Trypanosoma cruzi: explant organ cultures from mice with chronic Chagas' disease

Explants of 13 different organs obtained from C3H/HEN, Swiss-Webster, and C57Bl/6 mice chronically infected with Trypanosoma cruzi (Y strain) were cocultivated with mouse embryo fibroblasts to determine the organs that contain T. cruzi during the chronic infection. Explant cultures frequently yielded T. cruzi as late as 12 months after infection. Spleen and skeletal muscle were most frequently positive; heart cultures were rarely positive in any mouse strain. C3H/HEN mice had significantly more cultures positive than Swiss-Webster mice, as expected from relative susceptibility of C3H/HEN mice to acute infection. In contrast, C57Bl/6 mice, relatively resistant to acute infection, had significantly more cultures positive at 12 months of infection than Swiss-Webster mice. Also, C57Bl/6 mice had a significant increase in the number of positive cultures at 12 months of infection compared to 6 months of infection. These results show that organisms can be recovered routinely from some tissues during the chronic infection, that murine susceptibility to infection should differentiate between acute and chronic infection, and that C57Bl/6 mice may lose control of infection during the chronic infection.     

Isolation of Trypanosoma cruzi samples by xenodiagnosis and hemoculture from patients with chronic Chagas' disease

Fifty nine chronic chagasic patients were simultaneously submitted to xenodiagnosis and hemoculture for Trypanosoma cruzi samples isolations. The xenodiagnosis was done with 40 Panstrongylus megistus, Triatoma infestans and Dipetalogaster maximus nymphs, performing 120 triatomines. Groups of 10 insects per specie were dissecated and the intestinal content pooled and examined, after previous trituration and homogenization. The microscopically negative material was seed into LIT medium and examined after 20 days. Twenty nine patients were parasitologically proved, being 15 only by xenodiagnosis, 4 only by hemoculture and 10 by both methods. It was discussed the parasitological comprovation difficulties in chronic chagasic patients, the value of the simultaneous utilization of different triatomine species in xenodiagnosis and the hemoculture, in a favorable positive association to the sensitivity increase in the diagnosis' disease. The 49.2% of positivity obtained in this group, visualize approaches like clinic-therapeutic assay and or epidemiological (case-control) with the purpose to investigate a possible association with T. cruzi samples and different clinic forms in Chagas' disease.     

Follow-up of an Asymptomatic Chagas Disease Population of Children after Treatment with Nifurtimox (Lampit) in a Sylvatic Endemic Transmission Area of Colombia

Background

Chagas disease is an anthropozoonosis caused by Trypanosoma cruzi. Two drugs are currently used for the etiological treatment of the disease: Nifurtimox (Lampit) and Benznidazole. This study presents a quasi-experimental trial (non-control group) of sixty-two patients who were treated for Chagas disease with Nifurtimox (Lampit), and were then followed for 30 months post-treatment. The safety of Nifurtimox (Lampit) for Chagas disease in this group of children primarily between 4 and 19 years old was also evaluated.

Materials and methods

The 62 patients included in the study were selected when resulted seropositive for two out of three fundamentally different serological tests. All children were treated during two months according to protocols established by WHO. Monitoring was performed every twenty days to evaluate treatment safety. In 43 patients, two different serological tests: ELISA and IFAT; and two parasitological tests: blood culture, and real time PCR, (qPCR) were performed to assess therapeutic response, defined as post-treatment serological negativization.

Principal findings

All patients completed the treatment successfully, and six patients abandoned the post-treatment follow-up. Adverse effects occurred in 74% of patients, but only 4.8% of cases required temporary suspension to achieve 100% adherence to the 60-day treatment, and all symptoms reverted after treatment completion. Both parasite load (measured through qPCR) and antibodies (ELISA absorbance) evidenced a significant median reduction 6 months after treatment from 6.2 to 0.2 parasite equivalents/mL, and from 0.6 to 0.2 absorbance units respectively (p<0.001). Serological negativization by ELISA was evident since 6 months post-treatment, whereas by IFAT only after 18 months. Serological negativization by the two tests (ELISA and IFAT) was 41.9% (95%CI: 26.5–57.3) after 30 months post-treatment. qPCR was positive in 88.3% of patients pre-treatment and only in 12.1% of patients after 30 months. Survival analysis indicated that only 26.3% (95%CI: 15.5–44.8) persisted with negative qPCR during the whole follow-up period.

Conclusions

Nifurtimox was very well tolerated and successfully reduced parasite load and antibody titers. Re-infection, lysed parasites or a lack of anti-parasitic activity could explain these persistently positive qPCR cases.     

Cardiovascular risk factors in chronic Chagas' disease are associated with a different profile of putative heart-pathogenic antibodies

Given that cardiovascular risk factors (CRF), such as smoking, alcoholism and hypertension, may contribute to the development of heart lesions, chronically Trypanosoma cruzi-infected individuals were studied to explore the relationship between the presence of such CRF, cardiomyopathy and antibodies that have been proposed to play a pathogenetic role in Chagas' disease. The targets of these antibodies were T. cruzi antigens such as cruzipain (Cz), a P ribosomal antigen (P2), and a component of myelin sheaths also present in T. cruzi (sulphatide). Individuals were classified into four groups on the basis of specific serology and presence of CRF: subjects with T. cruzi infection and CRF; those with positive serology and no CRF; seronegatives with CRF; and seronegatives without CRF, were analysed. Seronegatives or seropositives with CRF showed a greater occurrence of heart involvement (chest X-ray and/or electrocardiogram abnormalities). Seropositives with CRF displayed significantly higher levels of antisulphatide antibodies than the three remaining groups and higher levels of antibodies against Cz and P2 compared to the seropositives without CRF. Increased amounts of anti-P2 and antisulphatide antibodies were also found in seropositives with marked heart involvement. The presence of CRF is associated with a different profile of antibody responses and degree of cardiac effects.     

Trypanosoma cruzi: antibodies to a MAP-like protein in chronic Chagas' disease cross-react with mammalian cytoskeleton

Trypanosoma cruzi lambda gt 11 library from epimastogote derived mRNA was screened with human chagasic sera or sera from chronically infected mice. Strong reactive recombinants were detected with both sera. Two recombinant clones were studied in more detail and shown to be composed of the same 114-bp repetitive sequence coding for a 38 amino acid repetition. This repetition is the same size and shares greater than 60% homology with the reported T. brucei microtubule associated protein (MAP) p320. The insert of one of these clones, K1-7 (228 bp), was subcloned into pMSgt11 and the soluble recombinant polypeptide expressed. Antibodies against the K1-7 fusion polypeptide recognized a major 110-kDa band from cytoskeleton. Anti K1-7 monospecific antibodies detected several cytoskeletal proteins from 3T3 fibroblasts and bovine brain microtubule preparations. Reciprocally, anti-MAP1b monoclonal antibodies raised against bovine brain microtubule reacted with the K1-7 polypeptide on Western blots. The protein identified by K1-7 antibodies may be one of the parasite molecules associated to molecular mimicry.