A versatile and sensitive lateral flow immunoassay for the rapid diagnosis of visceral leishmaniasis

Although dogs are considered the main reservoir of Leishmania infantum infection in endemic areas in Europe, the existence of other wild vertebrate reservoirs has been proposed as a possible cause of the lack of success of control measures. Evidence of L. infantum infection in European wildlife has been reported in carnivores, lagomorphs, and rodents. The red fox (Vulpes vulpes) received most attention, probably due to its taxonomic relationship with the dog and because it is the most abundant wild carnivore in Europe. Foxes and other wild carnivores often displayed high prevalences of infection but their infectiveness to the sandfly vector has never been demonstrated. However, xenodiagnosis demonstrated that black rats (Rattus rattus), are infectious to sandflies. This, together with their relative abundance, high rates of infection, and the fact that infected rats have been found on a Mediterranean island where dogs are not present, makes rats good candidate to be reservoirs of L. infantum. Recently, the Iberian hare (Lepus granatensis) has been recognized as the origin of a leishmaniosis outbreak in humans in Spain and xenodiagnosis showed that this species is also able to infect sandflies. In contrast, a recent survey in cave bats failed to detect infected individuals. In the future, the comparison of parasite isolates from humans, dogs and wildlife, xenodiagnosis studies in wild carnivores, and the study of other vertebrate taxonomic groups will help determine the current role of European wildlife in the epidemiology of leishmaniosis.

Leishmaniosis caused by Leishmania infantum is a vector-borne zoonotic disease endemic in southern Europe, but which is spreading northwards. Millions of dogs, cats and other non-conventional companion animals susceptible to L. infantum, living in European households, may develop a severe disease and contribute to the spread of leishmaniosis because of travelling or re-homing. Dogs are the main reservoir but other new reservoirs have recently been incriminated. Sand flies remain the sole proven vector and non-vectorial transmission has been reported at individual level and in areas where the vector is absent. Clinical disease affects only a proportion of infected dogs and a complex genetic background of immune response is responsible for this susceptibility. There is a wide range of serological and parasitological diagnostic tools available whose cost-effective use depends on a reasoned approach. Clinical response to treatment of sick dogs is variable. Clinical cure is often obtained but clinical recurrence can occur and post-therapy follow up should be maintained life-long. In Europe, vaccination can be combined with individual protection with pyrethroids as part of an integrated approach to prevention. L. infantum is the only species isolated from cats in Europe and xenodiagnosis substantiated that infected cats are infectious for sand flies. Feline infection may be frequent in endemic areas, but prevalence is generally lower than in dogs. When cats are tested by both serological and molecular techniques discordant results are often observed. Feline cases have been reported from endemic areas in Italy, France, Spain and Portugal, but four cases were also diagnosed in Switzerland in cats that had travelled to or been imported from Spain. Half of the cases were diagnosed in cats with impaired immune responses. Clinical manifestations compatible with feline leishmaniosis include lymph node enlargement, skin and mucocutaneous lesions, ocular lesions, chronic gingivostomatitis, hypergammaglobulinemia, and normocytic normochromic anemia. Cats have been empirically treated with some drugs used in dogs. Due to polymorphic clinical picture and the insidious progressive course, leishmaniosis can persist for a long time before dogs or cats are brought to a veterinarian and so diagnosis can be delayed. Exotic or new Leishmania spp. have been reported in humans, animals and vectors in Europe. This changing situation requires attention in Europe for designing epidemiological studies and control measures.

Background Speed Leish K® is used as a serological screening test for Leishmania infection prior to vaccination. Limited comparative serological studies with Speed Leish K® have been performed. The aim of this study was to evaluate the diagnostic performance of four commercially available serologic tests including ELISAs (Leiscan®, ID Screen® and Leishmania 96®), a rapid test (Speed Leish K®) and an in-house IFAT for the detection of specific antibodies against Leishmania infantum antigen in dogs in different states of infection. Methods Sick infected dogs (n = 36), healthy infected dogs (n = 18), L. infantum seropositive dogs with low to high levels of antibodies (n = 53), dogs seropositive to other pathogens (to evaluate cross reaction) (n = 14) and uninfected dogs from a non-endemic area (n = 50) and from an endemic area (n = 32) were analysed by the serological methods mentioned above. Results The sensitivity was as follows: ID Screen® (0.953), Leiscan® and Leishmania 96® (0.925), IFAT (0.869) and Speed Leish K® (0.636). The maximum specificity (1.000) was attained for all diagnostic tests except the Leishmania 96® (0.896) and IFAT (0.917). The accuracy was as follows: ID Screen® (0.975), Leiscan® (0.961), Leishmania 96® (0.911), IFAT (0.892) and Speed Leish K® (0.808). In relation to the area under the ROC curve (AUC-ROC), the maximum value was attained with the ID Screen® (0.993) closely followed by Leiscan® (0.990), then, Leishmania 96® (0.962), IFAT (0.926) and Speed Leish K® (0.818). For the Kappa index, the best result was obtained by the ID Screen® (0.951) followed by Leiscan® (0.921), Leishmania 96® (0.822), IFAT (0.783) and Speed Leish K® (0.622). Statistically significant differences were found between the AUC-ROC of quantitative serological tests and the only qualitative rapid test evaluated. There were also statistically significant differences between AUC-ROC of the ELISAs (ID Screen® and Leiscan®) and IFAT. Conclusions Leiscan® and ID Screen® had superior diagnostic performance measures than IFAT and all quantitative serological tests were superior when compared to Speed Leish K®. Thus, Speed Leish K® may be considered a less valuable screening test prior to vaccination as it may result in vaccination of seropositive dogs and in some cases seropositive sick dogs.