Health Outcome Predictive Evaluation for COVID 19 international registry (HOPE COVID-19), rationale and design

The disease produced by the new coronavirus known as SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), named COVID-19 (Coronavirus Disease-2019) has recently been classified as a pandemic by the World Health Organization (WHO). However, scarce clinical data is available and generally limited to the Chinese population due to the first cases were identified in Wuhan (Hubei, China).This article describes the rationale and design of the HOPE COVID-19 (Health Outcome Predictive Evaluation for COVID 19) registry (ClinicalTrials.gov Identifier: NCT04334291). With an ambispective cohort design, eligible patients are those discharged, deceased or alive, from any hospital center with a confirmed diagnosis or a COVID-19 high suspicion. With a current recruitment of more than 7000 cases, in 46 hospitals in 8 countries, since it is not possible to estimate the sample size based on literature reports, the investigators will try to get the maximum numbers of patients possible. The study primary objective is all cause mortality and aims to characterize the clinical profile of patients infected in order to develop a prognostic clinical score allowing, rapid logistic decision making. As secondary objectives, the analysis of other clinical events, the risk-adjusted influence of treatments and previous comorbidities of patients infected with the disease will be performed.The results of HOPE COVID-19 will contribute to a better understanding of this condition. We aim to describe the management of this condition as well as the outcomes in relation to the therapy chosen, in order to gain insight into improving patient care in the coming months. Clinical Trial registration: ClinicalTrials.gov. Unique identifier: NCT04334291

Dengue Type 3 Virus, Saint Martin, 2003–2004

We describe the spread of a dengue virus during an outbreak in Saint Martin island (French West Indies) during winter 2003–2004. Dengue type 3 viruses were isolated from 6 patients exhibiting clinical symptoms. This serotype had not been detected on the island during the preceding 3 years. Genome sequence determinations and analyses showed a common origin with dengue type 3 viruses isolated in Martinique 2 years earlier

Aquaglyceroporin-null trypanosomes display glycerol transport defects and respiratory-inhibitor sensitivity

Aquaglyceroporins (AQPs) transport water and glycerol and play important roles in drug-uptake in pathogenic trypanosomatids. For example, AQP2 in the human-infectious African trypanosome, Trypanosoma brucei gambiense, is responsible for melarsoprol and pentamidine-uptake, and melarsoprol treatment-failure has been found to be due to AQP2-defects in these parasites. To further probe the roles of these transporters, we assembled a T. b. brucei strain lacking all three AQP-genes. Triple-null aqp1-2-3 T. b. brucei displayed only a very moderate growth defect in vitro, established infections in mice and recovered effectively from hypotonic-shock. The aqp1-2-3 trypanosomes did, however, display glycerol uptake and efflux defects. They failed to accumulate glycerol or to utilise glycerol as a carbon-source and displayed increased sensitivity to salicylhydroxamic acid (SHAM), octyl gallate or propyl gallate; these inhibitors of trypanosome alternative oxidase (TAO) can increase intracellular glycerol to toxic levels. Notably, disruption of AQP2 alone generated cells with glycerol transport defects. Consistent with these findings, AQP2-defective, melarsoprol-resistant clinical isolates were sensitive to the TAO inhibitors, SHAM, propyl gallate and ascofuranone, relative to melarsoprol-sensitive reference strains. We conclude that African trypanosome AQPs are dispensable for viability and osmoregulation but they make important contributions to drug-uptake, glycerol-transport and respiratory-inhibitor sensitivity. We also discuss how the AQP-dependent inverse sensitivity to melarsoprol and respiratory inhibitors described here might be exploited

Phylogenetic history demonstrates two different lineages of dengue type 1 virus in Colombia

Background: Dengue Fever is one of the most important viral re-emergent diseases affecting about 50 million people around the world especially in tropical and sub-tropical countries. In Colombia, the virus was first detected in the earliest 70′s when the disease became a major public health concern. Since then, all four serotypes of the virus have been reported. Although most of the huge outbreaks reported in this country have involved dengue virus serotype 1 (DENV-1), there are not studies about its origin, genetic diversity and distribution. Results: We used 224 bp corresponding to the carboxyl terminus of envelope (E) gene from 74 Colombian isolates in order to reconstruct phylogenetic relationships and to estimate time divergences. Analyzed DENV-1 Colombian isolates belonged to the formerly defined genotype V. Only one virus isolate was clasified in the genotype I, likely representing a sole introduction that did not spread. The oldest strains were closely related to those detected for the first time in America in 1977 from the Caribbean and were detected for two years until their disappearance about six years later. Around 1987, a split up generated 2 lineages that have been evolving separately, although not major aminoacid changes in the analyzed region were found. Conclusion: DENV-1 has been circulating since 1978 in Colombia. Yet, the phylogenetic relationships between strains isolated along the covered period of time suggests that viral strains detected in some years, although belonging to the same genotype V, have different recent origins corresponding to multiple re-introduction events of viral strains that were circulating in neighbor countries. Viral strains used in the present study did not form a monophyletic group, which is evidence of a polyphyletic origin. We report the rapid spread patterns and high evolution rate of the different DENV-1 lineages

Characterization of Leishmania donovani Aquaporins Shows Presence of Subcellular Aquaporins Similar to Tonoplast Intrinsic Proteins of Plants

Leishmania donovani, a protozoan parasite, resides in the macrophages of the mammalian host. The aquaporin family of proteins form important components of the parasite-host interface. The parasite-host interface could be a potential target for chemotherapy. Analysis of L. major and L. infantum genomes showed the presence of five aquaporins (AQPs) annotated as AQP9 (230aa), AQP putative (294aa), AQP-like protein (279aa), AQP1 (314aa) and AQP-like protein (596aa). We report here the structural modeling, localization and functional characterization of the AQPs from L. donovani. LdAQP1, LdAQP9, LdAQP2860 and LdAQP2870 have the canonical NPA-NPA motifs, whereas LdAQP putative has a non-canonical NPM-NPA motif. In the carboxyl terminal to the second NPA box of all AQPs except AQP1, a valine/alanine residue was found instead of the arginine. In that respect these four AQPs are similar to tonoplast intrinsic proteins in plants, which are localized to intracellular organelles. Confocal microscopy of L. donovani expressing GFP-tagged AQPs showed an intracellular localization of LdAQP9 and LdAQP2870. Real-time PCR assays showed expression of all aquaporins except LdAQP2860, whose level was undetectable. Three-dimensional homology modeling of the AQPs showed that LdAQP1 structure bears greater topological similarity to the aquaglyceroporin than to aquaporin of E. coli. The pore of LdAQP1 was very different from the rest in shape and size. The cavity of LdAQP2860 was highly irregular and undefined in geometry. For functional characterization, four AQP proteins were heterologously expressed in yeast. In the fps1Δ yeast cells, which lacked the key aquaglyceroporin, LdAQP1 alone displayed an osmosensitive phenotype indicating glycerol transport activity. However, expression of LdAQP1 and LdAQP putative in a yeast gpd1Δ strain, deleted for glycerol production, conferred osmosensitive phenotype indicating water transport activity or aquaporin function. Our analysis for the first time shows the presence of subcellular aquaporins and provides structural and functional characterization of aquaporins in Leishmania donovani

Spatio-Temporal Tracking and Phylodynamics of an Urban Dengue 3 Outbreak in São Paulo, Brazil

The dengue virus has a single-stranded positive-sense RNA genome of ∼10.700 nucleotides with a single open reading frame that encodes three structural (C, prM, and E) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins. It possesses four antigenically distinct serotypes (DENV 1–4). Many phylogenetic studies address particularities of the different serotypes using convenience samples that are not conducive to a spatio-temporal analysis in a single urban setting. We describe the pattern of spread of distinct lineages of DENV-3 circulating in São José do Rio Preto, Brazil, during 2006. Blood samples from patients presenting dengue-like symptoms were collected for DENV testing. We performed M-N-PCR using primers based on NS5 for virus detection and identification. The fragments were purified from PCR mixtures and sequenced. The positive dengue cases were geo-coded. To type the sequenced samples, 52 reference sequences were aligned. The dataset generated was used for iterative phylogenetic reconstruction with the maximum likelihood criterion. The best demographic model, the rate of growth, rate of evolutionary change, and Time to Most Recent Common Ancestor (TMRCA) were estimated. The basic reproductive rate during the epidemics was estimated. We obtained sequences from 82 patients among 174 blood samples. We were able to geo-code 46 sequences. The alignment generated a 399-nucleotide-long dataset with 134 taxa. The phylogenetic analysis indicated that all samples were of DENV-3 and related to strains circulating on the isle of Martinique in 2000–2001. Sixty DENV-3 from São José do Rio Preto formed a monophyletic group (lineage 1), closely related to the remaining 22 isolates (lineage 2). We assumed that these lineages appeared before 2006 in different occasions. By transforming the inferred exponential growth rates into the basic reproductive rate, we obtained values for lineage 1 of R0 = 1.53 and values for lineage 2 of R0 = 1.13. Under the exponential model, TMRCA of lineage 1 dated 1 year and lineage 2 dated 3.4 years before the last sampling. The possibility of inferring the spatio-temporal dynamics from genetic data has been generally little explored, and it may shed light on DENV circulation. The use of both geographic and temporally structured phylogenetic data provided a detailed view on the spread of at least two dengue viral strains in a populated urban area

Impact of protozoan cell death on parasite-host interactions and pathogenesis

PCD in protozoan parasites has emerged as a fascinating field of parasite biology. This not only relates to the underlying mechanisms and their evolutionary implications but also to the impact on the parasite-host interactions within mammalian hosts and arthropod vectors. During recent years, common functions of apoptosis and autophagy in protozoa and during parasitic infections have emerged. Here, we review how distinct cell death pathways in Trypanosoma, Leishmania, Plasmodium or Toxoplasma may contribute to regulation of parasite cell densities in vectors and mammalian hosts, to differentiation of parasites, to stress responses, and to modulation of the host immunity. The examples provided indicate crucial roles of PCD in parasite biology. The existence of PCD pathways in these organisms and the identification as being critical for parasite biology and parasite-host interactions could serve as a basis for developing new anti-parasitic drugs that take advantage of these pathways

Instability of aquaglyceroporin (Aqp) 2 contributes to drug resistance in trypanosoma brucei

Defining mode of action is vital for both developing new drugs and predicting potential resistance mechanisms. Sensitivity of African trypanosomes to pentamidine and melarsoprol is predominantly mediated by aquaglyceroporin 2 (TbAQP2), a channel associated with water/glycerol transport. TbAQP2 is expressed at the flagellar pocket membrane and chimerisation with TbAQP3 renders parasites resistant to both drugs. Two models for how TbAQP2 mediates pentamidine sensitivity have emerged; that TbAQP2 mediates pentamidine translocation across the plasma membrane or via binding to TbAQP2, with subsequent endocytosis and presumably transport across the endosomal/lysosomal membrane, but as trafficking and regulation of TbAQPs is uncharacterised this remains unresolved. We demonstrate that TbAQP2 is organised as a high order complex, is ubiquitylated and is transported to the lysosome. Unexpectedly, mutation of potential ubiquitin conjugation sites, i.e. cytoplasmic-oriented lysine residues, reduced folding and tetramerization efficiency and triggered ER retention. Moreover, TbAQP2/TbAQP3 chimerisation, as observed in pentamidine-resistant parasites, also leads to impaired oligomerisation, mislocalisation and increased turnover. These data suggest that TbAQP2 stability is highly sensitive to mutation and that instability contributes towards the emergence of drug resistance

Phylogenetic reconstruction of dengue virus type 2 in Colombia

Background: Dengue fever is perhaps the most important viral re-emergent disease especially in tropical and subtropical countries, affecting about 50 million people around the world yearly. In Colombia, dengue virus was first detected in 1971 and still remains as a major public health issue. Although four viral serotypes have been recurrently identified, dengue virus type 2 (DENV-2) has been involved in the most important outbreaks during the last 20 years, including 2010 when the fatality rate highly increased. As there are no major studies reviewing virus origin and genotype distribution in this country, the present study attempts to reconstruct the phylogenetic history of DENV-2 using a sequence analysis from a 224 bp PCR-amplified product corresponding to the carboxyl terminus of the envelope (E) gene from 48 Colombian isolates. Results: As expected, the oldest isolates belonged to the American genotype (subtype V), but the strains collected since 1990 represent the American/Asian genotype (subtype IIIb) as previously reported in different American countries. Interestingly, the introduction of this genotype coincides with the first report of dengue hemorrhagic fever in Colombia at the end of 1989 and the increase of cases during the next years. Conclusion: After replacement of the American genotype, several lineages of American/Asian subtype have rapidly spread all over the country evolving in new clades. Nevertheless, the direct association of these new variants in the raise of lethality rate observed during the last outbreak has to be demonstrated