A. W. L. Joss

Do IgA, IgE, and IgG avidity tests have any value in the diagnosis of toxoplasma infection in pregnancy?

AIM: To determine the value of tests for specific IgA, IgE, and IgG avidity in diagnosing Toxoplasma gondii infection during pregnancy. METHODS: In a retrospective study, current serological tests (dye test and three IgM assays with different sensitivities) were compared with immunosorbent agglutination assays (ISAGA) for specific IgA and IgE and an IgG avidity enzyme linked immunosorbent assay (ELISA). Patient group 1 comprised six women with definite or probable infection during pregnancy determined by congenital toxoplasmosis or laboratory results. Group 2 comprised seven women infected during or before 11 pregnancies (two consecutive pregnancies in two patients and three in a third). RESULTS: One patient in group 1 seroconverted during pregnancy. IgA ISAGA and avidity confirmed acute infection when confirmatory IgM ELISA remained negative. In five of six patients from group 1, IgA and IgE ISAGA and avidity confirmed acute infection. In group 2, the dye test titre was raised in seven of 11 pregnancies (six of seven patients). Specific IgM and IgA were positive during all 11 pregnancies. IgE ISAGA was positive in only four of 11 pregnancies (three of seven patients), but negative results in the remainder may exclude acute infection. High avidity antibodies indicative of past infection were found in four of 11 pregnancies (two of seven patients). CONCLUSIONS: Each test improved diagnosis or timing of infection but no single test was ideal. The IgA ISAGA was sensitive and detected seroconversion. Positive IgE ISAGA and low avidity both confirmed infection, whereas negative IgE may exclude acute infection. High avidity diagnosed past infection but persistence of low avidity reduced its value to differentiate acute and past infection. Further studies with larger patient groups are needed to determine the optimum diagnostic strategy. These techniques are valuable in complementing existing tests.

Isolation of Borrelia burgdorferi from ticks in the Highlands of Scotland.

Borrelia burgdorferi, the causative agent of Lyme disease, was first isolated in 1982 and since then has been regularly isolated from ticks and clinical material in both Continental Europe and the USA. However, only three isolations have been reported in Britain. During the summer of 1997, 128 ticks were collected from two sites in the Highlands of Scotland and examined by the polymerase chain reaction (PCR) and culture. Eleven fresh isolates were obtained from culture and passed up to 22 times. Seven of the tick emulsions were also positive by flagellin gene PCR, and a further one was positive by PCR but negative on culture. All 11 isolate cultures were positive by the flagellin gene PCR. Further studies on four of these isolates confirmed their identity by immunofluorescence, but also detected possible differences between them and B. burgdorferi ACA-1 by enzyme profiles and by PCR with OspA gene primers. Culture of these new strains provides antigens that should improve diagnostic serological tests in Britain.

Comparison of relative uses of commercial assays for Toxoplasma gondii IgM antibodies.

AIMS: To compare the sensitivity and user friendliness of seven commercially available enzyme linked immunoabsorbent assay (ELISA) kits for toxoplasma specific IgM. METHODS: Five antibody capture assays supplied by Abbott, Mercia, Northumbria, Organon and Sorin, and two indirect ELISA assays from Biostat and Mast, were assessed. Using defined dilutions of Toxoplasma gondii specific IgM, the performance and sensitivity of each assay was established. They were further assessed on a panel of 27 sera with a range of dye test and IgM results (as determined by the Scottish Toxoplasma Reference Laboratory). All of the assays were performed by three experienced operators and assessed for user satisfaction. RESULTS: The Mast, Organon, and Abbott assays were of low sensitivity; the Mercia and Northumbria were of high sensitivity; and the Biostat and Sorin assays produced too many false positive results. The Mercia kit provided most user satisfaction; the Mast and Abbott assays were most difficult to use. CONCLUSIONS: Local laboratories investigating toxoplasma infection should have three tests: one IgG and two IgM (high and low sensitivity) to help in the timing of infection. Alternatively, one sensitive IgM assay, such as that of Mercia, could be used by selecting appropriate high and low thresholds.

Specificity and usefulness of an IgE immunosorbent agglutination assay for toxoplasmosis.

AIMS--To develop an immunosorbent agglutination assay for the detection of Toxoplasma gondii IgE antibodies (IgE-ISAGA); to assess its specificity; and to determine the role of specific IgE in the diagnosis of current toxoplasma infection. METHODS--Rabbit antihuman IgE capture antibody was adsorbed onto microtitre plates and formaldehyde fixed tachyzoites were used to identify specific antibody. Specificity was assessed in 513 serum samples (blood donor, potentially interfering and difficult, elevated and low total IgE and myeloma). Serum samples (n = 108) from 65 patients with documented toxoplasmosis were tested, as were 26 serum samples from nine pregnant women positive for specific IgM and 30 from 20 HIV positive patients. RESULTS--IgE-ISAGA was highly specific with only three of 513 (0.58%) positive reactions amongst the control groups, one of which (0.19%) was regarded as a false positive. Elevated total IgE did not influence specific IgE results nor did the presence of abnormal immunoglobulin concentrations. Sixty (92.3%) patients with toxoplasma associated lymphadenopathy had specific IgE in one or more samples. Positive or borderline results were obtained in 68 of 77 (88.3%) serum samples taken up to four months after onset and were also detected for up to 11 months in 21 of 31 (67.7%) sera. Of the nine pregnant women with detectable specific IgM, specific IgE was absent in five (12 specimens). Specific IgE was also detected in 10 of 30 (33.3%) serum samples from the 20 HIV positive patients, which was similar to the number with specific IgM. Neither the specific IgE nor IgM tests could distinguish symptomatic from asymptomatic HIV positive patients. CONCLUSIONS--IgE-ISAGA is sensitive, specific, and easy to perform. Although results suggest that specific IgE may be less helpful than previously claimed, specific IgE has a useful role in the diagnosis of current toxoplasma infection when used in conjunction with other tests.

Biotin-labelled antigen screening test for toxoplasma IgM antibody.

A method for the simple preparation of biotin-labelled toxoplasma antigen was used with avidin peroxidase in an IgM-capture enzyme linked immunosorbent assay (BAM-ELISA). Although the overall predictive value of a positive result was only 38%, its low cost and 100% sensitivity makes it a very suitable screening test. Positive results can be confirmed by an alternative assay, thus providing a more economical and effective diagnostic service than either screening all sera by a commercial test or selecting sera for IgM testing.

Do Toxoplasma gondii RH strain tachyzoites evolve during continuous passage

Aim: To examine three lineages of Toxoplasma gondii RH strain in terms of performance in the dye test, culture, and gene expression. Methods: Historical data (culture growth and performance in the dye test) from three lineages of RH strain tachyzoites (B, J, and Q) that had been continuously cultured in HeLa cells was assessed. Tachyzoite harvests obtained during continuous cell culture were retrieved from liquid nitrogen and cultured in HeLa cells, providing mRNA that was extracted and used to study gene expression using random amplified polymorphic DNA analysis at different stages of lineage adaptation to continuous culture. Results: The B and Q lineages consistently produced tachyzoites that were successfully used in the dye test and their gene expression was stable after multiple passages. The J lineage had unpredictable growth, tachyzoites unsuitable for use in the dye test, and changing gene expression with multiple passage. Conclusion: This study has explained some anomalies in the performance of different stocks of T gondii, and suggests that lineages that are still evolving in cell culture should be avoided.

Strategies for detecting toxoplasma immunity.

Abstract A strategy for identifying toxoplasma immunity in pregnancy must provide good evidence of immunity but not falsely reassure; that for immunocompromised patients should identify immunity and also the risk of reactivated toxoplasmosis. Using sera from both of these patient groups, the performance of an in-house IgG EIA and two commonly used commercial assays (Abbott AxSYM Toxo-G and Eiken latex test) were compared with the dye test. False-positive results were obtained using the IgG enzyme immunoassay (EIA) and AxSYM Toxo-G, and false negatives using all three screen tests. During pregnancy, positive results may falsely reassure, and patients should be tested for toxoplasma-specific IgM to differentiate between current infection and immunity. In immunocompromised patients, positive results indicate immunity but negative results do not exclude it; these should be tested by dye test. Despite these reservations, we have demonstrated that immunity screening can be performed within a district general hospital.

Differences exist in the immunoblotting profiles of cyst and trophozoite antigens of Pneumocystis carinii

The antigenic profiles of Pneumocystis carinii trophozoites and cysts were compared by immunoblotting with hyperimmune rat sera against cyst and trophozoite antigens. Strong bands corresponding to proteins of 50-60 kDa and 104 kDa were demonstrated in cyst and trophozoite antigens by all antisera. Additional prominent proteins of 81 and 63 kDa and less prominent proteins of 88, 73, 69 and 37 kDa were found only in trophozoite antigen. The latter proteins were recognised by anti-trophozoite and anti-cyst antisera but the 81- and 63-kDa proteins were associated specifically with trophozoites. With cyst-rich antigen, antibodies to the 50-60-kDa protein were detected in only two of 14 sera from P. carinii pneumonia (PCP)-positive rats. With trophozoite-rich antigen, 11 of 24 rats with PCP and one of 18 PCP-negative animals had antibodies to both the 50-60 kDa and 104-kDa antigens. Antibodies to the 81- or 63-kDa antigens were demonstrated in 15 of 24 PCP-positive animals and none of the PCP-negative animals. The use of trophozoites rather than cysts increased the sensitivity of immunoblotting. As trophozoites predominate in PCP, antibody to trophozoite-specific antigens rather than common cyst and trophozoite antigens is likely to be a more useful marker of current infection.

Pneumocystis carinii antibody testing.

Sera from blood donors and patients from all over Scotland were examined by indirect immunofluorescence using Pneumocystis carinii antigen from infected rat lung. Antibody was found in 76 of 488 (15.6%) of patients tested on clinical grounds but in only 13 of 148 (8.8%) blood donors. The antibody rates were higher in disease groups likely to have or develop P carinii pneumonia: in those with histologically confirmed or strongly suspected P carinii pneumonia the rate was 14 of 24 (58.3%); in those who had undergone transplantation eight of 24 (33.3%); in those who were immunosuppressed five of 16 (31.2%); in those who were human immunodeficiency virus antibody (HIV) positive 11 of 43 (25.6%); in those with malignancy 34 of 233 (14.6%); and in those with chest infection 10 of 85 (11.7%). P carinii pneumonia was confirmed or likely in four of 45 (8.8%) patients with titres of 1/8-1/16 and in three of seven (42.8%) in those with titres of greater than or equal to 1/128. Seroconversion or rising titre was detected in seven of 13 (53.8%) cases of confirmed or likely P carinii pneumonia compared with 10 of 93 (10.7%) in other patients. Diagnosis of P carinii infection can therefore be assisted by positive immunofluorescence results, but negative serology does not exclude infection.

How to detect current toxoplasma infection.

Abstract This study seeks to identify the best way to detect current toxoplasma infection for district general hospital laboratories. One hundred ‘ordinary’ and 174 ‘difficult’ sera are categorised into either an ‘evidence’ or ‘no evidence’ group for current toxoplasma infection. Twelve test protocols are investigated using different combinations of one whole antibody latex test (Eiken Toxoreagent), one in-house specific IgG enzyme-linked immunosorbent assay (ELISA) and three specific IgM assays (Toxo-ISAGA, in-house BAM ELISA IgM and Toxonostika ELISA M). The Eiken latex and in-house IgG assays produced significantly fewer false-negative results than were obtained with the single IgM test or the IgG and IgM test protocols (P<0.05), but a greater number of false-positive results (102/274 and 115/274, respectively). Of the IgM assay test protocols, the three IgM assays in combination produced the least number of false-negative results (1/274). However, a significantly greater number of false-positive results were produced than with one or two IgM tests or an IgG and any IgM test in combination (P<0.001). We recommend testing with three IgM tests, or a whole antibody (Eiken) or IgG-specific assay, and that positive or clinically important negative samples be referred to a reference laboratory for confirmation.