Evaluation of a new sulfadoxine sensitivity assay in vitro for field isolates of Plasmodium falciparum

SULFADOXINE-PYRIMETHAMINE

FOR FALCIPARUM

C. L. (1995). Response of PlQsmodiulPt f%ipanrm to chloroquine and Fansidar in vivo and chloroquine and amodiaquine in v&o in Uganda. East African Medical journal, 72, 349-354. Nwanyanwu, 0. C., Ziba, C., Kazembe, P., Chitsulo, L., Wirima. T., Kumwenda, N. & Redd, S. C. (1996). Efficacy of sulpdadoxine/pyrimeamine for- PIasmodium [falcipanr& malaria in Malawian children under five years of age. Tropical Medicine and Incernacional Health, 1,23 l-235. Olliaro, P., Nevill, C., LeBras, J,, Ringwald, P., Mussano, P., Garner, I?. & Brasseur, P. (1996). Systematic review of amodiaquine aeatment in uncomplicated malaria. Lancet, 348, 1196-1201. Onyiorah, E., Boele van Hensbroek, M., Jab, M. S. & Greenwood, B. (1996). Early clinical failures after pydmethaminesulfadoxine treatment of uncomplicated falcipamm malaria. Tranrac&ns of rhe Roval Sockni of Tmbical Medicine and xygiAe,90,3b7-308.c I_ ‘ Premji, Z., Minjas, J. N. & Shiff, C. J. (1993). Chloroquine resistant Plasmodium falcipancm in coastal Tanzania: a ihallenge to the continued strategy of village based chemotherapy for malaria control. TropicalMedicine and Parasitology, 45,4748. Ronn, A. M., Msageni, H. A., Mhina,J., Wemsdorfer, W. H. & Bygbjerg, I. C. (1996). High level of resistance of Plasmodium fulciparum to sulphadoxine-pyrimetamine in children in Tanzania, Transact&m ofrhe RoyalSociegof TropicalMedicine and Hygiene, 90,179-181. Sexton, J. D., Deloron, I’., Bugilimfuta, L., Ntilivamunda, A. & Neil& M. (1988). Parasitological and clinical efficacy of 25 mg/kg and 50 mg/kg of chloroquine for treatment of P&nod&z falciparum malaria in Rwandan children. AmericanJounza1 of Tropical Medicine artd Hygiene, 38,237-243. Targett, G. A. T. (1992). Malaria: drug use and the immune response. Purasitologv, 105, S6 1-S70. Trape, J. F., Pison, G., Preziosi, M. P., Enel, C., du Lou, A. D., Delaunay, V., Samb, B., Lagarde, E., Molez, J. F. & Simondon, F. (1998). Impact of chloroquine resistance on malaria mortality. Comptes Rendus de L’Academie des Sciences. Seti III, Sciences de la Vie, 321, 689-697. Van Agtmael, M. A., Eggelte, T. A. &van Boxtel, C. J. (1999). Artemisinin drugs in the treatment ofmalaria: from medicinal

TRANSACTIONS

55

MALARIA IN UGANDA

herb to registered medication. Trends in Pharmacological Sciences, 20;199-205. Verhoeff, F. H.. Brabin. B. I.. Masache, I?., Kachale. B., Kazekbe, I?. & van de; Kaay, H. J. (1997). Parasitoldgicai and haematological responses to treatment of Plasmodium SaZca’pancmmalaria with sulphadoxine-pyrimethamine in southern Malawi. AnnaLr of” TropicalMedicine and Parasitology, _ _. 91,133-140. Von Seidlein, L., Milligan, I?., Pinder, M., Bojang, K., Anyalebechi, C., Gosling, R., Coleman, R., Ude, J. I., Sadiq, A., Duraisingh, M., Warhurst, D., Alloueche, A., Targett, G., McAdam, K., Greenwood, B., Walraven, G., Olliaro, P. & Doherty, -T. (2000). Efficacy bf artesunaie pius pyrimethamineesulphadoxine for uncomplicated malaria in Gambian children: a double-blind, randomized, controlled trial. Lancet, 355, 352-357. Watkins, W. M., Percy, M., Crampton, J. M., Ward, S., Koech, D. & Howells, R. E. (1988). The changing response of Pkzsmodium falciparum to antimalarial drugs in East Africa. Transactions of rhe Rqyal Society of Tmpkal Medicine and Hygiene, 82,2i-26. WHO (1990). Severe and comDlicared malaria. TTamactions of the R&al &ciew of Tropical Medicine and Hygiene, X4 (supplement 2), l-65. WHO (1994). Anlimalatial drug policies: data requirement, treatment of uncomplicated malaria, and management of malaria in pregncsncy. Geneva, Switzerland: World Health Organization. WHOiMAu94.1070. WHO (1996). Assessment of #aerapeui efiacy of antimalarial drugs: for uncomplkatedfalciparum malaria in areas with intense transmission. Geneva, Switzerland: World Health Organization. WHOihlAu96.1077. Wolday, D., Kireab, T., Bukenya, D. & Hodes, R. (1995). Sensitivity of Plasmodium falcipanrtn in vivo to chloroquine and pyrimethamine-sulfadoxine in Rwandan patients in a refugee camp in Zaire. Transactions of rhe Royal Society of Tmpical Medicine and Hygiene, 89, 654-656.

Received 3 April 2000; revised 13 &ne 2000; accepted fur publication

20June

2000

OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND I-IYGIENE (2001) 95,55-57

Evaluation of a new sulfadoxine sensitivity assay in vitro for field isolates of Plasmodium falciparum Mathieu Ndounga, Leonardo K. Basco and Pascal Institut de Recherche pour k D.k&ppement Ringwald (IRt)) ~ Laboruroire de Recherche SW le Pa&d&e, Olgalaisarion de Coordinationpour EaLutte contre desEn&mies en Aj+ique Centrale (UCEAC), BP 288, Yaouna%, Cameroon Keywords: malaria, Plasmodium falciparum, drug resistance, sulfadoxine, Cameroon

chemotherapy,

Plasmodium falciparum ment of chioroquine-resistant infections. Although the drug combination remains generally effective in Central and West Africa, the declining clinical effectiveness of sulfadoxineipyrimethamine reported from East Africa (CURTIS et al., 1998) calls for regular surveillance of drug resistance. Sulfadoxine and pyrimethamine compete with the natural substrates (p-aminobenzoic acid (PABA) and dihydrofolate, respectively) and inhibit the enzymes of the folate biosynthetic pathway, dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR), respectively. Resistance of I? falciparum to these antifolate drugs is associated with the substitution of key amino acid residues, Ala437Gly and SerlOBAsn, of DHPS and DHFR,

respectively

Although

Introduction Sulfadoxine/pyrimethamine is currently used in Africa and in some parts ofAsia and South America as either the first-line or second-line antimalarial drug for the treat-

Address for correspondence: Dr Pascal Ringwald, Cluster of Communicable Diseases (CDS), Surveillance and Response (CSR), Anti-infective Drug Resistance Surveillance and Containment (DRS), World Health Organization, 1211 Geneva 27, Switzerland; e-mail ringwaldp@who,ch

(COWMAN,

1997). Additional

ami-

no acid substitutions that confer a higher level of drug resistance have been identified in both DHPS (positions 436, 540, 581, and 613) and DHFR (positions 16, 51, 59, and 164). the sensitivity

pyrimethamine

in vitro

of I? fakiparum

to

alone has been monitored (BASCO 8t NNGWALD, 2000), the activity in z&o of sulfadoxine alone and in combination with pyrimethamine has not been studied extensively in field isolates. The main reason underlying the relative scarcity of sensitivity data in virro for sulfadoxine is the poor solubility of the compound in water at neutral pH or in alcohol. WANG et al. (1997) developed a new assay procedure in V&O to determine the sulfadoxine sensitivity of reference clones of l? fdcapamm.Because of the preponderant position that sulfadoxine occupies in current antimalarial chemo-

56

therapy, we evaluated this novel assay system under field conditions with fresh clinical isolates. Methods The study was approved by the Cameroonian Ministry of Public Health and Cameroonian National Ethics Committee. Ninety isolates were obtained by venepuncture from patients with parasitaemia > O-1% and a negative Saker-Solomons urine test (MOUNT et al., 1989). Infected erythrocytes were separated from plasma within 2-3 h, suspended in 50 mL of phosphate-buffered saline (PBS) at room temperature for 20-30 min, and washed 3 times in folate-kee and PABA-free RPM1 1640 medium. If the initial parasitaemia was > 1 *O%, uninfected erythrocytes suspended in PBS overnight were added to adjust the parasitaemia to 0.6%. For sulfadoxine assays, the final suspension consisted of folate-free and PABA-free RPM1 1640,25 mM HEPES, 25 mM NaHCO,, 0.2% glucose, 1 pg/mL hypoxanthine, 50 pg/mL gentamycin, infected erythrocytes at 15% haematocrit, and either 10% non-immune AB+ human serum or 0.5% AlbumaxTM I (Gibco BRL Life Technologies, Cergy Pontoise, France) (WANG et al., 1997). For pyrimethamine assays, the same culture medium, without hypoxanthine and glucose, was used. For chloroquine assays, the standard RPM1 1640 containing 1 mg/L folate and 1 mg/L PABA, wirhout hypoxanthine and glucose, was used. Stock solutions and dilutions of sulfadoxine (final concentrations 7.8 to 32000 nM), pyrimethamine (O-3 - 51200 nM), and chloroquine (25 ~ 1600 no) w&e prepared in dðylsulnhoxide (DMSO). absolute ethanol. and distilled waier, respectively. -i-b test the activity df the sulfadoxineipyrimethamine combination, a fixed ratio of 25O:l was- histributed in the wells (SHAPIRA et al., 1986). Sulfadoxine il I.IL uer well) was distributed in 96well microtitre plate’s j&t before the assay was performed. Controls consisted of 3 wells without drug and solvent and 3 additional wells with 1 &of DMSO without drug. For pyrimethamine and chloroquine, the plates were pre-coated and air dried. All assays were performed in triplicate. After an initial 18 h incubation in 5% COZ at 37X, 1 uCi of r3Hlhvooxanthine (aaueous solution of lvonhil&d hypbxaL&ne diluted in &late-free and PAB*A:free RPMI for sulfadoxine assay, 50% ethanolic solution diluted in folate-free and PABA-&ee RPMI for pyrimethamine and chloroquine assays; Amersham Intemational) was added, and the plates were incubated for an additional 48 h period. The samples in the microtitre plates were processed as described previously (BASC~ & RINGWALD, 2000). The 50% inhibitory concentration (IC,,) was determined by non-linear regression analysis. The fkactional inhibitory concentration (FIC) was calculated as follows: (I&, of sulfadoxine in combination/ I&, of sulfadoxine alone)+(X& of pyrimethamine in combination/I&, of pyrimethamine alone) (BERENBAUM, 1978). Synergy, gdditivity, and antagonism were defined as FIC < 1. FIC = 1. and FIC > 1, resnectivelv. A fragment of the DHPS and DHFR domains carrying-5 codons that may influence sulfadoxine and pytimethamine sensitivity, respectively, was amplified by polymerase chain reaction and sequenced directly, as described previously (BASCO SzFQNGWALD, 1998,ZOOO). Results Adequate parasite growth, defined arbitrarily as incorporation of [3H]hypoxanthine > 1000 c.p.m in drugfree wells, was observed in 89 assays (99%) for chloroquine, 82 assays (9 1%) for pyrimethamine, and 76 assays (84%) for sulfadoxine. Of these assays with adequate growth, the proportions of interpretable assays (arbitrarily defined as >3-fold difference in the incorporation of [3H]hypoxanthine between drug-free wells and wells containing the highest drug concentration to allow the calculation of I& were as follows: 89 of 89 (100%) for

MATHEU

NDOUNGA

ETAL.

chloroquine, 64 of 82 (78%) for pyrimethamine, and 20 of 76 06%) for sulfadoxine. The interuretable results for sulfadoxink sensitivity, the corresponding pyrimethamine sensitivity, alone or in combination with sulfadoxine, and &ps genotypes are summarized in the Table. The sulfadoxine It& obtained withnon-immune serum and AlbumaxTM I were similar. The 250: 1 fixed combination of sulfadoxineipyrimethamine showed potentiation of pyrimethamine activity and, to a lesser extent, sulfadoxine activity. For the pyrimethamine-resistant isolates, the IC,,s of pyrimethamine combined with sulfadoxine (range 0.13 - 5.7 nM) were considerably below the threshold value for pyrimethamine resistance in -vitro (100 nM). Synergistic or additive interactions between sulfadoxine and pyrimethamine were observed in most isolates. The ICsOs of dhps mutant isolates with Gly437 were > 1000 nM. Wild-type isolates with Ala437 were characterized by ICsos < 1000 nM. Isolates with the wild-type dhfiwerk highly sensitive to pyrimethamine (mean I& = 1.15 nMI. whereas isolates with double or &iple mu&ions were characterized by ICsOs that were generally 100 to 3000 times greater than those of sensitive isolates with a wild-type dJ$ genotype. Discussion The toxic effect of DMSO largely accounted for the poor parasite growth. The solvent alone diminished the oarasite arowth from lo-50% in about one-third of our isolates aid > 50% in another one-third ofthe isolates. In addition, the absence of folic acid and PABA in the culture medium was associated with poor parasite growth, as evidenced by the comparison of the mean incorporation of [‘Hlhypoxanthine in the chloroquine assay (13 100 c.p.m.) and pyrimethamine assay (6850 c.p.m.). An additional factor that may explain the poor performance in the sulfadoxine assay is related to the presence of exogenous folate. The major sources of kxogenous folatek our study were infected erythrocytes and human serum. Incubation of red blood cells in PBS for 20 min may be inadequate 10 deplete folate and PABA. A much longer incubation period, however, is deleterious for fresh &nical isolates. Although the use of AlbumaxTM I reduces exogenous folate and PABA to a negligible level, the result; obtained from assays using 10% human serum and AlbumaxTM I were similar. In a previous study on East African strains, the I&s for sulfadoxine ranged from 1620 to 6470 w using a modified RPM1 1640 medium containing low levels of folate (10 pg/L) and PABA (O-5 pg/L) (SHAPIRA et al., 1986). Since the ICsOs in the present study were about lOOO- to 6000-fold less than those obtained with RPMI containing low levels of folate and PABA, we may deduce that the se-&m added to our assays probably contained an insienificant amount of folate and PABA. Nevertheless. sin& even a trace of folate and PABA may influence the IC, of antifolate drugs, this factor probably explains the relatively high ICSOs for sulfadoxine obtained in our study, as compared to those of WANG et al. (1997). Similarly, the presence of exogenous folate may have interfered with the synergistic action of the sulfadoxinei pyrimethamine combination. This possibility is consistent with the highly synergistic action of the combination observed with folate-free and PABA-free RPM1 medium, but less consistent with the synergy or additivity obtained with EWMI containing low levels of folate and PABA (SHAPIRA et al., 1986;lW~~KI~s et al., 1997). Other factors, such as the addition of glucose and unlabelled hypoxanthine, did not seem to influence our results. This novel assay system may be useful for laboratory experiments on culture-adapted strains of P. falcipamm, but it does not seem to be applicable to field isolates. There is currently no standardized assay in vkro for sulfadoxine that yields reproducible results. Further improvements are necessary to provide a relatively

SULFADOXINE

SENSITIVITY

ASSAY FOR PLASMODIUM

Table. Activity in v&o ofstidoxine falciparum, and parasite genotypes

57

FALCIPARUM

and pyrimethamine,

alone and in combination,

against Plasmodium

50% inhibitory concentration (I(&; nmol/L)

Genotype” Isolate

dhps

dhfi

34198 03199 5 l/99 23198 32198 33198 5 1198 76198 94198 01199 04199 08199 26199 45198 80198 26198 90198 18199 45199 30198

S-436, A-437 S-436, A-437 S-436, A-437 A-436, A-437 A-436, A-437 A-436, A-437 A-436, A-437 A-436, A-437 A-436, A-437 A-436, A-437 A-436, A-437 A-436, A-437 A-436, A-437 S/A-436, A-437 S/A-436, A-437 S-436, G-437 S-436, G-437 S-436, G-437 S-436, G-437 ND

Wild-type R-59, N-108 Mixed I-51, R-59, N-108 I-51, R-59, N-108 Wild-type R-59, N- 108 Mixed I-51, R-59, N-108 Mixed Wild-type I-51, R-59, N-108 R-59, N-108 I-51, R-59, N-108 Mixed Wild-type I-51, R-59, N-108 I-51, R-59, N-108 I-51, R-59, N-108 ND

Sulfadoxine 30.9 98.0 518 222 318 314 768 539 179 76.1 264 591 611 101 211 1304 1580 1310 1334 658

Pyrimethamine 0.89 141 144 1500 447 0.32 854 1790 323 89.2 330 639 79.6 3800 48.9 0.39 916 1150

653 0.24

Sulfadoxine/ pyrimethamineb 35-410.14 (l-30) 31.4/O-13 (0.32) 33211.33 (0.65) ND 66.410426 38411.5 50512.0 16910.67

(1.02) (0.50) (0.94) (0.95)

98*7039 (0.50) 42611.7 (0.72) 37611.5 (O-68) 1581Oi3 (0.76) 1430,;‘: (0.91) 80013.2 (0.61) 1054/4.2 (0.80) -

“The genotype of dihydropteroate synthase (DHPS) differed in codons 436 (S, Ser; A, Ala) and 437 (A, Ala; G, Gly). The other three

codons (Lys-540, Ala-581, and Ala-613) were invariant. Isolates 45/98 and SO/98had mixed dhps alleles. The genotype based on

dihydrofolate reductase (&fi) was wild-type (Asn-5 1, Cys-59, &r-108), mixed with alternative codons at positions 51,59, and 108, or mutant type with double mutations at positions 59 (R, Arg) and 108 (N, Asn) or triple mutations at positions 59, 108, and 51 (I, Ilc). Two codons (Ala-16 and Ile-164) were invariant. ND, not determined. bThe results of drug combination (sulfadoxineipyrimetamine at 250: 1 fixed combination) are expressed asthe IC,, ofpyrimethaminei I& ofsulfadoxine. Fractional inhibitory concentrations are given in parentheses. Dashes (-) denote uninterpretable results. ND, not determined.

simple and srandardized tool for the regular monitoring in vitro of sulfadoxine resistance in the field. Acknowledgements

We thank Sisters Solange Menard and Marie-Solange Oko and their nursing and laboratory staff at the Nlongkak Catholic missionary dispensary, YaoundC, Cameroon, for recruiting malaria-infected patients. This investigation was supported by grants from Agence Universitaire de la Francophonie (AUF) and the French Ministry of Co-operation and Development. Mathieu Ndounga was supported by a training grant from the French Ministry of Co-operation and Development. References

Basco, L. K. & Ringwald, P. (1998). Molecular epidemiology of malaria in YaoundP, Cameroon II. Baseline frequency of point mutations in the dihydropteroate synthase gene of

Phzwnodium falciparwn. American Journal of Ttwpical Medicine and Hygiene, 58,374-377. Basco, L. K. & Ringwald, P. (2000). Molecular epidemiology of

malaria in YaoundC, Cameroon VI. Sequence variations in the Plusmodiumj&iparum dihydrofolate reductase-thymidylate synthase gene and in nritruresistance to pyrimethamine and cycloguanil. American $urnd of Tropical Medicine and

Hygiene, 62,271-276.

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resistance in malaria. In: Molecular Genetics of Drug Resistance, Hayes, J. D. & Wolf, C. R. (editors). Chur, Switzerland: Harwood Academic Publications, pp. 221-246. Curtis, J., Duraisingh, M. T. & Warhurst, D. C. (1998). In viva selection for a specific genotype of dihydropteroate synthetase of Phnodium fakipawm by pyrimethamine-sulfadoxine but not chlorptoguanil-dapsone treatment. Juurnal of Infectious Diseases, 177, 1429-1433. Mount, D. L., Nahlen, B. L., Patchen, L. C. & Churchill, F. C. (1989). Adaptations of the Saker-Solomons test: simple, reliable calorimetric field assays for chloroquine and its metabolites in urine. Bulletin ojthe World Health Organization, 67,295-300. Shapira, A., Bygbjerg, I. C., Jepsen, S., Flachs, H. & Bentzon, M. W. (1984). The susceptibility of Phmodzum falciparum to sulfadoxine and pyrimethamine: correlation of in oieroand in vitro results. American 3ounzalof TwpicaI Medicine and Hygiene, 35,239-245. Wang, l’., Sims, P. F. G. & Hyde, J. E. (1997). A modified in v&o sulfadoxine susceptibility assayfor Plasmodium fulciparurn suitable for investigating Fansida? resistance. Parasitology, 115,223-230. Watkins, W. M.. Mbem. E. K.. Winstanlev. A. & Plowe. C. V. (1997). The &icacy df ant&late antimalarial combinations in Africa: a predictive model based on pbarmacodynamic and pharmacokinetic analyses. Parasitology Today, 13, 459-464.

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Received 14June 2000; acceptedforpublication

25July 2000