Zoonotic Threats: The (Re)emergence of Cercarial Dermatitis, Its Dynamics, and Impact in Europe.

Cercarial dermatitis (CD), or "Swimmer's itch" as it is also known, is a waterborne illness caused by a blood fluke from the family Schistosomatidae. It occurs when cercariae of trematode species that do not have humans as their definitive host accidentally penetrate human skin (in an aquatic environment) and trigger allergic symptoms at the site of contact. It is an emerging zoonosis that occurs through water and is often overlooked during differential diagnosis. Some of the factors contributing to the emergence of diseases like CD are related to global warming, which brings about climate change, water eutrophication, the colonization of ponds by snails susceptible to the parasite, and sunlight exposure in the summer, associated with migratory bird routes. Therefore, with the increase in tourism, especially at fluvial beaches, it is relevant to analyze the current epidemiological scenario of CD in European countries and the potential regions at risk.

Whole-Genome Characterization of Rotavirus G9P[6] and G9P[4] Strains That Emerged after Rotavirus Vaccine Introduction in Mozambique.

Mozambique introduced the Rotarix® vaccine into the National Immunization Program in September 2015. Following vaccine introduction, rotavirus A (RVA) genotypes, G9P[4] and G9P[6], were detected for the first time since rotavirus surveillance programs were implemented in the country. To understand the emergence of these strains, the whole genomes of 47 ELISA RVA positive strains detected between 2015 and 2018 were characterized using an Illumina MiSeq-based sequencing pipeline. Of the 29 G9 strains characterized, 14 exhibited a typical Wa-like genome constellation and 15 a DS-1-like genome constellation. Mostly, the G9P[4] and G9P[6] strains clustered consistently for most of the genome segments, except the G- and P-genotypes. For the G9 genotype, the strains formed three different conserved clades, separated by the P type (P[4], P[6] and P[8]), suggesting different origins for this genotype. Analysis of the VP6-encoding gene revealed that seven G9P[6] strains clustered close to antelope and bovine strains. A rare E6 NSP4 genotype was detected for strain RVA/Human-wt/MOZ/HCN1595/2017/G9P[4] and a genetically distinct lineage IV or OP354-like P[8] was identified for RVA/Human-wt/MOZ/HGJM0644/2015/G9P[8] strain. These results highlight the need for genomic surveillance of RVA strains detected in Mozambique and the importance of following a One Health approach to identify and characterize potential zoonotic strains causing acute gastroenteritis in Mozambican children.

Improved dsRNA isolation and purification method validated by viral dsRNA detection using novel primers in Saccharomyces cerevisiae.

Accurate genomic sequencing demands high-quality double-stranded RNA (dsRNA). Existing methods for dsRNA extraction from yeast, fungi, and plants primarily rely on cellulose, suitable only for small volume extractions, or the time-consuming lithium chloride precipitation. To streamline the traditional phenol-chloroform-based dsRNA extraction method, the main challenge is the reduction of mitochondrial DNA (mtDNA) and Single Stranded RNA (ssRNA) to no detectable levels after gel electrophoresis. This challenge is successfully addressed through the modified approach described here, involving phenol extraction at low pH, followed by the addition of ammonium sulfate to the aqueous buffer. The dsRNA isolated using this novel method exhibits comparable quality to that obtained through cellulose purification, and it is readily amenable to RT-PCR. Moreover, a single batch of yeast cell RNA isolation requires only 2-3 h of hands-on time, thus simplifying and expediting the process significantly.•Buffers were redesigned from [32,33,35].•No DNASE, Ribonuclease A or beads were used during the purification.•Simple and inexpensive dsRNA extraction and purification method is described.

In vitro and in vivo behaviour of sympatric Leishmania (V.) braziliensis, L. (V.) peruviana and their hybrids.

Leishmania (Viannia) braziliensis is the main cause of highly disfiguring mucocutaneous leishmaniasis (MCL) in South America. The related species L. (V.) peruviana has only been identified in simple cutaneous lesions (CL). Hybrids between L. braziliensis and L. peruviana have been reported although genetic exchange in Leishmania is considered to be rare. Here we compared growth in vitro, adaptive capacity under thermal and oxidative stress and behaviour in a hamster model, of L. braziliensis, L. peruviana, and their putative hybrids. At 24°C, the optimal temperature for in vitro growth, L. braziliensis had the highest growth rate. In in vitro studies hybrid clones presented heterogeneous phenotypes, from slower growth rates, similar to L. peruviana, to higher growth rates, as observed in L. braziliensis. Hamsters infected with hybrid strains, presented the highest parasite densities and aggressive relapses at a later stage of infection. Hybrids generally presented higher plasticity and phenotypic diversity than the putative parental species, with potential eco-epidemiological implications, including an impact on the success of disease control.

First report of an Onchocercidae worm infecting Psychodopygus carrerai carrerai sandfly, a putative vector of Leishmania braziliensis in the Amazon.

Sandflies are insects of public health interest due to their role as vectors of parasites of the genus Leishmania, as well as other pathogens. Psychodopygus carrerai carrerai is considered an important sylvatic vector of Leishmania (Viannia) braziliensis in Amazonia. In this study, sandflies were collected in a forested area in the Xapuri municipality, in the State of Acre (Northern Brazil). Two Ps. carrerai carrerai females were found parasitized with a larval form of a filarial worm, one in the labium of the proboscis, the other after the head was squashed, suggesting they were infective larvae. Sandflies were identified through morphological characters as well as amplification and sequencing of the cytochrome oxidase gene (COI). This was the first sequence obtained for Ps. carrerai carrerai for this marker. The obtained nematodes were also characterized through direct sequencing of a fragment of COI and 12S genes, both mitochondrial, and ITS1, a nuclear marker. Phylogenetic analyses revealed that the filarial nematodes belong to a species without sequences for these markers in the database, part of family Onchocercidade and closely related to genus Onchocerca (12S tree). Although sandfly infection with nematodes including members of the Onchocercidae has been reported in the Old World, this is the first report of sandfly infection by a member of the Onchocercidae family in the New World, to the best of our knowledge. Considering that the phylogenetic relationships and location in the insect, it can be expected that this is a parasite of mammals and the transmission cycle should be clarified.

Molecular Epidemiology of Rotavirus A Strains Pre- and Post-Vaccine (Rotarix®) Introduction in Mozambique, 2012-2019: Emergence of Genotypes G3P[4] and G3P[8].

Group A rotavirus (RVA) remains the most important etiological agent associated with severe acute diarrhea in children. Rotarix® monovalent vaccine was introduced into Mozambique's Expanded Program on Immunization in September 2015. In the present study, we report the diversity and prevalence of rotavirus genotypes, pre- (2012-2015) and post-vaccine (2016-2019) introduction in Mozambique, among diarrheic children less than five years of age. Genotyping data were analyzed for five sentinel sites for the periods indicated. The primary sentinel site, Mavalane General Hospital (HGM), was analyzed for the period 2012-2019, and for all five sites (country-wide analyses), 2015-2019. During the pre-vaccine period, G9P[8] was the most predominant genotype for both HGM (28.5%) and the country-wide analysis (46.0%). However, in the post-vaccine period, G9P[8] was significantly reduced. Instead, G3P[8] was the most common genotype at HGM, while G1P[8] predominated country-wide. Genotypes G9P[4] and G9P[6] were detected for the first time, and the emergence of G3P[8] and G3P[4] genotypes were observed during the post-vaccine period. The distribution and prevalence of rotavirus genotypes were distinct in pre- and post-vaccination periods, while uncommon genotypes were also detected in the post-vaccine period. These observations support the need for continued country-wide surveillance to monitor changes in strain diversity, due to possible vaccine pressure, and consequently, the effect on vaccine effectiveness.

Global genome diversity of the Leishmania donovani complex.

Protozoan parasites of the Leishmania donovani complex - L. donovani and L. infantum - cause the fatal disease visceral leishmaniasis. We present the first comprehensive genome-wide global study, with 151 cultured field isolates representing most of the geographical distribution. L. donovani isolates separated into five groups that largely coincide with geographical origin but vary greatly in diversity. In contrast, the majority of L. infantum samples fell into one globally-distributed group with little diversity. This picture is complicated by several hybrid lineages. Identified genetic groups vary in heterozygosity and levels of linkage, suggesting different recombination histories. We characterise chromosome-specific patterns of aneuploidy and identified extensive structural variation, including known and suspected drug resistance loci. This study reveals greater genetic diversity than suggested by geographically-focused studies, provides a resource of genomic variation for future work and sets the scene for a new understanding of the evolution and genetics of the Leishmania donovani complex.

Development of a multilocus microsatellite typing approach for discriminating strains of Leishmania (Viannia) species.

A multilocus microsatellite typing (MLMT) approach based on the analysis of 15 independent loci has been developed for the discrimination of strains belonging to different Viannia species. Thirteen microsatellite loci were isolated de novo from microsatellite-enriched libraries for both Leishmania braziliensis and L. guyanensis. Two previously identified markers, AC01 and AC16, were modified and added to our marker set. Markers were designed to contain simple dinucleotide repeats flanked by the minimal possible number of nucleotides in order to allow variations in repeat numbers to be scored as size variations of the PCR products. The 15 markers in total were amplified for almost all of the strains of Viannia tested; one marker did not amplify from the two L. peruviana strains included in the study. When 30 strains of L. braziliensis, 21 strains of L. guyanensis, and 2 strains of L. peruviana were tested for polymorphisms, all strains except two strains of L. guyanensis had individual MLMT types. Distance-based analysis identified three main clusters. All strains except one strain of L. guyanensis grouped together. Two clusters consisted of strains of L. braziliensis according to their geographical origins. The two strains of L. peruviana grouped together with strains of L. braziliensis from Peru and the adjacent Brazilian state of Acre. MLMT has proven capable of individualizing strains even from the same areas of endemicity and of detecting genetic structures at different levels. MLMT is thus applicable for epidemiological and population genetic studies of strains within the subgenus Viannia.

Phylogenetic Studies.

Phylogenetics is an important component of the systems biology approach. Knowledge about evolution of the genus Leishmania is essential to understand various aspects of basic biology of these parasites, such as parasite-host or parasite-vector relationships, biogeography, or epidemiology. Here, we present a comprehensive guideline for performing phylogenetic studies based on DNA sequence data, but with principles that can be adapted to protein sequences or other molecular markers. It is presented as a compilation of the most commonly used genetic targets for phylogenetic studies of Leishmania, including their respective primers for amplification and references, as well as details of PCR assays. Guidelines are, then, presented to choose the best targets in relation to the types of samples under study. Finally, and importantly, instructions are given to obtain optimal sequences, alignments, and datasets for the subsequent data analysis and phylogenetic inference. Different bioinformatics methods and software for phylogenetic inference are presented and explained. This chapter aims to provide a compilation of methods and generic guidelines to conduct phylogenetics of Leishmania for nonspecialists.

Leishmaniases in the Mediterranean in the era of molecular epidemiology.

Molecular tools are used increasingly for descriptive epidemiological studies in different Mediterranean foci of visceral and cutaneous leishmaniases. Several molecular markers with different resolution levels have been developed to address key epidemiological questions related to the (re-)emergence and spread of leishmaniases, as well as its risk factors: environmental changes, immunosuppression and treatment failure. Typing and analytical tools are improving but are not yet addressing all epidemiological issues satisfactorily. There is an urgent need for better cooperation between laboratory scientists and epidemiologists and for regional epidemiological surveillance of these infectious diseases that affect all Mediterranean countries.

Comparative efficacy, toxicity and biodistribution of the liposomal amphotericin B formulations Fungisome® and AmBisome® in murine cutaneous leishmaniasis.

Fungisome® (F), a liposomal amphotericin B (AmB) product, is marketed in India as a safe and effective therapeutic for the parasitic infection visceral leishmaniasis. Its potential in the treatment of cutaneous leishmaniasis (CL), a disfiguring form of the disease affecting the skin, is currently unknown. Here, we report the evaluation of the efficacy of F in the Leishmania major BALB/c murine model of CL, including a head-to-head comparison with the standard liposomal AmB formulation AmBisome® (A). Upon intravenous administration at dose levels of 5, 10 and 15 mg/kg of body weight (on days 0, 2, 4, 6 and 8), F showed clear signs of toxicity (at 15 mg/kg), while A did not. After complete treatment (day 10), the tolerated doses of 5 and 10 mg/kg F had significant antileishmanial activity (ED50 = 4.0 and 12.8 mg/kg for qPCR-based parasite load and lesion size, respectively), although less than that of A at identical doses (ED50 = 3.0 and 8.8 mg/kg). The efficacy of F was inferior compared to A because lower levels of the active agent AmB accumulated within the infected lesion. In conclusion, despite possibly being less safe and efficacious than A at equivalent doses, the moderate in vivo activity of F could indicate a role in the systemic pharmacotherapy of CL.

Glycoprotein 63 (gp63) genes show gene conversion and reveal the evolution of Old World Leishmania.

Species of the subgenus Leishmania (Leishmania) cause the debilitating disease leishmaniasis on four continents. Species grouped within the Leishmania donovani complex cause visceral leishmaniasis, a life-threatening disease, often associated with poverty, and affecting some 0.5 million people each year. The Leishmania glycoprotein GP63, or major surface protease, is a metalloprotease involved in parasite survival, infectivity and virulence. Here, we show that evolution of the gp63 multigene family is influenced by mosaic or fragmental gene conversion. This is a major evolutionary force for both homogenisation and for generating diversity, even in the absence of sexual reproduction. We propose here that the high GC content at the third codon position in the gp63 family of Old World Leishmania may be higher in multicopy regions, under the biased gene conversion model, because increased copy numbers may lead to increased rates of recombination. We confirm that one class of gp63 genes with an extended 3'end signal, gp63(EXT), reveals genetic groups within the complex and gives insights into evolution and host associations. Gp63(EXT) genes can also provide the basis for rapid and reliable genotyping of strains in the L. donovani complex. Our results confirmed that a more stringent definition of Leishmania infantum is required and that the species Leishmania archibaldi should be suppressed.

The Leishmania donovani complex: genotypes of five metabolic enzymes (ICD, ME, MPI, G6PDH, and FH), new targets for multilocus sequence typing.

Flagellates of the Leishmania donovani complex are causative agents of human cutaneous and visceral leishmaniasis. The complex is comprised of L. donovani, Leishmania infantum and Leishmania archibaldi, although the latter is not now considered to be a valid species. Morphological distinction of Leishmania species is impractical, so biochemical, immunological and DNA-based criteria were introduced. Multilocus enzyme electrophoresis (MLEE) is the present gold standard. We have sequenced the genes encoding five metabolic enzymes used for MLEE, both to resolve the DNA diversity underlying isoenzyme mobility differences and to explore the potential of these targets for higher resolution PCR-based multilocus sequence typing. The genes sequenced were isocitrate dehydrogenase, malic enzyme, mannose phosphate isomerase, glucose-6-phosphate dehydrogenase, and fumarate hydratase, for 17 strains of L. infantum, seven strains of L. donovani, and three strains of L. archibaldi. Protein mobilities predicted from amino acid sequences did not always accord precisely with reported MLEE profiles. A high number of heterozygous sites was detected. Heterozygosity was particularly frequent in some strains and indirectly supported the presence of genetic exchange in Leishmania. Phylogenetic analysis of a concatenated alignment based on a total of 263 kb protein-coding sequences showed strong correlation of genotype with geographical origin. Europe and Africa appear to represent independent evolutionary centres.

Molecular characterization of Dirofilaria spp. circulating in Portugal.

BACKGROUND: Dirofilariosis is a potentially zoonotic parasitic disease, mainly transmitted by mosquito vectors in many parts of the world. Data concerning the canine Dirofilaria species currently circulating in Portugal is scarce. Thereby, a large-scale study was conducted to determine the Dirofilaria spp. present in Portugal, based on a molecular approach, and also to optimize a reliable and highly sensitive species-specific polymerase chain reaction (PCR) assay that could be used for the simultaneous detection and differentiation of Dirofilaria immitis, Dirofilaria repens, and other concurrent filarial species in animal reservoirs. METHODS: Blood samples were collected from three districts of Portugal (Coimbra, Santarém and Setúbal) between 2011 and 2013. Samples were tested using rapid immunomigration tests (Witness® Dirofilaria), modified Knott's technique and acid phosphatase histochemical staining. In addition, molecular analysis was performed by amplification of the internal transcribed spacer (ITS) region using two different PCR protocols, specific for molecular screening of canine filarial species. RESULTS: Of the 878 dogs sampled, 8.8% (n = 77) were positive for D. immitis circulating antigen and 13.1% (n = 115) positive for microfilariae by the modified Knott's technique. Of the 134 samples tested by acid phosphatase histochemical staining, 100 (74.6%) were positive for D. immitis. Overall, 13.7% (n = 120) were positive by PCR for D. immitis by ITS2, of which 9.3% (67/720) were also positive by ITS1. ITS2 PCR was the most sensitive and specific method, capable of detecting mixed D. immitis and A. reconditum infections. Heterozygosity, in the form of double peaks, was detected by sequencing of both ITS regions. No D. repens was detected by any of the diagnostic methods. CONCLUSIONS: The present study confirmed D. immitis as the dominant species of the genus Dirofilaria infecting Portuguese dogs, based on sequencing of ITS1 and ITS2 PCR fragments. Additionally, ITS2 PCR was the most adequate method for diagnosis and prevalence estimation.

Towards multilocus sequence typing of the Leishmania donovani complex: resolving genotypes and haplotypes for five polymorphic metabolic enzymes (ASAT, GPI, NH1, NH2, PGD).

Multilocus enzyme electrophoresis is the gold standard for identification of Leishmania species and strains. Drawbacks include: only amino acid polymorphisms affecting electrophoretic mobility are detected; distinct allozymes can have coincident mobilities; few characters are available; and parasites must be cultured in bulk. So far, thousands of Leishmania strains have been phenotyped by multilocus enzyme electrophoresis. Here, we sequence enzyme-coding genes to provide a PCR-based higher resolution equivalent of multilocus enzyme electrophoresis, particularly for Leishmania infantum. Of 15 enzymes used for multilocus enzyme electrophoresis (MON typing) we have sequenced aspartate aminotransferase, glucose-6-phosphate isomerase, nucleoside hydrolase 1, nucleoside hydrolase 2 and 6-phosphogluconate dehydrogenase. Heterozygous alleles were common, with multiple heterozygous sites within a single locus for several of the genes. Haplotypes were resolved by allele-specific PCR and allele-specific sequencing. Heterozygous haplotypes conformed to the haplotypes of putative parents. One strain appeared to be hybrid across two genetic groups of the Leishmania donovani complex. In most cases, a single amino acid polymorphism was responsible for change in enzyme mobility. Some indistinguishable phenotypes were produced by distinct genotypes. Silent genetic polymorphisms provided enhanced discrimination over multilocus enzyme electrophoresis, for example, by subdividing the zymodeme MON-1. The PCR-based genotyping that we describe could be applied directly to clinical samples or to small volume cultures and in a multilocus sequence typing format. Furthermore, it can be used to detect recombination indirectly and for population genetics studies.

Leishmania major: genetic heterogeneity of Iranian isolates by single-strand conformation polymorphism and sequence analysis of ribosomal DNA internal transcribed spacer.

Protozoan parasites of Leishmania major are the causative agents of cutaneous leishmaniasis in different parts of Iran. We applied PCR-based methods to analyze L. major parasites isolated from patients with active lesions from different geographic areas in Iran in order to understand DNA polymorphisms within L. major species. Twenty-four isolates were identified as L. major by RFLP analysis of the ribosomal internal transcribed spacer 1 (ITS1) amplicons. These isolates were further studied by single-strand conformation polymorphism (SSCP) analysis and sequencing of ITS1 and ITS2. Data obtained from SSCP analysis of the ITS1 and ITS2 loci revealed three and four different patterns among all studied samples, respectively. Sequencing of ITS1 and ITS2 confirmed the results of SSCP analysis and showed the potential of the PCR-SSCP method for assessing genetic heterogeneity within L. major. Different patterns in ITS1 were due to substitution of one nucleotide, whereas in ITS2 the changes were defined by variation in the number of repeats in two polymorphic microsatellites. In total five genotypic groups LmA, LmB, LmC, LmD and LmE were identified among L. major isolates. The most frequent genotype, LmA, was detected in isolates collected from different endemic areas of cutaneous leishmaniasis in Iran. Genotypes LmC, LmD and LmE were found only in the new focus of CL in Damghan (Semnan province) and LmB was identified exclusively among isolates of Kashan focus (Isfahan province). The distribution of genetic polymorphisms suggests the existence of distinct endemic regions of L. major in Iran.

Application of kDNA as a molecular marker to analyse Leishmania infantum diversity in Portugal.

Around the Mediterranean basin Leishmania infantum is an important parasite causing canine leishmaniasis and visceral and cutaneous clinical forms in both immunocompetent and immunocompromised humans. Efficient monitoring and evaluation of epidemiology with discriminatory molecular markers are required. We investigated the genetic diversity of L. infantum in Portugal by polymerase chain amplification and restriction fragment length polymorphism analysis of kinetoplastid DNA, as molecular marker. We analysed 120 Portuguese isolates of L. infantum plus 16 other non-Portuguese isolates (as a reference group) from humans, dogs and sand flies. The Portuguese population showed a high degree of polymorphism with a total of 13 profiles identified. The predominant profile was A, which was only detected in the Portuguese samples. The kinetoplastid DNA PCR-RFLP assay described here was suitable for use directly with biological samples and the profiles obtained were stable during long-term growth in vitro and in laboratory animals.

Analysis of ribosomal DNA internal transcribed spacer sequences of the Leishmania donovani complex.

To understand phylogenetic relationships of species and strains within the Leishmania donovani complex, we have analyzed the ribosomal DNA internal transcribed spacer (ITS) sequences of 27 Leishmania infantum, 2 Leishmania chagasi, 18 L. donovani and 5 Leishmania archibaldi strains of different zymodemes and geographical origin. Eight ITS sequence types were found. All detected sequence variation within ITS1 and ITS2 was based on 12 polymorphic microsatellites. The L. infantum strains from the Mediterranean region, China and L. chagasi from the New World formed a phylogenetic group well separated from the second main group including all strains from East Africa and India. Within the latter group three distinct phylogenetic subgroups could be differentiated: (1) L. donovani (Sudan/Ethiopia, China) + L. archibaldi (Sudan), (2) L. donovani (Sudan/Ethiopia) + L. infantum (Sudan) + L. archibaldi (Sudan/Ethiopia), and (3) L. donovani (Kenya, India). These groups are not consistent with previous species definitions based on isoenzyme analyses, e.g. L. infantum is polyphyletic and L. archibaldi is not supported as a distinct species. Two groups of Indian strains could be differentiated, one of which has an identical sequence type to the strains from Kenya. Three main lineages of strains can thus be differentiated in East Africa: two quite distantly related groups of strains from Sudan/Ethiopia, and a third group including all strains from Kenya, which is more closely related to part of the Indian strains than to any of the Sudanese/Ethiopian groups. The ITS sequence analysis presented here supports the need for revision of the taxonomy of the L. donovani complex.

Localized cutaneous leishmaniasis due to Leishmania donovani and Leishmania tropica: preliminary findings of the study of 161 new cases from a new endemic focus in himachal pradesh, India.

Localized cutaneous leishmaniasis (LCL) in India is due mostly to Leishmania tropica. It is mainly endemic in the deserts of Rajasthan. Recently, Himachal Pradesh has been identified as a new endemic focus for the disease. In the last few years, the number of new cases has been increasing almost to epidemic proportions. This report presents the preliminary findings of clinico-epidemiologic and investigative results of 161 new localized cases of LCL seen between May 2001 and December 2003. The study populaton was composed of 80 males and 81 females between 10 months and 75 years of age. All were indigenous to the sub-alpine valley along the Satluj River in the mountainous region of the Kinnaur District (altitude = 700-2,900 meters). Most patients were seen from April to September and had 1-8 lesions (duration = 1-6 months) that involved mainly the face. Tissue smears were positive for amastigotes in 37% and histopathology showed non-caseating epitheloid cell granuloma in 77% of the cases. Analysis by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the ribosomal gene region of 10 biopsy specimens showed amplicons indistinguishable from L. donovani in eight cases and L. tropica in two cases. Leishmania was cultured on modified Nicole-Novy-McNeal (NNN) medium containing RPMI 1640 medium and heat-inactivated fetal bovine serum from 13 of 38 biopsy samples. Three of these isolated strains were identified as L. donovani while a fourth was L. tropica by PCR-RFLP of the ribosomal internal transcribed spacer region. One strain had a gp63 sequence identical to that of east African strains. Another strain had a unique gp63 sequence that has not been found in L. donovani complex strains. Sand flies trapped in the cattle sheds of a few patients were identified as Phlebotomus longiductus (Parrot 1928). Treatment with intralesional sodium stibogluconate was effective in all patients without any major side effects. One patient developed lupoid leishmaniasis that responded to higher dose of sodium stibogluconate. Though rarely reported as a cause of LCL, L. donovani seems to be the predominant pathogen in this new focus of cutaneous leishmaniasis. Phlebotomus longiductus is a possible vector, albeit based on circumstantial evidence.

Dirofilariasis by Dirofilaria repens: an imported case and a brief review.

Human infections caused by Dirofilaria repens, a cosmopolitan zoonotic parasitosis endemic in Southern and Eastern Europe and Asia still is an underdiagnosed infection due to parasite identification difficulties. Here, we report the first human case of subcutaneous dirofilariasis by D. repens diagnosed in Portugal. This was probably an imported case from India, as judged by epidemiological and clinical data. With this presentation we aim to alert clinicians for the emergence of vector-borne zoonoses associated with global warming and international travel. This case showed that differential diagnosis of D. repens in subcutaneous nodules is needed, in order to avoid further complications.