Effect of four different types of single-dose treatment with chloroquine and with chloroquine and pyrimethamine on Plasmodium falciparum infections in a semi-immune population in northern Nigeria.

BEHAVIOUR OF SCHISTOSOMA HAEMATOBIUM MIRACIDIA FROM IRAQ 117 The strain of S. haematobium from the Summer region of Iraq does not develop in the Moroccan strain of B. truncatus (Wajdi, unpublished data). Sectioning of these snails after exposure to miracidia has failed to show any penetrated larvae and it is possible that they protect the target snails as a result of some of the miracidia becoming exhausted in their attempts to enter the non-susceptible Bulinus. REFERENCES Barbosa, F. S. (1960) Bol. Fund. G. Moniz, 15, 1-54 Chernin, E. (1968) J. Parasit., 54, 509-516 Chemin, E. & Dunanan, C. A. (1962) Amer. J. trop. Med. Hyg., 11, 455-471 Ingals, J. W. (1949) J. Parasit., 35, 147-151 Richards, C. S. (1963) Amer. J. trop. Med. Hyg., 12, 26-33 Shiff, C. J. (1969) J. Parasit, 55, 108-110 Sudds, R. H. (1960) J. Elisha Mitchell Sci. Soc., 76, 121- 133 Takahashi, T., Mori, K. & Shigeta, Y. (1961) Jap. J. parasit., 10, 668-691 Wajdi, N. (1963) Penetration by the miracidia of S. man- soni into the snail host. Thesis, London University. Wright, C. A. (1962) The significance intraspecific taxo- nomy in bilharziasis. In: Wolstenholme, G. E. W. & O'Connor M., ed., Bilharziasis, London, Churchill (Ciba Foundation Symposium). Wright, C. A. (1966) J. Helminth., 40, 403-412 Effect of four different types of single-dose treatment with chloroquine and with chloroquine and pyrimethamine on Plasmodium falciparum infections in a semi-immune population in northern Nigeria R. KUSNECOV,1 J. STOREY 2 & P. LIETAERT 2 Single-dose treatment of malaria is often employed in tropical Africa for reducing morbidity and mortality. However, the drugs employed are subject to local availability, the dosages used for attaining these aims differ from country to country,3 and seldom have preliminary trials been carried out under local conditions to evaluate the therapeutic values of these drugs and dosages in the different age groups of the population. The present trial was undertaken in a savanna area of northern Nigeria where malaria was hyperen- demic, to assess the response of Plasmodium falci- parum infection to four types ofsingle-dose treatment, with chloroquine alone or with chloroquine and 1 Acting Team Leader, Malaria Field Research Project, Kano, Nigeria. Present address: Institute of Tropical Medi- cine and Medical Parasitology, Malaja Pirogovskaja 20, Moscow, USSR. ' WHO Malaria Field Research Project, P.O. Box 765, Kano, Nigeria. ' Lupascu, G. (1965) Report on the application of malaria chemotherapy and chemoprophylaxis in certain Africa coun- tries, Geneva (unpublished document Mal/Exp. Com. 12/WP/6). 2786B pyrimethamine, that are currently employed or might be used in the treatment of a semi-immune African population. As, in this area, there was virtually no transmission during the time of the trial, it was also possible to assess the suppressive, as well as the apparent " radical cure " effect obtained after the single-dose treatment. In this context, " radical cure " means that there was no reappearance of P. falciparum trophozoites in the peripheral blood after the initial clearance following treatment and up to day 60. All previous field trials on the effects of single-dose treat- ment, with the exception of those of Clyde (1961), were carried out in areas where the risk of reinfection was constant and therefore only the initial response of the infection to treatment could be assessed with certainty. Methods Selection of area and villages. The villages of Koda, Fakuwa, and Tama, with a total population of 1935, situated in the driest part of the savanna NOTES area in the Province of Katsina (near Kankiya town), North Central State, were selected for the trial. The crude parasite rates (children, 2-9 years of age) at the commencement of the trial were 60.0 %, 76.0 %, and 60.5 %, for the three villages, respectively. Previous parasitological surveys had shown that, in this area, malaria was stable with a high prevalence of P. falciparum (about 97 %); P. malariae and P. ovale were the next most prevalent parasites. Epidemiological situation in the villages at the time of the trial. The trial took place from March to the end of May 1969, during the hottest part of the dry season when surface water was reduced to a mini- mum. The densities of adult and larval mosquitos were regularly assessed in the villages during the trial and were so low that malaria transmission could hardly have been expected. The probability of malaria transmission taking place was even further reduced by weekly larviciding of all known water col- lections within a radius of 2.5 km of each village. At the beginning of the trial, a single dose of chloro- quine and pyrimethamine (adult dose, 450 mg of chloroquine and 45 mg of pyrimethamine) was administered to all known positive cases of malaria in the population excluded from the experiment. Selection of subjects for treatment. After a preli- minary examination of blood samples (thick smear) taken from the entire population of Koda and Fakuwa, and from all the children (1-16 years of age) of Tama, only the subjects having P. falciparum trophozoites in their blood were selected. The per- sons selected in Koda and Fakuwa were divided into two groups (group I and group 4) of approximately the same size, and having similar age ranges and levels of parasitaemia. The subjects selected in Tama were similarly divided into groups 2 and 3. Drug administration and follow-up observations. In groups 1, 2, and 3, the dosages of chloroquine base administered alone were adjusted, respectively, to body weight range (group 1), to surface area of subject (group 2), and to standard dosage related to age group (group 3). In group 4 a combination of chloroquine diphosphate and pyrimethamine was given at a standard dosage in relation to age. The drugs and groups of dosages were employed as indicated in Table 1. Prior to treatment, solid food (biscuits) was given to each subject. The drug was swallowed with water and its ingestion was carefully checked. Haskin's test for assessing the presence of chloroquine in the Table 1. Dosages used. The dosages in Group 2 are those suggested by the WHO Scientific Group on Resistance of Malaria Parasites to Drugs (1965). The dosages in Group 3 are those recommended in the report of the Technical Meeting on Malaria Eradication in Africa (unpublished WHO document AFRO/MAL/30) and those in Group 4 were used in aWHO field research projectin 1966-68 in Northern Nigeria Group 1 Group 2 Dose Dose adjusted Body weight adjusted to Body weight to weight range surface area range (mg base (kg) (mg base (kg) chloroquine) chloroquine) 600 51-60 600 51-60 500 41-50 530 41-50 400 31-40 460 31-40 300 21-30 380 21-30 200 11-20 290 11-20 100 5-10 180 5-10 Group 3 Group 4 Dose Dose (mg base) (mg base Age group Chioro Pyri- Age groupchloro- Chor- metha-quine) quine mine 150 3 months-3 years 75 7.5 3-1 1 months 300 4-9 years 150 15 1-3 years 450 >10 years 300 30 4-9 years 450 45 >10 years urine was carried out on samples collected in Tama from every child of 2-4 years of age and from every person who had diarrhoea at the time of the drug administration. Treatment took place on day " 0 " and thereafter blood films were taken daily from all subjects on days 1-5, every 5 days from day 5 to day 30, and then every 10 days until day 60. The presence or absence of diarrhoea and vomiting 2-3 hours after treatment and within 12 hours thereafter was noted. Blood examination. Giemsa-stained thick smears were examined for 20 min. During this time, about 400 microscopic fields, each containing an average of 10-15 leucocytes, were examined. In order to cal- culate the density of asexual parasites and of game- tocytes per mm3, trophozoites and/or gametocytes 118 EFFECIT OF SINGLE-DOSE TREATMENTS ON P. FALCIPARUM INFECIIONS IN NIGERIA Table 2. Effects of four different types of single-dose treatment with chloroquine (groups 1, 2, and 3) and chloroquine plus pyrimethamine (group 4) on P. falciparum infections in semi-immune subjects _ _ Age groups (years) Characteristic Group 1 Group 2 Group Group 4 1-9 | >14 1-9 10-16 1-9 1-9 >14 no. of cases followed up from day 0 to day 60 79 49 77 24 45 89 62 parasite rate on day 3 6.4 6.1 5.2 4.2 7.0 4.6 3.3 on day 5 1.3 2.1 2.6 0.0 2.3 3.4 3.3 mean clearance time (days) 2.08 1.7 2.18 2.0 2.25 2.0 1.5 mean clearance time (days) heavy infections a 2.3 2.5 2.5 light infections b 2.0 2.2 1.9 percentage of cases relapsing between day 10 and 7.6 4.1 6.5 0.0 11.1 6.9 9.7 day 60 a More than 1 000 falciparum trophozoites per mm3 of blood. b1 Less than 1 000 falciparum trophozoites per mm3 of blood. were counted against 500 leucocytes on the basis of an expected average number of 8 000 leucocytes per mm3 of blood. The 20-min blood examinations were necessary as the very low level of residual parasitaemia frequently found would often have been undetected by a 5-min examination (100 fields). This is borne out by a series of observations. The results of microscopic examinations in Koda and Fakuwa villages were recorded after 5, 10, 15, and 20 min. Of 566 slides found positive after 20 min of examination, only 22% had been detected after 5 min, 45% after 10 min, and 77% after 15 min. Results Of the 522 P. falciparum carriers initially selected for treatment, 425 were retained throughout the trial, the others being discarded for reasons such as absence for more than two successive examinations, for receiving unspecified treatment, for vomiting within two hours after taking the drug, for spitting out the drug, etc. The effects of treatment on those followed from days 0 to 60 are summarized in Table 2. In all four groups the parasite rates declined to levels of 3.3-7% on day 3 and by day 5 to only 1.3-3.4% of the original level. The mean clearance time of trophozoites in child- ren (1-9 years) in each of the four groups was similar, but in every case it was longer than that observed in the older age group. Although the difference in clearance time between light and heavy infections is not statistically signifi- cant in each treatment group (aged 1-9), this becomes significant if all the groups are pooled, in the sense that the mean duration of parasitaemia of all originally heavy infections in all the children aged 1-9 years is longer (by 0.5 day) than the mean duration of all light infections. Two subjects found positive for at least the first six examinations, and those who missed one or more examinations between day 1 and day 5, were not included in the calculation of the mean clearance time. It is worth noting that the percentage recrudescence among subjects with diarrhoea (10.8%) at the time of medication is slightly higher than that among those without diarrhoea (7.1 %) and that in the 14 years and above age-group, recrudescence was less in group 1 (4.1 %) than in group 4 (9.7 %), in 119 120 NOTES which the adult dosage was limited to a standard 450 mg of chloroquine base. Of the 32 detected cases that became positive again between day 10 and day 60, malaria parasites were found on only one occasion in 21 of them, and twice in two cases. In all these cases the parasitaemia was extremely low and often the diagnosis was made on a single parasite found after a long search. Of the remaining 9 cases, the parasitaemia continued from day 5 in two of them, and in the other 7 parasitaemia reappeared on days 15-20 and persisted almost con- tinuously until day 60. The second stage of the WHO field test for de- termining the strain sensitivity to a standard dose of chloroquine (WHO Scientific Group on Resistance of Malaria Parasites to Drugs, 1965) was carried out on six persons in Tama village who continued to show asexual parasites on day 10, but who had been discarded from the trial as they had not received the appropriate dose of drug (but still more than 10 mg of base per kg of body weight). In all cases, parasites disappeared from the blood within the first 2-3 days after treatment. Following drug administration, the gametocyte rates increased in all groups, but the rise in the 14-year and above age group was less pronounced. The average gametocyte rate for all the children under 10 years of age increased from 13.0% to 29.0% within a week after the treatment and was still considerably higher than the initial value in all groups on day 15. In the group of children who received a single dose of chloroquine and pyri- methamine, the rise of the gametocyte rate was less than in the other three groups. In 266 cases where no trophozoites were found after day 5, it was still possible to find gametocytes on day 30 (six cases) and on day 60 (one case). The gametocyte density index remained at nearly the same level from day 2 to day 10 and it fell gradually after day 10. Side-effects. No side-effects other than vomiting were reported. Vomiting was observed mainly in young children (less than 3 years of age); in group 2, children up to 6 years of age were affected. The percentage of children that vomited within two hours after the treatment was 2.5% for group 1, 8.0% for group 2, 4.4% for group 3, and 4.5% for group 4. Discussion The trial took place during a period, and under conditions, in which malaria transmission could be excluded. The dosages of chloroquine, with the exception of adults in group 4, were in many cases higher than the 10 mg of base per kg of body weight normally used in field trials. The peripheral blood was not cleared of trophozoites on day 5 in seven children aged 1-9 years (2.4%) and in three persons 10 years of age and over (2.2 %). The parasite rates on day 5 were comparable with those observed by Lelijveld (1970) but lower than those reported by Bruce-Chwatt (1951), by Shute & Dowling,' and by Beausoleil (1968); probably be- cause most of the infections in the present trial were not recently acquired infections. It should also be noted that 400 microscope fields were examined as compared to 100-200 fields examined by the first three authors quoted above. In all the groups of children, the mean clearance time was just over 2.0 days while in adults it was 2.0 days or less, even though the dosages of chloroquine base per kg of body weight were generally higher for children than for adults. Clyde (1961) and Lelijveld (1970) also observed a mean clearance time in adults below 2.0 days. In this trial, the mean clearance time was longer in the group of heavy infections than in the group of light infections and this corresponds with the findings of Charles (1958). It was observed that a single-dose treatment with chloroquine, in dosages of 10 mg of base per kg body weight or higher, failed to prevent the reappearance of parasites within 2 months of treatment in 6.5- 11.1 % of the children in the 1-9-year age group. These results are at variance with those reported by Clyde (1961) on 12 African children who, after a single-dose treatment of chloroquine and during a follow-up for 3 months in an area where malaria transmission was not occurring, did not show re- crudescence; however, neither the number of follow- up examinations nor the number of microscope fields examined was stated. In almost all cases in which parasitaemia was dis- covered between day 10 and day 60 after administra- tion of chloroquine, parasites were found only once or twice and always in very small numbers. Slightly better results were observed in the group of children treated with a combined single dose of chloroquine and pyrimethamine. This may have been caused by the longer persistence of pyrimethamine in the blood; I Shute, G. T. & Dowling, M. A. C. (1966) Effect of sul- phorthodimethoxine on parasites of Plasmodium falciparum and Plasmodium malariae on semi-immune schoolchildren in the western regiontofNigeria, Geneva (unpublished document WHO/MAL/66.544). EFFECT OF SINGLE-DOSE TREATMENTS ON P. FALCIPARUM INFECTIONS IN NIGERIA 121 the persistance of pyrimethamine may also account for the late reappearance of parasites (20 days or more after treatment)-in those cases where this occurred-compared with the groups treated with chloroquine alone in which parasites reappeared in the peripheral blood usually 10-20 days after treatment. An increase in the gametocyte rate above the initial level during the first 2-3 weeks following a regular 3-day course of treatment had already been noticed by other authors. A rise in the gametocyte rate after the administration of a single-dose treat- ment was observed by Bruce-Chwatt (1951) and by Charles (1958), although Miller (1954) and Shute & Dowling 1 observed a steep fall in gametocyte rate after treatment with chloroquine. In our trials, a rise in gametocyte rate occurred in all age groups after the administration of any type of single-dose treatment. The presence of gametocytes in the blood of semi-immune persons 60 days after treatment, in the absence of asexual parasitaemia, may be of epidemiological importance. When comparing the results of the present trial with others, attention should be paid to the fact that in previous trials of the same type (Bruce-Chwatt, 1951; Charles, 1958; Clyde, 1961; Lelijveld, 1970; Miller, 1954) the num- ber of microscope fields examined varied from 100 to 200 and this, according to present observation, may reveal only some 45 % of the total number of positives that would be found after a 20-min examination. Side-effects, such as vomiting, were observed in 2.5-4.5% of the children below 3 years of age in groups, 1, 3, and 4. In group 2, where dosages were adjusted to surface area and were as a rule higher than in the other groups, the percentage of subjects (up to 6 years of age) who vomited 2 hours after the treatment was as high as 8 %. In group 4, where the dosage was the same as in a trial carried out in 1967-68 in Kankiya district, but in which it was administered after ingestion of a biscuit, the per- centage of subjects vomiting (4.5%) was half that reported in Kankiya (Najera, unpublished data, 1968). Conclusions All the four types of treatment with dosages of 10 mg of chloroquine base per kg of body weight or 1 Shute, G. T. & Dowling, M. A. C. (1966) Effect of sul- phorthodimethoxine on parasites of Plasmodium falciparum and Plasmodium malariae on semi-immune schoolchildren in the western region of Nigeria, Geneva (unpublished document WHO/MAL/66.544). above proved, by and large, to be equally effective in all age groups as regards rapid reduction of parasite rates, parasite clearance time, and reduction of parasite density. In the absence of transmission, the reappearance of trophozoites between days 10 and 60 after single- dose treatment was detected in 4.7-11.1 % of the cases in the course of one or more of eight follow-up blood examinations of 20 min each (400 microscope fields). In general, the trophozoites reappeared more frequently in children than in adults except in the group 4 where a combined dosage of chloroquine and pyrimethamine was used and where the dosage of chloroquine given to adults weighing more than 45 kg was less than 10 mg of base per kg of body weight. The necessity for prolonged examination of each blood film in trials of drugs is indicated by the fact that the routine 5-minute examinations usually em- ployed in a trial of this type reveal only 22% of the positive cases found by a 20-min examination. A considerable rise in the fakiparum gametocyte rates was consistently observed for 3 weeks after a single-dose treatment with chloroquine, indicating the need to supplement this drug with gametocyto- cides in projects aiming at reduction of malaria transmission. The percentage of side-effects (vomiting) observed in children given a single dose of chloroquine adjusted to surface area of the body makes this type of dosage, using uncoated tablets, unsuitable under field conditions. Dosages of chloroquine adjusted to body weight range would appear preferable and could be used by health posts and in mass drug administration schemes covering selected groups of population. When using a standard treatment in relation to age, a single dose of 600 mg of base for persons above 16 years would appear to be desirable, except for ethnic groups of slight build. ACKNOWLEDGEMENTS The authors acknowledge the help and co-operation afforded to the team by the District Heads of Kankiya, Kaura, and Bindawa Districts. Their thanks are also due to Dr F. Y. Cheng, Mr G. R. Shidrawi, Mr D. P. Thomas, Mr T. 0. Crisp, Mr J. R. Boulzaguet, and Mr J. Petrides for their most valuable technical assistance. They also extend their thanks to Mr P. G. Shute and Miss M. Maryon at the Horton Malaria Reference Laboratory, Epsom, England, for so kindly checking some of the slides. 122 NOTES REFERENCES Beausoleil, E. G. (1968) Bull. Wld Hlth Org., 38, 488 Bruce-Chwatt, L. J. (1951) Trans. Roy. Soc. trop. Med. Hyg., 44, 563 Charles, L. J. (1958) Ann. trop. Med. Parasit., 52, 51 Clyde, D. F. (1961) Amer. J. trop. Med., 10, 1 Lelijveld, J. & Mzoo, F. (1970) Bull. Wld Hlth Org., 42, 471 Miller, M. J. (1954) Amer. J. trop. Med. Hyg., 3, 458 WHO Scientific Group on Resistance of Malaria Parasites to Drugs (1965) Wld Hlth Org. techn. Rep. Ser., No. 296 An apparatus for the study of the behaviour patterns of mosquitos under the influence of insecticides * T. GHEORGHIU,1 E. M. UNGUREANU, & C. GARRETT-JONES 2 The apparatus described here has been constructed to assess simultaneously the behaviour of mosquitos in the absence of, and under the influence of, insecti- cides in a multiple-response test. The principle of the apparatus is to offer mosquitos the opportunity to enter freely either a treated or an untreated baited compartment, to bite, and to leave the compartment before or after feeding. Construction of the apparatus. The apparatus is composed of the following parts: (1) Three cages made of plywood or plastic sheets (Fig. 1 a, 1 b); one of the cages serves as a release chamber (C) and communicates with the two other bait chambers (A and B). One of the baited cham- bers (B) can be lined partially or completely with brown paper sprayed with any desired concentration of an insecticide. The tops of the cages are removable and can be made of glass or transparent plastic. (2) Two exit traps (ET) into which the mosquitos can fly before or after feeding (Fig. 1 b). (3) One small release cage (SRC) (Fig. 1 b, 2 c) in which the mosquitos are first introduced and from which they can fly through a hole in the large release chamber (C). (4) Two large slides (S) made of thin board, which are used to cover the openings between the release cage and the baited cages (Fig. 1 a). * From the Institute of Hygiene, IaSi, Romania. This study was supported by the World Health Organization. 1 Entomologist, Institute of Hygiene, Ia$i. 2 Hope Department of Entomology, University Museum, Oxford, England. 2786c (5) Two medium-sized slides (S') made of thin board, which are used to close the openings between the baited cages and the exit traps (Fig. 1 b). Baited chambers. The dimensions of these cages are 50 x 50 x 50 cm (Fig. 1; A, B). A round hole 20 cm in diameter is cut in one of the walls (Fig. 1) of each cage to allow the mosquitos to enter the exit trap. A triangular hole (TH) is cut in the wall between each baited cage and the release cage C to allow mosquitos to enter cages A or B (Fig. 1 a). The dimensions of the triangular hole are 33 cm along the base and 14 cm in height (Fig. 2 a). A cone (Fig. 1 b; EC) made of board is fitted over each hole. The small aperture of the cone (Fig. 1 b) facing the bait is 6 cm in diameter. A number of small round holes 2 cm in diameter can be cut in the walls of the cage around the triangular hole and covered with mosquito net (Fig. 2 a; SH) in order to increase the attractiveness of the baited cages A and B. Release chamber (Fig. 1; C). The dimensions are 40 x 50 x 50 cm. On the walls facing the baited cham- bers are cut triangular holes corresponding to those in chambers A and B. A round hole is cut on the same side as those for the exit traps in chambers A and B and is covered with Nylon mosquito net. A cylindrical tube (Fig. 1 b; T) of the same diameter as that of the hole can be attached. Through this tube a stream of air can be drawn from inside the apparatus by means of a suction fan. If necessary, baffles may be fitted inside the cylindrical tube (Fig. 1 b; BL). Opposite this opening a hole 15 cm in diameter is cut and covered with a sleeve through which the mosqui- tos are introduced into the apparatus (Fig. 2 b; SL).