探针 qPCR 可替代改良的 Knott's 试验，用于联合抗原检测试验筛查犬心丝虫（Dirofilaria immitis）感染。
Probe-based qPCR as an alternative to modified Knott's test when screening dogs for heartworm (Dirofilaria immitis) infection in combination with antigen detection tests.
机构信息
Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, 4467 TAMU, College Station, TX, 77843-4467, USA.
出版信息
Parasit Vectors. 2022 Aug 29;15(1):306. doi: 10.1186/s13071-022-05372-x.
BACKGROUND
Current recommendations for diagnosis of Dirofilaria immitis infection in dogs rely on the detection of antigen produced largely by adult females coupled with the visualization of microfilariae (mf) in the circulation via a microfilaria detection test (MFDT). It is hypothesized that qPCR assays used in parallel with antigen detection tests will perform better in detecting mf than modified Knott's test (MK), when combined with antigen detection. This study compares probe-based qPCR and MK techniques for mf detection used in parallel with the DiroCHEK antigen test to screen for heartworm infection in shelter dogs.
METHODS
Matching blood and serum samples were collected from 300 shelter dogs in Brazos and Harris counties, Texas, USA. Blood was assessed for the presence of mf via MK and the presence of D. immitis DNA by a species-specific probe-based qPCR assay. Serum samples were tested for the presence of heartworm antigen using DiroCHEK before and after immune complex dissociation (ICD) via heat treatment. In addition, the performance of each diagnostic test was evaluated via Chi-square test, Cochran's Q test, and post hoc analysis.
RESULTS
Qualitatively, MK detected mf in 22.0% (66/300) of samples, 55 of which were morphologically identified as D. immitis and 11 as Acanthocheilonema reconditum. The range of heartworm mf was 28 to 88,803 mf/ml (median: 6627.5). Real-time PCR detected D. immitis DNA in 20.7% (62/300) of samples. Heartworm antigen was detected in 24.7% (74/300) of samples pre-ICD, and in 29.3% (88/300) post-ICD. When comparing tests, the Chi-square and McNemar's tests showed that the difference between positive and negative proportions was statistically significant. The Cochran test showed the difference in the distributions of cases and non-cases was significant when individual tests were combined (χ = 62.3, df = 3, P < 0.0001) and when parallel methods were combined (χ = 43.1, df = 4, P < 0.0001).
CONCLUSION
Considering individual and combined test performances, practicality, and efficient use of bench time, this heartworm-specific probe-based qPCR method is a viable option as a mf detection test to be used in parallel with antigen tests for canine heartworm infection in diagnostic and research settings.
背景
目前，犬心丝虫感染的诊断推荐方法依赖于检测主要由雌性成虫产生的抗原，同时通过微丝蚴检测试验（MFDT）检测循环中的微丝蚴（mf）。据推测，当与抗原检测联合使用时，qPCR 检测与改良 Knott 检测（MK）相比，在检测 mf 方面表现更好。本研究比较了探针 qPCR 和 MK 技术在与 DiroCHEK 抗原检测联合使用时，用于筛查收容所犬心丝虫感染的 mf 检测性能。
方法
从美国德克萨斯州布拉索斯县和哈里斯县的 300 只收容所犬中采集匹配的血液和血清样本。通过 MK 检测血液中 mf 的存在情况，并通过种特异性探针 qPCR 检测法检测犬心丝虫 DNA 的存在情况。使用 DiroCHEK 检测血清样本中的心丝虫抗原，然后通过热处理进行免疫复合物解离（ICD）前后检测。此外，通过卡方检验、Cochran Q 检验和事后分析评估每个诊断检测的性能。
结果
定性地，MK 在 22.0%（66/300）的样本中检测到 mf，其中 55 个经形态学鉴定为犬心丝虫，11 个为 Acanthocheilonema reconditum。心丝虫 mf 的范围为 28 至 88803 mf/ml（中位数：6627.5）。实时 PCR 在 20.7%（62/300）的样本中检测到犬心丝虫 DNA。在 ICD 前，74%（74/300）的样本中检测到心丝虫抗原，在 ICD 后，88%（88/300）的样本中检测到心丝虫抗原。在比较检测时，卡方检验和 McNemar 检验表明阳性和阴性比例之间的差异具有统计学意义。Cochran 检验表明，当单独测试时（χ=62.3，df=3，P<0.0001）和当平行方法联合时（χ=43.1，df=4，P<0.0001），病例和非病例的分布差异具有统计学意义。
结论
考虑到个体和联合检测性能、实用性以及在实验台上的有效利用时间，这种特定于心丝虫的探针 qPCR 方法是一种可行的选择，可作为抗原检测联合使用的 mf 检测方法，用于诊断和研究环境中犬心丝虫感染。
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