A newly developed droplet digital PCR for <i>Ehrlichia canis</i> detection: comparisons to conventional PCR and blood smear techniques

Abstract

Canine monocytic ehrlichiosis caused by Ehrlichia canis infection is a life-threatening vector-borne disease in dogs worldwide. Routine blood smear has very low sensitivity and cannot accurately provide a quantitative result. Conventional PCR (cPCR) and real-time PCR (qPCR) are widely used as molecular methods for E. canis detection. qPCR is quantitative but relies on standard curves of known samples.To overcome this difficulty, this study developed a new E. canis quantitative detection method, using droplet digital polymerase chain reaction (ddPCR). ddPCR was evaluated against cPCR and blood smears. PCR amplicons and genomic DNA (gDNA) from 12 microscopic positive samples were used to identify the limits of detection (LODs) in ddPCR and cPCR. Our ddPCR was assessed in 92 field samples, it was compared with cPCR and blood smears. ddPCR showed LOD = 1.6 copies/reaction, or 78 times more sensitive than cPCR (LOD = 126 copies/reaction), using PCR amplicons as a template, whereas both ddPCR and cPCR had equal LODs at 0.02 ng gDNA/reaction. In addition, ddPCR had 100% sensitivity and 75% specificity for E. canis detection compared to cPCR and no cross-reaction with other blood pathogens was observed. ddPCR identified more positive samples than cPCR and blood smear. ddPCR improved the overall performance of E. canis detection, with a better LOD and comparable sensitivity and specificity to cPCR. The technique might be helpful for diagnosis of E. canis in light infection, evaluating the number of E. canis and follow-up after treatment.

Detección de material genético mediante reacción en cadena de polimerasa en muestras de caninos seropositivos a Ehrlichia canis

“…La mayoría de diagnósticos para estas enfermedades se realizan mediante técnicas de inmunodiagnóstico, visualización microscópica, tests rápidos y pocas veces se utilizan técnicas de diagnóstico molecular (Dávalos & Melchiade, 2018). El estudio de Journal MQRInvestigar 1196 Wichianchot et al, (2022) aplicó dos técnicas de PCR y frotis sanguíneos a 92 muestras de campo positivas a Ehrlichia canis mediante cPCR las cuales se muestran que el mayor número de casos positivos se encontraron mediante PCR digital en gota (ddPCR) en un 50% (46/92), mientras que en la cPCR sólo produjo un 34% (31/92) y los frotis sanguíneos sólo un 13% (12/92). La técnica de ddPCR es una herramienta poderosa para evaluar con precisión el número de E. canis debido a que tiene el límite de detección (LODs) mucho más bajo que la cPCR.…”

History and Current Status of Mediterranean Spotted Fever (MSF) in the Crimean Peninsula and Neighboring Regions along the Black Sea Coast

“…It is expected that the broader availability of nucleic acid-based methods and their use in regular clinical practice will improve routine diagnosis of spotted fever group rickettsioses and permit more effective treatment of these diseases in the region. Droplet digital PCR is one of the possible approaches and may be most useful for multiplex testing [ 167 ]. Next-generation sequencing provides powerful methodology permitting relatively quick identification of the etiological agent, especially for patients with atypical clinical manifestations and unclear epidemiologic evidence of a tick bite or exposure; however, this platform is not fully validated with rickettsioses and may pose challenges to complete identification when only a small number of Rickettsia reads are generated and other abundant microbial flora are present in the clinical sample [ 9 , 168 ].…”

Molecular detection and phylogeny of Ehrlichia canis and Anaplasma platys in naturally infected dogs in Central and Northeast Thailand

“…Blood smears were prepared on the day of collection. Positive control was made up of a sample that was positive for E. canis in a blood smear and confirmed using PCR as described by Wichianchot et al [29]. Sterile distilled water was used as a negative control.…”