Serological reactivity against cyst and tachyzoite antigens of Toxoplasma gondii determined by FAST-ELISA.

AIMS: To obtain quantitative data on the human serological response to Toxoplasma gondii tachyzoite and bradyzoite antigens. METHODS: Serum samples from 30 patients who had positive antibody titres against T gondii and from 14 who were seronegative, together with sequential serum samples from four infected individuals, were screened by FAST-ELISA. RESULTS: Serum samples from the 30 seropositive patients showed high IgG and IgM titres against the T gondii tachyzoite antigen but very low responses to cyst antigen. This result was borne out in sequential serum samples from patients with toxoplasmosis. CONCLUSION: Antibody recognition of the cystic stage of T gondii is low, implying that either this stage is poorly immunogenic or that the antigen load is low.

Toxoplasmosis in England and Wales 1981 to 1992.

We have examined laboratory reports of toxoplasmosis received by the PHLS Communicable Disease Surveillance Centre between January 1981 and December 1992 in order to describe epidemiological trends in the three main clinical manifestations of toxoplasmosis-lymphadenopathy, eye disease, and neurological disease; and the two most important risk groups-fetuses and people whose immunity is impaired. The total numbers of reports each year did not change significantly between 1981 and 1992 and were similar to the numbers between 1976 and 1980, but different trends emerged in each subgroup. Reports of acute lymphadenopathic toxoplasmosis declined in children and young adults and eye disease associated with toxoplasmosis fell markedly in all age groups. Reports of neurological disease and severe toxoplasmosis complicating impaired immunity, due mainly to HIV infection or transplant surgery, rose. Reports of infections diagnosed in pregnancy rose steeply in the late 1980s although reports of congenital toxoplasmosis were no more common than in 1975 to 1980. Reports of acute toxoplasmosis came mainly from southern England. The emergence of these diverse trends from apparently unchanging totals emphasises the importance of surveillance systems capable of discrimination.

Toxoplasma gondii: detection of antibodies in human saliva and serum.

Saliva samples from 27 patients with a recent toxoplasma infection were tested for specific IgG, IgM and IgA antibodies to Toxoplasma gondii. Thirteen of the 27 saliva samples were positive for IgG anti-T. gondii by direct agglutination and 8 of the 27 were positive for IgM anti-T. gondii by an immunosorbent agglutination assay. Twenty of the 27 saliva samples were positive for IgG antibody on toxoplasma immunoblots with three major immunodominant antigens; 38, 30 and 35 kDa. IgA results on toxoplasma immunoblots were positive for all three groups tested, recently infected patients, chronically infected and seronegative adults without distinguishing between them. The 35 and 43 kDa antigens were the most frequently detected proteins. IgM in saliva gave negative or very weak reactions. None of the eight seronegative or the 17 chronically infected adults gave positive results in any of the tests performed to detect IgG or IgM in saliva. Serial saliva and serum samples from a laboratory-infected patient were collected and tested for toxoplasma-specific IgG, IgM and IgA. IgG in saliva was detected by 27 days post infection (p.i.) and was negative by 81 days p.i.; it detected mainly the 38 and 30 kDa antigens. IgM in saliva was detected by 11 days p.i. and was negative by 81 days p.i., with no reaction on immunoblots.

Toxoplasma infection and systemic lupus erythematosus: analysis of the serological response by immunoblotting.

AIMS: To examine the serological response of patients with systemic lupus erythematosus (SLE) and toxoplasma infection and to compare the blot profiles with those from immunocompetent subjects of similar immune response. METHODS: Forty serum samples from patients with SLE were tested for toxoplasma antibodies using the dye and indirect haemagglutination tests. Specific IgM was measured by mu-capture enzyme linked immunosorbent assay (ELISA). The sera were immunoblotted using antigen strips prepared from the RH strain of Toxoplasma gondii. For comparison, control blots were prepared from pooled sera from immunocompetent subjects with serological evidence of acute (pool 1), or chronic (pool 2) toxoplasma infection, or with no evidence of infection (pool 3). RESULTS: Some of the blot profiles from the patients with SLE were compatible with the corresponding serology but others showed considerable variation, particularly among the IgM blots. The blots from sera with low dye test titres suggested that the latter could be false positive results. CONCLUSIONS: Toxoplasma infection may enhance the production of autoantibodies which, when combined with the high titres characteristic of SLE, might interfere in the dye test and other serological tests. Immunoblotting could prove useful in the immunocompromised for confirming the presence of specific toxoplasma antibodies and for the staging of infection in those with positive serology.

Sequential reactivity of serum against cyst antigens in Toxoplasma infection.

AIMS: To compare the recognition of Toxoplasma gondii tachyzoites and cysts by sera, from 10 patients. METHODS: Recognition of antigens from purified tachyzoites (RH strain) and bradyzoites (18691 strain) was compared using western immunoblotting. Sequential serum samples from 10 patients and one laboratory acquired RH infection were used. RESULTS: Recognition of cyst antigens was relatively low and occurred late in infection. The two stages were antigenically distinct with only a few shared bands. CONCLUSION: Immunological recognition of the cystic stage of T gondii is low. This implies that either cysts are poorly immunogenic or that cyst antigen is not available for processing and presentation.

Prevalence of antibodies to Neospora caninum in a population of urban dogs in England.

Of 163 dogs, randomly selected from those examined at the University of Liverpool Small Animal Hospital, 12.9 per cent had antibody titres > or = to 1/200 to Neospora caninum in an indirect fluorescent antibody test. None was apparently suffering clinical neosporosis. There was no association between the occurrence of neospora antibodies and either toxoplasma antibodies measured by the dye test, sex, age, type of feeding or the presence of other dogs in the household. Antibody was detected at titres > or = to 1/200 in nine breeds, suggesting that there is a substantial level of subclinical infection in British dogs.

Use of the polymerase chain reaction to detect Toxoplasma gondii in human blood samples.

AIMS: To assess the value of detecting Toxoplasma gondii in human blood samples using the polymerase chain reaction (PCR). METHODS: Blood samples in lithium heparin were examined from 34 patients with suspected toxoplasmosis, and six healthy volunteers with or without the addition of doubling dilutions of toxoplasma tachyzoites. Products of a nested PCR, using oligonucleotide primers of the B1 gene, were analysed by electrophoresis and stained by ethidium bromide. The primary product was 194 base pairs in length; the nested products were 160 or 97 base pairs. RESULTS: When toxoplasma tachyzoites were added to the leucocytes of six different volunteers, eight to 16 parasites were detected by nested PCR in one experiment and one to four parasites in eight experiments. All nine experiments were negative in samples without added tachyzoites. Of 34 patients, PCR was negative in 13 with recent lymphadenopathy; nine with persisting IgM, including two pregnant patients; four with reactivated infection due to immunodeficiency; and five with no evidence of active infection. Positive PCR results were found in three patients with reactivated infection. There was only one discrepancy between PCR and animal culture results; this was in an immunocompromised patient with a positive PCR and negative culture. CONCLUSIONS: The PCR technique was rapid, sensitive, and specific in human blood samples. Negative PCR results in patients with persisting IgM suggested that the fetus was not at risk, or that treatment was not indicated in myalgic encephalomyelitis-like illness. PCR results in immunocompromised patients permitted appropriate management--no treatment if negative, treatment if positive.

Toxoplasmosis and systemic lupus erythematosus.

The toxoplasma serological status of 50 patients with systemic lupus erythematosus (SLE) was compared with that of 50 healthy controls; high titres of toxoplasma antibody were significantly more common in the patients with SLE. These titres did not correlate with any of the routinely measured indices in SLE nor with the patients' prior treatment. A case history is used to illustrate the difficulty in diagnosing toxoplasmosis in the presence of SLE.

Five commercial enzyme linked immunosorbent assay kits for toxoplasma specific IgM antibody.

Five commercially available enzyme linked immunosorbent assay (ELISA) kits for the detection of specific IgM against Toxoplasma gondii were evaluated in a three centre study and results compared with those of the Public Health Laboratory Service ELISA for Toxoplasma IgM (PHL IgM ELISA). Fifty selected sera were tested by all the methods (Toxo-M, Captia Toxo-M EIA, Toxo Enz M EIA, Toxonostika IgM EIA, Sopazyme Toxo IgM EIA) at the three reference centres in England and Wales and 177 routine sera by all the methods in one or other of the centres. Ten of the 50 selected sera contained autoimmune antibodies but no specific IgM and 29 had toxoplasma specific IgM detectable by the PHL IgM ELISA. The kits were assessed for their specificity and sensitivity compared with the PHL IgM ELISA, and the percentage coefficient of variation for binding to the solid phases was determined. They were also rated subjectively by the staff performing the assays and an overall impression of each kit was gained by allocating scores of several criteria. There was quite close agreement among the results obtained with all five commercial assays and the PHL IgM ELISA, although some of the sera pre-selected as being potentially problematic showed the limitations of some of the assays.

Toxoplasmosis in heart and heart and lung transplant recipients.

Of the first 250 heart and 35 heart and lung transplant recipients at Papworth Hospital, Cambridge, who survived for more than one month after transplantation, 217 heart and 33 heart and lung patients were investigated serologically for evidence of Toxoplasma gondii infection. Six patients acquired primary T gondii infection, most probably from the donor organ. Five patients experienced T gondii recrudescence, two of whom had recovered from primary infection a few years earlier. Two patients died from primary T gondii infection and the severity of symptoms in the other patients with primary infection was related to the amount of immunosuppressive treatment. Prophylaxis with pyrimethamine (25 mg a day for six weeks) was introduced for T gondii antibody negative transplant recipients who received a heart from a T gondii antibody positive donor after the first four cases of primary toxoplasmosis. Of the seven patients not given pyrimethamine, four (57%) acquired primary T gondii infection. This compared with two of the 14 patients (14%) given prophylaxis.

Non-Hodgkin's lymphoma presenting as 'chronic active toxoplasmosis'.

We report the case of a 58 year old man in whom an unusually prolonged infection with Toxoplasma gondii was the presenting feature of an underlying non-Hodgkin's lymphoma. This case demonstrates the particular usefulness of the IgM ELISA test in monitoring disease activity.

Serologic diagnosis of whooping cough by enzyme-linked immunosorbent assay.

Pernasal swabs were obtained on 3 consecutive days from 146 children referred for hospital admission with suspected whooping cough, and immunoglobulin A and immunoglobulin M antibodies to Bordetella pertussis were measured by enzyme-linked immunosorbent assay. The clinical features in 113 of the children were considered consistent with the diagnosis. Sixty-four cases were confirmed by serology, which showed a greater sensitivity (57% vs. 35%) than pernasal swab culture with no loss of specificity (100%). Paired serum samples were necessary for diagnosis in 30 (47%) of these 64 cases. Seventeen (43%) of 40 cases confirmed by pernasal swab culture had negative serologic results. Most of these were young infants who showed a less reliable antibody response. Detection of antibodies to B. pertussis by enzyme-linked immunosorbent assay can be a valuable additional test in the differential diagnosis of whooping cough but is not appropriate as the sole diagnostic test.

Prevalence of chlamydia, toxoplasma, toxocara and ringworm in farm cats in south-west England.

The prevalence of infection with Chlamydia psittaci, Toxoplasma gondii, Toxocara cati and Microsporum canis was examined in 51 cats on 22 sheep farms in the Bristol area. Serum antibody to C psittaci and T gondii was present in 45 per cent and 47 per cent of cats, respectively. At the time of sampling C psittaci was isolated from 6 per cent of the cats, T cati was identified in 63 per cent of faecal samples but neither T gondii nor M canis was isolated. When examined according to the farm of origin, 22.7 per cent of farms had cat populations with no evidence of infection with C psittaci or T gondii. Of the remainder, 45.5 per cent supported cat populations with evidence of both infections and 31.8 per cent had evidence of T gondii infection alone. None of the farms had cat populations with evidence of C psittaci infection alone. Two of the cats infected with C psittaci were excreting viable organisms in the faeces. The possible significance of this to the epidemiology of ovine enzootic abortion is discussed.

Use of monoclonal antibodies in an ELISA to detect IgM class antibodies specific for Toxoplasma gondii.

Two monoclonal antibodies CH6 and C1E3 were used in an antibody class capture assay for the detection of IgM antibodies specific for Toxoplasma gondii. CH6 was used on the solid phase to capture human IgM. After a Toxoplasma gondii antigen had been added, specifically bound material was detected using C1E3 coupled to horseradish peroxidase. The assay was compared with an established system using polyclonal antisera at both the capture and antigen detection stages. A good correlation was found, with 97.3% (125 of 128) of sera giving the same classification in both assays. Three sera were positive only in the polyclonal system. No false positive results were found when 118 negative sera were examined. The two monoclonal antibodies provide a viable alternative to the use of polyclonal sera at the capture and antigen detection stages in the antibody class capture assay for the measurement of specific IgM against T gondii.

Public Health Laboratory Service enzyme linked immunosorbent assay for detecting Toxoplasma specific IgM antibody.

An enzyme linked immunosorbent assay (ELISA) based on the antibody class capture method for the detection of specific IgM against Toxoplasma gondii, using the microtitre plate format, was developed. Antigen binding was detected using a monoclonal antibody, CIE3, conjugated to horseradish peroxidase. Prior mixing of the conjugate and antigen improved the stability of these reagents as well as removing an incubation stage from the assay. The incubation time of less than four hours permits a rapid throughput of specimens. Using the assay, a total of 163 sera were examined in a three centre study and good agreement was found. Results were expressed as arbitrary enzyme immunoassay units (EIUs) against a freeze dried standard. Throughout the study the standard serum showed a coefficient of variation less than 10% across the microtitre plate. By measuring IgM titres in patients having toxoplasmic lymphadenopathy with a known date of onset, IgM class antibodies were shown to peak at two months, persisting for about six months. In addition, a case of laboratory acquired toxoplasmosis was monitored. Sera shown to contain rheumatoid factor and antinuclear factor did not give false positive results. This rapid, robust, and simplified assay is used by the Public Health Laboratory Service Toxoplasma Reference Units and will provide a standard with which other assays can be compared.

Problems with serological diagnosis of Toxoplasma gondii infections in heart transplant recipients.

Of the first 128 patients to receive either heart or heart and lung transplants at Papworth Hospital, four developed Toxoplasma gondii infections acquired from the donor heart and two died. Six patients had passively acquired T gondii antibody as a result of blood transfusions around the time of transplantation. Eight patients developed antibodies against T gondii, which were detectable by changes in the latex agglutination test titres but not by those of the dye test. These false positive latex agglutination reactions occurred simultaneously with cytomegalovirus infection and were associated with the IgM serum fraction in the patients' sera. These reactions were not associated with cytomegalovirus specific IgM, Paul-Bunnell antibody, nor detectable rheumatoid factor. These findings emphasise the need for T gondii dye test confirmation of latex agglutination test titre rises in heart transplant recipients.

Detection of specific IgG and IgM antibodies to Toxoplasma gondii with a commercially available enzyme immunoassay kit system.

A total of 138 serum samples submitted for toxoplasma serology have been examined by enzyme immunoassay using kits produced by Labsystems Oy for the detection of specific antibodies of the IgG and IgM class. Results were compared with the dye test, an indirect haemagglutination test, and an indirect immunofluorescence test for specific IgM. The enzyme immunoassay was less sensitive than the dye test, but by running both IgG and IgM enzyme immunoassays, 92.4% sensitivity was achieved. The specificity of the enzyme immunoassay was good, with only one dye test negative serum giving a positive (but weak) IgG enzyme immunoassay reaction. Thirty serum samples from patients with no evidence of exposure to Toxoplasma gondii gave negative results in the IgM enzyme immunoassay. Enzyme immunoassay results were expressed in enzyme immunoassay units, as a percentage value of a standard serum. This convention will be of value in the direct comparison of assay systems and in the application of quality control procedures.