Research Brief Limit of detection of sedimentation and counting technique (SCT) for Echinococcus multilocularis diagnosis, estimated under experimental conditions Jacek Karamon * , Jacek Sroka, Tomasz Cencek Department of Parasitology and Invasive Diseases, National Veterinary Research Institute in Pulawy, Al. Partyzantow 57, 24-100 Pulawy, Poland article info Article history: Received 11 March 2009 Received in revised form 2 September 2009 Accepted 3 September 2009 Available online 8 September 2009 Keywords: Echinococcus multilocularis Diagnosis Limit of detection Sedimentation and counting technique abstract The aim of this study was to estimate the limit of detection of sedimentation and counting techniques (SCT) in Echinococcus multilocularis diagnosis. Samples of small intestines, experimentally enriched with known numbers of E. multilocularis tapeworms, were used. Forty intestinal samples containing 2, 5, 10, and 30 E. multilocularis tapeworms (10 samples for each level) were prepared and examined according to SCT. E. multilocularis was detected in 30%, 40%, 60%, and 100% in samples enriched with 2, 5, 10, and 30 tapeworms, respectively. The limit of detection was estimated at 10 E. multilocularis tapeworms per sample of intestine (for 60% probability of obtaining positive results). There was a wide dispersion of counting results; these were observed in samples containing the same numbers of tapeworms, which indicates the low repeatability of the method. The limitations of SCT determined in this experiment should be considered when analysing the prevalence of E. multilocularis in carnivores. Ó 2009 Elsevier Inc. All rights reserved. 1. Introduction Echinococcus infection is one of the most dangerous zoonoses and remains a significant public health problem worldwide. People can be the accidental host for these parasites and the developing larvae may even bring about their death. Echinococcus eggs pro- duced by adult tapeworms are found in the intestines of a defini- tive host – carnivores (mainly foxes and dogs) are the source of infection for people. In order to estimate the infection risk, a prev- alence study must be conducted in the population of foxes and dogs, especially in endemic regions. There are some techniques for the detection of Echinococcus infection in definitive hosts: dif- ferent post mortem methods, coproantigen detection by ELISA (CA-ELISA) or copro-DNA detection by different PCR techniques (Craig et al., 1995; Deplazes and Eckert, 1996; Dinkel et al., 1998; Van der Giessen et al., 1999). Among them, the most sensitive is a post mortem examination of the small intestines by the sedimen- tation and counting technique (SCT). This method was first used in the diagnosis of echinococcosis by Rausch et al. (1990) and was been modified by Hofer et al. (2000). This method, together with the intestinal scraping technique (IST) and ‘‘shaking in the vessel” technique (SVT), is recommended by the World Organisation for Animal Health (OIE, 2008) for post mortem echinococcosis diagno- sis in carnivores. It has also been regarded as the ‘‘gold standard”, and characteristics of other diagnostics techniques were often as- sessed by a comparison with SCT (Eckert, 2003). However, the real limit of detection of SCT has not been properly evaluated. For a proper estimation into the SCT limit of detection, intestinal sam- ples with known numbers of Echinococcus tapeworms are needed, and they need to be examined in several replicates. This is impos- sible with the use of intestines from naturally infected animals where the real number of parasites is unknown. Therefore, the aim of this study was to determine a limit of detection of SCT by the use of samples of intestines experimentally enriched with known numbers of E. multilocularis tapeworms. 2. Material and methods 2.1. Echinococcus tapeworms Echinococcus multilocularis tapeworms were obtained from intestines of naturally infected foxes during an examination with the use of SCT (OIE, 2008). Before the experiment was started, tapeworms were collected, put in 70% ethanol and stored at 4 °C to preserve them against degradation. On the day of experiment, known numbers of isolated E. multilocularis tapeworms were put into tubes with about 3 ml of physiological saline (0.9% NaCl), in order to prepare the doses for experimental enrichment. Only adult gravid tapeworms were used. Forty tubes containing 2, 5, 10, and 30 tapeworms (10 tubes for each dose) were prepared. 2.2. Negative intestines (without Echinococcus) Negative intestines were selected by examination using SCT. However, after an examination by SCT, the intestine is not suitable 0014-4894/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2009.09.007 * Corresponding author. Fax: +48 81 886 25 95. E-mail address: J.Karamon@piwet.pulawy.pl (J. Karamon). Experimental Parasitology 124 (2010) 244–246 Contents lists available at ScienceDirect Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr