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Simultaneous Detection and Quantification of Ancylostoma duodenale, Necator americanus, and Oesophagostomum bifurcum in Fecal Samples Using Multiplex Real-Time PCR

A multiplex real-time PCR was developed and evaluated for the simultaneous detection of Ancylostoma duodenale, Necator americanus, and Oesophagostomum bifurcum in fecal samples. Using well-defined control samples (N = 150), known positive fecal samples (N = 50), and fecal samples from an area in Ghana where human infections with all 3 nematode species are endemic (N = 339), the method proved to be highly specific and sensitive. Cycle threshold (Ct) values, reflecting parasite-specific DNA load, showed significant correlation with the intensity of infection as measured by microscopy using Kato–Katz fecal smears or by species specific third-stage larval count after coproculture. The multiplex real-time PCR described combined with the simple fecal sample collection procedure and the potential for high throughput makes this approach a powerful diagnostic tool to study species-specific transmission patterns of human hookworm-like infections. Moreover, this procedure facilitates monitoring of intervention programs and allows species-specific detection of treatment failure following rounds of mass treatment.

Received December 21, 2006. Accepted for publication June 3, 2007.

Acknowledgments: Collection of data and material would not have been possible without the tremendous efforts of field team workers Bayita Albano, Daniel Laar, Leonard Yelifari, Mohammed Awel, Moses Kolan, and Mathilda Abugri and Dutch students Welmoed Silvis, Bram Diederen, Gabrielle van Ramshorst, Esther Hamoen, and Rutger van der Meer. In addition, we are most grateful to Robert ten Hove and Maarten Eldering for laboratory assistance. The Severe Anaemia Project team of the Malawi–Liverpool–Wellcome Trust Clinical Research Program is kindly acknowledged for providing A. duodenale-positive fecal samples. The authors thank Gilbert Dery and Dr. Von Asigri (Parasitic Diseases Research Center, Tamale, Ghana) for logistical assistance and Coby Blotkamp for training and advising the field team. The Ghana Health Service in Bolgatanga and Tamale are gratefully acknowledged for their support. The authors also thank Dr. C. Graham Clark from the London School of Hygiene and Tropical Medicine for his advice and comments in the final version of the manuscript.

Financial support: Financial support was provided by the Dutch Foundation for the Advancement of Tropical Research (WOTRO-NWO) and the Oesophagostomum Research Intervention Project in Northern Ghana.

^* Address correspondence to Jaco J. Verweij, Leiden University Medical Center, Department of Parasitology, Postbus 9600, Leiden 2300 RC, The Netherlands. E-mail: j.j.verweij{at}lumc.nl

Authors’ addresses: Jaco J. Verweij, Eric A. T. Brienen, Anton M. Polderman, and Lisette Van Lieshout, Leiden University Medical Center, Department of Parasitology, Leiden, The Netherlands, Telephone: +31-71-5265080, E-mail: j.j.verweij{at}lumc.nl. Juventus Ziem, University for Development Studies, School of Medicine and Health Sciences, Tamale, Ghana, Telephone: +233-24-4753335. Lawrence Yelifari, Ministry of Health, Upper East Region, Bolgatanga, Ghana.