This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lachaud, L.
Right arrow Articles by Bastien, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lachaud, L.
Right arrow Articles by Bastien, P.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, February 2001, p. 613-617, Vol. 39, No. 2
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.2.613-617.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Comparison of Various Sample Preparation Methods for PCR Diagnosis of Visceral Leishmaniasis Using Peripheral Blood

Laurence Lachaud,1 Elisabeth Chabbert,1 Pascal Dubessay,2 Jacques Reynes,3 Jacques Lamothe,4 and Patrick Bastien*,1

Laboratoire de Parasitologie-Mycologie et Centre National de Référence sur les Leishmanioses (Prof. J. P. Dedet)1 and Service des Maladies Infectieuses et Tropicales,3 Centre Hospitalier Universitaire, and CNRS UMR5093 "Génome des Protozoaires Parasites," Faculté de Médecine,2 Montpellier, and Clinique Vétérinaire de Carros, Carros,4 France

Received 17 May 2000/Returned for modification 7 August 2000/Accepted 16 November 2000

We have compared various sample preparation methods for the PCR diagnosis of visceral leishmaniasis (VL) using peripheral blood samples and tested the influence of these protocols upon sensitivity. Four methods of lysis-DNA extraction were used with two types of blood samples: whole blood (WB) and buffy coat (BC). Comparisons were first carried out with seeded samples at various parasite concentrations. At high concentrations (>= 1,000 parasites/ml), there were no significant differences in PCR sensitivity among the methods tested. At concentrations of <= 100 parasites/ml, proteinase K (PK)-based methods proved clearly superior to guanidine-EDTA-based methods. Moreover, a 10-fold increase in sensitivity was observed for BC over that for WB. Thus, the best sensitivity was obtained with the BC prepared with PK-based methods. With this combination, the PCR reliably detected 10 parasites/ml but was inconsistent when the sample contained 1 parasite/ml of blood. The methods that yielded the highest sensitivities were evaluated with seven dogs and four human VL patients. Again, the utilization of the BC prepared with PK-based methods gave the best results. The optimization of each step of the assay (sample preparation, DNA extraction, and PCR conditions) yielded a highly sensitive tool for the diagnosis of VL using patient blood, thus avoiding more invasive diagnostic procedures and allowing the detection of low parasitemia during posttherapeutic follow-up.

* Corresponding author. Mailing address: Laboratoire de Parasitologie-Mycologie, Centre Hospitalier Universitaire, 163 Rue A. Brousssonet, F-34090 Montpellier, France. Phone: 33-4-67-63-27-51. Fax: 33-4-67-63-00-49. E-mail: genpara{at}sc.univ-montp1.fr.

Journal of Clinical Microbiology, February 2001, p. 613-617, Vol. 39, No. 2
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.2.613-617.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Alborzi, A., Pourabbas, B., Shahian, F., Mardaneh, J., Pouladfar, G. R., Ziyaeyan, M. (2008). Detection of Leishmania infantum Kinetoplast DNA in the Whole Blood of Asymptomatic Individuals by PCR-ELISA and Comparison with Other Infection Markers in Endemic Areas, Southern Iran. Am J Trop Med Hyg 79: 839-842 [Abstract] [Full Text]  
  • ALLAHVERDIYEV, A. M., BAGIROVA, M., UZUN, S., ALABAZ, D., AKSARAY, N., KOCABAS, E., KOKSAL, F. (2005). THE VALUE OF A NEW MICROCULTURE METHOD FOR DIAGNOSIS OF VISCERAL LEISHMANIASIS BY USING BONE MARROW AND PERIPHERAL BLOOD. Am J Trop Med Hyg 73: 276-280 [Abstract] [Full Text]  
  • Mary, C., Faraut, F., Lascombe, L., Dumon, H. (2004). Quantification of Leishmania infantum DNA by a Real-Time PCR Assay with High Sensitivity. J. Clin. Microbiol. 42: 5249-5255 [Abstract] [Full Text]  
  • Chabbert, E., Lachaud, L., Crobu, L., Bastien, P. (2004). Comparison of Two Widely Used PCR Primer Systems for Detection of Toxoplasma in Amniotic Fluid, Blood, and Tissues. J. Clin. Microbiol. 42: 1719-1722 [Abstract] [Full Text]  
  • Reithinger, R., Espinoza, J. C., Courtenay, O., Davies, C. R. (2003). Evaluation of PCR as a Diagnostic Mass-Screening Tool To Detect Leishmania (Viannia) spp. in Domestic Dogs (Canis familiaris). J. Clin. Microbiol. 41: 1486-1493 [Abstract] [Full Text]  
  • Reithinger, R., Quinnell, R. J., Alexander, B., Davies, C. R. (2002). Rapid Detection of Leishmania infantum Infection in Dogs: Comparative Study Using an Immunochromatographic Dipstick Test, Enzyme-Linked Immunosorbent Assay, and PCR. J. Clin. Microbiol. 40: 2352-2356 [Abstract] [Full Text]  
  • Lachaud, L., Marchergui-Hammami, S., Chabbert, E., Dereure, J., Dedet, J. P., Bastien, P. (2002). Comparison of Six PCR Methods Using Peripheral Blood for Detection of Canine Visceral Leishmaniasis. J. Clin. Microbiol. 40: 210-215 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»