An optimized procedure for detecting enterocytozoon intestinalis and encephalitozoon bieneusi using polymerase chain reaction technology

Authors: Jenkins, Mark; Obrien, Celia; Parker, Carolyn

Submitted to: Journal of Parasitic Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/16/2018
Publication Date: 12/4/2018
Citation: Jenkins, M.C., O'Brien, C.N., Parker, C.C. 2018. An optimized procedure for detecting enterocytozoon intestinalis and encephalitozoon bieneusi using polymerase chain reaction technology. Journal of Parasitic Diseases. 43(1):75-82. https://doi.org/10.1007/s12639-018-1060-5
DOI: https://doi.org/10.1007/s12639-018-1060-5

Interpretive Summary: Microsporidiosis is an emerging diarrheal disease in humans and other mammals, particularly dairy calves. The most often identified microsporidia in human and animal infections are in the genus Encephalitozoon and Enterocytozoon. Infection occurs by a fecal-oral route, and is most prevalent in the young who have incompletely developed immune systems or in immunosuppressed individuals, such as AIDS patients. Detecting the parasites in patient stool has improved through the use of monoclonal antibodies that can be used in epifluorescence microscopy to identify spores. However, the small size of the spores (around 1 um) makes it difficult to reliably diagnose infection. In the present study, an improved PCR technique was developed that is not only extremely sensitive, but also includes an internal standard that can control for false negative reactions. The presence of inhibitory substances in fecal material has compromised the reliability of PCR-based methods for detecting microsporidia in patient samples. In this study, it was found that the improved PCR methods for two types of microsporidia, namely Encephalitozoon and Enterocytozoon, were in excellent agreement with immunofluorescence detection methods. The advantage of the PCR methods is that it is less subjective than microscopy and does not require highly trained individuals to diagnose infection.

Technical Abstract: The purpose of this study was to optimize a sensitive molecular assay for detecting the microsporidia Encephalitozoon and Enterocytozoon in fecal samples from dairy calves and compare this method to immunofluorescence assays (IFA) based on commercially available monoclonal antibodies specific for outer wall proteins of Enc. intestinalis or Ent. bieneusi. Fecal samples were collected from 15 dairy calves and processed by molecular sieving followed by salt floatation. An aliquot of the final supernatant was applied to glass slides for IFA testing; another aliquot was extracted for total DNA using a QIAamp Stool Mini-Kit for primary and nested Encephalitozoon- and Enterocytozoon-specific PCR analysis. An internal standard was generated for both primary PCR assays to control for false negative reactions due to the presence of inhibitors commonly found in fecal samples. Using the commercial MicrosporIFA as the gold standard, the optimized Encephalitozoon PCR method provided 85.7 % sensitivity and 100 % specificity with a kappa value = 0.865. Likewise, using the commercial BienusiGlo IFA as the gold standard, the optimized Enterocytozoon PCR method provided 83.3 % sensitivity and 100 % specificity with a kappa value = 0.857. Sequencing of amplicons from both PCR assays confirmed the presence of Encephalitozoon or Enterocytozoon. In conclusion, our optimized PCR methods for detecting Encephalitozoon or Enterocytozoon in fecal smears from dairy calves provides a valuable alternative to traditional IFA methods and improves upon existing molecular detection techniques.