Evaluation of a Prototype Flow Cytometry Test for Serodiagnosis of Canine Visceral Leishmaniasis

Abstract

g Diagnosing canine visceral leishmaniasis (CVL) is a critical challenge since conventional immunoserological tests still present some deficiencies. The current study evaluated a prototype flow cytometry serology test, using antigens and fluorescent antibodies that had been stored for 1 year at 4°C, on a broad range of serum samples. Noninfected control dogs and Leishmania infantum-infected dogs were tested, and the prototype test showed excellent performance in differentiating these groups with high sensitivity, specificity, positive and negative predictive values, and accuracy (100% in all analyses). When the CVL group was evaluated according to the dogs' clinical status, the prototype test showed outstanding accuracy in all groups with positive serology (asymptomatic II, oligosymptomatic, and symptomatic). However, in dogs which had positive results by PCR-restriction fragment length polymorphism (RFLP) but negative results by conventional serology (asymptomatic I), serological reactivity was not observed. Additionally, sera from 40 dogs immunized with different vaccines (Leishmune, Leish-Tec, or LBSap) did not present serological reactivity in the prototype test. Eighty-eight dogs infected with other pathogens (Trypanosoma cruzi, Leishmania braziliensis, Ehrlichia canis, and Babesia canis) were used to determine cross-reactivity and specificity, and the prototype test performed well, particularly in dogs infected with B. canis and E. canis (100% and 93.3% specificities, respectively). In conclusion, our data reinforce the potential of the prototype test for use as a commercial kit and highlight its outstanding performance even after storage for 1 year at 4°C. Moreover, the prototype test efficiently provided accurate CVL serodiagnosis with an absence of false-positive results in vaccinated dogs and minor cross-reactivity against other canine pathogens.

Laboratory tests for diagnosing and monitoring canine leishmaniasis

“…There are techniques such as Western blotting that is highly accurate but not available in routine practice, while others have been proposed but are not extensively used, such as the latex agglutination test or detection of antibodies through immunosensors or flow cytometry. [120][121][122][123] The most common techniques used to detect antileishmanial antibodies are based on 3 analytic principles: immunofluorescent antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA), and immunochromatographic test (ICT). The latter method is the basis of all rapid in-clinic assays, which only provide a qualitative result (ie, presence/absence of specific reactive bands or spots).…”

Improving the serodiagnosis of canine Leishmania infantum infection in geographical areas of Brazil with different disease prevalence

“…In addition, it can be also employed for characterizing the diagnostic capacities of different antigens to further develop qualitative immune-chromatographic rapid tests that do not require laboratory equipment for their use (Travi et al, 2018). Different antigenic sources are employed in these diagnostic methods, including SLA, as well as different parasite antigenic fractions, individual recombinant proteins or small peptides containing defined antigenic determinants (Travi et al, 2001; Coelho et al, 2009; Solano-Gallego et al, 2009; Ker et al, 2013; Rodriguez-Cortes et al, 2013; Solano-Gallego et al, 2017). Some of these antigens are parasite-specific proteins like the kinetoplastid-membrane protein of 11 kDa (KMP-11) (Berberich et al, 1997), or members of intracellular protein families such as histones (Soto et al, 1999), heat shock proteins (HSP) (Angel et al, 1996; Quijada et al, 1996a; Oliveira et al, 2011), ribosome related factors including the acidic ribosomal protein family (Soto et al, 2009) or the recombinant K39 protein that contains an extensive repetitive domain located in the C-terminal region of the leishmanial kinesin protein (Scalone et al, 2002).…”

Comparison of a qualitative immunochromatographic test with two quantitative serological assays for the detection of antibodies to Leishmania infantum in dogs

“…A potential limitation of the present study was the lack of samples from dogs serologically reactive to other pathogens, particularly different Leishmania spp. or Trypanosoma cruzi, which are both protozoa that belong to the Trypanosomatidae family and share various antigens that may produce cross-reactions [14, 15]. All serum samples were collected from dogs from the city of Zaragoza, a region in Spain where T. cruzi is not present and L. infantum is the parasite responsible for canine leishmaniosis in Europe.…”