Evaluation of the co-agglutination test in diagnosis of experimental microsporidiosis

Experimental Parasitology 128 (2011) 18–25

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Evaluation of the co-agglutination test in diagnosis of experimental microsporidiosis Maha R. Gaafar Parasitology Department, Faculty of Medicine, Alexandria University, Alexandria, Egypt

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Article history: Received 14 June 2010 Received in revised form 20 January 2011 Accepted 24 January 2011 Available online 4 February 2011 Keywords: Encephalitozoon intestinalis Cyclospora cyatenensis Cryptosporidium parvum Co-A test

a b s t r a c t Microsporidiosis is an emerging and opportunistic infection associated with wide range of clinical syndromes in humans. Confirmation of the presence of microsporidia in different samples is laborious, costly and often difficult. The present study was designed to evaluate the utility of the Co-agglutination test (CoA test) for detection of urinary, fecal and circulating microsporidial antigens in experimentally infected mice. One hundred and twenty male Swiss albino mice were divided into non infected control and infected experimental groups which were further subdivided into two equal subgroups; immunosuppressed and immunocompetent. Microsporidial spores were isolated from human stools and identified to be Encephalitozoon intestinalis by the molecular methods. They were used to infect each subgroup of mice, then their urine, stools and sera were collected at the 1st, 3rd, 5th, 7th and 9th days post-infection (PI). Co-A test, using prepared hyperimmune serum, was used to detect antigens in all samples collected. The cross reactivity of microsporidial hyperimmune sera with antigens of Cyclospora cyatenensis and Cryptosporidium parvum was investigated by Co-A test. The results showed that Co-A test was effective in detecting microsporidial antigen in stool of immunosuppressed infected mice from the 1st day PI, and in urine and serum from the 3rd day PI till the end of the study. In the immunocompetent subgroup, Co-A test detected microsporidial antigens in stool, serum and urine of mice from the 1st day, 3rd day and the 5th day PI, respectively till the end of the study, without cross reactivity with C. cyatenensis or C. parvum in both subgroups. Co-A test proved to be simple and suitable tool for detecting microsporidial antigen in different specimens and did not need sophisticated equipment. It is very practical under field or rural conditions and in poorly equipped clinical laboratories. Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction Microsporidia are small obligate intracellular spore-forming micro-organisms, first recognized over 100 years ago and infect a wide variety of vertebrate and invertebrate hosts. Among the numerous microsporidial genera seven have been described as causing human diseases; Enterocytozoon bieneusi (E. bieneusi), Encephalitozoon species (E. cuniculi, E. hellem and Encephalitozoon intestinalis), Nosema, Pleistophora, Trachipleistophora, Vittaforma and Brachiola. E. intestinalis and E. bieneusi are the most frequently identified human parasites (Franzen and Müller, 1999; Weiss, 2000). Microsporidia spp. have a worldwide distribution. Cases of microsporidiosis have been reported in both developed and developing countries and among both immunosuppressed and immunocompetent individuals. Currently, most cases of microsporidiosis are reported in immunosuppressed individuals, and studies have found that E. bieneusi infection of small intestinal enterocytes is detected in 15–34% of patients with AIDS with chronic diarrhea and E-mail address: [email protected] 0014-4894/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2011.01.017

no other identified causes. Moreover, reports of E. bieneusi infections are increasing among travelers and residents of tropical countries who do not have human immunodeficiency virus (HIV) infection (Garcia, 2002; Didier et al., 2004; Didier and Weiss, 2006). Hosts are commonly infected by ingestion or inhalation of spores passed in the faeces or urine (Didier, 2005). Infection with microsporidia spp. is usually asymptomatic, and may cause nausea, low grade fever, abdominal cramps, anorexia and watery motions. In immunocompetent subjects, the illness is generally self limiting. However, such a heavy loss of electrolytes due to diarrhea can be an important cause of morbidity and mortality in immunocompromised patients and needs special consideration (Tuli et al., 2010). Furthermore, several clinical syndromes have been reported in HIV-infected individuals including enteropathy, keratoconjunctivitis, sinusitis, tracheobronchitis, encephalitis, interstitial nephritis, hepatitis, cholecystitis, osteomyelitis, and myositis (Wasson and Peper, 2000; Jones et al., 2004). E. bieneusi is the commonest microsporidia causing diarrhea and malabsorption, followed by E. intestinalis which is associated with disseminated disease (Kotler and Orenstein, 1998). Traditionally, the diagnosis of this infection has relied upon microscopic detection of spores in stool specimens

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Fig. 1. Microsporidial spores in patient’s stool stained with modified trichrome stain (1000).

using modified trichrome stain (MTS). However, these methods are laborious, require experience, are time consuming, have low sensitivity and are dependant on the ability of the observer, and hence are restricted for routine practice (Franzen and Müller, 1999). Unfortunately, sensitive, reliable, and easily performed methods for identification and speciation of microsporidia are generally not available (Lujan et al., 1998). This problem can potentially be overcome if immunological assays, which can be performed by none expert laboratory technicians, are developed. Immunological diagnosis is based on detection of circulating antibodies directed against the parasite using ELISA, indirect immunofluorescence (IFAT) or carbon immunoassay (CIA), the most commonly used serological methods for diagnosis in these species (Akerstedt, 2002). However, detection of antibodies does not differentiate between past and recent infection (Michel et al., 2000). Antigen detection assays have been developed using monoclonal antibodies (MAbs) which are determined by Western blot and have proved their efficacy (Lujan et al., 1998). The Co-agglutination test (Co-A test) is a simple and rapid slide agglutination test that can be performed in a routine laboratory without any need for trained personnel, expensive reagents, or equipment. The test is based on the immunological reaction between specific parasite antibodies bound to protein A-bearing Staphylococcus aureus Cowan I strain (SAPA) cells and parasite antigen (Karki and Parija, 1999). Some applications have already been found in parasitology for detection of antigens in water, serum, urine and stool (Michel et al., 2000; Ravinder et al., 2000; Devi and Parija, 2003; Parija and Reddy, 2006). However no reports are available on its efficacy for the detection of antigen in microsporidial infections. The present study aimed to evaluate the utility of Co-A test for detection of microsporidial antigen in different specimens (urine, stool and serum) of experimentally infected animals.

2. Materials and methods Stool samples were collected from 118 immunosuppressed patients with chronic diarrhea, in the Alexandria University Hospital and Fever Hospital from August 2009 to April 2010. Informed consent obtained taken from the patients. All specimens were microscopically screened by the conventional diagnostic method using direct wet saline smear, iodine smear and ether sedimentation technique. Furthermore, samples were stained by modified trichrome stain (MTS) in order to select 20 positive cases of microsporidia with heavy infection (Fig. 1) (Garcia and Bruckner, 1997).

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The spores were isolated from the 20 stool samples by Lumb’s technique. In short, each stool sample was mixed with 10 ml of distilled water, and then filtered through coarse sterile gauze. The homogenate was then centrifuged at 2500g for 5 min. The supernatant fluid was discarded, and the sediment was washed twice in 1 ml of phosphate buffer saline (PBS), with centrifugation at 13,000g for 2 min. After repeated washing followed by centrifugation, faecal debris was totally eliminated. Approximately half the spores obtained were preserved in 2.5% potassium dichromate and stored at 4 °C until use for infection and for antigen preparation (Lumb et al., 1993). The remaining spores were fixed in equal volumes of glycerol and PBS, and stored at 20 °C until used for molecular studies (Carnevale et al., 2000). DNA extraction for microsporidia was done for the 20 positive stool samples using DNA Zol reagent (GIBco BRL), 5 ll of the extracted DNA from each sample was loaded on 1% agarose gel, and the rest was stored at 20 °C until used for polymerase chain reaction (PCR) amplification (Silva et al., 1996). PCR amplification used two 16S-rDNA PCR universal primers, the forward primer (C1) was complementary to bases 1 to 18 of each microsporidial species, while the reverse primer (C2) was complementary to bases 1169 to 1186 of E. intestinalis (accession No. U09929) and bases 1152 to 1170 of E. bieneusi (accession No. L16868). PCR amplification was performed using an automated thermal cycler (PTC-200 MJ Research). Amplicons were electrophoresed through 1% agarose gel, stained with ethidium bromide in Tris/borate EDTA buffer (Breitenmoser et al., 1999). The primer pair used in this study resulted in efficient amplification in only 14 of the 20 stool samples. Restriction fragment length polymorphism analysis of the PCR products (PCR–RFLP) was applied with the help of two restriction endonucleases Hind III and Hinf I, which were used to digest the 16S-rDNA amplified fragments using Qiaquick spin columns (Qiagen). The fragments were separated by electrophoresis on 2% agarose gel and visualized with ethidium bromide (Fig. 2). The fragments in each pattern were compared for all samples (Raynaud et al., 1998). By comparing the fingerprint patterns of all samples, they could be classified into four different species. Subsequently, sequencing was performed in order to identify the species using an ABI PRISM dye terminator cycle sequencing kit with Ampli Taq DNA polymerase. All sequences were analyzed using the CHECK-CHIMERA and the SIMILARITY-RANK programs of the Ribosomal Database Project (Liguory et al., 2000). The sequence analysis identified eight parasite isolates as E. intestinalis when comparing their nucleotide sequences with the available database sequences in the GenBank. Three out of the eight samples preserved in 2.5% potassium dichromate, proven to be E. intestinalis by molecular methods, were pooled, and found to be enough for completion of the study. 2.1. Preparation of soluble antigen The E. intestinalis spores, washed twice in PBS, were sonicated for 30 min. The number of spores before and after disruption was counted in a haematocytometer to ensure that at least 95% of the spores were disrupted in the homogenate. After centrifugation (10 min at 1500g), the protein content of the supernatant was measured according to Loury’s method Loury et al., 1951). With the protocol used for antigen preparation, approximately 108 of microsporidia spores per ml gave a protein concentration in the final antigen solution of 0.3 mg/ml. The soluble antigen solution was aliquoted and was stored at 20 °C until use (Akerstedt, 2002). 2.2. Preparation of hyperimmune sera Hyperimmune sera were produced in two New-Zealand white male albino rabbits, aged 9–10 weeks, weighing 1600–1800 g.

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Fig. 2. Agarose gel (2%) electrophoresis analysis showing the digestion of 16S-rDNA amplified products by restriction endonuclease Hinf I.

They were housed in well ventilated large cages, supplied with water and leafy vegetables such as lettuce and potatoes. Bedding was changed every day. Rabbit stools were examined by direct wet saline smear, iodine and Sheather’s sugar flotation method to exclude the presence of any parasite (Garcia and Bruckner, 1997). Using the prepared microsporidial antigen, hyperimmune sera were produced following a pilot study using a dose of 5  104 spores/rabbit every 2 weeks for 6 weeks. One week after the final dose, exsanguination of the rabbits was performed after an overdose of diethyl-ether. The serum was separated from the blood by centrifugation at 2000g for 10 min (Turnen, 1983). Collected sera were stored at 20 °C. This animal study was approved by the Ethics Committee of Alexandria University. The hyperimmune serum titre was detected by direct agglutination test (DAT), as described by Silva et al. (2005) and Abou Elnaga et al. (2008). In brief, the test was conducted in 96 well round bottom microtitre plates. Twenty-five microlitres serum diluted 1:5 in PBS was mixed thoroughly with 75 ll antigen solution. The plate was carefully shaken, covered with a lid, and incubated overnight at 37 °C in a CO2 incubator. The agglutination reactions were read the next morning. Central discrete opaque dot or buttons was scored as negative and diffuse opacity across the entire diameter of the well as positive reaction. The reaction was positive with titre of 1:640.

of the blood and intestines at the 1st, 3rd, 5th, 7th and 9th day PI. Each time twelve uninfected mice and twelve of the experimental infected mice were sacrificed (six of Subgroup IIa and six of Subgroup IIb, respectively). Urine samples were collected in Eppendorf tubes follow ng stimulation of the animal by reflexes as sudden kinking of the tail and abdominal massage. The tubes were put into a tube holder and placed into a boiling water bath for 5 min, and then allowed to cool to ambient temperature. After centrifugation at 1400g for 10 min, the supernatant was collected and preserved at 20 °C until use (Rench et al., 1984; Rajalakshmi et al., 2002; Sundar et al., 2005). The urine was tested initially at three different concentrations; undiluted, 1:5 and 1:10 dilution. The optimal concentration was undiluted urine. Stool samples were centrifuged at 1400g for 10 min and the supernatant fluids were tested immediately for the antigen or stored at 4 °C for later testing (Pai et al., 1985). In a pilot study, the stool was tested at three different concentrations; undiluted, 1:5 and 1:10 dilution. The optimal concentration was stool diluted at 1:5 with PBS. After collecting blood, serum was separated by centrifugation at 2000g for 10 min. Serum was preserved at 20 °C until use (Turnen, 1983). Serum was initially tested at three different concentrations; undiluted, 1:2 and 1:4 dilution. The optimal concentration was serum diluted 1:4 with PBS.

2.3. Experimental mice 2.5. Co-agglutination test Animals used in this work were male Swiss albino mice, aged 3– 5 weeks, weighing 20–25 g. They were housed in well ventilated cages with perforated covers, supplied with standard pellet food and water. Bedding was changed every day. The mice were allowed to adapt to the laboratory environment for 1 week before the experiment (El-Fakhry et al., 1998), and their stools were examined by direct wet saline smear, iodine and Sheather’s sugar flotation method to exclude presence of parasites (Garcia and Bruckner, 1997). This animal study was approved by the Ethics Committee of Alexandria University. The mice were divided into two equal groups of 60 mice each; control non-infected group (Group I) and experimental infected group (Group II). The experimental group was further subdivided into two equal subgroups; immunosuppressed infected group (Subgroup IIa), and immunocompetent infected group (Subgroup IIb). Each mouse in subgroup IIa was immunosuppressed with cyclophosphamide (endoxan), using two doses of 70 mg/kg intraperitoneally, given a week apart. The last dose was given 48 h before infection (Sherwood et al., 1982). Each mouse of group II was inoculated orally with 0.1 ml of the isolated E. intestinalis spores at a dose of 105 spores/ml. Ingestion was performed by gastric gavage, using a 23-gauge needle tipped with plastic tubing (Allam et al., 1999; Gaafar, 2007).

Co-agglutination test (Co-A test) was used to detect microsporidial antigens in urine, stool and blood samples. S. aureus (Cowans’ strain I) bearing protein A cells (SAPA cells; Sigma No. 9151) were sensitized by mixing 1 ml 10% SAPA cell suspension with 0.5 ml antimicrosporidia rabbit hyperimmune serum, and incubation for 3 h at 37 °C. The Co-A test was performed on a glass slide divided by a glass marking pen into two halves. A drop of the sample was placed on each half of the glass slide. Volumes of 2% sensitized and unsensitized SAPA cell suspensions were added to one half of the glass slide, respectively. Unsensitized SAPA cells were added as a cell control. The slide was rotated manually for 2 min and inspected. For positive reactions, specific parasite antigen present in the sample agglutinated the sensitized SAPA cells, resulting in visible clumping. Agglutination was graded from 1 (+) (weak positive reaction) to 4 (+) (strong positive reaction) based on size of the clumps and clarity of the background: fine granularity with milky background (+), small clumps with milky background (++), small and large clumps with clear background (+++), and large clumps with clear background (++++). Microsporidial antigen was used as a positive control. Normal serum, urine and stool samples were used as negative controls (Karki and Parija, 1999; Devi and Parija, 2003).

2.4. Sampling

2.6. Cross-reactivity of the Co-A test

First, urine and stool were collected. Mice were then sacrificed by cervical incision using overdose of diethyl-ether for collection

In order to evaluate the cross-reactivity of the Co-A test, samples from mice infected with Cyclospora caytenensis (C. caytenensis)

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and Cryptosporidium parvum (C. parvum) were tested. Their oocysts were detected in patients’ stool by modified Ziehl Neelsen stain, and were preserved in 2.5% potassium dichromate. Sporulation for Cyclospora oocysts was done by putting the oocysts in small covered Petri dish at room temperature for 7–10 days. Frequent examinations were done till sporulation occurred (Sadaka and Zoheir, 2001). Mice were then infected with each of the sporulated oocysts of C. caytenensis and C. parvum at a dose of 104 oocysts/ml (Gaafar, 2007). Urine, stool and sera were collected, stored, and tested by the Co-A as previously described. 2.7. Microscopic examination Undiluted stool samples from all groups of mice were stained by MTS and examined by microscopy to detect microsporidial spores (Garcia and Bruckner, 1997). After sacrifice, the small intestines were dissected from both control and experimental groups of mice, fixed in 10% formalin, embedded in paraffin sections and stained by haematoxylin and eosin stain (H&E) (Drury and Wallington, 1980) and MTS (Weber et al., 1992). The sections were examined for parasites and for pathological lesions. 2.8. Calculations Mean and standard deviation were calculated for microsporidial spores in stool samples and intestinal sections according to Knapp and Miller, 1992. 3. Results The mean number of microsporidial spores in the stool and intestinal sections, and the results of the Co-A test in detection of antigen in the urine, stool and serum of the experimentally infected mice are summarized in Tables 1 and 2. In immunosuppressed mice (subgroup IIa), the Co-A test detected microsporidial antigens in the stool samples from the 1st day post-infection (PI), and in the serum and urine samples from the

3rd day PI, but there was more antigen in serum (++) than in urine (+). After the 3rd day, microsporidial antigen was detectable till the end of the study. In immunocompetent mice (subgroup IIb), the Co-A test detected a low level of microsporidial antigen in the stool samples from the 1st day PI, which increased with the duration of the study. In the serum samples from subgroup IIb, microsporidial antigen was detected from the 3rd day PI, and in the urine samples from the 5th day PI. After that, the amount of detected antigen increased and reached its maximum at the 7th day PI (++++), then started to decrease at the 9th day PI. No microsporidial antigen was detected in the control group (Figs. 3 and 4 [A–E]). The positive and negative controls were strong positive (++++) and negative ( ), respectively. Microsporidial spores were only detected in the stool samples stained by MTS in subgroup IIa and subgroup IIb at the 3rd day PI and the 5th day PI respectively until the end of the study. In addition, microsporidial spores were detected in the intestinal sections of both subgroups IIa and IIb as groups of intracellular organisms located within enterocytes, between the nuclei and the brush border at the 3rd day PI until the end of the study, which confirm the infection. No pathological changes were observed in these intestinal sections. The specificity of the Co-A test was investigated using urine, stool and sera from mice infected with Cyclospora cyatenensis or C. parvum oocysts. The test was negative for these samples. Thus no crossreactivity was detected.

4. Discussion Through increased awareness and improved diagnostics, microsporidiosis has now been identified in a broader range of human populations that includes not only immunosuppressed hosts, but also includes travelers, children, and the elderly (Didier and Weiss, 2006). Over the years, detection of microsporidia has been a challenge. Several methods have been used, but many of these techniques are cumbersome and time consuming. Moreover, microsporidia were usually missed by microscopy and staining. Therefore, rapid and sensitive techniques are needed to provide an early diagnosis of this infection which can influence therapeutic

Table 1 Detection of microsporidial spores in stool and intestinal sections and antigens in urine, stool and sera of infected immunosuppressed mice at different times post-infection. Time post-infection (PI)

Microsp. spores number (MTS count/ OIL in stool samples) mean ± SD

Microsp. spores number (MTS count/ OIL in intestinal sections) mean ± SD

1th day 3rd day 5th day 7th day 9th day Positive control Negative control

ND 17.4 ± 1.3 19.6 ± 5.14 22.7 ± 3.36 25.6 ± 1.25

ND 20.64 ± 1.83 22.07 ± 6.11 28.01 ± 7.46 31.71 ± 5.16

Co-agglutination test results Urine

Stool

Sera

+ ++ +++ ++++ ++++

++ +++ ++++ ++++ ++++ ++++

++ +++ ++++ ++++ ++++

+, ++, +++, +++ positive, graded from weak (+) to strong (++++) reaction negative, ND not determined, MTS modified trichrome stain, OIL oil immersion lens.

Table 2 Detection of microsporidial spores in stool and intestinal sections and antigens in urine, stool and sera of immunocompetent infected mice at different times post-infection. Time post-infection (PI)

Microsp. spores number (MTS count/ OIL in stool samples) mean ± SD

Microsp. spores number (MTS count/ OIL in intestinal sections) mean ± SD

1th day 3rd day 5th day 7th day 9th day Positive control Negative control

ND ND 16.6 ± 4.54 19.7 ± 1.33 22.6 ± 1.72

ND 16.3 ± 2.52 19.4 ± 8.63 21.8 ± 5.57 25.7 ± 4.14

+, ++, +++, +++ positive, graded from weak (+) to strong (++++) reaction,

Co-agglutination test results Urine

Stool

Sera

++ ++++ ++ ++ ++

+ ++ + ++ ++ ++++ +++ ++++

++ +++ ++++ +++ ++++

negative, ND not determined, MTS modified trichrome stain, OIL oil immersion lens.

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Fig. 3. Negative and positive reactions of the Co-A test on a microscopic glass slide.

intervention (Tuli et al., 2008). In this study, we report a simple and economic slide agglutination test for detecting microsporidial antigen in urine, stool and sera of both immunosuppressed and immunocompetent experimentally infected mice. The study of experimentally infected mice enabled us to determine the exact day of infection and to follow the course of the disease in different immune states, which could also be used in human cases as proposed by Michel et al. (2000). In the infected animals, Co-A test was able to detect the antigen in stool samples on the 1st day PI which were negative by MTS. This could be explained by intermittent shedding of the spores in the stool as their excretion is cyclic (Garcia and Shimizu, 1993). Additionally, no pathological changes caused by microsporidia were observed in infected intestinal sections. This agrees with Curry and Smith (1998), who explained that the intestinal infection by microsporidia could be often missed, as the organisms stain poorly with conventional histological stains and the intestine is completely normal. Microsporidial antigen in the present work was detected in urine, stool and serum samples of the infected animals. This may be explained by the wide spread of the E. intestinalis infections that often involve multiple body sites. This is supported by Garcia (2002), who stated that detection of microsporidial spores in any clinical specimen should be followed by examination of other body tissues and fluids, especially in patients whom disseminated microsporidiosis is suspected. Also, he added that Encephalitozoon spores are often shed sporadically in urine specimens, thus, they should be submitted for examination. A single negative urine specimen would not rule out the possibility of infection with microsporidia. On the first day of the infection, the antigen was not detected in urine or serum samples of all groups of mice that were positive by Co-A test on stool samples. Also, in immunosuppressed group (subgroup IIa), the antigen was detected in both serum and urine samples from the 3rd day PI, but the antigen in the serum was higher than that of the urine. This could be attributed to the presence of the organism in the intestine early in the infection; thus, the antigen was absent from the circulation, hence not secreted in the urine. By the 3rd day PI, the organism had started to disseminate, the serum antigen appeared, and was then secreted in the urine in lower amount. This was supported by Michel et al. (2000), who suggested that the intestinal parasite stayed in the intestine in the beginning of the infection, and dissemination occurred later. In immunocompetent mice (subgroup IIb), the negative Co-A test in the urine of animals at the third day of infection in comparison to positivity in the serum may be due to low levels of antigen in the urine which could not be detected by the Co-A test as suggested by Ravinder et al. (2000).

Furthermore, in immunocompetent mice, the Co-A test detected high levels of microsporidial antigen in all tested samples at the 7th day PI, which was followed by decrease in the antigen at the 9th day PI. This could be explained by the appearance of antibodies which react with the antigen resulting in a decrease in its level. This agreed with Salat et al. (2001) who suggested that the specific antibodies against E. cuniculi could be demonstrated from day 9 PI in the BALB/c infected mice sera. Moreover, Omalu et al. (2007) reported that microsporidia antibodies were first detected in sera of rabbits and mice approximately 7–10 days PI. On the other hand, the results of the Co-A test for the immunosuppressed group of mice was different as all the detected antigens were raised until the end of the study on the 9th day PI. This may be attributed to the deficient immune state and different immune reaction in this group. In the present study, there was no cross reactivity between microsporidia and C. cyatenensis or C. parvum by Co-A test. This was agrees with Abou Elnaga et al. (2008), who stated that there was no cross reactivity between microsporidia and C. cyatenensis or C. parvum by direct agglutination and fast agglutination screening tests. Furthermore, Kili et al. (2008) did not show any cross reactivity in the agglutination test for visceral leishmaniasis with other protozoal diseases as malaria. A variety of serologic testing methods have been used to detect immunoglobulin G and immunoglobulin M antibodies to microsporidia (primarily E. cuniculi) in animals (Akerstedt, 2002; Garcia, 2002). Lujan et al. (1998) reported the production of 21 MAbs specific to spore antigens of several species of microsporidia. Immunoelectron microscopy and Western blot confirmed the reactivity of these specific MAbs to the spore wall or with a few antigens. These MAbs were useful in the diagnosis and speciation of microsporidia, and detection of these antigens. Thus, currently, the available serologic data are interesting, but simple, reliable and suitable technique for rapid screening of human microsporidial infection in clinical practice is not yet available. The data presented in this study showed that Co-A using a SAPA cell suspension coated with microsporidial antisera was able to detect the presence of urinary, fecal and serum antigen in specimens from experimental animals with microsporidiosis. The Co-A test for detecting antigens offers many advantages. The technique is extremely simple, and even paramedical health personnel in a rural health center, or in a poorly equipped laboratory can perform this test on a glass microscope slide. The test does not require any special equipment or technically trained or skilled manpower. The infected persons residing in these rural areas cannot afford to pay for high-technology approaches and expensive assays such

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Fig. 4. Grades of Co-A test reactions: (A) no clumping (negative reaction [ ]). (B) Fine granularity with milky background (weak positive reaction [+]). (C) Small clumps with milky background [++]. (D) Small and large clumps with clear background [+++]. (E) Large clumps with clear background (strong positive reaction [++++]).

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as immunofluorescence, enzymelinked immunosorbent assay, or radioimmunoassay (Shariff and Parija, 1993; Karki and Parija, 1999). In addition, the Co-A test is rapid; the result can be obtained within minutes of performing the test. The use of SAPA cells as the principal Co-A reagent makes the test economical to use in many of the laboratories in developing countries. The SAPA cells can be prepared with a simple centrifuge and once prepared can be stored for weeks in a refrigerator (+4 °C) without any loss of sensitivity (Ravinder et al., 2000; Parija and Reddy, 2006). Urine and stool, in comparison to blood, appears to be ideal specimens for diagnosis. First and foremost, urine and stool can be collected easily and frequently without causing any inconvenience to the patient. Second, as urine and stool are collected by non-invasive procedure, the risk of blood-borne infections such as human immunodeficiency virus and hepatitis B virus, associated with the collection of blood samples is avoided. Detection of urinary and stool antigen might be particularly useful with the elderly, children, and other persons unwilling to provide blood for tests (Durigon et al., 1991; Islam et al., 1995; Parija, 1998; Ravinder et al., 2000). In conclusion, Co-A test proved to be a rapid and suitable tool for detecting microsporidial antigen in different types of specimen. It provided a good alternative to the invasive and labor intensive methods that are needed for parasitological diagnosis, particularly in resource-poor countries. Thus, this test is very practical in epidemiological surveys and in poorly equipped routine clinical laboratories. Nevertheless, we should bear in mind the limitations of comparing data from animal models to human situation. It is now necessary to undertake similar studies directly with microscopically proven human infection material in order to validate the test for human diagnosis. Acknowledgement The molecular work was supported by the Genetic Engineering and Biotechnology Research Institute, Mubarak City for Scientific Research and Technology applications. I also appreciate the technical assistance provided by Professor Doctor Desouky Ahmed Abd El-Haleem. References Abou Elnaga, I.F., Gaafar, M.R., El-Zawawy, L.A., El-Said, D., Mossallam, S.F., 2008. The utility of direct agglutination (DAT) and fast agglutination screening (FAST) tests in serodiagnosis of experimental microsporidiosis. Journal of Egyptian Society of Parasitology 38, 903–918. Allam, S.R., Sadaka, H.A., Eissa, M.M., Baddour, N.M., 1999. A novel macrolide in mixed protozoal infection in immunosuppressed mice. Journal of Medical Research Institute 20, 149–159. Akerstedt, J., 2002. An Indirect ELISA for detection of Encephalitozoon cuniculi infection in farmed blue foxes (Alopex lagopus). Acta Veterinaria Scandinavica 43, 211–220. Breitenmoser, A.C., Mathis, A., Burgf, E., Weber, R., Deplazes, P., 1999. High prevalence of Enterocytozoon bieneusi in swine with four genotypes that differ from those identified in humans. Parasitology 118, 447–453. Carnevale, S., Velasquez, J.N., Labbe, J.H., Chertcoff, A., Cabrera, M.G., Rodriguez, M.I., 2000. Diagnosis of Enterocytozoon bieneusi by PCR in stool samples eluted from filter paper disks. Clinical Diagnostic Laboratory and Immunology 7, 504–506. Curry, A., Smith, H.V., 1998. Emerging pathogens: Isospora, Cyclospora and microsporidia. Parasitology 117 S., S143–59. Devi, C.S., Parija, S.C., 2003. A new serum hydatid antigen detection test for diagnosis of cystic echinococcosis. American Journal of Tropical Medicine and Hygiene 69, 525–528. Didier, E.S., 2005. Microsporidiosis: an emerging and opportunistic infection in humans and animals. Acta Tropica 94, 61–76. Didier, E.S., Stovall, M.E., Green, L.C., 2004. Epidemiology of microsporidiosis: sources and modes of transmission. Veterinary Parasitology 126, 145. Didier, E.S., Weiss, L.M., 2006. Microsporidiosis: current status. Current Opinion of Infectious Disease 19, 485–492. Drury, R.A.B., Wallington, E.A., 1980. Carleton’s histo-logical technique, 5th ed. Oxford University Press, Oxford, New York, Toronto.

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