Performance of a real time PCR for leishmaniasis diagnosis using a L. (L.) infantum hypothetical protein as target in canine samples.

Visceral leishmaniasis represents an important public health issue in different parts of the world, requiring that measures be put in place to control the spread of the disease worldwide. The canine leishmaniasis diagnosis is not easy based on clinical signs, since dogs may not develop the infection with recognizable signs. Thus, the laboratorial diagnosis is essential to ascertain the incidence and prevalence of canine leishmaniasis especially in areas with major control efforts. Although, the diagnosis can be performed by the use of different approaches, the molecular methods such as PCR have become an indispensable tool for leishmaniases diagnosis. A TaqMan assay for real-time PCR (Linj31-qPCR) was developed to determine the parasite occurrence in clinical cases of leishmaniasis. The assay targets an L. (L.) infantum hypothetical protein region. The specificity of the assay was verified by using Leishmania World Health Organization reference strains including parasites belonging to subgenus L. (Leishmania), subgenus L. (Viannia), other Leishmania species and Trypanosoma cruzi. The sensitivity was verified by using isolates of L. (L.) amazonensis and L. (L.) infantum. The usefulness of the assay for diagnosis was ascertained by testing 277 samples from dogs in regions endemic for visceral and/or cutaneous leishmaniasis and from regions in which leishmaniasis was not endemic in São Paulo State, Brazil. Diagnosis of canine visceral leishmaniasis (CVL) was determined on these animals by conventional PCR and three serological tests. The dog samples were divided into four groups. I, dogs with CVL (n = 101); II, dogs with other diseases and without CVL (n = 97); III, dogs with American cutaneous leishmaniasis (n = 7), and, IV, dogs without CVL (n = 72) from areas where leishmaniasis was not endemic as control group. Results indicated that Linj31-qPCR was able to identify parasites belonging to subgenus L. (Leishmania) with no cross-amplification with other parasite subgenera. The Linj31-qPCR detected Leishmania parasites DNA in 98% of samples from Group I. In conclusion this methodology can be used as routine diagnostic tools to detect parasites from subgenus Leishmania.

Cytokine profile and parasite load in lymph nodes of dogs naturally infected with Leishmania infantum from distinct epidemiological scenarios in São Paulo State, Brazil.

Visceral leishmaniasis (VL) is an important zoonotic vector-borne disease and domestic dogs are considered the main domiciliary and peri-domiciliary reservoir of Leishmania (Leishmania) infantum in South America. Distinct eco-epidemiological scenarios associated to the prevalence of the disease, clusters of parasite genotypes and chemotypes of vectors population are described in Brazil, especially in the state of São Paulo (SP). In this context, the purpose of the present study is to evaluate the clinical signs, histopathological lesions, parasite load and cytokine profile by immunohistochemistry (IHC) in popliteal lymph nodes of canines naturally infected with L. infantum, from different municipalities of the state of SP. Eighty-three dogs with VL, 61 from northwest SP (NWSP) and 22 from southeast SP (SESP), were clinically classified in stage II, with no babesiosis and ehrlichiosis. Subcapsular inflammatory infiltration and histiocytosis were significantly higher in the SESP group (p = 0.0128; 0.0077, respectively). On the other hand, dogs from NWSP revealed 4.6-fold significantly higher parasite burden (p = 0.0004) and higher IHC scores of IL-1ß (p = 0.0275) and IL-4 (p = 0.0327) in the popliteal lymph node tissues, which may be associated with the susceptibility and progression of the disease in these dogs. Differences in immune response profile associated with higher parasite load in dogs can also contribute to explain the distinct eco-epidemiological patterns of VL in specific geographic regions.

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.

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.

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.

Real-time quantitative PCR in cerebral toxoplasmosis diagnosis of Brazilian human immunodeficiency virus-infected patients.

Cerebral toxoplasmosis is the most common cerebral mass lesion in AIDS patients in Brazil, and results in high mortality and morbidity, despite free access to HAART (highly active antiretroviral treatment). Molecular diagnosis based on conventional PCR (cnPCR) or real-time quantitative PCR (qrtPCR) has been indispensable for definitive diagnosis. We report here the evaluation of qrtPCR with blood and cerebrospinal fluid (CSF) samples from AIDS patients in Brazil. This prospective study was conducted for 2 years, analysing DNA samples extracted from 149 AIDS patients (98 blood and 51 CSF samples) with confirmed clinical and radiological diagnosis. The laboratory diagnosis included cnPCR (with the B22/B23 primer set) and indirect immunofluorescence (IF). For qrtPCR, two primer sets were simultaneously designed based on described genes and using a 6-carboxyfluorescein dye-labelled TaqMan MGB (minor groove binder) probe. One was B1Tg, which amplified a sequence from the B1 gene. The other was the RETg, which amplified a PCR product of the 529 bp sequence. The overall cnPCR and qrtPCR results were: positive results were observed in 33.6% (50) patients. The sensitivities were 98% for cnPCR (B22/B23), and 86 and 98% for qrtPCR (B1Tg and RETg, respectively). Negative reactions were observed in 66.4% patients. The specificities were 97% for cnPCR and qrtPCR (B1Tg), and 88.8 % for RETg. These data show that RETg PCR is highly sensitive as it amplifies a repeat region with many copies; however, its specificity is lower than the other markers. However, B1Tg PCR had good specificity, but lower sensitivity. Among the patients, 20 had blood and CSF collected simultaneously. Thus, their results permitted us to analyse and compare molecular, serological and clinical diagnosis for a better understanding of the different scenarios of laboratorial and clinical diagnosis. For nine patients with confirmed cerebral toxoplasmosis diagnosis, four scenarios were observed: (i) and (ii) negative molecular diagnosis for CSF and positive for blood with variable IF titres for the sera and CSF (negative or positive); (iii) positive molecular diagnosis with CSF and negative with blood; and (iv) positive molecular diagnosis in both samples. In the latter two situations, normally the IF titres in sera and CSF are variable. Other opportunistic infections were shown in 11 patients. Despite the IF titres in sera and CSF being variable, all of them had negative molecular diagnosis for both samples. qrtPCR allows for a rapid identification of Toxoplasma gondii DNA in patient samples; in a minority of cases discrepancies occur with the cnPCR.

A community-based survey of human toxoplasmosis in rural Amazonia: seroprevalence, seroconversion rate, and associated risk factors.

IgG antibodies to Toxoplasma gondii were detected in, March-April 2004, in 65.8% (95% confidence interval, 60.8-70.8%) of 342 systematically sampled subjects 5-90 years of age (87.5% of the eligible) living in a rural settlement in Amazonia, with a seroconversion rate of 9% over 1 year of follow-up of 99 seronegative subjects. Multiple logistic regression analysis identified age as the only significant independent predictor of seropositivity at the baseline. Each additional year of age increases the odds of being seropositive by 6%, and 76.8% of the subjects are expected to be seropositive at 30 years of age. A single high-prevalence spatial cluster, comprising 11.9% of the seropositive subjects, was detected in the area; households in the cluster were less likely to have dogs as pets and their heads had a lower education level, when compared with households located outside the cluster. The challenges for preventing human toxoplasmosis in tropical rural settings are discussed.

Toxoplasma gondii: genotyping of strains from Brazilian AIDS patients with cerebral toxoplasmosis by multilocus PCR-RFLP markers.

This study investigated the genetic characteristics of the Toxoplasma gondii strains isolated from 87 patients with cerebral toxoplasmosis and AIDS, treated in Sao Paulo State, Brazil. The laboratorial diagnosis of cerebral toxoplasmosis was based on positive serological exams and PCR of blood and/or cerebrospinal fluid. Four markers (5'-SAG2, 3'-SAG2, SAG3 and GRA6) were chosen to analyze the samples. Each having clear resolution to distinguish the three clonal lineages after PCR amplified targets were treated with restriction enzyme digestion (PCR-RFLP). The genotyping provided the following results: 40 patients (46%) were infected with strains classified as type I; 4 (4%), as type III; 13 (15%) were infected with polymorphic strains (unusual genotype); 6 patients with type I or II alleles; and 15 (17%) patients had strains not classified for any marker. PCR-RFLP, also classified 9 (11%) clinical isolates as type II, which is uncommon in South America. However, the sequencing of the nested-PCR products (of SAG3 marker) of type II and polymorphic isolates (of 5'-SAG2, SAG3 and GRA6 markers) showed a nucleotide polymorphism compared with the archetypal clonal genotypes (types I, II and III) and these isolates were considered as polymorphic strains. The markers used here were inappropriate to distinguish the most isolates considered as polymorphic strains. These data confirm other studies showing the high rate of genetic polymorphism in T. gondii strains isolated in Brazil.