Incipient resistance of Plasmodium falciparum to chloroquine among a semi-immune population of the United Republic of Tanzania

Bulletin of the World Health Organization, 60 (6): 899-906 (1982) Incipient resistance of Plasmodium falciparum to chloroquine among a semi-immune population of the United Republic of Tanzania 2. The impact of chloroquine used as a chemosuppressant on the immune status of the population E. ONORI,' B. GRAB,2 P. AMBROISE-THOMAS,3 & J. THELU4 Decreased sensitivity and incipient resistance of Plasmodium falciparum strains to chloroquine have been reported from Mto-wa-Mbu, in the north-east of the United Republic of Tanzania. In this locality the population had been exposed to chloroquine pressurefor about two decades, in theform ofmedicated salt and through easy availability of the drug itself. In an attempt tofind out whether such chemosuppression had influenced the immune response of the population, two seroepidemiological surveys were carried out in March 1981 and March 1982; the second survey was performed to confirm the results obtained in the first one. The humoral immunological response was measured by the immunofluorescent antibody technique. In the absence of information on the immuno- logical profile that existed in the area prior to the introduction of chloroquine in 1960, the results of the present surveys were compared with those obtained in another locality in the north-east of the United Republic of Tanzania in 1967, and in the West Kiang district of Gambia in 1965. The two areas usedfor comparison exhibited a malaria endemicity similar to that prevailing in Mto-wa-Mbu prior to the introduction of the medicated salt. The resultsfrom Mto-wa-Mbu showed a significantly lowerproportion ofsubjects with positive titres and a lower geometric mean titre in all age groups. A reduction in the humoral immunological response might be explained by the drug pressure that has been exerted in the areafor many years. The depressed immune response found at Mto-wa-Mbu, however, was so marked that otherfactors may have contributed to its establishment. In view of the importance of these findings, it is recommended that further, longi- tudinal serological studies be conducted in the field to assess the effects ofchemosuppres- sion on the immune response of the protected populations. The decreased sensitivity of strains of Plasmodium falciparum to chloroquine in Mto-wa-Mbu, a village in the north-east of the United Republic of Tanzania which had been exposed to chloroquine pressure for about two decades, and the presence of strains resis- tant to chloroquine at RI level have been confirmed by in vivo and in vitro studies and reported in a previous paper (1). ' Medical Officer, Chief, Epidemiological Methodology and Evaluation, Malaria Action Programme, World Health Organiz- ation, Geneva, Switzerland. 2 Formerly Statistician, Health Statistical Methodology, World Health Organization, Geneva, Switzerland. 3 Professor of Parasitology, Faculty of Medicine, University of Grenoble, France. 4 Scientist, Department of Parasitology, Faculty of Medicine, University of Grenoble, France. At the time these studies were carried out, blood specimens were taken from a sample of the population in order to determine the serological profile of the local residents. This was in order to find out whether the chemosuppression exerted by the chloroquinized salt for many years and the easy access to chloroquine tablets at the local dispensary and village shops had affected the immune status of the population. A reduced immunological defence might have been partly responsible for the change in the sensitivity of the local P.falciparum strains to chloroquine. A second seroepidemiological survey was carried out in Mto-wa-Mbu in March 1982. The results of the two surveys are presented and discussed in the present paper. 4242 899- E. ONORI ET AL. METHODS At both surveys, blood samples were taken from the same 300 persons who had been examined to assess the malaria prevalence in the area. The blood was taken using microhaematocrit tubes and trans- ferred to filter paper strips. These were dried and conveyed to a specialized laboratory 3 weeks later, where one of the present authors (P.A.T.) carried out the indirect immunofluorescent antibody (IFA) test, as described previously (2). The antigen was prepared using red cells parasitized with schizonts of P.falci- parum. The parasite had been isolated from a patient who had contracted the disease in Gambia, and had been maintained in culture for some years following the technique of Trager & Jensen (3). Synchron- ization by sorbitol was carried out prior to the preparation of the antigenic slides. The seroepidemiological results obtained at Mto- wa-Mbu have been compared with those from Muheza-Ubembe, another locality in the north- eastern part of the United Republic of Tanzania (4), and with those from the West Kiang district of Gambia (5). In the studies in Muheza-Ubembe, P.fieldi was used as antigen for the IFA technique (6). In Gambia, thin blood films from patients with P.falciparum parasitaemia of not less than 30 000 trophozoites per mm3 constituted the antigen (5). RESULTS Table 1 shows the parasite rates and the IFA titres found in residents in Mto-wa-Mbu, in March 1981. It appears that at the time of the examination, the prevalence of the disease in the area was high, indicating a hyperendemic situation. However, the percentage of patients with a positive IFA titre was not as high as might be expected in an area with high malaria endemicity, and the geometric mean titre was consistently low in all the age groups. The results of the IFA test were more striking when they were analysed according to the results of parasitological examination, separately for children and adults. From Table 2, it can be seen that the geometric mean antibody titre was very low for the children, with practically no difference between those with positive or negative slides. Among the adults (15 years of age and over), the geometric mean titre was again low, but with a slightly higher value among subjects with a negative blood film. Fig. 1 shows the percentage of each age group who were parasitologically or serologically positive, while Fig. 2 shows the titre distribution in the two age groups, separately for those with and without parasitaemia. The percentage of children with a negative titre (< 1: 20) was surprisingly high, both for subjects with a positive, and those with a negative blood film, and the percentage with a low positive titre (1:20-1:80) was much greater than the percent- age with a higher value () 1:160). In the adult group, the percentage with a negative immunological response was lower than that in the children's group and almost identical for subjects with a positive or negative blood film. The large number of immuno- negative subjects was unexpected in a hyperendemic area, where the humoral immunological response is usually positive, confirming that practically all sub-jects have been in contact with the parasite. Low anti- body titres were more prevalent than high values, even among the adults. Because of these unexpected findings, the survey was repeated under the same conditions exactly one year later in March 1982. The results of this second survey are presented in Tables 3 and 4. The parasito- Table 1. Parasite rate and results of IFA tests on blood samples taken from residents of Mto-wa-Mbu, United Republic of Tanzania, March-April 1981 No. of IFA titre Percentage GeometricAge group people Paaiewith ma(years) examined rate positive titre<20 20 40 80 160 320 640 1280 titre < 1 20 50.0 12 5 1 1 1 40.0 16.2 1 1 1 45.5 6 2 2 1 45.5 20.0 2-4 39 53.8 17 7 8 4 1 1 1 56.4 24.3 5-9 48 66.7 29 7 8 3 1 39.6 20.0 10-14 34 52.9 12 2 7 3 3 4 2 1 64.7 47.1 > 15 148 37.8 24 13 24 31 21 19 15 1 83.8 74.9 Total 300 47.3 100 36 42 47 29 26 18 2 66.7 42.8 900i CHLOROQUINE RESISTANCE OF P. FALCIPARUM IN AFRICA 901 Table 2. IFA test results in residents of Mto-wa-Mbu, according to the results of the parasitological examination, March-April 1981 Parasito- No. of IFA titre Percentage GeometricAge group logical people poitive ea(years) result examined < 20 20 40 80 160 320 640 1280 titre titre 0-14 Positive 85 39 15 10 11 4 5 1 54.1 25.5 Negative 67 37 8 8 5 4 2 2 1 44.8 23.8 Total 152 76 23 18 16 8 7 3 1 50.0 24.8 > 15 Positive 56 9 6 9 11 10 8 3 83.9 68.1 Negative 92 15 7 15 20 11 11 12 1 83.7 79.4 Total 148 24 13 24 31 21 19 15 1 83.8 74.9 logical and serological age profiles observed at the second survey are also shown in Fig. 1. The similarity of the findings of the two surveys is evident, and in particular, the surprisingly low number of serologi- cally positive individuals observed in the first survey is not invalidated by the results of the second survey. 100 90 go . ISko-lil~Mte~bu, 1961 80 -s Mw-Mbu, 1982 70 so 'Z40I 20 20 10 A Parasitological test OL I 0 1 2 5 10 15 Age (years) 100 90 _ go f 70' 60 / 050 -01 W-Mhza.-Ub.enb., 1967 S - Msto-Mbu. 1981o40 0 Mtwe-s Mbu. 1962 Fig. 1. Percentage of subjects with a positive parasito- logical or serological result, according to age, in Mto-wa- Mbu, 1960, 1981, and 1982, and in Muheza-Ubembe, 1967. ClHILDREN (O-14 years) 5 BLOOD FILM POSITIVE 5 <20 N0 PA iii, 320 640 1200 g 0 . im *9 _| [lLOODFILMA NEGTIVE 10 20 10 O1 ---- ------- ~~d el a - 20 D24S 019 32 U 120 02is101as 312 IFA tite IFA tk. Fig. 2. IFA titre distribution in adults and children in Mto- wa-Mbu, March-April 1981. DISCUSSION Chemosuppression of malaria, especially among vulnerable groups of the population, is considered an important activity which may help reduce malaria morbidity, especially in areas where vector control measures are precluded by the lack of human and financial resources. Although this activity has been carried out in the past, either in the form of medicated salt projects (7-10) or through regular administration of drugs to selected groups of the population (11-13), key questions such as whether and how chemotherapy influences or depends on the immune response and its role in selecting drug-resistant parasites, remain to be answered. In hyperendemic areas, fluorescent antibody levels are usually high in newborn infants, then fall in the weeks following birth and remain low during the first E. ONORI ET AL. Table 3. Parasite rate and results of IFA tests on blood samples taken from residents of Mto-wa-Mbu, March 1982 No. of IFA titre Percentage GeometricAge group people Paaiewith ma(years) examined rate positive titean < 20 20 40 80 160 320 640 1280 titre tir < 1 15 40.0 12 1 2 20.0 12.6 1 16 68.8 9 4 3 43.8 15.4 2-4 29 62.1 14 9 5 1 51.7 17.3 5-9 49 59.2 26 9 9 2 2 1 46.9 19.7 10-14 45 60.0 16 13 10 3 2 1 64.4 23.7 > 15 146 39.0 37 33 30 20 20 3 3 74.7 35.4 Total 300 49.3 114 69 59 25 25 3 4 1 62.0 25.7 Table 4. IFA test results in residents of Mto-wa-Mbu, according to the results of the parasitological examination, March 1982 Parasito- No. of IFA titre Percentage GeometricAge group logical people with mean (years) rsl exmndpositive titree ult amined < 20 20 40 80 160 320 640 1280 titre 0-14 Positive 91 32 25 24 5 3 1 1 64.8 23.6 Negative 63 45 11 5 2 28.6 13.8 Total 154 77 36 29 5 5 1 1 50.0 18.9 > 15 Positive 57 9 11 15 10 11 1 84.2 43.6 Negative 89 28 22 15 10 9 3 2 68.5 30.9 Total 146 37 33 30 20 20 3 3 74.7 35.4 year of life (5). With advancing age and increased exposure to a high degree of transmission, there is a progressive rise in the IFA titre (5, 14, 15). However, the results presented in this paper indicate that there was a reduced immunological response among the population of Mto-wa-Mbu. Such a statement could be fully substantiated only by comparing the parasite rates and the immuno- logical status of the population before and after the introduction of the medicated salt. Unfortunately, although parasite rates are available, the immuno- logical status of the population prior to the intro- duction of the drug in 1961 is unknown. An attempt has been made to circumvent this diffi- culty by using data obtained in other hyperendemic areas. In 1967, parasitological and seroepidemiologi- cal surveys were carried out in Muheza-Ubembe, a highly endemic malarious area in the north-east of the United Republic of Tanzania (4). The results of these surveys have been compared with those of the para- sitological survey carried out at Mto-wa-Mbu in 1960 (Table 5), and it appears that the endemicity of malaria in Mto-wa-Mbu was certainly equal to and probably higher than that found in Muheza-Ubembe. Both areas exhibited perennial transmission with seasonal fluctuations, with the main malaria vectors being Anopheles gambiae s.l. and A.funestus, and with P.falciparum being the most prevalent species. Thus, a comparison of the percentages of subjects with positive titres in Muheza in 1967 and in Mto-wa- Mbu in 1981 would appear to be justifiable. Fig. 1(A) displays the age-specific parasite rates observed in Mto-wa-Mbu in 1960, before the intro- duction of chloroquine, and in 1981 and 1982. The observed decrease in the level of parasitaemia in children and increase of the parasite rate in adults (1) are consistent with the serological findings, as shown in Fig. 1(B). The proportion of each age group with a positive titre was substantially and constantly lower in Mto-wa-Mbu in 1981 and 1982 than in Muheza in 1967; the humoral immunological response in Mto- wa-Mbu remained unusually low even in the adults. In Mto-wa-Mbu, the highest parasite rate and the lowest proportion of serologically positive subjects 902 CHLOROQUINE RESISTANCE OF P. FALCIPARUM IN AFRICA Table 5. Results of the parasitological survey in Mto-wa-Mbu in 1960, and the parasitological and serological surveys in Muheza-Ubembe in 1967 Mto-wa-Mbu, 1960 Muheza-Ubembe, 1967 Age group No. of people Parasite Age group No. of people Parasite Percentage with (years) examined rate (years) examined rate positive titre <1 34 64.7 <1 25 36.0 71 1 74 68.9 1 21 42.9 84 2-5 91 76.9 2-4 102 61.8 85 6-10 180 71.7 5-9 124 48.4 92 11-15 84 65.5 10-19 120 45.0 92 > 16 243 24.7 20 403 22.4 98 Total 706 54.8 795 35.9 94 were found in the 5-9 year age group; the reasons for this are not known. A comparison was also made of the titre distribu- tion among children less than 5 years old in Mto-wa- Mbu with that found in a similar group from the West Kiang district of Gambia in 1965. This latter area was hyperendemic and no chemosuppressive measures had been implemented. The results are shown in Fig. 3, separately for children with and without parasitaemia. In subjects with positive blood films, 40% of the Mto-wa-Mbu group had a negative IFA titre compared with only 1.7% of those from the West Kiang district. High titres were significantly much more prevalent in West Kiang ( > 1: 100) than in Mto- wa-Mbu () 1:80). As regards the children with negative blood films, 57.1% of the Mto-wa-Mbu group were seronegative, compared with 30.4070 of MTO.WA-MfU. I*t WEST KIANG DISTRICT, 19g5 IFA tit, IFA itie Fig. 3. Comparison of titre distributions among children under 5 years old, in Mto-wa-Mbu, 1981, and in the West Kiang district of Gambia, 1965. the subjects from the West Kiang district. It therefore appears that the child population of Mto-wa-Mbu exhibited a much reduced humoral immunological response. Several factors may have been responsible for the different immunological profiles obtained in the three areas, namely (a) differences in the level of endemicity prevailing in the areas at the time the serological tests were performed; (b) discrepancies in the IFA results, which were obtained using different antigens; or (c) reduced sensitivity of the IFA test system used at Mto- wa-Mbu. It has already been noted that the level of malaria endemicity found at Mto-wa-Mbu in 1960 was similar to that measured at Muheza-Ubembe in 1967. It may further be assumed that the epidemiological situ- ations in Mto-wa-Mbu (1960) and the West Kiang district of Gambia (1965) were also comparable, since they had similar prevalence, both areas had perennial transmission with a seasonal increase during the wet months, and P.falciparum was the most prevalent malaria species in both areas. The endemic situation in Mto-wa-Mbu does not appear to have changed in recent years. Parasitologi- cal surveys carried out in 1978, 1979, 1980, and 1982 did not show significant differences from the 1981 findings. With regard to the antigens used in the different areas, the highest FA response would have been expected with the P. falciparum (local strains) antigen used at West Kiang, followed by the cultured P. falciparum antigen used in Mto-wa-Mbu, and the P. fieldi in Muheza. It has already been demon- strated, however, that the average FA response with P. fieldi is very similar to that obtained with P. fakciparum (16-18). Although the two different antigens may have had some bearing on the results, the FA response found in Mto-wa-Mbu was signi- 903 904 E. ONORI ET AL. ficantly lower than that observed in Muheza-Ubembe despite the fact that the homologous and, therefore, more specific antigen was used. The difference in the humoral immunological response observed in Mto- wa-Mbu and the West Kiang district was, on the other hand, very marked; it is unlikely that this could be related only to different antigen specificity. There is no reason to suspect that the sensitivity of the IFA assay system used in Mto-wa-Mbu was at fault. The IFA test is carried out routinely in the refer- ence laboratory, not only for seroepidemiological studies but also for diagnostic purposes and for the screening of healthy blood donors. The sensitivity of the antigen and of the reagents are checked frequently in order to verify the validity and reliability of the test. During the second serological survey in March 1982, a number of precautionary measures were taken to avoid all possible causes of error. The results obtained confirmed to a large extent the original findings. There is not, as yet, an immunological technique that can unquestionably relate antibody levels to protective immunity. The IFA technique is recognized as the best and most valid tool for the determination of the presence of antibodies to human malaria and has proved useful in estimating the development, persistence, and specificity of these antibodies. It is however not yet possible to say whether the humoral immunological response measured with the IFA tech- nique is directly related to protective immunity. It can only be assumed that any significant change in the level of IFA antibodies may correspond to a similar change in protective immunity. Previous longitudinal studies have proved that chemosuppression reduces the immunological re- sponse. It has been shown in the Gambia that after administration of antimalarial drugs to mothers and their infants, from the time of their birth for a period of 1 year, the FA levels of the mothers were markedly reduced and the children were serologically negative (19). In another study in Senegal, conducted among the child population of one village, this observation was confirmed.a In a research project in northern a MATTERN, P. ET AL. Chimioprophylaxie antipalustre et anti- corps fluorescents. WHO unpublished document, WHO/MAL/ 67.609, 1967. Nigeria, studies were carried out on the humoral immune response to malaria before, during, and after the application of control measures (spraying of residual insecticide and mass drug administration). At the end of the intervention phase, which lasted two years, the P.falciparum IFA titres in the protected population had decreased. A comparison between antibody titres in the protected and unprotected population showed a very significant difference in all age groups, and especially in the younger subjects (20). During the post-intervention phase there was convincing epidemiological evidence that the inter- vention was followed by a loss of parasitological immunity (21). The results of the investigations carried out at Mto- wa-Mbu seem to provide further evidence that drugs, when given for long periods, have a negative influence on the immunological response of the population. However, the humoral immunological response of Mto-wa-Mbu's population was exceptionally low. Seronegative reactions have often been observed in parasite carriers in hyperendemic malarious areas, and several possible reasons have been suggested. One explanation is that occasionally there may be a complete or partial immunological unresponsiveness (22). Any attempt to relate the lack of immunological response in Mto-wa-Mbu to the drug pressure exerted for many consecutive years can only be speculative, though in vivo studies have proved that the sensitivity of the P.falciparum strains in the locality has greatly diminished after many years of chemosuppression (1). It seems logical to suppose that the increased amount of chloroquine now required to eliminate the parasite from the peripheral blood circulation is the result of a certain loss of protective immunity. This loss may have been due to reduced antigenic stimulation as a consequence of the effect of the drug, such stimulation being necessary to maintain high levels of humoral antibodies (23). The significance and interpretation of the results obtained in Mto-wa-Mbu are open to discussion; however, it is clear that more field studies are urgently required, particularly in view of the practical impli- cations of the findings presented here. ACKNOWLEDGEMENTS The authors wish to thank Professor I.A. McGregor, Liverpool School of Tropical Medicine and Hygiene, for his invaluable advice and encouragement to report the results of this investigation. Thanks are also due to Mr David Payne, WHO Technical Officer, for having carried out the microscopic parasitological examination of slides collected at Mto-wa- Mbu during the second survey, and to Dr E.E. Moshi, Regional Medical Officer, Arusha, for logistic support during the second survey. The excellent collaboration of Mr Abdalla Matongo, village chairman, and of the whole community of Mto-wa-Mbu on the occasion of the two surveys is acknowledged with thanks. CHLOROQUINE RESISTANCE OF P. FALCIPARUM IN AFRICA 905 RESUME APPARITION D'UNE RESISTANCE DE PLASMODIUMFALCIPARUM A LA CHLOROQUINE AU SEIN D'UNE POPULATION SEMI-IMMUNE DE LA REPUBLIQUE-UNIE DE TANZANIE. 2. IMPACT DE LA CHLOROQUINE UTILISEE COMME MEDICAMENT CHIMIOPROPHYLACTIQUE SUR L'STAT IMMUNITAIRE DE LA POPULATION Une dimunition de la sensibilite et un debut de resistance a la chloroquine des souches de P.falciparum ont et signales a Mto-wa-Mbu, une localite du nord-est de la Republique- Unie de Tanzanie. Dans cette localit6 l'emploi de sels medicamenteux a base de chloroquine et l'acces facile a ce remede pour le traitement du paludisme remontaient a une vingtaine d'ann&es. Deux enquetes sero-epidemiologiques ont et executees en mars 1981 et mars 1982 afin de mesurer par la technique d'immunofluorescence (IF) l'influence 6ventuelle de la chimioprophylaxie sur la reponse immunitaire de la popula- tion. En l'absence d'information sur le profil immuno- logique de la population de cette region avant l'introduction de la chloroquine (1960), les resultats des deux enquetes ont et compares a ceux obtenus en 1967 dans une autre localite du nord-est de la Republique-Unie de Tanzanie et en 1965 dans le district de West Kiang en Gambie. Dans ces deux territoires de reference le niveau d'endemicite paludienne etait en effet le meme qu'a Mto-wa-Mbu avant l'introduc- tion du sel medicamenteux. Par comparaison avec ce profil immunologique de ces deux territoires, les r6sultats obtenus en 1981 et 1982 a Mto-wa-Mbu ont r6v6l6 une diminution hautement significative de la proportion des sujets a titre positif ainsi que des titres moyens systematiquement inf& rieurs dans tous les groupes d'age. Une reduction de la reponse immunitaire pourrait s'expli- quer par la pression que le medicament a exerc6e sans inter- ruption dans le territoire pendant de nombreuses ann&es. Cependant une chute aussi importante depassait toute attente et il n'est pas exclu que d'autres facteurs aient pu y contribuer. Etant donne l'importance epidemiologique de ces obser- vations, il est hautement recommande d'entreprendre des etudes serologiques longitudinales sur le terrain afin d'eva- luer les effets possibles de la chimioprophylaxie sur la reponse immunitaire des populations protegees. REFERENCES 1. ONORI, E. ET AL. 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