Development and evaluation of a molecular diagnostic method to rapidly detect Histoplasma capsulatum var. farciminosum, the causative agent of epizootic lymphangitis, in equine clinical samples.



Scantlebury, CE ORCID: 0000-0002-0761-9872, Pinchbeck, G ORCID: 0000-0002-5671-8623, Loughnane, P, Aklilu, N, Ashine, T, Stringer, A, Gordon, L, Marshall, M, Christley, R ORCID: 0000-0001-9250-3032 and McCarthy, A
(2016) Development and evaluation of a molecular diagnostic method to rapidly detect Histoplasma capsulatum var. farciminosum, the causative agent of epizootic lymphangitis, in equine clinical samples. Journal of Clinical Microbiology, 54 (12). 2990 - 2999.

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Abstract

Histoplasma capsulatum var. farciminosum (HCF), the causative agent of epizootic lymphangitis (EZL), is endemic in parts of Africa. Diagnosis based on clinical signs and microscopy lacks specificity, and is a barrier to further understanding this neglected disease. Here, a nested PCR method targeting the ITS region of the rRNA operon was validated for application to equine clinical samples. Twenty-nine horses with signs of EZL, from different climatic regions of Ethiopia, were clinically examined. Blood samples and aspirates of pus from cutaneous nodules were taken, along with blood from a further 20 horses with no cutaneous EZL lesions. HCF was confirmed in DNA extracts of pus and blood samples from 25 and 17 horses, respectively, of the 29 suspected EZL cases. Positive PCR results were also obtained from heat-inactivated pus (24 horses) and blood (23 horses) spotted onto Whatman FTA cards. Two positives were obtained among blood samples from 20 horses that did not exhibit clinical signs of EZL. These are the first reports of the direct detection of HCF in equine blood, and at high frequency amongst horses exhibiting cutaneous lesions. The nested PCR outperformed conventional microscopic diagnosis, as characteristic yeast cells could only be observed in 14 pus samples. HCF DNA was confirmed by sequencing the cloned PCR products, and while alignment of the ITS amplicons showed very little sequence variation, there was preliminary SNP-based evidence for the existence of two subgroups of HCF. This molecular diagnostic method now permits investigation of the epidemiology of EZL.

Item Type: Article
Depositing User: Symplectic Admin
Date Deposited: 06 Oct 2016 14:20
Last Modified: 31 May 2021 22:10
DOI: 10.1128/JCM.00896-16
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URI: https://livrepository.liverpool.ac.uk/id/eprint/3003641