背景:目前关于诊断犬丝虫感染的建议依赖于检测主要由成年女性产生的抗原,并通过微丝虫检测试验(MFDT)观察循环中的微丝虫(mf)。假设与抗原检测测试并行使用的qPCR分析在检测mf方面比改良的Knott's测试(MK)更好,当结合抗原检测。这项研究比较了基于探针的qPCR和MK技术,用于与DiroCHEK®抗原测试并行使用的mf检测,以筛选收容所犬的心丝虫感染。
方法:从布拉索斯县和哈里斯县的300只收容所犬中收集了匹配的血液和血清样本,德州,美国。通过MK评估血液中mf的存在和通过基于物种特异性探针的qPCR分析评估D.immitisDNA的存在。在经由热处理的免疫复合物解离(ICD)之前和之后,使用DiroCHEK®测试血清样品中的心丝虫抗原的存在。此外,通过卡方检验评估每个诊断测试的性能,Cochran的Q测试,和事后分析。
结果:定性,MK在22.0%(66/300)的样品中检测到mf,其中55个在形态学上被鉴定为D.immitis,11个为Acanthocheilonemareconditum。心虫病mf范围为28至88,803mf/ml(中位数:6627.5)。实时PCR在20.7%(62/300)的样品中检测到D.immitisDNA。在ICD前的样本中,有24.7%(74/300)检测到心虫抗原,和29.3%(88/300)后ICD。比较测试时,卡方检验和McNemar检验表明,阳性和阴性比例之间的差异有统计学意义。Cochran检验显示,单项检验组合时(χ2=62.3,df=3,P<0.0001)和平行方法组合时(χ2=43.1,df=4,P<0.0001),病例与非病例的分布差异有统计学意义。
结论:考虑到单独和组合测试性能,实用性,和有效利用工作台时间,这种基于心丝虫特异性探针的qPCR方法作为mf检测试验是一种可行的选择,可在诊断和研究环境中与犬心丝虫感染的抗原检测同时使用.
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 (χ2 = 62.3, df = 3, P < 0.0001) and when parallel methods were combined (χ2 = 43.1, df = 4, P < 0.0001).
CONCLUSIONS: 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.