Comparative Assessment of In-House Real-Time PCRs Targeting Enteric Disease-Associated Microsporidia in Human Stool Samples

Konstantin Tanida,Fred Stephen Sarfo,Andreas Hahn,Veronica Di Cristanziano,Ulrike Loderstädt,Torsten Feldt,Kirsten Alexandra Eberhardt,Olfert Landt,Simone Kann,Egbert Tannich,Hagen Frickmann

doi:10.3390/pathogens10060656

Abstract

Microsporidiosis is an infection predominantly occurring in immunosuppressed patients and infrequently also in travelers. This study was performed to comparatively evaluate the diagnostic accuracy of real-time PCR assays targeting microsporidia with etiological relevance in the stool of human patients in a latent class analysis-based test comparison without a reference standard with perfect accuracy. Thereby, two one-tube real-time PCR assays and two two-tube real-time PCR assays targeting Enterocytozoon bieneusi and Encephalocytozoon spp. were included in the assessment with reference stool material (20), stool samples from Ghanaian HIV-positive patients (903), and from travelers, migrants and Colombian indigenous people (416). Sensitivity of the assays ranged from 60.4% to 97.4% and specificity from 99.1% to 100% with substantial agreement according to Cohen’s kappa of 79.6%. Microsporidia DNA was detected in the reference material and the stool of the HIV patients but not in the stool of the travelers, migrants, and the Colombian indigenous people. Accuracy-adjusted prevalence was 5.8% (n = 78) for the study population as a whole. In conclusion, reliable detection of enteric disease-associated microsporidia in stool samples by real-time PCR could be demonstrated, but sensitivity between the compared microsporidia-specific real-time PCR assays varied.

Highlights

Microsporidiosis is a disease caused by intracellular pathogens related to fungi as indicated by phylogenetical analyses [1]
Microsporidiaspecific DNA was detected in diagnostic samples from the diagnostic department of the Bernhard Nocht Institute in Hamburg and in the samples from the HIV patients from Ghana only, while no microsporidia DNA was identified in stool of any other assessed population
There was no potentially interfering positivity of PCR 6 with specificity for the non-target fungal agents Microsporidium spp. observed; all samples remained negative in PCR 6

Summary

Introduction

Microsporidiosis is a disease caused by intracellular pathogens related to fungi as indicated by phylogenetical analyses [1]. Microsporidia were already known at the beginning of the 20th century [2], they had initially been misidentified as primitive protozoa and only later were assigned to the phylum of fungi [3], while the phylogenetic assignment remained controversial [4]. More recent assessments consider microsporidia as “intracellular parasites . Related to fungi” [1,5]. The genera Nosema, Vittaforma, Brachiola, Pleistophora, Encephalitozoon, Enterocytozoon, Septata (reclassified to Encephalitozoon), and Trachipleistophora have been associated with human disease [2]. Thereby, the four species Enterocytozoon bieneusi, Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis are considered as the quantitatively most relevant ones with particular focus on Enterocytozoon bieneusi [7]. Therapeutic options are scarce and poorly standardized, comprising tubulin-inhibiting albendazole (Encephalocytozoon spp.)

Methods

Results

Discussion

Conclusion