Vzv

Fungal infection Lymphoma or metastatic tumor Brain abscess Progressive multifocal leukoencephalopathy Fungal or mycobacterial disease

Only in macrophages, may see budding

Only in macrophages, amastigote has kinetoplast

Amastigotes only in myocytes, kinetoplasts seen

Only in macrophages, morphology characteristic

Distinguish by serologic tests

Distinguish by serologic tests

Distinguish by serologic tests

Trypomastigotes seen in blood film

Distinguish by serologic tests

Distinguish by serologic tests

Biopsy of tissue

Biopsy of tissue and culture

Biopsy of tissue

Biopsy of tissue

Distinguish by culture

Distinguish by serologic tests

Distinguish by culture of virus

Distinguish by culture of virus

Distinguish by culture and serologic tests

Distinguish by serologic tests

Distinguish by culture

Distinguish by serologic tests

Distinguish by hematological tests

Distinguish by culture

Distinguish by serologic tests

Distinguish by biopsy

Distinguish by serologic and antigen tests

Distinguish by culture or PCR of virus

Distinguish by serologic tests

Distinguish by culture or PCR

Distinguish by culture or PCR

Distinguish by culture

Distinguish by tissue biopsy

Distinguish by culture

Distinguish by PCR

Distinguish by biopsy and culture

Interpretation of Serological Tests

The humoral immune response to T. gondii is rapid and intense, and forms the basis of useful diagnostic tests for the various forms of the disease. Antibodies may be produced to a number of T. gondii antigens, but the immunodominant antigen is the 30kDa major surface antigen, SAG-1. IgG antibodies are a reliable and sensitive indicator of exposure to T. gondii, but do not establish the chronicity of the infection. Several methods are available for the determination of anti-Toxoplasma IgG, and all are sensitive and specific. The historical gold standard assay is the Sabin-Feldman dye test, which relies on the ability of complement-fixing IgG or IgM antibody in the patient serum to produce changes in permeability of live T. gondii and allow dye to enter the parasite. This test is no longer widely available, but is still useful clinically because it has a very high specificity and can be used for comparison with other IgG assays. Indirect immunofluorescence assays and various forms of ELISA and microparticle immunoassays all correlate well with the Sabin-Feldman dye test. All of these tests are reliable to establish past infection with T. gondii. If IgG tests are negative and there is no evidence of IgM antibody, the diagnosis of toxoplasmosis can be considered very unlikely. If the IgG assay is

Fig. 5.13 Intracellular and extracellular tachyzoites (arrows) in a smear of peritoneal exudate cells of an infected mouse. Wright's stain. Bar=10 ^m

equivocal, another specimen should be tested after 2-3 weeks to establish whether a real seroconversion has taken place. Once present, IgG antibody usually persists for the life of the host, even at high levels for years. Clinical settings in which it would be useful to perform IgG serology include before conception or very early in gestation, before therapeutic immuno-suppression, and early after the primary diagnosis of HIV.

Determination of the time of infection is more difficult. Tests for IgM do not have ideal performance characteristics to allow straightforward interpretation of the time of primary infection. Commercially available IgM solidphase assays may produce both false negative and false positive results in some situations (Anonymous, 1997). Most available assays of IgM antibody lack sensitivity to detect the low avidity IgM antibodies synthesized by neonates and young infants. If specific IgG antibody is present and an IgM assay is equivocal or positive, further investigation is necessary to establish with confidence that the individual has been recently infected. This is best done at a large public health laboratory or reference laboratory with experience and availability of alternative assays that can be performed to support the diagnosis. If an IgG assay is positive and IgM is truly negative, the infection has most likely been established for more than 1 year. Specific anti-Toxoplasma IgM may be detected for 18 months or more with sensitive assays (Wilson and McAuley, 1999). IgM ELISA assays in the 'capture' format, which trap serum IgM on the solid phase in the first step, are useful to indicate the relative subset of IgM that recognizes Toxoplasma antigen. This method is less likely to be interfered with by excess non-specific IgM or rheumatoid factor (Naot and Remington, 1980; Siegal and Remington, 1983).

Tests useful as adjuncts to confirm an equivocal or positive IgM titer include the IgA and IgE ELISA assays. The IgA ELISA is useful to confirm IgM determinations and can be elevated in acute disease and, although it is not a sensitive test in congenital disease, it may be useful in confirmation of IgM in that setting (Foudrinier et al, 1995; Stepick-Biek et al, 1990). IgE antibodies may be present in the acute phase of disease and disappear rapidly (Wong et al., 1993). The IgE ELISA is therefore useful in dating infection and confirming congenital infection. There is some evidence that IgE antibodies also appear in reactivation episodes associated with toxoplasmic encephalitis and retino-choroiditis (Wong et al., 1993). A differential agglutination test has been developed to distinguish the pattern of antibodies seen in recent acute infection from those seen in more distant infections (Dannemann et al., 1990). Tachyzoites fixed with acetone or methanol (AC antigen) display antigens that are recognized by antibodies made early in primary infection, and can be agglutinated by serum diluted at alkaline pH (Suzuki et al., 1988b). Tachyzoites fixed in formalin (HS antigen) are agglutinated by antibodies produced later in primary infection. The AC:HS agglutination titer ratio can be used to assign acute and non-acute patterns to sera. Among persons known to be infected for more than 2 years, only 13% showed an acute pattern with the AC:HS test (Dannemann et al., 1990). The IgM immunosorbent agglutination assay (ISAGA) has better sensitivity than IgM ELISA assays, and is useful in detection of IgM in congenitally infected infants (Desmonts et al., 1981). The avidity of IgG antibodies produced early in infection is low (Hedman et al., 1989). Avidity can be used to help differentiate between recent and long-standing infection in pregnancy (Cozon et al., 1998; Jenum et al., 1997; Lappalainen et al., 1993).

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