Development and Evaluation of a Molecular Diagnostic Method for Rapid Detection of Histoplasma capsulatum var. farciminosum, the Causative Agent of Epizootic Lymphangitis, in Equine Clinical Samples

Abstract

Histoplasma capsulatum var. farciminosum, 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 internal transcribed spacer (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. Among the 29 horses with suspected cases of EZL, H. capsulatum var. farciminosum was confirmed by extraction of DNA from pus and blood samples from 25 and 17 horses, respectively. Positive PCR results were also obtained with heat-inactivated pus (24 horses) and blood (23 horses) spotted onto Whatman FTA cards. Two positive results 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 H. capsulatum var. farciminosum in equine blood and at high frequency among horses exhibiting cutaneous lesions. The nested PCR outperformed conventional microscopic diagnosis, as characteristic yeast cells could be observed only in 14 pus samples. The presence of H. capsulatum var. farciminosum DNA was confirmed by sequencing the cloned PCR products, and while alignment of the ITS amplicons showed very little sequence variation, there was preliminary single nucleotide polymorphism-based evidence for the existence of two subgroups of H. capsulatum var. farciminosum. This molecular diagnostic method now permits investigation of the epidemiology of EZL.

Epidemiology of Epizootic Lymphangitis of cart horses in Northern Ethiopia Using Conventional diagnostic Methods and Nested Polymerase Chain Reaction

“…The DNA isolated from buffy coat of the 70 presumably EL positive cases, 88.6% (62/70) were PCR positive which was much higher than the performance of the test (63%; 17/27) on DNA isolated from whole blood samples of presumably EL positive animals (13). Considering the intracellular nature of HCF particularly in neutrophils and macrophages, the buffy coat centrifugation could allow aggregation of the infected cells and probably the yeast fungi in the buffy coat layer.…”

“…In this study, we evaluated the diagnostic accuracy of clinical observation in comparison with nested PCR primer sets for diagnosis of HCF infection developed by (13). Clinical signs used for diagnosis of EL showed a sensitivity of 73.81% and specificity 92.52%.…”

“…Taking PCR as a gold standard/reference test (13), the results obtained with the 191 buffy coat samples and 32 pus smears/gram stain of clinically suspected animals and the diagnostic tests were represented in a contingency Table (Table 4). Sensitivities and specificities of clinical observation and gram stain using PCR as reference test are shown in Table 5.…”

“…There are asymptomatic early cases and potential carriers that cannot be detected. Thus, evaluation using the applications of PCR based protocols to rapidly identify HCF directly from equine clinical specimens is of paramount importance to understand the molecular epidemiology of the disease (13). The aim of this study was to investigate the epidemiology of EL in northern Ethiopia using the conventional methods and PCR and to evaluate the inherent diagnostic capacity of conventional tests (clinical sign and microscopic examination) for diagnosis of EL in horses taking PCR as a gold standard.…”

Re-drawing the Maps for Endemic Mycoses

“…There is an additional variety, H. capsulatum var. farciminosum , which has predominantly been described as an equine pathogen but, based on molecular analyses, may have a broader host range which could include humans [ 16 , 17 ]. Histoplasmosis was first described by Samuel Darling in a worker during construction of the Panama Canal in 1906 [ 18 ].…”