Centro de Desenvolvimento da Tecnologia Nuclear (CDTN), Cidade Universitária-Campus da UFMG, 31120-970, Belo Horizonte, MG, Brazil.
Vet Parasitol. 2010 Jun 24;170(3-4):201-6. doi: 10.1016/j.vetpar.2010.02.020. Epub 2010 Feb 20.
The efficacy of conjunctival swab (CS) as a sampling method for visceral leishmaniasis (VL) diagnosis by PCR of asymptomatic dogs was evaluated. The CS was compared to blood samples (B) and skin biopsies (SB), two less invasive samples potentially useful for massive screening of dogs. Thirty asymptomatic dogs, with serological and parasitological positive tests, were used. The samples were analyzed by two PCR methods: kDNA PCR-hybridization and ITS-1 nPCR. The DNA sample volume used was of 1.0 microL and 10.0 microL respectively. Using CS samples the kDNA PCR-hybridization was able to detected parasite DNA in 24/30 dogs (80%) using the right conjunctiva (RC) and 23/30 dogs (76.6%) with the left conjunctiva (LC), 17/30 dogs (56.7%) by means of SB and 4/30 dogs (13.3%) with B. The CS positivity obtained combining RC and LC results was of 90% (27/30 dogs). The assay of CS samples by ITS-1 nPCR revealed that 25/30 dogs (83.3%) were positive when using RC and 20/30 dogs (66.6%) were positive when using LC. Via the same method 15/30 dogs (50.0%) were positive by SB and 17/30 dogs (56.7%) with B. The CS positivity obtained by ITS-1 nPCR combining RC and LC was of 83.3%. The CS positivities for RC and LC were significantly higher (p<0.05) than SB and B for kDNA PCR-hybridization method. Statistical difference in relation to SB and B was verified by ITS-1 nPCR only for RC. The kDNA PCR-hybridization and ITS-1 nPCR methods showed similar sensitivities for CS and SB samples. On the other hand, for blood samples, the positivity of ITS-1 nPCR was significantly higher than the one obtained by the kDNA PCR-hybridization, indicating that sensitivity of PCR methods can vary according to the biological sample examined. Our results showed that CS was suitable to detect Leishmania DNA in asymptomatic animals when comparing to other low-invasive samples. The CS sensitivities obtained in this study were similar to the ones observed in other studies for VL diagnosis in symptomatic dogs. We concluded that the use of CS for regular screenings of dogs by PCR should be considered.
评价了结膜拭子 (CS) 作为聚合酶链反应 (PCR) 诊断无症状犬内脏利什曼病 (VL) 的采样方法的疗效。CS 与血液样本 (B) 和皮肤活检样本 (SB) 进行了比较，这两种样本的侵入性较小，可用于对大量犬进行大规模筛查。使用了 30 只具有血清学和寄生虫学阳性检测结果的无症状犬。样本使用两种 PCR 方法进行分析：kDNA PCR-杂交和 ITS-1 nPCR。分别使用 1.0 μL 和 10.0 μL 的 DNA 样本量。使用 CS 样本，kDNA PCR-杂交法能够在 30 只狗中的 24 只（80%）右眼结膜（RC）和 30 只狗中的 23 只（76.6%）左眼结膜（LC）中检测到寄生虫 DNA ，17 只（56.7%）通过 SB 检测，4 只（13.3%）通过 B 检测。结合 RC 和 LC 结果，CS 呈阳性的比例为 90%（30 只狗中的 27 只）。CS 样本的 ITS-1 nPCR 检测显示，RC 检测时 30 只狗中有 25 只（83.3%）呈阳性，LC 检测时 30 只狗中有 20 只（66.6%）呈阳性。通过相同的方法，SB 检测时 15 只（50.0%）呈阳性，B 检测时 30 只狗中有 17 只（56.7%）呈阳性。RC 和 LC 的 CS 阳性率分别为 83.3%。RC 和 LC 的 CS 阳性率明显高于 kDNA PCR-杂交法的 SB 和 B（p<0.05）。仅在 RC 中，ITS-1 nPCR 验证了与 SB 和 B 相比存在统计学差异。kDNA PCR-杂交法和 ITS-1 nPCR 法对 CS 和 SB 样本的敏感性相似。另一方面，对于血液样本，ITS-1 nPCR 的阳性率明显高于 kDNA PCR-杂交法，表明 PCR 方法的敏感性可能因所检查的生物样本而异。我们的结果表明，CS 适用于检测无症状动物中的利什曼原虫 DNA，与其他低侵入性样本相比。本研究中获得的 CS 敏感性与其他研究中用于诊断有症状犬内脏利什曼病的敏感性相似。我们得出结论，应考虑使用 CS 对犬进行常规 PCR 筛查。
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