The Mediterranean basin is an endemic region for canine leishmaniosis (CanL), where it represents a major veterinary problem and raises human health concerns. However, the distribution of the disease is heterogeneous and not all countries and locations have been equally studied and characterized. This work describes the situation of CanL in Girona province (Catalonia, Spain), for which no data has been previously reported, and presents a relevant study to exemplify other areas with similar characteristics across the region. Four cross-sectional seroprevalence surveys were performed from 2012 to 2016 throughout the province, including 36 sampling stations in 26 localities and a total of 593 dogs. For each animal, individual and location variables were also collected. Additionally, each dog owner answered a questionnaire about their knowledge of CanL and preventive methods used. Blood samples were analysed by an in-house ELISA and a mixed logistic regression model was used to assess the relationship between pre-determined variables and dog seropositivity. A Spearman's correlation was used to assess the association between dog owners' perceived risk of CanL and Leishmania infantum seropositivity in dogs at a given location. The overall true seroprevalence estimated for Girona province was 19.5% (95%CI: 15.5-23.5), of which only 6.8% (10/146) were considered symptomatic. Age of the dog [OR = 1.21 (95%CI: 1.11-1.31); p < 0.001] and altitude [OR = 0.02 (95%CI: 0.001-0.19); p = 0.001] were identified as risk factors for the infection. The results obtained in this study are expected to aid in the implementation of directed control programmes in CanL endemic areas throughout Europe, as well as to provide suitable data for the design of better risk assessment maps of the disease.

Leishmaniasis is a transmissible disease caused by Leishmania protozoa. Spain is endemic for both visceral and cutaneous leishmaniasis, the autochthonous aetiological agent being Leishmania infantum. Around the world, the L. donovani complex is associated with visceral symptoms, while any species of the Leishmania or Viannia subgenera affecting human can produce tegumentary forms. In a context of growing numbers of imported cases, associated with globalisation, the aim of this study was to analyse the aetiological evolution of human tegumentary leishmaniasis in a region of Spain (Catalonia). Fifty‐six Leishmania strains, isolated from 1981 to 2018, were analysed using MLEE, gene sequencing (hsp70, rpoIILS, fh and ITS2) and MALDI‐TOF. The utility of these different analytical methods was compared. The results showed an increase in leishmaniasis over the two last decades, particularly imported cases, which represented 39% of all cases studied. Leishmania infantum, L. major, L. tropica, L. braziliensis, L. guyanensis and L. panamensis were identified. The combination of molecular and enzymatic methods allowed the identification of 29 different strain types (A to AC). Strain diversity was higher in L. (Viannia), whilst the different L. major types were relatable with geo‐temporal data. Among the autochthonous cases, type C prevailed throughout the studied period (39%). Minor types generally appeared within a short time interval. While all the techniques provided identical identification at the species complex level, MALDI‐TOF and rpoIILS or fh sequencing would be the most suitable identification tools for clinical practice, and the tandem hsp70‐ITS2 could substitute MLEE in the epidemiological field.

Chagas disease (CD), caused by Trypanosoma cruzi and endemic in Latin America, has become an emergent health problem in non-endemic countries due to human migration. The United States (US) is the non-Latin American country with the highest CD burden and cannot be considered as non-endemic, since triatomine vectors and reservoir animals have been found. Populations of T. cruzi are divided into genetic subdivisions, which are known as discrete typing units (DTUs): TcI to TcVI and TcBat. Autochthonous human T. cruzi infection in the US is sporadic, but it may change due to environmental factors affecting the geographic distribution of triatomines. We aimed to perform a literature review of the genetic diversity of T. cruzi in triatomine vectors and mammalian hosts, including human cases, in the US. The 34 analyzed studies revealed the presence of T. cruzi in 18 states, which was mainly concentrated in Texas, Louisiana and New Mexico. TcI and TcIV were the principal DTUs identified, being TcI the most genotyped (42.4%; 917/2164). This study represents a first attempt to compile the molecular epidemiology of T. cruzi in the US, which is fundamental for predicting the progression of the infection in the country and could be of great help in its future management.

Leishmaniasis is a parasitic disease with clinical presentations that vary from asymptomatic infection to cutaneous, mucocutaneous, or visceral disease. Global change, with migratory movements and travels, among others, has had an impact on the presentation of the clinical forms of leishmaniasis in a given area, hindering its diagnosis. The traditional parasitological techniques have limited sensitivity, and currently, there is no reference or gold-standard molecular diagnostic method. The aim of this study was to evaluate the effectivity of the VIASURE Leishmania Real-Time PCR Detection Kit prototype (CerTest Biotec, Zaragoza, Spain) for the diagnosis of autochthonous and imported leishmaniasis in comparison with two other commercialized molecular kits: STAT-NAT Leishmania spp. (Sentinel, Milano, Italy) and Leishmania spp. Real-TM PCR Kit (Sacace Biotechnologies, Como, Italy). Four species of Leishmania, L. infantum, L. major, L. braziliensis, and L. panamensis, were targeted to assess analytical sensitivity, whereas diagnostic sensitivity was evaluated by studying a panel of 49 DNA samples from patients with suspected or confirmed Leishmania infection. The prototype could detect all the New and Old World species studied and achieved a limit of detection of 510⁻⁵ng DNA/μL in all species. Also, it allowed the diagnosis of autochthonous and imported cases of cutaneous and visceral leishmaniasis (VL). Diagnostic sensitivity was 81.8 for the prototype and 100 for the Sacace kit (27 and 33 positive samples detected, respectively). The STAT-NAT kit failed to detect Vianna species. The VIASURE Leishmania Real-Time PCR Detection Kit prototype was found to have good analytical and diagnostic sensitivity. Using a simple protocol and ready-to-use reagents, results are obtained quickly and are easy to interpret. The evaluation results indicate that the test is a promising candidate for routine diagnosis of cutaneous leishmaniasis and VL in endemic countries, but more studies are necessary to address its sensitivity and specificity.

Leishmaniasis is caused by protozoans of the Leishmania genus, which includes more than 20 species capable of infecting humans worldwide. In the Americas, the most widespread specie is L. braziliensis, present in 18 countries including Bolivia. The taxonomic position of the L. braziliensis complex has been a subject of controversy, complicated further by the recent identification of a particular subpopulation named L. braziliensis atypical or outlier. The aim of this study was to carry out a systematic analysis of the L. braziliensis complex in Bolivia and to describe the associated clinical characteristics. Forty‐one strains were analyzed by sequencing an amplified 1245 bp fragment of the hsp70 gene, which allowed its identification as: 24 (59%) L. braziliensis, 16 (39%) L. braziliensis outlier, and one (2%) L. peruviana. In a dendrogram constructed, L. braziliensis and L. peruviana are grouped in the same cluster, whilst L. braziliensis outlier appears in a separate branch. Sequence alignment allowed the identification of five non‐polymorphic nucleotide positions (288, 297, 642, 993, and 1213) that discriminate L. braziliensis and L. peruviana from L. braziliensis outlier. Moreover, nucleotide positions 51 and 561 enable L. peruviana to be discriminated from the other two taxa. A greater diversity was observed in L. braziliensis outlier than in L. braziliensis‐L. peruviana. The 41 strains came from 32 patients with tegumentary leishmaniasis, among which 22 patients (69%) presented cutaneous lesions (11 caused by L. braziliensis and 11 by L. braziliensis outlier) and 10 patients (31%) mucocutaneous lesions (eight caused by L. braziliensis, one by L. braziliensis outlier, and one by L. peruviana). Nine patients (28%) simultaneously provided two isolates, each from a separate lesion, and in each case the same genotype was identified in both. Treatment failure was observed in six patients infected with L. braziliensis and one patient with L. peruviana.

In 2009, a large outbreak of leishmaniasis, associated with environmental changes, was declared near Madrid (Spain), in which Phlebotomus perniciosus was the vector, whereas the main reservoirs were hares and rabbits. Analysis of isolates from humans, vectors and leporids from the focus identified the Leishmania infantum ITS-Lombardi genotype. However, multilocus enzyme electrophoresis (MLEE), the reference technique for Leishmania typing, and sequencing of the hsp70 gene, a commonly used marker, were not performed. In the present study, 19 isolates from P. perniciosus (n = 11), hares (n = 5) and rabbits (n = 3) from the outbreak area, all characterized as ITS-Lombardi in previous studies, were analysed by MLEE and hsp70 sequencing. The hsp70 results confirmed that all the analysed strains are L. infantum. However, by MLEE, 4 different zymodemes of L. infantum were identified based on variable mobilities of the NP1 enzyme: MON-34 (NP1 100 , n = 11), MON-80 (NP1 130 , n = 6), MON-24 (NP1 140 , n = 1) and MON-331 (NP1 150 , n = 1). The relative frequency of these zymodemes does not correspond to their usual occurrence in Spain. Moreover, MON-34 and MON-80 were found in P. perniciosus, hares and rabbits for the first time. These findings continue to provide insights into the outbreak and call for further studies with a higher number of strains.