Limits...
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

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus

Light micrograph of a pus impression smear from an EZL case horse stained with Giemsa and examined for the presence of Histoplasma yeast cells. The impression smear of pus aspirated from an unruptured subcutaneous nodule was viewed at ×1,000 magnification. Arrowheads, clusters of ovoid to lemon-shaped yeast cells (diameter, 4 to 5 μm) with a characteristic refractive cell wall. For comparison, an equine neutrophil is approximately 12 to 15 μm in diameter.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5121390&req=5

Figure 2: Light micrograph of a pus impression smear from an EZL case horse stained with Giemsa and examined for the presence of Histoplasma yeast cells. The impression smear of pus aspirated from an unruptured subcutaneous nodule was viewed at ×1,000 magnification. Arrowheads, clusters of ovoid to lemon-shaped yeast cells (diameter, 4 to 5 μm) with a characteristic refractive cell wall. For comparison, an equine neutrophil is approximately 12 to 15 μm in diameter.

Mentions: Impression smears were prepared from a total of 27 pus samples and 28 blood samples from the 29 case horses (in 2 horses with cutaneous lesions, it was not possible to aspirate nodules, and for 1 horse, only a pus sample was taken). Yeast cells were apparent in 14 (52%) pus smear preparations, as determined by light microscopy (Fig. 2). No yeast cells were visible on any of the blood smear preparations.


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
Light micrograph of a pus impression smear from an EZL case horse stained with Giemsa and examined for the presence of Histoplasma yeast cells. The impression smear of pus aspirated from an unruptured subcutaneous nodule was viewed at ×1,000 magnification. Arrowheads, clusters of ovoid to lemon-shaped yeast cells (diameter, 4 to 5 μm) with a characteristic refractive cell wall. For comparison, an equine neutrophil is approximately 12 to 15 μm in diameter.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5121390&req=5

Figure 2: Light micrograph of a pus impression smear from an EZL case horse stained with Giemsa and examined for the presence of Histoplasma yeast cells. The impression smear of pus aspirated from an unruptured subcutaneous nodule was viewed at ×1,000 magnification. Arrowheads, clusters of ovoid to lemon-shaped yeast cells (diameter, 4 to 5 μm) with a characteristic refractive cell wall. For comparison, an equine neutrophil is approximately 12 to 15 μm in diameter.
Mentions: Impression smears were prepared from a total of 27 pus samples and 28 blood samples from the 29 case horses (in 2 horses with cutaneous lesions, it was not possible to aspirate nodules, and for 1 horse, only a pus sample was taken). Yeast cells were apparent in 14 (52%) pus smear preparations, as determined by light microscopy (Fig. 2). No yeast cells were visible on any of the blood smear preparations.

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus