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Year : 2008  |  Volume : 51  |  Issue : 1  |  Page : 137-138
Evaluation of an enzyme-linked immunoassay for the detection of Cryptosporidium antigen in fecal specimens of HIV/AIDS patients

1 Department of Microbiology, PSG Institute of Medical Sciences and Research, Coimbatore, India
2 Department of Dermatology and Venereology, Coimbatore Medical College, Coimbatore, India

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Cryptosporidium parvum, a protozoan parasite, causes severe diarrhea in immunodeficient hosts like HIV/AIDS patients, leading to significant morbidity and mortality. Diagnosis of the Cryptosporidium oocyst in the stool of these patients by conventional microscopy is labor intensive and time consuming. Therefore, we planned to evaluate the usefulness of a stool ELISA test in detecting Cryptosporidial antigen. About 89 stool specimens obtained from HIV-seropositive patients with diarrhea were subjected to an ELISA test and modified acid-fast staining (gold standard), on both direct and formol ether-concentrated specimens. The prevalence of Cryptosporidial diarrhea was found to be 12.4% (11/89). Other enteric pathogens detected were Isospora belli (3), Giardial cyst (3), Entamoeba coli cyst (2), and Entamoeba histolytica cyst (1). Dual infection with Cryptosporidium and Isospora belli was seen in two patients. Concentration technique improved identification by microscopy. The sensitivity and specificity for stool ELISA were found to be 90.9% and 98.7% respectively. The results of stool ELISA indicate that this simple, rapid, reliable, and standardized immunoassay test is sensitive and specific for routine diagnosis and may be useful for large-scale epidemiological studies of Cryptosporidiosis.

Keywords: Cryptosporidium spp., human immunodeficiency virus, isospora belli, stool ELISA

How to cite this article:
Jayalakshmi J, Appalaraju B, Mahadevan K. Evaluation of an enzyme-linked immunoassay for the detection of Cryptosporidium antigen in fecal specimens of HIV/AIDS patients. Indian J Pathol Microbiol 2008;51:137-8

How to cite this URL:
Jayalakshmi J, Appalaraju B, Mahadevan K. Evaluation of an enzyme-linked immunoassay for the detection of Cryptosporidium antigen in fecal specimens of HIV/AIDS patients. Indian J Pathol Microbiol [serial online] 2008 [cited 2016 Oct 26];51:137-8. Available from:

   Introduction Top

Cryptosporidium spp. are protozoan parasites classified as emerging pathogens by the Center for Disease Control and Prevention (CDC). The organism infects the gastrointestinal epithelium to produce a diarrhea that is self limited in immunocompetent persons but potentially life threatening in immunocompromised persons, especially those with HIV/AIDS. [1] Clinical diagnosis of Cryptosporidial infections is primarily based on the detection of oocysts in stool, which is labor intensive and relies on stool concentration, with subsequent staining and microscopy. The purpose of this study was to evaluate the clinical utility of an ELISA test in detecting Cryptosporidial antigen in stool.

   Materials and Methods Top

After obtaining Institutional Ethical Committee's approval and informed consent from the patients, a total of about 89 stool specimens collected from HIV-seropositive patients (confirmed by western blot) with diarrhea, at PSG Hospitals and Coimbatore Medical College, Coimbatore, were subjected to routine stool examination, which included saline and iodine mount to screen for helminthic ova, protozoan cyst, and trophozoites.

Modified acid-fast staining [2] was performed on both the direct stool smears and stool samples concentrated by formol ether sedimentation technique; and screening was done for Cryptosporidium oocyst, which were spherical to oval, measuring 4 µm to 6 µm in diameter, stained bright pink against a green background.

An ELISA test (RIDA screen Cryptosporidium , R - BioPharma, Germany) was performed on the unconcentrated stool samples according to the manufacturer's instructions.

   Results and Analysis Top

Out of the 89 stool samples screened by microscopy (gold standard) [3] after staining by modified acid-fast stain, 11 (12.4%) samples were positive for Cryptosporidium oocyst, and 3 (3.4%) samples showed the presence of Isospora belli . Concentration technique improved the detection of 3 Cryptosporidium parvum samples and 2 Isospora belli samples and also demonstrated the presence of the second pathogen in 2 patients with dual infection with Cryptosporidium and Isospora [Figure - 1].

Ten out of the 11 samples that were positive for Cryptosporidium by microscopy were positive by stool ELISA. One negative stool sample was wrongly identified as positive by ELISA [Table - 1]. The sensitivity and specificity of the test were found to be 90.9% and 98.7% respectively.

Routine stool examination by saline and iodine preparation identified the presence of Giardial cyst (3), Entamoeba coli cyst (2), and Entamoeba histolytica cyst(1).

   Discussion Top

Cryptosporidiosis is now recognized as an important component of HIV-related diarrhea. Failure to diagnose cryptosporidiosis early in these patients may interfere with therapeutical procedures, leading to high morbidity and mortality. [4] An ELISA test with high sensitivity and specificity for the coprodiagnosis of Cryptosporidial antigen, which is not dependent on the skill of the technicians for the morphological detection and identification of the oocyst, offers a diagnostic alternative to the laborious conventional direct microscopy. [5]

In the present study, 1 sample each was misidentified as false positive and false negative by the ELISA test respectively. In samples with fewer than five oocysts per 0.01 mL of concentrated stool, the ELISA fails to detect because the antigen is inaccessible or not recognized by the detecting polyclonal antibodies. [6] Experimental studies have shown that ELISA-positive specimens lost their antigenicity and gave false-negative results by 37 days (earliest) to 158 days (latest) after a minimum of three freeze-thaw cycles. [3] False-positive results are seen in patients recovering from Cryptosporidiosis and may represent the continued presence of Cryptosporidial antigens in the stool without the presence of whole oocysts when the parasite's integrity is compromised. [6]

The prevalence of Cryptosporidium in the study population was found to be 12.4% and was the predominant parasite isolated; earlier reported rates ranged from 6% to 37%. [7] Higher prevalence of Isospora belli compared to that of Cryptosporidium parvum was reported by various workers. [8],[9],[10] However, in our study Isospora belli was found to have a prevalence of only 3.4%. Other emerging intestinal parasites such as Cyclospora cayetanensis , which is also acid fast, were not detected by modified acid-fast stain, and Microsporidia was screened only among the last 22 samples using modified trichrome stain [11] and was therefore not included in this study. The presence of other intestinal parasites did not alter the ELISA results for Cryptosporidial antigen.

With sensitivity and specificity of 90.9% and 98.7% respectively, this simple, reliable, and less subjective test can be very useful in routine diagnosis and for screening a large number of specimens in short time, particularly in large-scale epidemiological surveys. However, in HIV-seropositive patients, the utility of stool microscopy to detect the presence of various other intestinal pathogens that cause diarrhea cannot be underestimated.

   References Top

1.Chien XM, Janet S, Keithly, Paya CV, LaRusso NF. Cryptosporidiosis. N Engl J Med 2002;346:1723-31.  Back to cited text no. 1    
2.Sehgal R, Malla N. Collection, tranportation and examination of faecal samples for parasites. Pre-congress CME cum workshop on parasitic diseases: Newer techniques for the next millennium. XXIII IAMM: 1999. p. 24-6.  Back to cited text no. 2    
3.Newman RD, Jaeger KL, Wuhib T, Lima AA, Guerrant RL, Sears CL. Evaluation of an antigen capture enzyme-linked immunosorbent assay for detection of cryptosporidium oocysts. J Clin Microbiol 1993;31:2080-4.  Back to cited text no. 3  [PUBMED]  [FULLTEXT]
4.Silva CV, Ferreira MS, Gonηalves-Pires Mdo R, Costa-Cruz JM. Detection of cryptosporidium-specific coproantigen in human immunodeficiency virus/acquired immunodeficiency syndrome patients by using a commercially available Immunoenzymatic assay. Mem Inst Oswaldo Cruz 2003;98:1097-9.  Back to cited text no. 4    
5.Mehta P. Laboratory diagnosis of cryptosporidiosis. J Postgrad Med 2002;48:217.  Back to cited text no. 5  [PUBMED]  [FULLTEXT]
6.Ungar BL. Enzyme-linked immunoassay for detection of Cryptosporidium antigens in fecal specimens. J Clin Microbiol 1990;28:2491-5.  Back to cited text no. 6  [PUBMED]  [FULLTEXT]
7.Mohandas K, Sehgal R, Sud A, Malla N. Prevalence of intestinal parasitic pathogens in HIV-seropositive individuals in Northern India. Jpn J Infect Dis 2002;55:83-4.  Back to cited text no. 7    
8.Mukhopadhya A, Ramakrishna BS, Kang G, Pulimood AB, Mathan MM, Zacharian A, et al. Enteric pathogens in southern Indian HIV-infected patients with and without diarrhoea. Indian J Med Res 1999;109:85-9.  Back to cited text no. 8    
9.Prasad KN, Nag VL, Dhole TN, Ayyagari A. Identification of enteric pathogens in HIV- Positive patients with diarrhea in Northern India. J Health Popul Nutr 2000;8:23-6.  Back to cited text no. 9    
10.Satheesh KS, Ananthan S, Lakshmi P. Inmtestinal parasitic infection in HIV infected patients with diarrhea in Chennai. Indian J Med Microbiol 2002;20:88-91.  Back to cited text no. 10    
11.Garcia LS. Laboratory Identification of the Microsporidia. J Clin Microbiol 2002;40:1892-901.  Back to cited text no. 11  [PUBMED]  [FULLTEXT]

Correspondence Address:
J Jayalakshmi
Department of Microbiology, PSG Institute of Medical Sciences and Research, Coimbatore - 641 004
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0377-4929.40427

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  [Figure - 1]

  [Table - 1]

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