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Journal of clinical microbiology2016; 54(12); 2990-2999; doi: 10.1128/JCM.00896-16

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.
Copyright © 2016 Scantlebury et al.
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Publication Date: 2016-10-05 PubMed ID: 27707938PubMed Central: PMC5121390DOI: 10.1128/JCM.00896-16Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This study evaluates a diagnostic method (nested PCR) for the rapid detection of Histoplasma capsulatum var. farciminosum, a fungus causing epizootic lymphangitis (EZL) in horses.

Objective of the Research

  • The research aimed at developing and evaluating a molecular diagnostic method, the nested PCR, which targets the internal transcribed spacer (ITS) region of the rRNA operon of Histoplasma capsulatum var. farciminosum.
  • This method was intended to be a more effective and reliable detection method for EZL in horses because existing diagnostic methods, such as clinical symptom observation and microscopy, lack specificity and hinder understanding of this disease.

Methodology and Results

  • Participants of the study were horses from diverse climatic regions of Ethiopia. Twenty-nine of these horses showed signs of EZL, and blood samples and pus aspirates from nodules were collected.
  • The PCR method validated the presence of H. capsulatum var. farciminosum DNA in 25 and 17 of the respective pus and blood samples.
  • Additionally, positive PCR results were obtained from heat-inactivated pus and blood spotted onto Whatman FTA cards, which are used for collecting and preserving nucleic acids at room temperature.
  • Horses without cutaneous EZL symptoms had two positive blood results.
  • This research marks the first progress in directly detecting H. capsulatum var. farciminosum in equine blood, especially in horses showing skin lesions.

Comparison with Traditional Methods

  • The nested PCR outperformed the conventional microscopic diagnosis as only 14 pus samples displayed characteristic yeast cells.

Insights for Further Research

  • Through sequence alignment, the research confirmed the presence of H. capsulatum var. farciminosum DNA and identified very little sequence variation.
  • The research further reveals a preliminary single nucleotide polymorphism, indicating the potential existence of two subgroups of H. capsulatum var. farciminosum.
  • This breakthrough in molecular diagnostic methodology now allows for more in-depth investigation of the epidemiology of EZL.

Cite This Article

APA
Scantlebury CE, Pinchbeck GL, Loughnane P, Aklilu N, Ashine T, Stringer AP, Gordon L, Marshall M, Christley RM, McCarthy AJ. (2016). 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. J Clin Microbiol, 54(12), 2990-2999. https://doi.org/10.1128/JCM.00896-16

Publication

Journal of clinical microbiology
ISSN: 1098-660X
NlmUniqueID: 7505564
Country: United States
Language: English
Volume: 54
Issue: 12
Pages: 2990-2999

Researcher Affiliations

Scantlebury, C E
  • Department of Epidemiology and Population Health, Institute of Infection and Global Health, University of Liverpool, Neston, Wirral, United Kingdom claire.scantlebury@liverpool.ac.uk aj55m@liverpool.ac.uk.
  • Microbiology Research Group, Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom.
Pinchbeck, G L
  • Department of Epidemiology and Population Health, Institute of Infection and Global Health, University of Liverpool, Neston, Wirral, United Kingdom.
Loughnane, P
  • Microbiology Research Group, Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom.
Aklilu, N
  • SPANA Ethiopia, College of Veterinary Medicine and Agriculture, Addis Ababa University, Addis Ababa, Ethiopia.
Ashine, T
  • SPANA Ethiopia, College of Veterinary Medicine and Agriculture, Addis Ababa University, Addis Ababa, Ethiopia.
Stringer, A P
  • SPANA UK, London, United Kingdom.
Gordon, L
  • Microbiology Research Group, Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom.
Marshall, M
  • Microbiology Research Group, Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom.
Christley, R M
  • Department of Epidemiology and Population Health, Institute of Infection and Global Health, University of Liverpool, Neston, Wirral, United Kingdom.
  • NIHR Health Protection Research Unit in Emerging and Zoonotic Infections, Liverpool, United Kingdom.
McCarthy, A J
  • Microbiology Research Group, Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom claire.scantlebury@liverpool.ac.uk aj55m@liverpool.ac.uk.

MeSH Terms

  • Animals
  • Blood / microbiology
  • Corynebacterium pseudotuberculosis / isolation & purification
  • DNA, Ribosomal Spacer / genetics
  • Diagnosis, Differential
  • Ethiopia
  • Histoplasma / classification
  • Histoplasma / isolation & purification
  • Histoplasmosis / diagnosis
  • Histoplasmosis / microbiology
  • Histoplasmosis / veterinary
  • Horse Diseases / diagnosis
  • Horse Diseases / microbiology
  • Horses
  • Lymphangitis / diagnosis
  • Lymphangitis / microbiology
  • Lymphangitis / veterinary
  • Molecular Diagnostic Techniques / methods
  • Polymerase Chain Reaction / methods
  • Suppuration / microbiology

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