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

Abstract

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). 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. Graphical Abstract

High prevalence of canine heartworm, Dirofilaria immitis, in pet dogs in south Texas, USA, with evidence of Aedes aegypti mosquitoes contributing to transmission

“…The American Heartworm Society recommends both antigen testing and microfilariae testing of all canine samples [33], but microfilariae tests are not routine procedure. Consistent with our own previous results [16,42], antigen and microfilaria testing by qPCR in the present study gave different estimates of prevalence. Antigen tests work well at diagnosing infection with higher numbers of female D. immitis worms but are less sensitive for male-only infection with ≤ 3 females [40].…”

“…Moreover, antigen tests are more effective at identifying female worms that are > 6 months of age, making infection difficult to diagnosis in dogs aged < 6 months as it takes 50-58 days post-infection for L3 to develop into adults [41]. qPCR is a reliable and specific diagnostic tool for D. immitis [42] and the use of multiple diagnostic tests has been shown to improve diagnostic performance [29,43,44]. Two earlier studies found that qPCR detected a higher prevalence rate than antigen testing alone [29,45], similar to our results as three dogs in the present study were considered to be D. immitis microfilaria positive and NAD before and after ICD.…”

Application of a novel rectangular filtering microfluidic device for microfilarial detection

“…It will be interesting if we can improve the potential of the chip on in-chip identification. Moreover, purifiedlife microfilariae isolation and concentration are the device's advantages over other conventional methods (21,22). These might be very impactful for further research.…”

The long-distance relationship between Dirofilaria and the UK: case report and literature review

“…These may be divided into parasitological techniques for mfs detection in the host’s blood (e.g., fresh blood smear, buffy coat examination, filtration or modified Knott’s test), serological (i.e., enzyme-linked immunosorbent assays (ELISA), rapid immunochromatographic tests (ICT) and molecular approaches (i.e., probe-based quantitative polymerase chain reaction (qPCR), conventional PCR (cPCR), duplex qPCR for the simultaneous detection of both Dirofilaria spp. )), together with fine-needle aspirate (FNA) and histological examination of nodules when present ( 3 , 12 , 17 , 18 ). Serological tests are available as reference laboratory assays and also point-of care (POC) tests ( 3 , 19 – 21 ).…”

“…Indeed, studies on the incidence and/or prevalence of Dirofilaria spp. infection in the same canine population have shown differing results according to the diagnostic method adopted ( 15 , 18 , 19 , 30 ). For this reason, diagnosis of animal dirofilariosis must rely on the integration of results from at least two methods in order to reduce the risk of false positives and negatives, as recommended by the AHS ( 24 ).…”