Untersuchung der in-vitro Effektivität von verschiedenen Photosensibilisatoren bei der photodynamischen Inaktivierung von Leishmania major

Unter Leishmaniose versteht man eine Gruppe von Erkrankungen, die durch humanpathogene Arten des Parasiten Leishmania hervorgerufen werden. Es werden drei Hauptgruppen unterschieden: Die viszerale, die kutane und die mukokutane Leishmaniose. Die Erkrankung wird zu den „Neglected Tropical Diseases“ gezählt, da es Schätzungen nach 1 - 2 Millionen Neuerkrankungen und 40.000 Todesfälle pro Jahr gibt. Über 350 Millionen Menschen leben in Risikogebieten, in denen die Erkrankungsfälle momentan noch zunehmen. Dabei ist die kutane Leishmaniose hauptsächlich in Südamerika, dem Mittelmeerraum (Nordafrika und Südeuropa), dem Mittleren Osten und in Zentralasien verbreitet. Die viszerale Leishmaniose kommt vor allem im indischen Subkontinent sowie in Ostafrika und Brasilien vor. Die Therapie der Leishmaniose ist komplex und es gibt momentan keine optimale Therapiestrategie für die Behandlung dieser infektiösen Erkrankung. Es gibt nur wenig wirksame Medikamente, die viele Nebenwirkungen haben und je nach Region und Spezies unterschiedlich effektiv sind. Außerdem können bei allen Formen der Leishmaniose Rückfälle nach Beginn der Behandlung auftreten. Obwohl einige Versuche unternommen wurden, gibt es zudem im Moment keinen wirksamen Impfstoff gegen die Leishmaniose. Es besteht also die Notwendigkeit zur Verbesserung der Leishmaniose-Therapie sowie zur Entwicklung neuer Therapien. Für die Behandlung der kutanen Leishmaniose beispielsweise könnte die photodynamische Inaktivierung (PDI) eine vielversprechende neue nichtmedikamentöse, lokale Therapieoption darstellen. Das Prinzip der PDI ist, dass ein nicht toxischer Farbstoff, der als Photosensibilisator (PS) bezeichnet wird, in Anwesenheit von Sauerstoff, Licht im sichtbaren Wellenlängenbereich absorbiert und dadurch angeregt wird. Im angeregten Zustand interagiert der PS mit umliegenden Molekülen und es kommt zur Bildung von reaktiven Sauerstoffspezies und hochreaktivem Singulettsauerstoff, welche wiederum die verschiedensten Biomoleküle schädigen können und so zur Apoptose von Zellen aber auch zur Abtötung von pathologischen Mikroorganismen führen. Die bereits vielversprechende therapeutische Anwendung des Prinzips in einigen Bereichen der Medizin sowie die durch Studien belegte Wirksamkeit der PDI in vielen neuen Anwendungsgebieten, machen die PDI zu einem Verfahren mit großem Potenzial. In diesem Zusammenhang wurde aufgrund der in klinischen Studien bereits effektiv durchgeführten Behandlung der kutanen Leishmaniose mit δ-Aminolävulinsäure (ALA)-PDI und Methyl-Aminolävulinat (MAL)-PDI, in dieser Arbeit untersucht, ob auch andere PS als Porphyrin-Derivate zur Therapie der Erkrankung geeignet wären. Dabei wurde die photodynamische Effektivität der exogenen PS TMPyP, SAPyR und FLASH-07a bei der Inaktivierung von L. major Promastigoten und L. major Amastigoten in-vitro getestet und miteinander verglichen. Zusätzlich wurde die Zytotoxizität der PDI auf die Wirtszellen der Leishmanien in-vitro evaluiert und mit der photodynamischen Wirkung der PS gegenüber dem Parasiten L. major verglichen. Dabei wurde zunächst die nicht infizierte J774A.1-Makrophagenzelllinie und in nachfolgenden Experimenten mit L. major infizierte Peritoneal-Exsudat-Makrophagen, die aus C57BL/6 Mäusen isoliert wurden, untersucht. Insgesamt zeigten die Ergebnisse dieser Arbeit, dass die drei verwendeten Farbstoffe in Kombination mit sichtbarem Licht zu einer effizienten Inaktivierung von L. major Promastigoten führen. Dabei nahm die Effektivität der Inaktivierung in folgender Reihenfolge zu: FLASH-07a < TMPyP < SAPyR. Die PDI der intrazellulär, amastigoten Form von L. major mit SAPyR war ebenfalls effektiv. Gleichzeitig hatte die Photosensibilisierung aber auch auf Wirtszellen, wie die untersuchte J774A.1-Makrophagenzelllinie oder Peritoneal-Exsudat-Makrophagen, eine toxische Wirkung. Daher ist die Frage, ob ein „Therapeutisches Fenster“, indem die Vitalität der Leishmanien deutlich abnimmt, die Wirtszellen aber unbeschadet bleiben wichtig ist, oder ob eine potentiell lokal toxische Wirkung auf die Immunzellen nur eine geringe Auswirkung auf den menschlichen Organismus hat, von zentraler Bedeutung, um abschließend zu klären, ob die hier verwendeten PS sicher beim Menschen angewendet werden könnten. Zudem ist die weitere Klärung des genauen Wirkmechanismus der hier verwendeten PS bei der PDI der kutanen Leishmaniose wichtig. Ferner sollte die PDI optimiert werden, um unerwünschte Nebenwirkungen auf nichtinfizierte Zellen und Gewebe zu minimieren und gleichzeitig die Effektivität zu erhöhen. Die Ergebnisse der Experimente dieser Arbeit können somit als Grundlage für weitere in-vivo Tierexperimente und unter der Vorraussetzung, dass die PS als sicher für die Anwendung beim Menschen angesehen werden auch als Grundlage für klinische Studien angesehen werden. Es sollten unbedingt auch andere neue phototoxische Substanzen getestet werden, um die PDI als vielversprechenden neuen Ansatz in der Therapie der kutanen Leishmaniose weiter zu etablieren

Μελέτη του ρόλου της κινάσης LinDYRK1 του οαρασίτου Leishmania infantum

Ο όρος λεϊσμανίαση μια ασθένεια που οφείλεται σε ενδοκυτταρικά πρωτoζωϊκά παράσιτα του γένους Leishmania, περικλείει μία ομάδα νοσημάτων όπως τη δερματική, τη βλεννογονοδερματική και τη σπλαγχνική, η τελευταία είναι και η πλέον σοβαρή αφού προκαλεί βέβαιο θάνατο χωρίς τη χορήγηση θεραπείας. Τα παράσιτα Leishmania μεταδίδονται στο τελικό ξενιστή με το τσίμπημα μολυσμένης σκνίπας του γένους Phlebotomus or Lutzomyia. Ο βιολογικός κύκλος του παρασίτου Leishmania απαιτεί την παρουσία δύο ξενιστών. Οι φλεβοτόμοι-διαβιβαστές φιλοξενούν την εξωκυτταρική μορφή εντερικών μαστιγωτών (flagellate), τη λεγόμενη προμαστιγωτή, ενώ τα θηλαστικά φιλοξενούν την ενδοκυτταρική μορφή που ονομάζεται αμαστιγωτή, η οποία ζει και πολλαπλασιάζεται στο όξινο ενδολυσσόσωμα των μακροφάγων. Η απόκριση στα ερεθίσματα της διαφοροποίησης της θερμοκρασία (26--&gt;37οC), του pH(7,4--&gt;5,5) στο ανοσοποιητικό σύστημα του ξενιστή είναι καθοριστικά για την εξέλιξη της νόσου. Η λεϊσμανίαση, αν και εκτιμάται ότι προκαλεί το ένατο μεγαλύτερο φορτίο νόσου όλων των λοιμωδών νοσημάτων, η επίπτωσή της στη Δημόσια Υγεία υποεκτιμάται σε μεγάλο βαθμό, λόγω της πολύπλοκης επιδημιολογίας/οικολογίας της, της έλλειψης αποτελεσματικών μέτρων αντιμετώπισης και της ανεπαρκούς επιδημιολογικής επιτήρησής της. Παράλληλα, τα λίγα φάρμακα που χορηγούνται σήμερα παρουσιάζουν σημαντικούς περιορισμούς, όπως ανάπτυξη αντοχής, μειωμένη αποτελεσματικότητα, υψηλή τοξικότητα και μεγάλο κόστος. Οι περιορισμοί αυτοί έχουν καταστήσει επιτακτική ανάγκη την ανακάλυψη νέων και πιο αποτελεσματικών θεραπειών. Για αυτό το λόγο, γίνονται προσπάθειες για την ταυτοποίηση μορίων-στόχων. Mια πολύ καλή πηγή φαρμακευτικών στόχων είναι οι πρωτεϊνικές κινάσες αφού ρυθμίζουν σχεδόν όλα τα μονοπάτια του κυττάρου. Οι πρωτεϊνικές κινάσες DYRK είναι κινάσες διπλής ειδικότητας και αποτελούν κεντρικούς ρυθμιστές του κυτταρικού κύκλου και της κυτταρικής διαφοροποίησης. Το παράσιτο Leishmania κωδικοποιεί 8 DYRK κινάσες και ανάμεσά τους τη DYRK1. Στην συγκεκριμένη εργασία διερευνήθηκε η επίδραση της πλήρης εξάλειψης ή της υπερέκφρασης της πρωτεϊνικής κινάσης DYRK1 του παρασίτου Leishmania infantum (Lin) και πως επηρεάζουν την ανάπτυξη, τον κυτταρικό κύκλο και τη μολυσματικότητα του παρασίτου . Επιπλέον μελετήθηκε η συμπεριφορά των διαγονιδιακών παρασίτων σε στρεσσογόνες συνθήκες που συναντά το παράσιτο μέσα στο τελικό ξενιστή όπως η μεταβολή της θερμοκρασίας (26--&gt;37οC) και του όξινου pH (7,4--&gt;5,5). Τα αποτελέσματα μας δείχνουν ότι η υπό μελέτη κινάση είναι ρυθμιστής του κυτταρικού κύκλου καθώς η υπερέκφραση του επιμηκύνει τη G1 φάση. Επίσης δείξαμε ότι διαδραματίζει σημαντικό ρόλο στην επιβίωση του παρασίτου σε στρεσσογόνες συνθήκες καθώς και σε συνθήκες διαφοροποίησης, αφού η εξάλειψη της προκαλεί αυξημένη ευαισθησία και κυτταρικό θάνατο, στη στατική φάση (μολυσματική) της προμαστιγωτής μορφής και σε θερμικό σοκ (26--&gt;37οC). Επομένως δείξαμε ότι η LinDYRK1 είναι ένας αρνητικός ρυθμιστής του κυτταρικού κύκλου που βοηθά το παράσιτο να διαφοροποιηθεί και να επιβιώσει στο τελικό ξενιστή. Συμπερασματικά η LinDYRK1 θεωρείται ένας πιθανός φαρμακευτικός στόχος για χημειοπροφύλαξη. Mελλοντικές μελέτες σκοπεύουν να διερευνήσουν το ρόλο της κινάσης ύστερα από τη διαφοροποίηση του παρασίτου σε αμαστιγωτή μορφή και αναμένεται να μας διαφωτίσουν αν μπορεί να ταυτοποιηθεί ως φαρμακευτικός στόχος για τη χημειοθεραπεία της λεϊσμανίασης.Τhe term leishmaniasis, caused by protozoan parasites of the genus Leishmania is an umbrella term of diseases with multiple manifestations including cutaneous and mucocutaneous forms, and visceral, the latter of which is lethal if left untreated. Leishmaniasis is spread by the bite of sandflies of the genera Phlebotomus or Lutzomyia. Leishmania has a digenic life-cycle and undergoes cellular differentiation from the sandfly promastigote form into the amastigote form, which survives and and replicates in the acidic endolysosomal environment of mammalian macrophages. Τhe adaptation of the parasite to the alternating environmental stimuli (temperature upshift (26→37oC), pH change and the immune system of the host is critical for the completion of its life cycle and the outcome of the disease. Leishmaniasis, although it is estimated to cause the ninth largest disease burden of all infectious diseases, with two million new cases occurring worldwide each year, the impact on public health is underestimated largely due to its complex epidemiology/ecology, the lack of effective countermeasures and poor epidemiological surveillance. The control of leishmaniasis relies mainly on chemotherapy and the armory of available treatments today is very small with only a handful of antileishmanial drugs in the market. Moreover current chemotherapy has important limitations, including increase of drug resistance, toxicity and high cost. To this end, there is an urgent need to discover new treatments for combating leishmaniasis. Thus many research efforts today, focus on the identification of molecular drug targets. Eukaryotic protein kinases are considered important drug targets for the treatment of leishmaniasis, as they are central mediators of almost all cellular processes in the lifecycle of the parasite. Dual-specificity tyrosine-regulated kinases (DYRKs) comprise a family of protein kinases that are central regulators of cell cycle and differentiation. Interestingly, the parasite Leishmania encodes for 8 members of the DYRK family. Herein, we examined how deletion and over-expression of Leishmania infantum (Lin) DYRK1 affects the growth, cell cycle and infectivity of Leishmania infantum. We also studied the phenotype of LinDYRK1 transgenic parasites and null mutants under the differentiation stress conditions,which the parasite encounters in the host environment, namely increased temperature (26--&gt;37οC) and acidic pH (7,4--&gt;5,5). Our results showed that the kinase LinDYRK1 delays growth, as it prolongs the G1 phase of the cell-cycle. Moreover we showed that LinDYRK1 is essential for the establishment of stationary phase promastigotes (the infective stage of the parasite) and has a protective role under a temperature upshift (26--&gt;37οC), the natural condition the parasite encounters in the host essential for differentiation. Thus, LinDYRK1 is negative regulator of cell cycle but also a prosurvival kinase required for promastigote to amastigote differentiation and for parasite infectivity. Overall from this study, we have showed is a validated drug target for prophylactic therapy. Future studies investigating the role of LinDYRK1 after the promastigote to amastigote differentiation are required to enlighten us on the role of this kinase as a chemotherapeutic drug target for the treatment of leishmaniasis

Seasonal dynamics of canine antibody response to Phlebotomus perniciosus saliva in an endemic area of Leishmania infantum

Background: Canine leishmaniosis (CanL) is an important zoonotic parasitic disease, endemic in the Mediterranean basin. In this region, transmission of Leishmania infantum, the etiological agent of CanL, is through the bite of phlebotomine sand flies. Therefore, monitoring host- vector contact represents an important epidemiological tool, and could be used to assess the effectiveness of vector-control programmes in endemic areas. Previous studies have shown that canine antibodies against the saliva of phlebotomine sand flies are specific markers of exposure to Leishmania vectors. However, this method needs to be further validated in natural heterogeneous dog populations living in CanL endemic areas. Methods: In this study, 176 dogs living in 12 different locations of an L. infantum endemic area in north-east Spain were followed for 14 months. Blood samples were taken at 5 pre-determined time points (February, August and October 2016; January and April 2017) to assess the canine humoral immune response to whole salivary gland homogenate (SGH) and to the single salivary 43 kDa yellow-related recombinant protein (rSP03B) of Phlebotomus perniciosus, a proven vector of L. infantum naturally present in this region. Simultaneously, in all dogs, L. infantum infection status was assessed by serology. The relationship between anti-SGH and anti-rSP03B antibodies with the sampling month, L. infantum infection and the location was tested by fitting multilevel linear regression models. Results: The dynamics of canine anti-saliva IgG for both SGH and rSP03B followed the expected trends of P. perniciosus activity in the region. Statistically significant associations were detected for both salivary antigens between vector exposure and sampling month or dog seropositivity to L. infantum. The correlation between canine antibodies against SGH and rSP03B was moderate. Conclusions: Our results confirm the frequent presence of CanL vectors in the study area in Spain and support the applicability of SGH- and rSP03B-based ELISA tests to study canine exposure to P. perniciosus in L. infantum endemic areas. Keywords: Canine leishmaniosis, Phlebotomus perniciosus, Saliva proteins, Markers of exposure, Longitudinal study, Northeast Spai

Efectividad in vitro de tres fármacos aprobados y su interacción sinérgica contra Leishmania infantum

Introduction: Leishmaniasis remains one of the neglected tropical diseases. Repurposing existing drugs has proven to be successful for treating neglected tropical diseases while combination therapy is a strategic alternative for the treatment of infectious diseases.Auranofin, lopinavir/ritonavir, and sorafenib are FDA approved drugs used in the treatment of diverse diseases by acting on different essential biological enzymes.Objective: To evaluate the effects of monotherapy and combined therapies with the three drugs against Leishmania infantum.Materials and methods: We compared the leishmanicidal effects of the three drugs on promastigotes in vitro as regards the parasite count, the drug concentration providing a half-maximal response, and the ultrastructural changes of the parasite. We determined the fractional inhibitory concentration index of combined drugs in two ways, as well as the activity of the three drugs together to establish their synergetic effect.Results: The monotherapy with the three drugs was effective with auranofin showing the best leishmanicidal effect (EC50=1.5 μM), whereas sorafinib reduced parasite growth at EC50=2.5 μM. The scanning electron microscopy of promastigotes from all treated media showed distortion in the shape with loss of flagella and bleb formation. Acidocalcinosis was evident by transmission electron microscopy with all treatments suggesting apoptosis. Treatment with lopinavir/ritonavir showed signs of autophagy. The two-way combination of the drugs led to additive interactions while the combination of the three drugs showed synergistic action.Conclusion: Each drug when used as monotherapy against Leishmania spp. was effective, but the combination therapy was more effective than the individual drugs due to the additive or synergistic effects.Introducción. La leishmaniasis sigue siendo una de las enfermedades tropicales desatendidas. La reutilización de medicamentos existentes ha demostrado ser exitosa para tratar enfermedades tropicales desatendidas y la terapia combinada es una alternativa estratégica para el tratamiento de enfermedades infecciosas. Auranofin, lopinavir/ritonavir y sorafenib son medicamentos aprobados por la Food and Drug Administration (FDA) de Estados Unidos utilizados en el tratamiento de diversas enfermedades, pues actúan sobre diferentes enzimas biológicas esenciales.Objetivo. Evaluar los efectos terapéuticos de la monoterapia y de los tres fármacos combinados contra Leishmania infantum.Materiales y métodos. Los efectos leishmanicidas de los tres fármacos sobre los promastigotes se compararon in vitro en cuanto al recuento de parásitos, la concentración del fármaco que proporcionara una respuesta semimáxima y los cambios ultraestructurales del parásito. Se calculó el índice de concentración inhibitoria de fracciones de fármacos combinados de dos maneras y la actividad de los tres fármacos juntos para determinar el efecto sinérgico.Resultados. La monoterapia con los tres medicamentos fue efectiva, pero la auranofina tuvo el mejor efecto antileishmanicida con un CE50 de 1,5 μM, en tanto que el sorafinib redujo el crecimiento del parásito con un CE50 de 2,5 μM. La microscopía electrónica de barrido de promastigotes de todos los medios tratados mostró una distorsión en la forma, con pérdida de flagelos y formación de ampollas. La acidocalcinosis fue evidente por microscopía electrónica de transmisión con todos los tratamientos, lo que sugiere apoptosis. El tratamiento con lopinavir/ritonavir mostró signos de autofagia. La combinación de dos medicamentos condujo a interacciones aditivas, mientras que la combinación de las tres drogas produjo una acción sinérgica.Conclusión. Los tres medicamentos usados como monoterapia contra Leishmania spp. fueron efectivos, pero el tratamiento combinado lo fue en mayor medida debido a los efectos aditivos o sinérgicos

Antigen-Specific T Cells and Cytokines Detection as Useful Tool for Understanding Immunity against Zoonotic Infections

Zoonoses include a broad range of diseases, that are becoming of great interest, due to the climate changing, that cause the adaptation of vectors to new niches and environments. Host immune responses play a crucial role in determining the outcome of infections, as documented by expansion of antigen-specific T cells during several zoonotic infections. Thus, understanding of the contribution of antigen-specific T-cell subsets in the host immune response is a powerful tool to evaluate the different immunological mechanisms involved in zoonotic infections and for the development of effective vaccines. In this paper we discuss the role of T cells in some eukaryotic and prokaryotic infectious models

The Effects Of Rurality And Cost Of Transportation On Time Of Evolution And Disease Complexity At Diagnosis For Cutaneous Leishmaniasis In Colombia

Patients in Colombia affected by cutaneous leishmaniasis (CL) face significant geopolitical and socioeconomic barriers to accessing diagnosis and treatment. This study aimed to understand how the geosocial characteristics of a patient’s area of residence affect time to medical consultation and disease status at consultation. An observational cross-sectional study was performed of individuals with CL who consulted at a local clinical referral center (CIDEIM) in Cali and Tumaco, Colombia. Descriptive, bivariate, and multivariable simple and ordinal logistic regression analyses were done to examine how area of residence (urban or rural) and cost of transportation to the nearest urban center may influence complexity and time of evolution of the disease at first medical visit. Two separate disease complexity classifications – cplexA and cplexB - were created based on factors such as mucosal involvement, lymphatic dissemination, and varying number and size of lesions; a composite score of both definitions was also constructed. CL Patients who had to pay greater than $60,000 COP for transportation were more likely to have a complex disease state at consultation based on the cplexA criteria (aOR = 2.11 [95% CI: 1.11 – 4.02]) and had higher odds of presenting with more complex disease (ordinal logistic regression; aOR = 1.78 [95% CI: 1.09 – 2.92]). A protective association between rurality and the time of evolution for the first lesion was also seen, showing that patients who lived in rural regions were less likely to have longer wait times before consulting at CIDEIM when compared to those who lived in urban centers (aOR = 0.66 [95% CI: 0.45 – 0.97]). High cost of transportation to nearby urban centers remains a significant barrier to equitable access of medical services for CL patients living in rural Colombia. Additionally, patients with CL in living non-endemic areas suffer extended time of evolution of the disease before consulting at a local referral center, signifying a need for heighted clinical suspicion and services in urban areas

Genetic tools discriminate strains of leishmania infantum isolated from humans and dogs in Sicily, Italy

Background Leishmaniasis is one of the most important vector-borne diseases and it represents a seri-ous world health problem affecting millions of people. High levels of Leishmania infections, affecting both humans and animals, are recognized among Italian regions. Among these, Sicily has one of the highest prevalence of Leishmania infection. Methodology/Principal Findings Seventy-eight Leishmania strains isolated from human and animal samples across Sicily, were analyzed for the polymorphic k26-gene and genotypes were assigned according to the size of the PCR products. A multilocus microsatellite typing (MLMT) approach based on the analysis of 11 independent loci was used to investigate populations structure and genetic diversity of the isolated strains. Six L. infantum reference strains were included in the analysis for comparison. Bayesian clustering analysis of microsatellite data showed that all the isolated strains clustered in two genetically distinct populations, corresponding to human and canine isolates respectively. A further subdivision was observed between the two main groups, giving a good correlation between human strains and their geographic origin, conversely canine population showed a great genetic variability diffused in the territory. Conclusions/Significance Among the 78 Leishmania isolates, K26 analysis detected 71 samples (91%) as MON-1 zymodeme, confirming it as the predominant strain in Mediterranean area and 7 human samples (9%) as non-MON-1. MLMT gives important insights into the epidemiology of leish-maniases and allows characterization of different strains to a higher resolution than possible with zymodeme typing. Two main populations presented a strong correlation respect to the different hosts, exhibiting a co-circulation of two distinct populations of L. infantum. The population found in infected humans exhibited a correlation with geographic origin. These clusters could represent a geographically restricted population of strains with the same or related genotypes. This study can contribute to an understanding of Leishmania epidemiol-ogy, including the spread of reservoirs and sand fly vectors in the different foci of infection, characterizing parasites within the different hosts

Clinical aspects of visceral leishmaniasis caused by L. infantum in adults. Ten years of experience of the largest outbreak in Europe: what have we learned?

BACKGROUND: An outbreak of leishmaniasis caused by Leishmania infantum was declared in the southwest of the Madrid region (Spain) in June 2009. This provided a unique opportunity to compare the management of visceral leishmaniasis (VL) in immunocompetent adults (IC-VL), patients with HIV (HIV-VL) and patients receiving immunosuppressants (IS-VL). METHODS: A cohort of adults with VL, all admitted to the Hospital Universitario de Fuenlabrada between June 2009 and June 2018, were monitored in this observational study, recording their personal, epidemiological, analytical, diagnostic, treatment and outcome variables. RESULTS: The study population was made up of 111 patients with VL (10% HIV-VL, 14% IS-VL, 76% IC-VL). Seventy-one percent of the patients were male; the mean age was 45 years (55 years for the IS-VL patients, P = 0.017). Fifty-four percent of the IC-VL patients were of sub-Saharan origin (P = 0.001). Fever was experienced by 98% of the IC-VL patients vs 73% of the LV-HIV patients (P = 0.003). Plasma ferritin was > 1000 ng/ml in 77% of the IC-VL patients vs 17% of the LV-HIV patients (P = 0.007). Forty-two percent of patients fulfilled the criteria for haemophagocytic lymphohistiocytosis. RDT (rK39-ICT) serological analysis returned sensitivity and specificity values of 45% and 99%, respectively, and ELISA/iIFAT returned 96% and 89%, respectively, with no differences in this respect between patient groups. Fourteen (13.0%) patients with VL experienced treatment failure, eight of whom were in the IC-VL group. Treatment with < 21 mg/kg (total) liposomal amphotericin B (LAB) was associated with treatment failure in the IC-VL patients [P = 0.002 (OR: 14.7; 95% CI: 2.6-83.3)]. CONCLUSIONS: IS-VL was more common than HIV-VL; the lack of experience in dealing with IS-VL is a challenge that needs to be met. The clinical features of the patients in all groups were similar, although the HIV-VL patients experienced less fever and had lower plasma ferritin concentrations. RDT (rK39-ICT) analysis returned a good specificity value but a much poorer sensitivity value than reported in other scenarios. The patients with HIV-VL, IS-VL and IC-VL returned similar serological results. Current guidelines for treatment seem appropriate, but the doses of LAB required to treat patients with HIV-VL and IS-VL are poorly defined.This work received financial support from the Red de Investigación Cooperativa en Enfermedades Tropicales (RICET) (RD12/0018/0008; RD16/0027/0017) (ISCIII) and the FEDER.S

Assessment of Vaccine-Induced Immunity Against Canine Visceral Leishmaniasis

Canine visceral leishmaniasis is an increasingly important public health problem. Dogs infected by Leishmania infantum are the main domestic reservoir of the parasite and play a key role in its transmission to humans. Recent findings have helped in the development of novel diagnostic methods, and of control measures such as vaccines, some of which are already commercially available. However, quantitative procedures should be followed to confirm whether these vaccines elicit a cell-mediated immune response. The present work describes the need for this evaluation, and the techniques available for confirming this type of immune response.The author thank the Instituto de Salud Carlos III which provided funding via the Red de Enfermedades Tropicales, Subprograma RETICS del Plan EstataldeI+D+I2013-2016, which is co-funded by FEDER Una manera de hacer Europa funds, projects RD16CIII/0003/0002.S

Leishmania infantum

Las leishmaniasis pertenecen a un grupo de enfermedades parasitarias, causadas por diferentes especies de protozoos flagelados del género Leishmania, que son morfológicamente indistinguibles por observación directa (Cavalier-Smith, 1998; Alvar et al., 2004). El género Leishmania fue descripto simultáneamente por Charles Donovan y William Leishman a partir de biopsias viscerales y cutáneas en pacientes enfermos de la India, y en conmemoración se le otorga el nombre de Leishmania donovani al complejo de especies que conforman el género y Leishmaniosis Visceral (LV) o Kala-azar (del sánscrito: Kala “negra, impura” y azar “fiebre”) a la enfermedad. A nivel mundial, la principal especie implicada en producir Leishmaniosis canina (LC) y LV en el hombre es Leishmania infantum (Syn. L. infantum chagasi) (Ross, 1903; Alvar et al., 2004).Facultad de Ciencias Veterinaria