Evaluation of staining techniques, antigen detection and nested PCR for the diagnosis of cryptosporidiosis in HIV seropositive and seronegative patients

https://doi.org/10.1016/j.actatropica.2008.02.007Get rights and content

Abstract

The study was designed to determine the efficacy of modified Ziehl–Neelsen (ZN), safranine methylene blue (SM) staining, antigen detection ELISA and a nested PCR assay (specific for Cryptosporidium parvum) for detection of Cryptosporidium in HIV seropositive and seronegative patients with diarrhoea.

Cryptosporidium was detected in 10 (4.9%), 9 (4.4%), 39 (18.9%) and 27 (13.1%) of 206 HIV seropositive and 7 (4.6%), 6 (3.9%), 21 (13.7%) and 17 (11.1%) of 153 HIV seronegative patients by ZN staining, SM staining, antigen detection ELISA and PCR, respectively. None of the 50 apparently healthy control subjects was found to be infected with Cryptosporidium by any of the techniques.

Based on the criteria of ‘true positive’ samples positive by at least any two techniques out of ZN staining, antigen detection and PCR, sensitivity of ZN and SM staining techniques was 37% and 33.3% in HIV seropositive and 41.2% and 35.3% in seronegative patients, respectively. Sensitivity of antigen detection ELISA was 92.6% and 94.1% in HIV seropositive and seronegative patients, respectively, while sensitivity of PCR was 100% each in HIV seropositive and seronegative patients.

Specificity of all three techniques, i.e. ZN, SM staining and PCR was 100% in both HIV seropositive and seronegative patients while specificity of antigen detection was 92.2% and 96.3% in HIV seropositive and seronegative patients, respectively. The staining techniques were found less sensitive as compared to antigen detection and PCR for detection of Cryptosporidium in HIV seropositive patients with CD4 count >200 cells/μl.

Introduction

Cryptosporidiosis caused by Cryptosporidium has been increasingly reported world-wide both in immunocompetent and immunocompromised individuals causing a spectrum of diseases ranging from asymptomatic carrier state to severe diarrhoea. Infection with this parasite results in severe but self-limiting diarrhoea in immunocompetent and often lethal diarrhoea in immunocompromised individuals, most notably patients with AIDS (Fayer et al., 2000, Chen et al., 2002).

Laboratory diagnosis relies on the recognition of the Cryptosporidium oocysts in stool specimen after modified acid-fast staining. Conventional microscopy, however, is time-consuming, tedious and requires experienced microscopists to accurately identify the oocysts. In addition, the detection limit of conventional diagnostic techniques can be as low as 50,000–500,000 oocysts g−1 faeces (Weber et al., 1991). Immunological based detection methods have been developed for use in both clinical and environmental monitoring. However, antigenic variability within clinical isolates of Cryptosporidium can result in some infections remaining undetected (Griffin et al., 1992) and antigen detection with varying degrees of sensitivity and specificity of ELISA has been reported (Kehl et al., 1995, Graczyk et al., 1996). In addition, different studies evaluating identical assays have been reported to show diverse sensitivity values. Two separate evaluations (Katanik et al., 2001, Johnston et al., 2003) of the ProSpectT Cryptosporidium microplate assay reported sensitivity values of 70% and 100%, respectively. PCR assays have been introduced as a very sensitive and specific method to detect Cryptosporidium in environmental and clinical specimens and to define the taxonomic status, as well as the existence of genotypes within C. parvum (Higgins et al., 2001). The sensitivity of 97–100% and specificity of 100% has been reported for diagnosis of Cryptosporidium by PCR (Morgan et al., 1998, Bialek et al., 2002). However, need of expertise and availability of reagents has hampered its routine use in the diagnosis.

Given the low sensitivity of microscopy and varying sensitivity and specificity of antigen detection ELISA, the present study was aimed to evaluate two staining techniques (ZN and SM), antigen detection ELISA and a nested PCR assay for the detection of Cryptosporidium in HIV seropositive and seronegative patients. Among patients who have AIDS and in whom cryptosporidiosis can be detected early, improvement in immune function with effective antiretroviral therapy can result in dramatic improvement in diarrhoea (Carr et al., 1998, Foudraine et al., 1998). The early detection of Cryptosporidium in HIV seropositive patients may further add to the knowledge for clinical management of the disease.

Section snippets

Patients and controls

Study group included 409 individuals which comprised of 206 HIV seropositive patients, 153 HIV seronegative patients with diarrhoea and 50 HIV seronegative, apparently healthy individuals without any history suggestive of cryptosporidiosis. HIV-seropositivity was defined as positivity based on three different ELISA kits, as per national policy for diagnosis of HIV infection (NACO, 2003). Two hundred and six HIV seropositive patients were selected, based on HIV-seropositivity status attending

Demographics

Demographic characteristics of the subjects are shown in Table 1.

On retrospective analysis, CD4 count was available for all 206 HIV seropositive patients. Sixty-seven (32.5%), 115 (55.8%) and 24 (11.7%) patients had CD4 count of <200, 200–500 and >500 cells/μl, respectively. In HIV seropositive patients with CD4 count <200 cells/μl, the number of patients with diarrhoea was significantly higher (74.6%) as compared with patients without diarrhoea (25.3%) (p < 0.001) (Table 2).

Intestinal parasites

Intestinal parasites

Discussion

Studies comparing four techniques, i.e. modified ZN staining, SM staining, antigen detection ELISA and PCR for the detection of Cryptosporidiosis in HIV seropositive or seronegative individuals are scarce. The present study was an attempt to evaluate these four diagnostic techniques on large number of HIV seropositive and seronegative individuals so as to find out the best possible diagnostic marker for the diagnosis of cryptosporidiosis.

In the present study, highest positivity was shown by

Acknowledgement

The authors wish to thank Dr. Boris Striepen, Center for Tropical & Emerging Global Diseases, University of Georgia, Athens, GA for providing the genomic DNA of Cryptosporidium parvum Iowa strain.

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