Molina N, Polverino D, Minvielle M, Basualdo J

Language: English
References: 29
Page: 5-10
PDF size: 201.18 Kb.

ABSTRACT

Giardia lamblia (syn G. intestinalis, G. duodenalis) is the intestinal protozoan producing non-bacterial diarrhea most prevalent in the world. PCR genotype classification of Giardia in feces depends on the quality and quantity of purified DNA and the removal of a great number of inhibitors. The aim of this study was to adapt a PCR protocol to the amplification of the triosephosphate isomerase (tpi) gene of Giardia lamblia in formalin-fixed feces. The tpi gene of G. lamblia was amplified in 28 of the 34 analyzed samples (82.35%) and the B genotype was obtained in all cases. Two major modifications were implemented to improve the performance of PCR from formulated fecal matter. One of these improvements was the use of polyvinylpyrrolidone (PVP) and the other was the addition of bovine serum albumin (BSA). The PCR protocol used in this study showed an amplification percentage exceeding the values reported by other authors with high sensibility and specificity.

REFERENCES

1. Adam, R.D. 2001. Biology of Giardia lamblia. Clin. Microbiol. Rev. 14: 447-475.

2. Ali, S.A. & D.R. Hill. 2003. Giardia intestinalis. Curr. Opin. Infect. Dis. 16: 453-460.

3. Al-Soud, W.A. & P. Rådström. 1998. Capacity of Nine Thermostable Polymerases To Mediate DNA Amplification in the Presence of PCR-Inhibiting Samples. Appl. Environ. Microbiol. 64: 3748-3753.

4. Al-Soud, W.A. & P. Rådström. 2000. Effects of Amplification Facilitators on Diagnostic PCR in the Presence of Blood, Feces and Meat. J. Clin. Microbiol. 38: 4463-4470.

5. Amar, C.F.L., P.H. Dear, S. Pedraza-Díaz, N. Looker, E. Linnane & J. McLauchlin. 2002. Sensitive PCR-Restriction Fragment Length Polymorphism Assay for Detection and Genotyping of Giardia duodenalis in Human Feces. J. Clin. Microbiol. 40: 446-452.

6. Amar, C.F.L., P.H. Dear & J. McLauchlin. 2003. Detection and genotyping by real-time PCR/RFLP analyses of Giardia duodenalis from human faeces. J. Med. Microbiol. 52: 681-683.

7. Bienz, M., M. Siles-Lucas, P. Wittwer & N. Müller. 2001. vsp Gene Expression by Giardia lamblia Clone GS/M-83-H7 during Antigenic Variation In Vivo and In Vitro. Infect. Immun. 69: 5278-5285.

8. Carnevale, S., J.N. Velásquez, J.H. Labbé, A. Chertcoff, M.G. Cabrera & M.I. Rodríguez. 2000. Diagnostic of Enterocytozoon bieneusi by PCR in Stool Samples Eluted from Filter Paper Disks. Clin. Diagn. Lab. Inmunol. 7: 504-506.

9. Dowd, S.E., C.P. Gerba, F.J. Enriquez & I.L. Peper. 1998. PCR Amplification and Species Determination of Microsporidia in Formalin-Fixed Feces after Immunomagnetic Separation. Appl. Environ. Microbiol. 64: 333-336.

10. Eligio García, L., S.C. Galván & E. Jiménez Cardoso. 2002. Distancia filogenética de aislados de Giardia intestinalis de niños sintomáticos y asintomáticos. Rev. Invest. Clin. 54: 113-118.

11. Eligio García, L., A. Cortes-Campos & E. Jiménez Cardozo. 2005. Genotype of Giardia intestinalis isolates from children and dogs and its relationship to host origin. Parasitol. Res. 97: 1-6.

12. El-Shazly, A.M., N. Mowafy, M. Soliman, M. El-Bendary, A.T. Morsy, N.I. Ramadan & W.A. Arafa. 2004. Egyptian genotyping of Giardia lamblia. Egypt. Soc. Parasitol. 34: 265-280.

13. Ghosh, S., A. Debnath, A. Sil, S. De, D.J. Chattopadhyay & P. Das. 2000. PCR detection of Giardia lamblia in stool: targeting intergenic spacer region of multicopy rRNA gene. Mol. Cell. Probes, 14: 181-189.

14. Guy, R.A., P. Payment, U.J. Krull & P.A. Horgen 2003. Real-Time PCR for Quantification of Giardia and Cryptosporidium in Environmental Water Samples and Sewage. Appl. Environ. Microbiol. 69: 5178-5185.

15. Homan, W.L. & T.G. Mank. 2001. Human giardiasis: genotype linked differences in clinical symptomatology. Int. J. Parasitol. 31: 822-826.

16. Lawson, A.J., D. Linton, J. Stanley & R.J. Owen. 1997. Polymerase chain reaction and speciation of Campylobacter upsaliensis and C. helveticus in human feces and comparison with culture techniques. J. Appl. Microbiol. 83: 375-380.

17. Monis, P.T., H.A. Ross, G. Mayrhofer & P.L. Ey. 1999. Molecular Systematics of the Parasitic Protozoan Giardia intestinalis. Mol. Biol. Evol. 16: 1135-1144.

18. Morgan, U.M., L. Pallant, B.W. Dwyer, D.A. Forbes, G. Rich & R.C.A. Thompson. 1998. Comparison of PCR and Microscopy for Detection of Cryptosporidium parvum in Human Fecal Specimens: Clinical Trial. J. Clin. Microbiol. 36: 995-998.

19. Ng, C.T., C.A. Gilchrist, A. Lane, S. Roy, R. Haque & E.R. Houpt. 2005. Multiplex Real-Time PCR Assay Using Scorpion Probes and DNA Capture for Genotype-Specific Detection of Giardia lamblia on Fecal Samples. J. Clin. Microbiol. 43: 1256-1260.

20. Ponce-Macotela, M., G.E. Peralta-Abarca & M.N. Martínez- Gordillo. 2005. Giardia intestinalis and other zoonotic parasites: Prevalence in adult dogs from the southern part of Mexico City. Vet. Parasitol. 131:1-4.

21. Polverino, D., N.B. Molina, M.C. Minvielle, M.E. Lozano & J.A. Basualdo. 2004. Técnicas de purificación y ruptura de Giardia spp. Rev. Arg. Microbiol. 36: 97-100.

22. Read, C.M., P.T. Monis & R.C.A. Thompson. 2004. Discrimination of all genotypes of Giardia duodenalis at the glutamate dehydrogenase locus using PCR-RFLP. Infect. Genet. Evol. 4:125-130.

23. Rimhanen-Finne, R., A. Hörman, P. Ronkainen & M.L. Hänninen. 2002. An IC-PCR method for detection of Cryptosporidium and Giardia in natural surface waters in Finland. J. Microbiol. Methods, 50: 299-303.

24. Rochelle, P.A., R. De Leon, M.H. Stewart & R.L. Wolfe. 1997. Comparison of Primers and Optimization of PCR Conditions for Detection of Cryptosporidium parvum and Giardia lamblia in Water. Appl. Environ. Microbiol. 63: 106-114.

25. Sincinschi, L.A., A. Pelayo Correa, L.E. Bravo & B.G. Schneider. 2003. Detection and typing of Helicobacter pylori cagA/vacA genes by radioactive, one-step polymerase chain reaction in stool samples from children. J. Microbiol. Methods, 52: 197-207.

26. Thompson, R.C.A., R.M. Hopkins & W.L. Homan. 2000. Nomenclature and Genetic Groupings of Giardia Infecting Mammals. Parasitol. Today, 16: 210-213.

27. Traub, R.J., P.T. Monis, I. Robertson, P. Irwin, N. Mencke & R.C.A. Thompson. 2004. Epidemiological and molecular evidence supports the zoonotic transmission of Giardia among humans and dogs living in the same community. Parasitology, 128: 253-262.

28. Troll, H., M. Hanspeter & N. Weiss. 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.

29. Wilson, I.G. 1997. Inhibition and Facilitation of Nucleic Acid Amplification. Appl. Environ. Microbiol. 63: 3741-3751.