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Abstract
Parasitic infections, including helminthes and protozoa, have made a substantial contribution to the morbidity, mortality, social problems and economic losses of the world’s population. Consequently, reaching the correct diagnosis is a great important deal. The effective diagnosis of parasitic infection is required both for administration of drugs to infected individuals and for monitoring of control programs.
The aim of the present study is to highlight the antigenic components of different parasites, and to clarify the role of antigen detection in diagnosis of the corresponding parasitic infections in Egypt.
Parasitic antigens are classified either according to immunological, parasitological or biochemical criteria. Immunologically, they are classified into immunopathologic, immunodiagnostic, parasite-protective or host-protective antigens. Parasitologically, they are classified into surface, somatic or excretory/secretory antigens according to their origin and location; or into species-specific, strain-specific or stage-specific antigens according to parasite population and life cycle. While biochemically, they are classified according to their composition (protein, carbohydrate, etc.), chain structure or function (enzyme, metabolite, receptor, etc.).
In human body, parasitic antigens could be detected as circulating antigens in blood, serum, urine, CSF or saliva. They could be detected also as coproantigens in stool samples or in the fluids at the site of tissue pathology like in the aspirate of an amoebic liver abscess.
Common methods of antigen analysis include the use of monoclonal antibodies or Western blot that often follows SDS-PAGE. However, enzyme-linked immunosorbent assays (ELISA like direct ELISA, dot-ELISA, as well as sandwich ELISA), direct immunofluorescence (DFA), direct radioimmunoassay (RIA), agglutination techniques (like latex agglutination and co-agglutination) and more recently the immunochromatographic (IC) tests and dipstick antigen-capture assays are commonly used for parasitic antigens detection.
The ideal target antigen for antigen detection assays should be specific and expressed by most of life cycle stages of the parasite in abundance in different body fluids and should be cleared and not persist after parasitaemia disappears. On the other hand, the ideal antigen detection test should be sensitive, specific, cheap, simple, rapid, easy to perform and to interpret, qualitative and to some extent quantitative.
Almost all antigen detection assays detect early, pre-patent or active parasitic infections and help assessment and follow up of efficacy of therapy. Some of the assays could offer a qualitative and quantitative assessment for disease intensity. They are more suitable than antibodies detection for the diagnosis of concomitant parasitic infections in immunocompromized individuals. The rapid formats that include dipsticks and immunochromatographic assays, represent a new generation of rapid, simple and reliable tests that appear promising both in diagnosis and epidemiological use. However, most of antigen detection test suffer from being expensive and therefore, they are not cost effective for their use in the developing countries where most of parasitic diseases are endemic.
Schistosomes have a very complicated antigenic structure. Some of these antigens show cross reactivity between different life cycle stages of the parasite and some show mimicry with host antigens. The most important antigens as regard antigen detection tests are the gut-associated CCA and the CAA. Most of the antigen detection tests depend on the development of a MCA that can detect antigens in serum or urine. The introduction of assays for detection of circulating antigens in urine offered an easier non invasive approach which has its clinical utilities in certain situations. These assays in general show high sensitivity and specificity. However, this is not the truth in every time as the standardization of the chosen test and type of specimen may lead to variable results of lower sensitivity and/or specificity. These assays offer a tool to correlate with intensity of infection and hence the degree of morbidity or cure after treatment.
Fasciola species have a large number of antigens. Some of these antigens like FhSAP-2 protein showed cross reactivity with Schistosoma mansoni antigens. Fh12-fatty acid binding protein shows homology to 14 kDa S. mansoni antigen, a property that led to its trial as a cross protective immunogen in vaccination against both parasites. Important major E/S antigens of Fasciola species that received much attention are cysteine proteases. Antigen detection tests have been improved to detect circulating antigens in sera as well as coproantigens in stool. The major advantage of these assays is their ability to detect the pre-patent infection that could potentially replace the parasitological examination in acute fascioliasis outbreak studies.
The main source of antigenicity as regard human cystic echinococcosis is the hydatid cyst fluid and especially antigen B and antigen 5 which are detected as circulating antigens. However, the strain type, anatomic location of the cyst, cyst wall structure, and the speed and type of growth may influence the level of circulating antigen. Regarding the anatomic location of the cyst, the 16 and 21 kDa fractions of antigen B are more efficient in detecting antibodies in patients with hepatic hydatid cyst, whereas the 8 kDa antigen is the most reactive band in patients with pulmonary hydatid cyst. Circulating antigen detection has been proved useful prior to direct detection via fine needle aspiration. While, in conjunction with direct detection via fine needle aspiration, antigen detection in the cyst fluid is a confirmatory bedside test especially in case of sterile cysts.
W. bancrofti has a diversity of antigens. A 132 kDa microfilarial antigen could aid the diagnosis of filariasis at the early stage of tropical pulmonary eosinophilia. Antigens could be detected in serum, urine and hydrocoele fluid. Monoclonal antibodies represent the mainstay for antigen detection assays in bancroftian filariasis like in case of TropBio ELISA. The ICT appears promising as a rapid convenient test, but it can not replace the conventional microscopic detection of microfilariae.
E. histolytica has a less complicated life cycle with less complicated antigenic diversity. Among which, lectins are the most important group of antigens as they are used in antigen detection tests and are different between E. histolytica and E. dispar, thus helps differentiation between the two species. Coproantigen detection tests today offer a practical, sensitive, and specific method for the clinical laboratory to detect intestinal E. histolytica. All of the current tests suffer from the fact that the antigens detected are denatured by fixation of the stool specimen, limiting testing to fresh or frozen (not preserved) samples. Thus, these tests might be a useful addition to, but not a substitute for microscopical methods. Detection of circulating antigen in the serum and saliva is a promising yet still under research as an approach to the diagnosis of amoebic liver abscess.
Giardia lamblia has a dimorphic life cycle as it exists in trophozoite and cyst forms. Each stage has surface and excretory/secretory antigens. Trophozoite surface shows variant surface proteins that represents an immune-evasion strategy for G. lamblia. Giardins are immunodiagnostically important as they are detected by commercial immunoassays. The 65 kDa ESA is also important as it has been detected as coproantigen by ELISA.
The most studied antigens of C. parvum are those of sporozoite and oocyst. Disulfide isomerase of Cryptosporidium is targted by MCA in an immunochromatographic assay. The 200 kDa antigen of the oocyst is the target antigen of DFA. DFA is the most antigen detection assay for both giardiasis and cryptosporidiosis as it shows high sensitivity and specificity reaching 100% in most cases; however, it requires the presence of the expensive fluorescent microscopy. The simultaneous detection of both G. lamblia and C. parvum by immunofluorescence and immunochromatographic assays are the most reliable approach for diagnosis as the co-infection by these two diarrheogenic parasites is not uncommon.
Trophozoites and sporozoites are the stages of interest in the life cycle of T. gondii for studying the antigenic make-up of this parasite. The antigenic components of the tachyzoites are still of more interest because of the ease in obtaining such stage of the parasite. Examples of the important Toxoplasma antigens that related to the ultrastructure of its trophozoite are surface antigens (SAG1-3), micronemal proteins (MIC1-10), rhoptry proteins (ROP1-4) and dense granule antigens (GRA1-8). Antigen detection tests have been developed to detect T. gondii antigens in serum and urine in acute toxoplasmosis. The detection of T. gondii circulating antigens appears useful in the diagnosis of opportunistic reactivated toxoplasmosis as the antibodies levels are usually affected in immunocompromized patients. The literature and studies about the detection of T. gondii antigens in different body fluids including urine are still in need to be more enriched.
Plasmodium shows a very wide variety of polymorphic antigens that are not only different among species or life cycle stages only, but also different among isolates of the same species of the parasite. Moreover, the single parasite shows antigenic variability and switching during the course of infection that help the parasite to evade the immune response. Therefore, studying these antigens is not an easy issue. The most important antigens that are detected by antigen detection assays are the HRP-2, aldolase and pLDH. Nowadays, the RDTs have almost replaced the other diagnostic techniques like ELISA and RIA in the field of malarial antigen detection tests. These RDTs carry several advantages, but on the other hand they also suffer from certain drawbacks that can not be neglected. Therefore, RDTs can be a valuable adjunct for diagnosis but can not be the only diagnosis-dependent technique. Thick blood film examination is still the standard method for diagnosing malaria because it detects all Plasmodium species and offers the clear distinctions between parasite growth stages, which are essential for therapeutic decisions.
Frankly, all these tests can not replace the direct detection of parasites via conventional microscopy. It also appears that no single method can be optimal in all circumstances. The estimated prevalence of a certain disease in a certain area, clinical picture of the patient, the need for a rapid diagnosis, financial resources, availability of laboratory facilities, the number of samples likely to be processed, expertise and trained persons available in the laboratory and the local logistical frame, plus, of course, the overall costs, will determine whether parasitological examination, antigen detection, antibody detection or PCR will be dominant in practical usage. Consequently, the selection of a particular diagnostic kit and the approach for incorporation into the work flow should be the responsibility of each laboratory.
Briefly, in all cases, the combination of microscopic examination with serological tests (antibody detection), antigen detection and/or PCR, offers the best approach to diagnosis. If the results of the first test are negative and the patient remains symptomatic, additional testing can then be performed.