Diagnosis of Gambian trypanosomiasis in man by isolating trypanosomes from blood passed through DEAE-cellulose*

Bull. Org. mond. Sante ) 1971, 45, 13-19Bull. Wid Hith Org. Diagnosis of Gambian Trypanosomiasis in Man by Isolating Trypanosomes from Blood Passed Through DEAE-Cellulose* D. G. GODFREY 1, ' & SHEILA M. LANHAM1 A method is described whereby small numbers of trypanosomes in blood can be con- centrated free of blood cells on to a slide. Infected blood is passed through a column of DEAE-cellulose, which adsorbs the blood cells, and the organisms are separatedfrom the eluate by centrifugation. By this technique trypanosomes were detected in 24 % of 41 Nigerian patients with suspected Gambian trypanosomiasis, but whose cervical lymph glands were not infected; of these, only 5 % were positive in thick bloodfilms. Trypano- somes were also found in 91 % of 21 gland-positive patients, of which only 19 % were positive in thick blood-films. The organisms could not be found in 5 patients recently treated with either pentamidine or suramin-sodium and melarsen, and disappeared within two hours from the blood of another patient given melarsoprol. Trypanosomes were not found in 3 samples ofinfected cerebrospinalfluidpassed through DEAE-cellulose. Although 2 gland-positive patients and I patient with a patent parasitaemia failed to give positive results, column-separation of suspected blood is a potentially valuablr aid in the parasito- logical diagnosis of Gambian trypanosomiasis. Parasitaemia in Gambian trypanosomiasis of man is usually so low that the infecting organisms can rarely be found in a blood film examined under the microscope for a reasonable length of time. Demonstration of the parasites by animal inocula- tion is unreliable because of the low infectivity of Trypanosoma brucei gambiense for laboratory ro- dents; diagnosis by in vitro cultivation still requires proper assessment but nevertheless may be uncertain in field conditions because of contamination with other micro-organisms (Weinman, 1963). Normally, parasitological diagnosis relies on finding trypanosomes in the fluid of the cervical lymph glands or in the cerebrospinal fluid. The organisms cannot be found, however, in many suspected cases, and diagnosis relies on indirect evidence such as clinical deterioration, an increased erythrocyte sedimentation rate, and increases in protein and the number of white cells in the cerebro- * The expense of a visit by the authors to Nigeria was borne by a grant from the World Health Organization. 1 Trypanosomiasis Group (Overseas Development Admin- istration), Lister Institute of Preventive Medicine, Chelsea Bridge Road, London, England. ' External Scientific Staff, Medical Research Council. spinal fluid; none of these changes, however, is pathognomonic of trypanosomiasis. An increased level of serum IgM appears to be indicative of Gambian trypanosomiasis and recently IgM estimations have been successfully used in screening populations at risk (Mattem, 1964; Binz et al., 1968); however, increases may also be caused by other pathological conditions (Lumsden, 1970). Thus a reliable parasitological method for diag- nosing T. b. gambiense in man is needed, if only to assess correctly the value of the convenient indirect methods. This need may be fulfilled by Lanham's (1968) technique for separating trypano- somes from infected blood. The flagellates are eluted from a column of DEAE-cellulose on to which the blood cells and platelets are adsorbed; trypanosomes present at microscopically subpatent levels are separable and are readily found after centrifuging the eluate (Lanham, 1968; Godfrey & Lanham, 1969; Lanham & Godfrey, 1970). This paper describes the attempts, in co-operation with the Nigerian Institute for Trypanosomiasis Research at Vom and Kaduna, Nigeria (NITR), to isolate trypanosomes by column-separation and 2699 -13 2 D. G. GODFREY & S. M. LANHAM centrifugation from the blood of sleeping sickness patients. Unfortunately, facilities were not available in this preliminary trial for a proper comparison with the indirect diagnostic methods. MATERIALS AND METHODS Phosphate-buffered saline-glucose The buffers used for diluting blood, and for equilibrating and washing the anion-exchange columns (Lanham & Godfrey, 1970) were as follows: Na2HPO4 (anhydr.) NaH2PO4.2H20 NaCl Glucose H20 to PSG-J 8.09 g 0.47 g 2.55 g It 0( pH (approx.) Ionic strength (Long, 1961) The buffers PSG-2 an modified, where indicate 25 g glucose per 1 000 m] Blood samples All the patients exami definite or suspected cas Service in the areas of I (south of the Jos Plateau) nite cases had trypanosc glands (gland-positive), (CSF-positive), or in boti diagnosed mainly on clini were examined at either ti ratory of the Nigerian In Research (NITR); the re Gboko, near the River Be Samples of 5 or 10 ml PSG-2 6.74 g 0.39 g 2.13 g PSG-3 5.39g 0.31 g 1.70 g with HCl; indicator papers were sufficiently accurate to judge the correct pH value. After standing for about 20 min, the supernatant, containing the very fine particles, was discarded. The sediment was resuspended in 3 litres of buffer and the supernatant again discarded after settling for a further 20 min. This washing with PSG was repeated 4 more times. The final slurry of DEAE-cellulose was either used immediately or stored deep-frozen (- 25° C) in plastic bottles. On the visit to Gboko, frozen material was transported in the bottles inside large vacuum flasks (4-litre capacity) and was still in a frozen state 3 days later. The frozen slurry was thawed by immersing the plastic bottles in hot water. Preparation of columns I.wU g IU.WU g lU.W g Glass Buchner funnels with slit-sieve plates )0 ml 1000 ml I 000 ml (Lanham & Godfrey, 1970) were fitted with rubber 8.0 8.0 8.0 tubing and screw-clips to control flow. A funnel with a plate diameter of 5.5 cm and a height of 0.22 0.18 0.14 5.5 cm was used for a 10-ml blood sample, and a funnel 4.0 cm x 4.0 cm for a 5-ml sample. A d PSG-3 were occasionally volume of the slurry equivalent to about 30 g of the d in the results, to contain pre-swollen DEAE-cellulose was used for the 5-ml 1. samples of blood and a volume equivalent to 60 g for the 10-ml samples. The slit-sieve plate was thoroughly wetted by flooding with buffer and then ned had been diagnosed as drained. A filter-paper (preferably Whatman No. 41) es by the Sleeping Sickness of the correct diameter was seated on the plate. 3ida, Bauchi, Barakin Ladi The equilibrated slurry of DEAE-cellulose was ), Gboko and Wamba. Defi- poured into the funnel and the excess buffer was)mes in the cervical lymph slowly run off until the required volume of slurry in the cerebrospinal fluid had entered the funnel. After the adsorbent had h; suspected cases had been settled, the supematant was run off to leave a firm ical grounds. Most patients horizontal upper surface 1-2 cm below the rim of ie Kaduna or the Vom labo- the funnel; about another 10 ml of buffer was run stitute for Trypanosomiasis through until only a few millimetres depth of ,mainder were examined at liquid remained on top. Just before use, the excess nue in Benue-Plateau State. buffer was run off to leave a moist but firm surface iere taken by venepuncture of anion exchanger. into 250 IU of heparin (0.5 ml of 500 IU/ml in 0.9% NaCl). as an anticoagulant. A thick film for micros- copy was made with the non-heparinized blood re- maining in the needle. Erythrocyte sedimentation rates (ESR) on citrated blood were estimated by NITR staff, using the Westergren method. Equilibration of DEAE-cellulose Pre-swollen DEAE-cellulose (Whatman Chro- media DE 52) in 500-g batches was suspended in the appropriate buffer and the pH reduced to 8.0 Column separation The column-separation was carried out at ambient temperatures that ranged from 25°C to 36°C. The blood was immediately diluted to 4 times its original volume with buffer containing 10 IU of heparin per ml, applied drop by drop to the column and flow was started. The unrestricted flow rate from most columns was too fast and had to be retarded to give an even and horizontal front to the adsorbed erythrocytes; occasionally the flow was too slow and gentle suction was applied from a water-pump 14 DIAGNOSIS OF GAMBIAN TRYPANOSOMIASIS IN MAN or an aspirator. When all the diluted blood had entered the DEAE-cellulose, twice its volume of PSG was used to wash the blood container and then this was also processed on the column. When all this first washing had entered the anion exchanger, the column was washed a second time with the same amount of buffer. The initial void volume of buffer from the column was discarded. The amount was about 25 ml for the 10-ml sample size, but was usually precisely determined for each separation by testing sample drops of the eluate with 10% trichloroacetic acid. A precipitate indicated when the plasma proteins, and consequently any trypanosomes, began to appear in the eluate. The subsequent eluate, about 40 ml for each 5 ml of blood, was collected in 50-ml polycarbonate centrifuge tubes. Preparation offilms for microscopy The eluate was centrifuged in an angle-head bench centrifuge at 2 500 rev/min (900 g) for 20 min. After removal of the supernatant, the last 3-4 ml were trans- ferred to 15-ml polycarbonate centrifuge tubes, the deposits from the two tubes being pooled when neces- sary. The large tubes were washed 2 or 3 times with a total of about 10 ml of PSG and the washings added to the small centrifuge tube, which was spun for 10 min at 2 500 rev/min (800 g) in an angle-head centrifuge. Most of the supernatant fluid was removed, except for the last few drops, which were mixed with a fine pipette; care was taken to scrape and wash the sides near the bottom since the tubes had been spun at an angle. Occasionally, a wet film of this fluid was examined under the 40 x objective of the microscope, but usually the centrifuged deposit was dried in air on a slide, the 10-20 drops being superimposed one on another in order to avoid spreading the film too much. Difficulties encountered in this drying process are described in the results. Slides were fixed in methanol and stained face downward for 30 minutes in 10% Giemsa stain diluted in buffer at pH 7.2 (Na2HPO4, 3.0 g; KH2PO4, 1.2 g; H20 to 1 000 ml). The thick blood films were prefixed in 0.5 % aqueous methylene blue for a few seconds (Maclennan, 1957), lysed in the pH 7.2 buffer, and stained in Giemsa stain. A hundred fields of each blood film were examined with the 40 x dry or the 45 x oil-immersion micro- scope objective; uncertain findings of trypanosomes were checked with the 90 x oil-immersion objective. RESULTS The observations on each group of patients are shown in Table 1 and the combined results are summarized in Table 2. Table 1. Summary of results from the different groups of patients with suspected T. B. gambiense infection Area No.Area examined Bida Bauchi Barakin Ladi Gboko Wamba 8 3 1 1 18 22 Glands Positive 8 Positive 2 Negative 1 Positive 4 Negative 7 Positive 5 Negative 13 Positive 2 Negative 20 Positive blood films 2 0 0 1 0 O Positive CSF a NE 0 0 NE NE 3 3 NE NE a NE = not examined. Positive eluate-film 8 2 0 4 2 3 3 2 5 15 D. G. GODFREY & S. M. LANHAM Table 2. Summaries of the combined observations on the blood of patients with suspected T. B. gambiense infection Blood film Eluate Glands No. examined Positive Negative Positive Negative Positive 21 (34 %) 4 (19 %) 4 (19 %) 0 17 (81 %) 15 (72 %) 2 (9 %) Total - - 19 (91 %) 2 (9 %) Negative 41 (66 %) 2 (5 %) 1 (2 %) 1 (2 %) 39 (95 %) 9 (23 %) 30 (73 %) Total - - 10 (24 %) 31 (76 %) Grand total 62 6 (10 %) - 29 (47 %) - __I Bida area, Kwarra State Nine patients, reported as gland-positive 3-5 weeks previously, were sampled over a period of 3 days, to determine the optimal volume of blood and buffer conditions for separation of trypanosomes. Thick blood films from 2 patients were positive. From 2 patients, 20 ml of blood were divided into 10-ml, 5-ml, and 2-ml portions, and diluted with PSG-1 for examination. All eluates were grossly contaminated with red blood cells. With similar volumes of blood from 3 other patients, a lower ionic strength buffer, PSG-2 with an additional 1.5% glucose to maintain osmolality, was used. The eluates were free of erythrocytes but a deep band of adsorbed red cells, with a diffuse front, indicated poor adsorption. Nevertheless, trypanosomes were found in both wet and stained films of the centrifuged eluates. Since 5-ml samples were strongly positive, this volume was used for all subsequent gland-positive patients. All 9 patients were re-examined, low ionic strength buffer, PSG-3 with an extra 1.5% glucose, being used to ensure good retention of blood cells. Trypanosomes were found in the stained eluates in 6 cases. However, the organisms in some eluate- films were grossly distorted, especially in 2 dried rapidly in direct sunlight. The following day, 2 of the 3 apparently negative patients were re-examined with PSG-2, the only buffer available at the time, and were positive. The other apparently negative patient was re- examined clinically and by gland puncture. No trypanosomes were found in the gland fluid and Dr K. D. B. Thomson (NITR) considered that there were no clinical symptoms of trypanosomiasis; this patient complained only of general malaise whereas the other 8 reported one or more symptoms such as listlessness, inability to work, and bodily pain. The ESR was normal-3 mm at 30 min, 12 mm at 60 min-whereas the others had values ranging from 39-120 mm at 30 min to 77-126 mm at 60 min. It was concluded that the patient was not suffering from trypanosomiasis and that the original positive gland puncture was wrong. The patient could not be classed as clinically suspicious and is excluded from the tables. The tables record the final conclusions, after re-examination, on the other 8 patients. Bauchi area, North-Eastern State Two 5-ml samples from each of 2 gland-positive cases were processed, one with PSG-3 containing 1.0% glucose and the other with PSG-3 containing 2.5% glucose. Trypanosomes were readily found in good condition in all the stained elnates. It was concluded that the buffer with 1.0% glucose was more satisfactory since, with 2.5% glucose the fluid was viscous during the last stages of drying. PSG-3 with 1.0% glucose was used for all subsequent exam- inations. The thick blood films were negative. In both of these patients ESR was increased (34 and 134 mm at 30 min, 65 and 139 mm at 60 min). The cell count in the CSF was raised (7.5 and 20.0 per ml; normal 0-5 per ml) but the protein con- centration was within normal limits and no trypano- somes were found. 16 DIAGNOSIS OF GAMBIAN TRYPANOSOMIA-SIS IN MAN A third gland-negative patient appeared to be a clinically advanced case, although the CSF cell count (3.5 per ml) was normal and the CSF protein level, 41 mg per 100 ml (normal: <40 mg per 100 ml), was possibly slightly increased. No trypanosomes were found in the eluate, even though 10 ml of blood were examined. The blood film was also negative. Barakin Ladi area, Benue-Plateau State Eleven patients from areas to the south and at the southern edge of the Jos Plateau were examined; 4 were gland-positive and, of these, 1 was also blood-film positive. The 7 clinically suspicious but gland-negative patients had been diagnosed about a month previously; their glands were not palpable at the time of our examination. Eluate films from 5-ml samples of blood from the 4 gland-positive patients were strongly positive. Of the 10-ml samples from the 7 gland-negative patients, 2 were positive, although few trypanosomes were seen. None were blood-film positive. Trypano- somes were not found in the 2 gland-negative patients who had previously completed courses of suramin-sodium-tryparsamide, one in 1961 and the other in August 1969. Gboko area, Benue-Plateau State At Gboko, where there were 19 patients, 5ml of blood were taken from each of 4 gland- positive patients, 3 ml were taken from another gland-positive patient, and 10 ml were taken from each of 14 gland-negative cases. One gland-nega- tive patient, from whom trypanosomes were not iso- lated, was re-examined and considered not to be clinically suspicious, and is not included in the tables. The thick blood films from 1 gland-negative and I gland-positive patient were positive. Of the 5 gland-positive patients, 2 were eluate- negative and were also CSF-positive. One other CSF-positive was both gland- and eluate-positive. Of the 13 gland-negative cases, 3 were positive; 3 that were CSF-positive were eluate-negative. One patient with infected CSF had a positive blood film, although both the glands and eluate were negative; this was the only instance during our investigations of a negative eluate from a patient with a patent parasitaemia. Of the gland-negative cases, 5 were suspected relapses from drug treatment before 1970. One was eluate-positive and had received suramin-sodium- tryparsamide in 1964 and two courses of melarsen in 1965 and 1968. Five recently treated patients, not included in the tables, were also examined; 4 had been treated with pentamidine during the 6 weeks previous to our sampling and the other with a course of suramin- sodium and melarsen that had finished 12 days beforehand. All were eluate-negative. One of the gland- and eluate-positive patients was given 2.5 ml of melarsoprol intravenously by Dr H. Whittle (Ahmadu Bello University). The eluate from blood taken 55 min before the injection was strongly positive, but only a few distorted trypanosomes were seen in the eluate from blood taken 20 minutes later, and none were found in the eluate from blood taken 2 hours after the treatment. Trypanosomes were demonstrated by Miss H. Pope (Ahmadu Bello University) in centrifuged CSF from 3 patients, but none was detected in eluates from further amounts (3-5 ml) of the samples that were diluted and processed, in the same manner as blood, through DEAE-cellulose; wet films were examined. Wamba area, Benue-Plateau State Altogether 22 patients, of whom 2 were gland- positive, and in whom Dr W. Ronga (NITR) had found increased levels of serum IgM in the last half of 1969, were examined. Eluates were positive from 5-ml samples of blood from the 2 gland-positive patients, and from 5 of the 10-ml samples from the 20 gland-negative patients; 1 of the eluate-positives was also blood-film positive. Microfilariae were seen in 13 (59.1 %) of the 22 blood films. The eluate films from 12 gland-negative patients were difficult to search as they were extremely dirty, because of contamination from the prevailing northerly wind, characteristically laden with dust from the Sahara. In this group only 1 positive eluate was found. Films from the remaining eluates were dried in an air-conditioned room and were much cleaner. The 2 gland-positive cases were readily detected and eluates were positive from 4 of the 8 gland-negative patients. DISCUSSION The demonstration of trypanosomes in a host provides the most certain diagnosis of trypano- somiasis, but is often difficult in many suspected T. b. gambiense infections in man. We have now partly overcome this difficulty by using Lanham's 17 D. G. GODFREY & S. M. LANHAM (1968) method for separating trypanosomes from blood by the adsorption of the blood cells on to DEAE-cellulose. The efficiency of the method, however, is un- certain since trypanosomes were detected in only 24% of gland-negative, but clinically suspect, cases. One patent parasitaemia was missed by column- separation; the infection was also missed by gland puncture on the same day although the glands had been positive one month previously. Two gland- positive cases were also negative by our method. There are several possible reasons why the organ- isms were not found in many of the suspects. Firstly, some patients may not have been suffering from trypanosomiasis; no symptoms are pathogno- monic and rural Africans are beset by many other diseases. The uncertainties of clinical diagnosis are illustrated by the 2 suspected sufferers who were subsequently rejected, on clinical grounds, as sleeping sickness patients. Secondly, the parasitaemia may have declined below detectable limits at the time of blood sampling. Alternation of exacerbation and recession of para- sitaemia is well known in most trypanosome infec- tions, and if this occurs with T. b. gambiense in man, which is characteristically subpatent at any time, then extremely few organisms would occur when parasitaemia declined. It is interesting that trypanosomes were not isolated from the blood of 5 CSF-positive cases, 2 of whom were also gland- positive; perhaps a dramatic decrease in parasitaemia occurs when the central nervous system is invaded, as apparently occurred in a chimpanzee infected with T. b. brucei (Godfrey & Killick-Kendrick, 1967). Although these reasons may be valid for our failure to find trypanosomes in many suspected patients, we feel that more trypanosomes would be found if the method for concentrating the flagellates on microscope slides were improved. Without doubt the failure to isolate the organisms from 1 patent infection must have been due to faulty pre- paration. The passage through the column and the 2 centrifugations at high ambient temperatures appeared not to affect the trypanosomes, since those eluted and then examined in a wet film had normal activity. When the final eluate deposit was dried, however, care had to be taken to prevent disintegra- tion; too rapid drying certainly caused the organisms to break up. Previous laboratory experience in drying T. b. brucei in this manner without damage (Godfrey & Lanham, 1970) suggests that T. b. gambiense is the more fragile organism; T. b. gam- biense in cerebrospinal fluid is reputed to be particu- larly fragile-which may account for our failure to isolate it from infected cerebrospinal fluid. Another problem was the dust that collected on the slides during drying, which, as suggested by results from one group of patients, may have obscured the organisms. Even the staining was hazardous as parts of the eluate-film sometimes became detached, particularly in films stained soon after drying; films kept overnight, unfixed, were less likely to become detached during staining. The preparation of the films from the eluates was also the most time-consuming part of the technique. Even in field conditions, one person could process and centrifuge 6 eluates from 10-ml blood samples in about 75 min and, while these were centrifuging, start a further series of samples. However, each centrifuged deposit had to be dried a small drop at a time to avoid a widespread film, which would have entailed a lengthy examination. Drying the film in this way was time-consuming, each slide taking at least an hour and very much longer in conditions of high humidity. It is unfortunate that the serum IgM level could not be estimated when our samples were taken. However, the patients from the Wamba area were reported to have increased IgM levels in their sera 5 months previously. Trypanosomes were found in only 32% of them but some negative results may have been due to faulty technique, particularly dirty eluate films. Despite the limitations of the column-separation technique, trypanosomes were found in 23% (9 out of 39 in Table 2) of the patients in whom the organ- isms could not be found either by gland puncture or by blood-film examination. Our technique by itself found more infections (47%) than were found by gland puncture alone (34%) or by blood films alone (10 %). However, each method failed to find one or more infections found by one of the other methods. Used with these other methods, column-separation should provide a much better measure of the efficiency of the indirect methods of diagnosis. Column-separation should also be invaluable in the search for human non-clinical and animal reservoirs of T. b. gambiense and, since viable organisms are isolated, for immunopathological studies of the human disease. It must be emphasized that with animal blood, the ionic strength of the buffer must be correctly chosen for the species of host (Lanham & Godfrey, 1970). 18 DIAGNOSIS OF GAMBIAN TRYPANOSOMIASIS IN MAN 19 ACKNOWLEDGEMENTS This survey would have been impossible without the advice, wholehearted assistance, and facilities extended to us by Dr A. A. Amodu, Director of the Nigerian Institute for Trypanosomiasis Research. We are espe- cially grateful for the personal co-operation of many of his staff, especially Dr K. D. B. Thomson and Dr W. Ronga, together with their Sleeping Sickness Survey staff, and Dr A. R. Gray, Mr N. Mitchell, and Mallam H. M. Yesufu. It is also our pleasure to record the help given at Gboko by Dr H. Whittle and Miss Helen Pope of the Department of Medicine at Ahmadu Bello Uni- versity. RESUME DIAGNOSTIC DE LA TRYPANOSOMIASE A TRYPANOSOMA GAMBIENSE CHEZ L'HOMME PAR ISOLEMENT DES TRYPANOSOMES APRES PASSAGE DU SANG SUR DEAE-CELLULOSE Les auteurs decrivent une methode qui permet de concentrer, en vue de 1'examen sur lame, les trypano- somes presents en petit nombre dans le sang. Apres passage sur DEAE-cellulose, le sang est debarrasse de ses cellules et des plaquettes et les micro-organismes contenus dans 1'eluat sont recueillis par centrifugation. Par cette technique, on a decele les trypanosomes chez 24% de 41 malades nigerians presumes atteints d'une infection a Trypanosoma gambiense, mais chez lesquels 1'examen du suc ganglionnaire n'avait pas montre la presence de parasites; la goutte epaisse etait positive chez 5% seulement d'entre eux. Des trypanosomes ont aussi ete decouverts, grace a cette technique, chez 91 % de 21 malades a suc ganglionnaire positif, mais chez lesquels la goutte epaisse n'etait positive que dans la proportion de 19%. T. gambiense n'a pas &te retrouve chez 5 patients recemment traites par la pentamidine ou par la suramine sodique et le melarsene. Chez un sujet positif, le parasite a disparu du sang 2 heures apres une injection de melarsoprol. Apres passage sur DEAE- cellulose, on n'a pas deceld de parasites dans trois dchan- tillons de liquide c6phalo-rachidien infecte. Bien que la methode ait ete utilisee sans succes chez deux trypanoses a suc ganglionnaire positif et chez un patient presentant une parasitemie nette, on estime qu'elle offre un intdret certain pour le diagnostic parasitologique de la trypanosomiase a T. gambiense. REFERENCES Binz, G., Timperman, G. & Hutchinson, M. P. (1968) Bull. Wld Hlth Org., 38, 523-545 Godfrey, D. G. & Killick-Kendrick, R. (1967) Trans. roy. Soc. trop. Med. Hyg., 61, 781-791 Godfrey, D. G. & Lanham, S. M. (1969) Trans. roy. Soc. trop. Med. Hyg., 64, 159 Lanham, S. M. (1968) Nature (Lond.), 218, 1273-1274 Lanham, S. M. & Godfrey, D. G. (1970) Exp. Parasit., 28, 521-534 Long, C. (1961) Biochemist's handbook, London, Spon Lumsden, W. H. R. (1970) Advanc. Parasit., 8, 227- 249 Maclennan, K. J. R. (1957) Trans. roy. Soc. trop. Med. Hyg., 51, 301-302 Mattern, P. (1964) Ann. Inst. Pasteur, 107, 415-421 Mattern, P. (1968) Bull. Wld Hlth Org., 38, 1-8 Weinman, D. (1963) Bull. Wld. Hlth. Org., 28, 731- 743