The Rapid Test Based on <i>Leishmania infantum</i> Chimeric rK28 Protein Improves the Diagnosis of Canine Visceral Leishmaniasis by Reducing the Detection of False-Positive Dogs

<div><p>Visceral Leishmaniasis (VL) has spread to many urban centers worldwide. Dogs are considered the main reservoir of VL, because canine cases often precede the occurrence of human cases. Detection and euthanasia of serologically positive dogs is one of the primary VL control measures utilized in some countries, including Brazil. Using accurate diagnostic tests can minimize one undesirable consequence of this measure, culling false-positive dogs, and reduce the maintenance of false-negative dogs in endemic areas. In December 2011, the Brazilian Ministry of Health replaced the ELISA (EIE CVL) screening method and Indirect Immunofluorescence Test (IFI CVL) confirmatory method with a new protocol using the rapid DPP CVL screening test and EIE CVL confirmatory test. A study of diagnostic accuracy of these two protocols was done by comparing their performance using serum samples collected from a random sample of 780 dogs in an endemic area of VL. All samples were evaluated by culture and real time PCR; 766 out of the 780 dogs were tested using the previous protocol (IFI CVL + EIE CVL) and all 780 were tested using the current protocol (DPP CVL + EIE CVL). Performances of both diagnostic protocols were evaluated using a latent class variable as the gold standard. The current protocol had a higher specificity (0.98 vs. 0.95) and PPV (0.83 vs. 0.70) than the previous protocol, although sensitivity of these two protocols was similar (0.73). When tested using sera from asymptomatic animals, the current protocol had a much higher PPV (0.63 vs. 0.40) than the previous protocol (although the sensitivity of either protocol was the same, 0.71). Considering a range of theoretical CVL prevalences, the projected PPVs were higher for the current protocol than for the previous protocol for each theoretical prevalence value. The findings presented herein show that the current protocol performed better than previous protocol primarily by reducing false-positive results.</p></div

Percent of positive results of five CVL diagnostic tests performed on canine sera samples from an endemic area of Camaçari.

<p>Percent of positive results of five CVL diagnostic tests performed on canine sera samples from an endemic area of Camaçari.</p

Evaluating the Accuracy of Molecular Diagnostic Testing for Canine Visceral Leishmaniasis Using Latent Class Analysis

<div><p>Host tissues affected by <i>Leishmania infantum</i> have differing degrees of parasitism. Previously, the use of different biological tissues to detect <i>L. infantum</i> DNA in dogs has provided variable results. The present study was conducted to evaluate the accuracy of molecular diagnostic testing (qPCR) in dogs from an endemic area for canine visceral leishmaniasis (CVL) by determining which tissue type provided the highest rate of parasite DNA detection. Fifty-one symptomatic dogs were tested for CVL using serological, parasitological and molecular methods. Latent class analysis (LCA) was performed for accuracy evaluation of these methods. qPCR detected parasite DNA in 100% of these animals from at least one of the following tissues: splenic and bone marrow aspirates, lymph node and skin fragments, blood and conjunctival swabs. Using latent variable as gold standard, the qPCR achieved a sensitivity of 95.8% (CI 90.4–100) in splenic aspirate; 79.2% (CI 68–90.3) in lymph nodes; 77.3% (CI 64.5–90.1) in skin; 75% (CI 63.1–86.9) in blood; 50% (CI 30–70) in bone marrow; 37.5% (CI 24.2–50.8) in left-eye; and 29.2% (CI 16.7–41.6) in right-eye conjunctival swabs. The accuracy of qPCR using splenic aspirates was further evaluated in a random larger sample (n = 800), collected from dogs during a prevalence study. The specificity achieved by qPCR was 76.7% (CI 73.7–79.6) for splenic aspirates obtained from the greater sample. The sensitivity accomplished by this technique was 95% (CI 93.5–96.5) that was higher than those obtained for the other diagnostic tests and was similar to that observed in the smaller sampling study. This confirms that the splenic aspirate is the most effective type of tissue for detecting <i>L. infantum</i> infection. Additionally, we demonstrated that LCA could be used to generate a suitable gold standard for comparative CVL testing.</p></div

Prevalence of latent classes and conditional probabilities to the LCA model for <i>L. infantum</i> infection detection in dogs.

<p>Prevalence of latent classes and conditional probabilities to the LCA model for <i>L. infantum</i> infection detection in dogs.</p

Response patterns^a of Camaçari dogs for LCA model with 2 latent classes for diagnosis of CVL.

a<p>Response patterns of all samples tested using the three techniques.</p><p>*Estimation based on only one animal sample presenting this pattern.</p><p>N: Negative; P: Positive.</p

Parasitic loads detected in different canine tissue types from a total of 20 dogs from the endemic area of Jequié.

a<p>number of parasites normalized by the established reference amplification value for the housekeeping gene 18S rRNA in 100 mg of host tissue DNA.</p><p>*<i>p</i><0.05 Friedman’s together with Dunn’s multiple comparisons test of splenic aspirates and swab of right or left eye and splenic aspirates and bone marrow.</p

Distribution of parasitic load according to number of clinical signs in dogs from the prevalence study.

<p>Distribution of parasitic load according to number of clinical signs in dogs from the prevalence study.</p