Improving the reference standard for the diagnosis of canine visceral leishmaniasis: a challenge for current and future tests

Abstract

BACKGROUND Studies aimed at validating canine visceral leishmaniasis diagnostic tests present heterogeneous results regarding test accuracy, partly due to divergences in reference standards used and different infection evolution periods in animals. OBJECTIVE This study aimed to evaluate the accuracy of the rapid test-dual path platform (TR-DPP) (Biomanguinhos®), EIE-Leishmaniose-Visceral-Canina-Biomanguinhos (EIE-LVC) (Biomanguinhos®), enzyme-linked immunosorbent assay (ELISA) rK39 (in-house), and the direct agglutination test (DAT- Canis ) against a reference standard comprising parasitological and molecular techniques. METHODS A phase II/III validation study was carried out in sample sera from 123 predominantly asymptomatic dogs living in an area endemic for visceral leishmaniasis. FINDINGS Sixty-nine (56.1%) animals were considered infected according to the reference standard. For each test, the sensitivity and specificity, respectively, were as follows: TR-DPP, 21.74% [confidence interval (CI)95% 13.64% to 32.82%] and 92.59% (CI95% 82.45% to 97.08%); EIE-LVC, 11.59% (CI95% 5.9% to 21.25%) and 90.74% (CI95% 80.09% to 95.98%); ELISA rK39, 37.68% (CI95% 27.18% to 49.48%) and 83.33% (CI95% 71.26% to 90.98%); and DAT- Canis , 18.84% (CI95% 11.35% to 29.61%) and 96.30% (CI95% 87.46% to 98.98%). CONCLUSION We concluded that improving the sensitivity of serum testing for diagnosing asymptomatic dogs must constitute a priority in the process of developing new diagnostic tests to be used in the visceral leishmaniasis control program in Brazil.

A cross-sectional approach including dog owner characteristics as predictors of visceral leishmaniasis infection in dogs

“…The under-detection of cases of asymptomatic canine infection caused by the lack of sensitivity of the methods recommended for routine examination has already been highlighted by other authors as a possible factor associated with the lack of effective CVL control measures currently recommended in Brazil. (27,32,33,34) There was no significant difference between the clinical signs scores for infected and noninfected dogs, and there were dogs with suggestive symptoms that were not infected with leishmaniasis but were possibly affected by other diseases that were not specifically investigated in this study. This observation is consistent with the perception that the clinical syndrome associated with CVL is nonspecific.…”

“…Diagnostic tests procedures -Direct parasitology; parasite culture; PCR amplification of the ITS1 target; RT DPP serology (Biomanguinhos®) and ELISA serology (EIE-CVL) (Biomanguinhos®), sequentially; TR DPP serology (Biomanguinhos®) and ELISA serology (Rk39), sequentially, were performed according to the methodology described in the study by Teixeira et al (27) When the tests were performed, the professionals were not aware of the identity of the source animals or of the results obtained for the other tests.…”

“…(22,29) This discrepancy is most likely due to the combined use of a larger number of diagnostic techniques in the present study, especially PCR, which enabled the detection of a larger number of infected animals because of its greater sensitivity. (27,30) The difference of 12.5% between the prevalence of infection (26.25%; 95% CI: 20.05 to 33.57) and the prevalence of the disease (13.75%; 95% CI: 9.26 to 19.94) may have resulted from the greater sensitivity of the combination of diagnostic methods used for the detection of infected dogs. The detection of asymptomatic-infected dogs is important in terms of epidemiological surveillance, especially because autochthonous cases of human VL have been reported in this region.…”

“…The modified clinical score was validated in the field through a pilot study in which 20 evaluations were performed by two veterinarians. The final evaluation applied in the study had a maximum score of 46 points, instead of 87 as in the original evaluation according to Teixeira et al (27) Collection, transport, and storage of biological samples -Peripheral blood (cephalic vein, saphenous vein or jugular vein) was collected using a 3 mL syringe and a 25 × 0.7 mm needle. The collected blood was stored in two tubes, one with EDTA anticoagulant for molecular tests and the other without anticoagulant to perform serological tests.…”

Molecular Methodology for the Detection of the Leishmania Genus in Different Biological Samples Extracted from Dogs

“…In the present study, 19/50 (38%) of the dogs were under these conditions and among them only two were not detected the presence of the parasite DNA in the three tests performed. The result demonstrates that even without the presence of symptoms, the dog may be in the pathway of transmission of the disease, since the parasite is present in the blood tissue [1,22].…”