Abstract
Microsporidia are obligate intracellular parasitic protozoa infecting the wide variety of hosts and are commonly known as a cause of chronic diarrhea particularly in immunocompromised individuals. Molecular-based tests have high sensitivity and specificity in disease diagnosis. However, these tests’ performance relies on the isolation of DNA in a good concentration. The standard procedures of commercial DNA extraction kits are usually insufficient for this purpose due to the tough walls of spores. This study aimed to test the significance of pretreatments by glass beads and freeze-thawing processes in DNA isolation from microsporidia spores. The parasite was cultured in growing Vero cells and seven serial dilutions were prepared from the collected spores. DNA purification was performed according to different tissue kits and stool kit procedures with and without any pretreatment. Concentration of isolated DNA samples were evaluated by real-time PCR. As a result of this study, the detectable amount of spores is minimum 10 spores in each 100 μl sample according to the different tissue kits’ standard protocols. However, according to the DNA stool mini kit, the detectable amount of spores was found to be 1,000 spores/100 μl of stool sample when pretreated with both the freeze-thawing and glass beads methods.In conclusion, the current study demonstrated that further pretreatments are an essential process for DNA extraction from the stool specimens in order to avoid possible false negativity in the diagnosis of microsporidiosis.
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References
Ariefdjohan, M.W., Savaiano, D.A., Nakatsu C.H. 2010. Comparison of DNA extraction kits for PCR-DGGE analysis of human intestinal microbial communities from fecal specimens. Nutrition Journal, 9, 23. DOI: 10.1186/1475-2891-9-23
Babaei, Z., Oormazdi, H., Rezaie, S., Rezaeian, M., Razmjou E. 2011. Giardia intestinalis: DNA extraction approaches to improve PCR results. Experimental Parasitology, 128, 159–162. DOI: 10.1016/j.exppara.2011.02.001
Cetinkaya, U., Yazar, S., Kuk, S., Sivcan, E., Kaynar, L., Arslan, D., Sahin I. 2016. The high prevalence of Encephalitozoon intestinalis in patients receiving chemotherapy and children with growth retardation and the validity of real-time PCR in its diagnosis. Turkısh Journal Of Medıcal Scıences, 46, 1050–1058. DOI:10.3906/sag-1504-69
Curry A. 2005. Microsporidiosis. In: Cox FEG, Wakelin D, Gillespie SH, Despommier, DD. (Eds Topley and Wilson’s) Microbiology and Microbial Infections. Washington, DC: ASM Press, 529–555
Franzen, C., Muller A. 2001. Microsporidiosis: human diseases and diagnosis. Microbes and Infection, 3, 389–400
Garcia L.S. (Ed.) 2007. Diagnostic medical parasitology. Washington DC: ASM Press, pp. 33–46
Ghosh, K., Weiss L.M. 2009. Molecular diagnostic tests for microsporidia. Interdisciplinary Perspectives on Infectious Diseases, 2009, 926521. DOI:10.1155/2009/926521
Hawash Y. 2014. DNA Extraction from Protozoan Oocysts/Cysts in Feces for Diagnostic PCR. Korean Journal of Parasitology, 52, 263–271. DOI: 10.3347/kjp.2014.52.3.263
Joseph, J., Sharma S. 2009. In vitro culture of various species of microsporidia causing keratitis: evaluation of three immortalized cell lines. Indian journal of medical microbiology, 27, 35–39
Lelu, M., Gilot-Fromont, E., Aubert, D., Richaume, A., Afonso, E., Dupuis, E., et al. 2011. Development of a sensitive method for Toxoplasma gondii oocyst extraction in soil. Veterinary Parasitology, 183, 59–67. DOI: 10.1016/j.vetpar.2011.06.018
Mirjalali, H., Mohebali, M., Mirhendi, H., Gholami, R., Keshavarz, H., Meamar, A.R., Rezaeian M. 2014. Emerging Intestinal Microsporidia Infection in HIV(+)/AIDS Patients in Iran: Microscopic and Molecular Detection. Iranian Journal of Parasitology, 9, 149–154
Paulos, S., Mateo, M., de Lucio, A., Hernandez-de Mingo, M., Bailo B., Saugar, J.M., et al. 2016. Evaluation of five commercial methods for the extraction and purification of DNA from human faecal samples for downstream molecular detection of the enteric protozoan parasites Cryptosporidium spp., Giardia duodenalis, and Entamoeba spp. Journal of Microbiological Methods, 127, 68–73. DOI: 10.1016/j.mimet.2016.05.020
Pessoa E.S.R., Mendonca Trajano-Silva, L.A., Lopes da Silva, M.A., da Cunha Goncalves-de-Albuquerque, S., de Goes, T.C., Silva de Morais, R.C., et al. 2016. Evaluation of urine for Leishmania infantum DNA detection by real-time quantitative PCR. Journal of Microbiological Methods, 131, 34–41. DOI: 10.1016/j.mimet.2016.10.002
Pinar, A., Akyon, Y., Alp, A., Erguven S. 2010. Adaptation of a sensitive DNA extraction method for detection of Entamoeba histolytica by real-time polymerase chain reaction. Mikrobiyoloji Bulteni, 44, 453–159
Polley, S.D., Boadi, S., Watson, J., Curry, A., Chiodini P.L. 2011. Detection and species identification of microsporidial infections using SYBR Green real-time PCR. Journal of Medical Microbiology, 60, 459–466. DOI: 10.1099/jmm.0.026781-0
Procop G.W. 2007. Molecular diagnostics for the detection and characterization of microbial pathogens. Clinical Infectious Diseases, 45, 99–111
Qi, M., Wang, H., Jing, B., Wang, R., Jian, F., Ning, C., Zhang L. 2016. Prevalence and multilocus genotyping of Giardia duodenalis in dairy calves in Xinjiang, Northwestern China. Parasites & Vectors, 9, 546. DOI: 10.1186/s13071-016-1828-3
Subrungruang, I., Mungthin, M., Chavalitshewinkoon-Petmitr, P., Rangsin, R., Naaglor, T., Leelayoova S. 2004. Evaluation of DNA extraction and PCR methods for detection of Enterocytozoon bienuesi in stool specimens. Journal of Clinical Microbiology, 42, 3490–3494
Weber, R., Bryan, R.T., Schwartz, D.A., Owen R.L. 1994. Human microsporidial infections. Clinical Microbiology Reviews, 7, 426–461
Weiss, L.M., Vossbrinck C.R. 1998. Microsporidiosis: molecular and diagnostic aspects. Advances in Parasitology, 40, 351–395
Yazar, S., Koru, O., Hamamcı B., Cetinkaya, U., Karaman, U., Kuk S. 2013. Microsporidia and microsporidiosis. Turkiye Parazitoloji Dergisi, 37, 123–134. DOI: 10.5152/tpd.2013.28
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Çetinkaya, Ü., Charyyeva, A., Sivcan, E. et al. Evaluation of four commercial DNA extraction kits for the detection of Microsporidia and the importance of pretreatments in DNA isolation. Acta Parasit. 63, 386–392 (2018). https://doi.org/10.1515/ap-2018-0044
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DOI: https://doi.org/10.1515/ap-2018-0044