1887

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Volume 60, Issue 4

Detection and species identification of microsporidial infections using SYBR Green real-time PCR Free

Abstract

Diagnosis of microsporidial infections is routinely performed by light microscopy, with unequivocal non-molecular species identification achievable only through electron microscopy. This study describes a single SYBR Green real-time PCR assay for the simultaneous detection and species identification of such infections. This assay was highly sensitive, routinely detecting infections containing 400 parasites (g stool sample), whilst species identification was achieved by differential melt curves on a Corbett Life Science Rotor-Gene 3000. A modification of the QIAamp DNA tissue extraction protocol allowed the semi-automated extraction of DNA from stools for the routine diagnosis of microsporidial infection by real-time PCR. Of 168 stool samples routinely analysed for microsporidian spores, only five were positive by microscopy. By comparison, 17 were positive for microsporidial DNA by real-time analysis, comprising 14 , one and two separate species infections.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): CI, confidence limit , Ct, cycle threshold , SSU, small subunit and TEM, transmission electron microscopy
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2011-04-01
2024-04-24
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References

  1. Abe N., Kimata I., Iseki M. 2003; Identification of genotypes of Giardia intestinalis isolates from dogs in Japan by direct sequencing of the PCR amplified glutamate dehydrogenase gene. J Vet Med Sci 65:29–33 [CrossRef]
     [Google Scholar]
  2. Aviles H., Belli A., Armijos R., Monroy F. P., Harris E. 1999; PCR detection and identification of Leishmania parasites in clinical specimens in Ecuador: a comparison with classical diagnostic methods. J Parasitol 85:181–187 [CrossRef]
     [Google Scholar]
  3. Berry A., Fabre R., Benoit-Vical F., Cassaing S., Magnaval J. F. 2005; Contribution of PCR-based methods to diagnosis and management of imported malaria. Med Trop (Mars 65:176–183
     [Google Scholar]
  4. Bertrand I., Gantzer C., Chesnot T., Schwartzbrod J. 2004; Improved specificity for Giardia lamblia cyst quantification in wastewater by development of a real-time PCR method. J Microbiol Methods 57:41–53 [CrossRef]
     [Google Scholar]
  5. Chabchoub N., Abdelmalek R., Mellouli F., Kanoun F., Thellier M., Bouratbine A., Aoun K. 2009; Genetic identification of intestinal microsporidia species in immunocompromised patients in Tunisia. Am J Trop Med Hyg 80:24–27
     [Google Scholar]
  6. Chupp G. L., Alroy J., Adelman L. S., Breen J. C., Skolnik P. R. 1993; Myositis due to Pleistophora (microsporidia) in a patient with AIDS. Clin Infect Dis 16:15–21 [CrossRef]
     [Google Scholar]
  7. da Silva A. J., Slemenda S. B., Visvesvara G. S., Schwartz D. A., Wilcox C. M., Wallace S., Pieniazek N. J. 1997a; Detection of Septata intestinalis (microsporidia) Cali et al. 1993 using polymerase chain reaction primers targeting the small subunit ribosomal RNA coding region. Mol Diagn 2:47–52 [CrossRef]
     [Google Scholar]
  8. da Silva A. J., Bornay-Llinares F. J., del Aguila de la Puente Cdel. A., Moura H., Peralta J. M., Sobottka I., Schwartz D. A., Visvesvara G. S., Slemenda S. B., Pieniazek N. J. 1997b; Diagnosis of Enterocytozoon bieneusi (microsporidia) infections by polymerase chain reaction in stool samples using primers based on the region coding for small-subunit ribosomal RNA. Arch Pathol Lab Med 121:874–879
     [Google Scholar]
  9. De Groote M. A., Visvesvara G., Wilson M. L., Pieniazek N. J., Slemenda S. B., daSilva A. J., Leitch G. J., Bryan R. T., Reves R. 1995; Polymerase chain reaction and culture confirmation of disseminated Encephalitozoon cuniculi in a patient with AIDS: successful therapy with albendazole. J Infect Dis 171:1375–1378 [CrossRef]
     [Google Scholar]
  10. Didier E. S., Rogers L. B., Brush A. D., Wong S., Traina-Dorge V., Bertucci D. 1996; Diagnosis of disseminated microsporidian Encephalitozoon hellem infection by PCR-Southern analysis and successful treatment with albendazole and fumagillin. J Clin Microbiol 34:947–952
     [Google Scholar]
  11. Dieterich D. T., Lew E. A., Kotler D. P., Poles M. A., Orenstein J. M. 1994; Treatment with albendazole for intestinal disease due to Enterocytozoon bieneusi in patients with AIDS. J Infect Dis 169:178–183 [CrossRef]
     [Google Scholar]
  12. Ditrich O, Kucerová Z., Koudela B. 1994; In vitro sensitivity of Encephalitozoon cuniculi and E. hellem to albendazole. J Eukaryot Microbiol 41:37S
     [Google Scholar]
  13. Fabre R., Berry A., Morassin B., Magnaval J. F. 2004; Comparative assessment of conventional PCR with multiplex real-time PCR using SYBR Green I detection for the molecular diagnosis of imported malaria. Parasitology 128:15–21 [CrossRef]
     [Google Scholar]
  14. Field A. S., Marriott D. J., Milliken S. T., Brew B. J., Canning E. U., Kench J. G., Darveniza P., Harkness J. L. 1996; Myositis associated with a newly described microsporidian, Trachipleistophora hominis , in a patient with AIDS. J Clin Microbiol 34:2803–2811
     [Google Scholar]
  15. Franzen C., Schwartz D. A., Visvesvara G. S., Müller A., Schwenk A., Salzberger B., Fätkenheuer G., Hartmann P., Mahrle G. other authors 1995; Immunologically confirmed disseminated, asymptomatic Encephalitozoon cuniculi infection of the gastrointestinal tract in a patient with AIDS. Clin Infect Dis 21:1480–1484 [CrossRef]
     [Google Scholar]
  16. Gama B. E., Silva-Pires F. E., Lopes M. N., Cardoso M. A., Britto C., Torres K. L., de Mendonça Lima L., de Souza J. M., Daniel-Ribeiro C. T., Ferreira-da-Cruz M. F. 2007; Real-time PCR versus conventional PCR for malaria parasite detection in low-grade parasitemia. Exp Parasitol 116:427–432 [CrossRef]
     [Google Scholar]
  17. Garcia L. S. 2002; Laboratory identification of the microsporidia. J Clin Microbiol 40:1892–1901 [CrossRef]
     [Google Scholar]
  18. Ghosh K., Weiss L. M. 2009; Molecular diagnostic tests for microsporidia. Interdiscip Perspect Infect Dis 2009926521
     [Google Scholar]
  19. Grau A., Valls M. E., Williams J. E., Ellis D. S., Muntané M. J., Nadal C. 1996; Myositis caused by Pleistophora in a patient with AIDS. Med Clin (Barc) 107:779–781
     [Google Scholar]
  20. Gumbo T., Gangaidzo I. T., Sarbah S., Carville A., Tzipori S., Wiest P. M. 2000; Enterocytozoon bieneusi infection in patients without evidence of immunosuppression: two cases from Zimbabwe found to have positive stools by PCR. Ann Trop Med Parasitol 94:699–702
     [Google Scholar]
  21. Guy R. A., Xiao C., Horgen P. A. 2004; Real-time PCR assay for detection and genotype differentiation of Giardia lamblia in stool specimens. J Clin Microbiol 42:3317–3320 [CrossRef]
     [Google Scholar]
  22. Helmy M. M., Abdel-Fattah H. S., Rashed L. 2009; Real-time PCR/RFLP assay to detect Giardia intestinalis genotypes in human isolates with diarrhea in Egypt. J Parasitol 95:1000–1004 [CrossRef]
     [Google Scholar]
  23. Joseph J., Sharma S., Murthy S. I., Krishna P. V., Garg P., Nutheti R., Kenneth J., Balasubramanian D. 2006; Microsporidial keratitis in India: 16S rRNA gene-based PCR assay for diagnosis and species identification of microsporidia in clinical samples. Invest Ophthalmol Vis Sci 47:4468–4473 [CrossRef]
     [Google Scholar]
  24. Katakai Y., Chiabchalard R., Komaki-Yasuda K., Kawazu S., Singhasivanon P., Krudsood S., Looareesuwan S., Kano S. 2004; Application of real-time polymerase chain reaction (PCR) analysis for detection and discrimination of malaria parasite species in Thai patients. Southeast Asian J Trop Med Public Health 35:S10–S14
     [Google Scholar]
  25. Katakura K., Kawazu S., Naya T., Nagakura K., Ito M., Aikawa M., Qu J. Q., Guan L. R., Zuo X. P. other authors 1998; Diagnosis of kala-azar by nested PCR based on amplification of the Leishmania mini-exon gene. J Clin Microbiol 36:2173–2177
     [Google Scholar]
  26. Koudela B., Lom J., Vítovec J., Kucerová Z, Ditrich O, Trávnícek J. 1994; In vivo efficacy of albendazole against Encephalitozoon cuniculi in SCID mice. J Eukaryot Microbiol 41:49S–50S
     [Google Scholar]
  27. Kumar S. S., Ananthan S., Joyee A. G. 2005; Detection of Enterocytozoon bieneusi (Microsporidia) by polymerase chain reaction (PCR) using species-specific primer in stool samples of HIV patients. Indian J Med Res 121:215–219
     [Google Scholar]
  28. Lubell Y., Reyburn H., Mbakilwa H., Mwangi R., Chonya K., Whitty C. J., Mills A. 2007; The cost-effectiveness of parasitologic diagnosis for malaria-suspected patients in an era of combination therapy. Am J Trop Med Hyg 77:S128–S132
     [Google Scholar]
  29. Mangold K. A., Manson R. U., Koay E. S., Stephens L., Regner M., Thomson R. B. Jr, Peterson L. R., Kaul K. L. 2005; Real-time PCR for detection and identification of Plasmodium spp. J Clin Microbiol 43:2435–2440 [CrossRef]
     [Google Scholar]
  30. Mathis A., Deplazes P. 1995; PCR and in vitro cultivation for detection of Leishmania spp. in diagnostic samples from humans and dogs. J Clin Microbiol 33:1145–1149
     [Google Scholar]
  31. McGlade T. R., Robertson I. D., Elliot A. D., Thompson R. C. 2003; High prevalence of Giardia detected in cats by PCR. Vet Parasitol 110:197–205 [CrossRef]
     [Google Scholar]
  32. Menotti J., Cassinat B., Porcher R., Sarfati C., Derouin F., Molina J. M. 2003a; Development of a real-time polymerase-chain-reaction assay for quantitative detection of Enterocytozoon bieneusi DNA in stool specimens from immunocompromised patients with intestinal microsporidiosis. J Infect Dis 187:1469–1474 [CrossRef]
     [Google Scholar]
  33. Menotti J., Cassinat B., Sarfati C., Liguory O., Derouin F., Molina J. M. 2003b; Development of a real-time PCR assay for quantitative detection of Encephalitozoon intestinalis DNA. J Clin Microbiol 41:1410–1413 [CrossRef]
     [Google Scholar]
  34. Müller A., Stellermann K., Hartmann P., Schrappe M., Fätkenheuer G., Salzberger B., Diehl V., Franzen C. 1999; A powerful DNA extraction method and PCR for detection of microsporidia in clinical stool specimens. Clin Diagn Lab Immunol 6:243–246
     [Google Scholar]
  35. Niesters H. G. 2002; Clinical virology in real time. J Clin Virol 25:S3–S12
     [Google Scholar]
  36. Niesters H. G. 2004; Molecular and diagnostic clinical virology in real time. Clin Microbiol Infect 10:5–11 [CrossRef]
     [Google Scholar]
  37. Notermans D. W., Peek R., de Jong M. D., Wentink-Bonnema E. M., Boom R., van Gool T. 2005; Detection and identification of Enterocytozoon bieneusi and Encephalitozoon species in stool and urine specimens by PCR and differential hybridization. J Clin Microbiol 43:610–614 [CrossRef]
     [Google Scholar]
  38. Perandin F., Manca N., Calderaro A., Piccolo G., Galati L., Ricci L., Medici M. C., Arcangeletti M. C., Snounou G. other authors 2004; Development of a real-time PCR assay for detection of Plasmodium falciparum , Plasmodium vivax , and Plasmodium ovale for routine clinical diagnosis. J Clin Microbiol 42:1214–1219 [CrossRef]
     [Google Scholar]
  39. Ramos F., Zurabian R., Morán P., Ramiro M., Gómez A., Clark C. G., Melendro E. I., García G., Ximénez C. 1999; The effect of formalin fixation on the polymerase chain reaction characterization of Entamoeba histolytica . Trans R Soc Trop Med Hyg 93:335–336 [CrossRef]
     [Google Scholar]
  40. Rozen S., Skaletsky H. 2000; Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365–386
     [Google Scholar]
  41. Sak B., Kucerova Z., Kvac M., Kvetonova D., Rost M., Secor E. W. 2010; Seropositivity for Enterocytozoon bieneusi , Czech Republic. Emerg Infect Dis 16:335–337 [CrossRef]
     [Google Scholar]
  42. Tosoni A., Nebuloni M., Ferri A., Bonetto S., Antinori S., Scaglia M., Xiao L., Moura H., Visvesvara G. S. other authors 2002; Disseminated microsporidiosis caused by Encephalitozoon cuniculi III (dog type) in an Italian AIDS patient: a retrospective study. Mod Pathol 15:577–583 [CrossRef]
     [Google Scholar]
  43. Troll H., Marti H., Weiss N. 1997; Simple differential detection of Entamoeba histolytica and Entamoeba dispar in fresh stool specimens by sodium acetate-acetic acid-formalin concentration and PCR. J Clin Microbiol 35:1701–1705
     [Google Scholar]
  44. van Gool T., Vetter J. C., Weinmayr B., Van Dam A., Derouin F., Dankert J. 1997; High seroprevalence of Encephalitozoon species in immunocompetent subjects. J Infect Dis 175:1020–1024 [CrossRef]
     [Google Scholar]
  45. Verweij J. J., Schinkel J., Laeijendecker D., van Rooyen M. A., van Lieshout L., Polderman A. M. 2003; Real-time PCR for the detection of Giardia lamblia . Mol Cell Probes 17:223–225 [CrossRef]
     [Google Scholar]
  46. Verweij J. J., Blangé R. A., Templeton K., Schinkel J., Brienen E. A., van Rooyen M. A., van Lieshout L., Polderman A. M. 2004; Simultaneous detection of Entamoeba histolytica , Giardia lamblia , and Cryptosporidium parvum in fecal samples by using multiplex real-time PCR. J Clin Microbiol 42:1220–1223 [CrossRef]
     [Google Scholar]
  47. Verweij J. J., Ten Hove R., Brienen E. A., van Lieshout L. 2007; Multiplex detection of Enterocytozoon bieneusi and Encephalitozoon spp. in fecal samples using real-time PCR. Diagn Microbiol Infect Dis 57:163–167 [CrossRef]
     [Google Scholar]
  48. Visvesvara G. S., da Silva A. J., Croppo G. P., Pieniazek N. J., Leitch G. J., Ferguson D., de Moura H., Wallace S., Slemenda S. B. other authors 1995; In vitro culture and serologic and molecular identification of Septata intestinalis isolated from urine of a patient with AIDS. J Clin Microbiol 33:930–936
     [Google Scholar]
  49. Wang Z., Orlandi P. A., Stenger D. A. 2005; Simultaneous detection of four human pathogenic microsporidian species from clinical samples by oligonucleotide microarray. J Clin Microbiol 43:4121–4128 [CrossRef]
     [Google Scholar]
  50. Weber R., Deplazes P., Flepp M., Mathis A., Baumann R., Sauer B., Kuster H., Lüthy R. 1997; Cerebral microsporidiosis due to Encephalitozoon cuniculi in a patient with human immunodeficiency virus infection. N Engl J Med 336:474–478 [CrossRef]
     [Google Scholar]
  51. Weiss L. M., Michalakakis E., Coyle C. M., Tanowitz H. B., Wittner M. 1994; The in vitro activity of albendazole against Encephalitozoon cuniculi . J Eukaryot Microbiol 41:65S [CrossRef]
     [Google Scholar]
  52. Wolk D. M., Schneider S. K., Wengenack N. L., Sloan L. M., Rosenblatt J. E. 2002; Real-time PCR method for detection of Encephalitozoon intestinalis from stool specimens. J Clin Microbiol 40:3922–3928 [CrossRef]
     [Google Scholar]
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Detection and species identification of microsporidial infections using SYBR Green real-time PCR
J. Med. Microbiol. 60, 459 (2011); https://doi.org/10.1099/jmm.0.026781-0
/content/journal/jmm/10.1099/jmm.0.026781-0
/content/journal/jmm/10.1099/jmm.0.026781-0
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