- Email: [email protected]
EFFECTIVENESS OF AMODIAQUINE AS TREATMENT FOR CHLOROQUINE-RESISTANT PLASMODIUM FALCIPARUM INFECTIONS IN KENYA
357
Furthermore, this criticism cannot be extended to the breath
hydrogen test, which measures lactose absorption independently of the blood glucose levels. In the present investigation the results of the breath hydrogen test, both in controls and in patients with idiopathic cataract, were similar to those obtained by measuring the rise in blood glucose after oral lactose administration. No significant difference in the milk-drinking habits was found between lactose absorbers and malabsorbers or between controls and cataract patients. In conclusion, in a population with a high frequency of primary adult lactose malabsorption, lactose absorbers are more common among adults with cataract than in the general population. These results, if confirmed by other surveys in other populations with a high frequency of lactose malabsorption in adult life, suggest that adults able to absorb galactose from a lactose-containing diet are more susceptible to senile or presenile cataract. Whether lactose absorbers with cataract have a lower peripheral utilisation of blood galactose derived from the intestinal hydrolysis of lactose-either as a consequence oflower levels ofblood insulin in response to the rise in blood glucoseor as a result of lower activities of the enzymes involved in galactose metabolism12-should now be ascertained. We thank Dr Pietro Vajro for his continuing interest and support in the evaluation of the breath hydrogen test data.
EFFECTIVENESS OF AMODIAQUINE AS TREATMENT FOR CHLOROQUINE-RESISTANT PLASMODIUM FALCIPARUM INFECTIONS IN KENYA W. M. WATKINS H. C. SPENCER D. M. KARIUKI
D. G. SIXSMITH D. A. BORIGA T. KIPINGOR D. K. KOECH
Clinical Research Centre, Kenya Medical Research Institute, Nairobi, Kenya; University of Nairobi, Department of Pharmacy; Division of Parasitic Diseases, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia; Division of Vector-borne Diseases, Kenya Ministry of Health
conducted in Malindi, Kenya, the response of Plasmodium falciparum to chloroquine and amodiaquine in vivo (by an extended 14-day test) and in vitro (with the Rieckmann micro test). In-vivo resistance was demonstrated in 19 of 69 (28%) infections treated with chloroquine, but in only 2 of 60 (3·3%) of those treated with amodiaquine (p<0·001). Invitro resistance to chloroquine was demonstrated in 15 of 23 (65%) tests. In contrast, 22 of the same 23 isolates were sensitive to amodiaquine in vitro. Effective concentrations by probit analysis for 50% and 99% (EC50 and EC99) inhibition, respectively, were 180·7 and 4319·6 nmol/l for chloroquine and 12·2 and 147·0 nmol/l for amodiaquine. The results suggest that amodiaquine is effective for the treatment of chloroquine-resistant falciparum malaria in Kenya.
Summary
Studies
to
were
assess
Introduction SINCE 1978, chloroquine-resistant Plasmodiumfalciparum infections have been reported in non-immune visitors to East Africa.l,2 Resistant falciparum malaria is rare among Kenyan Africans,3 but widespread in the Afriean population of neighbouring Tanzania and Zanzibar.1,4 Amodiaquine, a 4-aminoquinoline structurally similar to chloroquine, is more active than chloroquine against chloroquine-resistant strains
Correspondence should be addressed to S. A., Department of Clinical Paediatrics, II Faculty of Medicine, University of Naples, Via Sergio Pansini 5, 80131 Naples, Italy. REFERENCES 1. Simoons FJ. A
geographic approach to senile cataracts: Possible links with milk consumption, lactase activity and galactose metabolism. Dig Dis Sci 1982; 27:
257-64. 2. Auricchio S, Rubino
A, Semenza G, Landolt M, Prader A. Isolated intestinal lactase deficiency in the adult. Lancet 1963; ii: 324-26. 3. Simoons FJ. The geographic hypothesis and lactose malabsorption. A weighting ofthe evidence. Dig Dis Sci 1978; 23: 963-80. 4. American Academy of Pediatrics Committee on Nutrition. The practical significance of lactose intolerance in children. Pediatrics 1978; 62: 240-45. N, Vajro P, Dioguardi G, Mensitieri R, Longo D. Gas chromatographic quantitation of breath hydrogen and carbon monoxide for clinical investigation in adults and in children. J Chromatogr Biomed Appl (in press). Auricchio S, Rubino A, Tosi R, Semenza G, Landolt K, Kilster H, Prader A. Disaccharidase activities in human intestinal mucosa. Enzymol Biol Clin 1963; 3: 193-208. Asp NG, Dahlqvist A. Human small intestine &bgr;-galactosidases: Specific assay of three different enzymes. Analyt Biochem 1972; 47: 527-38. Asp NG, Berg O, Dahlqvist A, Jussila J, Salmi H. The activity of three different smallintestinal &bgr;-galactosidases in adults with and without lactase deficiency. Scand J Gastroenterol 1971; 6: 755-62. De Ritis F, Balestrieri GG, Ruggiero G, Filosa E, Auricchio S. High frequency of lactase activity deficiency of small bowel of adults in the Neapolitan area. Enzymol Biol Clin 1970; 11: 263-67. Editorial. Epidemiology of cataract. Lancet 1982; i: 1392-93. Courpotin C, Bordas M, Kragen MT, Etienne G, Lestradet H. Metabolisme et utilization du galactose an cours de l’ingestion du lactose. Arch Franc Pédiatr 1980; 37: LVIII-LIX. Skalka HW, Prchal JT. Presenile cataract formation and decreased activity of galactosemic enzymes. Arch Ophthalmol 1980; 98: 269-73.
5. Sannolo
6.
7.
8.
9.
10. 11.
12.
falciparum in the Aotus monkey5 and man.In a recent study in Kisumu, Kenya, amodiaquine was an effective of P
antimalarial, and all isolates
were
more
senstitive
to
chloroquine in vitro.7 The purpose of amodiaquine our study was: (1) to document the degree of chloroquineresistance after a standard therapeutic regimen in semiimmune individuals at the Kenyan coast, and (2) to compare the effectiveness of chloroquine and amodiaquine as P than
to
falciparum schizonticides. Methods In-vivo Tests The study was conducted at Kakuyuni School, Malindi, on the Indian Ocean coast of Kenya, where malaria is hyperendemic according to Ministry of Health data. 423 school children aged 6-17 years were screened and were included in the study if a blood film was positive for only P falciparum, there was no history of antimalarial drug ingestion during the previous fortnight, the DillGlazko urine test for 4-aminoquinolines8was negative, and consent to participate in the study was obtained from parents or teachers. Subjects were treated with either chloroquine or amodiaquine, 25 mg base per kg, over 3 days. Both drugs were within pharmacopoeial potency limits on assay.9 The Dill-Glazko test was repeated on day 1 or 2 to confirm drug absorption. Thick blood films were examined on days 1-7, inclusive, and on day 14. Since recrudescence of parasitaemia cannot be distinguished from reinfection, we followed-up patients for only 14 days. The incubation period for P falciparum is at least 9 days (average 12 days)r° and in our observations of over 300 extended in-vivo tests, done with the same regimen of chloroquine as used in the present study, in drug-sensitive areas of Kenya, no reinfections occurred within 14 days (Spencer HC, unpublished). Reappearance of parasitaemia within 14 days after the absorption of a therapeutic dose of chloroquine is therefore indicative of recrudescence and not reinfection. ’
In-vitro Tests Before treatment 4 ml blood samples were obtained from 29 subjects (15 from the amodiaquine treatment group, 14 from the chloroquine group) with initial parasitaemia of 25 per 300 leucocytes. (The 29 subjects tested were not the only ones with 25
358 TABLE I-IN-VIVO RESPONSE TO
parasites/300 WBC, but more tests were impracticable under field conditions.) All these samples were tested in Malindi for amodiaquine and chloroquine resistance by the Rieckmann micro in-vitro tests. IIRPMI medium 1640 was supplemented with 25 mmol/1 NaHC03, 25 mmol/l HEPES buffer, and gentamicin 10 pg/ml. Blood containing parasites was diluted 1/10 with medium, and 50 1 portions of the mixture were added to wells of a 96-well microculture plate supplied by the World Health Organisation and predosed to give final concentrations in the test of 0, 20, 40, 80, 114, 160, 320, and 640 nmol/1 of chloroquine and 0, 5, 10, 20, 40, 80, 160, and 320 nmol/1 of amodiaquine. The microculture plate was placed in a gas-tight box (Bellco Glass Inc., Vineland, New Jersey, USA), which was flushed with a mixture of 3% CO2, 5% O2, and 92% N2
OF
129
CHLOROQUINE OR AMODIAQUINE FALCIPARUM MALARIA, MALINDI,
KENYAN CHILDREN WITH
KENYA, 1983
before incubation at 37-38°C for 24 h. After incubation, thick films were prepared and the number of schizonts per 300 leucocytes counted. A successful test was one in which schizont maturation in the control well was 5% predose parasitaemia. The minimum inhibitory concentration (MIC) was defined as the lowest drug concentration associated with <1% of the number of schizonts in drug-free control wells. Proven chloroquine-sensitive P falciparum isolates are inhibited at a concentration of 114 nmol/1.11 Effective concentrations for 50% and 99% inhibition (EG;o and ECg9) were obtained by probit analysis of log dose/response by the method of Grab and Wernsdorfer World Health Geneva, Organisation, (unpublished; WHO/MAL/83 -990). Blood samples obtained from 6 subjects with recurrent parasitaemia on day 14 were sent to Nairobi by air, where Rieckmann micro tests were performed. All patients with recurrent parasitaemia were subsequently treated with pyrimethamine/
* WHO definition.6 tFisher’s exact test.
* x:
test
with Yates’ correction.
TABLE II-IN-VITRO RESPONSE OF PLASMODIUMFALCIPARUM ISOLATES TO CHLOROQUINE AND AMODIAQUINE, MALINDI, KENYA, 1983
’
sulfadoxine (’Fansidar’).
Results
Study Population Of the 423 children initially screened, 188 (44%) had positive thick films. 59 children did not fulfill study requirements or were lost to follow-up, leaving a final study population of 129. The amodiaquine group consisted of 34 boys and 26 girls with a mean weight of 29’9 kg (range 18-52), a mean age of 10’6 years (range 6-17), and a mean asexual parasite count before treatment of 92 (range 2-1362) per 300 leucocytes. The chloroquine group consisted of 35 boys and 34 girls with a mean weight of 28 -3 kg (range 19-50), a mean age of 10 -5years (range 6-16), and a mean asexual parasite count before treatment of 53 (range 1-576) per 300 leucocytes. Comparison by Student’s t test showed no significant differences (p<0 1) between the two groups in
weight,
age,
or
initial
parasitaemia.
In-vivo Tests
During the first 2 days the rate of parasite clearance was similar in the two treatment groups. In the amodiaquine group the mean asexual parasite count per 300 leucocytes was 44 (range 0-729) on day 1, and 2 (range 0-53) on day 2. Corresponding figures for the choloroquine group were 41 (range 0-633) on day 1 and 5 (range 0-131) on day 2. Thereafter the patterns in the two groups progressively became dissimilar (table I). In the amodiaquine group, thick films became negative by day 5 and remained negative up to day 7 in all subjects. In the chloroquine group thick blood films from 3 patients either did not become negative (2 cases) or remained negative for only 1 day (1 case), indicating RII resistance.6 In 5 subjects treated with chloroquine parasites reappeared by day 7 (RI resistance, early recrudescence). In 2 subjects treated with amodiaquine and 11 treated with chloroquine, parasites reappeared by day 14 (RI, delayed recrudescence). Resistance to either drug at the RIII level was not detected.
*Lowest drug concentration at which schizont development free controls; Rieckmann micro test. NT =not tested.
was
1%
of drug-
In-vitro Tests
In 23 of 29 patients in-vitro tests were successful for both drugs-12 from the amodiaquine treatment group and 11 from the chloroquine group. All 23 isolates were inhibited by 80 nmol/1 of amodiaquine. Only 7 of 23 isolates were inhibited by chloroquine at < 114 nmol/l. The proportion of chloroquine-resistant isolates in the two treatment groups was not significantly different (7/12 vs 9/11, p=0’22, Fisher’s exact test, table n). Probit analysis of the chloroquine in-vitro results gave values of 180-77 and 4319-6 nmol/1 for EC50 and EC99, respectively. Comparative values for amodiaquine were 12 - 2 and 147-0 nmol/1. In-vitro tests performed on the 6 blood samples obtained during the follow-up phase were successful, despite the lapse of at least 10 h between blood collection in Kakayuni and testing in Nairobi. The five isolates from chloroquine-treated patients had MIC values of >320 nmol/1 to chloroquine, but C40 nmol/1 to amodiaquine. The single isolate from an amodiaquine-treated patient had an MIC of 40 nmol/1 to amodiaquine and >640 nmol/1 to chloroquine. A comparison of the in-vivo and the in-vitro results for the chloroquine-treatment group is given in table II. Discussion This study shows the presence of chloroquine resistance of the RI and RII types in one village on the Kenya coast. 28% patients given a standard therapeutic regimen of 25 mg chloroquine base per kg over 3 days had persistent or
359 TABLE III-RESULTS OF IN-VITRO AND IN-VIVO TESTS ON 11 PATIENTS TREATED WITH CHLOROQUINE, MALINDI, KENYA, 1983
confirmation in other areas. However, we believe that the results justify a consideration of the use of amodiaquine in place of chloroquine for the treatment of falciparum malaria along the Kenyan coast. We thank the director of medical services, Kenya Ministry of Health for permission to publish these results; the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, and the Kenya Council for Science and Technology for their support; Mr J. Ogeto, department of pharmacy, University of Nairobi, for doing the drug assays; and Dr J. Chulay, US Army Medical Research Unit, Kenya, for advice in the preparation of the manuscript.
Correspondence should be addressed to H. C. S., Division of Parasitic Diseases, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 30333, USA. *
REFERENCES
Patient’s age
(years) given in parentheses. -MIC =minimum inhibitory concentration. $All three patients with RI responses had negative positive smears on day 14.
1. Peters W. Antimalarial smears on
day
7 and
recurrent parasitaemia during the next 14 days. In contrast all those given amodiaquine, also at a dose of 25 mg base/kg for 3 days, became clear of parasites initially, with only 3% recurrence within 14 days. In-vitro tests by both MIC and probit analyses confirmed the greater susceptibility of P falciparum in the study area to amodiaquine. For chloroquine, all isolates with an MIC 320 nmol/1 were sensitive to the drug in vivo. This contrasts with results in non-immune individuals, in whom isolates with an MIC of 160 nmol/1 are often resistant in-vivo." In addition, two isolates with MIC values of >640 nmol/ were from subjects classified as sensitive in the in-vivo test (table III). These discrepancies emphasise the importance of host immunity in our semi-immune subjects from a hyperendemic of parasitaemia Recurrence after malarious area. amodiaquine treatment occurred in 2 subjects, and an in-vitro test in 1 of these cases gave an MIC for amodiaquine of 40 nmol/1. Since 96% of isolates were sensitive to this concentration of amodiaquine in vitro, it is possible that parasite reappearance in this subject resulted from either reinfection or incomplete eradication of the original parasite population, possibly because of poor drug absorption. Although this is the first study to report substantial in-vivo chloroquine resistance amongst Kenyans, the results accord with those obtained by periodic in-vitro tests in Malindi: over the past 3 years the pattern of chloroquine susceptibility as portrayed by the Rieckmann micro test has changed from entirely sensitive to predominantly resistant. 12 The chloroquine resistance we observed gives cause for concern on two accounts: (1) chloroquine is the drug of choice for the treatment of falciparum malaria throughout Kenya and a considerable failure rate in therapy with this drug is predictable in coastal and possibly other regions of the country, and (2) there is the possibility that chloroquine resistance may spread rapidly. The double biological advantage of the resistant parasite (its ability to outgrow sensitive strains, and the comparative facility with which infection is established in mosquitoes feeding on chloroquine-dosed individuals I) suggests that the prevalence of chloroquine-resistance in Kenya will increase, especially with continued chloroquine usage and inadequate vector control. Although cross-resistance occurs, chloroquine-resistant P falciparum infections frequently are either cured by or less resistant to amodiaquine,13 and our results suggest that amodiaquine may be a useful treatment for a limited period in Kenya. Our study was confined to one village and requires
drug resistance: an increasing problem. Br Med Bull 1982; 38:
187-92.
Weniger BC, Blumberg RS, Campbell CC, Jones TC, Mount DL, Friedman SM. High level chloroquine resistance of Plasmodium falciparum malaria acquired in Kenya. N Engl J Med 1982; 307: 1560-62. 3. Spencer HC, Kariuki DM, Koech DK. Chloroquine resistance in Plasmodium falciparum from Kenyan infants. Am J Trop Med Hyg 1983; 32: 922-25. 4. Schwartz IK, Payne D, Campbell CC, Khatib OJ. In vivo and in in vitro assessment of chloroquine-resistant Plasmodium falciparum malaria in Zanzibar. Lancet 1983; i: 2.
1003-05. 5. Schmidt LH,
Vaughan D, Mueller D, Crosby R, Hamilton R. Activities of various 4-aminoquinolines against infection with chloroquine-resistant strains of
Plasmodium falciparum. Antimicrob Ag Chemother 1977; 11: 826-43. 6. World Health Organisation. Chemotherapy of malaria and resistance to antimalarials: Report ofa WHO Scientific Group. WHO Tech Rep Ser 1973; no 529: 46. 7. Spencer HC, Kipengor T, Agure R, Koech DK, Chulay JD. Plasmodium falciparum in Kisumu, Kenya: Differences in sensitivity to amodiaquine and chloroquine in vitro. J Infect Dis 1983; 148: 732-36. 8. Lelijveld J, Kortman H. The eosin colour test of Dill and Glazko: a simple field test to detect chloroquine in urine. Bull WHO 1970; 42: 477-79. 9. British Pharmacopoeia. London: Pharmaceutical Press, 1973: 28. 10. Bruce-Chwatt LJ Essential malariology. London: Heinemann, 1980: 35 11. Rieckmann KH, Sax LJ, Campbell GH, Mrema JE. Drug sensitivity of Plasmodium falciparum: An in vitro micro technique Lancet 1978; i: 22-23. 12. Sixsmith DG, Spencer HC, Watkins WM, Koech DK, Chulay JD. Changing in vitro response to chloroquine of Plasmodium falciparum in Kenya. Lancet 1983; ii. 1022. 13. Hall AP, Segal HE, Pearlman EJ, Phintuyothin P, Kosakal S. Amodiaquine-resistant falciparum malaria in Thailand. Am J Trop Med Hyg 1975; 24: 574-80.
METHOTREXATE SYSTEMIC CLEARANCE INFLUENCES PROBABILITY OF RELAPSE IN CHILDREN WITH STANDARD-RISK ACUTE LYMPHOCYTIC LEUKAEMIA* WILLIAM E. EVANS CLINTON F. STEWART CHEN-HSIN CHEN
JOSEPH
WILLIAM R. CROM W. PAUL BOWMAN MINNIE ABROMOWITCH V. SIMONE
Pharmacokinetics and Pharmacodynamics Section, Pharmaceutical Division, Division of Hematology and Oncology, and Biostatistics Section, St Jude Children’s Research Hospital, Memphis, Tennessee, USA
Summary
108
children
with
standard-risk
acute
lymphocytic leukaemia (ALL) were randomised to a post-induction treatment protocol including 15 doses of intermediate-dose methotrexate (1000 mg/m2) in addition to conventional oral therapy of mercaptopurine and low-dose methotrexate. After median follow-up of 26 months, 22 patients have had relapses. Among the 108 patients, rates of methotrexate systemic clearance ranged from 44· 7 to 132 ml/min/m2. When the group
was
divided into three
patients’ rates of methotrexate clearance, statistical analysis of the Kaplan-Meier curves estimating the probability of complete remission showed significant differences (p=0·016) among the subgroups, subgroups according
to
the
*Presented in part to the American California, May, 1983.
Society of Clinical Oncology, San Diego,
Furthermore, this criticism cannot be extended to the breath
hydrogen test, which measures lactose absorption independently of the blood glucose levels. In the present investigation the results of the breath hydrogen test, both in controls and in patients with idiopathic cataract, were similar to those obtained by measuring the rise in blood glucose after oral lactose administration. No significant difference in the milk-drinking habits was found between lactose absorbers and malabsorbers or between controls and cataract patients. In conclusion, in a population with a high frequency of primary adult lactose malabsorption, lactose absorbers are more common among adults with cataract than in the general population. These results, if confirmed by other surveys in other populations with a high frequency of lactose malabsorption in adult life, suggest that adults able to absorb galactose from a lactose-containing diet are more susceptible to senile or presenile cataract. Whether lactose absorbers with cataract have a lower peripheral utilisation of blood galactose derived from the intestinal hydrolysis of lactose-either as a consequence oflower levels ofblood insulin in response to the rise in blood glucoseor as a result of lower activities of the enzymes involved in galactose metabolism12-should now be ascertained. We thank Dr Pietro Vajro for his continuing interest and support in the evaluation of the breath hydrogen test data.
EFFECTIVENESS OF AMODIAQUINE AS TREATMENT FOR CHLOROQUINE-RESISTANT PLASMODIUM FALCIPARUM INFECTIONS IN KENYA W. M. WATKINS H. C. SPENCER D. M. KARIUKI
D. G. SIXSMITH D. A. BORIGA T. KIPINGOR D. K. KOECH
Clinical Research Centre, Kenya Medical Research Institute, Nairobi, Kenya; University of Nairobi, Department of Pharmacy; Division of Parasitic Diseases, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia; Division of Vector-borne Diseases, Kenya Ministry of Health
conducted in Malindi, Kenya, the response of Plasmodium falciparum to chloroquine and amodiaquine in vivo (by an extended 14-day test) and in vitro (with the Rieckmann micro test). In-vivo resistance was demonstrated in 19 of 69 (28%) infections treated with chloroquine, but in only 2 of 60 (3·3%) of those treated with amodiaquine (p<0·001). Invitro resistance to chloroquine was demonstrated in 15 of 23 (65%) tests. In contrast, 22 of the same 23 isolates were sensitive to amodiaquine in vitro. Effective concentrations by probit analysis for 50% and 99% (EC50 and EC99) inhibition, respectively, were 180·7 and 4319·6 nmol/l for chloroquine and 12·2 and 147·0 nmol/l for amodiaquine. The results suggest that amodiaquine is effective for the treatment of chloroquine-resistant falciparum malaria in Kenya.
Summary
Studies
to
were
assess
Introduction SINCE 1978, chloroquine-resistant Plasmodiumfalciparum infections have been reported in non-immune visitors to East Africa.l,2 Resistant falciparum malaria is rare among Kenyan Africans,3 but widespread in the Afriean population of neighbouring Tanzania and Zanzibar.1,4 Amodiaquine, a 4-aminoquinoline structurally similar to chloroquine, is more active than chloroquine against chloroquine-resistant strains
Correspondence should be addressed to S. A., Department of Clinical Paediatrics, II Faculty of Medicine, University of Naples, Via Sergio Pansini 5, 80131 Naples, Italy. REFERENCES 1. Simoons FJ. A
geographic approach to senile cataracts: Possible links with milk consumption, lactase activity and galactose metabolism. Dig Dis Sci 1982; 27:
257-64. 2. Auricchio S, Rubino
A, Semenza G, Landolt M, Prader A. Isolated intestinal lactase deficiency in the adult. Lancet 1963; ii: 324-26. 3. Simoons FJ. The geographic hypothesis and lactose malabsorption. A weighting ofthe evidence. Dig Dis Sci 1978; 23: 963-80. 4. American Academy of Pediatrics Committee on Nutrition. The practical significance of lactose intolerance in children. Pediatrics 1978; 62: 240-45. N, Vajro P, Dioguardi G, Mensitieri R, Longo D. Gas chromatographic quantitation of breath hydrogen and carbon monoxide for clinical investigation in adults and in children. J Chromatogr Biomed Appl (in press). Auricchio S, Rubino A, Tosi R, Semenza G, Landolt K, Kilster H, Prader A. Disaccharidase activities in human intestinal mucosa. Enzymol Biol Clin 1963; 3: 193-208. Asp NG, Dahlqvist A. Human small intestine &bgr;-galactosidases: Specific assay of three different enzymes. Analyt Biochem 1972; 47: 527-38. Asp NG, Berg O, Dahlqvist A, Jussila J, Salmi H. The activity of three different smallintestinal &bgr;-galactosidases in adults with and without lactase deficiency. Scand J Gastroenterol 1971; 6: 755-62. De Ritis F, Balestrieri GG, Ruggiero G, Filosa E, Auricchio S. High frequency of lactase activity deficiency of small bowel of adults in the Neapolitan area. Enzymol Biol Clin 1970; 11: 263-67. Editorial. Epidemiology of cataract. Lancet 1982; i: 1392-93. Courpotin C, Bordas M, Kragen MT, Etienne G, Lestradet H. Metabolisme et utilization du galactose an cours de l’ingestion du lactose. Arch Franc Pédiatr 1980; 37: LVIII-LIX. Skalka HW, Prchal JT. Presenile cataract formation and decreased activity of galactosemic enzymes. Arch Ophthalmol 1980; 98: 269-73.
5. Sannolo
6.
7.
8.
9.
10. 11.
12.
falciparum in the Aotus monkey5 and man.In a recent study in Kisumu, Kenya, amodiaquine was an effective of P
antimalarial, and all isolates
were
more
senstitive
to
chloroquine in vitro.7 The purpose of amodiaquine our study was: (1) to document the degree of chloroquineresistance after a standard therapeutic regimen in semiimmune individuals at the Kenyan coast, and (2) to compare the effectiveness of chloroquine and amodiaquine as P than
to
falciparum schizonticides. Methods In-vivo Tests The study was conducted at Kakuyuni School, Malindi, on the Indian Ocean coast of Kenya, where malaria is hyperendemic according to Ministry of Health data. 423 school children aged 6-17 years were screened and were included in the study if a blood film was positive for only P falciparum, there was no history of antimalarial drug ingestion during the previous fortnight, the DillGlazko urine test for 4-aminoquinolines8was negative, and consent to participate in the study was obtained from parents or teachers. Subjects were treated with either chloroquine or amodiaquine, 25 mg base per kg, over 3 days. Both drugs were within pharmacopoeial potency limits on assay.9 The Dill-Glazko test was repeated on day 1 or 2 to confirm drug absorption. Thick blood films were examined on days 1-7, inclusive, and on day 14. Since recrudescence of parasitaemia cannot be distinguished from reinfection, we followed-up patients for only 14 days. The incubation period for P falciparum is at least 9 days (average 12 days)r° and in our observations of over 300 extended in-vivo tests, done with the same regimen of chloroquine as used in the present study, in drug-sensitive areas of Kenya, no reinfections occurred within 14 days (Spencer HC, unpublished). Reappearance of parasitaemia within 14 days after the absorption of a therapeutic dose of chloroquine is therefore indicative of recrudescence and not reinfection. ’
In-vitro Tests Before treatment 4 ml blood samples were obtained from 29 subjects (15 from the amodiaquine treatment group, 14 from the chloroquine group) with initial parasitaemia of 25 per 300 leucocytes. (The 29 subjects tested were not the only ones with 25
358 TABLE I-IN-VIVO RESPONSE TO
parasites/300 WBC, but more tests were impracticable under field conditions.) All these samples were tested in Malindi for amodiaquine and chloroquine resistance by the Rieckmann micro in-vitro tests. IIRPMI medium 1640 was supplemented with 25 mmol/1 NaHC03, 25 mmol/l HEPES buffer, and gentamicin 10 pg/ml. Blood containing parasites was diluted 1/10 with medium, and 50 1 portions of the mixture were added to wells of a 96-well microculture plate supplied by the World Health Organisation and predosed to give final concentrations in the test of 0, 20, 40, 80, 114, 160, 320, and 640 nmol/1 of chloroquine and 0, 5, 10, 20, 40, 80, 160, and 320 nmol/1 of amodiaquine. The microculture plate was placed in a gas-tight box (Bellco Glass Inc., Vineland, New Jersey, USA), which was flushed with a mixture of 3% CO2, 5% O2, and 92% N2
OF
129
CHLOROQUINE OR AMODIAQUINE FALCIPARUM MALARIA, MALINDI,
KENYAN CHILDREN WITH
KENYA, 1983
before incubation at 37-38°C for 24 h. After incubation, thick films were prepared and the number of schizonts per 300 leucocytes counted. A successful test was one in which schizont maturation in the control well was 5% predose parasitaemia. The minimum inhibitory concentration (MIC) was defined as the lowest drug concentration associated with <1% of the number of schizonts in drug-free control wells. Proven chloroquine-sensitive P falciparum isolates are inhibited at a concentration of 114 nmol/1.11 Effective concentrations for 50% and 99% inhibition (EG;o and ECg9) were obtained by probit analysis of log dose/response by the method of Grab and Wernsdorfer World Health Geneva, Organisation, (unpublished; WHO/MAL/83 -990). Blood samples obtained from 6 subjects with recurrent parasitaemia on day 14 were sent to Nairobi by air, where Rieckmann micro tests were performed. All patients with recurrent parasitaemia were subsequently treated with pyrimethamine/
* WHO definition.6 tFisher’s exact test.
* x:
test
with Yates’ correction.
TABLE II-IN-VITRO RESPONSE OF PLASMODIUMFALCIPARUM ISOLATES TO CHLOROQUINE AND AMODIAQUINE, MALINDI, KENYA, 1983
’
sulfadoxine (’Fansidar’).
Results
Study Population Of the 423 children initially screened, 188 (44%) had positive thick films. 59 children did not fulfill study requirements or were lost to follow-up, leaving a final study population of 129. The amodiaquine group consisted of 34 boys and 26 girls with a mean weight of 29’9 kg (range 18-52), a mean age of 10’6 years (range 6-17), and a mean asexual parasite count before treatment of 92 (range 2-1362) per 300 leucocytes. The chloroquine group consisted of 35 boys and 34 girls with a mean weight of 28 -3 kg (range 19-50), a mean age of 10 -5years (range 6-16), and a mean asexual parasite count before treatment of 53 (range 1-576) per 300 leucocytes. Comparison by Student’s t test showed no significant differences (p<0 1) between the two groups in
weight,
age,
or
initial
parasitaemia.
In-vivo Tests
During the first 2 days the rate of parasite clearance was similar in the two treatment groups. In the amodiaquine group the mean asexual parasite count per 300 leucocytes was 44 (range 0-729) on day 1, and 2 (range 0-53) on day 2. Corresponding figures for the choloroquine group were 41 (range 0-633) on day 1 and 5 (range 0-131) on day 2. Thereafter the patterns in the two groups progressively became dissimilar (table I). In the amodiaquine group, thick films became negative by day 5 and remained negative up to day 7 in all subjects. In the chloroquine group thick blood films from 3 patients either did not become negative (2 cases) or remained negative for only 1 day (1 case), indicating RII resistance.6 In 5 subjects treated with chloroquine parasites reappeared by day 7 (RI resistance, early recrudescence). In 2 subjects treated with amodiaquine and 11 treated with chloroquine, parasites reappeared by day 14 (RI, delayed recrudescence). Resistance to either drug at the RIII level was not detected.
*Lowest drug concentration at which schizont development free controls; Rieckmann micro test. NT =not tested.
was
1%
of drug-
In-vitro Tests
In 23 of 29 patients in-vitro tests were successful for both drugs-12 from the amodiaquine treatment group and 11 from the chloroquine group. All 23 isolates were inhibited by 80 nmol/1 of amodiaquine. Only 7 of 23 isolates were inhibited by chloroquine at < 114 nmol/l. The proportion of chloroquine-resistant isolates in the two treatment groups was not significantly different (7/12 vs 9/11, p=0’22, Fisher’s exact test, table n). Probit analysis of the chloroquine in-vitro results gave values of 180-77 and 4319-6 nmol/1 for EC50 and EC99, respectively. Comparative values for amodiaquine were 12 - 2 and 147-0 nmol/1. In-vitro tests performed on the 6 blood samples obtained during the follow-up phase were successful, despite the lapse of at least 10 h between blood collection in Kakayuni and testing in Nairobi. The five isolates from chloroquine-treated patients had MIC values of >320 nmol/1 to chloroquine, but C40 nmol/1 to amodiaquine. The single isolate from an amodiaquine-treated patient had an MIC of 40 nmol/1 to amodiaquine and >640 nmol/1 to chloroquine. A comparison of the in-vivo and the in-vitro results for the chloroquine-treatment group is given in table II. Discussion This study shows the presence of chloroquine resistance of the RI and RII types in one village on the Kenya coast. 28% patients given a standard therapeutic regimen of 25 mg chloroquine base per kg over 3 days had persistent or
359 TABLE III-RESULTS OF IN-VITRO AND IN-VIVO TESTS ON 11 PATIENTS TREATED WITH CHLOROQUINE, MALINDI, KENYA, 1983
confirmation in other areas. However, we believe that the results justify a consideration of the use of amodiaquine in place of chloroquine for the treatment of falciparum malaria along the Kenyan coast. We thank the director of medical services, Kenya Ministry of Health for permission to publish these results; the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, and the Kenya Council for Science and Technology for their support; Mr J. Ogeto, department of pharmacy, University of Nairobi, for doing the drug assays; and Dr J. Chulay, US Army Medical Research Unit, Kenya, for advice in the preparation of the manuscript.
Correspondence should be addressed to H. C. S., Division of Parasitic Diseases, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 30333, USA. *
REFERENCES
Patient’s age
(years) given in parentheses. -MIC =minimum inhibitory concentration. $All three patients with RI responses had negative positive smears on day 14.
1. Peters W. Antimalarial smears on
day
7 and
recurrent parasitaemia during the next 14 days. In contrast all those given amodiaquine, also at a dose of 25 mg base/kg for 3 days, became clear of parasites initially, with only 3% recurrence within 14 days. In-vitro tests by both MIC and probit analyses confirmed the greater susceptibility of P falciparum in the study area to amodiaquine. For chloroquine, all isolates with an MIC 320 nmol/1 were sensitive to the drug in vivo. This contrasts with results in non-immune individuals, in whom isolates with an MIC of 160 nmol/1 are often resistant in-vivo." In addition, two isolates with MIC values of >640 nmol/ were from subjects classified as sensitive in the in-vivo test (table III). These discrepancies emphasise the importance of host immunity in our semi-immune subjects from a hyperendemic of parasitaemia Recurrence after malarious area. amodiaquine treatment occurred in 2 subjects, and an in-vitro test in 1 of these cases gave an MIC for amodiaquine of 40 nmol/1. Since 96% of isolates were sensitive to this concentration of amodiaquine in vitro, it is possible that parasite reappearance in this subject resulted from either reinfection or incomplete eradication of the original parasite population, possibly because of poor drug absorption. Although this is the first study to report substantial in-vivo chloroquine resistance amongst Kenyans, the results accord with those obtained by periodic in-vitro tests in Malindi: over the past 3 years the pattern of chloroquine susceptibility as portrayed by the Rieckmann micro test has changed from entirely sensitive to predominantly resistant. 12 The chloroquine resistance we observed gives cause for concern on two accounts: (1) chloroquine is the drug of choice for the treatment of falciparum malaria throughout Kenya and a considerable failure rate in therapy with this drug is predictable in coastal and possibly other regions of the country, and (2) there is the possibility that chloroquine resistance may spread rapidly. The double biological advantage of the resistant parasite (its ability to outgrow sensitive strains, and the comparative facility with which infection is established in mosquitoes feeding on chloroquine-dosed individuals I) suggests that the prevalence of chloroquine-resistance in Kenya will increase, especially with continued chloroquine usage and inadequate vector control. Although cross-resistance occurs, chloroquine-resistant P falciparum infections frequently are either cured by or less resistant to amodiaquine,13 and our results suggest that amodiaquine may be a useful treatment for a limited period in Kenya. Our study was confined to one village and requires
drug resistance: an increasing problem. Br Med Bull 1982; 38:
187-92.
Weniger BC, Blumberg RS, Campbell CC, Jones TC, Mount DL, Friedman SM. High level chloroquine resistance of Plasmodium falciparum malaria acquired in Kenya. N Engl J Med 1982; 307: 1560-62. 3. Spencer HC, Kariuki DM, Koech DK. Chloroquine resistance in Plasmodium falciparum from Kenyan infants. Am J Trop Med Hyg 1983; 32: 922-25. 4. Schwartz IK, Payne D, Campbell CC, Khatib OJ. In vivo and in in vitro assessment of chloroquine-resistant Plasmodium falciparum malaria in Zanzibar. Lancet 1983; i: 2.
1003-05. 5. Schmidt LH,
Vaughan D, Mueller D, Crosby R, Hamilton R. Activities of various 4-aminoquinolines against infection with chloroquine-resistant strains of
Plasmodium falciparum. Antimicrob Ag Chemother 1977; 11: 826-43. 6. World Health Organisation. Chemotherapy of malaria and resistance to antimalarials: Report ofa WHO Scientific Group. WHO Tech Rep Ser 1973; no 529: 46. 7. Spencer HC, Kipengor T, Agure R, Koech DK, Chulay JD. Plasmodium falciparum in Kisumu, Kenya: Differences in sensitivity to amodiaquine and chloroquine in vitro. J Infect Dis 1983; 148: 732-36. 8. Lelijveld J, Kortman H. The eosin colour test of Dill and Glazko: a simple field test to detect chloroquine in urine. Bull WHO 1970; 42: 477-79. 9. British Pharmacopoeia. London: Pharmaceutical Press, 1973: 28. 10. Bruce-Chwatt LJ Essential malariology. London: Heinemann, 1980: 35 11. Rieckmann KH, Sax LJ, Campbell GH, Mrema JE. Drug sensitivity of Plasmodium falciparum: An in vitro micro technique Lancet 1978; i: 22-23. 12. Sixsmith DG, Spencer HC, Watkins WM, Koech DK, Chulay JD. Changing in vitro response to chloroquine of Plasmodium falciparum in Kenya. Lancet 1983; ii. 1022. 13. Hall AP, Segal HE, Pearlman EJ, Phintuyothin P, Kosakal S. Amodiaquine-resistant falciparum malaria in Thailand. Am J Trop Med Hyg 1975; 24: 574-80.
METHOTREXATE SYSTEMIC CLEARANCE INFLUENCES PROBABILITY OF RELAPSE IN CHILDREN WITH STANDARD-RISK ACUTE LYMPHOCYTIC LEUKAEMIA* WILLIAM E. EVANS CLINTON F. STEWART CHEN-HSIN CHEN
JOSEPH
WILLIAM R. CROM W. PAUL BOWMAN MINNIE ABROMOWITCH V. SIMONE
Pharmacokinetics and Pharmacodynamics Section, Pharmaceutical Division, Division of Hematology and Oncology, and Biostatistics Section, St Jude Children’s Research Hospital, Memphis, Tennessee, USA
Summary
108
children
with
standard-risk
acute
lymphocytic leukaemia (ALL) were randomised to a post-induction treatment protocol including 15 doses of intermediate-dose methotrexate (1000 mg/m2) in addition to conventional oral therapy of mercaptopurine and low-dose methotrexate. After median follow-up of 26 months, 22 patients have had relapses. Among the 108 patients, rates of methotrexate systemic clearance ranged from 44· 7 to 132 ml/min/m2. When the group
was
divided into three
patients’ rates of methotrexate clearance, statistical analysis of the Kaplan-Meier curves estimating the probability of complete remission showed significant differences (p=0·016) among the subgroups, subgroups according
to
the
*Presented in part to the American California, May, 1983.
Society of Clinical Oncology, San Diego,