Immunological Methods of Common Intestinal Protozoa: Comparison
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Intestinal protozoan infection is a persistent public health problem affecting the populations of developing countries in tropical and subtropical regions. The diagnosis of intestinal protozoa remains a challenge especially in developing countries due to a shortage of laboratory facilities, limited health funding, and the remoteness of communities. Despite still being widely used, conventional diagnoses using microscopy and staining methods pose important limitations, particularly due to their low sensitivities and specificities. The selection of diagnostic methods needs to be carefully considered based on the objective of examination, availability of resources, and the expected parasite to be found. Immunodiagnostic tests are generally inexpensive, user-friendly, and enable fast-obtained results. Antibody and antigen detection tests, such as indirect hemagglutination (IHA), indirect immunofluorescence (IIF), enzyme-linked immunosorbent assay (ELISA), direct fluorescent antibody (DFA), rapid enzyme immune assay (EIA), immunochromatographic test (ICT), or latex agglutination, are commercially available via several different platforms. The combination of antibody and fecal antigen detection assays is more sensitive and specific than microscopy for the diagnosis of several intestinal protozoan infections.

  • Entamoeba histolytica
  • Giardia spp.
  • Cryptosporidium spp.
  • immunodiagnosis
  • molecular diagnosis

1. Immunodiagnostic Methods for Amoebiasis

For the diagnosis of E. histolytica infections, indirect hemagglutination (IHA) and indirect immunofluorescence (IIF) and IIF methods can be performed. However, one of the most popular and widely used platforms is the enzyme-linked immunosorbent assay (ELISA)ELISA. Variations in this platform can be used to detect either antigens during intestinal amoebiasis or anti-Entamoeba antibodies during amoebic liver abscess (ALA) [1], with several ELISA kits being commercially available. In addition to fecal specimens, serum or liver abscess aspirates can also be subjected to this method [2][3].
Several studies developed monoclonal antibody-based platforms using various E. histolytica antigens, such as lectin-rich surface antigen, lipophosphoglycan [4], and the 170-kDa amoebic adherence lectin [5]. In areas of the world where E. histolytica infection is endemic or if intestinal amoebiasis is specifically suspected by a physician, antigen-based tests can be performed. These tests usually employ monoclonal antibodies against the E. histolytica adhesin Gal/GalNAc lectin. This antigen has also been reported to be detectable in the sera and saliva of patients, although further evaluations are required [3].
Nevertheless, not all commercially available antigen tests can differentiate between E. histolytica and E. dispar. The sensitivity of the E. histolytica antigen detection tests ranges from 80% to 94% compared to that of polymerase chain reaction (PCR), with one study reporting a commercial, ELISA-based platform to be less sensitive than microscopy. Although antigen-detection tests proved to be useful for intestinal amoebiasis detection, they possess some crucial limitations, including the need for fresh or unpreserved fecal samples and the inability to differentiate E. histolytica from E. dispar and E. moshkovskii [6][7].
When evaluating patients from E. histolytica-endemic areas, it is important to notice that immunoassays detecting anti-E. histolytica antibodies turn negative earlier following the treatment of extraintestinal amoebiasis compared to IHA-based tests, which remain positive for at least 6 months following treatment [8]. An in-house point-of-care antibody-detecting test using a dipstick platform has also been developed with a claimed sensitivity of 98.1% compared to a commercial latex agglutination test [9]. Overall, antibody detection tests have shown good performance in the diagnosis of extraintestinal amoebiasis but are less practical for the detection of intestinal amoebiasis, patients in endemic areas with a high baseline antibody, and immunocompromised patients.
A rapid IC assay to simultaneously detect E. histolytica/E. dispar, G. duodenalis, and C. parvum was also commercially available. This strip assay used monoclonal antibodies against proteins of these protozoa and claimed a nearly perfect sensitivity and specificity [10]. However, like several aforementioned immunodiagnostic platforms, it failed to distinguish between the pathogenic E. histolytica and the non-pathogenic E. dispar and has been discontinued from the market.

2. Immunodiagnostic Methods for Giardiasis

The detection of Giardia duodenalis has been enhanced using antigen detection methods. Some immunoassays for Giardia are commercially available and widely used in clinical laboratories. The ELISA platform for Giardia duodenalis that has been approved by the World Health Organization is a rapid, sensitive, specific, and inexpensive method of confirming Giardia duodenalis coproantigens even in the absence of live parasites in the fecal samples [11].
Several previous studies found that the commercial direct fluorescent antibody (DFA)DFA test used to detect Giardia duodenalis showed a sensitivity of 96–99% and specificity of 100%. This test utilizes fluorescein-labeled antibodies directed against the cell wall proteins of Giardia duodenalis cysts and allows visualization of the intact parasites, thus, providing a definitive diagnosis with a greater sensitivity than the conventional permanent smears [12][13].
For laboratories with limited capacity for diagnostic complexity, simple enzyme immune assays (EIAs) and immunochromatographic tests (ICTs) and ICTs are commercially available for the detection of Giardia duodenalis. Rapid immunoassays based on immunochromatographic lateral flow for Giardia duodenalis have become a popular diagnostic tool because they do not require trained microscopists, expensive equipment and can be completed very quickly. Meanwhile EIA-based tests might be more appropriate for screening in high-prevalence areas [12]. A study comparing four EIAs found sensitivities ranging between 63% and 91% and specificities of 95%. Another study demonstrated 94% to 100% sensitivity and 100% specificity when five Giardia duodenalis EIAs were evaluated [12][13]. Although the aforementioned methods are able to detect the Giardia duodenalis species with prominent sensitivities and specificities, no immunological test to date can differentiate the Giardia duodenalis assemblages in clinical samples.

3. Immunodiagnostic Methods for Cryptosporidiosis

Direct immunofluorescence microscopy, ELISA, and ICTs are three methods that have been successful in the immunological detection of Cryptosporidium spp. oocyst antigens, and a number of commercial kits are available. Compared to traditional stains, immunofluorescence kits are more sensitive and specific in detecting Cryptosporidium spp. oocysts in fecal smears. Cryptosporidium spp., using direct immunofluorescence from fecal samples using fluorescein isothiocyanate (FITC)-labelled monoclonal antibodies, works against surface-exposed epitopes of oocysts. It was reported to have nearly perfect sensitivity and specificities against this protozoan, although it could not distinguish different species of Cryptosporidium [13][14][15][16][17].
Several studies evaluated the sensitivities and specificities of the available kits for cryptosporidiosis and found overall similar performance levels for EIA- and DFA-based methods (90% sensitivity; 95% specificity) [12]. Some commercially available immunoassays allow simultaneous and rapid detection of Giardia duodenalis and Cryptosporidium spp. These tests, including EIA, ICT, and DFA assays, are favorable since coinfections of both protozoa are commonly found [13][14][15][16]. Since HIV-infected and immunocompromised individuals are particularly at risk for serious complications from these coccidian parasites, clinicians should consider routinely suggesting at least DFA and molecular testing, if available, for patients with suspected cryptosporidiosis [12].
An ELISA test is performed to detect the presence of soluble Cryptosporidium spp. coproantigens. Depending on the commercial kit, a combination of monoclonal and polyclonal antibodies is used to collect and identify Cryptosporidium spp. coproantigens. These tests were developed to identify antigens from C. parvum in fecal samples, but they can also identify common epitopes from infections with other Cryptosporidium species [17].
Rapid, ICT-based methods for cryptosporidiosis are significantly less sensitive, with a multi-institutional study reporting a sensitivity of between 50.1% and 86.7% depending on the test manufacturer [12]. However, a rapid immunochromatographic assay kit for the detection of both Giardia duodenalis and Cryptosporidium spp. is also available with superior specificities and sensitivities. This monoclonal, antibody-based platform is quick, easy, and simple to interpret [13][14][15][16].

References

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  11. Singhal, S.; Mittal, V.; Khare, V.; Singh, Y.I. Comparative analysis of enzyme-linked immunosorbent assay and direct microscopy for the diagnosis of Giardia intestinalis in fecal samples. Indian J. Pathol. Microbiol. 2015, 58, 69–71.
  12. McHardy, I.H.; Wu, M.; Shimizu-Cohen, R.; Couturier, M.R.; Humphries, R.M. Detection of intestinal protozoa in the clinical laboratory. J. Clin. Microbiol. 2014, 52, 712–720.
  13. Johnston, S.P.; Ballard, M.M.; Beach, M.J.; Causer, L.; Wilkins, P.P. Evaluation of three commercial assays for detection of Giardia and Cryptosporidium organisms in fecal specimens. J. Clin. Microbiol. 2003, 41, 623–626.
  14. Gotfred-Rasmussen, H.; Lund, M.; Enemark, H.L.; Erlandsen, M.; Petersen, E. Comparison of sensitivity and specificity of 4 methods for detection of Giardia duodenalis in feces: Immunofluorescence and PCR are superior to microscopy of concentrated iodine-stained samples. Diagn. Microbiol. Infect. Dis. 2016, 84, 187–190.
  15. Vanathy, K.; Parija, S.C.; Mandal, J.; Hamide, A.; Krishnamurthy, S. Cryptosporidiosis: A mini review. Trop. Parasitol. 2017, 7, 72–80.
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  17. World Organisation for Animal Health. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 2022; WOAH: Paris, France, 2022; p. 17.
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