Impact of the emergence and re-emergence of different dengue viruses' serotypes in Rio de Janeiro, Brazil, 2010 to 2012.

BACKGROUND: Rio de Janeiro (RJ) has been of major importance for the epidemiology of dengue viruses (DENVs) in Brazil. After the DENV 1-4 introductions in 1986, 1990, 2000 and 2011, respectively, the state has suffered explosive epidemics. We aimed to describe laboratorial, epidemiological and clinical aspects due to the emergence and re-emergence of distinct DENV in a 2-year period. METHODS: Suspected dengue cases (n=2833), including 190 fatal cases, were submitted to virus isolation, RT-PCR and non-structural 1 (NS1) antigen capture ELISA, IgM antibody-capture (MAC)-ELISA and IgG-ELISA. RESULTS: Case confirmation was 47.5%. MAC-ELISA confirmed 32.6% of the cases, RT-PCR confirmed 56.3%; DENV was recovered in 33.1% of samples inoculated and NS1 ELISA confirmed 27.5% of the cases. DENV-2 was prevalent in 2010, DENV-1 in 2011 and DENV-4 in 2012. Individuals infected by DENV-3 and over 65 years-old, and children 15 years-old and under infected by DENV-2 had a significantly higher risk of developing a severe disease. Fatal cases confirmed (n=67) were due to DENV-1 (26.8%), DENV-2 (14.9%), DENV-3 (2.9%) and DENV-4 (7.4%). CONCLUSIONS: It has been shown here that viral emergences or re-emergences may play different roles in the disease epidemiology, especially when many serotypes co-circulate.

First report of multiple lineages of dengue viruses type 1 in Rio de Janeiro, Brazil.

BACKGROUND: In Brazil dengue has been a major public health problem since DENV-1 introduction and spread in 1986. After a low or silent co-circulation, DENV-1 re-emerged in 2009 causing a major epidemic in the country in 2010 and 2011. In this study, the phylogeny of DENV-1 strains isolated in RJ after its first introduction in 1986 and after its emergence in 2009 and 2010 was performed in order to document possible evolutionary patterns or introductions in a re-emergent virus. FINDINGS: The analysis of the E gene sequences demonstrated that DENV-1 isolated during 2009/2010 still belong to genotype V (Americas/Africa) but grouping in a distinct clade (lineage II) of that represented by earlier DENV-1 (lineage I). However, strains isolated in 2011 grouped together forming another distinct clade (lineage III). CONCLUSIONS: The monitoring of DENV is important to observe the spread of potentially virulent strains as well to evaluate its impact over the population during an outbreak. Whether explosive epidemics reported in Brazil caused mainly by DENV-1 was due to lineage replacement, or due the population susceptibility to this serotype which has not circulated for almost a decade or even due to the occurrence of secondary infections in a hyperendemic country, is not clear. This is the first report of multiple lineages of DENV-1 detected in Brazil.

Autologous bone-marrow mononuclear cell transplantation after acute myocardial infarction: comparison of two delivery techniques.

The objective of this study was to investigate safety and feasibility of autologous bone marrow mononuclear cells (BMMNC) transplantation in ST elevation myocardial infarction (STEMI), comparing anterograde intracoronary artery (ICA) delivery with retrograde intracoronary vein (ICV) approach. An open labeled, randomized controlled trial of 30 patients admitted with STEMI was used. Patients were enrolled if they 1) were successfully reperfused within 24 h from symptoms onset and 2) had infarct size larger than 10% of the left ventricle (LV). One hundred million BMMNC were injected in the infarct-related artery (intra-arterial group) or vein (intravenous group), 1% of which was labeled with Tc(99m)-hexamethylpropylenamineoxime. Cell distribution was evaluated 4 and 24 h after injection. Baseline MRI was performed in order to evaluate microbstruction pattern. Baseline radionuclide ventriculography was performed before cell transfer and after 3 and 6 months. All the treated patients were submitted to repeat coronary angiography after 3 months. Thirty patients (57 +/- 11 years, 70% males) were randomly assigned to ICA (n = 14), ICV (n = 10), or control (n = 6) groups. No serious adverse events related to the procedure were observed. Early and late retention of radiolabeled cells was higher in the ICA than in the ICV group, independently of microcirculation obstruction. An increase of EF was observed in the ICA group (p = 0.02) compared to baseline. Injection procedures through anterograde and retrograde approaches seem to be feasible and safe. BMMNC retention by damaged heart tissue was apparently higher when the anterograde approach was used. Further studies are required to confirm these initial data.

Molecular characterization of cytosolic and mitochondrial tryparedoxin peroxidase in Trypanosoma cruzi populations susceptible and resistant to benznidazole.

Antioxidant defense in Trypanosomatids has been indicated as a potential target for chemotherapy. Tryparedoxin peroxidase (TXNPx) participates in this defense by metabolizing hydrogen peroxide to water molecules. In this work, genes encoding both cytosolic (cTcTXNPx) and mitochondrial (mTcTXNPx) TXNPx were characterized in 15 benznidazole-susceptible and resistant Trypanosoma cruzi strains. Northern blot and real-time RT-PCR analyses revealed that the levels of cTcTXNPx and mTcTXNPx mRNA were two-fold higher in the in-vitro-induced resistant 17 LER T. cruzi population than its drug-susceptible counterpart 17 WTS. The mRNA levels for both genes were similar among the other T. cruzi samples studied. No amplification of these genes was observed in the parasite genome. In silico analyses indicated that cTcTXNPx and mTcTXNPx genes present eight and two copies, respectively, dispersed in the parasite genome. By western blot analysis, anti-cTcTXNPx and anti-mTcTXNPx polyclonal antibodies recognized a 23- and 25-kDa peptide, respectively, in all T. cruzi samples analyzed. The expression levels of these native proteins were similar for all samples except 17 LER, which displayed two-fold greater expression. In addition, the oxidized mTcTXNPx protein (50 kDa) demonstrated 5.5-fold greater expression in the 17 LER population than 17 WTS. Our findings demonstrate increased expression of the cytosolic and mitochondrial TcTXNPx in the T. cruzi population with in-vitro-induced resistance to benznidazole.

Differential gene expression in Trypanosoma cruzi populations susceptible and resistant to benznidazole.

Differential gene expression in three pairs of Trypanosoma cruzi populations or clones susceptible or resistant to benznidazole (BZ) was investigated by differential display (DD) and representation of differential expression (RDE). GenBank searches of 14 genes selected by DD showed that four sequences corresponded to different hypothetical proteins and the others were very similar to T. cruzi genes encoding mucin (TcMUC), dihydrolipoamide dehydrogenase (TcLipDH), the hexose transporter (TcHT), or a ribosomal protein. Sequence analysis was performed on 34 clones obtained by RDE; approximately half of these clones encoded 14 different hypothetical proteins and the other half encoded proteins involved with stress response, antioxidant defence, metabolism, transporter proteins, surface proteins, ribosomal proteins and others. The mRNA levels of eight T. cruzi genes obtained by RDE and DD were analysed by northern blotting to confirm the differential expression of these sequences. For six of the eight genes, TcLipDH, TcHT, TcFeSOD-A (iron superoxide dismutase-A), TcHSP70, TcHSP100 (heat shock protein) and Tc52 (thiol-transferase), mRNA levels in the drug-resistant T. cruzi population were at least twice those in the susceptible population. Further analysis of TcHSP70 showed that although the levels of TcHSP70 mRNA were four-fold higher in T. cruzi BZ-resistant population, no corresponding increase was observed in the levels of TcHSP70 protein expression. The results suggest that TcHSP70 is not directly associated with the T. cruzi drug resistance phenotype.

Trypanosoma cruzi: characterisation of the gene encoding tyrosine aminotransferase in benznidazole-resistant and susceptible populations.

Various biochemical differences exist between mammalian tyrosine aminotransferase (TAT) and its analogue in Trypanosoma cruzi (TcTAT), the causative agent of Chagas disease. Moreover, TcTAT is over-expressed in strains of the parasite that are resistant to benznidazole (BZ), a drug currently used in chemotherapy. TAT has thus been indicated as a potential target for the development of new chemotherapeutic agents. In the present study, the TcTAT gene has been characterised in 14 BZ-resistant and susceptible strains and clones of T. cruzi. A unique transcript of 2.0kb and similar levels of TcTAT mRNA were observed in all parasite populations. TcTAT gene is organized in a tandem multicopy array and is located on 8 chromosomal bands that vary from 785-2500kb. No amplification of TcTAT was observed in the parasite genome. A 42kDa protein expressed by TcTAT was present in all T. cruzi samples. The results suggest that TcTAT is not directly associated with the T. cruzi drug resistance phenotype. However, it may act as a general secondary compensatory mechanism or stress response factor rather than as a key component of the specific primary resistance mechanism in T. cruzi.

Increased expression of iron-containing superoxide dismutase-A (TcFeSOD-A) enzyme in Trypanosoma cruzi population with in vitro-induced resistance to benznidazole.

Superoxide dismutase (SOD) removes excess superoxide radicals via dismutation to oxygen and hydrogen peroxide. In this work, we have characterized TcFeSOD-A gene from 25 Trypanosoma cruzi populations and clones susceptible, naturally resistant or with in vitro-induced (17 LER) or in vivo-selected resistance to benznidazole (BZR). In the 17 LER T. cruzi population, the levels of TcFeSOD-A mRNA were at least 3-fold higher than its drug-susceptible counterpart 17 WTS. The levels of TcFeSOD-A mRNA were similar among the other T. cruzi populations and clones regardless of the drug-resistance phenotype. We determined whether the increase in mRNA levels was due to gene amplification using Southern blot analysis of the T. cruzi populations and clones. We found that the number of TcFeSOD-A gene copies was similar for all samples tested, except for 17 LER that presented twice as many copies. The chromosomal location of the TcFeSOD-A gene and polymorphisms detected in nucleotide and amino acid sequences of TcFeSOD-A were associated with the zymodeme of the T. cruzi strain but not with drug-resistance phenotype. We observed a 23 kDa TcFeSOD-A polypeptide in all analysed T. cruzi strains. The level of this polypeptide was increased only in the 17 LER population. Specific enzyme activity analysis of TcFeSOD in the T. cruzi samples revealed a correlation between expression and activity. Our findings show an increased expression of the TcFeSOD-A enzyme in the T. cruzi population with in vitro-induced resistance to benznidazole.