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The choice of cost-effective molecular methods for diagnosing and monitoring of Chagas disease before and after treatment is crucial in endemic countries with high patients' demand and limited financial resources. To this end, a kDNA was compared to a satellite real-time quantitative PCR (sat-qPCR), both amplifications using Sybr Green instead of Taqman hydrolysis probes. Non-isogenic Swiss albino mice were infected with a small inoculum of the highly virulent and partially resistant to benznidazole Y strain, belonging to T. cruzi discrete typing unit II (DTU-II) that predominates in Atlantic and Central Brazil. DNA from EDTA-blood samples and 10 organs of mice containing high, moderate and low parasite load levels were extracted by a highly used commercial kit and tested in triplicate, showing no disagreements between the two qPCRs. The melting temperature of positive samples was 79.8 °C ± 1 °C for satellite-DNA and 78.1 °C ± 1 °C for kDNA. DNA from genetically-related parasites such as Leishmania sp. showed no cross-reactions, but the sympatric T. rangeli was detected by both qPCRs, more effectively by kDNA than the satellite system, which required the equivalent of 50 parasites to give a positive result. Samples from infected mice, regardless of the type of biological matrix (blood or organ samples) or the parasite load gave positive results by both qPCRs. The sensitivity of sat-qPCR was 2 × 10−3 parasite or 240 target copies, and for kDNA, 2 × 10−4 parasite or 24 target copies. Regarding repeatability and reproducibility, the coefficient of variation (CV) was always < 25% in both assays; linearity of sat-qPCR was 0.991 (±0.002) and 0.991 (±0.008) for kDNA qPCR. In most collection times, the median Ct values found in blood and organs provided by sat-DNA and kDNA qPCRs were similar. In conclusion, although kDNA qPCR achieved a better analytical sensitivity, sat-qPCR gave better specificity results. Nevertheless, further research is intended to test other T. cruzi DTUs and chagasic patients' samples before these cost-effective techniques are incorporated into diagnostic routines. • kDNA and satellite DNA Sybr Green real-time PCR were similar to T. cruzi detection. • kDNA qPCR achieved a better analytical sensitivity. • sat-qPCR gave better specificity results. [ABSTRACT FROM AUTHOR]

Abstract: Analyses of MLEE, RAPD and LSSP-PCR were used to compare the panel of american tegumentary leishmaniasis (ATL) isolates obtained from lesions of patients with rare clinical manifestations of the disease and typical lesions. All of the 34 samples analyzed by MLEE demonstrated similar electromorphic profiles with Leishmania (Viannia) braziliensis reference strain. Through the RAPD analysis, nine genetic profiles (genotypes) were identified. LSSP-PCR corroborates the initial screening and phenetic analysis has grouped the isolates into two major clusters comprising the nine different genotypes. Prevalent genotype defined as LbmtDNAgen1 was detected in the largest number of isolates. There was no association between genotypes and clinical symptoms. However, two different genotypes could be identified in the initial (LbmtDNAGen9) and reactivated lesion (LbmtDNAGen3) of the same patient. Our results support the idea of a less pronounced genotypic diversity among L. (V.) braziliensis circulating in the State of Rio de Janeiro and demonstrate the useful application of these molecular markers in genetics variability studies. [Copyright &y& Elsevier]

Abstract: Topoisomerase II of kinetoplastid parasites plays an important role in the replication of unusual networks of kinetoplast DNA (kDNA) and is a very useful target for antiparasitic drugs. In this study, we cloned full-length Crithidia fasciculata mitochondrial topoisomerase II gene into pFastBac-HTc vector and successfully expressed an active recombinant full-length mitochondrial topoisomerase II in Bac-to-Bac baculovirus expression system. A rapid and simple purification strategy was established by incorporating a FLAG-tag at the C-terminus of the protein. The purified recombinant topoisomerase II showed a major single band on SDS–PAGE (>96% purity) and was verified through Western blot analysis. The recombinant full-length mitochondrial topoisomerase II exhibited decatenation, catenation and relaxation activity of type II topoisomerase as well as various sensitivities to a series of known topoisomerase inhibitors. These studies explore new way and lay groundwork to express all other similar full-length kinetoplastid topoisomerases, it will also facilitate further elucidation of X-ray structure, catalysis mechanism of kinetoplastid topoisomerases and design of new antiparasitic drugs targeting kinetoplastid topoisomerases. [Copyright &y& Elsevier]

Abstract: DNA replication mechanisms are poorly understood in most of trypanosomatids, in particular the replication of the peculiar mitochondrial DNA, the kinetoplast DNA (kDNA). To contribute to the knowledge on the mechanism of kDNA replication in Trypanosoma cruzi, we have previously characterized the Universal Minicircle Sequence Binding Protein of this parasite (TcUMSBP), which was first called PDZ5 [E.R. Coelho, T.P. Urmenyi, J. Franco da Silveira, E. Rondinelli, R. Silva, Identification of PDZ5, a candidate universal minicircle sequence binding protein of Trypanosoma cruzi, Int. J. Parasitol. 33 (2003) 853–858]. In this work, we describe two highly polymorphic alleles of the TcUMSBP locus in the T. cruzi reference clone CL Brener and the differential expression pattern of these alleles. A 62bp sequence in the TcUMSBP upstream intergenic region in one of its alleles affects the efficiency of polycistronic RNA processing and the polyadenylation sites, and therefore regulates the differential expression of TcUMSBP alleles of this locus. [Copyright &y& Elsevier]

In rats, CL-Brener clone caused high mortality, severe acute myocarditis, and myositis that subsided completely in surviving animals. Accordingly, no parasite kDNA could be amplified in several organs after 4 months. The monoclonal JG strain caused null mortality, acute predominantly focal myocarditis, discrete and focal myositis, and a chronic phase with sparse inflammatory foci. Double infection with both Trypanosoma cruzi populations turned mortality very low or null. At the end of the acute phase, the heart exhibited only JG strain kDNA (LSSP-PCR), while skeletal muscles and rectum exhibited only CL-Brener kDNA. Molecular and histopathological findings were accordant. In double infection chronic phase, JG strain remains in heart and appeared in organs previously parasitized by CL-Brener clone. Understanding the virulence and histotropism shifts now described could be important to clarify the variable clinical course and epidemiological peculiarities of Chagas’ disease.Index Descriptors and Abbreviations: kDNA, kinetoplast DNA; PCR, polymerase chain reaction; LSSP-PCR, low-stringency single-specific primer-PCR [Copyright &y& Elsevier]