Antimalarial drug response of Plasmodium falciparum from Zaria, Nigeria

422 TRANSACTIONS OFWE ROYALSOCIETY OF TROPICALMEDICINEANDHYGIENE(1995) 89, 422-425

Antimalarial

drug response

of Plasmodium

falciparum

from Zaria, Nigeria

I. S. Adagu’, D. C. Warhurst’, W. N. Ogala’, I. Abdu-Aguye3, L. I. Audu2, F. 0. Bamgbola2 and U. B. Ovwigho2 ‘Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, Keppel Street, London, WClE 7HT, UK; 2Department of Paediatrics, Ahmadu Belle University Hospital, Zaria, Nigeria; 3Department of Pharmacology and Clinical Pharmacy, Ahmadu Bello University, Zaria, Nigeria

Abstract The sensitivity of Zaria strains of Plasmodium falciparum to chloroquine, mefloquine, quinine and sulphadoxineipyrimethamine was investigated 5 years after the appearanceof in vivoiin vitro chloroquine resistance in urban Zaria. Infections in 36143children (83.7%) treated with chloroquine were sensitive while those in 7 (16.3%) were resistant. 8/13 isolates cultured (61.5%) were sensitive in vitro to chloroquine and 5 (38.5%) were resistant. Of the cultured isolates, 13113(loo%), 12/13 (92.3%) and 517(71.4%) showed mefloquine, quinine and sulphadoxine/pyrimethamme sensitivity, respectively. The results confirmed chloroquine and sulphadoxineipyrimethamine resistancein urban Zaria and revealed emerging quinine resistance. Resistance to chloroquine and sulphadoxineipyrimethamine is at RI level and chloroquine should continue to be the first-line drug for the treatment and prevention of P. falciparum infection in the Zaria area of northern Nigeria. We suggest that, while quinine servesas second-line drug, mefloquine should be reserved for infections resistant to chloroquine, quinine and sulphadoxine/pyrimethamine. Keywords:

malaria,Plasmodiumfalciparum,drug sensitivity,Nigeria

Introduction Resistance of Plasmodium falciparum to chloroquine (CQ) has been well documented in many parts of Africa (WERNSDORFER & PAYNE. 1991). The first sentinel case of CQ resistance in Nigeria occurred in 1985. Investigation in the following year in south-western Nigeria revealed the first indigenous P. falciparum resistance in vitro to CQ and a decline in sensitivity in vivo (SALAKO & ADEROUNMU, 1987). Three years later, reports from Zaria,, northern Nigeria, indicated CQ resistance in vivoizn vitro (LEGE-OGUNTOYE et al., 1989). These findings prompted investigations on the sensitivity of P. falciparum to other commonly used antimalarial drugs in the Zaria area (ADAGU et al., 1990, 1991). Resistance to sulphadoxineipyrimethamine combination (SDXPYR) was subsequently reported (LEGE-OGUNTOYE et al., 1990). Despite these reports, CQ is still the recommended drug for outpatient treatment of P. falciparum malaria. In Maria an-array of drugs, ranging from the traditional herbal doeo yaro (Azadirachta indica) to svnthetic agents like CQ, SDXiPYR, quinine (QN), halofantrine (HF), and pyrimethamine (PYR), are also available for the treatment and prevention of malaria. These drugs are available over the counter even without prescription, and casesof incomplete treatment courses and sub-standard drugs are not rare. In this study, we reassessedthe sensitivity in vivolin vitro of P. falciparum to CQ, 5 years after the emergence of CQ resistance in vivoiin vitro. A parallel test in vitro of the sensitivity of the isolates to mefloquine (MQ), QN and SDXPYR was also carried out. We found that the sensitivity of Zaria strains of P. falciparum to CQ had not changed significantly over the last 5 years. Coupled with the results of the parallel tests in vitro, these observations have important implications for the treatment and prevention of malaria in Zaria. Materials and Methods Study area

This study was carried out in Zaria, an areain the Guinea Savannah belt of northern Nigeria, where malaria is holoendemic and affects all age groups with serious consequencesin primigravidae and children under the ageof 5 years. Malaria transmission in this area is not entirely season-specific; peak transmission occurs during the oeriod of heaw rainfall, Mav-December. P. falciParum ls the predominant parasite in this area, although-an insignificant number of casesof mixed infections with P. makzriaehave been observed. The study was conducted at the Paediatrics Out-Patient Department and Clinical Pharmacology Unit of Ahmadu Bello University Teaching Hospital, Zaria. Earlier studies in this area have

shown that children presenting with malaria have a high prevalence of suitable parasitaemia (> 1000 parasites/mm3;WHO, 1973). Test in vivo Patients. From July to December 1993, children aged

7 months to 11 years nresenting febrile illness and uncomplicated -malaria -with an axillary temperature 237.5”C, and a blood film with asexual forms of P. falcipar-urn only, were enrolled in the study. Those who had positive urine tests (5173 of the parasitaemic children (6.8%); Saker Solomon/Dill-Glazko tests) or concomitant diseasewere excluded, as were children who had taken antimalarial drugs within the previous 14 d. Informed consent was obtained from parents before enrolment. This study was approved by the ethical committees of Ahmadu Bello Universitv Teaching Hosnital, Zaria and the London School of Hygiene and Tropical Medicine, London, UK. Study procedure. The 28 d extended field test (WHO, 1973) was used. Children enrolled were clinically evaluated not to be too ill to be treated orally. All children received 25 mg CQ base/kg (May and Baker, Dagenham, UK) in 3 doses: 10 mg on day 0 and day 1 and 5 mg on day 2. Children whose parasitaemia did not clear after CQ treatment were re-treated with the standard dose of SDXPYR tablets (500 mg SDX plus 25 mg PYR, Roche Products, Welwyn Garden City, UK) equivalent to 35 mgikg sulphadoxine and 1.75 mgikg pyrimethamine. Those whose parasitaemia reappeared after SDXiPYR treatment were again treated with QN (200 mg QN sulphate tablets, Cox Pharmaceutical, Barnstaple, UK), 30 mg/kg in 3 divided doses for 7 d. Clinical signs and symptoms were monitored from day 0 to day 28. Urine sampleswere tested for 4-aminoquinolines on days 0, 1, 2, 5, 7, 14, 21 and 28. Thick and thin blood films were prepared every 24 h for the first 3 d and thereafter on the days of urine tests. Parasite counts were made on the Giemsa-stainedthick blood film bv counting: the number of asexual forms of P. falciparum relative to”the numbers of leucocytes and assuming a standard leucocyte count of 6000/mms of blood. The treatment responsewas graded according to WHO (1973) criteria. Parasites were considered sensitive if the child’s thick blood film became negative at least by day 7 after admission into the study and remained parasite-free for the remaining days of follow-up. Low grade resistance (RI) was defined as disappearanceof parasites from peripheral blood in the first 7 d, followed by recrudescence/reappearanceby days 14, 21 or 28. High grade resistance (RIVRIII) was defined as the presenceof asexual parasitesuntil day 7.

423 Test in vitro

The WHO in vitro microtest (mark II) (WHO, 1990a) was used to assessthe susceptibility of P. falciparum isolates to CQ, MQ, QN and SDXIPYR. Standard test kits were obtained from the World Health Organization, Geneva, Switzerland. A computer probit inhibition-dose response regression program (Epson QXlO@) was used to determine the inhibitory concentrations (ICsa, ICso, I&I) which prevented 50%, 90% and 99% of the parasites from maturing to schizonts (a3 nuclei for CQ, MQ, QN and 28 nuclei for SDXIPYR). Schizont growth at 8 pmol CQiwell (1.6 umol/L of blood), 64 pmol MQiwell (12.8 un-~ol/L of blood), and 256 pmol QNiwell (51.2 ymol/L of blood) were taken to indicate resistance. For SDXIPYR, the threshold was 90% inhibition of schizonts with 28 nuclei. Results Test in vivo Parasitological response. Forty-three children completed the 28 d test (Table); 36 (83.7%) becameaparasi-

Table. Clinical and parasitological data for 43 children with P. fakiparztm infection in Zaria, Nigeria No. examined No. infected No. who completed the 28 d test Age (years)a Wei ht (kg)a Ax11 3 ary temperature (“C)” Parasitaemia on day 0 (per mm3)b

256 ii 5.2+3 (7112-11) 17.556.8 7-30) 38.4k1.2 i 36-40~5) 20629(1000-55353)

aMean?standard deviation (range in parentheses). bGeometric mean (range in parentheses) taemic by day 2 or 3 and parasitaemia did not reappear within the remaining test period. The infections in these children were therefore sensitive to CQ. Although the mean parasite density for these children had declined only to 73.4% of the pretreatment value at the end of day 1, by days 2 and 3 it had declined to 1.7% and 0% respectively. After initial clearance on day 3 (6 cases)and day 5 (1 case), parasitaemia in these 7 children (16.3%) reappeared on day 7 (2 cases), 14 (2 cases),21 (1 case), and 28 (2 cases).Although reinfection could not be ruled out for the reappearanceson day 28, these results indicate CQ resistance at RI level. Only 4 of these children became aparasitaemic after re-treatment with the standard dose of SDXIPYR; the remaining 3 (42.8%) became parasite-free after a QN course. The mean parasite clearance time was 2.48kO.7 d (SD) in 33 children whose follow-up was completed. Clinical response. All the 36 children whose infection was sensitive responded promptly to CQ treatment and none had clinical evidence of malaria on or after day 3. Although clinical signs of malaria were resolved within the first 5 d in the resistant cases,these signs reappeared unon recrudescence. The children becameclinicallv well after SDXPYR or QN re-treatment. The mean-fever clearance time was 1.4kO.5 d in the 43 children. Pruritus was the only adverse effect, recorded in 32.6% of the children treated with CQ. Test in vitro P. falciparum isolates from 32 (71.1%) of the 43 children enrolled were tested in vitro: 28 187.5%) of the iso-

lates underwent schizogony. bf these, 13 (46.4%) showed 210% schizont maturation in the control well and thus permitted interpretation of the test result. Parasitaemia in these children varied from 9963 to 55 353imma of blood. Although the I&, ICso and IC99 values were obtained for all the drugs tested, the determination of resistance is based on the IC99 values. None of the 8 CQ sensitive isolates underwent schizogony at the cut-off concentration of 1.6 moVL of blood; 2 of the 8 were highly sensitive to Ca”, schizogony being in-

hibited at 0.4 umol/L of blood. The ICaa value of 0.6 umol/L of blood (r=0.7; P0.745 umol/L of blood. In the study, 1.6 umol/L of blood achieved complete growth inhibition in about 50% of the isolates tested, suggesting that the ICso cut-off point for MQ could be set in this region (160 nmol/L actual concentration). The QN sensitivitv in vitro reported here shows that the sensitive isolates had an I&-value of 17.34 umol/L of blood. well below the 51.2 umollL threshold. HOWever, one isolate was clearly resistant to QN (IC99=71.060 umol/L of blood). Interestingly,

although

the resistant isolate was resistant in vitro to CQ, in vivo this drug was effective. Considering the effectiveness of QN in clearing parasitaemia in children whose infection was resistant to CO and SDXPYR, ON is, as observed elsewhere, a valuable reserve drug. _ . This study has once more demonstrated the presence of in vivoiin vitro SDX/PYR resistancein Zaria; 42.8% of the 5 CQ resistant infections in children did not respond

to SDXPYR treatment. The assessmentin vitro indicated 28.6% resistance. This is in contrast with the mevious study in which all the 8 isolates tested were resistant to the drug combination (LEGE-OGUNTOYE et aE., 1990). The IC99 value of 11.996 umol/L of blood for the sensitive isolates represents very high sensitivity in vitro. On the other hand, the IC99 value for SDXPYR of 3014 umol/L of blood obtained for the resistant isolates is a clear indication of very high resistance to the drug combination in the study area. The Zaria isolates of P. fakiparum studied were sensitive to both MQ and QN. Therefore either of these drugs could be used to treat infections resistant to CQ and SDXIPYR. MQ would be a better choice as its single doseregimen avoids patient compliance problems associated with QN; it is, however, very expensive and not

within easy reach for an average patient. Its safety in pregnancy is not certain, but reports of its teratogenic effects in rodents (WHO, 1990b) suggest that the drug may not be safe in early pregnancy. These drawbacks may not permit its use in an area where QN is almost equally effective against P. falciparum which is resistant to CQ and SDXPYR. Although QN requires multiple dosing, it is cheaper, better understood, and safein pregnancy and young children. Resistance to QN is, however, gradually emerging in urban Zaria as shown by the results in vitro; therefore, to delay full-scale resistance and to avoid cross resistance, QN should be used with caution in the Zaria area of northern Nigeria. The proportion of children which responded to CQ treatment was very remarkable. This, coupled with the proportion that was sensitive in vitro, suggestthat CQ should remain the first-line drug for the treatment and prevention of malaria infection in Zaria. Although QN is relatively more toxic than SDXPYR, it would be the preferred second-line drug in view of resistance to the drug combination. MQ should be reserved for infections that fail to respond to CQ, QN, or SDXIPYR. Acknowledgements This work was supported by the WHOiUNDIVWorld Bank Special Programme for Research and Training in Tropical Diseases. We thank Chief J. 0. Omuya for transporting a spare cryobank from London to us in Nigeria. D. C. W. is supported by the Public Health Laboratory Service, UK. I. S. A. is in receipt of a WHOiUNDPIWorld Bank Special Programme award. References Adagu, I. S., Lege-Oguntoye, L., Ugbode, R. O., Okoyeh, J. N.. Asuku, Z. A. & Slotboom. A. B. (1990). In z&/in nirro sensitivity of P. falciparum to halofantrine. I@estAfricanJournal of Pharmacology and Drug Research, 4, E-15.

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Received 29 November 1994; revised 2 February 1995; accepted for publication 3 February 1995

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