Immunological assays are available for measuring the humoral immune response after parasitic infections. For the detection and quantitation of antibodies or antigens an immunoassay has to fulfill three basic requirements: highly specific, highly sensitive, and reproducible. The field of application and the quality of antigen/antibody available are the main variables which determine the choice for one specific assay. A test system may be adequate for population screening or for individual diagnosis. Ideally, it is a single definitive test which can equally detect antibodies in low-responders, in people with recently contracted infection or with high titers and clinical signs, and be suitable for a posttreatment follow-up.
The stability of the antigen-antibody complex (avidity) determines the success of all immunological assays.
IFA, EIA, WB
The indirect immunofluorescence (fluorescent antibody) assay (test) (IIFA, IFA, IIFAT, IFT) has long been the best choice in parasitology. By using cryosections from different developmental stages of helminths or free protozoa as antigens the test provides a satisfactory discrimination between specific and non-specific reactions. The differential binding of antibodies to different structures of the parasite or cell is used to distinguish between specific and nonspecific reactivity. Its disadvantage is subjective reading and labour intensiveness. Screening of a large population is practically impossible. The indirect haemagglutination test (IHAT, IHA) is applied for fast screening at low costs of several parasitic infections. A broad application of the indirect enzyme-linked immunosorbent (enzyme immunosorbent) assay ( ELISA, EIA) has long been delayed by the use of nondefined, crude parasite extract antigens, resulting in a high degree of cross-reactivity. This disadvantage is now partly overcome by the availability of secretory/excretory (E/S) antigens, synthetic peptides and recombinant antigens. Still, the sensitivity and specificity of the ELISA vary considerably but it is an easy and fast alternative especially for population and herd screening and epidemiological studies. The western blot (WB, enzyme immunoblot, EIB), which provides a species or stage specific diagnosis is a confirmation test and applied as the final diagnostic step when screening results are positive.
Other Test Systems
Today, only few laboratories still use immunodiffusion (ID), gel precipitation, complement fixation test (CFT), Sabin Feldman dye test (DT), counterimmunoelectrophoresis (CIE) or radioimmunoassay (RIA) in routine serodiagnosis. These test systems consume much antigen in comparison to the ELISAs, are less sensitive or use radiolabelled conjugates. Other test systems like card agglutination test (CATT), direct agglutination test (DAT), latex agglutination test (LA), carbon immunoassay (CIA), dot blot, and dip stick antigen assay were developed for a rapid, low cost screening under field conditions.
The rapidly increasing knowledge on immunology and molecular biology may result in a large scale production of characterized parasitic antigens applicable in different test systems. However, the suitability of each product for diagnostic purposes depends on the dynamics of the hosts immune response to that particular antigen.
IIFAT results depend not only on the kind of antigen used but also on its processing. Fixation is one of the most important steps (unfixed, formalin, aceton, methanol, heat, and others) to enhance or decrease the sensitivity or specificity of the test. The reading of the IIFAT requires a specific microscope. Reading of the slides by inexperienced staff may lead to different end-point titers due to subjective impressions. A new generation of IHATs using selected red blood cells of the 0 Rh- type for antigen cross-linking reduces the non-specific agglutination of erythrocytes and improves the specificity of this fast test system. ELISA is a suitable test system for the detection of both antigen and antibody. It is very sensitive and economic for testing a large number of serum samples within a relatively short time. The test principle allows many variations in the performance of the assay and the use of detection systems. Direct and indirect, capture and dip stick and many other ELISA systems are described as efficient in different diagnostic fields. Reading of the test is normally by use of a photometer. In highly specific assays a discrimination between positive and negative results is also possible visually. Indirect ELISAs, which are predominately used in parasitology for antibody detection, measure antibody binding and not antibody levels and therefore provide only a limited quantitative information. The WB analysis demonstrates serum reaction patterns with antigen fractions of either a complex mixture or predefined extracts of parasites. It is used to demonstrate either an individual stage specific antibody response in follow-up studies or species specific reaction for the discrimination of infection with closely related parasites. The CATT is an easily and fast to perform low-cost system which uses the surface antigen of different Trypanosoma-species for antibody detection. It is applied as a screening tool for the (West) African sleeping sickness and animal trypanosomiasis caused by T. brucei/congolense and T. evansi.
Standardisation of immunological assays is a crucial problem in parasitology. The evaluation of the specificity, sensitivity, and negative/positive predictive value of an assay requires either a “gold standard” or a population based evaluation of numerous defined serum samples. Only the DT is an accepted “gold standard” for toxoplasmosis serology. For other parasitic infections either parasite detection, disease, or some other immunological assay with proven diagnostic value are used as “gold standard.” Many home made or commercial test systems still work with undefined antigens. Also, reference sera are not available for all parasitic infections. Hopefully, standardization will improve with the availability of defined antigens.
The clinical value of an immunoassay depends on its ability to allow the identification of an infected individual on the results of single test. Ideally, the producer provides information on the positive and negative predictive value for his immunoassay. Due to a comparatively low demand for parasitological immunoassays in the low incidence “developed” countries test systems are often produced in parasitological laboratories. At present, only the serodiagnosis of toxoplasmosis is to a high degree commercialized and, in Germany, submitted to an official, external quality control. The choice of a suitable test system is therefore essential for a good diagnosis. For individual diagnosis a quantitative and specific measurement of the serum antibody concentration is required. The approximate antibody concentration is given either by end-point titers (IIFAT, IHAT), units, or absorbances (ELISA). The WB pattern can provide only semiquantitative, but highly specific results. In any case, an interlaboratory comparison of test results is only possible when identical assays are performed. The application of intra- and interassay quality controls by use of defined reference material is obligatory. For screening purposes a sensitive assay is preferred for primary diagnosis, followed by a more specific test for definitive diagnosis. Qualitative test results (positive/negative) will be sufficient for population screening or herd control. When interpreting test results it has to be considered that there is not only a population-based variability of the antibody responses to parasitic infections but also a great variability of the individual response depending on the parasite load and stage of infection. Finally, antibodies show variation among different techniques due to differences in multivalent binding.
The use of different test systems for seroepidemiological studies may result in different rates of seroprevalence achieved. These differences may reflect basic differences in the sensitivity or specificity between test systems. However, when direct parasite detection as “gold standard” for test evaluation is used, the resulting sensitivity depends not only on the experience of the investigator but also the frequency of sampling and the parasite load of the individuals tested. When disease is used as “gold standard” for test evaluation, a comparatively high seropositivity will result when the infective parasite exhibits a long incubation period and/or low morbidity rate. The clinical outcome of parasitic infections depends also on the genetic predisposition of the infected individual. A positive test result in one immunoassay may therefore provide evidence for a yet unknown immunological recognition of the parasite, which is undetectable in other assays. Discrepant rates in the prevalence of Toxocara canis-antibodies in human serum were reported when either CIEP and IIFAT or E/S-ELISA was applied. The interpretation of unresolved positive values is carried out by considering the incubation period and possible pathological effect of each parasite.