glucuronide (MUG) as the substrate;

a-amylase as the enzyme and 2-chloro-4-nitrophenyl-alpha-maltotrioside as the substrate; • β-lactamase as the enzyme and chromogenic cephalosporin (CENT A) as the substrate;

• luciferase as the enzyme and D-(-)-luciferin as the substrate.

Considerations when choosing an enzyme-substrate pair include cost, availability, and stability of the reagents. In a preferred embodiment, the enzyme is horseradish peroxidase and the substrate is TMB.

[0049] The RDT-Sh results are preferably observed between 0 and 20 minutes of applying the substrate to the filter. In one preferred embodiment, the signal is detected within about 1 minute of applying the substrate to the filter. In another preferred embodiment, the signal is detected between about 1-2 minutes after applying the substrate to the filter. Heavier infections will produce a positive result almost instantaneously, while lighter infections can take longer to produce a positive result. The RDT-Sh is more sensitive, but less specific, as the time between applying the substrate and observing the results is increased.

[0050] The methods of the invention can optionally include a wash step between the step of passing the sample through the filter and applying the substrate to the filter. The wash solution can be any suitable liquid, including water, saline, or buffer. The wash step may improve the specificity of the RDT-Sh by reducing false positives.

[0051] Optionally, the methods of the invention can include pre-filtration of the sample prior to passing the sample through the filter to collect the S. haematobium eggs (Fig. 4). For example, the sample can first be passed through a filter having a pore size of about 200 microns to about 1,000 microns, preferably about 400 microns. The first filter can be made of any suitable material. Preferably, the first filter is comprised of synthetic material that will bind non-specific proteins, which can cause false positive results. The pre-filtration will also reduce the likelihood that the egg-collection filter will get clogged and break, and will allow for faster filtration.

[0052] In addition to anti-human immunoglobulin antibodies, antibodies to S.

haematobium eggs can optionally be added to the urine samples to increase the amount of antibodies bound to the eggs. The anti- S. haematobium antibodies can be conjugated to the same or a different enzyme as the anti-human immunoglobulins. If a different enzyme is used, a second substrate is passed through the filter concomitantly with or sequentially to the substrate for the conjugated anti-human immunoglobulins. In a preferred embodiment, the anti-iS*. haematobium antibodies are conjugated to the same enzyme as the anti-human immunoglobulin antibodies.

[0053] The following Examples demonstrate that the RDT-Sh can detect S. haematobium eggs by detecting the presence of human IgG in urine filtrate. The RDT-Sh is inexpensive, quick, easy, non-technical to perform, and can be done in the field. The RDT-Sh has excellent sensitivity for all but the lightest of infections. By utilizing the RDT-Sh on only those samples without hematuria, the overall sensitivity can be increased beyond either RDT- Sh or hematuria independently. Additionally, evaluating the RDT-Sh filter microscopically for eggs on only those samples negative for both hematuria and the RDT-Sh test at one minute improves the sensitivity to about 100%.

EXAMPLES

[0054] The following examples are meant to illustrate the methods and materials of the present invention and are not intended to limit the invention in any way.

EXAMPLE 1. STUDY DESIGN

[0055] This study was conducted in the Kwale District on the coast of Kenya that is endemic for schistosomiasis and soil-transmitted helminth infections. A survey conducted in July 2007 in six schools across the district found that the prevalence of S. haematobium was 44% (3-75%).

[0056] Urine was collected between 10:00am-2 :00pm on 177 school children, of which 160 were between the ages of 8-17 and qualified for use in data analysis. Data collection was done at two different schools. The age and sex of all study subjects was recorded. Urine from each participant was noted to be yellow/clear, turbid, or grossly bloody. The urine was then analyzed for protein and blood using a Rapid Response™ urine dipstick (BTNX Inc., Markham, Ont., Canada). Two lOmL urine samples were removed and each was filtered using Nuclepore™ polycarbonate 12 micron filters (Whatman Ltd., Maidstone, Kent, UK). The two filters were then placed on a glass microscope slide and fixed. The number of eggs on each filter was determined with microscopy, and the average between the two samples was used to estimate the average number of eggs per l OmL urine. The remainder of the urine sample was analyzed by the RDT-Sh. All data collection, including the RDT-Sh, was done in the field, with the exception of the urine microscopy, which was read in the laboratory within 24 hours of preparing the slide.

[0057] Importantly, the RDT-Sh used in this study only cost about $0.45 per reaction, and the cost could likely be reduced even further if wholesale or bulk pricing is available.. Urine dipsticks for detection of microscopic hematuria and proteinuria cost about $0.15 apiece.

EXAMPLE 2. DETECTION OF HUMAN IMMUNOGLOBULINS BOUND TO S. HAEMATOBIUM EGGS

[0058] RDT-Sh filters were prepared in advance. A standard sized hole-punch was used to create paper disks of wet strengthened grade 113 cellulose with a 30 micron pore size (Whatman GE Healthcare Life Sciences, Piscataway, NJ). Using a hot glue gun, the paper disks were glued onto one end of a polycarbonate female-female luer lock coupling device (Cole-Parmer, Vernon Hills, IL).

[0059] To prepare the antibody used in the RDT-Sh, commercially available anti-human IgG-conjugated to horseradish peroxidase(Promega Corp., Madison, WI) was diluted to 400ng/mL in unsterile commercially available bottled drinking water. The antibody was diluted on the day of the experiment and kept at room temperature. 0.25mL of the diluted anti-human IgG antibody was added to each urine sample and mixed.

[0060] Up to 60 mL urine containing the anti-human IgG conjugated antibody was drawn up into a syringe, which was attached to the open end of the female luer lock coupling device. The other end of the luer device contained the glued filter paper. The urine was filtered, and S. haematobium eggs remained trapped on the inner aspect of the filter paper. The empty syringe was removed, and a syringe containing 3,3'5,5'-tetramethylbenzidine base (TMB) was attached to the open female luer lock coupling device. Approximately 0.25mL of TMB was pushed through the filter. Since TMB turns from a pale yellow liquid to blue in the presence of horseradish peroxidase, the filter paper changed color to blue if the RDT-Sh was positive and remained white if the RDT-Sh was negative. A positive RDT-Sh result was noted to occur immediately, within 30 seconds, 60 seconds, or within 20 minutes of adding the TMB. By 24 hours, previously positive (blue) RDT-Sh filters where indistinguishable from negative RDT-Sh filters (all where white).

[0061] The results of the RDT-Sh were independently compared with those of urine microscopy in a double-blinded manner. All subjects, regardless of the urine microscopy or RTD-Sh results, received Praziquantel for treatment of S. haematobium, as directed by the Kenyan Ministry of Health. The results are summarized in Table 1.

Table 1. Comparison of Detection of Infection by Microscopy Versus RDT-Sh

[0062] The RDT-Sh was positive in 89%> of urine samples containing >1 egg/10 mL (58/65 samples) and in 97% of urine samples containing >11 eggs/10 mL (35/36 samples). The RDT-Sh was negative in 79% of cases where no eggs were seen using microscopy. Instances of a positive result using RDT-Sh where microscopy was negative could be due to the fact that up to three times more urine was used for RDT-Sh than for urine microscopy, thus providing a chance to capture more eggs on the filter where infection was light. Because the RDT-Sh filters were attached to the coupling device using glue, it was not possible to analyze the same filters using microscopy.

[0063] LCA is a method of statistical analysis that is useful when the gold-standard method of diagnosing the infection is imperfect, such as urine microscopy for S. haematobium infection; LCA has been used in other S. haematobium studies (Koukounari et al. 2009). When LCA is used to analyze the data incorporating urine microscopy, RDT-Sh, protein in the urine, and microscopic hematuria results into the analysis, the sensitivity is 97% and the specificity 78% when the RDT-Sh is read at one minute; the sensitivity is 96% and the specificity 44% when the RDT-Sh is read at 20 minutes [Table 2].

Table 2. Sensitivity and Specificity of RDT-Sh

Microscopic hematuria 96% (75-100%) 99% (91-100%)

Protein in the urine 59% (38-80%) 89% (83-95%)

[0064] The RDT-Sh is clearly better when more eggs are present on the filter. The higher the concentration of eggs/1 OmL urine was associated with the RDT-Sh becoming positive faster. When more than 1 1 eggs/ 1 OmL urine was present, which is still considered to be a "light infection", the RDT-Sh was positive in 29 out of 30 samples (97% sensitive) at 1 minute and 30 out of 30 samples (100% sensitive) at 20 minutes.

[0065] The number of eggs present on the RDT-Sh filter can be estimated by taking the number of eggs per milliliter of urine seen on urine microscopy and multiplying it by the volume of urine filtered for the RDT-Sh. Those results are summarized in Table 3.

Table 3. Estimate of Eggs Per Filter

5 samples with incomplete data and not analyzed

[0066] The RDT-Sh was less likely to be positive when very few eggs were likely to be found in the filter. The average volume of urine filtered on the RDT-Sh when different concentrations of eggs were present on the corresponding urine microscopy is shown in Table 4.

Table 4. Egg Count Relative to Average Volume of Urine Filtered

[0067] A few of the urine samples contained visible sediment and obvious foreign bodies such as dirt and grass, clogging the RDT-Sh filter 39 out of 160 times (24%). In some cases, this limited the amount of urine available for filtration. In addition, the filter broke under the filtering process 15 out of 160 times (9%), although this did not necessarily affect the results. In general, grossly blood or cloudy urine resulted in lower RDT-Sh filtration volumes.

[0068] The ability of microscopic hematuria and proteinuria (as detected by urine dipstick) and the RDT-Sh to detect S. haematobium infection, as determined by urine microscopy is summarized in Table 5.

Table 5. Comparison of Microscopy and Dipstick Tests

[0069] Three study subjects had microscopic hematuria on urine dipstick and no S.

haematobium eggs on microscopy; one subject was a 10-year old female who had a negative RDT-Sh. The other two subjects were 9- and 16-year old males who both had positive RDT- Sh results within one minute.

[0070] The presence of hematuria indicates S. haematobium infection with good specificity in endemic areas, but the sensitivity is modest. The RDT-Sh has good sensitivity for diagnosing S. haematobium infection. The data in the RDT-Sh study was analyzed using the following algorithm (Fig. 5):

• All grossly bloody urine samples were considered positive for S. haematobium.

• All urine samples negative for gross hematuria were evaluated for microscopic hematuria.

• Those that were positive for microscopic hematuria were considered positive for S.

haematobium, and those negative for microscopic hematuria were evaluated using RDT- Sh.

• Those positive by RDT-Sh were considered infected with S. haematobium and those

negative by RDT-Sh were considered negative.

[0071] This algorithm would correctly identify 85% (66 of 78) of samples with 0.5 to > 1000 eggs/10 mL, as observed using urine microscopy, and would identify as negative 78% (64 of 82) samples with no eggs observed using microscopy. If all samples with no hematuria and negative RDT-Sh were examined microscopically for the presence of eggs, and those filters with eggs were considered positive for S. haematobium infection, the algorithm would have 100% sensitivity, 84% specificity. This would require microscopic examination of only 76 samples (to identify the 12 samples in the data set with no hematuria, a negative RDT-Sh, and eggs seen on urine microscopy), compared with examination of 320 filters if each of the original 160 urine specimens had two lOmL urine samples filtered and examined. This algorithm could be especially helpful in field settings where all but the lightest S. haematobium infections could be quickly and easily detected and treated. Where the algorithm is used, samples negative for both hematuria and the RDT-Sh could have their RDT-Sh filters examined microscopically at a later time (i.e. back in the lab or clinic) to detect nearly 100% of urine samples with S. haematobium eggs.

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Claims

CLAIMS I claim:

1. A method for detecting a Schistosoma haematobium infection in a human subject, the method comprising:

(a) passing a sample through a filter, wherein the sample comprises (i) urine from the human subject and (ii) an anti-human immunoglobulin antibody linked to an enzyme;

(b) applying to the filter a substrate for the enzyme, wherein reaction of the enzyme with the substrate produces a detectable signal;

whereby detection of the signal indicates a Schistosoma haematobium infection in the subject.

2. The method of claim 1, wherein the filter has a pore size of at least about 10 microns.

3. The method of claim 1, wherein the filter has a pore size of about 40 microns or less.

4. The method of claim 1, wherein the sample comprises at least about 10 mL of urine.

5. The method of claim 1, wherein the human immunoglobulin is IgG.

6. The method of claim 1, wherein the enzyme is horseradish peroxidase.

7. The method of claim 6, wherein the substrate is 3,3 '5,5 '- tetramethylbenzidine base (TMB).

8. The method of claim 1 , wherein the sample is passed through the filter using a syringe.

9. The method of claim 1 , wherein the sample is passed through a first filter having a pore size of about 200 microns to about 1,000 microns prior to step (a).

10. The method of claim 9, wherein the first filter has a pore size of about 400 microns.

11. The method of claim 1 , further comprising passing a wash solution through the filter between step (a) and step (b).

12. The method of claim 1, wherein the detectable signal is produced by a colorimetric reaction.

13. The method of claim 1, wherein the signal is detected within twenty minutes of applying the substrate to the filter.

14. A kit comprising (i) a syringe, (ii) a filter on or in a housing that is capable of being connected to the syringe, (iii) a first reagent comprising an anti- human immunoglobulin antibody linked to an enzyme, and (iv) a second reagent comprising a substrate for the enzyme.

15. The kit of claim 14, wherein the filter has a pore size of about 40 microns or less.

16. The kit of claim 14, further comprising a second filter having a pore size of about 200 microns to about 1,000 microns, wherein the second filter is on or in a housing that is capable of being connected to the syringe.

17. The kit of claim 16, wherein the second filter has a pore size of about 400 microns.

18. The kit of claim 14, wherein the human immunoglobulin is IgG.

19. The kit of claim 14, wherein the enzyme is horseradish peroxidase.

20. The kit of claim 19, wherein the substrate is 3,3'5,5'- tetramethylbenzidine base (TMB).