Diagnostic Value of Conjunctival Swab Sampling Associated with Nested PCR for Different Categories of Dogs Naturally Exposed to Leishmania infantum Infection

Abstract

The objective of the present study was to evaluate the diagnostic performance of a noninvasive assay, conjunctival swab (CS) nested-PCR (n-PCR), for diagnosing canine leishmaniasis (CanL) in different stages of infection in comparison to the performance of the indirect immunofluorescence antibody test (IFAT), lymph node microscopy, and buffy coat n-PCR. To this end, we performed a cross-sectional survey among 253 nonselected dogs in areas of endemicity in central Italy. We also performed a longitudinal study of CS n-PCR among 20 sick dogs undergoing antileishmanial treatment. In the first study, among the 72 animals that were positive by at least one test (28.45%), CS n-PCR showed the best relative performance (76.38%), with a high concordance in comparison to standard IFAT serology ( ‫؍‬ 0.75). The highest positivity rates using CS n-PCR were found in asymptomatic infected dogs (84.2%) and sick dogs (77.8%); however, the sensitivity of the assay was not associated with the presence of clinical signs. In the follow-up study on treated sick dogs, CS n-PCR was the most sensitive assay, with promising prognostic value for relapses. The univariate analysis of risk factors for CanL based on CS n-PCR findings showed a significant correlation with age (P ‫؍‬ 0.012), breed size (P ‫؍‬ 0.026), habitat (P ‫؍‬ 4.9 ؋ 10 ؊4 ), and previous therapy (P ‫؍‬ 0.014). Overall, the results indicated that CS n-PCR was the most sensitive assay of the less invasive diagnostic methods and could represent a good option for the early and simple diagnosis of CanL infection in asymptomatic animals and for monitoring relapses in drug-treated dogs.

Detection of canine visceral leishmaniasis by conjunctival swab PCR

“…Accordingly, blood and conjunctival swab PCR were also not signifi cantly correlated (p > 0.05). These results are summarized in Table 2, and are in line with published results (12) . Notably, statistical analysis (Table 3) indicated that a positive result on conjunctival swab PCR is significantly associated with the presence of clinical signs (p < 0.05), while a positive result on blood PCR is signifi cantly associated with the absence of clinical signs (p < 0.05).…”

“…Similarly, nested PCR has been used to enhance sensitivity, but is more time consuming and is more susceptible to contamination (11) . In line with previous results (12) , the adjusted kappa index between indirect immunofl uorescence and conjunctival swab PCR was 0.53, suggesting moderate (9) , but statistically signifi cant agreement (p < 0.05, Table 2). However, the number of seronegative dogs that tested positive on conjunctival swab PCR (n = 11) was similar to the number of seropositive dogs that tested negative on conjunctival swab PCR (n = 12).…”

“…Notably, statistical analysis (Table 3) indicated that a positive result on conjunctival swab PCR is significantly associated with the presence of clinical signs (p < 0.05), while a positive result on blood PCR is signifi cantly associated with the absence of clinical signs (p < 0.05). Indeed, conjunctival swab PCR has been demonstrated to have high sensitivity for Leishmania in clinically diseased (12) (13) , and asymptomatic dogs (2) . We note, however, that higher sensitivity was achieved in asymptomatic dogs by other groups (2) .…”

Laboratory tests for diagnosing and monitoring canine leishmaniasis

“…When there are no lesions eligible for fine‐needle aspiration or biopsy (eg, when the only prevalent clinical symptoms include anemia or proteinuric nephropathy), bone marrow and/or lymph nodes and spleen are the tissues with the highest potential prevalence for detection by PCR, especially in sick dogs, provided that the quality of the sample is adequate. Recent studies demonstrated that conjunctival and, to a lesser extent, oral and nasal swabs are very sensitive for the detection of Leishmania DNA and, in addition, can provide positive results earlier than other tissues . Whole blood or buffy coats may also be used for conventional or quantitative PCR analysis.…”

Application of qPCR in conjunctival swab samples for the evaluation of canine leishmaniasis in borderline cases or disease relapse and correlation with clinical parameters

“…In this context, molecular techniques can improve the diagnosis of leishmaniasis. In particular, qualitative and quantitative PCR (qPCR) may play an important role in parasite detection [13] and therapy monitoring [14,15], in both human and in veterinary medicine. In molecular methods, the constant region of Leishmania minicircles has been widely used as a specific target because of its very high number of target sequences [16][17][18][19].…”