Cristina da Silva Meira

Contribution of laboratory methods in diagnosing clinically suspected ocular toxoplasmosis in Brazilian patients

This prospective study evaluated the value of laboratorial diagnosis in ocular toxoplasmosis analyzing peripheral blood samples from a group of Brazilian patients by immunologic and molecular methods. We analyzed blood samples from 184 immunocompetent patients with ocular disorders divided into 2 groups: Group I, composed of samples from 49 patients with ocular toxoplasmosis diagnosed by clinical features; Group II, samples from 135 patients with other ocular diseases. Samples were assayed by conventional polymerase chain reaction (cnPCR), real-time PCR (qPCR) for Toxoplasma gondii, indirect immunofluorescence reaction (IF), avidity test (crude tachyzoite lysate as antigen), and excreted-secreted tachyzoite proteins as antigen (ESA-ELISA). cnPCR and qPCR profiles were concordant in all samples. Positive PCR was shown in 40.8% of group I patients. The majority of the positive blood samples (75%) were taken from patients with toxoplasmic retinochoroiditis scars, and the others (25%), from patients with retinal exudative lesions. Despite that 86 of the 135 patients from Group II had asymptomatic toxoplasmosis, all DNA blood samples had negative PCR. Concordant results were shown in the data obtained by serologic methods. Around 24% of the patients with ocular toxoplasmosis had high antibody titers determined by ESA-ELISA and IF. Anti-ESA antibodies are shown principally in patients with active infection. Collectively, these data demonstrate the presence of tachyzoites in the blood of patients with chronic infection, supporting the idea of recurrent disease. Circulating parasites in blood of immunocompetent individuals may be associated with the reactivation of the ocular disease.

Use of the serum reactivity against Toxoplasma gondii excreted-secreted antigens in cerebral toxoplasmosis diagnosis in human immunodeficiency virus-infected patients

Despite the development of serological and molecular methods in recent years, the diagnosis of cerebral toxoplasmosis in human immunodeficiency virus (HIV)-infected patients still presents difficulties. In the present study, we investigated whether cerebral toxoplasmosis induced changes in the reactivity of serum toward Toxoplasma gondii excreted-secreted antigens (ESA) in order to develop an assay for evaluating HIV-infected patients with cerebral toxoplasmosis. The antigen selection was based on those produced by tachyzoites, since it is the form of the organism responsible for disseminating the infection, as well as stimulation of the humoral and cellular immune responses. By using an ELISA containing pooled ESA recovered from infected culture supernatants with tachyzoites-RH strain (ESA-ELISA), we found that ESA had a high specificity for sera from patients with cerebral toxoplasmosis. The reactions were compared with an ELISA using crude tachyzoites antigen, widely used in traditional serology. The assays were performed on 293 serum samples separated as follows: 100 sera from patients with cerebral toxoplasmosis and AIDS (symptomatic), 99 sera from individuals with chronic toxoplasmosis (asymptomatic) and 94 sera from healthy individuals without toxoplasmosis (control). The crude tachyzoite antigen in ELISA was able to distinguish both groups of sera with toxoplasmosis, as similar reactivity were observed in sera from patients with cerebral toxoplasmosis and those from chronic individuals. In contrast, ESA-ELISA distinguished sera from symptomatic and asymptomatic individuals (three times more reactive in the former group, 12.6 versus 4.2). The assays were reproducible based on immunoblotting and statistical analysis. These data suggest the utility of ESA-ELISA in the diagnosis of cerebral toxoplasmosis in HIV-infected patients, since it provided clear evidence that anti-ESA antibodies are present principally in patients with active infection. The absence of a significant amount of antibodies distinguished the patients without clinical symptoms of infection.

Evaluation of immunization with tachyzoite excreted–secreted proteins in a novel susceptible mouse model (A/Sn) for Toxoplasma gondii

Toxoplasma gondii is an important food-borne parasite transmitted primarily from animals to humans through meat consumption, mainly pork and lamb, as well as through oocysts shed by cats. Infection in humans can cause severe neonatal malformations, ocular complications or encephalitis. Toxoplasmosis infection during pregnancy, especially in sheep, often results in abortion, representing considerable economic loss. The aim of this study was to investigate whether Toxoplasma gondii pooled excreted-secreted antigens (ESA), recovered from infected culture supernatants with tachyzoites used as immunogen, can protect experimental mice against T. gondii infection. For immunization experiments, we evaluated A/Sn inbred mice, a novel susceptible mouse model for T. gondii and a virulent strain (RH) for challenge experiments. The antigen selection was based on those produced by tachyzoites since they are responsible for disseminating the infection as well as stimulating the humoral and cellular immune responses. ESA were recovered from VERO cell-culture supernatants infected with virulent RH strain tachyzoites harvested after 48 h. Groups of 5 female mice were intraperitoneally (i.p.) immunized with 4 doses at 2 week intervals with 20 microg of ESA adsorbed to 0.5 mg of alum. The control group received only the adjuvant in PBS on the same dates. Pooled serum collected from chronically infected mice was used as positive control. Blood samples were collected from tail veins 14 days after each immunization. Antibody was detected using ELISA, indirect immunofluorescence and immunoblotting. Anti-ESA antibodies were also evaluated by agglutination, complement-mediated lysis and antibody-mediated cellular toxicity. Fifteen days after the last immunization, both groups were challenged (i.p.) with 1 x 10(3) RH strain tachyzoites. The parasitemia was evaluated by PCR, and survival was followed daily. The results showed an increase of antibody levels after each immunization. Anti-ESA antibodies also reacted with a crude tachyzoite antigen and bonded on the parasite surface, with particularly high intensity at the apical region. Anti-ESA antibodies were also able to agglutinate and kill tachyzoites in vitro through interactions with complement and cellular pathways. Even though the tachyzoite challenge was lethal to the mice, PCR results suggested that immunized mice had lower parasitemia as well as longer survival (72 h) than mice from the control group.

Risk factors for ocular toxoplasmosis in Brazil

SUMMARY The aim of this study was to investigate risk factors for ocular toxoplasmosis (OT) in patients who received medical attention at a public health service. Three hundred and forty-nine consecutive patients, treated in the Outpatient Eye Clinic of Hospital de Base, São José do Rio Preto, São Paulo state, Brazil, were enrolled in this study. After an eye examination, enzyme-linked immunosorbent assay (ELISA) was used to determine anti-Toxoplasma gondii antibodies. The results showed that 25·5% of the patients were seronegative and 74·5% were seropositive for IgG anti-T. gondii antibodies; of these 27·3% had OT and 72·7% had other ocular diseases (OOD). The presence of cats or dogs [odds ratio (OR) 2·22, 95% confidence interval (CI) 1·24–3·98, P = 0·009] and consumption of raw or undercooked meat (OR 1·77, 95% CI 1·05–2·98, P = 0·03) were associated with infection but not with the development of OT. Age (OT 48·2 ± 21·2 years vs. OOD: 69·5 ± 14·7 years, P < 0·0001) and the low level of schooling/literacy (OT vs. OOD: OR 0·414, 95% CI 0·2231–0·7692, P = 0·007) were associated with OT. The presence of dogs and cats as well as eating raw/undercooked meat increases the risk of infection, but is not associated with the development of OT.

Cerebral and ocular toxoplasmosis related with IFN-γ, TNF-α, and IL-10 levels.

This study analyzed the synthesis of Interferon gamma (IFN-γ), Tumor Necrosis Factor alpha (TNF-α), and Interleukin 10 (IL-10) in chronically infected patients which developed the symptomatic disease as cerebral or ocular toxoplasmosis. Blood from 61 individuals were divided into four groups: Cerebral toxoplasmosis/AIDS patients (CT/AIDS group) (n = 15), ocular toxoplasmosis patients (OT group) (n = 23), chronic toxoplasmosis individuals (CHR group) (n = 13) and healthy individuals (HI group) (n = 10). OT, CHR, and HI groups were human immunodeficiency virus (HIV) seronegative. The diagnosis was made by laboratorial (PCR and ELISA) and clinical subjects. For cytokine determination, peripheral blood mononuclear cells (PBMC) of each patient were isolated and stimulated in vitro with T. gondii antigen. IFN-γ, TNF-α, and IL-10 activities were determined by ELISA. Patients from CT/AIDS and OT groups had low levels of IFN-γ when were compared with those from CHR group. These data suggest the low resistance to develop ocular lesions by the low ability to produce IFN-γ against the parasite. The same patients, which developed ocular or cerebral toxoplasmosis had higher TNF-α levels than CHR individuals. High TNF-α synthesis contribute to the inflammatory response and damage of the choroid and retina in OT patients and in AIDS patients caused a high inflammatory response as the TNF-α synthesis is not affected since monocytes are the major source this cytokine in response to soluble T. gondii antigens. IL-10 levels were almost similar in CT/AIDS and OT patients but low when compared with CHR individuals. The deviation to Th2 immune response including the production of anti-inflammatory cytokines, such as IL-10 may promote the parasites survival causing the tissue immune destruction. IL-10 production in T. gondii-infected brains may support the persistence of parasites as down-regulating the intracerebral immune response. All these indicate that OT and CT/AIDS patients produced low levels of IL-10 (Th2 response) and IFN-γ (Th1 response). They produced high TNF-α suggesting a high inflammatory response triggered by the parasite.

Immunodiagnosis in cerebrospinal fluid of cerebral toxoplasmosis and HIV-infected patients using Toxoplasma gondii excreted/secreted antigens ☆

Cerebral toxoplasmosis is the most common neurologic opportunistic infection in HIV-infected patients. Excretory-secretory antigens (ESA) are the majority of the circulating antigens in sera from hosts with acute toxoplasmosis, and their usefulness as antigens has been shown. This study considered whether it could find anti-ESA antibodies in cerebrospinal fluid (CSF) and whether these antibodies can be markers of active infection. Samples of CSF from 270 HIV-infected patients were analyzed and divided into 3 groups according to the presence or absence of active toxoplasmosis. Group I: 99 patients with cerebral toxoplasmosis; group II: 112 patients with other opportunistic neurologic diseases and seropositive for toxoplasmosis; and group III: 59 patients with other opportunistic neurologic diseases and seronegative for toxoplasmosis. Toxoplasma gondii ESA and a crude tachyzoite antigen were used as antigens using ELISA and immunoblotting. The statistical analysis was done using the F test and unpaired Students t test. Crude tachyzoite antigen: mean ELISA-relative values ± standard error for CSF of groups I and II were 7.0 ± 0.27 and 3.9 ± 0.19, respectively. Variance analysis revealed that results of both groups of patients were statistically different (1.80, P = 0.0025). The difference between the mean results was 3.0 ± 0.3, and the Students t test value was 9.41 (P = 0.0001). Samples from groups I and II were reactive by immunoblotting, with similar intensities. In ESA-ELISA, the mean for group I was 9.0 ± 0.39. Group II showed a mean value of 2.7 ± 0.12. Both groups were statistically different (9.16, P < 0.001). However, in ESA, the difference between the mean results was higher (6.2 ± 0.39) and the Students t test value was 16.04 (P < 0.0001). Similar results were shown in immunoblotting where a CSF sample from group I reacted well with ESA, and the sample from a group II patient failed to do so. The mean ELISA-relative value of the control group (group III) was 0.5 ± 0.09 for the first antigen and 0.4 ± 0.22 for the second. ESA-ELISA and/or immunoblotting of CSF samples can be used for diagnosis of cerebral toxoplasmosis in association with clinical, serologic, and radiological information, thus providing a simple straightforward methodology, particularly suitable in countries with high prevalence of latent toxoplasmosis in the general population.

Toxoplasma gondii antigens: Recovery analysis of tachyzoites cultivated in Vero cell maintained in serum free medium

Vero cells have been used successfully in Toxoplasma gondii maintenance. Medium supplementation for culture cells with fetal bovine serum is necessary for cellular growth. However, serum in these cultures presents disadvantages, such as the potential to induce hypersensitivity, variability of serum batches, possible presence of contaminants, and the high cost of good quality serum. Culture media formulated without any animal derived components, designed for serum-free growth of cell lines have been used successfully for different virus replication. The advantages of protozoan parasite growth in cell line cultures using serum-free medium remain poorly studied. Thus, this study was designed to determine whether T. gondii tachyzoites grown in Vero cell cultures in serum-free medium, after many passages, are able to maintain the same antigenic proprieties as those maintained in experimental mice. The standardization of Vero cell culture in serum-free medium for in vitro T. gondii tachyzoite production was performed establishing the optimal initial cell concentration for the confluent monolayer formation, which was 1×10(6) Vero cell culture as initial inoculum. The total confluent monolayer formatted after 96 h and the best amount of harvested tachyzoites was 2.1×10(7) using parasite inoculum of 1.5×10(6) after 7 days post-infection. The infectivity of tachyzoites released from Vero cells maintained in serum-free medium was evaluated using groups of Swiss mice infected with cell-culture tachyzoites. The parasite concentrations were similar to those for mice infected with tachyzoites collected from other infected mice. The data from both in vivo and in vitro experiments showed that in at least 30 culture cell passages, the parasites maintained the same infectivity as maintained in vivo. Another question was to know whether in the several continued passages, immunogenic progressive loss could occur. The nucleotide sequences studied were the same between the different passages, which could mean no change in their viability in the lysate antigen. Thus, the antigen production by cell culture has clear ethical and cost-saving advantages. Moreover, the use of culture media formulated without any human or animal derived components, designed for serum-free growth of cell lines, successfully produced tachyzoites especially for antigen production.

Anti-Toxoplasma gondii antibodies in pregnant women and their newborn infants in the region of Sao Jose do Rio Preto, Sao Paulo, Brazil

CONTEXT AND OBJECTIVE Toxoplasmosis transmission during pregnancy can cause severe sequelae in fetuses and newborns. Maternal antibodies may be indicators of risk or immunity. The aim here was to evaluate seropositivity for anti-Toxoplasma gondii (anti-T. gondii) immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies and IgG avidity in pregnant women and their newborn infants. DESIGN AND SETTING Cross-sectional study in a high-risk pregnancy outpatient clinic. METHODS Serum samples from pregnant women (n = 87) and their respective newborns (n = 87) were evaluated for anti-T. gondii antibodies using indirect immunofluorescence (IIF) (IgM and IgG), enzyme-linked immunosorbent assay (ELISA) (IgG) and an avidity test. RESULTS Anti-T. gondii antibodies were identified in 64.4% of the serum samples from the mothers and their infants (56/87). Except for two maternal serum samples (2.3%), all others were negative for anti-T. gondii IgM antibodies, using IIF. The results showed that 92.9% of the pregnant women had high IgG avidity indexes (> 30%) and four samples had avidity indexes between 16 and 30%. Two women in the third trimester of pregnancy were positive for anti-T. gondii IgM antibodies; their babies had avidity indexes between 16 and 30%. The avidity indexes of serum from the other 83 newborns were similar to the results from their mothers. CONCLUSIONS The results showed that 2% of the pregnant women were at risk of T. gondii transmission during the gestational period. These data seem to reflect the real situation of gestational toxoplasmosis in the northwestern region of the state of São Paulo.

IgG4 specific to Toxoplasma gondii excretory/secretory antigens in serum and/or cerebrospinal fluid support the cerebral toxoplasmosis diagnosis in HIV-infected patients

Cerebral toxoplasmosis is the most common neurological opportunistic disease manifested in HIV infected patients. Excretory/secretory antigens (ESA) are serological markers for the diagnosis of reactivation of the infection in HIV-infected patients with cerebral toxoplasmosis. Immunosuppressed patients develop high antibody titers for ESA. However, little is known about the humoral response for these antigens. The present study analyzed the profile of antibody recognition against ESA in comparison with tachyzoite lysate antigen (TLA) in 265 sera and 270 cerebrospinal fluid (CSF) samples from infected patients with Toxoplasma gondii and or HIV and in sera of 50 healthy individuals. The samples of sera and CSF were organized in 8 groups. The sera sample groups were: Group I - Se/CT/AIDS (patients with cerebral toxoplasmosis/AIDS) with 58 samples; Group II - Se/ONinf/AIDS/PosT (patients with AIDS/other neuroinfections/positive toxoplasmosis) with 49 samples; Group III - Se/ONinf/AIDS/NegT (patients with AIDS/other neuroinfections/negative toxoplasmosis) with 58 samples; Group IV - Se/PosT/NegHIV (individuals with asymptomatic toxoplasmosis/negative HIV) with 50 samples and Group V - Se/NegT/NegHIV (healthy individuals/negative toxoplasmosis and HIV) with 50 samples. The CSF sample groups were: Group VI - CSF/CT/AIDS (patients with cerebral toxoplasmosis/AIDS) with 99 samples; Group VII - CSF/ONinf/AIDS/PosT (patients with AIDS/other neuroinfections/positive toxoplasmosis) with 112 samples, and Group VIII - CSF/ONinf/AIDS/NegT (patients with AIDS/other neuroinfections/negative toxoplasmosis) with 59 samples. Levels of IgM, IgA, IgE, IgG and subclasses were determined by ELISA against TLA and ESA antigens. IgM, IgA or IgE antibodies against ESA or TLA were not detected in sera from patients with toxoplasmosis suggesting that all patients were in chronic phase of the infection. High levels of IgG1 against TLA were found in sera samples from groups I, II and IV and in CSF samples from groups VI and VII; whereas IgG2, IgG3 and IgG4 levels were not detected in the same sera or CSF sample groups. However, patients from groups I and VI, that had tachyzoites circulating in blood and CSF respectively, produced a mix of IgG1 and IgG4 antibodies against ESA. IgG2 against ESA were predominant in serum from patients with the latent (non-active) T. gondii infection/HIV negative and in CSF samples from patients with other neuroinfections and positive toxoplasmosis (groups IV and VII, respectively). IgG4 levels against ESA were found to be significantly (P<0.05 and P<0.005) higher in patients with cerebral toxoplasmosis (groups I and VI, respectively) in comparison with groups II, IV and VII. This data suggest that IgG4 can be valuable for supporting the diagnosis of focal brain lesions, caused by T. gondii infection, in HIV-infected patients. This approach might be useful, mainly when molecular investigation to detect parasites is not available.

Asymptomatic cryptococcal antigen prevalence detected by lateral flow assay in hospitalised HIV-infected patients in São Paulo, Brazil

To determine the prevalence of asymptomatic cryptococcal antigen (CRAG) using lateral flow assay (LFA) in hospitalised HIV‐infected patients with CD4 counts <200 cells/μl.