Experimental Parasitology 124 (2010) 244–246
Contents lists available at ScienceDirect
Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
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
a r t i c l e
i n f o
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
a b s t r a c t 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 produced by adult tapeworms are found in the intestines of a definitive host – carnivores (mainly foxes and dogs) are the source of infection for people. In order to estimate the infection risk, a prevalence 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: different 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 sedimentation 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 diagnosis in carnivores. It has also been regarded as the ‘‘gold standard”, and characteristics of other diagnostics techniques were often assessed by a comparison with SCT (Eckert, 2003). However, the real * Corresponding author. Fax: +48 81 886 25 95. E-mail address:
[email protected] (J. Karamon). 0014-4894/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2009.09.007
limit of detection of SCT has not been properly evaluated. For a proper estimation into the SCT limit of detection, intestinal samples with known numbers of Echinococcus tapeworms are needed, and they need to be examined in several replicates. This is impossible 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
J. Karamon et al. / Experimental Parasitology 124 (2010) 244–246
for a repeated examination; the method for obtaining negative samples in our investigation had to be modified. E. multilocularis tapeworms are usually not found only in the anterior part of intestine – it has been shown that when these tapeworms occurred in the anterior part, they were always observed distally (Karamon et al., 2008; Tackmann et al., 2006). Therefore, the negative intestines in our investigation were obtained as follows: After 2-week-long storage at 80 °C, the small intestines of foxes were divided on three equal parts (anterior, middle and posterior). The middle and posterior parts of each intestine were examined with the use of SCT (OIE, 2008). When the Echinococcus tapeworms were not detected in these parts, the anterior part of intestine was considered as negative. Overall, 40 of such negative intestines were frozen (at 20 °C) and used in investigations. 2.3. The course of investigation The limit of detection for the method was determined with the use of experimentally enriched samples. Forty samples containing 2, 5, 10, and 30 E. multilocularis tapeworms (10 samples per each level) were prepared and examined according to the following procedure. The negative intestine was incised longitudinally and cut into about 20 cm long segments. These pieces were then transferred to a polypropylene 1-L bottle containing physiological saline solution (0.9% NaCl). An appropriate dose of E. multilocularis tapeworms was added into the bottle. The bottle was shaken vigorously for a few seconds and the pieces of intestine were removed. The superficial mucosal layer was stripped by exerting pressure between the arms of tweezers. The bottle was left for 15 min for sedimentation to occur; supernatant was then decanted. The bottle was refilled with a physiological saline solution. This procedure was repeated three times. The sediment fraction was examined in small portions of about 5–10 ml in plastic Petri dishes under stereomicroscope (12–40). The entire sediment was then checked and the detected E. multilocularis tapeworms were counted. 2.4. Statistical analysis The limit of detection – the lowest number of parasites, which can be detected in the samples without precise determination of their quantity – was estimated as the level of worms at which 50% of the replicates are positive (Lombard, 2006). All the data were analysed by the use of Microsoft Excel 2002. 3. Results and discussion All 10 replicates were E. multilocularis positive in the examination of samples enriched with 30 tapeworms. In samples containing smaller numbers of tapeworms, the following results were obtained: 60% positive results for samples with 10 tapeworms, 40% for five tapeworms, and 30% for samples with only two tapeworms (Table 1). Because a precise 50% of positive results were not obtained, the limit of detection was estimated for nearest level in which at least
Table 1 Results of examination of intestinal samples (containing different number of E. multilocularis) with the use of SCT. No. of E. multilocularis worms in sample
No. of examined samples
% Of positive results
Mean no. of detected worms (range)
SD (CV%)
2 5 10 30
10 10 10 10
30 40 60 100
0.3 0.6 1.3 8.4
0.48 0.84 1.64 4.84
(0–1) (0–2) (0–5) (2–14)
SD, standard deviation; CV%, coefficient of variation.
(161.02) (140.55) (125.88) (57.56)
245
50% of samples were positive. Therefore, the limit of detection was estimated at 10 E. multilocularis tapeworms per sample of intestine (for 60% probability of obtaining positive results). These results generally confirmed the relatively high efficacy of SCT in echinococcosis diagnosis and the ability to detect low numbers of tapeworms (Eckert, 2003; Hofer et al., 2000). However, these results also showed that the efficacy of SCT is not perfect and this method, regarded as the ‘‘gold standard”, has some limitations. It must be emphasised that, despite making efforts to simulate the natural conditions of examination, our investigation is an experimental model and does not fully represent the examination of naturally infected intestines. Naturally occurring Echinococcus worms are localised between the villi, and are firmly fixed within the mucosal layer; therefore isolation of these tapeworms is probably less effective than in samples artificially enriched with ethanol preserved tapeworms. Moreover, tapeworms chosen for the experiment were in good condition, whereas in routine SCT examinations they were often deformed and fragmented, which makes the detection more difficult. For these reasons, it may be expected that the limit of detection in the examination of naturally infected intestines is probably worse (higher) than in our investigation. The mean number of E. multiloculasris found by SCT was several times lower than the real content of these tapeworms in samples (Table 1). This is caused most probably by ‘‘losses” connected with the preparation of the intestinal sample before examination: a possibility of removing some tapeworms from a bottle together with intestinal fragments or with supernatant during decantation. The other reason for detection of a low number of tapeworms was overlooking them during microscopic examinations (difficulties connected with alimentary particles, mucus, hairs, etc.). Probably, this is of lesser importance because the method of sample preparation before microscopic investigation allows optimal conditions for identification and enables a thorough examination and correct differentiation of the suspicious material. A wide variation in counts were observed in samples containing the same numbers of tapeworms (Table 1). The smallest variation was noted in samples enriched with the highest number of tapeworms (30), but even then the coefficient of variation (CV%) was very high (57.86%). This demonstrates the low repeatability of the method. In conclusion, the limit of detection of the sedimentation and counting technique (SCT) in E. multilocularis diagnosis was determined for the first time. Some limitations of this method (regarded as ‘‘gold standard”) determined in this experiment ought to be taken into consideration in the analysis of results concerning the prevalence of E. multilocularis in carnivores, especially in populations with low intensity of infection. References Craig, P.S., Gasser, R.B., Parada, L., Cabrera, P., Parietti, S., Borgues, C., Acuttis, A., Agulla, J., Snowden, R., Paolillo, E., 1995. Diagnosis of canine echinococcosis: comparison of coproantigen and serum antibody tests with arecoline purgation in Uruguay. Veterinary Parasitology 56, 293–301. Deplazes, P., Eckert, J., 1996. Diagnosis of Echinococcus multilocularis infection in final hosts. Applied Parasitology 37, 245–252. Dinkel, A., von Nickisch-Rosenegk, M., Bilger, B., Merli, M., Lucius, R., Romig, T., 1998. Detection of Echinococcus multilocularis in the definitive host: coprodiagnosis by PCR as an alternative to necropsy. Journal of Clinical Microbiology 35, 1871–1876. Eckert, J., 2003. Predictive values and quality control of techniques for the diagnosis of Echinococcus multilocularis in definitive hosts. Acta Tropica 85, 157–163. Hofer, S., Gloor, S., Muller, U., Mathis, A., Hegglin, D., Deplazes, P., 2000. High prevalence of Echinococcus multilocularis in urban red foxes (Vulpes vulpes) and voles (Arvicola terrestris) in the city of Zurich, Switzerland. Parasitology 120, 135–142. Karamon, J., Ziomko, I., Cencek, T., Sroka, J., Zie˛ba, P., 2008. Prevalence of Echinococcus multilocularis in red foxes in the Lublin voivodeship, Poland: preliminary study. Medycyna Weterynaryjna 64, 1237–1239. Lombard, B., 2006. Estimation of measurement uncertainty in food microbiology: the ISO approach. Accreditation and Quality Assurance 11, 94–100.
246
J. Karamon et al. / Experimental Parasitology 124 (2010) 244–246
OIE (Office International des Epizooties), 2008. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (mammals, birds and bees), sixth ed. World Organisation for Animal Health, Paris, pp. 175–189. Rausch, R.L., Fay, F.H., Williamson, F.S., 1990. The ecology of Echinococcus multilocularis (Cestoda: Taenidae) on St. Lawrence Island, Alaska. II. Helminth population in the definitive host. Annales de Parasitologie Humaine et Comparee 65, 131–140.
Tackmann, K., Mathis, R., Conraths, F.J., 2006. Detection of Echinococcus multilocularis in foxes: evaluation of a protocol of the intestinal scraping technique. Journal of Veterinary Medicine B 53, 395–398. Van der Giessen, J.W., Rombout, Y.B., Franchimont, J.H., Limper, L.P., Homan, W.L., 1999. Detection of Echinococcus multilocularis in foxes in The Netherlands. Veterinary Parasitology 82, 49–57.
