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.     

Performance of a Western Blot Assay to Compare Mother and Newborn Anti-Toxoplasma Antibodies for the Early Neonatal Diagnosis of Congenital Toxoplasmosis

 The aim of this study was to retrospectively evaluate the performance of a Western blot assay to compare mother and newborn anti-Toxoplasma gondii antibodies for the early neonatal diagnosis of congenital toxoplasmosis. Since specific anti-Toxoplasma IgM or IgA is detected inconstantly at birth in the neonate, the diagnosis of congenital toxoplasmosis is often delayed until 6–9 months, after IgG titers have been observed persistently. In this study, 81 paired samples from 60 mother/child pairs were tested for IgG and IgM patterns. All mothers had (or were strongly suspected to have) acquired toxoplasmosis during pregnancy. Specific IgM and IgA were simultaneously detected by immunocapture tests, and IgG was titrated. A serological and clinical follow-up of infants was conducted during the first year of life until the diagnosis of congenital toxoplasmosis could be either confirmed or ruled out. Seventeen of the 60 newborns were congenitally infected. Specific IgM or IgA was detected by immunocapture at birth in 76.5% and 70.6% of cord sera from infected neonates, respectively, with an equal specificity of 77.5%. Comparative Western blot allowed the detection of neosynthesized IgG and IgM in the cord blood of 50% and 78.6% of infected infants, respectively, with a specificity of 100%. The combination of IgA and IgM immunocapture tests, the analysis of IgG and IgM Western blot patterns, and the combination of both techniques allowed the detection of 94%, 94%, and 100% of cases within the first 3 months of life, respectively. In conclusion, Western blotting seems to be a useful complementary tool for the early postnatal diagnosis of congenital toxoplasmosis.     

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.     

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.     

<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.     

Recombinant proteins in the diagnosis of toxoplasmosis

Kotresha D, Rahmah N. Recombinant proteins in the diagnosis of toxoplasmosis. APMIS 2010; 118: 529–42. Toxoplasma gondii is an important human pathogen with a worldwide distribution. It is primarily of medical importance for pregnant women and immunocompromised patients. Primary infection of the former is often associated with fetal infection, which can lead to abortion or severe neonatal malformation. Immunocompromised patients are at risk of contracting the severe form of the disease that may be fatal. Thus, detection of T. gondii infection with high sensitivity and specificity is crucial in the management of the disease. Toxoplasmosis is generally diagnosed by demonstrating specific immunoglobulin M (IgM) and IgG antibodies to toxoplasma antigens in the patient’s serum sample. Most of the commercially available tests use T. gondii native antigens and display wide variations in test accuracy. Recombinant antigens have great potential as diagnostic reagents for use in assays to detect toxoplasmosis. Thus in this review, we address recent advances in the use of Toxoplasma recombinant proteins for serodiagnosis of toxoplasmosis.     

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).     

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.     

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     

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.     

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.     

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     

Toxoplasma gondii surface antigen-1 in sera of HIV-infected patients as an indicator of reactivated toxoplasmosis

The major surface antigen from the proliferative form ofToxoplasma gondii (P-30 or SAG-1) was chosen as a target for exploration ofToxoplasma gondii reactivation in sera from immunocompromised patients. Samples were obtained from 37 HIV-infected subjects with lymphocyte levels of CD4+ <200/mm³. The prevalence of IgG antibodies toToxoplasma gondii was 64.9 %. Ten patients had clinical symptoms of reactivated toxoplasmosis; eight of these hadToxoplasma encephalitis. The SAG-1 epitopes were found as circulating antigen in five cases with an immunocapture enzyme immunoassay (EIA). The EIA was improved with an IgG1 monoclonal antibody to SAG-1 and a streptavidinbiotin amplification. The sensitivity, specificity and positive predictive value were 30, 92 and 60 %, respectively. The SAG-1 levels were compared with different biological parameters such as HIV p24 antigen, ₂ microglobulin, CD4+ cell count and IgG antibodies toToxoplasma gondii. The levels of SAG-1 in these patients were significantly higher than those in the 75 healthy control persons with or without a chronicToxoplasma gondii infection. Therefore, SAG-1 may be involved as a marker of reactivated toxoplasmosis in HIV-infected patients.     

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.     

Toxoplasmosis in heart transplant recipients

In cardiac transplant recipients, infection withToxoplasma gondii may be transmitted with the transplanted organ to immunosuppressed recipients or may be due to reactivation under immunosuppression in cases of pretransplant infection. In the present study the incidence of infection withToxoplasma gondii and the clinical presentation of the infection in 121 consecutive heart transplant recipients were investigated. Data on IgG and IgM antibodies forToxoplasma gondii measured by a semiquantitative microparticle immunoassay of donors and recipients were collected prospectively in 121 patients. Infection withToxoplasma gondii was defined as IgM seroconversion with proven pretransplant seronegativity (primary infection) or at least a fourfold increase of IgG antibodies (reactivation). Infection withToxoplasma gondii occurred in 16 of 121 patients (13%), whereas overt clinical disease occurred in 5 of 121 patients (4%). Organ-transmitted infection was more frequent (11/18, 61%) and more often associated with acute disease than reactivation of latent infection (5/69 patients, 7%) (p < 0.01), although one case ofToxoplasma retinochoroiditis occurred in a patient with recrudescence of latent pretransplant infection. Treatment with pyrimethamine and sulfadiazine was efficient in all patients with acute disease and in controlling disease in patients with evidence of acute infection.     

Analysis of the antibodies anti-<Emphasis Type="Italic">Toxoplasma gondii</Emphasis> by ELISA based on two diagnostic antigens: rSAG1 and rBAG1

Schizophrenia is a serious neuropsychiatric disease of uncertain etiology. Previous studies have demonstrated that antibodies to Toxoplasma gondii infection are associated with an increased risk of schizophrenia. The objective of this study was to analyze anti-T. gondii antibodies in 477 Chinese schizophrenia patients using an enzyme-linked immunosorbent assay (ELISA) based on recombinant surface antigen 1 (rSAG1), recombinant bradyzoite antigen 1 (rBAG1) and the soluble tachyzoite antigens (STAg) of T. gondii RH strain. Results showed that among the sero-positives (IgG and/or IgM) for T. gondii infection examined in schizophrenia patients, sero-positive samples for rSAG1, rBAG1 and STAg were 20.5% (98/477), 20.5% (98/477) and 23.5% (112/477) respectively, while compared to 210 blood donors, sero-positive (IgG and/or IgM) samples for these antigens (rSAG1, rBAG1 and STAg) were only 5.7% (12/210), 6.2% (13/210) and 5.7% (12/210), respectively. Furthermore, when IgG antibody reaction in the schizophrenia sera was compared with the rBAG1 and rSAG1, results demonstrated that beside the cases which can be detected by both rSAG1 and rBAG1, some sero-positive for T. gondii in schizophrenia sera can only be detected either by rSAG1 or rBAG1. This phenomenon was also observed in the detection of IgM with rSAG1 and rBAG1. 5.9% (28/477) of cases of schizophrenia which are positive for IgG or IgM by rSAG1 are negative for STAg, while 9.2% (44/477) of the schizophrenia cases which are positive for IgG or IgM by rBAG1 are negative for STAg. Although STAg can also be used to diagnose T. gondii infection from schizophrenia patients, it may not actually indicate the infection as some positive samples may be mistakenly considered to be negative. In conclusion, our results demonstrate that the sero-positive rate for T. gondii in the Chinese schizophrenia patients was higher than blood donors. More importantly, our results provide evidence that the combination of rSAG1 and rBAG1 antigens in the diagnosis of T. gondii infection could closely reflect the actual infection of this parasite in schizophrenia patients.     

Role of gamma interferon inToxoplasma gondii infection

Toxoplasma gondii has emerged as an important pathogen in the ever increasing numbers of patients with disorders of the immune system. Better understanding of the mechanisms of resistance of the host against this protozoan is important for development of safe, effective alternative treatment regimens for toxoplasmosis. Gamma interferon is the cytokine that plays a central role in protection againstToxoplasma gondii. The purpose of this review is to highlight the current knowledge of the role of gamma interferon inToxoplasma gondii infection.     

Detection of toxoplasma antibodies in sera of Salmonidae by ELISA

Toxoplasma gondii is a protozoan parasite with a worldwide distribution. It is capable of infecting all warm-blooded animals. Toxoplasmosis was not considered a waterborne zoonoses, but recently, it has been reported in many marine mammals. Coastal pollution by sewage from humans and pets has been suggested as a source for toxoplasma infection in these animals. Recent reports of toxoplasmosis in marine mammals raise concern that cold-blooded marine animals are potential sources of T. gondii infection. Conversely, the increasing proclivity for eating fish, crabs, shrimp, and mollusks—raw, undercooked, smoked, or dried—facilitates zoonoses infections caused by protozoan microorganisms; and one of them is toxoplasma. Detection of antibodies against T. gondii can be achieved by different serological tests such as enzyme-linked immunosorbent assay (ELISA). To determine whether toxoplasmosis has a role in Salmonidae infection, which is the most common seafood in Shahrekord district, this research was carried out on 50 Salmonidae aged 4 months (weight 700 ± 200 g). ELISA was performed on serum samples for detecting T. gondii-specific immunoglobulin M (IgM) and immunoglobulin G (IgG). As a result, toxoplasmic IgM antibody was detected in five of 50 samples (cut-off value of ≥0.183). Based on these findings, we believe that Salmonidae may be susceptible to primary T. gondii infection. While there is still no evidence of T. gondii transmission from cold-blooded sea animals to human via consuming their meat or other products, further research can be done to prove the possibility of this hypothesis.     

Evaluation of a New Protocol for Retrospective Diagnosis of Congenital Toxoplasmosis by Use of Guthrie Cards

The aim of this study was to assess the diagnostic value of IgM Western blotting (WB), IgA enzyme immunoassay (EIA), and DNA amplification by real-time PCR on Guthrie cards to retrospectively establish the diagnosis of congenital toxoplasmosis (CT). To this purpose, Guthrie cards were collected from 18 infants born to mothers with primary Toxoplasma gondii infection during pregnancy. Moreover, the analytical sensitivity of T. gondii PCR was assessed by testing mock dried blood specimens set up with several known DNA dilutions. IgM WB was demonstrated to be the most sensitive method. When the results of T. gondii DNA detection and specific IgM recovery were combined, retrospective CT diagnosis by using Guthrie cards was established in 3 out of 6 infected infants (sensitivity, 50%; 95% confidence interval, 26.8% to 73.2%). No positive PCR or serologic results were found in the group of 12 uninfected infants, demonstrating the excellent specificity of the three methods (95% confidence interval, 78.1% to 99.5%). The findings of the present study suggest that, in cases of missed diagnosis of CT at birth, analysis of Guthrie cards for children with compatible clinical findings after the perinatal period, in particular the combination of recovery of specific IgM antibodies and T. gondii DNA amplification, could be helpful. Nevertheless, since suboptimal conditions of storage of dried blood specimens can seriously affect sensitivity, negative results cannot rule out CT diagnosis. In contrast, because of the excellent specificity shown by IgM serologic testing and T. gondii DNA amplification on Guthrie cards, positive results obtained by either of the two methods should be considered diagnostic.     

Diagnosis ofToxoplasma gondii infection in AIDS patients by a tissue-culture technique

Toxoplasma gondii infection was investigated in 14 AIDS patients with neurological involvement who showed clinical and computer tomographic scan signs suggestive of toxoplasmic encephalitis or extraneuronal localization suggestive of toxoplasmic infection. Blood, lung secretion (bronchoalveolar lavage and/or induced sputum sample) and cerebral spinal fluid (CSF) samples were cultured, and the results compared with the results of direct examination by Giemsa and by immunofluorescence of lung secretions and CSF.Toxoplasma gondii were observed directly in only four patients by immunofluorescence in bronchoalveolar lavage, induced sputum and CSF samples, and in three of these patients by Giemsa staining of bronchoalveolar lavage and CSF smears. In contrast, parasites were detected after 48 h in blood cultures from 11 of the 14 AIDS patients, in CSF cultures from eight of them and also in cultures of bronchoalveolar lavage and induced sputum from the six patients with respiratory and radiological features indicative of lung tissue damage. The findings indicate the value of tissue culture for diagnosis of toxoplasmosis as well as for monitoring the effects of treatment and also indicate that culture of induced sputum could be a powerful tool in establishing the incidence of pulmonary toxoplasmosis in AIDS patients.