Detection of the first incidence of Akodon paranaensis naturally infected with the Jabora virus strain (Hantavirus) in Brazil

We characterised hantaviruses circulating in different Akodon rodent species collected in midwestern Santa Catarina (SC), southern Brazil, where the Jabora hantavirus (JABV) strain was first identified in Akodon montensis. Genetic and phylogenetic analyses based on a partial S segment indicated that, in SC, Akodon paranaensis and A. montensis carried the same type of hantavirus. Additionally, we conducted the first genomic characterisation of the complete S segment from the Brazilian JABV strain. This is the first report of A. paranaensis infected with the JABV.

Molecular characterisation of dengue virus type 1 reveals lineage replacement during circulation in Brazilian territory

Dengue fever is the most important arbovirus infection found in tropical regions around the world. Dispersal of the vector and an increase in migratory flow between countries have led to large epidemics and severe clinical outcomes, such as dengue haemorrhagic fever and dengue shock syndrome. This study analysed the genetic variability of the dengue virus serotype 1 (DENV-1) in Brazil with regard to the full-length structural genes C/prM/M/E among 34 strains isolated during epidemics that occurred in the country between 1994-2011. Virus phylogeny and time of divergence were also evaluated with only the E gene of the strains isolated from 1994-2008. An analysis of amino acid differences between these strains and the French Guiana strain (FGA/89) revealed the presence of important nonsynonymous substitutions in the amino acid sequences, including residues E297 (Met→Thr) and E338 (Ser→Leu). A phylogenetic analysis of E proteins comparing the studied isolates and other strains selected from the GenBank database showed that the Brazilian DENV-1 strains since 1982 belonged to genotype V. This analysis also showed that different introductions of strains from the 1990s represented lineage replacement, with the identification of three lineages that cluster all isolates from the Americas. An analysis of the divergence time of DENV-1 indicated that the lineage circulating in Brazil emerged from an ancestral lineage that originated approximately 44.35 years ago.

Dengue virus type 4 in Niterói, Rio de Janeiro: the role of molecular techniques in laboratory diagnosis and entomological surveillance

In Niterói, state of Rio de Janeiro, dengue virus type 4 (DENV-4) was isolated for the first time in March 2011. We analysed the laboratory findings of the first cases and evaluated the use of molecular techniques for the detection of DENV-4 in Aedes aegypti that were field-caught. Conventional reverse transcriptase-polymerase chain reaction (RT-PCR) and SimplexaTM Dengue real-time RT-PCR confirmed DENV-4 infection in all cases. Additionally, DENV-4 was confirmed in a female Ae. aegypti with 1.08 x 10³ copies/mL of virus, as determined by quantitative real-time RT-PCR. This is the first time the SimplexaTM Dengue real-time assay has been used for the classification of cases of infection and for entomological investigations. The use of these molecular techniques was shown to be important for the surveillance of dengue in humans and vectors.

Environmental influences on antibody-enhanced dengue disease outcomes

Because an enriched environment (EE) enhances T-cell activity and T-lymphocytes contribute to immunopathogenesis during heterologous dengue virus (DENV) infections, we hypothesised that an EE increases dengue severity. To compare single serotype (SS) and antibody-enhanced disease (AED) infections regimens, serial intraperitoneal were performed with DENV3 (genotype III) infected brain homogenate or anti-DENV2 hyperimmune serum followed 24 h later by DENV3 (genotype III) infected brain homogenate. Compared AED for which significant differences were detected between the EE and impoverished environmental (IE) groups (Kaplan-Meyer log-rank test, p = 0.0025), no significant differences were detected between the SS experimental groups (Kaplan-Meyer log-rank test, p = 0.089). Survival curves from EE and IE animals infected with the AED regimen were extended after corticoid injection and this effect was greater in the EE than in the IE group (Kaplan-Meyer log-rank test, p = 0.0162). Under the AED regimen the EE group showed more intense clinical signs than the IE group. Dyspnoea, tremor, hunched posture, ruffled fur, immobility, pre-terminal paralysis, shock and death were associated with dominant T-lymphocytic hyperplasia and presence of viral antigens in the liver and lungs. We propose that the increased expansion of these memory T-cells and serotype cross-reactive antibodies facilitates the infection of these cells by DENV and that these events correlate with disease severity in an EE.

Vector biology prospects in dengue research

We argue that using more natural blood feeding methods to study mosquito vector competence for dengue viruses and exploring the effect of viral infection on other mosquito life-history traits that influence vectorial capacity will significantly advance our understanding of dengue epidemiology.

Chloroquine use improves dengue-related symptoms

Dengue is the most important arboviral disease in the world. As chloroquine, an antimalarial agent, has shown some antiviral effects, this study evaluated its effect in patients with dengue. A randomised, double-blind study was performed by administering chloroquine or placebo for three days to 129 patients with dengue-related symptoms. Of these patients, 37 were confirmed as having dengue and completed the study; in total, 19 dengue patients received chloroquine and 18 received placebo. There was no significant difference in the duration of the disease or the degree and days of fever. However, 12 patients (63%) with confirmed dengue reported a substantial decrease in pain intensity and a great improvement in their ability to perform daily activities (p = 0.0004) while on the medication and the symptoms returned immediately after these patients stopped taking the medication. The same effect was not observed in patients with diseases other than dengue. Therefore, this study shows that patients with dengue treated with chloroquine had an improvement in their quality of life and were able to resume their daily activities. However, as chloroquine did not alter the duration of the disease or the intensity and days of fever, further studies are necessary to confirm the clinical effects and to assess the side effects of chloroquine in dengue patients.

Potential biomarkers for the clinical prognosis of severe dengue

Currently, several assays can confirm acute dengue infection at the point-of-care. However, none of these assays can predict the severity of the disease symptoms. A prognosis test that predicts the likelihood of a dengue patient to develop a severe form of the disease could permit more efficient patient triage and treatment. We hypothesise that mRNA expression of apoptosis and innate immune response-related genes will be differentially regulated during the early stages of dengue and might predict the clinical outcome. Aiming to identify biomarkers for dengue prognosis, we extracted mRNA from the peripheral blood mononuclear cells of mild and severe dengue patients during the febrile stage of the disease to measure the expression levels of selected genes by quantitative polymerase chain reaction. The selected candidate biomarkers were previously identified by our group as differentially expressed in microarray studies. We verified that the mRNA coding for CFD, MAGED1, PSMB9, PRDX4 and FCGR3B were differentially expressed between patients who developed clinical symptoms associated with the mild type of dengue and patients who showed clinical symptoms associated with severe dengue. We suggest that this gene expression panel could putatively serve as biomarkers for the clinical prognosis of dengue haemorrhagic fever.

Evidence of an increased incidence of day 3 parasitaemia in Suriname: an indicator of the emerging resistance of Plasmodium falciparum to artemether

The emerging resistance to artemisinin derivatives that has been reported in South-East Asia led us to assess the efficacy of artemether-lumefantrine as the first line therapy for uncomplicated Plasmodium falciparum infections in Suriname. This drug assessment was performed according to the recommendations of the World Health Organization in 2011. The decreasing number of malaria cases in Suriname, which are currently limited to migrating populations and gold miners, precludes any conclusions on artemether efficacy because adequate numbers of patients with 28-day follow-up data are difficult to obtain. Therefore, a comparison of day 3 parasitaemia in a 2011 study and in a 2005/2006 study was used to detect the emergence of resistance to artemether. The prevalence of day 3 parasitaemia was assessed in a study in 2011 and was compared to that in a study in 2005/2006. The same protocol was used in both studies and artemether-lumefantrine was the study drug. Of 48 evaluable patients in 2011, 15 (31%) still had parasitaemia on day 3 compared to one (2%) out of 45 evaluable patients in 2005/2006. Overall, 11 evaluable patients in the 2011 study who were followed up until day 28 had negative slides and similar findings were obtained in all 38 evaluable patients in the 2005/2006 study. The significantly increased incidence of parasite persistence on day 3 may be an indication of emerging resistance to artemether.

Construction and characterisation of a complete reverse genetics system of dengue virus type 3

Dengue virulence and fitness are important factors that determine disease outcome. However, dengue virus (DENV) molecular biology and pathogenesis are not completely elucidated. New insights on those mechanisms have been facilitated by the development of reverse genetic systems in the past decades. Unfortunately, instability of flavivirus genomes cloned in Escherichia coli has been a major problem in these systems. Here, we describe the development of a complete reverse genetics system, based on the construction of an infectious clone and replicon for a low passage DENV-3 genotype III of a clinical isolate. Both constructs were assembled into a newly designed yeast- E. coli shuttle vector by homologous recombination technique and propagated in yeast to prevent any possible genome instability in E. coli . RNA transcripts derived from the infectious clone are infectious upon transfection into BHK-21 cells even after repeated passages of the plasmid in yeast. Transcript-derived DENV-3 exhibited growth kinetics, focus formation size comparable to original DENV-3 in mosquito C6/36 cell culture. In vitro characterisation of DENV-3 replicon confirmed its identity and ability to replicate transiently in BHK-21 cells. The reverse genetics system reported here is a valuable tool that will facilitate further molecular studies in DENV replication, virus attenuation and pathogenesis.

Aedes aegypti on Madeira Island (Portugal): genetic variation of a recently introduced dengue vector

The increasing population of Aedes aegypti mosquitoes on Madeira Island (Portugal) resulted in the first autochthonous dengue outbreak, which occurred in October 2012. Our study establishes the first genetic evaluation based on the mitochondrial DNA (mtDNA) genes [cytochrome oxidase subunit I (COI) and NADH dehydrogenase subunit 4 (ND4)] and knockdown resistance ( kdr ) mutations exploring the colonisation history and the genetic diversity of this insular vector population. We included mosquito populations from Brazil and Venezuela in the analysis as putative geographic sources. The Ae. aegypti population from Madeira showed extremely low mtDNA genetic variability, with a single haplotype for COI and ND4. We also detected the presence of two important kdr mutations and the quasi-fixation of one of these mutations (F1534C). These results are consistent with a unique recent founder event that occurred on the island of Ae. aegypti mosquitoes that carry kdr mutations associated with insecticide resistance. Finally, we also report the presence of the F1534C kdr mutation in the Brazil and Venezuela populations. To our knowledge, this is the first time this mutation has been found in South American Ae. aegypti mosquitoes. Given the present risk of Ae. aegypti re-invading continental Europe from Madeira and the recent dengue outbreaks on the island, this information is important to plan surveillance and control measures.

Incidence of liver damage of uncertain origin in HIV patients not co-infected with HCV/HBV.

BACKGROUND AND AIMS Several studies have reported that a significant number of HIV patients not co-infected with HCV/HBV develop liver damage of uncertain origin (LDUO). The objective of our study was to evaluate the incidence of and risk factors for the development of LDUO in HIV infected patients not co-infected with HCV/HBV. METHODS Prospective longitudinal study that included HIV-infected patients free of previous liver damage and viral hepatitis B or C co-infections. Patients were followed up at 6-monthly intervals. Liver stiffness was measured at each visit. Abnormal liver stiffness (ALS) was defined as a liver stiffness value greater than 7.2 kPa at two consecutive measurements. For patients who developed ALS, a protocol was followed to diagnose the cause of liver damage. Those patients who could not be diagnosed with any specific cause of liver disease were diagnosed as LDUO and liver biopsy was proposed. RESULTS 210 patients matched the inclusion criteria and were included. 198 patients completed the study. After a median (Q1-Q3) follow-up of 18 (IQR 12-26) months, 21 patients (10.6%) developed ALS. Of these, fifteen patients were diagnosed as LDUO. The incidence of LDUO was 7.64 cases/100 patient-years. Histological studies were performed on ten (66.6%) patients and all showed liver steatosis. A higher HOMA-IR value and body mass index were independently associated with the development of LDUO. CONCLUSION We found a high incidence of LDUO in HIV-infected patients associated with metabolic risk factors. The leading cause of LDUO in our study was non-alcoholic fatty liver disease.

Evaluation of the WHO classification of dengue disease severity during an epidemic in 2011 in the state of Ceará, Brazil

In 2009, the World Health Organization (WHO) issued a new guideline that stratifies dengue-affected patients into severe (SD) and non-severe dengue (NSD) (with or without warning signs). To evaluate the new recommendations, we completed a retrospective cross-sectional study of the dengue haemorrhagic fever (DHF) cases reported during an outbreak in 2011 in northeastern Brazil. We investigated 84 suspected DHF patients, including 45 (53.6%) males and 39 (46.4%) females. The ages of the patients ranged from five-83 years and the median age was 29. According to the DHF/dengue shock syndrome classification, 53 (63.1%) patients were classified as having dengue fever and 31 (36.9%) as having DHF. According to the 2009 WHO classification, 32 (38.1%) patients were grouped as having NSD [4 (4.8%) without warning signs and 28 (33.3%) with warning signs] and 52 (61.9%) as having SD. A better performance of the revised classification in the detection of severe clinical manifestations allows for an improved detection of patients with SD and may reduce deaths. The revised classification will not only facilitate effective screening and patient management, but will also enable the collection of standardised surveillance data for future epidemiological and clinical studies.

Incidence and transmission patterns of tuberculosis among indigenous populations in Brazil

Approximately 10% of the Brazilian indigenous population lives in the state of Mato Grosso do Sul (MS), where a large number of new cases of tuberculosis (TB) are reported. This study was conducted to assess TB occurrence, transmission and the utility of TB diagnosis based on the Ogawa-Kudoh (O-K) culture method in this remote population. The incidence of TB was estimated by a retrospective review of the surveillance data maintained by the Notifiable Diseases Surveillance System for the study region. The TB transmission pattern among indigenous people was assessed by genotyping Mycobacterium tuberculosis isolates using the IS 6110restriction fragment length polymorphism (RFLP) technique. Of the 3,093 cases identified from 1999-2001, 610 (~20%) were indigenous patients (average incidence: 377/100,000/year). The use of the O-K culture method increased the number of diagnosed cases by 34.1%. Of the genotyped isolates from 52 indigenous patients, 33 (63.5%) belonged to cluster RFLP patterns, indicating recently transmitted TB. These results demonstrate high, on-going TB transmission rates among the indigenous people of MS and indicate that new efforts are needed to disrupt these current transmissions.