Ricardo Parreira

Dengue virus serotype 4 and chikungunya virus coinfection in a traveller returning from Luanda, Angola, January 2014.

A concurrent dengue virus serotype 4 and chikungunya virus infection was detected in a woman in her early 50s returning to Portugal from Luanda, Angola, in January 2014. The clinical, laboratory and molecular findings, involving phylogenetic analyses of partial viral genomic sequences amplified by RT-PCR, are described. Although the circulation of both dengue and chikungunya viruses in Angola has been previously reported, to our knowledge this is the first time coinfection with both viruses has been detected there.

Intestinal parasites in dogs and cats from the district of Évora, Portugal.

Intestinal parasites, both helminths and protozoa, are commonly found in domestic animals, and the possible transmission of enteric parasites from dogs and cats to humans may constitute a global potential health risk worldwide. In the present study, we analysed 148 stool samples from dogs (n=126) and cats (n=22) collected from animal shelters and veterinary clinics, in the district of Évora, Portugal. Microscopic examination confirmed that Giardia was the most frequent parasite in the studied population (34/148; 23%). Other parasites such as Ancylostoma sp., Isospora spp., Toxocara, Trichuris spp., Toxascaris and Toxoplasma were also found. Furthermore, molecular characterization of Giardia duodenalis analysis targeting the small subunit ribosomal RNA (ssu-rRNA) was performed revealing the presence of host-specific (C and D) and zoonotic assemblages (A and B). This work points out to the importance of protozoan parasites in companion animals, and reanalyses the need for parasite prophylaxis.

Genetic characterization of an insect-specific flavivirus isolated from Culex theileri mosquitoes collected in southern Portugal

Highlights ► A new insect flavivirus (CTFV) was isolated from Culex theileri mosquitoes. ► CTFV does not replicate in Vero cells. ► Phylogenetic analyses place CTFV among Culex-associated flaviviruses. ► CTFV seems to be dispersed in the Iberian Peninsula. ► CTFV sequences were not found in theirs hosts’ genome.

Molecular detection of Leishmania DNA and identification of blood meals in wild caught phlebotomine sand flies (Diptera: Psychodidae) from southern Portugal

BackgroundZoonotic visceral leishmaniasis caused by Leishmania infantum which is transmitted by phlebotomine sand flies (Diptera, Psychodidae) is endemic in the Mediterranean basin. The main objectives of this study were to (i) detect Leishmania DNA and (ii) identify blood meal sources in wild caught female sand flies in the zoonotic leishmaniasis region of Algarve, Portugal/Southwestern Europe.MethodsPhlebotomine sand flies were collected using CDC miniature light traps and sticky papers. Sand flies were identified morphologically and tested for Leishmania sp. by PCR using ITS-1 as the target sequence. The source of blood meal of the engorged females was determined using the cyt-b sequence.ResultsOut of the 4,971 (2,584 males and 2,387 females) collected sand flies, Leishmania DNA was detected by PCR in three females (0.13%), specifically in two specimens identified on the basis of morphological features as Sergentomyia minuta and one as Phlebotomus perniciosus. Haematic preferences, as defined by the analysis of cyt-b DNA amplified from the blood-meals detected in the engorged female specimens, showed that P. perniciosus fed on a wide range of domestic animals while human and lizard DNA was detected in engorged S. minuta.ConclusionsThe anthropophilic behavior of S. minuta together with the detection of Leishmania DNA highlights the need to determine the role played by this species in the transmission of Leishmania parasites to humans. In addition, on-going surveillance on Leishmania vectors is crucial as the increased migration and travelling flow elevate the risk of introduction and spread of infections by Leishmania species which are non-endemic.

Negeviruses found in multiple species of mosquitoes from southern Portugal: Isolation, genetic diversity, and replication in insect cell culture

In this report, an RT-PCR approach based on the use of degenerate primers allowed the identification of negeviruses in four different species of mosquitoes (Ochlerotatus caspius, Culex pipiens, Cx. theileri and Cx. univittatus) collected in southern Portugal. The genomes of two of these viruses, sequenced to full completion, were shown to encode all the proteins encoded by previously described negeviruses. One of these viruses induces exuberant cytopathic effect in insect cell culture, with no obvious signs of apoptosis induction, replicating very rapidly and allowing for the detection of viral genomes in the infected culture supernatant as soon as 4h post-infection. This virus was also shown to use a dsRNA intermediate, which was found to be fully formed and active 3h after infection. Phylogenetic analysis of two products encoded by the viral ORF1 placed both viruses among Negev virus cluster, in the recently proposed Nelorpivirus taxon.

Echinococcus granulosus in Portugal: The first report of the G7 genotype in cattle

Although cystic echinococcosis (CE) has been a recognized public health problem in Portugal, molecular data regarding the types and prevalence of infecting strains of its etiological agent (Echinococcus granulosus) are still scarce. Over the last years we have been evaluating the prevalence of CE in the country, and in this report we determined the parasite genotypes infecting sheep, goats, cattle and human in Portugal, based on 209 hydatid cysts recovered from liver (n=96), lung (n=95), pancreas (n=17) and kidney (n=1) samples obtained between 2008 and 2011. Protoscoleces or germinal layers were collected from individual cysts, DNA was extracted, and a part of the mitochondrial DNA encoding the cytochrome c oxidase subunit 1 was amplified by PCR. Overall, the results confirm the overall dominance of the G1-G3 cluster of strains, which are particularly prevalent in southern Portugal in livestock ruminants. Unexpectedly, one parasite sequence with cattle origin was found to correspond to E. granulosus G7 genotype (also known as E. intermedius), here reported for the first time in bovine, in Portugal.

Angola's 2013 dengue outbreak: clinical, laboratory and molecular analyses of cases from four Portuguese institutions.

INTRODUCTIONnDengue virus (DENV) is the arbovirus with the widest impact on human health. In Africa in general, and in Angola in particular, the epidemiology and public health impact of DENV is far from clear. However, rapid population growth, unplanned urbanization, increased international travel, and the presence of virus major vector (Aedes aegypti) in the country suggest that DENV transmission may occur.nnnMETHODOLOGYnIn parallel to the occurrence of a dengue outbreak affecting the capital of Angola, between March and July 2013 four Portuguese institutions diagnosed dengue infection in 146 individuals returning to Portugal. Clinical presentation, laboratory findings, and molecular analyses of partial viral genomic segments were performed.nnnRESULTSnThe mean age of the individuals included in this study was 42 years old, the majority being men of Portuguese nationality, reporting various lengths of stay in Angola. Fever was the most reported clinical sign, being frequently associated (61.0%) with myalgia and headache. Hematological values, including hematocrit, white-blood cell and platelets counts, correlated with the absence of severe or complicated cases, or coagulation disorders. No deaths were observed. Viral NS1 was detected in 56.2% of the samples, and all NS1 negative cases had anti-dengue IgM antibodies. RT-PCR indicated the presence of DENV1, which was confirmed by phylogenetic analysis of 25 partial NS5 viral sequences.nnnCONCLUSIONnThe DENV cases analyzed conformed to classical and uncomplicated dengue, caused by the suggested exclusive circulation of a genetically homogeneous DENV1 of genotype III, apparently with a single origin.

Exploring the utility of phylogenetic analysis of cytochrome oxidase gene subunit I as a complementary tool to classical taxonomical identification of phlebotomine sand fly species (Diptera, Psychodidae) from southern Europe.

Phlebotomine sand flies (Diptera, Psychodidae) are known to be vectors of several pathogens such as Leishmania and Phlebovirus genera. The identification of phlebotomine sand fly species is currently based on morphological characters, and requires considerable taxonomic expertise and skilfulness, but may be complemented by DNA-based analyses for (i) accurate species identification and (ii) for estimating sand fly diversity. The aim of this study was to evaluate the utility of mitochondrial cytochrome oxidase gene subunit I (cox1) sequence analysis as a complementary tool to classical taxonomical for the identification of the most prevalent phlebotomine sand fly species from southern Europe (i.e. Phlebotomus ariasi, P. perniciosus, P. sergenti and Sergentomyia minuta). Phylogenetic analyses of cox1 sequences allowed conclusive assignment of most of the sand flies into individual species, and revealed the genetic heterogeneity that characterizes some of the identified genetic clusters. Nevertheless, it showed some limitations, as it failed to (i) allocate correctly all of all species of a given subgenus to a single lineage, or (ii) conclusively identify sequences amplified from individuals classified morphologically as P. ariasi. A more extensive analysis of cox1 sequences together with morphometric characterization of specimens from different geographic areas/regions might be useful for the correct assessment of the phylogenetic relationship within the P. ariasi/P. chadlii cluster and/or help to ascertain the usefulness of cox1 for molecular taxonomy of sand flies.

Dengue fever in Europe: could there be an epidemic in the future?

Dengue virus (DENV) is the arbovirus with the widest impact on human health. Although its dispersal is partially conditioned by environmental constraints that limit the distribution of its main vector (Aedes aegypti), DENV has been spreading geographically in recent times, but mostly afflicting tropical and subtropical regions. With no prophylactic vaccine or specific therapeutics available, vector control remains the best alternative to restrain its circulation. Moreover, the establishment of thriving vector populations in peri urban environments brings humans and viruses together, opening the possibility for the occurrence of unexpected outbreaks. Europe is no exception: such was the case of Madeira in 2012. In addition to its impact on the health of the local population, health services, and economy, this outbreak revealed how difficult it may be to control the circulation of pathogenic arboviruses, especially taking into consideration that Europe is already partially colonized by another DENV vector, Aedes albopictus.

Detection of Leishmania DNA and blood meal sources in phlebotomine sand flies (Diptera: Psychodidae) in western of Spain: Update on distribution and risk factors associated

Leishmaniosis caused by Leishmania infantum is present in Mediterranean countries, with high prevalence in areas of the center and south of Spain. However, in some regions such as Extremadura (in southwest of Spain), data has not been updated since 1997. The aim of this work was (i) to provide information about the distribution of phlebotomine sand fly species in western of Spain (Extremadura region), (ii) to determine risk factors for the presence of sand fly vectors and (iii) to detect Leishmania DNA and identify blood meal sources in wild caught females. During 2012-2013, sand flies were surveyed using CDC miniature light-traps in 13 of 20 counties in Extremadura. Specimens were identified morphologically and females were used for molecular detection of Leishmania DNA by kDNA, ITS-1 and cyt-B. In addition, blood meals origins were analyzed by a PCR based in vertebrate cyt b gene. A total of 1083 sand flies of both gender were captured and identified. Five species were collected, Phlebotomus perniciosus (60.76%), Sergentomyia minuta (29.92%), P. ariasi (7.11%), P. papatasi (1.48%) and P. sergenti (0.74%). The last three species constitute the first report in Badajoz, the most southern province of Extremadura region. Leishmania DNA was detected in three out of 435 females (one P. pernicious and two S. minuta). Characterization of obtained DNA sequences by phylogenetic analyses revealed close relatedness with Leishmania tarentolae in S. minuta and L. infantum in P. perniciosus. Haematic preferences showed a wide range of hosts, namely: swine, humans, sheep, rabbits, horses, donkeys and turkeys. The simultaneous presence of P. perniciosus and P. ariasi vectors, the analysis of blood meals, together with the detection of L. infantum and in S. minuta of L. tarentolae, confirms the ideal conditions for the transmission of this parasitosis in the western of Spain. These results improve the epidemiological knowledge of leishmaniosis and its vectors in this part of Spain, highlighting the need for ongoing entomological and parasitological surveillance.