All neonates suspected of having a congenital infection should have a complete blood cell count (20), including a platelet count to screen for thrombocytopenia (40,41) and eosinophilia (42) (although the latter has not been a common finding in many infected infants), and liver function tests. A lumbar puncture should be done to determine if there is a disproportional increase in cerebrospinal fluid (CSF) protein relative to the degree of pleocytosis (25,43,44) and a computed axial tomographic scan of the brain with contrast to detect diffuse cerebral calcifications (27). Every infant suspected to be congenitally infected (especially those who are asymptomatic) should have a thorough ophthalmologic examination to detect any ocular complications (i.e., chorioretinitis) (Table 1) (1).
Serological Diagnosis of Congenital Toxoplasma Infection
Although there are a number of direct and indirect assays available to detect infection with Toxoplasma, serological testing remains the most common means of diagnosing toxoplasmosis (1,3,4,24-26). The results of such testing, however, must be interpreted carefully. The diagnosis of acute acquired infection can be made by finding the simultaneous presence of Toxoplasma IgM and IgG antibodies or the demonstration of a fourfold rise in IgG titers (1). The sensitivity of the commonly employed Diagnostic Clinical Laboratory techniques for these antibodies is adequate for many cases of acquired infection but not all, especially when it involves the confirmation of primary infection during pregnancy. These same assays are usually inadequate in cases of congenital infection.
In both of these scenarios, the appropriate clinical specimens should be submitted to a reference laboratory with expertise in the serological diagnosis of toxoplasmosis to either confirm or refute the presence of infection. Table 3 contains a brief description of the various methods that have been developed to detect Toxoplasma-specific antibodies. That numerous assays employing different methodologies have had to be developed hints at the difficulties previously encountered with serological testing. The antigenic structure of T. gondii is extremely complex, composed of several proteins (both membrane and cytoplasmic) from different phases of the life cycle. This may partially explain the discrepancy in the ability of different assays to detect Toxoplasma-specific antibodies. Certain antigens cross-react with normal human proteins, as evidenced by the presence of "natural" anti-Toxoplasma IgM antibodies in the sera of uninfected patients.
Furthermore, methodologic difficulties and lack of standardization have prevented a single assay from being widely adopted. The continuing improvements in various ELISA assays (i.e., double-sandwich [DS] ELISAs for Toxoplasma-specific IgM and IgA) and the further development of immunosorbent agglutination assays for IgM, IgA, and IgE, with their greater sensitivity and specificity, should lend impetus to the need to standardize these tests. Once standardized, it is hoped they will then become more widely available in many diagnostic laboratories in the United States.
Toxoplasma-specific IgG antibodies typically achieve a peak concentration 1-2 months following acute infection and remain positive for life following both acute and congenital infection. Although the Sabin-Feldman dye test is the standard (45,46) against which all other assays should be judged, many clinical laboratories have adopted various commercially available Toxoplasma IgG ELISA assays. The IgG indirect immunofluorescence assay (IFA) measures the same antibody as the dye test and is easier to perform, but it is highly laboratory dependent. False-negative results may occur in sera with low titers, and sera that contain antinuclear antibodies may yield false-positive results. The results from IgG ELISA assays correlates well with the dye test and the IFA, but these have not been standardized as yet (47).
Toxoplasma-specific IgG declines continuously (approximate half-life 30 days) in the uninfected infant as the transplacentally acquired maternal antibody is catabolized. It usually becomes undetectable sometime between 6 and 12 months of age (48,49). Among congenitally infected infants, depending on the timing of in utero infection, the Toxoplasma IgG may decline initially and not begin to rise until 3-4 months of
Serological Assays for Toxoplasma gondii-Specific Antibodies and the Diagnosis of Congenital Infection
Sabin Feldman Dye Test: The dye test is the gold standard for the detection of Toxoplasma-specific IgG but requires the availability of a mouse facility to generate the fresh tachyzoites used in this assay. It is available only in reference laboratories. Following incubation in normal saline and methylene blue, the parasite swells and stains deep blue. Pretreatment of the parasites with antibody containing sera and complement produces lysis. Lysed organisms appear thin and distorted and fail to stain with the dye. The IgG titer reported is that dilution of serum at which half of the organisms are not killed (appear stained). The titers are determined by comparison to a reference serum and expressed in international units (IU) per milliliter of serum.
Indirect Hemagglutination Test: Red cells tagged with T. gondii agglutinate when sera containing specific antibodies are added. It is not recommended for the diagnosis of primary infection during pregnancy because of the delay in the rise in T. gondii IgG titers. It is also not recommended for the diagnosis of congenital toxoplasmosis as false-negative reactions have been observed in cases with high dye test titers.
Complement Fixation Test: Complement-fixing IgG antibodies appear later than those demonstrable by the dye test. The antigen preparations used in this test have not been standardized; therefore, this test cannot be recommended for routine use for infants.
Agglutination Test: This method is very sensitive in detecting specific IgM and is routinely used as part of the mouse inoculation assay. It has primarily been used in Europe to screen for T. gondii IgM and IgG but is not widely available in the United States. The differential agglutination method (HS/AC) has been demonstrated to be capable of differentiating between acute vs chronic infection.
IFA Test: Slide preparations of killed T. gondii are incubated with serial dilutions of the patient's serum. A positive reaction is detected by the fluorescence of the organisms when examined under the microscope. Correlation with the dye test in detecting IgG antibodies is excellent, but reliable and reproducible titers are difficult to obtain. False-positive results occur with sera that contain antinuclear antibodies. The IFA test has been adapted for the demonstration of IgM antibodies to T. gondii, but the sensitivity and specificity of this assay when used to diagnose congenital infection is poor. The sensitivity is only 25%; the specificity is marred by the occurrence of false-positive reactions. Thus, although IFA may detect Toxoplasma-specific IgM, it should not be used as the only means of diagnosing congenital infection.
Conventional ELISA: This method has largely replaced other tests in the routine clinical laboratory because of its availability, rapid turnaround time, simplicity, reliability in most clinical situations, and capability of performing tests on multiple clinical specimens at the same time. It has been used successfully to detect Toxoplasma-specific IgG, IgM, IgA, and IgE in the pregnant woman, fetus, and neonate. IgG ELISA titers correlate well with the results of the dye test, but the conventional IgM ELISA (IgM-EIA) detects IgM in only 50% of congenitally infected infants. False-positive reactions have also been a problem. Commercial kits are presently available to detect Toxoplasma IgG and IgM.
Table 3 (Continued)
Serological Assays for Toxoplasma gondii-Specific Antibodies and the Diagnosis of Congenital Infection
Double-Sandwich or Capture Enzyme-Linked Immunosorbent Assay(DS-ELISA or DS-EIA): These assays differ from the conventional ELISA in that they employ plates with wells that are coated with antihuman IgM or IgA antibodies (rather than antigen) to bind these specific antibodies. The IgM DS-EIA has become the most widely used method for the demonstration of T. gondii IgM. It avoids false-positive results (related to rheumatoid factor) and false-negative results (secondary to competition from maternal IgG) previously seen with the IFA. The sensitivity of the IgM DS-EIA among congenitally infected infants approaches 73% during the neonatal period and 81% during the first month of life. The DS-ELISA for the detection of T. gondii-specific IgA is more sensitive than either the IgM-EIA or the IgM ISAGA for the diagnosis of congenital infection when the commercial assay is appropriately standardized.
Enzyme-Linked Immunofiltration Assay: Preliminary data suggest that this assay may hold great promise as a means of detecting IgA and IgE in congenitally infected infants, although it is not available commercially. It employs a Millipore filter as a solid-phase, immunopre-cipitation to determine antibody specificity, and immunofiltration to determine antibody isotypes. Pinon et al. (64) were able to diagnose 94% of congenitally infected infants by 3 months of age using a combination of IgA enzyme-linked immunofiltration and IgM ISAGA.
ISAGA: The ISAGA combines the sensitivity and specificity of the direct agglutination and the DS-ELISA for T. gondii IgM, IgA, and IgE. The sensitivity and specificity of the ISAGA for IgM is superior to both the IgM IFA and the IgM ELISA and detects T. gondii IgM both earlier and later than other assays. The ISAGA has also been used to detect T. gondii IgA and IgE. The IgM ISAGA has been widely used by several investigators. A commercial assay is available from Bio-Meriux (Lyon, France).
Immunoblot: Although a modification of the Western blot assay has been useful in identifying some congenitally infected infants (based on the presence of select antigen-antibody bands not found in maternal sera ), it is primarily a research tool.
IgG Avidity Testing: Based on the increasing avidity of IgG antibodies during the transition from acute to chronic infection, this assay (which correlates well with the HS/AC agglutination assay) has proven to be useful in excluding acute infection during the earlier part of pregnancy. However, because of a lack of consensus on a standard procedure and the lack of availability, it cannot be recommended presently for routine use in the diagnosis of congenital infection.
age. If the mother and her infant are infected close to birth, the infant's Toxoplasma IgG may not be detectable until a few months of age. Consequently, the diagnosis may be missed. This phenomena may also explain why the most sensitive serological assays for Toxoplasma IgM (i.e., IgM immunosorbent agglutination [ISAGA], etc.) only have a sensitivity of 75-80% for detecting congenital infection.
Two additional clinical scenarios in which the Toxoplasma-specific antibody may be impaired relate to congenitally infected infants who are also perinatally infected with HIV or whose anti-Toxoplasma therapy is begun perinatally or soon after birth. Such treatment, if it is begun early in infection, reduces antigenic challenge by killing tachyzoites, thus blunting antibody synthesis. Serological rebound occurs in a majority of such infants 2-6 months following cessation of therapy (49-53). Typically, once established the Toxoplasma IgG titer will remain positive for life. Congenitally infected infants who have rapidly progressive HIV-1 disease may not be able to synthesize Toxoplasma-specific antibodies (38).
Typically, IgM antibodies can be detected 1-2 weeks following acute infection, peak in 1 month, and decline thereafter. In many cases, they may become undetectable within 6-9 months. The IgM antibody, however, may persist for several more months. Although the IgM IFA and the conventional IgM ELISA can usually detect Toxoplasma-specific IgM following acute infection (54-56), the IgM DS-ELISA and IgM ISAGA are both more sensitive and specific and should be used to screen for Toxoplasma IgM associated with congenital infection (47,57-60). Antinuclear antibodies (61) and rheumatoid factor (62) may cause false-positive results with the IgM IFA. The sensitivity of the IgM double-sandwich enzyme immunoassay (DS-EIA) among congenitally infected infants is 73% during the neonatal period and 81% during the first months of life. The sensitivity of the IgM ISAGA approaches 75-80% among congenitally infected infants and usually detects Toxoplasma IgM both earlier and later than many other assays (63).
Toxoplasma-specific IgA and IgE usually decline to undetectable levels sooner than IgM and are helpful in determining the timing of acute infection during pregnancy (especially when the initial infection pre-dated conception and the Toxoplasma IgM is still positive). IgA production parallels IgM production, with peak levels occurring approx 2 months following initial infection and then declining quickly (48,52). Toxo-plasma-specific IgE as detected by the IgE ISAGA has a similar pattern of development but decreases more rapidly than do IgA antibodies (64).
As with Toxoplasma IgM, the documentation of Toxoplasma IgA or IgE in neonatal sera is diagnostic of congenital infection (unless a materno-fetal transfusion has occurred). Follow-up testing for these antibodies should probably be performed at more than 2 weeks of age to confirm their presence if the initial assays are reactive. Prior reports have suggested that the sensitivity of the Toxoplasma IgA and IgE assays for detecting congenital infection may in fact be superior to that previously noted with IgM testing (52,64), and although the development of commercial kits for Toxoplasma IgA is encouraging, they are as yet not reliable (65,66). There are as yet no commercial kits to test for Toxoplasma IgE (64).
Isolation of this obligate intracellular parasite from amniotic fluid or neonatal blood provides unequivocal proof of infection (67-69) but requires the availability of a clinical laboratory that is proficient in these procedures. Furthermore, the results may not be available for a period of weeks. An attempt to isolate the parasite from cord or peripheral blood of the newborn should, however, be made as a definitive serological diagnosis may not be possible during the first weeks or months of infection. Blood (in a serum tube) should ideally be drawn from the infant within the first week of life as the frequency of detectable parasitemia decreases after this point. Specimens of CSF and other bodily fluids as indicated (i.e., peritoneal fluid, amniotic fluid in the case of prenatal diagnosis, etc.) should also be submitted for testing. Although the specimen should be processed and inoculated as soon as possible to prevent the death of the organism, specimens should be kept at 4°C if short-term storage or shipping is required. Freezing should be avoided (1).
Because the organism is most likely to reside within the white blood cells in patients with parasitemia, the buffy coat layer should be separated from the plasma and processed for subsequent inoculation. The separation of the buffy coat from the plasma should also lessen the chance of T. gondii-specific antibodies interfering with the detection of the organism (1). If the blood clot from a serum tube is used, the clot should be triturated in a small amount of normal saline and drawn into a syringe. Placental tissue from all infants suspected of congenital infection (stored at 4°C without fixative) should also be processed by trypsin digestion and inoculated into mice (1,24). Positive results have previously been found to correlate well with congenital infection.
Isolation of T. gondii by Tissue Culture Inoculation
Although isolation by tissue culture inoculation has been used by certain investigators with some success, the consensus seems to be that this method is a less-sensitive method than mouse inoculation (67,70). Inoculated cultures of human embryonic fibroblasts grown on coverslips are incubated for 72-96 hours, fixed, and processed for indirect immunofluorescence using anti-T. gondii-specific antibody. Although this method would be most useful when an early diagnosis is needed (positive results may be obtained in 4-5 days), its relative insensitivity and lack of availability have lessened its importance as a diagnostic tool.
Mouse intraperitoneal inoculation has been considered the gold standard for demonstration of this organism (1). At 5-10 days after injection, peritoneal fluid should be examined for the presence of tachyzoites. Demonstration of the organism is proof of infection (67-69). If no organisms are recovered, mouse serum should be tested for T. gondii antibodies 6 weeks postinjection (the preferred method of testing is agglutination as this requires only a small amount of serum). Confirmation of infection once antibodies are found should be performed by examination of the brain for the presence of cysts (1). Although earlier serological testing of the mice may detect antibodies sooner, definitive results from this assay are usually not available for at least 3-6 weeks.
The development of a PCR assay capable of detecting T. gondii has significantly enhanced the ability to detect congenital infection. Grover (71) demonstrated the utility of a PCR assay (employing primers for the repetitive B1 gene) as a means of making a rapid prenatal diagnosis. In this study, PCR correctly identified T. gondii in amniotic fluid samples from 7 of 9 congenitally infected infants studied. No false positives were reported.
Subsequent reports (72) have documented that PCR testing of amniotic fluid have equivalent sensitivity, specificity, and positive and negative predictive valves as conventional assays (i.e., mouse inoculation and serological assays) (44,73-77). PCR testing will most likely become the preferred method of testing because of its rapid turnaround time, reliability, safety, simplicity, and low cost. PCR has also been shown to be capable of detecting T. gondii in CSF, ascitic fluid, blood, urine, and placental tissue (78,79). There is as yet insufficient data about the utility of these sources compared to amniotic fluid. The use of PCR on amniotic fluid without having to use fetal blood samples represents perhaps the greatest advancement in prenatal diagnosis of T. gondii infection in the fetus.
PCR will most likely replace the other two methods of isolation of the parasite described above, although there are disadvantages. These include the following: (1) PCR's primary utility as a means of making the diagnosis prenatally, (2) limited availability of PCR at this time; (3) occasional false-negative assays (false-positive assays appear to be rare), and (4) like any of the other conventional assays, failure to detect materno-fetal transmission occurring after amniocentesis close to partuition.
Detection of T. gondii-Specific Antigens and Cell-Mediated Immunity
Serological detection of T. gondii antigens by ELISA and demonstration of a specific cell-mediated immune response as assessed by antigen-specific lymphocyte transformation have both been used previously to detect congenital infection. There is as yet insufficient data regarding their respective sensitivity and specificity to warrant their inclusion as part of the standard diagnostic workup.
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