Nasal, Oral and Ear Swabs for Canine Visceral Leishmaniasis Diagnosis: New Practical Approaches for Detection of <i>Leishmania infantum</i> DNA

<div><p>Background</p><p>The aim of this study was to evaluate the potential use of nasal, oral, and ear swabs for molecular diagnosis of canine visceral leishmaniasis (CVL) in an endemic urban area in Brazil.</p> <p>Methodology/Principal Findings</p><p>Sixty-two naturally infected and ten healthy dogs were enrolled in this study. Bone marrow aspirates, peripheral blood, skin biopsy, and conjunctival, nasal, oral, and ear swabs were collected. All samples, except blood, were submitted to conventional PCR (cPCR) and quantitative real time PCR (qPCR) to detect and quantify <i>Leishmania infantum</i> DNA, respectively. All dogs were submitted to thorough clinical analysis and were included based on a combination of serological (ELISA immunoassay and immunofluorescent antibody test) and parasitological methods. The cPCR positivity obtained from nasal swab samples was 87% (54/62), equivalent to those from other samples (P>0.05). Positive results were obtained for 79% (22/28) in oral swabs and 43% (12/28) in ear swab samples. A significant difference was observed between these data (P = 0.013), and the frequency of positive results from oral swab was equivalent to those from other samples (P>0.05). The use of ear swab samples for cPCR assays is promising because its result was equivalent to skin biopsy data (P>0.05). The qPCR data revealed that parasite loads in mucosal tissues were similar (P>0.05), but significantly lower than the parasite burden observed in bone marrow and skin samples (P<0.05).</p> <p>Conclusions</p><p>Nasal and oral swab samples showed a high potential for the qualitative molecular diagnosis of CVL because their results were equivalent to those observed in samples collected invasively. Considering that mucosae swab collections are painless, noninvasive, fast and practical, the combination of these samples would be useful in massive screening of dogs. This work highlights the potential of practical approaches for molecular diagnosis of CVL and human leishmaniasis infections.</p> </div

El Compostelano : diario independiente: Ano XIX Número 5478 - 1938 agosto 26

*<p>Only the left nostril was considered;</p>**<p>Only the left conjunctiva was considered.</p><p>The <i>P</i> values were calculated using the chi square test.</p

Estimated parasite loads in different clinical samples from 28 naturally infected dogs.

<p>Data distributions with distinct letters are significantly different according to the Mann-Whitney <i>U</i> test. Nasal swab and bone marrow (P = 0.0024); Nasal swab and skin biopsy (P = 0.019); Oral swab and bone marrow (P = 0.0013); Oral swab and skin biopsy (P = 0.0058); Conjunctival swab and bone marrow (P = 0.0009); Conjunctival swab and skin biopsy (P = 0.0057) *Only the left nostril was considered. **Only the left conjunctiva was considered.</p

Conventional PCR positivity obtained from combination of clinical samples collected using mucosal swabs.

<p>NS: nasal swab; CS: conjunctival swab; OS: oral swab; n: number of naturally infected dogs.</p

Paired comparisons between cPCR results obtained from oral or ear swab and clinical samples obtained invasively.

<p>The <i>P</i> values were calculated using Fisher's exact test.</p

Estimated parasite loads in different clinical samples from 62 naturally infected dogs.

<p>Data distributions with distinct letters are significantly different according to the Mann-Whitney <i>U</i> test. Nasal swab and bone marrow (P = 0.0004); nasal swab and skin biopsy (P = 0.0074); conjunctival swab and bone marrow (P = 0.0076); conjunctival swab and skin biopsy (P = 0.04). *Only the left nostril was considered. **Only the left conjunctiva was considered.</p

Frequency of clinical signs detected in ear, eyes, mouth and nose in naturally infected dogs.

<p>Frequency of clinical signs detected in ear, eyes, mouth and nose in naturally infected dogs.</p

Reciprocal of serological titers obtained by IFAT for 80 naturally infected dogs.

<p>Group 1: 40 dogs with no clinical signs of CVL; Group 2: 40 dogs with clinical manifestations of CVL. The horizontal line in each titer distribution represents the geometric mean. Cut-off = 1∶40.</p

Parasite load in clinical samples of naturally infected dogs.

<p>(A): asymptomatic dogs; (B): dogs with clinical manifestations suggestive of VL. (a): No statistical difference among the measures indicated. (b) The parasite load in the skin was higher than those estimated in conjunctival swab and bone marrow samples (P<0.0001).</p

Comparison between paired clinical samples according to the results obtained by PCR/hybridization test in group 2.

<p>The statistical significance shown for each comparison is based on the χ² test.</p