Adelina Riarte

Outcome of infection with different strains of Trypanosoma cruzi in mice lacking CD4 and/or CD8

Mice lacking CD4 and/or CD8 gene expression, generated by embryonic stem-cell technology, were used to study the role of CD4+ and CD8+ cells in the resistance to the acute infection with virulent (Tulahuén and RA) or mild (CA-I) strains of Trypanosoma cruzi. The presence of both CD4+ and CD8+ cells contributed to the survival of mice infected with T. cruzi, and each T-cell subtype was able to sustain protective functions in the absence of the other one. However, in certain host-parasite combinations, CD8+ cell-independent mechanisms were able to control the parasite load. Moreover, CD8- mice chronically infected with a low virulent strain of T. cruzi were protected from an otherwise lethal challenge with the parasite. A different organ distribution of parasite nests was observed when mutant (but not wild type) animals infected with different parasite strains were compared. CD4- mice produced high levels of IgG antibodies against peptide antigens or a whole homogenate from the parasite after infection with CA-I strain. A dramatic enhancement of IgG1- and IgG2a-specific antibodies was observed.

Chagas disease in bone marrow transplantation: an approach to preemptive therapy

Summary:The efficacy of preemptive therapy was evaluated in bone marrow transplantation (BMT) recipients associated with Chagas disease (CD). The criterion to include patients in the protocol was the serological reactivity for CD in recipients and/or donors before transplant. After BMT, the monitoring was performed using the direct Strout method (SM), which detects clinical levels of Trypanosome cruzi parasitemia, and CD conventional serological tests. Monitoring took place during 60 days in ABMT and throughout the immunosuppressive period in allogeneic BMT. Reactivation of CD was diagnosed by detecting T. cruzi parasites in blood or tissues. In primary T. cruzi infection, an additional diagnostic criterion was the serological conversion. A total of 25 CD-BMT patients were included. Two ABMT and four allogeneic BMT recipients showed CD recurrences diagnosed by SM. One patient also showed skin lesions with T. cruzi amastigotes. Benznidazole treatment (Roche Lab), an antiparasitic drug, was prescribed at a dose of 5 mg/kg/day during 4–8 weeks with recovery of patients. Primary T. cruzi infection was not observed. This report proves the relevance of monitoring CD in BMT patients and demonstrates that preemptive therapy was able to abrogate the development of clinical and systemic disease.

Organ Transplantation and Chagas Disease

1. Stephan BC, Matthews FE, McKeith IG, Bond J, Brayne C; Medical Research Council Cognitive Function and Aging Study. Early cognitive change in the general population. J Am Geriatr Soc. 2007;55(10):1534-1540. 2. Boustani M, Callahan C, Unverzagt F, et al. Implementing dementia screening and diagnosis in primary care. J Gen Intern Med. 2005;20(7):572-577. 3. UK National Screening Committee. http://www.nsc.nhs.uk. Accessed January 7, 2008. 4. Boustani M, Fox C, Katona C, et al. Patients’ perceptions about dementia screening in the United Kingdom and the United States. Alzheimers Dement. 2006; 2(3)(suppl 1):S587. 5. Borson S, Scanlan JM, Hummel J, Gibbs K, Lessig M, Zuhr E. Implementing routine cognitive screening of older adults in primary care. J Gen Intern Med. 2007; 22(6):811-817. 6. Brodaty H, Low LF, Gibson L, Burns K. What is the best dementia screening instrument for general practitioners to use? Am J Geriatr Psychiatry. 2006;14 (5):391-400.

Congenital Trypanosoma cruzi Infection. Efficacy of Its Monitoring in an Urban Reference Health Center in a Non-Endemic Area of Argentina

Congenital transmission (CT) has acquired relevance in Chagas disease (CHD). A cohort of pregnant CHD women (4,355) and their babies were studied in the period 1994-2004. Children were excluded when they had received blood transfusions, or were born or had been in endemic areas; CT rate was 6.1%. Babies were diagnosed between months 1 and 5 in 68.9% of the cases and between months 6 and 12 in 31.1%. In the latter group, parasitemia was detected in 94% and serology in 74.7%. Between months 6 and 9, parasitemia diagnosed 36.2% (P = 0.000) more cases than serology. If serology had been the diagnosis method, those children would have been considered CT free. Taking the overall outcomes, 38.1% of babies were CT free, and 55.8% did not complete the follow-up. Establishing CT as a public health priority and improving first-line health service, congenital CHD coverage could be more efficient in endemic countries.

Recipients and donors of bone marrow transplants suffering from Chagas' disease: management and preemptive therapy of parasitemia.

We report the clinical course of five adult patients with chronic Chagas’ disease (Cd) who underwent BMT. Two patients with non-Hodgkin’s lymphoma and one with ALL received an ABMT. Allogeneic BMT was performed in two patients with AML and CML respectively. One donor had chronic Cd. Samples of peripheral blood for parasite investigation by the Strout method, blood culture, and immunological studies by indirect immunofluorescent assay, ELISA and indirect hemagglutination tests were performed weekly from the start of chemotherapy until day +60 for ABMT and during the period of immunosuppression for allogeneic BMT. No prophylaxis was given to any of these patients. In only one ABMT patient were trypomastigotes detected early by blood culture without symptoms of reactivation. Benznidazole as preemptive treatment was administered at 5–8 mg/kg/daily for 30 days. Parasitemia was rapidly cleared and at the end of therapy xenodiagnosis was negative. The other Cd patients showed no evidence of relapse of parasitemia or signs and symptoms of reactivation. In brief, evidence of Cd should be sought in all BMT patients coming from endemic areas because parasitemia and reactivation are potential complications during the period of neutropenia and immunosuppression. The strategy used for early detection and treatment of parasitemia and reactivation was safe and effective.

Humoral immune response to cruzipain and cardiac dysfunction in chronic Chagas disease.

The humoral immune response to epitopes expressed on cruzipain was evaluated in 31 Chagas disease patients (CDP) with different degrees of cardiac dysfunction. We took advantage of the availability of anti-Trypanosoma cruzi microsomal fraction monoclonal antibodies (MoAbs) reactive with epitopes that are recognized (5A9B11) or not recognized (1A10C11) by CDP sera. The 5A9B11- and 1A10C11-like epitopes are expressed on cruzipain. The reactivity of 5A9B11 against cruzipain was completely inhibited by sera of severe cardiopathy patients while a partial inhibition was found with sera from chagasic patients with mild disease. CDP sera did not block cruzipain recognition by 1A10C11. The antigenic determinants recognized by CDP sera appeared to be linear and carbohydrate free. When the overall anti-cruzipain immune response was evaluated, 70% of CDP with severe disease showed cruzipain titers higher than 1/800 while none of them displayed titers lower that 1/400. This report shows for the first time that the humoral immune response against epitopes expressed on cruzipain appeared to be related with the severity of chronic Chagas disease.

Increased expression and secretion of ICAM‐1 during experimental infection with Trypanosoma cruzi

In the present study we demonstrate that spleens and hearts from BALB/c mice infected with the virulent Tulahuén or the low virulent CA‐I strains of Trypanosoma cruzi, contain substantially higher ICAM‐1 transcripts than uninfected controls. ICAM‐1 expression in heart cells was also increased at the protein level, as measured by flow cytometry, ELISA and immunohistochemistry. The adhesive receptor was observed not only on inflammatory cells but also on sarcolemma of cardiac myocytes from T. cruzi infected mice. ICAM‐1 expression was higher during the acute phase than in the chronic phase of infection, and paralleled the density of inflammatory leukocytes. Elevated titres of soluble ICAM‐1 (s‐ICAM‐1) were detected in sera from mice during the acute phase of infection with CA‐I or Tulahuén parasites. Cytokines, including IFN‐γ, IL‐1α, IL‐6 and TNF‐α have been shown to modulate expression of ICAM‐1. Spleens and hearts from mice infected with CA‐I or Tulahuén strains showed increased accumulation of mRNAs specific for these cytokines, which peaked during the acute phase of infection. However, IFN‐γ activity was not necessary for ICAM‐1 induction, as its levels were also increased during infection in IFN‐γ receptor knock‐out (IFN‐γR−) mice. Upregulation of ICAM‐1 expression might be a direct consequence of parasite infection, since its density on cell lines of different lineages was enhanced after 24 or 48 h of infection with T. cruzi

REACTIVATION OF CHRONIC CHAGAS' DISEASE FOLLOWING ALLOGENEIC BONE MARROW TRANSPLANTATION AND SUCCESSFUL PRE-EMPTIVE THERAPY WITH BENZNIDAZOLE

This report shows the early detection of reactivation of chronic Chagas’ disease (CCd) in a 27‐year‐old man with chronic myelogenous leukemia undergoing allogeneic bone marrow transplantation (ABMT). Pre‐emptive therapy with benznidazole during a period of 7 weeks led to a rapid recovery of the patient, who remains free of parasitemia 2 years after the bone marrow transplantation.

Immunoprotection of mice against Trypanosoma cruzi with a lyophilized flagellar fraction of the parasite plus adjuvant

The immunization with the flagellar (F) fraction from epimastigotes of Trypanosoma cruzi has been shown to protect mice against a challenge of bloodstream trypomastigotes of the parasite, both in terms of mortality and decrease in parasitemia. We have compared the immunoprotective properties of the fresh F fraction with those of a lyophilized F (LF) fraction, alone or together with Bordetella pertussis (Bp) as adjuvant. The best results were obtained with LF + Bp: after challenge with 1 X 10(3) metacyclic trypomastigotes, 100% of the mice immunized with LF + Bp survived, and 60% of them showed no signs of parasitemia. Only the animals in which patent parasitemia was demonstrated presented heart and muscle infiltrates.

Predictive role of polymerase chain reaction in the early diagnosis of congenital Trypanosoma cruzi infection.

The efficacy of specific chemotherapy in congenital Chagas disease before the first year of life ranges between 90 and 100%. Between this age and 15 years of age, the efficacy decreases to around 60%. Therefore, early infection detection is a priority in vertical transmission. The aim of this work was to assess whether polymerase chain reaction (PCR) plays a predictive role in the diagnosis of congenital Chagas disease as compared to conventional parasitological and serological methods. To this end, we studied a total of 468 children born to Trypanosoma cruzi seroreactive mothers came from Argentina, Bolivia and Paraguay, who lived in the city of Buenos Aires and suburban areas (Argentina), a non-endemic area of this country. These children were assessed by PCR from 2004 to 2009 with the specific primers Tcz1 and Tcz2, and 121 and 122. PCR allowed detecting 49 T. cruzi-positive children. Eight of these 49 children were excluded from the analysis: six because they did not complete follow-up and two because the first control was performed after 12 months of age. Parasitological methods allowed detecting 25 positive children, 7 of whom had been earlier diagnosed by PCR (1.53±2.00 vs. 6.71±1.46 months; p=0.0002). Serological methods allowed detecting 16 positive children, 12 of whom had been earlier diagnosed by PCR (1.46±1.48 vs. 11.77±4.40 months; p<0.0001). None of the children negative by PCR was positive by serological or parasitological methods. This study shows that PCR allows early diagnosis in congenital Chagas disease. At present, an early positive PCR is not indicative for treatment. However, a positive PCR would alert the health system to search only those infected infants diagnosed by early PCR and thus generate greater efficiency in the diagnosis and treatment of congenital T. cruzi infection.