Evaluation of staining techniques, antigen detection and nested PCR for the diagnosis of cryptosporidiosis in HIV seropositive and seronegative patients
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.
References (46)
- et al.
Comparison of fluorescence, antigen and PCR assays to detect Cryptosporidium parvum in faecal specimens
Diagn. Microbiol. Infect. Dis.
(2002) - et al.
Treatment of HIV-1 associated microsporidiosis and cryptosporidiosis with combination antiretroviral therapy
Lancet
(1998) - et al.
Epidemiology of Cryptosporidium transmission, detection and identification
Int. J. Parasitol.
(2000) - et al.
Real-time PCR for the detection of Cryptosporidium parvum
J. Microbiol. Methods
(2001) - et al.
Occurrence and significance of Cryptosporidium infection in Calcutta
Trans. R. Soc. Trop. Med. Hyg.
(1989) - et al.
Evaluation of lacto-phenol cotton blue (LPCB) for detection of Cryptosporidium. Cyclospora and Isospora in the wet mount preparation of stool
Acta Trop.
(2003) - et al.
Cryptosporidium and Isospora belli diarrhoea in immunocompromised hosts
Ind. J. Cancer
(1999) Acid fast staining versus ELISA for detection of Cryptosporidium in stool
J. Commun. Dis.
(1998)- et al.
The development and performance of a simple, sensitive method for the detection of Cryptosporidium oocysts in faeces
J. Hyg. (Lond.)
(1984) - et al.
Intestinal protozoa in HIV-infected patients in Apulia, South Italy
Epidemiol. Infect.
(1999)
Laboratory diagnosis of cryptosporidiosis
J. Clin. Pathol.
An Enzyme immunoassay for detecting cryptosporidia in faecal and environmental samples
J. Med. Microbiol.
Cryptosporidiosis
N. Engl. J. Med.
Intestinal parasites in HIV-seropositive Zambian children with diarrhoea
J. Trop. Paed.
Prevalence of intestinal protozoans in French patients infected with HIV
J. Acquir. Immune. Defic. Syndr.
Molecular characterization of Cryptosporidium spp. from children in Kolkata, India
J. Clin. Microbiol.
High Cryptosporidium prevalences in healthy Aymara children from the northern Bolivian Altiplano
Am. J. Trop. Med. Hyg.
Improvement of chronic diarrhoea in patients with advanced HIV-1 infection during potent antiretroviral therapy
AIDS
Beyond Normality: The Predictive Value and Efficiency of Medical Diagnosis
Commercial assay for detection of Giardia lamblia and Cryptosporidium parvum antigens in human faecal specimens by rapid solid-phase qualitative immunochromatography
J. Clin. Microbiol.
Evaluation of commercial enzyme immunoassay (EIA) and immunofluorescent antibody (IFA) test kits for detection of Cryptosporidium oocysts of species other than Cryptosporidium parvum
Am. J. Trop. Med. Hyg.
Antigenic diversity among oocysts of clinical isolates of Cryptosporidium parvum
J. Protozool. Res.
Asymptomatic Cryptosporidium hominis Infection Among Human Immundeficiency Virus–Infected Patients In Tanzania
Am. J. Trop. Med. Hyg.
Cited by (72)
Cyclosporiasis in an immunocompromised patient who had undergone renal allograft transplant-A rare case report
2022, Indian Journal of Medical MicrobiologyCryptosporidium
2022, Encyclopedia of Infection and ImmunityCryptosporidium spp. diagnosis and research in the 21<sup>st</sup> century
2021, Food and Waterborne ParasitologyCitation Excerpt :Thus, in order to improve diagnostic sensitivity, faecal specimens are often collected over three different days, adding to the laboratory workload (Goñi et al., 2012; van Gool et al., 2003). Overall, this time consuming and tedious staining technique demands an experienced microscopist, but exhibits poor sensitivity (37-100%) (Abou El-Naga and Gaafar, 2014; Chalmers et al., 2011; Kaushik et al., 2008; Tuli et al., 2010; Zaglool et al., 2013). Unsurprisingly, given the variable levels of sensitivity associated with brightfield and fluorescent staining techniques, a number of oocyst concentration methods have been developed in order to maximise oocyst yields from faecal samples (Garcia et al., 1983; Weber et al., 1991).
Comparison of current methods used to detect Cryptosporidium oocysts in stools
2018, International Journal of Hygiene and Environmental HealthCitation Excerpt :qPCR is generally accepted to be the most sensitive laboratory assay for the detection and enumeration of Cryptosporidium oocysts in faecal samples from different hosts (Elwin et al., 2012; Hadfield et al., 2011; Yang et al., 2015). Its superior sensitivity was reported when compared to MAF (Fathy et al., 2014; Ignatius et al., 2016; Kaushik et al., 2008; Khurana et al., 2012; Martín-Ampudia et al., 2012; Morgan et al., 1998; Yang et al., 2013, 2014; Zaidah et al., 2008). When compared with nested PCR, qPCR provides rapid, cost effective and accurate screening, identification and quantification of C. parvum and C. hominis.
Evaluation of enzyme linked immunosorbent assay for stool antigen detection for the diagnosis of cryptosporidiosis among HIV negative immunocompromised patients in a tertiary care hospital of northern India
2018, Journal of Infection and Public HealthCitation Excerpt :Stool antigen shedding continues, which is likely to be missed on microscopy and diagnosed better by antigen detection techniques [13]. The prevalence of cryptosporidiosis reported by microscopy by Indian studies ranges between 3.7%–36.22% [6,7,14,15] for immunocompromised patients and between 2.70% and 4.6% [12,16] for non-immunocompromised patients. Detection of Cryptosporidium copro-antigen has been tried by various methods.
Pediatric gastrointestinal tract infections
2023, Chronic Disease and Disability: The Pediatric Gastrointestinal Tract, Second Edition. Medical and Surgical Perspectives including Infection and Pain