Journal Article

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

Saleh, Meriam N., Verocai, Guilherme G., Negron, Veronica, Luksovsky, Joe L., Sobotyk, Caroline, Harvey, Tatiani V.

2022

Overview

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 2  = 62.3, df  = 3, P  < 0.0001) and when parallel methods were combined ( χ 2  = 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

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: Acanthocheilonema - genetics / adults / Animals / antigen detection / Antigen testing / antigen-antibody complex / Antigens / Antigens, Helminth / Bias / Biomedical and Life Sciences / Biomedicine / Blood / blood serum / Canine filariosis / chi-square distribution / Chi-square test / Comparative analysis / Detection / Diagnostic systems / Diagnostic tests / Dipetalonema reconditum / Dirofilaria immitis / Dirofilaria immitis - genetics / Dirofilariasis - diagnosis / Dissociation / DNA / Dog Diseases - diagnosis / Dogs / Entomology / Female / Heart / Heartworm disease / heartworms / Heat treatment / Heat treatments / Infections / Infectious Diseases / Laboratories / Methods / microfilariae / Microfilariae - genetics / Microfilariae detection test / Mitochondrial DNA / Molecular diagnostics / Morphology / Nematodes / Nucleotide sequence / Parasite antigens / Parasitology / PCR / Polymerase chain reaction / quantitative polymerase chain reaction / Real-Time Polymerase Chain Reaction - veterinary / Sediments / Serum / Shelters / Statistical analysis / Testing / Tests / Texas / Tropical Medicine / Veterinary Medicine/Veterinary Science / Virology