A differential filtration technique for the measurement of schistosome cercarial densities in standing waters

Bulletin of the World Health Organization, 57 (6): 971-975 (1979) A differential filtration technique for the measurement of schistosome cercarial densities in standing waters ANDRE THERON1 The technique of differential filtration allows the detection of schistosome cercariae, with a recovery greater than 80%, in natural waters with a high concentration of microorganisms. This technique makes use ofa new type ofmonofilamentpolyamide filter Nytrel- TI, with a pore size of 40 Rm, allowing filtration of turbid water without clogging of the filter. A three-level prefiltration column (1000, 500 and 200 Jim), which retains organisms larger than cercariae, facilitates reading of the filter. The apparatus used weighs less than 3.5 kg; it requires no power supply and can easily be used in the field. Several techniques have been proposed (1-12) for indirect or direct measurement of cercarial densities. None appears to be entirely satisfactory for filtration and detection of cercariae in turbid water, such as is often found in stagnant transmission sites. To over- come this problem, we have developed the method described in this paper. APPARATUS The filtration apparatus (Fig. 1) consists of four main components (Fig. 2): the prefiltration column; the connecting piece; the recovery filter; and the filter support. This apparatus can be adapted to fit a vessel for recovery of filtered water (20-30 litres). The prefiltration column (1 in Fig. 2) is composed of a series of steel sieves of decreasing pore size. This column is intended to trap particles larger than the cercariae. The retention of debris and organisms at different levels of the column facilitates both the water flow and the counting of cercariae on the final filter. After many trials, we found that three sieves provided good recovery with easy reading of the filter. The first filter of 1000 iim pore size retains large plant debris. The second and third filters, of 500 and 200 iim pore size, retain mostly zooplankton (Copepoda, Ostracoda, etc.), as well as some of the phytoplankton that is abundant in stagnant waters. The pore size of the third filter (200 ,tm) is large enough to allow easy 1 Attache de Recherche au C.N.R.S., Departement de Biologie Animale, Universite, 66025 Perpignan Cedex, France. passage of schistosome cercariae. It should be noted that this choice is suitable for a reasonable range of size of cercariae ; forexample, with this three-filter system, we have obtained satisfactory recovery (greater than 70 %) with large cercariae such as Ribeiroia marini (Faust et Hoffman, 1934), whose body size is 445 x 175 lim. We therefore conclude that it is the diameter of the cercarial body that should determine the selection of the pore size of the third filter. The connecting piece (2 in Fig. 2) is funnel-shaped. It ensures a tight connexion between the prefiltra- tion column and the filter support. The 200-[lm filter is fitted into the upper part. The lower part rests on the filter support. The recovery filter (3 in Fig. 2) is made from a monofilament polyamide material Nytrel-TI, with a pore size of 40 [im.a This material has two advan- tages over traditional glass fibre filters in that it allows more rapid filtration and it is reusable after washing with water. The filter has a diameter of 120 mm. It is attached to a circular rubber washer, 15 mm wide and 1 mm thick, on which rests the lower edge of the connect- ing piece. The area of filtration, marked in squares to facilitate reading, has a diameter of 90 mm. Two factors were considered in selecting the pore size of the monofilament polyamide filter: first, prevention of clogging, which makes the filter unreadable, and second, a satisfactory percentage of recovery of cercariae. a Supplier: U.G.B., 42360 Panissieres, France. Cost: US$ 15.00 per m2 (1 filter costs US$ 0.25). 3916 971 A. THERON Fig. 1. Differential filtration apparatus: general view. We have determined that (1) for pore size of 40 [Im or greater there is no blockage of the filter, and (2) the recovery of schistosome cercariae is less than 50 % for pore size greater than 40 rim, whereas it exceeds 80% at or below 40 [tm pore size. Consequently, a pore size of 40 1.m was chosen. This allows filtration on a single filter of more than 10 litres of water with a high density of microorgan- isms and suspended organic matter, whilst assuring easy reading of the filter. Filters of pore size less than 40 pm, which give an even better recovery, can be used for filtration of clear water. The filter support (4 in Fig. 2) is a polypropylene Buchner funnel, which is fitted to both the connect- ing piece and the container for recovery of the water. PROCEDURE Filtration Filtration of water samples on a Nytrel-TI filter does not require suction. The vacuum pump and generator essential for filtration on glass fibre filters Fig. 2. Schematic cross-section of the differential filtra- tion apparatus: 1. Prefiltration column 2. Connecting piece 3. Re- covery filter 4. Filter support. are, therefore, unnecessary. The weight of the apparatus is considerably reduced (maximum 3.5 kg) so that it can easily be used in the field, the filtration being done later in the laboratory. Water samples are carried in plastic jars, to each of which is added a little formol (about 1 ml/litre). Filtration of cercariae can be carried out several hours after sampling with no alteration in the measured values of density compared with immediate filtration in the field (Table 1). In the field or in the laboratory, at least 5 and preferably 10 litres of water are necessary for accurate measurement of cercarial density. This volume can be reduced when cercarial density is high. Reading of the filter After filtration of the samples, the filtration col- umn is washed with clean water. The Nytrel-TI filter is removed from its support and placed on a What- man GF/A glass fibre filter. It is stained in a solution of Lugol, which colours the cercariae red. Cercariae 972 SCHISTOSOME CERCARIAL DENSITIES Table 1. Comparison of cercarial densities obtained from several biotopes by immediate or delayed filtra- tion Method No. of cercariae in 10 litres Direct filtration in the field 98 193 110 140 56 57 Delayed filtration- 6 h later in the laboratory 94 192 120 145 62 52 are counted under a binocular microscope. The Nytrel-TI filters are then placed in a detergent solution for several hours, after which they are rinsed for reuse. Reading of filters can be delayed by introducing an intermediate step. The Nytrel-TI filters are washed with formolated water and the washings collected in a small plastic bottle. This can be stored for subsequent refiltration and staining with Lugol as described. This treatment results in a loss of about 5 % of the cercariae. EVALUATION Limit imposed by turbidity Although the method allows filtration of water with a high concentration of microorganisms, there is a limit above which the filter becomes clogged, making the counting of cercariae difficult. This limit is about 200 Jackson turbidity units (JTU). For waters with a turbidity of 150-200 JTU, filtration is slower but can still be carried out without suction. It is likely that if the particles causing the turbidity were similar in size to the cercariae (between 40 and 200 glm) the apparatus would be ineffective. Such conditions were never encountered during our trials in the field. Rate of filtration We compared the time required for filtration of different volumes of clear water through Nytrel-TI polyamide filters of 40 lim pore size (under gravity) and through Whatman GF/A glass fibre filters (under suction). Table 2 shows that filtration is about twice as fast on Nytrel-TI filters. Recovery of cercariae Recovery of cercariae (Schistosoma mansoni) was measured on Whatman GF/A and Nytrel-TI filters, with and without a prefiltration column. The test consisted of filtration of 20 samples of 5 litres of Table 2. Filtration times of different volumes of water on Nytrel-TI and Whatman GF/A filters Filtration times (seconds) Filtration volumes (litres) On Nytrel-TI On Whatman GF/A under gravity under suction 1 9 15 2 15 25 3 22 45 4 28 60 5 37 80 6 42 95 7 45 106 8 50 111 9 63 120 10 65 130 water (10 containing 20 cercariae, 10 containing 60 cercariae). One series of tests was carried out using clear water, a second using turbid water; the latter was collected from a pool in Guadeloupe that is rich in zoo- and phytoplankton but contains no cercariae. The results presented in Table 3 allow the follow- ing conclusions: 1. In clear water, the recovery of cercariae on Nytrel-TI 40-,m filters is very satisfactory (78.80 ±4.88% to 84.20 ±4.00%), even though slightly lower than that obtained with glass fibre filters (85.83 ± 2.98 % to 91.5 ± 2.29 %). Here, the Table 3. Recovery of S. mansoni cercariae on What- man GF/A glass filters and Nytrel-TI polyamide filters, with or without prefiltration, in clear and turbid water Prefiltration Cercarial recovery Water Filter column from 20 analyses (>m) (% ± SE) Whatman GF/A 0 91.50 ± 2.29 glass fibre with suction 1000 + 500 + 200 85.83 ± 2.98 Clear Nytrel-TI 0 84.20 ± 4.00 polyamide, 40 gm 1000 + 500 + 200 78.80 ± 4.88 Whatman GF/A 0 unreadable glass fibre with suction 1000 + 500 + 200 unreadable Turbid Nytrel-TI 0 unreadable polyamide, 40 ,Im 1000 + 500 + 200 86.35 ± 2.73 973 A. THERON advantage of the polyamide filter lies in its more rapid filtration and easier operation. 2. In turbid water, only the apparatus combining the Nytrel-TI filter with the prefiltration column gives satisfactory recovery of cercariae: 86.35 ± 2.73 %. Without prefiltration, the filter is unreadable, while the Whatman GF/A filter is blocked during passage of the first few litres, even with prefiltration. CONCLUSION The apparatus and methods developed for detec- tion of schistosome cercariae by differential filtra- tion are excellently suited for use in the field. In comparison with results obtained using glass fibre filters, the use of 40 ,um polyamide Nytrel-TI filter allows more rapid filtration in clear water, and filtration of greater volumes of turbid water, with easier reading of the filters. In both cases, a high recovery of cercariae is obtained. In addition to these advantages, our apparatus presents two characteristics that seem to be extreme- ly useful in any field survey: (a) it is compact and light and is easy to use, even for unqualified staff; and (b) it is autonomous in operation, requiring no power supply. ~~~44~~~~~~~~~.4I;X:~~~~~~~~~~~~~~~~~~~~~ -Q- - - - - $- - - .. - - - ---- 4~~~~~~~~~~~~1 41 5 l*101520. 253 [ 5 0 ,:. : 0t,.,'"5 l0 40 70|";' ' f-ti~~~~~~~~~~~~<9WlIZ 1p-__llg'i~ S ii | - E ' s .,. s .~~~~~~~~~~~~~~~~~~4 z Fig. 3. Recovery of cercariae of S. mansoni in clear water as a function of pore size of Nytrel-TI filters. Shaded areas indicate pore sizes at which filtration or reading of the filter is difficult with very turbid water. A pore size of 40 Itm ensures both easy reading of the filter and highly satisfactory recovery of cercariae. For filters with pore size greater than 40 gim, recovery of the cercariae is inadequate. ACKNOWLEDGMENTS This work was supported by the De'legation G&n6ra1e A la Recherche Scientifique et Technique (DGRST), the Institut National de la Sant6 et de la Recherche M6dica1e (INSERM), and the World Health Organization. Field work was carried out in Guadeloupe at the zoological station of the Institut National de la Recherche Agronomique (INRA) under the auspices of the "action concerte'e Bilharziose "'. 974 SCHISTOSOME CERCARIAL DENSITIES 975 RtSUMt TECHNIQUE DE FILTRATION DIFFE-RENTIELLE POUR LA MESURE DES DENSITES CERCARIENNES DE SCHISTOSOMES DANS LES EAUX STAGNANTES La technique de filtration differentielle permet de re- trouver, avec un taux de recuperation depassant 80%, les cercaires de schistosomes dans des eaux naturelles tres chargees en micro-organismes. Cette technique fait inter- venir un nouveau type de filtre Nytrel-TI en tissu monofila- ment polyamide d'une ouverture de maille de 40 [m permettant la filtration d'eau turbide sans colmatage du filtre. Une colonne de prefiltration a 3 niveaux (1000, 500 et 200 [tm) facilite la lecture du filtre en retenant les organismes de taille superieure a celle des cercaires. L'appareillage employe pese moins de 3,5 kg; il ne necessite aucune source d'energie et peut etre facilement utilise sur le terrain. REFERENCES 1. BARRET, P. D. & ELLISON, P. R. A continuous flow centrifuge for testing the presence of Bilharzia cer- cariae in water. The Central African journal of medicine, 11: 338-340 (1965). 2. BUTLER, J. M. ET AL. New field device for quantitative recovery of Schistosoma mansoni cercariae. Public health reports, 82: 250-252 (1967). 3. KLOCK, J. W. A method for the direct quantitative recovery of Schistosoma mansoni cercariae from natural waters of Puerto Rico. Bulletin of the World Health Organization, 25: 738-740 (1961). 4. OLIVIER, L. J. A continuous flow centrifuge for concentration of Schistosoma cercariae. American journal of tropical medicine and hygiene, 15: 875-881 (1966). 5. PESIGAN, T. P. ET AL. Studies on Schistosoma japonicum infection in the Philippines. 2. The mollus- can host. 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