Eur J Clin Microbiol Infect Dis (2002) 21:624–625 DOI 10.1007/s10096-002-0778-1
CONCISE ARTICLE
M.T. Llorente · A. Clavel · M. Varea · S. Olivera F.J. Castillo · J. Sahagún · M.C. Rubio R. Gómez-Lus
Evaluation of an Immunochromatographic Dip-Strip Test for the Detection of Cryptosporidium Oocysts in Stool Specimens Published online: 10 August 2002 © Springer-Verlag 2002
Abstract The study presented here examined the efficacy of a commercially available qualitative immunochromatographic assay for detecting Cryptosporidium oocysts in stool samples. A total of 75 samples were tested, including 50 positive for Cryptosporidium spp. by acid-fast stain, 20 positive for other parasites (Blastocystis hominis, Endolimax nana, Entamoeba coli, Giardia lamblia, Ascaris lumbricoides, Strongyloides stercoralis and Trichuris trichiura), and five negative samples. The observed sensitivity was 98%, while specificity was 100%; the detection threshold was near 1,000 oocysts/ml. Correctly diagnosed positive samples included Cryptosporidium parvum genotypes 1 and 2, whereas the single falsenegative sample corresponded to a Cryptosporidium meleagridis infection.
Introduction Cryptosporidium parvum is an intestinal protozoan that infects the microvillus border of the gastrointestinal epithelium of a wide range of mammalian hosts, including humans. It is recognized as an agent of diarrhoeal disease worldwide [1]. Laboratory diagnosis of cryptosporidiosis is usually achieved by microscopic detection of Cryptosporidium oocysts in stool specimens; staining techniques include acid-fast stains and immunofluorescence [2]. Microscopy is time-consuming and requires an experienced observer to identify the organism. Other diagnostic methods, such as enzyme immunoassay or immunochromatography, are quicker, less labor-intensive, and easier to interpret [3, 4]. Presented here are the results of a study designed to evaluate the Crypto-Strip (Coris BioConM.T. Llorente · A. Clavel (✉) · M. Varea · S. Olivera F.J. Castillo · J. Sahagún · M.C. Rubio · R. Gómez-Lus Servicio de Microbiología y Parasitología, Hospital Clínico Universitario “Lozano Blesa”, C/San Juan Bosco 15, 50009 Zaragoza, Spain e-mail:
[email protected] Tel.: +34-976-556400 Ext. 4336, Fax: +34-976-761664
cept, Belgium), a commercially produced immunochromatographic test for the detection of Cryptosporidium parvum oocysts in unconcentrated stool samples.
Materials and Methods In the Crypto-Strip immunochromatographic test, liquid sample migrates by capillary action up the test strip, first rehydrating a specific anti-Cryptosporidium, gold-conjugate, monoclonal mouse antibody and then reaching a nitrocellulose membrane. A first line in the membrane (detection line) presents anti-Cryptosporidium monoclonal antibodies. A second line (control line) displays antimouse-immunoglobulin antibodies. On reaching the second line, the remaining conjugate is blocked (Fig. 1A). Gold-conjugate antibody fixed on either line appears as a pink/purple colour. In order to perform the test, approximately 50 mg of stool sample is added to a tube containing 0.5 ml of a dilution buffer provided with the kit. After mixing and waiting for 1–2 min, one test strip is dipped into the stool suspension for 5–10 min at room temperature before being read. A negative test yields one pink line (control line), while a positive strip shows two pink lines (detection line and control line) (Fig. 1B, C).
Fig. 1 A Scheme of the Crypto-Strip: line 1, plastic backing; lines 2 and 7, absorbent; line 3, nitrocellulose membrane; line 4, control antibody; line 5, specific antibody; line 6, gold-conjugate specific antibody. B Negative result strip showing one line (1). C Positive result strip showing two lines (1, 2). D Weakly positive result
625 In our study, 75 human stool samples were processed. Fifty were positive for Cryptosporidium oocysts, as determined by a modified Ziehl-Neelsen stain. Twenty samples contained other parasites: five Blastocystis hominis, two Endolimax nana, two Entamoeba coli, five Giardia lamblia, two Ascaris lumbricoides, one Strongyloides stercoralis and three Trichuris trichiura. Five samples presented no parasites. All samples were stored at 4°C without preservatives. Five positive specimens were mixed separately, in serial twofold dilutions, with Cryptosporidium-negative stools suspended in specimen dilution buffer, and these dilutions were tested again. For each sample, the concentration of Cryptosporidium oocysts in the last detected and the first undetected dilution was estimated by counting the number of oocysts present in 20 µl of the remaining sample suspension after direct immunofluorescence staining (Merifluor C-G; Meridian Diagnostics, USA). All Cryptosporidium isolates tested were genotyped in the course of another project by means of a polymerase chain reaction-restriction fragment length polymorphism assay. DNA was extracted as described elsewhere [5]. Briefly, oocysts were lysed by shaking with 0.5 mm zirconia-silica beads in a 10 M guanidinium thiocyanate buffer. DNA was then adsorbed on silica particles, purified with polyvinyl-pyrrolidone, and stored at –20°C in Tris-EDTA buffer. The typing protocol used involved amplification of the small-subunit ribosomal RNA gene, digestion of the amplicon by the restriction enzymes SspI (for species diagnosis) or VspI (for Cryptosporidium parvum genotype identification), and 2% agarose gel electrophoresis [6].
Results and Discussion Forty-nine of the 50 known positive samples tested positive with the Crypto-Strip test, whereas all 25 negative samples tested negative (98% sensitivity and 100% specificity, compared with modified Ziehl-Neelsen stain). Dilution of samples yielded progressively weaker detection lines (Fig. 1D); the lowest detectable dilution was 1/4 in one sample and 1/8 in four samples. The lowest concentration detected was 1,050 oocysts per millilitre of stool suspension, corresponding to approximately 10,500 oocysts per gram of stool. The highest undetected concentration was 540 oocysts/ml. The manufacturer claims this test is specific for Cryptosporidium parvum. In our study, all of the successfully diagnosed positive samples harboured Cryptosporidium parvum genotype 1 (29 samples) or 2 (20 samples), while the single false-negative sample contained Cryptosporidium meleagridis. This sample repeatedly tested negative, even though oocysts were abundant on the modified Ziehl-Neelsen slide and the Merifluor immunofluorescence assay was positive. Cryptosporidium meleagridis has been identified in about 1% of human cryptosporidial infections in the UK [7]; its frequency has been found to be higher in immunodeficient patients in France (5%) [8] and in children in Peru (8%) [9]. Testing of additional isolates of other Cryptosporidium spp.,
especially those known to be human pathogens, would be needed to assess the test's overall specificity. A modified Ziehl-Neelsen stain is not only useful for the diagnosis of Cryptosporidium spp., it can also identify Cyclospora cayetanensis and Isospora belli. Nevertheless, the oocysts of these other organisms, especially those of Isospora belli, are larger, and therefore more conspicuous in a routine examination of ova and parasites. Any institution considering replacing an acid-fast stain with a more specific test, such as the Crypto-Strip, should carefully weigh the relative advantages (i.e., decreased workload) with the disadvantages (especially a narrower diagnostic spectrum). Such an assay could be particularly useful as a confirmatory test, or in a potential outbreak scenario, where a large number of samples must be processed quickly [3].
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