Two lenses, one pathogen: Complementing microscopy with molecular investigation for accurate microsporidia diagnosis
DOI:
https://doi.org/10.63719/imrj.2026.01(SE).01Keywords:
Microsporidium spp., phylogenetic tree analysis, SSU rRNA, immunocompromised, E. bieneusiAbstract
Microsporidium is an intestinal protozoan parasite that infects a wide range of animal species and has emerged as an important pathogen in immunocompromised individuals. The most common species infecting humans include Enterocytozoon bieneusi, Encephalitozoon intestinalis, Encephalitozoon cuniculi, and Encephalitozoon hellem. Microscopic examination remains the primary diagnostic method as it is rapid and reliable; however, it is limited to genus-level identification. This study aimed to apply molecular techniques to complement microscopy in the identification of Microsporidium species. A total of 121 stool samples were re-analysed using polymerase chain reaction (PCR) targeting species-specific small subunit rRNA (SSU rRNA) genes, which include 109 samples previously identified as positive for Microsporidium spp. by Gram-chromotrope staining and 12 samples that were negative by the same method. The results showed that eight samples (6.6%) were PCR-positive for Microsporidium spp., all identified as E. bieneusi. Of these, 3 samples were previously positive by microscopy (3/109; 2.75%), while 5 were from microscopy-negative samples (5/12; 41.67%). The amplified products were sequenced for confirmation. All samples were negative for E. intestinalis, E. cuniculi, E. hellem, and Vittaforma corneae. Phylogenetic analyses using three different methods showed that the detected strains were closely related to isolates from immunocompromised patients in Malaysia and diarrheal patients in China. The discrepancy between PCR and microscopy results may be due to several factors and requires further investigation. A larger sample size and combined diagnostic methods are needed to evaluate the role of molecular techniques in Microsporidium detection. These findings highlight the importance of using both molecular and microscopic methods.
References
Franzen C, Müller A. Microsporidiosis: Human diseases and diagnosis. Microbes Infect. 2001; 3(5):389–400. doi:10.1016/S1286-4579(01)01395-8.
Ghoshal U, Khanduja S, Pant P, Prasad K, Dhole T, Sharma R, et al. Intestinal Microsporidiosis in renal transplant recipients: Prevalence, predictors of occurrence and genetic characterization. Indian J Med Microbiol. 2015;33(3):357–63. doi:org/10.4103/0255-0857.158551.
Galván-Díaz AL, Magnet A, Fenoy S, Henriques-Gil N, Haro M, Gordo FP, et al. Microsporidia detection and genotyping study of human pathogenic E. bieneusi in animals from Spain. PLoS One. 2014;9(3):1–5. doi: org/10.1371/journal.pone.0092289.
Nourrisson C, Moniot M, Tressol M, Lambert C, Fréalle E, Robert-Gangneux F, et al. Multicenter comparative study of Enterocytozoon bieneusi DNA extraction methods from stool samples, and mechanical pretreatment protocols evaluation. Sci Rep. 2024;14(1):1–9. doi:org/10.1038/s41598-024-66154-2.
Lobo ML, Xiao L, Antunes F, Matos O. Microsporidia as emerging pathogens and the implications for public health: A 10-year study on HIV-positive and -negative patients. Int J Parasitol. 2012;42(2):197–205. doi: 10.1371/journal.pone.0035239.
Mohamed Yusoff PS, Osman E, Raja Sabudin RZA. Disseminated Microsporidiosis: An underdiagnosed and emerging opportunistic disease. Malays J Pathol. 2021;43(1):9–18.
Tekeli NTF, Al-Jarjary SAA, Sheet OH. Molecular detection and phylogenetic analysis of Microsporidia in water and soil in Mosul city. Open Vet J. 2024;14(9):2421–32. doi: org/10.5455/ovj.2024.v14.i9.31.
Ruan Y, Xu X, He Q, Li L, Guo J, Bao J, et al. The largest meta-analysis on the global prevalence of Microsporidia in mammals, avian, and water provides insights into the epidemic features of these ubiquitous pathogens. Parasites and Vectors. 2021;14(1):1–14. doi: org/10.1186/s13071-021-04700-x
Lono A, Kumar GS, Chye TT. Prevalence of Microsporidia in an indigenous Orang Asli community in Pahang, Malaysia. Trans R Soc Trop Med Hyg. 2010;104(3):214–8. doi: 10.1016/j.trstmh.2009.07.006.
Nasarudin, SNS., Zainudin, NS., Bernadus, M., Nawi, AM., Hanafiah, A., & Osman, E. (2015). Loop-mediated isothermal amplification for rapid molecular detection of Enterocytozoon bieneusi in faecal specimens. Journal of Medical Microbiology, 64(11), 1329–1334. doi:10.1099/jmm.0.000156.
Norhayati M, Al-Mekhlafi HM, Azlin M, Nor Aini U, Shaik A, Sa’Iah A, et al. Intestinal Microsporidial infections among Orang Asli (aborigine) children from Malaysia. Ann Trop Med Parasitol. 2007;101(6):547–50. doi: 10.1179/136485907X193789.
Lono, A., Kumar, S., & Chye, TT. (2011). Detection of Microsporidia in local HIV-positive population in Malaysia. Transactions of the Royal Society of Tropical Medicine and Hygiene, 105(7), 409–413. doi: org/10.1016/j.trstmh.2011.03.006.
Hassan NA, Lim YAL, Mahmud R, Mohd-Shaharuddin N, Wan Sulaiman WY, Ngui R. Molecular diagnosis of Microsporidia among immunocompromised patients in Kuala Lumpur, Malaysia. Am J Trop Med Hyg. 2018;99(6):1562–6. doi: org/10.1016/j.ijheh.2018.03.007.
Mumcuoglu I, Cetin F, Dogruman Al F, Oguz I, Aksu N. Prevalence of Microsporidia in healthy individuals and immunocompetent patients with acute and chronic diarrhea. Infect Dis (Auckl). 2016;48(2):133–7. doi: 10.3109/23744235.2015.1094572.
Zahran F, Ibrahim A, Abou-Seri H. Evaluation of different laboratory techniques for diagnosis of intestinal Microsporidiosis in diarrheic children. Parasitol United J. 2020;13(2):93–8. doi: 10.21608/puj.2020.26216.1063.
Wei J, Fei Z, Pan G, Weiss LM, Zhou Z. Current Therapy and Therapeutic Targets for Microsporidiosis. Front Microbiol. 2022;13(March):1–14. doi: 10.3389/fmicb.2022.835390.
Valenčáková A, Sučik M. Alternatives in molecular diagnostics of Encephalitozoon and Enterocytozoon infections. J Fungi. 2020;6(3):1–18. doi:10.3390/jof6030114.
Visvesvara GS, Leitch GJ, da Silva AJ, Croppo GP, Moura H, Wallace S, et al. Polyclonal and monoclonal antibody and PCR-amplified small-subunit rRNA identification of a microsporidian, Encephalitozoon hellem, isolated from an AIDS patient with disseminated infection. J Clin Microbiol. 1994 Nov;32(11):2760–8. doi: 10.1128/JCM.32.11.2760-2768.1994.
Da Silva AJ, Slemenda SB, Visvesvara GS, Schwartz DA, Mel Wilcox C, Wallace S, et al. Detection of Septata intestinalis (microsporidia) using polymerase chain reaction primers targeting the small subunit ribosomal RNA coding region. Mol Diagnosis. 1997;2(1):47–52. doi: 10.1054/MODI00200047.
Müller A, Stellermann K, Hartmann P, Schrappe M, Fätkenheuer G, Salzberger B, et al. A powerful DNA extraction method and PCR for detection of Microsporidia in clinical stool specimens. Clin Diagn Lab Immunol. 1999;6(2):243–6. doi: 10.1128/CDLI.6.2.243-246.1999.
Ghosh K, Weiss LM. Molecular diagnostic tests for Microsporidia. Interdiscip Perspect Infect Dis. 2009; 2009:1–13. doi:10.1155/2009/926521.
Hasegawa M, Kishino H, Yano T. Dating of the human-ape splitting by a molecular clock of mitochondrial DNA. J Mol Evol. 1985;22(2):160–74. doi: 10.1007/BF02101694.
Nei, M. and Kumar S. Molecular evolution and phylogenetics. Oxford Univ Press New York. 2000. ISBN: 978-0195135855.
Felsenstein, J. (1985). Confidence limits on phylogenies: An approach using the bootstrap. Evolution, 39(4), 783–791. doi: 10.2307/2408678
Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018;35(6):1547–9. doi: 10.1093/molbev/msy096
Rambaut A. FigTree v1.4.4: Tree Figure Drawing Tool. Institute of Evolutionary Biology, University of Edinburgh, Edinburgh; 2018.
Behravan M, Yusuf MA, Solgi R, Haghighi A. Prevalence of intestinal Microsporidiosis by morphological and molecular methods in eastern Iran. Gene Reports. 2021;24(July):4–7. doi: 10.1016/j.genrep.2021.101296.
Colmer-Hamood JA. Fecal Microscopy: Artifacts mimicking ova and parasites. Laboratory Medicine. 2001;32(2):80–84. doi: 10.1309/0VTA-YUXK-F20G-T20E.
Stensvold CR, Nielsen HV. Comparison of microscopy and PCR for detection of intestinal parasites in Danish patients supports an incentive for molecular screening platforms. J Clin Microbiol. 2012;50(2):540–1. doi: 10.1128/JCM.06012-11.
Dowd SE, Gerba CP, Javier Enríquez F, Pepper IL. PCR amplification and species determination of Microsporidia in formalin-fixed feces after immunomagnetic separation. Appl Environ Microbiol. 1998;64(1):333–6 , doi: 10.1128/AEM.64.1.333-336.1998.
Jangpatarapongsa K, Saimuang K, Polpanich D, Thiramanas R, Techakasikornpanich M, Yudech P, et al. Increased sensitivity of enterotoxigenic Escherichia coli detection in stool samples using oligonucleotide immobilised magnetic nanoparticles. Biotechnol Reports. 2021;32: e00677. doi: 10.1016/j.btre. 2021.e00677
Lantz PG, Matsson M, Wadström T, Rådström P. Removal of PCR inhibitors from human faecal samples through the use of an aqueous two-phase system for sample preparation prior to PCR. J Microbiol Methods. 1997;28(3):159–67. Doi: 10.1016/S0167-7012(97)00912-9.
Morio F, Poirier P, Le Govic Y, Laude A, Valot S, Desoubeaux G, et al. Assessment of the first commercial multiplex PCR kit (ParaGENIE Crypto-Micro Real-Time PCR) for the detection of Cryptosporidium spp., Enterocytozoon bieneusi, and Encephalitozoon intestinalis from fecal samples. Diagn Microbiol Infect Dis. 2019;95(1):34–7. doi: 10.1016/j.diagmicrobio.2019.04.004
Wang SS, Wang RJ, Fan XC, Liu TL, Zhang LX, Zhao GH. Prevalence and genotypes of Enterocytozoon bieneusi in China. Acta Trop. 2018;183(February):142–52. Doi: 10.1016/j.actatropica.2018.04.017.
Wolk DM, Schneider SK, Wengenack NL, Sloan LM, Rosenblatt JE. Real-time PCR method for detection of Encephalitozoon intestinalis from stool specimens. J Clin Microbiol. 2002;40(11):3922–8. doi: 10.1128/JCM.40.11.3922-3928.2002.
Tersigni J, Tamim El Jarkass H, James EB, Reinke AW. Interactions between Microsporidia and other members of the microbiome. J Eukaryot Microbiol. 2024;(February):1–15. doi: 10.1111/jeu.13025
Josephson KL, Gerba CP, Pepper IL. Polymerase chain reaction detection of nonviable bacterial pathogens. Appl Environ Microbiol. 1993;59(10):3513–5. doi: 10.1128/AEM.59.10.3513-3515.1993.
Zeng D, Chen Z, Jiang Y, Xue F, Li B. Advances and challenges in viability detection of foodborne pathogens. Front Microbiol. 2016;7:1–12. doi: 10.3389/fmicb.2016.01833.
Ashikin A, Al-Mekhlafi HM, Moktar N, Anuar TS. Molecular detection and species identification of Enterocytozoon bieneusi isolated from immunocompetent Orang Asli in Malaysia. Parasitol Int. 2017;66(2):163–5. doi:org/10.1016/j.parint.2017.01.013.
Han B, Weiss LM. Therapeutic targets for the treatment of Microsporidiosis in humans. Expert Opin Ther Targets. 2018;22(11):903–15. doi: 10.3389/fmicb.2016.01833.
Maillard A, Scemla A, Laffy B, Mahloul N, Molina JM. Safety and efficacy of fumagillin for the treatment of intestinal Microsporidiosis. A French prospective cohort study. J Antimicrob Chemother. 2021;76(2):487–94. doi: 10.1093/jac/dkaa460.
Zhou L, Guan Z, Chen C, Zhu Q, Qiu S, Liu Y, et al. The successful treatment of Enterocytozoon bieneusi Microsporidiosis with nitazoxanide in a patient with B-ALL: A Case Report. Front Cell Infect Microbiol. 2023;12(January):1–7. doi: 10.3389/fcimb.2022.1097951.
Rijsman LH, Monkelbaan JF, Kusters JG. Clinical consequences of polymerase chain reaction-based diagnosis of intestinal parasitic infections. J Gastroenterol Hepatol. 2016;31(11):1808–15. doi: 10.1111/jgh.13356.
