PDF(Pubmed)
Abstract：
BACKGROUND: For decades, zinc sulfate centrifugal fecal flotation microscopy (ZCF) has been the mainstay technique for gastrointestinal (GI) parasite screening at veterinary clinics and laboratories. Elsewhere, PCR has replaced microscopy because of generally increased sensitivity and detection capabilities; however, until recently it has been unavailable commercially. Therefore, the primary aim of this study was to compare the performance of real-time PCR (qPCR) and ZCF for fecal parasite screening. Secondary aims included further characterization of markers for hookworm treatment resistance and Giardia spp. assemblages with zoonotic potential and qPCR optimization.
METHODS: A convenience sampling of 931 canine/feline fecal samples submitted to a veterinary reference laboratory for routine ZCF from the Northeast US (11/2022) was subsequently evaluated by a broad qPCR panel following retention release. Detection frequency and agreement (kappa statistics) were evaluated between ZCF and qPCR for seven GI parasites [hookworm/(Ancylostoma spp.), roundworm/(Toxocara spp.), whipworm/(Trichuris spp.), Giardia duodenalis, Cystoisospora spp., Toxoplasma gondii, and Tritrichomonas blagburni] and detections per sample. Total detection frequencies were compared using a paired t-test; positive sample and co-infection frequencies were compared using Pearson\'s chi-squared test (p ≤ 0.05 significant) and qPCR frequency for hookworm benzimidazole (BZ) resistance (F167Y) and zoonotic Giardia spp. assemblage markers calculated. Confirmatory testing, characterization, and qPCR optimization were carried out with Sanger sequencing.
RESULTS: qPCR detected a significantly higher overall parasite frequency (n = 679) compared to ZCF (n = 437) [p = < 0.0001, t = 14.38, degrees-of-freedom (df) = 930] and 2.6 × the co-infections [qPCR (n = 172) vs. ZCF (n = 66)], which was also significant (p = < 0.0001, X2 = 279.49; df = 1). While overall agreement of parasite detection was substantial [kappa = 0.74; (0.69-0.78], ZCF-undetected parasites reduced agreement for individual and co-infected samples. qPCR detected markers for Ancylostoma caninum BZ resistance (n = 5, 16.1%) and Giardia with zoonotic potential (n = 22, 9.1%) as well as two parasites undetected by ZCF (T. gondii/T. blagburni). Sanger sequencing detected novel roundworm species, and qPCR optimization provided detection beyond ZCF.
CONCLUSIONS: These results demonstrate the statistically significant detection frequency advantage offered by qPCR compared to routine ZCF for both single and co-infections. While overall agreement was excellent, this rapid, commercially available qPCR panel offers benefits beyond ZCF with detection of markers for Giardia assemblages with zoonotic potential and hookworm (A. caninum) BZ resistance.
METHODS: A convenience sampling of 931 canine/feline fecal samples submitted to a veterinary reference laboratory for routine ZCF from the Northeast US (11/2022) was subsequently evaluated by a broad qPCR panel following retention release. Detection frequency and agreement (kappa statistics) were evaluated between ZCF and qPCR for seven GI parasites [hookworm/(Ancylostoma spp.), roundworm/(Toxocara spp.), whipworm/(Trichuris spp.), Giardia duodenalis, Cystoisospora spp., Toxoplasma gondii, and Tritrichomonas blagburni] and detections per sample. Total detection frequencies were compared using a paired t-test; positive sample and co-infection frequencies were compared using Pearson\'s chi-squared test (p ≤ 0.05 significant) and qPCR frequency for hookworm benzimidazole (BZ) resistance (F167Y) and zoonotic Giardia spp. assemblage markers calculated. Confirmatory testing, characterization, and qPCR optimization were carried out with Sanger sequencing.
RESULTS: qPCR detected a significantly higher overall parasite frequency (n = 679) compared to ZCF (n = 437) [p = < 0.0001, t = 14.38, degrees-of-freedom (df) = 930] and 2.6 × the co-infections [qPCR (n = 172) vs. ZCF (n = 66)], which was also significant (p = < 0.0001, X2 = 279.49; df = 1). While overall agreement of parasite detection was substantial [kappa = 0.74; (0.69-0.78], ZCF-undetected parasites reduced agreement for individual and co-infected samples. qPCR detected markers for Ancylostoma caninum BZ resistance (n = 5, 16.1%) and Giardia with zoonotic potential (n = 22, 9.1%) as well as two parasites undetected by ZCF (T. gondii/T. blagburni). Sanger sequencing detected novel roundworm species, and qPCR optimization provided detection beyond ZCF.
CONCLUSIONS: These results demonstrate the statistically significant detection frequency advantage offered by qPCR compared to routine ZCF for both single and co-infections. While overall agreement was excellent, this rapid, commercially available qPCR panel offers benefits beyond ZCF with detection of markers for Giardia assemblages with zoonotic potential and hookworm (A. caninum) BZ resistance.
摘要：
背景：几十年来，硫酸锌离心粪便浮选显微镜（ZCF）已成为兽医诊所和实验室胃肠道（GI）寄生虫筛查的主要技术。在其他地方,PCR已经取代了显微镜，因为通常提高了灵敏度和检测能力；然而，直到最近，它一直没有商业。因此,本研究的主要目的是比较实时PCR(qPCR)和ZCF在粪便寄生虫筛查中的表现.次要目标包括进一步表征钩虫治疗抗性和贾第鞭毛虫的标记。具有人畜共患潜力和qPCR优化的组合。
方法：随后通过广泛的qPCR小组评估了从美国东北部（11/2022）提交给兽医参考实验室的常规ZCF的931个犬/猫粪便样本的方便采样。在ZCF和qPCR之间评估了7种GI寄生虫的检测频率和一致性（kappa统计）[钩虫/（Ancylostomaspp。),蛔虫/(弓形虫属),鞭虫/（鞭虫属。),十二指肠贾第虫,囊孢子菌属。,弓形虫,和Tritrichomonasblagburni]和每个样本的检测。使用配对t检验比较总检测频率；使用Pearson卡方检验（p≤0.05显着）和qPCR频率比较钩虫苯并咪唑（BZ）抗性（F167Y）和人畜共患贾第鞭毛虫的阳性样本和共感染频率。已计算组合标记。验证性测试,表征，用Sanger测序进行qPCR优化。
结果：qPCR检测到总体寄生虫频率（n=679）明显高于ZCF（n=437）[p=<0.0001，t=14.38，自由度（df）=930]和2.6×合并感染[qPCR（n=172）vs.ZCF(n=66)]，这也是显著的(p=<0.0001,X2=279.49;df=1)。虽然寄生虫检测的总体一致性是相当大的[kappa=0.74；（0.69-0.78]，未检测到的ZCF寄生虫降低了个体和共感染样品的一致性。qPCR检测到了犬科囊虫BZ抗性（n=5，16.1%）和具有人畜共患潜力的贾第鞭毛虫（n=22，9.1%）以及两种未被ZCF检测到的寄生虫（T。gondii/T.布拉布尔尼)。Sanger测序检测到新型蛔虫物种，qPCR优化提供了超越ZCF的检测。
结论：这些结果表明，与常规ZCF相比，qPCR在单一感染和共感染方面具有统计学上的显着检测频率优势。虽然总体协议非常好，这么快,市售的qPCR面板提供了超越ZCF的好处，可以检测具有人畜共患潜力和钩虫的贾第鞭毛虫组合的标记（A.caninum)BZ电阻。
方法：随后通过广泛的qPCR小组评估了从美国东北部（11/2022）提交给兽医参考实验室的常规ZCF的931个犬/猫粪便样本的方便采样。在ZCF和qPCR之间评估了7种GI寄生虫的检测频率和一致性（kappa统计）[钩虫/（Ancylostomaspp。),蛔虫/(弓形虫属),鞭虫/（鞭虫属。),十二指肠贾第虫,囊孢子菌属。,弓形虫,和Tritrichomonasblagburni]和每个样本的检测。使用配对t检验比较总检测频率；使用Pearson卡方检验（p≤0.05显着）和qPCR频率比较钩虫苯并咪唑（BZ）抗性（F167Y）和人畜共患贾第鞭毛虫的阳性样本和共感染频率。已计算组合标记。验证性测试,表征，用Sanger测序进行qPCR优化。
结果：qPCR检测到总体寄生虫频率（n=679）明显高于ZCF（n=437）[p=<0.0001，t=14.38，自由度（df）=930]和2.6×合并感染[qPCR（n=172）vs.ZCF(n=66)]，这也是显著的(p=<0.0001,X2=279.49;df=1)。虽然寄生虫检测的总体一致性是相当大的[kappa=0.74；（0.69-0.78]，未检测到的ZCF寄生虫降低了个体和共感染样品的一致性。qPCR检测到了犬科囊虫BZ抗性（n=5，16.1%）和具有人畜共患潜力的贾第鞭毛虫（n=22，9.1%）以及两种未被ZCF检测到的寄生虫（T。gondii/T.布拉布尔尼)。Sanger测序检测到新型蛔虫物种，qPCR优化提供了超越ZCF的检测。
结论：这些结果表明，与常规ZCF相比，qPCR在单一感染和共感染方面具有统计学上的显着检测频率优势。虽然总体协议非常好，这么快,市售的qPCR面板提供了超越ZCF的好处，可以检测具有人畜共患潜力和钩虫的贾第鞭毛虫组合的标记（A.caninum)BZ电阻。
