[Trypanosoma cruzi pathology. Strain dependent?]. / Patología por Trypanosoma cruzi. Cepa dependiente?

There is agreement today about the role that the characteristics of the population of Trypanosoma cruzi play in the pathogenesis of the different clinical forms of Chagas disease. In our laboratory, we have studied the outcome of the infection of mice with two populations with polar biological behaviour: RA and CA-I. We have demonstrated that the neuromuscular damage is, in part, mediated by different T cell subsets. We have also observed that the T cell phenotype responsible for the pathology and the targetted tissues depend on the parasite population. Although we found no differences regarding the reactivity of IgG to native nerve structures in sera from mice infected with either strain, it is presumed that the humoral response would play an additional role in the development of strain-dependent neuromuscular pathology since passive transfer of sera from mice infected with RA triggered alterations of the nerve action potential whereas sera from CA-I-infected mice did not. We have also detected a reduction in the fertility of female mice infected with CA-I/K98, whereas females infected with RA showed no difference in comparison with uninfected controls. However, congenital transmission was only observed in mice infected with RA. The differences observed in fertility, in newborn survival, and in the number of fetal resorptions in mice infected with the myotropic strain could be attributed to the uterine inflammatory response, since no estrous alterations were observed between infected and control groups.

An improved DNA isolation technique for PCR detection of Strongyloides stercoralis in stool samples.

Strongyloides stercoralis is a nematode that causes severe infections in immunocompromised patients. The low parasitic burden of chronically infected patients makes diagnosis difficult to achieve by conventional methods. Here, an in-house (IH) method for the isolation of parasite DNA from stools and a PCR assay for the molecular diagnosis of S. stercoralis were optimized. DNA yield and purity improved with the IH method which included a step of incubation of stool samples with a glycine-SDS buffer and mechanical disruption prior to DNA extraction. For the PCR assay, the addition of bovine serum albumin was required to neutralize inhibitors present in stool. The analytical sensitivity of the PCR using DNA as template, isolated with the IH method, was superior to the commercial one. This study demonstrates that a combined method that adds the step of glycine-SDS buffer incubation plus mechanical disruption prior to DNA isolation with the commercial kit increased PCR sensitivity to levels of the IH method. Finally, our assay was tested on 17 clinical samples. With the IH method for DNA isolation, a S. stercoralis specific band was detected by PCR in the first stool sample in all patients (17/17), while with the commercial kit, our S. stercoralis-specific band was only observed in 7 samples. The superior efficiency of the IH and combined methods over the commercial kit was demonstrated when applied to clinical samples with low parasitic burden. These results show that the DNA extraction procedure is a key to increase sensitivity of the S. stercoralis PCR assay in stool samples. The method developed here could help to improve the molecular diagnosis of S. stercoralis.

Chagas' disease: reactivity against homologous tissues induced by different strains of Trypanosoma cruzi.

We have previously reported that the mechanisms of neuromyopathic damage induced by Trypanosoma cruzi are mediated by T cells and are parasite-strain dependent. In the present study our aim was to determine whether the humoral response against muscle and nervous system is also parasite-strain dependent. Of the sera from mice infected with RA and CA-I. T. cruzi strains, 93% reacted against antigens of the nervous system (sciatic nerve, spinal cord and brain). No differences in the ability to recognize heart antigens were found between RA (48%) and CA-I (63%) antisera. Reactivity against skeletal muscle was only relevant in anti-CA-I sera at 270 days post-infection. Each of the antisera assayed in Western blots developed a particular antigenic pattern, but 3 antigens in the nervous system of molecular weight 48, 60 and 70 kDa were detected by 42, 28 and 23% of the sera, respectively. On the other hand, deposits of IgG were observed at the interstitial matrix in sciatic nerve and as endomisial and sarcolemmal patterns in skeletal muscle by IFAT for both RA and CA-I antisera. Absorption of sera with parasite antigens did not abolish the autoreactivity. Our results suggest that major serum autoreactivity from T. cruzi-infected mice is not parasite-strain dependent and does not arise from molecular mimicry.