Rita Cristina Bezerra

Real-Time PCR in HIV/Trypanosoma cruzi Coinfection with and without Chagas Disease Reactivation: Association with HIV Viral Load and CD4(+) Level

Background Reactivation of chronic Chagas disease, which occurs in approximately 20% of patients coinfected with HIV/Trypanosoma cruzi (T. cruzi), is commonly characterized by severe meningoencephalitis and myocarditis. The use of quantitative molecular tests to monitor Chagas disease reactivation was analyzed. Methodology Polymerase chain reaction (PCR) of kDNA sequences, competitive (C-) PCR and real-time quantitative (q) PCR were compared with blood cultures and xenodiagnosis in samples from 91 patients (57 patients with chronic Chagas disease and 34 with HIV/T. cruzi coinfection), of whom 5 had reactivation of Chagas disease and 29 did not. Principal Findings qRT-PCR showed significant differences between groups; the highest parasitemia was observed in patients infected with HIV/T. cruzi with Chagas disease reactivation (median 1428.90 T. cruzi/mL), followed by patients with HIV/T. cruzi infection without reactivation (median 1.57 T. cruzi/mL) and patients with Chagas disease without HIV (median 0.00 T. cruzi/mL). Spearmans correlation coefficient showed that xenodiagnosis was correlated with blood culture, C-PCR and qRT-PCR. A stronger Spearman correlation index was found between C-PCR and qRT-PCR, the number of parasites and the HIV viral load, expressed as the number of CD4+ cells or the CD4+/CD8+ ratio. Conclusions qRT-PCR distinguished the groups of HIV/T. cruzi coinfected patients with and without reactivation. Therefore, this new method of qRT-PCR is proposed as a tool for prospective studies to analyze the importance of parasitemia (persistent and/or increased) as a criterion for recommending pre-emptive therapy in patients with chronic Chagas disease with HIV infection or immunosuppression. As seen in this study, an increase in HIV viral load and decreases in the number of CD4+ cells/mm3 and the CD4+/CD8+ ratio were identified as cofactors for increased parasitemia that can be used to target the introduction of early, pre-emptive therapy.

Inconclusive results in conventional serological screening for Chagas’ disease in blood banks: evaluation of cellular and humoral response

Objective  To find the most reliable screening method for Trypanosoma cruzi infection in blood banks.

Elevada porcentagem de blastocistose em escolares de São Paulo, SP

As a part of medical assistance activities, parasitological examination of fecal samples from 227 school children from a public institution of São Paulo (SP) revealed a rather high proportion of results positive for Blastocystis hominis. Other protozoan and worm species were markedly scarcer, a peculiar situation according to our judgement. It is acknowledged that blastocystosis is still largely an indefinite and controversial subject, which deserves adequate analysis to avoid drawbacks in the sphere of action of public health and general medical assistance.

Microwave treatment of human milk to prevent transmission of Chagas disease

It is recognized that breast feeding is an alternative means of transmission of Chagas disease. However, thermal treatment of milk can prevent this occurrence. As domestic microwave ovens are becoming commonplace, the efficacy of microwave thermal treatment in inactivating Trypanosoma cruzi trypomastigotes in human milk was tested. Human milk samples infected with T. cruzi trypomastigotes (Y strain) from laboratory-infected mice, were heated to 63 degrees C in a domestic microwave oven (2,450 MHz, 700 W). Microscopical and serological examinations demonstrated that none of the animals inoculated orally or intraperitoneally with infected milk which had been treated, got the infection, while those inoculated with untreated, infected milk, became infected. It was concluded that the simple treatment prescribed, which can easily be done at home, was effective in inactivating T. cruzi trypomastigotes contained in human milk.

Methylene blue vital staining for Trypanosoma cruzi trypomastigotes and epimastigotes

The morphological identification of Trypanosoma cruzi is currently considered to have a high specificity, but its sensitivity, which depends on the volume of the sample examined, is rather low. Trypanosome developmental stages suspended in blood, reduviid feces, and culture media are routinely searched for by means of fresh film examination (about 2 microL). High speed centrifugation of blood samples separates the buffy coat, where most trypomastigotes concentrate. As the parasites are transparent and colorless, their detection is mostly dependent on their motility. The fluorescent vital stain acridine orange has been used to enhance image contrast, as exemplified by the QBC (Quantitative Buffy Coat) technique. Staining blood, buffy coat, reduviid feces, and culture media samples with methylene blue (also a vital dye) is a means of producing sharp, well contrasted images of motile or non-motile T. cruzi developmental stages, only standard laboratory microscopes being required. Slides previously coated with a thin layer of methylene blue are used to stain fresh blood films. Photomicrographs exemplify the results of methylene blue staining applied to living and fixed parasites.

Observações sobre Blastocystis hominis e Cyclospora cayetanensis em exames parasitológicos efetuados rotineiramente

We report some observations made from routine parasitological examinations on feces. The methods of Faust et al. and of spontaneous sedimentation in water are not enough to identify Blastocystis hominis. Significant percentage presence of this protozoan was found, especially when staining with iron hematoxylin was performed. Cyclospora cayetanensis was found in 0.7% of the cases, which suggests that this parasite should also routinely be investigated by appropriate techniques.

Avaliação da eficácia da azitromicina e pirimetamina em camundongos infectados por cepa cistogênica de Toxoplasma gondii

The efficacy of prolonged administration of azithromycin and pyrimethamine was evaluated in mice experimentally infected with cystogenic strain of Toxoplasma gondii. The animals were intraperitoneally inoculated with one cyst of T. gondii and after 20 days were allocated into four groups: GI, infected without treatment; GII, infected and treated with the association of pyrimethamine (12.5 mg/kg/day) and azithromycin (100 mg/kg/day); GIII, infected and treated with the same dose of pyrimethamine; and GIV, infected and treated in the same way with azithromycin. The oral treatment lasted 120 days, after this period all the animals were sacrificed and the count of cysts in the brain was done. The association of both drugs provided the best results, by diminishing the cyst count in the brain of the animals treated in this way.

Evaluation of the "symbiosys" immunoassay for the serological diagnosis of Chagas disease

infection is characterized by the presence of the parasite in the blood and not by clinical or epidemiologic findings, for example. As for the diagnosis in the chronic stage, serological tests are usually the first choice.In this context, new procedures are commonly proposed as new diagnosing tools, but it should be noted that they need to show further advances, especially in regard to sensitivity, specificity, ease of implementation, availability and cost.To carry out the serological studies exemplifying these interests we mention the use of purified, recombinant, synthetic or excretory-secretory antigens with different strains of

Role of T. cruzi exposure in the pattern of T cell cytokines among chronically infected HIV and Chagas disease patients

OBJECTIVES: The impact of Chagas disease (CD) in HIV-infected patients is relevant throughout the world. In fact, the characterization of the adaptive immune response in the context of co-infection is important for predicting the need for interventions in areas in which HIV and Chagas disease co-exist. METHODS: We described and compared the frequency of cytokine-producing T cells stimulated with soluble antigen of Trypanosoma cruzi (T. cruzi) using a cytometric assay for the following groups: individuals with chronic Chagas disease (CHR, n=10), those with Chagas disease and HIV infection (CO, n=11), those with only HIV (HIV, n=14) and healthy individuals (C, n=15). RESULTS: We found 1) a constitutively lower frequency of IL-2+ and IFN-γ+ T cells in the CHR group compared with the HIV, CO and healthy groups; 2) a suppressive activity of soluble T. cruzi antigen, which down-regulated IL-2+CD4+ and IFN-γ+CD4+ phenotypes, notably in the healthy group; 3) a down-regulation of inflammatory cytokines on CD8+ T cells in the indeterminate form of Chagas disease; and 4) a significant increase in IL-10+CD8+ cells distinguishing the indeterminate form from the cardiac/digestive form of Chagas disease, even in the presence of HIV infection. CONCLUSIONS: Taken together, our data suggest the presence of an immunoregulatory response in chronic Chagas disease, which seems to be driven by T. cruzi antigens. Our findings provide new insights into immunotherapeutic strategies for people living with HIV/AIDS and Chagas disease.

CHAGASIC MENINGOENCEPHALITIS IN AN HIV INFECTED PATIENT WITH MODERATE IMMUNOSUPPRESSION: PROLONGED SURVIVAL AND CHALLENGES IN THE HAART ERA

The reactivation of Chagas disease in HIV infected patients presents high mortality and morbidity. We present the case of a female patient with confirmed Chagasic meningoencephalitis as AIDS-defining illness. Interestingly, her TCD4+ lymphocyte cell count was 318 cells/mm3. After two months of induction therapy, one year of maintenance with benznidazol, and early introduction of highly active antiretroviral therapy (HAART), the patient had good clinical, parasitological and radiological evolution. We used a qualitative polymerase chain reaction for the monitoring of T. cruzi parasitemia during and after the treatment. We emphasize the potential value of molecular techniques along with clinical and radiological parameters in the follow-up of patients with Chagas disease and HIV infection. Early introduction of HAART, prolonged induction and maintenance of antiparasitic therapy, and its discontinuation are feasible, in the current management of reactivation of Chagas disease.