Annals of Tropical Medicine & Parasitology, Vol. 92, No. 2, 181-185 (1998)
The distribution of Schistosoma japonicum eggs in faeces and the effect of stirring faecal specimens BY X.-P. YE, C. A. DONNELLY*, R. M. ANDERSON The Wellcome Trust Centre for the Epidemiology of Infectious Disease, Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, U.K. Y.-L. FU Yujiang City Hospital of Schistosomiasis, Yujiang, Hunan, China AND A. AGNEW Department of Biology, University of Leeds, Leeds LS2 9]T, U.K.
Received 3 June 1997, Revised 18 November 1997, Accepted 20 November 1997 Experiments were performed to determine whether Schistosoma japonicum eggs are randomly dispersed in faeces and the effect of stirring faecal specimens prior to sampling. For each of 13 patients infected with S. japonicum, eggs were counted in 150 subsamples from a single stool specimen, using the Kato-Katz smear technique. Eggs were non-randomly distributed in all 13 stools, and showed an aggregated distribution. In most patients there were significant differences in the distribution of eggs between the centre and the surface of the stool. Stirring of the stool prior to sampling decreased the variability of counts.
There are three methods of obtaining egg counts from faecal samples: direct smear; dilution; and concentration (Stall and Hausheer, 1926; Scott, 1938; Beaver, 1949; Bell, 1963; Ashford et al., 1981). With each of these methods, especially for the Kato direct-smear technique, any irregularity in the distribution of helminth eggs within faeces creates difficulties, in that the use of a small sample of the faecal mass may lead to an inaccurate estimate of the number of eggs excreted (Martin, 1965; Anderson and May, 1985; Anderson and Schad, 1985). Martin (1965), using the dilution and direct-smear methods, found that helminth eggs such as those of Ascaris lumbricoides " Author to whom correspondence should be addressed. E-mail:
[email protected]; fax: + 44 (0)1865 281 245. 0003-4983/98/020181-05 $9.00 Carfax Publishing Ltd
and Trichuris trichiura, like beads and seeds, were randomly scattered m the stools, and the variability of counts was not affected by stirring of the stool before sampling. Martin and Beaver (1968) and Woodstock et al. (1971), using the Kato technique, also found eggs of Schistosoma mansoni to be randomly distributed throughout a single human faecal specimen. Retard et al. (1990) similarly revealed no significant differences between the centre and the surface, or the beginning and the end of the stools they examined, in terms of the numbers of S. mansoni eggs. However, Barreto et al. ( 1978) reported that S. mansoni eggs had a negative binomial distribution in single stools. The main aim of the present study was to see if the eggs of S. japonicum, which are laid in clumps by the female worms and not singly, are non-randomly distributed in stools. © 1998 Liverpool School of Tropical Medicine
182
YE ET AL.
The first goal was to describe the distribution of S. japonicum eggs in stool specimens from 13 patients. If S. japonicum eggs are irregularly distributed in the faecal mass, then there should be an advantage in stirring stool specimens before removing the sample for counting. The second goal was therefore to determine whether stirring decreased the variance of egg counts. PATIENTS AND METHODS Patients and Sampling Twenty-four-hour stool specimens appearing normal in form and consistency were collected from 13 patients who were infected with S. japonicum and lived in Yujiang City, Hunan province, China. Subsamples were selected from each specimen according to the following protocol: an outline of the stool was drawn on a card, and the area was divided, approximately evenly, to indicate 25 cross-sections. The sections were cut and each divided across the section thickness into two portions. A scoop was constructed to hold approximately 1.5 g faeces and used to prepare two Kato--Katz slides (one from the original surface and one from the centre) from each portion (Katz et al., 1970). Thus, 100 subsamples were taken from each specimen prior to stirring. Centre samples were collected with great care to avoid transferring any surface material to the centre of the stools. The whole stool was then thoroughly mixed by hand with a stirring rod for 15-20 min and the sampling procedure was repeated except that only one slide was prepared from each of the 50 portions. Each subsample was examined by the Kato technique (WHO, 1983) and egg counts recorded as number of eggs/ 42 mg of stool. Statistical Analysis The mean count and variance were calculated for each patient, for subsamples taken before stirring (from the surface, from the centre and combined) and for subsamples collected after stirring. The null hypothesis, that the distribution of egg counts was identical for samples
from the surface and the centre of the faecal mass, was tested for each patient using the Kuiper statistic (Press et al., 1992). This nonparametric test does not make any assumption about the shape of the egg-count distribution. The means and variances were compared by l test for the samples collected prior to stirring (surface and centre combined) and those taken after stirring. The Kuiper statistic was also used to test the null hypothesis that stirring had no effect on the distribution of eggs in the stool from each patient. Probability (P) values were adjusted for the high number of comparisons, using the Bonferroni method (Armitage and Berry, 1994). RESULTS The distributions of S. japonicum eggs in the subsamples collected from the surface and centre of the stool were significantly different for seven of the 13 stool specimens (see Table). In each of these seven cases, the mean egg count for surface subsamples was significantly greater than that for centre samples. The index of dispersion (see Table) reflects the extent to which the counts in each stool specimen were dispersed, compared with what can occur by random chance alone (a Poisson distribution). Eggs were shown to be non-randomly distributed in all of the 13 stool specimens (pooling surface and centre counts), indicating that the distribution of S. japonicum eggs in all the faeces sampled was contagious or aggregate. There were significant differences in the distribution of S. japonicum eggs before and after stirring for six of the 13 stool specimens. Counts from the stirred faeces were less variable than those from the unstirred faeces in all 13 stool specimens (see Table). DISCUSSION The present results show that the egg counts on the surface of the stool were greater than the mean value in the centre of the stool in seven of 13 stool samples contammg S. japonicum eggs. The even distribution of
TABLE
Mean centre counts and (variance)
0.94 (1.04) 2.48 (24.59) 147.26 (4334) 1.94 (2.63) 13.64 (65.26) 5.74 (12.56) 3.84 (41.97) 83.44 (1336) 25.84 (77.65) 44.30 (108.4) 120.82 (1081) 28.46 (67.44) 26.16 (107.6)
Mean surface counts and (variance)
2.80 (4.12) 2.32 (13.73) 225.72 (9687) 4.70 (16.87) 14.14 (83.84) 4.68 (6.47) 7.06 (579.8) 135.04 (5431) 44.82 (151.1) 60.86 (246.1) 195.84 (3843) 40.66 (76.80) 37.44 (197.1)
Sample
1 2 3 4 5 6 7 8 9 10 11 12 13
0.029 1.000 0.020 0.005 1.000 1.000 1.000 0.108 0.005 0.049 0.005 0.005 0.966
P-value for Kuiper statistic
Comparing surface and centre counts
1.86 (3.41) [ < 0.001] 2.40 (18.78) [
