Response of Plasmodium falciparum to dihydrofolate reductase inhibitors in Malindi, Kenya

Response

of Plasmodium falciparum to dihydrofolate inhibitors in Malindi, Kenya

reductase

WILLIAM W. WATKINS~, DAVID G. SIXSMITH~ AND DAVY K. K~EcH’~~ HARRISON C. SPENCER~“, ‘Clinical Research Centre, Kenya Medical Research Institute, Nairobi, Kenya; ‘Division of Parasitic Diseases, Center forr Infectious Diseases, Centers for Disease Control, Public Health Service, U.S. Department o{yeatth and Human Services, Atlanta, Georgia, USA; ‘Department of Pharmacy, University of Nairobi; Dtvzszon of Vector Borne Diseases, Kenya Ministry of Health

Abstract The response of Plasmodiumfalciparum isolates to dihydrofolate reductase inhibitors (DHFRI) was examined in Malindi, Kenya. All 20 infected children treated with pyrimethamineisulphadoxine responded. In contrast, after treatment with pyrimethamine, parasitaemia in 9 of 14 infections failed to clear or recrudesced during the seven-day follow-up. In a 48-hour in vitro test, five of six isolates resistant to pyrimethamine in vivo had a minimal inhibitory concentration (MIC) to pyrimethamine ~300 nmolesil compared with c 100 nmolesil for the four sensitive isolates; four isolates did not grow. MIC to M-B 35769, an experimental DHFRI structurally similar to pyrimethamine were the same (six isolates) or lo-fold lower (three isolates). In the laboratory four of five isolates adapted to in vitro culture had the same MICs as in the field while one isolate became less responsive to both drugs. Cycloguanil (the active metabolite of proguanil) was more active in vitro in the laboratorv than pyrimethamine or M-B 35769. Introduction As resistance to chloroquine continues to spread in East Africa, other antimalarial drugs are being used more frequently for treatment of Plasmodium falciparurn infections (PETERS, 1982). The combination of the dihydrofolate reductase inhibitor pyrimethamine with the sulphonamide sulphadoxine is the major alternative to the 4-aminoquinolines chloroquine and amodiaquine. Therefore, it is important to assess continuously the response of I’. falciparum to dihydrofolate reductase inhibitors in areas where they could be used as treatment for malaria. Pyrimethamine resistant isolates of I’. falciparum were first reuorted from Kenva in 1954 (WHO. 1961). However, there have been &w recent studies’ on th’e response to Kenyan P. falciparum isolates to pyrimethamine alone or to pyrimethamineisulphadoxine. In addition, in the 1970s pyrimethamine had been rarely used to treat malaria or as chemoprophylaxis in Kenyans. In 1981, most of the P. falciparum isolates examined in western Kenya proved resistant to pyrimethamine in vivo and in vitro (NGUYEN-DINH et al., 1982). In contrast all infections treated with pyrimethamineisulfadoxine responded. The coast is the area of Kenya from which most cases of chloroquine resistance have been reported (SIXSMITH ez al., 1983.; WATKINS et al., 1984). This paper reports on studies done on the response of P. falciparum isolates from Malindi, Kenya, to pyrimethamine, pyrimethamine/sulphadoxine, M-B 35769 and cycloguanil. M-B 35769 was tested because studies in the laboratory suggested that it is more active than either pyrimethamine or cycloguanil against pyrimethamine-resistant P. falciparum strains (SIXSMITH et al., 1984). Materials and Methods Study population The study was done in two schools in Malindi, Kenya, a hyperendemic malarious area along the Indian Ocean coast. In September 1982, schoolchildren were screened for malaria infection and included in the study if onlv P.

fulcipanm

was

KORTMANN

present, the Dill-Glazko test (LELIJVELD & for 4-aminoquinolines in the urine was

1970)

negative, parasitaemia was adequate (approximately 225 asexual parasites/300 leucocytes), there was no history of ingestion of antimalarials in the previous two weeks, and permission to participate in the study was given by teachers and/or parents. In vivo tests In vivo tests similar to the standard WHO 7-day test for chloroquine were done (WHO, 1973). Patients were weighed and treated on day 0 with a single dose of pyrimethamine 25 mg (one tablet) if <25 kg body-weight or two tablets if 25 to 5O%g, or with a single d&e oipyrðamine 25 mg plus sulohadoxine 500 me (one tablet) if t25 mn bodv-weight or two tablets if 25 to 56 kg. Patients were obs&ved ior 3
which failed to respond to treatment or recrudesced during the seven-dav follow-uo oeriod quine phosphate 25 mgbase per Dill-Glazko test was performed on day 1 to exclude concomitant

were treated with chlorokg given over three days. A on urine samples collected treatment with chloroquine.

Field in vitro tests A 48.hour field in vitro test for sensitivity of P. falciparum to pyrimethamine and to M-B 35769 was done as previously described with few modifications (NGUYEN-DINH & PAYNE, 1980). M-B 35769 is 2,4 diamino-5(phenoxypropyloxy[4’chlorophenylJ)6-methylpyrimidine hydrochloride and is a structural analogue of pyrimethamine. Before drug administration a 1 to 5 ml venous blood specimen was collected, placed in a heparinized, sterile tube and immediately put into a portable refrigerator. Within four hours 0.3 ml of the parasitized blood was added to 7.2 ml of RPM1 medium 1640 supplemented with 25 mM NaHC03, 25 mM HEPES, 10% AB+ non-immune human serum and gentamicin 10 pg/ml. Then 0.5 ml aliquots of the parasitized erythrocyte suspension were distributed into 16 mm wells of a flat-bottomed 24-well tissue culture plate pre-dosed with

Reprint

requests

to: Dr. Harrison C. Spencer, Division of Center for Infectious Diseases, Centres Control, Atlanta. GA 30333. USA

Parasitic Diseases, for

Disease

202

response

0F

P. falciparum

~0

DHFRI

Table I-Results 48-hour in vitro rest for sensitivity of Plasmodium falciparum isolates to pyrimethamine M-B 35769 (M-B), and to Cycloguanil (C)-Kenya, 1982 Pyrimethamine Inhibitory Concentration’

Miniem; Isolate No. M49 M60 M70 M73 MS1 M87 MS5 M71 M79 M81

In vivo response to pyrimethamine’

P

M-B

10

300

(nrnoles/l) Laboratory M-B

C

>lOOO

10

: fl 100

N3c0, 100 100

10

30

10

30

30 30

30

30

10

100

>lOOO

>lOOO

>lOOO

100

RR1 RI1

>lOOO > 1000

RI1 RI1

>lOOO

300 2

P

(P), to

3005

>lOOO

300

100 3

30

3005

’ Lowest concentration with complete inhibition of ring forms when 5000 erythrocytes examined. ’ According to WHO criteria-see text S = sensitive, R = resistant. 3 Isolates M60, M73, M87, M71, and M81 could not be adapted to in vitro culture in the laboratory. 4 No growth. ’ Concentration of 1000 nmoles/l not tested. either pyrimethamine or M-B 35769. Stock solutions of each drug in 70% (v/v) ethanol were made. To prepare plates, these were then diluted further in sterile distilled water. Final concentrations of each drug rested were 0,30,100,300 and 1000 nmol/l. The plates were agitated gently and placed in a gastighr box which was flushed with a gas mixture containing 3% CO*, 5% 02, and 92% Nz and incubated at 37°C for 48 hours. After incubation excess medium was removed and a thin blood smear made from the remaining erythrocyte suspension in each well. Smears were stained with Giemsa and parasites per 5000 erythrocytes counted. A successful test was one in which there was at least two-fold growth in the well containing no drug relative to the pre-incubation count. The minimal inhibitory concentration (ME) was taken to be the lowest drug concentration at which no ring stages were present. Pyrimethamine-sensitive isolates have an MIC<30 nmoles/l (NGUYEN-DINH & PAYNE, 1980; NGUYEN-DINH Laboratory in vitro tests

et al.,

1981).

Isolates were cryopreserved in liquid nitrogen using 10% dimethylsulphoxide (DMSO) (COLLINS & JEFFERY, 1963). In the laboratory, isolates successfdly rested in the field were thawed and an attempt made to adapt them to in vitro culture using the Trager-Jensen method (TRAGER & JENSEN, 1976). Cultures were maintained in 25 ml tissue culture flasks

flushed with a gas mixture of 3% CO*, 5% O2 and 92% Nz. If successfully adapted, the cultured isolates were tested for in vine response using the 48-hour method described above with two exceptions; gentamicin was not added fo the medium and response to cycloguanil (the active metabolite of proguanil) as well as to pyrimethamine and M-B 35769 was examined. Results S&y population All 20 infections treated with pyrimethaminei sulphadoxine were sensitive in the seven-day test; 18 patients cleared parasitaemia on day 2, one on day 3, and one on day 4. No parasites were detected on days 4 to 7. Mean parasite count on day 0 was 107 (range 17 to 702) per 300 leucocytes, on day 1 was 46 (range 2 to 246), on day 2 was 9 (range 0 to 178) and on day 3 was one (range 0 to 11). In contrast, 9 of 14 (64.3%) infections treated with pyrimethamine were resistant in vivo. In three infec-

tions parasitaemia cleared for 48 hours but recrudesced before the end of the seven-day follow-up period (RI resistance according to WHO criteria for chloroquine) (WHO, 1973) and in six infections parasitaemia decreased but did not clear (RI1 resistance). Mean initial parasitaemia in the pyrimethamine treatment group was 192 per 300 leucocytes (range 35-576).

In vitro tests Field: 48-hour in vitro tests were attempted on the parasites from the 14 patients in the pyrimethamine treatment group and grew in 10. MIC values are presented in Table I. In general, in vitro response to pyrimethamine was predictive of the in vivo result. All isolates sensitive in vivo had a pyrimethamine MIC
For three isolates (M87, M55, M79) the MIC to M-B 35769 was more than lo-fold lower than that to pyrimethamine (Table I). Laboratory: Five of the 10 isolates successfully tested in the field were adapted to in vitro culture and tested for sensitivity to pyrimethamine, M-B 35769, and cycloguanil using the 48-hour test (Table I). In general in vitro response patterns observed in the laboratory were similar to those found in the field; MICs were the same or varied by only one well. Isolate M49 was much less sensitive to pyrimethamine and M-B 35769 in the laboratory while isolate M55 exhibited a change in response to M-B 35769. Four of the five isolates had MICs to cycloguanil ~30 nmoles/l; in two of these (M49 and M79) the MIC to cycloguanil was much lower than that to pyrimethamine. Discussion These results are important in providing further information on the sensitivity of Kenyan P. falciparurn to dihydrofolate reductase inhibitors. The in vivo

H. c:. ~mrw:m

data presented here from the Kenya coast for response of P. falciparwn to pyrimethamineisulphadoxine and to pyrimethamine alone were similar to those found earlier in western Kenya, another malarious region (NGUYEN-DINH et al., 1982). More than 60% of the P. falciparum infections in this study failed to respond to pyrimethamine treatment initially or recrudesced within seven days. This resistance to pyrimethamine occurs in the absence of drug pressure since to our knowledge pyrimethamine is not readily available in this area and is not used for treatment of malaria. In contrast, all infected patients treated with pyrimethamineisulphadoxine cleared their parasitaemia and remained parasite-free for seven days. Follow-up was not

extended

beyond

seven

days

because

of

the

difficulty in distinguishing recrudescence from reinfection. Therefore RI resistance with parasite clearance followed by recrudescence after seven days could not be excluded. The high level of pyrimethamine resistance observed suggests that the effective half-life of the combination pyrimethamineisulphadoxine may not remain effective for long since the response of P. falciparum to the combination has been shown to be directly related to that to pyrimethamine alone (LAMONT & DARLOW. 1982: SPENCER et al.. 1984). ‘These data from the coast associated with the results of studies in other areas of Kenya emphasize that pyrimethamine alone should not be used for the prophylaxis of malaria in Kenya. fn vitro

results

in the 48-hour

test for

sensitivity

of

P. fakiparum to pyrimethamine correlated well with in vivo response as has been previously shown (NGUYEN-DINH et al., 1982). M-B 35769 exhibited equal or greater effectiveness in vitro against these isolates when compared with pyrimethamine. However, in viva data are needed to determine the effectiveness of M-B 35769. The relationship between in vivo response and in vitro MIC would be affected both by the blood levels achievable when the drug is given for treatment and by the quantity and type of metabolites which are active against malaria parasites formed in vivo but not in the in virro system. Cycloguanil appeared more effective than pyrimethamine in vitro, even against pyrimethamine-resistant isolates although

one isolate

in culture

was clearly

resistant

to

both drugs. In vitro, two isolates appeared sensitive to cycloguanil but resistant to pyrimethamine. The extent

and importance

of such

difference

between

the

response of P. falciparum to pyrimethamine and to cycloguanil need to be defined. Thus both M-B 35769 and proguanil deserve further testing in vivo. In conclusion, pyrimethamine no longer appears to be an effective antimalarial for chemoprophylaxis in Kenya. While P. falciparum isolates in Malindi respond to the combination drug pyrimethamine’ sulphadoxine, this antimalarial may not remain effective for long if used extensively because of the high degree of pyrimethamine resistance which exists in this area. M-E 35769 deserves further investigation in v& and in vitro but from these crude in vitro data, it may offer no significant advantage over pyrimethamine. In viva studies are also indicated to ascertain

the continued prophylaxis

effectiveness of proguanil of falciparum

malaria

along

for chemothe

Kenya

coast since there were differences observed in the in vitro responses of Kenyan P. falciparum isolates to

et al.

203

pyrimethamine and to cycloguanil. Because of the rapidly changing response of P. falciparum to antimalarial drugs, it is necessary that there be continuous monitoring of drug sensitivity patterns so that the existing antimalarials can be used wisely. Acknowledgements

This investigation received support from the UNDP/ World BankWHO Special Programme for Research and Training in Tropical Diseases and from the Kenya National Council for Science and Technology. We appreciate the excellent technical assistance of David Boriga Anyona and Daniel M. Kariuki. We thank the Director, Kenya Medical Research Institute. for nermission to oublish this 1naner. The r samples of pyrimethakine, cycloguaiil and M-B 35769 were donated by Burroughs Wellcome Co.. ICI and Mav & Baker respectively. References Collins, W. E. & Jeffery, G. M. (1963). The use of dimethyl sulfoxide in rhe low-temperature frozen preservation of experimental malarias. ,~ournal of Parasitology, 49, 524525. Lamont, G. & Darlow, B. (1982). Comparison of tn vtfro pyrimethamine assay and in viva response to sulphadoxine-pyrimerhamine in Plasmodium falcipanrm from Papua, New Guinea. Transactions of the Rqval Society of Tropical Medrcine and Hygiene, 76, 797-799. Lelijveld, J. & Kortmann, H. (1970). The eosin colour test of Dill and Glazko: a simple field test to detect chloroquine in the urine. Bulletin of the World Health Organizarlon, 42, 477-479. Nguyen-Dinh, l? & Payne, D. (1980). Pyrimethamme sensitivity in Plasmodium falcipancm: determination m virro by a modified 48-hour test. Builertn of the World Healrh Organization, 58, 909-912. Nguyen-Dinh, I’., Hobbs, J. & Campbell, C. C. (1981,1. Assessment of chloroquine sensitivity of Plasmodium falcipanrm in Chloureca, Honduras. Bulletin of the World Health Organization, 59, 641-646. Nguyen-Dinh, I’., Spencer, H. C.! Masaba, S. C. & Churchill, F. C. (1982). Suscepttbility of Plasmodium falciparum to pyrimethamine and sulfadoxine/pyrimethamine in Kisumu, Kenya. Lancer, i, 823-825. Peters, W. (1982). Antimalarial drug resistance: an increasing problem. British Medical Bulletin, 38, 187-192. Sixsmith, D. G., Spencer, H. C., Watkins, W. M., Koech, D. K. & Chulay, J. D. (1983). Changing in vitro response to chloroquine of Plasmodium faluparum in Kenya. Lancer, ii, 1022. Sixsmith, D. G., Watkins, W. M., Chulay, J. D. & Spencer, H. C. (1984). In vitro antimalarial acrivitv of retrahvdrofolaie degydrogenase inhibitors. Amer&n Journal it’ Tropical Medicme and Hygiene, 33, 772-776. Spencer, H. C., Watkins, W. M., Sixsmirh, D. G.. Koech, D. K. & Chulay, J. D. (1984). A new in vitro test for pyrimethamineisulfadoxine susceptibiliry of Plasmodium falclparum and its correlation with in vivo resistance in Kenya. Bulletin of the World Health Organizurton, 62, 615-621. Trager, W. & Jensen, J. B. (1976). Human malaria parasites in conrinuous culture. Science, 193, 673-675. Watkins, W. M., Sixsmith, D. G., Spencer, H. C., Boriga, D. A., Kariuki, D. M., Kipenjor, T. & Koech, D. K. (1984). Effectiveness of amodiaquine as treatment for chloroquine-resistant Plasmodium falciparum infections in Kenya. Lancet, i, 357-359. WHO (1961). Chemotherapy of malaria: report of a WHO sczenrific group. WHO Technical Report Series No. 226. WHO (1973). Chemotherapy of malaria and resistance to antimalarials: Report of a WHO Scientific Group. WHO Technical Report Series No. 529. Accepted

for publication

14th

March,

1985.