BackgroundVisceral leishmaniasis (VL), the most severe form of leishmaniasis, is endemic in Europe with Mediterranean countries reporting endemic status alongside a worrying northward spread. Serological diagnosis, including immunochromatographic test based on the recombinant antigen rK39 (rK39-ICT) and a direct agglutination test (DAT) based on the whole parasite antigen, have been validated in regions with high VL burden, such as eastern Africa and the Indian subcontinent. To date, no studies using a large set of patients have performed an assessment of both methods within Europe.Methodology/Principal findingsWe selected a range of clinical serum samples from patients with confirmed VL (including HIV co-infection), Chagas disease, malaria, other parasitic infections and negative samples (n = 743; years 2009–2015) to test the performance of rK39-ICT rapid test (Kalazar Detect Rapid Test; InBios International, Inc., USA) and DAT (ITM-DAT/VLG; Institute of Tropical Medicine Antwerp, Belgium). An in-house immunofluorescence antibody test (IFAT), was included for comparison. Estimated sensitivities for rK39-ICT and DAT in HIV-negative VL patients were 83.1% [75.1–91.2] and 84.2% [76.3–92.1], respectively. Sensitivity was reduced to 67.3% [52.7–82.0] for rK39 and increased to 91.3% [82.1–100.0] for DAT in HIV/VL co-infected patients. The in-house IFAT was more sensitive in HIV-negative VL patients, 84.2% [76.3–92.1] than in HIV/VL patients, 79.4% [73.3–96.2]. DAT gave 32 false positives in sera from HIV-negative VL suspects, compared to 0 and 2 for rK39 and IFAT, respectively, but correctly detected more HIV/VL patients (42/46) than rK39 (31/46) and IFAT (39/46).Conclusions/SignificanceThough rK39-ICT and DAT exhibited acceptable sensitivity and specificity a combination with other tests is required for highly sensitive diagnosis of VL cases in Spain. Important variation in the performance of the tests were seen in patients co-infected with HIV or with other parasitic infections. This study can help inform the choice of serological test to be used when screening or diagnosing VL in a European Mediterranean setting.

Leishmaniasis is endemic in southwest Europe. Recent data point to the spread and (re-)emergence of this disease in previously endemic and non-endemic European countries. A recent example is the urban community outbreak of cutaneous and visceral leishmaniasis in the southwest of Madrid autonomous community, Spain, which began on 1 July 2009. A total of 446 cases associated to this outbreak were reported up to 31 December 2012. We show molecular typing data for 73 Leishmania infantum isolates obtained from January 2008 to July 2012 from different areas of Madrid, including those affected by the outbreak. Seven different genotypes were identified by combining data from two targets: the ribosomal internal transcribed spacers (ITS)-1 and-2 and the haspb (k26) gene. The results contribute to a better understanding of the parasite population circulating in the region, and indicate that most of the outbreak-associated isolates (22/31) were infected by parasites with the same combined genotype. Additional data from 82 L. infantum isolates typed as either MON-1 or MON-24 by isoenzyme analysis indicate that far from concluding that the outbreak was caused by a 'new' emerging genotype, further molecular typing-based surveillance studies are required to better understand the epidemiology of leishmaniasis in the region.

Parasites of medical importance, such as Leishmania and Trypanosoma, are characterized by the presence of thousands of circular DNA molecules forming a structure known as kinetoplast, within the mitochondria. The maxicircles, which are equivalent to the mitochondrial genome in other eukaryotes, have been proposed as a promising phylogenetic marker. Using whole-DNA sequencing data, it is also possible to assemble maxicircle sequences as shown here and in previous works. In this study, based on data available in public databases and using a bioinformatics workflow previously reported by our group, we assembled the complete coding region of the maxicircles for 26 prototypical strains of trypanosomatid species. Phylogenetic analysis based on this dataset resulted in a robust tree showing an accurate taxonomy of kinetoplastids, which was also able to discern between closely related Leishmania species that are usually difficult to discriminate by classical methodologies. In addition, we provide a dataset of the maxicircle sequences of 60 Leishmania infantum field isolates from America, Western Europe, North Africa, and Eastern Europe. In agreement with previous studies, our data indicate that L. infantum parasites from Brazil are highly homogeneous and closely related to European strains, which were transferred there during the discovery of America. However, this study showed the existence of different L. infantum populations/clades within the Mediterranean region. A maxicircle signature for each clade has been established. Interestingly, two L. infantum clades were found coexisting in the same region of Spain, one similar to the American strains, represented by the Spanish JPCM5 reference strain, and the other, named “non-JPC like”, may be related to an important leishmaniasis outbreak that occurred in Madrid a few years ago. In conclusion, the maxicircle sequence emerges as a robust molecular marker for phylogenetic analysis and species typing within the kinetoplastids, which also has the potential to discriminate intraspecific variability.