January 09, 2019

Testing a New Molecular Assay for Ehrlichia Detection

Which is more useful in clinical practice—a newly designed TaqMan molecular diagnostic assay or commercially available technology?
 
By Ashley O. Bohn, PhD, MS, RVT, and Syeelah Stoessel, BS
Ehrlichiosis is a reportable, emerging zoonotic infectious disease spread primarily through tick bites. Over 900 cases are reported in humans annually, with at least 1 fatality each year. Ehrlichia spp are obligate intracellular parasites that usually infect the host’s immune cells. Rickettsial diseases, such as Ehrlichia spp infections and Rocky Mountain spotted fever, have similar clinical presentations and are important zoonotic diseases about which the veterinary team should be educated due to the possibility of transmission to humans.

Indirect fluorescent antibody assay, which detects antibodies to Ehrlichia spp, is considered the gold standard for diagnosing ehrlichiosis; however, additional testing, such as polymerase chain reaction (PCR), is needed to determine the specific organism in the blood. In a recent study published in Parasites & Vectors, a new real-time TaqMan PCR assay was compared with the commercially available PCRun assay to detect Ehrlichia canis and Ehrlichia minasensis from canine blood samples.

Study Design  
E canis and E minasensis are closely related according to phylogenetic analyses of 16S ribosomal RNA, although E minasensis is a newly identified species and little is known about its pathogenicity. E canis and E minasensis also share similarities in the citrate synthase (gltA) gene. The investigators in this study designed primers to amplify a 146 base-pair (bp) sequence of gltA. Multiple primers and PCR conditions were tested for optimal results. The optimized conditions were then tested for sensitivity using serial dilutions of clinical isolates of Ehrlichia spp in triplicate. The sensitivity of each assay was scored, and the amplification efficiency calculated via logarithmic regression analysis of the data.

Two clinical cohorts of canine whole blood samples were used for the evaluation. The first cohort contained 33 samples known to be positive for Ehrlichia spp from the United Kingdom. The second cohort consisted of 215 samples from dogs displaying signs of ehrlichiosis, including anorexia, fever, and thrombocytopenia, with known recent exposure to ticks in Israel.     

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Results
The efficiency of the new TaqMan assay was calculated to be 97%. The amplified PCR product was analyzed by gel electrophoresis and observed to have 1 product migrate at its predicted molecular weight (~150 bp). Similar sensitivities were reported by both TaqMan and PCRun during amplification of E canis DNA. The TaqMan assay was specific for E canis and E minasensis, whereas the PCRun method could detect all Ehrlichia- and Anaplasma-derived 16S sequences. Both tests correctly identified Ehrlichia spp, and the TaqMan assay achieved 100% sensitivity and specificity compared with the PCRun benchmark. No P values were generated in the data analyses.

Conclusion
According to the data presented, both TaqMan and PCRun are equally capable of detecting Ehrlichia spp. The TaqMan assay is slightly faster than PCRun and is highly specific for Ehrlichia spp, although it does require additional data interpretation and calculations. At this time, PCRun may be more suited for clinical use since the commercial assay is capable of performing multiple amplifications simultaneously and has broader applications due to its ability to detect both Ehrlichia and Anaplasma spp. Given that treatment for ehrlichiosis is not dependent on the species causing the infection, species identification is less important in clinical applications. The TaqMan assay is less suitable for clinical use without further development and optimization. 

This study demonstrated that gltA is conserved in Ehrlichia spp across the globe, and further development of diagnostic techniques using this conserved sequence is possible. 

Dr. Bohn received her PhD and MS from Georgia State University and has been a practicing veterinary nurse for nearly 20 years. Ms. Stoessel is a veterinary assistant and medical writing intern. They provide freelance medical writing services through Bohn Communications, LLC.
 

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