Successful treatment ofToxoplasma gondii myocarditis in an AIDS patient

Central nervous system disease due toToxoplasma gondii is a common cause of morbidity and mortality in patients with the acquired immunodeficiency syndrome. Cardiac toxoplasmosis, however, has been described in only a limited number of cases. In a 45-year-old patient with symptoms suggestive of myocarditis,Toxoplasma gondii was detected in myocardial tissue obtained by biopsy. After the institution of appropriate antiprotozoal therapy, the patient recovered. This patient is believed to be the first patient to survive biopsy-proven myocarditis caused byToxoplasma gondii. Cardiac toxoplasmosis should be ruled out in HIV-infected patients presenting with high fever and/or cardiorespiratory symptoms and exhibiting serologic evidence of prior exposure toToxoplasma gondii as determined by a positive IgG EIA, especially if the CD4+ count is low and no systemicPneumocystis carinii pneumonia prophylaxis has been administered. A high index of clinical suspicion and, if necessary, invasive diagnostic tests, including myocardial biopsies, are most important in making the correct diagnosis.     

Antibody response of HIV-infected patients to latent,cerebral and recently acquired toxoplasmosis

The aim of this longitudinal study with 626 HIV-infected patients was to evaluate the capability of serological tests in diagnosing the presence of Toxoplasma gondii infection in HIV-infected patients, as well as the potential impact of various treatment regimes on serological results. Low IgG antibody levels and stable or declining titres predominated. IgM positivity occurred in ten patients (one seroconversion, seven latent, two cerebral toxoplasmosis). Complement fixation test (CFT) titres ≥1:32 imply that the relative risk of cerebral toxoplasmosis is 6.84 (95% confidence interval [CI] 1.44–32.5) but with a predictive value of only 14.0% (95% CI 5.3–27.9). Values of specific antibodies are not biassed by antiretroviral treatment and/or prophylaxis for toxoplasmosis, and the detection of specific antibodies is very useful in the identification of T. gondii infection in the HIV-infected population, but the role of serology in predicting the clinical manifestation of T. gondii infection is limited.     

Prevalence of latent toxoplasmosis and serological diagnosis of active infection in HIV-positive patients

The seroprevalence of latentToxoplasma gondii infection was determined in a cohort of 715 HIV-positive patients followed up at an HIV outpatient clinic. Using indirect immunofluorescence and direct agglutination assays for detecting IgG, the prevalence of anti-Toxoplasma gondii antibodies was shown to be 50 %. During a four-year period, clinically apparent acute toxoplasmosis occurred in 47 patients (43 with cerebral, 3 with ocular and 1 with bone marrow toxoplasmosis) among the 360 patients positive for anti-Toxoplasma gondii IgG and in one patient (with cerebral toxoplasmosis) among the 355 patients who were serologically negative. A significant rise in IgG levels could be shown during acute toxoplasmosis episodes in only 30 % of patients, compared with 3 % of patients without active toxoplasmosis. During acute toxoplasmosis, IgM antibodies were detected in only two patients (6 %) by an immunosorbent agglutination assay and in one (3 %) by an enzymatic immunocapture assay. Specific IgA was detected by a non-enzymatic immunocapture assay in six patients (18 %) during acute episodes. The very high predictive value (99.7 %) of a negative IgG test remains the best serological parameter for excluding an acute episode of toxoplasmosis in HIV-positive patients.     

Evaluation of immunoblotting for the detection ofToxoplasma gondii immunoglobulin M antibodies

Immunoblot analysis was used to detect human IgM antibodies toToxoplasma gondii in 20 patients with recent toxoplasmosis, 30 immune individuals, 30 non-immune individuals, and 24 children less then two years old. Analysis of the IgM strips revealed that specific IgM antibodies detectable after a recentToxoplasma gondii infection react with the same antigens as the natural antibodies present in the sera of immune and non-immune individuals and in the sera of young children. These data indicate that immunoblotting is not useful as a reference method forToxoplasma gondii IgM detection, and suggest that improvement of the specificity of IgM detection will remain difficult.     

Enzyme-linked immunosorbent assay for detection of solubleToxoplasma gondii antigen in acute-phase toxoplasmosis

Sixty-two sera from 51 patients with lymphadenopathy presumed to be due to acute-phase toxoplasmosis were tested for specific IgM class antibodies by both the immunofluorescence antibody toToxoplasma gondii in sera were first dissociated in 3M NaSCN. Antigen attached to the solid phase was detected with enzyme-coupled IgG antibody toToxoplasma antibody toToxoplasma gondii in sera were first dissociated in 3M NaSCN. Antigen attached to the solid phase was detected with enzyme-coupled IgG antibody toToxoplasma gondii. Neither hepatitis B surface antigen nor antigen ofMycoplasma pneumoniae, rubella, cytomegalovirus or herpes simplex virus interfered with this ELISA. Soluble antigen was detected in 13(30%) of 42 IgM-positive acute-phase toxoplasmosis patients and in only one of 20 sera cleared of IgM. None of an additional 44 IgM-negative patients with low IgG titres had a positive result in the antigen ELISA. Follow-up studies in four acute-phase toxoplasmosis patients showed that the soluble antigen cleared in all cases before the specific IgM antibodies. Simultaneous detection of IgM antibodies toToxoplasma gondii and soluble antigen would thus seem to indicate an early stage of the infection.     

Induction of tolerance toToxoplasma gondii in newborn mice by maternal antibody

To develop an animal model for analysing the suppressed immune response toToxoplasma gondii in newborn humans with congenital toxoplasmosis, newborn mice from chronically infected mothers were inoculated intraperitoneally with bradyzoites of an avirulent strain. The newborn mice with maternalToxoplasma antibodies showed a marked delay in the production ofToxoplasma antibodies when infected after birth. Many mice (11/13; 85%) developed a state of tolerance toT. gondii after disappearance of the maternal antibody, demonstrable by the absence ofToxoplasma antibody in their sera despite the fact that they were infected. The duration of tolerance differed between individuals, with two mice showing the longest tolerant state of 8 weeks. This murine model might be suitable for analysing the mechanism of suppressed immune response toT. gondii that has been observed in many human cases of congenital toxoplasmosis.     

<Emphasis Type="Italic">Toxoplasma gondii</Emphasis> antibody profile in HIV-infected pregnant women and the risk of congenital toxoplasmosis

The purpose of this study was to investigate the antibodies to Toxoplasma gondii in human immunodeficiency virus (HIV)-infected pregnant women and to determine the association between serological profile and the risk of congenital toxoplasmosis. The study, conducted in a public maternity ward from May 2002 to April 2005, included all HIV-infected women who delivered live infants during the 36 months, and, as a control group, all HIV-negative women that delivered live infants in the first 12 months of the study. Antibodies to T. gondii were detected in 1,624 of 2,421 HIV-negative women (67%; 95% confidence interval [CI] 65–69%) and in 121 of 168 HIV-infected patients (72%; 95% CI 65–79%). A total of 547 HIV-negative and 103 HIV-infected patients were tested at delivery and had positive T. gondii-specific IgG. In HIV-negative women, the median of the specific IgG concentration was 79 (interquartile range 38–160), and in HIV-infected patients, it was 283 (interquartile range 94–704) (P < 0.001). In the group of co-infected women, the only infant with congenital toxoplasmosis was born to a mother with acute toxoplasmosis infection acquired during pregnancy who did not have a high specific IgG concentration or a positive result for specific IgM. We concluded that high T. gondii-specific IgG values were much more frequent among HIV-infected pregnant women, but it did not translate into an increased risk of maternal–fetal transmission of toxoplasmosis.     

Estimation of the avidity of immunoglobulin G for routine diagnosis of chronicToxoplasma gondii infection in pregnant women

Present serological methods differentiate poorly between acute and chronic toxoplasmosis in pregnant women, particularly when immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies toToxoplasma gondii are present simultaneously. In the present study, a simple test for discriminating between high-avidity antibodies, which are usually present in chronic infections, and low-avidity antibodies, typical of acute infection, was evaluated. Sera were evaluated forToxoplasma gondii antibodies using a commercial enzyme immunoassay, but a duplicate well was washed in 6M urea to disrupt lowavidity complexes. Results are expressed as the percentage of antibodies resisting elution by urea. Equivocal sera (n=493) containing both IgG and IgMToxoplasma gondii antibodies from 309 pregnant women whose status as chronically or acutely infected had been independently determined using standard methods were evaluated for antibody avidity. A value of >35% elution-resistant antibodies was always associated with chronic infection and could absolutely exclude a recent (<3 months) infectious incident. Values of <35% require repeat testing four weeks later to confirm the patient's status, since a proportion of individuals with chronic toxoplasmosis maintain low-avidity antibodies over long periods. This inexpensive, simple method can provide reassurance to clearly chronically infected individuals and avoids the need for repeated testing in these cases.     

High predictive value ofToxoplasma gondii IgG antibody levels in HIV-infected patients for diagnosis of cerebral toxoplasmosis

The results of serological tests forToxoplasma gondii IgG in 31 HIV-infected patients with toxoplasmic encephalitis (TE) and 49 HIV-infected patients seropositive forToxoplasma gondii but without TE were compared. All patients had a CD4+ lymphocyte count < 1 50/l. Of the TE patients, 22 (71%) were designated as having relatively high IgG levels on the basis of the followingToxoplasma IgG titre combination: Sabin-Feldman test 1256, indirect hemagglutination test 11024, direct agglutination test 114,580. Only 3 patients without TE had relatively high IgG titres. Relatively high IgG titres indicated TE with a positive predictive value of 88% in HIV-infected patients with CD4+ cell counts < 150/l, and could be observed in most patients several months prior to the first clinical and radiological signs of TE.     

Detection of specific antibodies toToxoplasma gondii by a competitive enzyme immunoassay using a monoclonal antibody against the P30 antigen

An inhibition EIA using a monoclonal antibody against the major P30Toxoplasma gondii surface protein was designed for detection of specific antibodies in human sera. The assay was based on the inhibition of binding of peroxidase labelled monoclonal antibody toToxoplasma gondii crude antigen coated plates by the corresponding antibodies present in human sera. This rapid and simple assay was compared to indirect immunofluorescence, direct agglutination and an immunosorbent agglutination assay using 435 human sera. The specificity and sensitivity were 100 % and 97 % respectively. This test was found to be as sensitive as the dye test.     

Unspecific Reactivity of IgM Directed Against the Low-Molecular-Weight Antigen of<Emphasis Type="Italic"> Toxoplasma gondii</Emphasis>

During routine serological survey, eight patients (5 pregnant women, 3 grafted patients) were positive for Toxoplasma gondii-specific IgM by enzyme-linked immunoassay but negative by a simultaneously performed immunosorbent agglutination assay. No clinical or biological symptoms of toxoplasmosis were observed later, despite the absence of treatment. Only one IgM-reactive band, which corresponded to the low-molecular-weight antigen of Toxoplasma gondii, was observed by Western blotting of these patients' sera. Dot blotting of lipid extracts of Toxoplasma gondii demonstrated that this reactivity was directed against sphingolipids or ceramides. This IgM positivity, which is unrelated to acute toxoplasmosis, raises strong concerns about the possibility of misleading results of this test in the diagnosis of toxoplasmosis in humans.     

Serodiagnosis of acute toxoplasmosis using a recombinant form of the dense granule antigen GRA6 in an enzyme-linked immunosorbent assay

We developed an indirect enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of acute toxoplasmosis that used the recombinant granule antigen GRA6-GST as diagnostic antigen for the detection of IgG antibodies to Toxoplasma gondii in human sera. A total of 431 sera obtained from 336 patients with acute and chronic toxoplasmosis and from patients who were not infected with T. gondii were tested. Sera from patients with acute T. gondii infection, chronic infection, and no infection showed different absorbance values. For discrimination between the presence and the absence of acute toxoplasmosis the assay reached a specificity of 99.6%. Only one of the sera without significant anti-T. gondii. IgM antibodies showed a positive reaction to rGRA6-GST. The assay showed good intra- and interassay reproducibility (CV 6%/14%). We included a glutathione S-transferase (GST)-IgG enzyme immunoassay as a control assay in this study. Only 7 (4%) of 159 random sample sera reacted positively with GST. Received: 22 November 1997 / Accepted: 26 March 1998     

Value of specific immunoglobulin a detection by two immunocapture assays in the diagnosis of toxoplasmosis

The diagnosis ofToxoplasma gondii infection is currently based on immunological tests, but tests for IgG and IgM antibodies alone are often insufficient to assess the risk of active disease, especially during pregnancy and in immunodeficient subjects. The supplementary diagnostic value of testing for antitoxoplasmic IgA in cases of acute, chronic, congenital and reactivated toxoplasmosis, relative to classical immunological tests, was evaluated using two immunocapture tests, one based on tachyzoite agglutination and the other on an immunoenzymatic complex recognizing the membrane protein P30 ofToxoplasma gondii. A total of 4,541 sera from 395 uninfected subjects, 468 immunized subjects with chronic infection, 117 subjects with acute infection and 403 children, 103 of whom had congenital toxoplasmosis, was tested. Specific IgA tests were negative in the nonimmune population, but tests for this immunoglobulin subtype became positive very rapidly during primary infection, and IgA disappeared more rapidly than IgM. In the children infected in utero, specific IgA was detected more frequently than IgM. In contrast, in a population of HIV-seropositive subjects with clinical toxoplasmosis, tests for IgA were poorly sensitive. The two tests for specific IgA produced similar results, except in the early stages of primary infection, in which immunoenzymatic testing for anti-P30 IgA was less sensitive than the agglutination method.     

Application of the polymerase chain reaction in the diagnosis of pulmonary toxoplasmosis in immunocompromised patients

Bronchoalveolar lavage (BAL) fluid from 47 immunocompromised patients (26 with AIDS and 21 patients on immunosuppressive therapy) was analysed for the presence ofToxoplasma gondii DNA by means of the polymerase chain reaction (PCR). Specific target DNA derived from the B1 and P30 gene ofToxoplasma gondii was detected in BAL fluids from three patients with AIDS (6.4%). Pneumonia as the presenting feature of disseminated toxoplasmosis was confirmed by both clinical findings and by detection ofToxoplasma gondii DNA in blood obtained from two patients. The findings indicate that PCR has potential value in the detection ofToxoplasma gondii as an etiologic agent of atypical pneumonia in immunocompromised patients.     

Comparison of a commercial enzyme immunoassay and an immunoblot technique for detection of immunoglobulin A antibodies toToxoplasma gondii

A commercial enzyme immunoassay (Platelia-Toxo IgA) and an immunoblot technique were compared with regard to their ability to detect IgA antibodies to the major surface protein P30(SAG1) ofToxoplasma gondii in 105 serum samples from patients with suspected or proven acquired toxoplasmosis. Comparison of the IgA-EIA with the immunoblot technique showed a concordance of 81.0 %, with a sensitivity of 92.6 % and a specificity of 78.4 %. Due to its high sensitivity the IgA-EIA might detect IgA antibodies againstToxoplasma gondii at an early stage of infection, although excessive sensitivity could lead to detection of IgA antibodies for an extended period of time following the onset of infection.     

The detection ofHammondia hammondi in Australia and the identification of a free-living intermediate host

Summary A cat fed commercially prepared cat food and free-living rats (Rattus rattus andR. norvegicus) passed coccidian oocysts which were morphologically similar toToxoplasma gondii. Mice dosed with these oocysts did not develop toxoplasmosis, but instead developed small, thin-walled parasitic cysts containing small bradyzoites in their striated muscles. These cysts measured 35 to 90 m by 9 to 40 m and they resembled cysts ofHammondia hammondi. UnlikeT. gondii this coccidian parasite was shown to have an obligatory two-host cycle and consequently it is considered to beH. hammondi. This represents the first recorded detection ofH. hammondi in Australia.The source of theH. hammondi infection was aRattus rattus. This represents the first record of a natural intermediate host forH. hammondi.     

Onset of ocular complications in congenital toxoplasmosis associated with immunoglobulin M antibodies toToxoplasma gondii

Four patients with congenital toxoplasmosis serologically diagnosed by the Sabin-Feldman test (SFT) and the IgM-indirect fluorescent antibody test (IgM-IFAT) in the first year of life presented with eye disease between the age of 21 months and ten years. Repeated serological testing revealed increasing levels of specific antibodies as measured by the SFT. IgM antibodies toToxoplasma gondii were detected in all four patients by the immunosorbent agglutination assay, in two by the IgM-IFAT and in three by the IgM-indirect haemagglutination test. Findings suggest that specific IgM antibodies reappear at the time of reactivation of congenital toxoplasmosis later in life, or possibly persist for an extraordinary long period (up to ten years).     

Serologic responses toRhodococcus equi in individuals with and without human immunodeficiency virus infection

Thirty healthy blood donors, 15 workers from horse-breeding farms, 69 human immunodeficiency virus (HIV)-negative persons at risk for HIV infection, 125 HIV-infected subjects withoutRhodococcus equi infection, and nine HIV-infected patients withRhodococcus equi pneumonia were evaluated in order to detect serum antibodies toRhodococcus equi precipitate-soluble antigen by an enzyme immunoassay (EIA). Whereas EIA values for healthy donors, horse farm workers, individuals at risk for HIV infection, and HIV-positive subjects withoutRhodococcus equi infection were comparable, HIV-infected patients with rhodococcal disease had significantly higherRhodococcus equi antibody levels (p<0.0001). The clinical outcome ofRhodococcus equi pneumonia was more severe in subjects who had low levels of specific antibodies, whereas patients who recovered had elevatedRhodococcus equi antibody levels over time. Immunoblot studies showed that bothRhodococcus equi-infected patients and foals recognized a protein band of approximately 60 kDa in theRhodococcus equi precipitate-soluble antigen. On the other hand, theRhodococcus equi-infected patients did not react with the diffuse 15 to 17 kDa virulence-associated proteins that represent important virulence factors both in mice and horses.     

Cellular Immunity to <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> in Congenitally Infected Newborns and Immunocompetent Infected Hosts

The aim of this study was to determine the frequency of anergy to Toxoplasma gondii in congenitally infected newborns and immunocompetent infected individuals. Specific anergy to Toxoplasma has been reported previously, especially in cases of congenital toxoplasmosis. Whole blood from 592 immunocompetent patients with suspected toxoplasmosis was cultured in the presence of soluble Toxoplasma antigen for 7 days. Activated T lymphocytes were detected by flow cytometry. In patients over 1 year of age, the percentage of soluble Toxoplasma antigen-stimulated T cells expressing the interleukin-2 receptor CD25 was higher in infected patients than in uninfected subjects (40.0±18.3% vs. 1.8±2.0%, P<0.0001). No differences were detected between seroconverters, i.e. those with recent rises in IgM and IgG antibodies, and those with acquired or congenital toxoplasmosis. Similar results were observed when congenitally infected (n=38) and uninfected (n=89) children under 1 year of age were compared (17.6±9.2% vs. 3.0±4.9%, P<0.0001). The sensitivity and specificity values of CD25 detection for diagnosis of congenital toxoplasmosis in infants were 95% and 89%, respectively, at a threshold value of 7% above control culture. The results show that specific cellular immunity is detectable in virtually all Toxoplasma-infected patients, including newborns. Detection of CD25 constitutes a simple, sensitive and specific test for diagnosis of congenital toxoplasmosis. Electronic Publication     

Antigenaemia and antibody response to Toxoplasma gondii in human immunodeficiency virus-infected patients

Toxoplasma encephalitis in immunocompromised patients results from reactivation of previously acquired (latent) infection. The aim of the study is to assess the antigenaemia and antibody response to Toxoplasma gondii in human immunodeficiency virus (HIV)-infected patients to determine the best marker for early diagnosis of toxoplasmosis in such patients. Indirect enzyme-linked immunosorbent assay (ELISA) for detection of IgG, IgM and IgA anti-toxoplasma antibodies and double-sandwich ELISA for toxoplasma antigen is carried out in serum samples collected from 100 HIV seropositive patients and 75 controls. Toxoplasma-specific IgG, IgM and IgA antibody response and antigenaemia were detected in 12%, 6%, 7% and 14% of HIV-infected patients, respectively. On retrospective analysis of 14 patients with antigenaemia only one had central nervous system (CNS) symptoms attributable to toxoplasma infection. In this patient, the CD4+ cell count was below 50/microL and none of the specific immunoglobulin isotype responses could be detected. The patient showed clinical improvement following specific chemotherapy for toxoplasmosis. In 25 HIV-negative and anti-toxoplasma IgG antibody-positive controls, IgM was detected in two (8%), IgA in five (20%) and antigenaemia in 10 (40%), while 50 HIV seronegative healthy controls were negative for both antigen and antibody responses. The study indicates that detection of toxoplasma antigen in addition to IgG antibody response may prove to be a useful indicator in the early diagnosis of reactivated toxoplasmosis in HIV/AIDS patients.