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Assessment of susceptibility of Plasmodium falciparum to chloroquine, quinine, mefloquine, sulfadoxine-pyrimethamine and artemisinin in southern Viet Nam
TRANSACTIONSOFTHEROYALSOCIETYOFTROPICALMEDICINEANDHYGIENE(2001)95,513-517
Assessment mefloquine,
of susceptibility of Plasmodium falciparum to chloroquine, quinine, sulfadoxine-pyrimethamine and artemisinin in southern Viet Nam
N. V. Thanh’, A. F. Cowman*, D. Hipgrave3p4, T. B. Kim’, B. Q. Phuc’, L. D. Gong’ and B.-A. Biggs4>5 ‘National Institute of Malariology, Parasitology and Entomology, Hanoi, Viet Nam; ‘The Walter and Eliza Hall Institute of Medical Research, The Royal Melbourne Hospital, Parkville, Victoria 3050, Australia; 3Vietnam Health Alliance, 5th fl57 ~c;;tgaT~~un~’ Ba Trung District, Hanoi, Viet Nam, . 4Macfarlane Burnet Centre of Medical Research, Fairfield, 3078, . ‘The Unzversaty of Melbourne, Department of Medicine, The Royal Melbourne Hospital, Parkville, Victoria’3050, Ausiralia Abstract Resistance to antimalarial chemotherapy is a major concern for malaria control in Viet Nam. In this study undertaken in 1998, 65 patients with uncomplicated Plasmodium falciparum malaria were monitored for 28 days after completion of a 5-day treatment course with artemisinin. Overall 36.9% (24/65) of patients had recurrent parasitaemia during the surveillance period. I? falciparum isolates were tested for sensitivity in vitro to chloroquine, mefloquine, quinine, sulfadoxine-pyrimethamine and results were compared to those from a similar study in 1995. Increased parasite sensitivity to sulfadoxine-pyrimethamine, chloroquine and quinine was demonstrated, with significantly lower mean EC,, and EC& values in 1998 compared to 1995. Parasite sensitivity to mefloquine did not differ significantly in the 2 surveys. Isolates were also tested for sensitivity in vitro to artemisinin in the 1998 survey. The mean E&, was 0.03 nmol/L and the EC,, was 0.94 nmol/L. Parasite sensitivity to artemisinin will need to be monitored in view of its increasing use in Viet Nam. malaria, Plasmodiumfalciparum, drug sensitivity, in vitro, chloroquine, mefloquine, quinine, sulfadoxinepyrimethamine, artemisinin, trends, Viet Nam
Keywords:
Introduction Malaria is still a major health concern in Viet Nam with over 500000 cases and 198 deaths reported in 1996 (HIEN et al., 1997). I? falciparum resistance to antimalarial drugs is an important problem and a number of local studies have described increasing resistance particularly to sulfadoxine-pyrimethamine, chloroquine and quinine (CAILIPBELL et al., 1973; HIEN et al., 1997). Artemisinin as a single dose, and in combination with mefloquine, has been successfully evaluated in the treatment of both non-complicated as well as severe complicated multidrug-resistant malaria in Viet Nam (HIEN et al., 1997). Additionally, other studies have analysed the efficacy of mefloquine, chloroquine and sulfadoxine-pyrimethamine (RYAN, 1975; SHCHERBAKOV et al., 1990) as well as the mutation pattern in genes involved in the mechanism of resistance to pyrimethamine (REEDER et al., 1996; ZINDROU et al., 1996). An important change in the management of clinical malaria in Viet Nam has been the introduction of artemisinin and its widespread availability as a first-line treatment. It is likely that this has been a major factor in the reduction in the number of deaths from severe malaria since 1991 (HIEN et al., 1997; ANONYMOUS, 1997). As yet, there have been no reports of clinical resistance to artemisinin and its derivatives. We report the findings of a survey undertaken in 1998 that examined the suscentibilitv of Z? falciaar-um parasites to artemisinin, chloroquine,< mefloquine, quinine, and sulfadoxine-pyrimethamine in an area to the north-east of Ho Chi Minh city. Comparisons are made with a similar survey undertaken in 199 5. Materials and Methods Field site and patients, 1998 study The study was conducted at the health care centre in the Phu Rieng rubber plantation, which is situated 130 km north-east of Ho Chi Minh city. This area has hyper-endemic I? falciparum malaria with peak transmission between September and December. This site
Address for correspondence: Dr Beverley-Ann Biggs, Department of Medicine, The Royal Melbourne Hospital, Parkville, 3050, Australia; phone 161 3 9342 7702, fax +61 3 9347 1863, e-mail [email protected]
has been used for malaria studies for several years and a laboratory has been established within the health centre. Patients with uncomplicated I? falciparum malaria who were aged >5 years-were recruited into the study during: Sentember to November 1998. The total number 07 slides examined during this period was 683. There were 141 positive slides. Among the positive slides, 95 showed I? falciparum (including children aged ~5 years), 41 showed Z? vivax and 5 slides showed mixed I? falciaarum and l? vivax. Sixtv-five patients (aged >5 year;) with I? falciparum alone’were enrolled in the study. All patients were informed about the study and gave verbal consent before participation. Individuals who had received quinine within the previous 7 days, 4-aminoquinolines within the previous 14 days, pyrimethamine or sulphonamides within the previous 28 days or mefloquine within the previous 63 days were excluded from the study. Participants were treated with artemisinin (20 mg/kg on dav 0, 10 me/kst on davs l-4) for 5 davs. Parasitaemia was monitored with a thick and thin blood smear at days 0, 1, 2, 3, 7, 14, 21, 28 and during every episode of fever after day 7. Patients were observed in the health clinic during the first 7 days and thereafter returned for review according to the above schedule or if they became unwell. .
_
-I
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Parasites Blood (5 mL) was collected by venepuncture into Vacutainer tubes (Becton Dickinson. UK) containing potassium oxalate and sodium fluoride. Thick and thin films were prepared, treated with 10% Giemsa stain, and examined by a trained microscopist. Blood (300 a) was transferred from Z?falcipanrm-positive samples into plastic Falcon tubes containing 2.7 mL of RPM1 1640 low p-aminobenzoic acid and low folic acid content culture medium (RIECKMANN et al., 1978) in preparation for drug assays. In-vitro drug sensitivity assays In-vitro microtest plates (WHO, 1990, 1997) containing chloroquine, mefloquine, quinine and sulfadoxine-pyrimethamine were used to measure schizont maturation inhibition in all samples with confirmed I? falciparum infection. The chloroquine plates were dosed at 1, 2, 4, 8, 16, 32, and 64 pmol/well. The mefloquine plates were dosed at 2, 4, 8, 16, 32, 64 and 128 pmol/well; the quinine plates at 4, 8, 16, 32, 64, 128
514
N. V. THANH ETAL.
and 256 pmol/well; the sulfadoxine-pyrimethamine plates with a mixture of sulfadoxine and pyrimethamine (80: 1) at pyrimethamine concentrations of 0.125, 0.375, 1.25, 3.75, 12.5, 37.5 and 125 pmol/well. Experimental plates containing artemisinin [prepared at the National Institute of Malariology, Parasitology and Entomology, Hanoi, Viet Nam (NIMPE) and dosed at 0.15, 0.5, 1.5, 5, 15, 50, and 150pmol/well] were also used, as the WHO artemisinin plates were found to be inactive. One of the 12 columns on each plate was used for each patient’s blood sample. All the wells of the appropriate column were dosed with 50 & of the blood-medium mixture (BMM, 1 : 9). Dosing started with the control (well A) and followed an increasing order of drug concentration ending at well H (the highest concentration). The plates were placed into a candle jar and incubated at 37°C (*0,5”C) for 24-36 h. After incubation thick films were made from the pellet in each well of the test plate and treated with 2% Giemsa stain for 30 min. The number of schizonts per 200 parasites was used to assessmaturation inhibition. The test was considered valid if 210% of the parasites in the control well had reached schizont stage (28 nuclei in sulfadoxine-pyrimethamine plates, 23 nuclei in the other plates) within 24-36 h. Parasite isolates were classified as sensitive or resistant to a particular drug according to the drug concentration at which schizont maturation was completely inhibited (Table) (WERNSDORFER & WERNSDORFER,
ology as in the 1998 study. Satisfactory artemisinin testing was not available at that time. Data analysis
Mean effective concentrations (EC) were calculated using a computer adapted probit analysis of log-dose responses (WERNSDORFER & WERNSDORFER, 1995) based on the methods of LITCHFIELD & WILCOXON (1949). 2 X 2 tables and x2 test (P value from x”) were used for comparison of the number of isolates showing resistance to chloroquine, mefloquine, quinine and compound sulfadoxine-pyrimethamine in 1995 and 1998 according to standard WHO criteria. Ethics
The project was part of an existing and ongoing programme of surveillance for drug resistance and received ethical approval through the NIMPE Ethics Review Committee. Results
In the 1998 study, 65 patients with uncomplicated I?
falciparum malaria completed a 5-day treatment course
with artemisinin. The mean fever clearance and parasite clearance times were 1.29 days and 1.75 days, respectively. The mean parasite count at day 0 was 12 308/pL (range 1360-102 564). Overall, 36.9% (24/65) patients had recrudescent parasitaemia during the 28-day surveillance period. The earliest recrudescence occurred on day 11 (6 patients had recrudescence between days 11 and 14, 13 patients between days 15 and 21, 5 patients between days 22 and 26). Twelve of the 24 patients had a further parasite recrudescence after retreatment with a 5-day course of artemisinin (2 occurred between days 11 and 14, 6 between days 15 and 2 1 and 4 on day 22 or 23). In-vitro drug sensitivity tests for Z?falciparum isolates collected during the 1998 study and also during a similar study in 1995 were performed for chloroquine, sulfadoxine-pyrimethamine, mefloquine, and quinine.
1995).
1995 Study
Results from a similar study conducted between June and September 1995 are shown for comparison (Table). The study was conducted by staff from the same Institute and patients were recruited from the Phu Rieng rubber plantation health care centre or a nearby hospital. Treatment and monitoring of patients, parasite isolate collection and drug sensitivity testing to chloroquine, mefloquine, quinine and sulfadoxine-pyrimethamine were carried out using the same method-
Table. Response of P. falciparum WHO Microtest) in 1995 and 1998
Drug Chloroquine Mefloquine Quinine SDXK’YR’ Artemisining
Year 1995
isolates
in southern
Viet Nam
to antimalarial
drugs
Number of Sensitive valid testsa isolatesb (n> (%I
in vitro
(using
P valued
8 66
0.27 0.04
0.83 0.13
2.20 0.18
13.70 0.36
29 35
100 97
0.09 0.08
0.25 0.20
0.33 0.26
0.57 0.41
NS
1995
19
100
1998
31
100
0.45 0.27
1.58 0.77
2.25 1.04
4.38 1.81
<0*05
1995
14
0.14
21.89
1998
25
0.04
0.34
92.37 0.60
137.00 1.80
1998
34
0.03
0.20
0.34
0.94
1998
$0”
1995 1998
2s
NRh
1
“For a valid test, schizont maturation in the control well must be 210%. bThe WHO classification of sensitivity and resistance of I? fulcipamm isolates to antimalarial drugs is used (WERNSDORFER & mixture (BMM); WERNSDORFER, 1995): chloroquine sensitive, complete schizont inhibition at ~0.08 pmol/L of blood-medium (I’yr)/L; sensitive, CO.025 pm01 pyrimethamine resistant, schizont formation at 30.16 pmol/L. Sulfadoxine-pyrimethamine resistant, 20.25 pmol I?yr/L. Mefloquine sensitive, ~0.32 pmol/L; resistant, 30.64 pmol/L. Quinine sensitive, G2.56 kmol/L; resistant, z5.12 pmol/L. ‘Values are the mean effective concentrations (EC) for P. fualciparum isolates (n) calculated for 50%, 90%, 95%, 99% schizont maturation inhibition. Units: pmol/L of BMM. d2 X 2 tables and x2 test (P value from x’) were used for comparison of the number of isolates showing resistance to chloroquine, mefloquine, quinine and compound sulfadoxine-pyrimethamine in 1995 and 1998 according to standard WHO criteria. eSulfadoxine-pyrimethamine. ‘These isolates showed borderline sensitivity, i.e., schizont inhibition occurred at a concentration of 0.075 ymol/L. gFresh microtest plates prepared at NIMPE were used in the artemisinin experiments. Standard WHO plates were used in other experiments. hNR, not relevant. Artemisinin resistance is undefined. In this assay 62% (21/34) isolates showed complete inhibition of schizont maturation at an artemisinin concentration of 0.1 kmol/L and 97% were inhibited at 0.3 pmol/L.
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Sixty-five patients were tested for all drugs in 1998 and the proportion of valid tests was 30/65 (chloroquine), 35/65 (mefloquine), 31/65 (quinine), 34/65 (artemisinin) and 25/65 (sulfadoxine-pyrimethamine). The results are summarized below and in the Figure and the Table.
pmol/L in 1998 (Table). This is an approximately 7fold decrease in the mean EC5,, and a 40-fold decrease from 1995 to 1998 and was significant in E& (P < 0.001) (Figure).
Mefloquine Chloroquine
The mean EC50 and EC,, for chloroquine was 0.27 pmol/L and 13.74 kmol/L respectively for 1995 whereas it had reduced to 0.04 (EC,,) and 0.36 (EC,,)
The mean EC50 and EC,, for mefloquine was 0.09 pmol/L and 0.57 Fmol/L, respectively, for 1995 which was similar to that seen in 1998 with an EC,, of 0.08 pmol/L and an EC,, of 0.41 pmol/L. There was no
99.9,
A 99 -
1998
90
1995
s VY tj 50 ;: 10 lOfi
0.1 SDXPYR
1 @mol/L)
0.1 Chloroquine
0
99.9
99.9 -
99
99 -
1 (pmol/L)
D
90 ,\” z
5010 -
I
I
0.1
1 Mefloquine @mol/L)
I
I
0.1 1.0 Quinine (pmol/L)
99.9 99
10 1
0
Artemisinin (mmol/L)
Figure. Sensitivity of l? fdciparum isolates in southern Viet Nam to chloroquine, mefloquine, quinine, sulfadoxine-pyrimethamine in 1995 and 1998 and artemisinin in 1998. SMI (y-axis), schizont maturation inhibition; SDX/PYR, sulfadoxine-pyrimethamine.
516
significant difference in sensitivity to mefloquine tween 1995 and 1998 isolates (Table, Figure).
N. V. THANH ETAL.
be-
derivatives in South-East Asia and Africa, and if this trend continues it will suggest that true resistance of I? falciparum to this drug family may be possible.
Quinine
For quinine the mean ECSo and EC,, was 0.45 pmol/L and 4.38 kmol/L, respectively, for 1995, with slightly decreased values for 1998 of ECI, 0.27 pmol/L and ECg9 1.81 pmol/L. This change represents a 2fold reduction in the level of resistance and is statistically significant (P < O.OS)(Figure).
Acknowledgements We are grateful to the Australian Agency for International Development and the Walter and Elisa Hall Institute of Medical Research for supporting this work, and to Michael Reed for technical and statistical advice. We thank Helen Dedman and Virginia de Crespigny for travel and accommodation arrangements and for administrative assistance.
Sulfadoxine-pyrimethamine
The mean ECSo and E& were 0.14 and 137.00 pmol/L in 1995 but decreased to 0.04 and 1.80 Fmol/L, respectively, in 1998 (Table). The 4-fold decrease of the mean EC&, and the 70-fold decrease of the E& from 1995 to 1998 was significant (P < 0.00 1) (Figure). Artemisinin
The mean ECso for artemisinin was 0.03 ymol/L and the EC& was 0.94 pmol/L. The number of isolates showing complete inhibition of schizont maturation increased from 62% (21/34) at an artemisinin concentration of 0.1 pmol/L to 100% (34/34) at 1.0 pmol/L. Discussion Malaria is still a major health problem in some parts of Viet Nam, although the overall incidence of this disease in 1995 was only 8.5 per 1000 (MORILLON et al., 1996). The spread of drug-resistant I? falciparum has made the choice of antimalarial therapy for control and treatment of this infection problematic. For example, in 1996 chloroquine resistance rates ranged from 41% to 62%, and resistance rates for sulfadoxinepyrimethamine ranged from 26% to 47% (MORILLON et al., 1996). The prevalence of chloroquine resistance in the study area for 1995 was 92%, and fell to 33% in 1998. A possible reason for this decrease in the level of chloroquine resistance is decreased drug use as artemisinin has been increasingly used for malaria treatment in this area since 1992 (HIEN, 1994; HIEN et al., 1997). It is a surprising result, as chloroquine resistance in I? falciparum tends to be relatively stable (PHILIPPS et al., 1998; LE BRAS, 1999). It will be important to conduct follow up in-vitro sensitivity studies in subsequent years to determine whether this decrease is stable. In contrast, both mefloquine resistance and quinine resistance were relatively stable with only small decreases from 1995 to 1998. The level of resistance to sulfadoxine-pyrimethamine also fell between 1995 and 1998 from 100% to 34%. The 1995 levels of resistance are higher than those reported previously in Viet Nam (MORILLON et al., 1996), and presumably reflect high levels of sulfadoxine-pyrimethamine use in the study area before 1995. The decreased resistance observed in 1998 is consistent with decreased use of this drug combination since 1995. Follow-up studies in this area of Viet Nam will determine whether this downward trend for sulfadoxine-pyrimethamine resistance is ongoing. The susceptibility of parasites to artemisinin observed in the 1998 survey was less than that reported by WONGSRICHANAUI et al. (19971, who tested l? falciparum isolates collected fro& pat&ts in 1995 in s&hem Viet Nam. Some studies undertaken in Africa report slightly higher E& levels than observed in the 1998 survey (PHILIPPS et al., 1998). Although the cutoff value for resistance to artemisinin remains unknown, differences in sensitivity of individual parasite isolates to the qinghaosu deri;atives have be& previouslv reoorted in vitro (BASCO & LE BRAS. 1994: PRAD~NE~ et al., 1998), ihcluding reduced se&itiviG after mutagenic treatment (INSELXJRG, 1985). It will be essential to monitor susceptibility to the qinghaosu
References
Anonymous (1997). National Malaria
Treatment Guidelines. Vietnam: Ministrv of Health. Tune 1997. Basco, L. K. & Le’ Bras, J. (iG94). In vitro susceptibility of Cambodian isolates of Plasmodium falcipawm to halofantrine, pyronaridine and artemisinin-desvatives. Annals of Tropical Medicine and Parasitology, 88, 137- 144. Campbell, C. H., Holiday, J., Biggs, J. C. & Backhouse, T. C. (1973). Chloroquine-resistant falciparum malaria acquired in Vietnam. MedicalJournal ofAustralia, 2, 729-734. Hien, T. T. (1994). An overview of the clinical use of artemisinin and its derivatives in the treatment of falciparum malaria in Viet Nam. Transactions of the Royal Socieg of Tropical Medicine and Hygiene, 88, supplement 1, S1/7-SIl8. Hien, T. T.. VinhChau. N. V.. Vinh. N. N.. Hung. N. T.. Phbng, k. Q., Toan; L. MI, Ma< P. I’.,’ Dun; N. T.; HoaiTam, D. T. & Arnold, K. (1997). Management of multiple drug-resistant malaria in Vietnam. Annals of Academic Medicine Singapore, 26, 659-663. Inselburg, J. (1985). Induction and isolation of artemisinineresistant mutants of Plasmodium falciparum. American Journal of Troaical Medicine and Hwiene, 34. 417-418. Le Bras, J. -(1999). Mechanismsand- dynamics of drug resistance in Plasmodium falciparum. Bulletin de la SociS de Pathologic Exotique, 92,236-241. Litchfield, J. T. & Wilcoxon, F. (1949). A simplified method for evaluating dose-effect. Journal of Pharmacology and Experimental Therapeutics, 96, 93- 113. Morillon, M., Baudon, D. & Dai, B. (1996). Malaria in Vietnam in 1996: brief synthesis of epidemiological data [in French]. Midecine Tropicale (Mars), 56, 197-200. Philipps, J., Radloff, P. D., Wernsdorfer, W. & Kremsner, P. G. (1998). Follow-up of the susceptibility of Plasmodium falcipancm to antimalarials in Gabon. American Journal of Tropical Medicine and Hygiene, 58, 612-618. Pradines, B., Rogier, C., Fusai, T., Tall, A., Trape, J. F. & Doury, J. C. (1998). In vitro activity of artemether against African isolates (Senegal) of Plasmodium falciparum in comparison with standard antimalarial drugs. American JournaZ of Tropical Medicine and Hygiene, 58, 354-357. Reeder, J. C., Rieckmann, K. H., Genton, B., Lorry, K., Wines, B. & Cowman, A. F. (1996). Point mutations in the dihydrofolate reductase and dihydropteroate synthase genes and in vitro susceptibility to pyrimethamine and cycloguanil of Plasmodium falciparum isolates from Papua New Guinea. American Journal of Tropical Medicine and Hygiene, 55, 209-213. Rieckmann, K. H., Campbell, G. H., Sax, L. J. & Mrema, J. E. (1978). Drug sensitivity to Plasmodium falciparum. An &-vitro microtechnique. La&et, i, 22-23. Ryan, B. P. (1975). Sulfadoxine and pyrimethamine in the treatment of falcioarum malaria in Vietnam. Medical .,7oumal OfAustralia, 1, lOi- 103. Shcherbakov, A. M., Nguyen, T. L., Chin, K. A., Chan, K. T.. Rabinovich, S. A., Nrmven, V. D.. Chan, V. F., Nguyen, T. L., Vu,- T. T. h Pidh;lt, H. (199b). The clinical efficacy of and tolerance for Lariam (mefloquine) in tropical malaria in the south of the Socialist Republic of Vietnam [in Russian]. Medical Parazitology (Mask), 5,57-59. Wernsdorfer, W. H. & Wernsdorfer, M. G. (1995). The valuation of in vitro test for the assessment of drug response in Plasmodium falciparum. Parasitology, 17, 22 l-227. WHO ( 1990). In vitro micro-test (Mark III) for the assessmentof the response of Plasmodium falciparum to chloroquine, me$i’oquine, quinine, suljadoxinelpyrimethamine and amodiaquine. Geneva, Switzerland: World Health Organization. WHO/ MAP/87.1. WHO (1997). In vitro micro-test (Mark III) for the assessmentof the response of Plasmodium falciparum to chloroquine, mefloand artemisinin. q&e, quinine, sulfadoxinelpyrimethamine Geneva, Switzerland: World Health Organization. WHO/ CTD/ti97.20.
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IN VIET
517
NAM
Wongsrichanalai, C., Nguyen, T. D., Trieu, N. T., Wimonwattrawatee, T., Sookto, I’., Heppner, D. G. & Kawamoto, F. (1997). In vitro susceptibility of Plasmodium falciparum isolates in Vietnam to artemisinin derivatives and other antimalarial% Actu Tropica, 63, 151-l 58. Zindrou S., Nguyen, P. D., Nguyen, D. S., Skold, 0. & Swedenberg, G. (1996). Plasmodiumfalciparxm: mutation pattern in the dihydrofolate reductase-thymidylate synthase
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Acute asymptomatic hepatitis in a healthy normal volunteer exposed to 2 oral doses of amodiaquine and artesunate C. Orrell’, W. R. J. Taylor2’3 and P. Olliar02’ Depanment of Pharmacology, University of Cape Town, Cape Town, South Africa; ’ UNDPIWorld BanklWHO Special Programme for Research and Training in Tropical Diseases (TDR), WHO, Geneva, Switzerland; 3The Centre for Infectious Diseases, Royal Free and University College Medical School, London, UK Abstract Combination antimalarial therapy is being explored to delay development of resistance to falciparum malaria. This report describes an unexpected drug-induced hepatitis in a previously healthy young woman exposed to 2 doses of amodiaquine and artesunate. Use of these combinations should be closely monitored. Keywords: antimalarial agents, amodiaquine, artesunate, adverse events, volunteer, liver function, hepatitis, South Africa
Multidrug-resistant Plasmodium falciparum is a global problem (WERNSDORFER & PAYNE, 1991). The use of antimalarial drug combinations with independent mechanisms of action is a rational approach to delay the onset of drug resistance and safeguard existing drugs (WHITE & OLLIARO, 1996). Current field research is assessing oral artesunate combined with standard antimalarials, e.g., sulfadoxine-pyrimethamine, amodiaquine, for treating acute uncomplicated falciparum malaria. Artesunate has a short half-life and produces a rapid and substantial reduction of malaria parasites; those that remain are then killed by the combinant drug acting over a longer time period. To complement the field studies, an interactive pharmacokinetic study of artesunate and amodiaquine was conducted in normal healthy volunteers at the University of Cape Town. We report the occurrence of asymptomatic hepatitis in a woman during this study. This single-dose 3-phase cross-over study was conducted in 15 volunteers, 10 male and 5 female, aged 19-42 years. All volunteers received 1 dose of oral artesunate (4 mg/kg) on day 0, followed, on day 7, by either 1 dose of oral amodiaquine (10 mg/kg) alone or combined with a single dose of artesunate (4 mg/kg). Subjects were given the alternative regimen on day 28. Blood for routine haematology and biochemistry was taken on days 0, 6,27 and 48.
genes of Vietnamese isolates, a novel mutation, and coexistence of two clones in a Thai patient. Experimental Parasitology,84, 56-57.
Received 13 October 2000; revised 5 April 2001; accepted for publication 9 May 2001
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(2001) 95,517-518
Our subject was a healthy 20-year-old South African woman of African-Caucasian descent, with no previous significant illnesses. During the study she did not take any prescribed or over-the-counter drugs, including paracetamol, and had no reported drug allergies. She did not drink alcohol and had no risk factors for hepatitis B. Her family history was unremarkable. Clinically, there were no abnormal physical signs and her baseline blood tests were all normal. She received artesunate (day 0), amodiaquine (day 7), followed by the combination (day 28). Six days after taking artesunate alone, her aspartate aminotransferase (AST) and total bilirubin were normal but she had a slight increase in alanine aminotransferase (ALT) of 31 IU/L (normal 1-25 IU/L) that was not considered clinically significant (Figure). On day 27, all blood tests were normal. On day 48, her transaminases were elevated, rising to peak values (AST 236 IU/L, ALT 483 IU/L) on day 69, decreasing thereafter to normal values over 45 days (day 124). Lactic dehydrogenase (LDH) and gamma glutamyl transpeptidase (yGT) were also elevated. She remained asymptomatic and afebrile throughout her illness. She did not develop any physical signs of either liver dysfunction or the possible aetiology of her hepatitis: in particular no rash, lymphadenopathy, splenomegaly, enlarged tonsils. Pertinent investigations were non-contributory and included a normal urea, creatinine, CPK, total white cell, neutrophil, lymphocyte and eosinophil counts, and prothrombin time. There were no atypical lymphocytes. Serologies for hepatitis A, B and C, and autoantibodies [anti-nuclear factor and smooth-muscle anti@NW, anti-mitochondrial bodies] were negative. A liver ultrasound on day 72 was normal. A liver biopsy was postponed after the first improved transaminase results. This normal, healthy female volunteer developed an asymptomatic rise in liver enzymes following the sequential administration of artesunate and amodiaquine which we believe was most probably caused by the amodiaquine alone. Other causes of asymptomatic hepatitis, e.g., Epstein-Barr virus, cytomegalovirus, toxoplasmosis were considered unlikely in the absence of other physical signs, the lack of lymphocytosis, her immune competent state (HSU et al., 1995). Furthermore toxoplasmosis is rare in Cape Town (Liver Unit, Universitv of Cane Town. nersonal communication). Amodiaquinelinduced -hepatitis during malaria prophylaxis is well described and may be associated with neutropenia (LARREY et al., 1986; NEFTEL et al., 1986; WOODTLI et al., 1986; CHARMOT & GOUJON, 1987; BERNUAU et al., 1988; RAYMOND et al., 1989). These side-effects are thought to be immune mediated but the pathogenesis remains unclear (CLARKE et al., 1991). Hepatitis has occurred from as early as 3 weeks (exposure to 3 weekly doses) to as late as 10 months of prophylaxis (CHARMOT & GOUJON, 1987). Clinically, reported cases have ranged from a mild, transient elevation of liver enzymes With few symptoms (LARREY et al., 1986; CHARMOT & GOUTON. 1987) to fulminant hepatitis with either slow recov&y of fun&ion (LARREY
Address for correspondence: Dr C. Orrell, Clinical Research Unit, Somerset Hospital, P.O. Box 50309, Waterfront, 8005, South Africa; phone +27 21 4026393, fax +27 21 4252021, e-mail [email protected]
Cape Town
Assessment mefloquine,
of susceptibility of Plasmodium falciparum to chloroquine, quinine, sulfadoxine-pyrimethamine and artemisinin in southern Viet Nam
N. V. Thanh’, A. F. Cowman*, D. Hipgrave3p4, T. B. Kim’, B. Q. Phuc’, L. D. Gong’ and B.-A. Biggs4>5 ‘National Institute of Malariology, Parasitology and Entomology, Hanoi, Viet Nam; ‘The Walter and Eliza Hall Institute of Medical Research, The Royal Melbourne Hospital, Parkville, Victoria 3050, Australia; 3Vietnam Health Alliance, 5th fl57 ~c;;tgaT~~un~’ Ba Trung District, Hanoi, Viet Nam, . 4Macfarlane Burnet Centre of Medical Research, Fairfield, 3078, . ‘The Unzversaty of Melbourne, Department of Medicine, The Royal Melbourne Hospital, Parkville, Victoria’3050, Ausiralia Abstract Resistance to antimalarial chemotherapy is a major concern for malaria control in Viet Nam. In this study undertaken in 1998, 65 patients with uncomplicated Plasmodium falciparum malaria were monitored for 28 days after completion of a 5-day treatment course with artemisinin. Overall 36.9% (24/65) of patients had recurrent parasitaemia during the surveillance period. I? falciparum isolates were tested for sensitivity in vitro to chloroquine, mefloquine, quinine, sulfadoxine-pyrimethamine and results were compared to those from a similar study in 1995. Increased parasite sensitivity to sulfadoxine-pyrimethamine, chloroquine and quinine was demonstrated, with significantly lower mean EC,, and EC& values in 1998 compared to 1995. Parasite sensitivity to mefloquine did not differ significantly in the 2 surveys. Isolates were also tested for sensitivity in vitro to artemisinin in the 1998 survey. The mean E&, was 0.03 nmol/L and the EC,, was 0.94 nmol/L. Parasite sensitivity to artemisinin will need to be monitored in view of its increasing use in Viet Nam. malaria, Plasmodiumfalciparum, drug sensitivity, in vitro, chloroquine, mefloquine, quinine, sulfadoxinepyrimethamine, artemisinin, trends, Viet Nam
Keywords:
Introduction Malaria is still a major health concern in Viet Nam with over 500000 cases and 198 deaths reported in 1996 (HIEN et al., 1997). I? falciparum resistance to antimalarial drugs is an important problem and a number of local studies have described increasing resistance particularly to sulfadoxine-pyrimethamine, chloroquine and quinine (CAILIPBELL et al., 1973; HIEN et al., 1997). Artemisinin as a single dose, and in combination with mefloquine, has been successfully evaluated in the treatment of both non-complicated as well as severe complicated multidrug-resistant malaria in Viet Nam (HIEN et al., 1997). Additionally, other studies have analysed the efficacy of mefloquine, chloroquine and sulfadoxine-pyrimethamine (RYAN, 1975; SHCHERBAKOV et al., 1990) as well as the mutation pattern in genes involved in the mechanism of resistance to pyrimethamine (REEDER et al., 1996; ZINDROU et al., 1996). An important change in the management of clinical malaria in Viet Nam has been the introduction of artemisinin and its widespread availability as a first-line treatment. It is likely that this has been a major factor in the reduction in the number of deaths from severe malaria since 1991 (HIEN et al., 1997; ANONYMOUS, 1997). As yet, there have been no reports of clinical resistance to artemisinin and its derivatives. We report the findings of a survey undertaken in 1998 that examined the suscentibilitv of Z? falciaar-um parasites to artemisinin, chloroquine,< mefloquine, quinine, and sulfadoxine-pyrimethamine in an area to the north-east of Ho Chi Minh city. Comparisons are made with a similar survey undertaken in 199 5. Materials and Methods Field site and patients, 1998 study The study was conducted at the health care centre in the Phu Rieng rubber plantation, which is situated 130 km north-east of Ho Chi Minh city. This area has hyper-endemic I? falciparum malaria with peak transmission between September and December. This site
Address for correspondence: Dr Beverley-Ann Biggs, Department of Medicine, The Royal Melbourne Hospital, Parkville, 3050, Australia; phone 161 3 9342 7702, fax +61 3 9347 1863, e-mail [email protected]
has been used for malaria studies for several years and a laboratory has been established within the health centre. Patients with uncomplicated I? falciparum malaria who were aged >5 years-were recruited into the study during: Sentember to November 1998. The total number 07 slides examined during this period was 683. There were 141 positive slides. Among the positive slides, 95 showed I? falciparum (including children aged ~5 years), 41 showed Z? vivax and 5 slides showed mixed I? falciaarum and l? vivax. Sixtv-five patients (aged >5 year;) with I? falciparum alone’were enrolled in the study. All patients were informed about the study and gave verbal consent before participation. Individuals who had received quinine within the previous 7 days, 4-aminoquinolines within the previous 14 days, pyrimethamine or sulphonamides within the previous 28 days or mefloquine within the previous 63 days were excluded from the study. Participants were treated with artemisinin (20 mg/kg on dav 0, 10 me/kst on davs l-4) for 5 davs. Parasitaemia was monitored with a thick and thin blood smear at days 0, 1, 2, 3, 7, 14, 21, 28 and during every episode of fever after day 7. Patients were observed in the health clinic during the first 7 days and thereafter returned for review according to the above schedule or if they became unwell. .
_
-I
-
Parasites Blood (5 mL) was collected by venepuncture into Vacutainer tubes (Becton Dickinson. UK) containing potassium oxalate and sodium fluoride. Thick and thin films were prepared, treated with 10% Giemsa stain, and examined by a trained microscopist. Blood (300 a) was transferred from Z?falcipanrm-positive samples into plastic Falcon tubes containing 2.7 mL of RPM1 1640 low p-aminobenzoic acid and low folic acid content culture medium (RIECKMANN et al., 1978) in preparation for drug assays. In-vitro drug sensitivity assays In-vitro microtest plates (WHO, 1990, 1997) containing chloroquine, mefloquine, quinine and sulfadoxine-pyrimethamine were used to measure schizont maturation inhibition in all samples with confirmed I? falciparum infection. The chloroquine plates were dosed at 1, 2, 4, 8, 16, 32, and 64 pmol/well. The mefloquine plates were dosed at 2, 4, 8, 16, 32, 64 and 128 pmol/well; the quinine plates at 4, 8, 16, 32, 64, 128
514
N. V. THANH ETAL.
and 256 pmol/well; the sulfadoxine-pyrimethamine plates with a mixture of sulfadoxine and pyrimethamine (80: 1) at pyrimethamine concentrations of 0.125, 0.375, 1.25, 3.75, 12.5, 37.5 and 125 pmol/well. Experimental plates containing artemisinin [prepared at the National Institute of Malariology, Parasitology and Entomology, Hanoi, Viet Nam (NIMPE) and dosed at 0.15, 0.5, 1.5, 5, 15, 50, and 150pmol/well] were also used, as the WHO artemisinin plates were found to be inactive. One of the 12 columns on each plate was used for each patient’s blood sample. All the wells of the appropriate column were dosed with 50 & of the blood-medium mixture (BMM, 1 : 9). Dosing started with the control (well A) and followed an increasing order of drug concentration ending at well H (the highest concentration). The plates were placed into a candle jar and incubated at 37°C (*0,5”C) for 24-36 h. After incubation thick films were made from the pellet in each well of the test plate and treated with 2% Giemsa stain for 30 min. The number of schizonts per 200 parasites was used to assessmaturation inhibition. The test was considered valid if 210% of the parasites in the control well had reached schizont stage (28 nuclei in sulfadoxine-pyrimethamine plates, 23 nuclei in the other plates) within 24-36 h. Parasite isolates were classified as sensitive or resistant to a particular drug according to the drug concentration at which schizont maturation was completely inhibited (Table) (WERNSDORFER & WERNSDORFER,
ology as in the 1998 study. Satisfactory artemisinin testing was not available at that time. Data analysis
Mean effective concentrations (EC) were calculated using a computer adapted probit analysis of log-dose responses (WERNSDORFER & WERNSDORFER, 1995) based on the methods of LITCHFIELD & WILCOXON (1949). 2 X 2 tables and x2 test (P value from x”) were used for comparison of the number of isolates showing resistance to chloroquine, mefloquine, quinine and compound sulfadoxine-pyrimethamine in 1995 and 1998 according to standard WHO criteria. Ethics
The project was part of an existing and ongoing programme of surveillance for drug resistance and received ethical approval through the NIMPE Ethics Review Committee. Results
In the 1998 study, 65 patients with uncomplicated I?
falciparum malaria completed a 5-day treatment course
with artemisinin. The mean fever clearance and parasite clearance times were 1.29 days and 1.75 days, respectively. The mean parasite count at day 0 was 12 308/pL (range 1360-102 564). Overall, 36.9% (24/65) patients had recrudescent parasitaemia during the 28-day surveillance period. The earliest recrudescence occurred on day 11 (6 patients had recrudescence between days 11 and 14, 13 patients between days 15 and 21, 5 patients between days 22 and 26). Twelve of the 24 patients had a further parasite recrudescence after retreatment with a 5-day course of artemisinin (2 occurred between days 11 and 14, 6 between days 15 and 2 1 and 4 on day 22 or 23). In-vitro drug sensitivity tests for Z?falciparum isolates collected during the 1998 study and also during a similar study in 1995 were performed for chloroquine, sulfadoxine-pyrimethamine, mefloquine, and quinine.
1995).
1995 Study
Results from a similar study conducted between June and September 1995 are shown for comparison (Table). The study was conducted by staff from the same Institute and patients were recruited from the Phu Rieng rubber plantation health care centre or a nearby hospital. Treatment and monitoring of patients, parasite isolate collection and drug sensitivity testing to chloroquine, mefloquine, quinine and sulfadoxine-pyrimethamine were carried out using the same method-
Table. Response of P. falciparum WHO Microtest) in 1995 and 1998
Drug Chloroquine Mefloquine Quinine SDXK’YR’ Artemisining
Year 1995
isolates
in southern
Viet Nam
to antimalarial
drugs
Number of Sensitive valid testsa isolatesb (n> (%I
in vitro
(using
P valued
8 66
0.27 0.04
0.83 0.13
2.20 0.18
13.70 0.36
29 35
100 97
0.09 0.08
0.25 0.20
0.33 0.26
0.57 0.41
NS
1995
19
100
1998
31
100
0.45 0.27
1.58 0.77
2.25 1.04
4.38 1.81
<0*05
1995
14
0.14
21.89
1998
25
0.04
0.34
92.37 0.60
137.00 1.80
1998
34
0.03
0.20
0.34
0.94
1998
$0”
1995 1998
2s
NRh
1
“For a valid test, schizont maturation in the control well must be 210%. bThe WHO classification of sensitivity and resistance of I? fulcipamm isolates to antimalarial drugs is used (WERNSDORFER & mixture (BMM); WERNSDORFER, 1995): chloroquine sensitive, complete schizont inhibition at ~0.08 pmol/L of blood-medium (I’yr)/L; sensitive, CO.025 pm01 pyrimethamine resistant, schizont formation at 30.16 pmol/L. Sulfadoxine-pyrimethamine resistant, 20.25 pmol I?yr/L. Mefloquine sensitive, ~0.32 pmol/L; resistant, 30.64 pmol/L. Quinine sensitive, G2.56 kmol/L; resistant, z5.12 pmol/L. ‘Values are the mean effective concentrations (EC) for P. fualciparum isolates (n) calculated for 50%, 90%, 95%, 99% schizont maturation inhibition. Units: pmol/L of BMM. d2 X 2 tables and x2 test (P value from x’) were used for comparison of the number of isolates showing resistance to chloroquine, mefloquine, quinine and compound sulfadoxine-pyrimethamine in 1995 and 1998 according to standard WHO criteria. eSulfadoxine-pyrimethamine. ‘These isolates showed borderline sensitivity, i.e., schizont inhibition occurred at a concentration of 0.075 ymol/L. gFresh microtest plates prepared at NIMPE were used in the artemisinin experiments. Standard WHO plates were used in other experiments. hNR, not relevant. Artemisinin resistance is undefined. In this assay 62% (21/34) isolates showed complete inhibition of schizont maturation at an artemisinin concentration of 0.1 kmol/L and 97% were inhibited at 0.3 pmol/L.
ANTIMALARIAL
SUSCEPTIBILITY
515
IN VIET NAM
Sixty-five patients were tested for all drugs in 1998 and the proportion of valid tests was 30/65 (chloroquine), 35/65 (mefloquine), 31/65 (quinine), 34/65 (artemisinin) and 25/65 (sulfadoxine-pyrimethamine). The results are summarized below and in the Figure and the Table.
pmol/L in 1998 (Table). This is an approximately 7fold decrease in the mean EC5,, and a 40-fold decrease from 1995 to 1998 and was significant in E& (P < 0.001) (Figure).
Mefloquine Chloroquine
The mean EC50 and EC,, for chloroquine was 0.27 pmol/L and 13.74 kmol/L respectively for 1995 whereas it had reduced to 0.04 (EC,,) and 0.36 (EC,,)
The mean EC50 and EC,, for mefloquine was 0.09 pmol/L and 0.57 Fmol/L, respectively, for 1995 which was similar to that seen in 1998 with an EC,, of 0.08 pmol/L and an EC,, of 0.41 pmol/L. There was no
99.9,
A 99 -
1998
90
1995
s VY tj 50 ;: 10 lOfi
0.1 SDXPYR
1 @mol/L)
0.1 Chloroquine
0
99.9
99.9 -
99
99 -
1 (pmol/L)
D
90 ,\” z
5010 -
I
I
0.1
1 Mefloquine @mol/L)
I
I
0.1 1.0 Quinine (pmol/L)
99.9 99
10 1
0
Artemisinin (mmol/L)
Figure. Sensitivity of l? fdciparum isolates in southern Viet Nam to chloroquine, mefloquine, quinine, sulfadoxine-pyrimethamine in 1995 and 1998 and artemisinin in 1998. SMI (y-axis), schizont maturation inhibition; SDX/PYR, sulfadoxine-pyrimethamine.
516
significant difference in sensitivity to mefloquine tween 1995 and 1998 isolates (Table, Figure).
N. V. THANH ETAL.
be-
derivatives in South-East Asia and Africa, and if this trend continues it will suggest that true resistance of I? falciparum to this drug family may be possible.
Quinine
For quinine the mean ECSo and EC,, was 0.45 pmol/L and 4.38 kmol/L, respectively, for 1995, with slightly decreased values for 1998 of ECI, 0.27 pmol/L and ECg9 1.81 pmol/L. This change represents a 2fold reduction in the level of resistance and is statistically significant (P < O.OS)(Figure).
Acknowledgements We are grateful to the Australian Agency for International Development and the Walter and Elisa Hall Institute of Medical Research for supporting this work, and to Michael Reed for technical and statistical advice. We thank Helen Dedman and Virginia de Crespigny for travel and accommodation arrangements and for administrative assistance.
Sulfadoxine-pyrimethamine
The mean ECSo and E& were 0.14 and 137.00 pmol/L in 1995 but decreased to 0.04 and 1.80 Fmol/L, respectively, in 1998 (Table). The 4-fold decrease of the mean EC&, and the 70-fold decrease of the E& from 1995 to 1998 was significant (P < 0.00 1) (Figure). Artemisinin
The mean ECso for artemisinin was 0.03 ymol/L and the EC& was 0.94 pmol/L. The number of isolates showing complete inhibition of schizont maturation increased from 62% (21/34) at an artemisinin concentration of 0.1 pmol/L to 100% (34/34) at 1.0 pmol/L. Discussion Malaria is still a major health problem in some parts of Viet Nam, although the overall incidence of this disease in 1995 was only 8.5 per 1000 (MORILLON et al., 1996). The spread of drug-resistant I? falciparum has made the choice of antimalarial therapy for control and treatment of this infection problematic. For example, in 1996 chloroquine resistance rates ranged from 41% to 62%, and resistance rates for sulfadoxinepyrimethamine ranged from 26% to 47% (MORILLON et al., 1996). The prevalence of chloroquine resistance in the study area for 1995 was 92%, and fell to 33% in 1998. A possible reason for this decrease in the level of chloroquine resistance is decreased drug use as artemisinin has been increasingly used for malaria treatment in this area since 1992 (HIEN, 1994; HIEN et al., 1997). It is a surprising result, as chloroquine resistance in I? falciparum tends to be relatively stable (PHILIPPS et al., 1998; LE BRAS, 1999). It will be important to conduct follow up in-vitro sensitivity studies in subsequent years to determine whether this decrease is stable. In contrast, both mefloquine resistance and quinine resistance were relatively stable with only small decreases from 1995 to 1998. The level of resistance to sulfadoxine-pyrimethamine also fell between 1995 and 1998 from 100% to 34%. The 1995 levels of resistance are higher than those reported previously in Viet Nam (MORILLON et al., 1996), and presumably reflect high levels of sulfadoxine-pyrimethamine use in the study area before 1995. The decreased resistance observed in 1998 is consistent with decreased use of this drug combination since 1995. Follow-up studies in this area of Viet Nam will determine whether this downward trend for sulfadoxine-pyrimethamine resistance is ongoing. The susceptibility of parasites to artemisinin observed in the 1998 survey was less than that reported by WONGSRICHANAUI et al. (19971, who tested l? falciparum isolates collected fro& pat&ts in 1995 in s&hem Viet Nam. Some studies undertaken in Africa report slightly higher E& levels than observed in the 1998 survey (PHILIPPS et al., 1998). Although the cutoff value for resistance to artemisinin remains unknown, differences in sensitivity of individual parasite isolates to the qinghaosu deri;atives have be& previouslv reoorted in vitro (BASCO & LE BRAS. 1994: PRAD~NE~ et al., 1998), ihcluding reduced se&itiviG after mutagenic treatment (INSELXJRG, 1985). It will be essential to monitor susceptibility to the qinghaosu
References
Anonymous (1997). National Malaria
Treatment Guidelines. Vietnam: Ministrv of Health. Tune 1997. Basco, L. K. & Le’ Bras, J. (iG94). In vitro susceptibility of Cambodian isolates of Plasmodium falcipawm to halofantrine, pyronaridine and artemisinin-desvatives. Annals of Tropical Medicine and Parasitology, 88, 137- 144. Campbell, C. H., Holiday, J., Biggs, J. C. & Backhouse, T. C. (1973). Chloroquine-resistant falciparum malaria acquired in Vietnam. MedicalJournal ofAustralia, 2, 729-734. Hien, T. T. (1994). An overview of the clinical use of artemisinin and its derivatives in the treatment of falciparum malaria in Viet Nam. Transactions of the Royal Socieg of Tropical Medicine and Hygiene, 88, supplement 1, S1/7-SIl8. Hien, T. T.. VinhChau. N. V.. Vinh. N. N.. Hung. N. T.. Phbng, k. Q., Toan; L. MI, Ma< P. I’.,’ Dun; N. T.; HoaiTam, D. T. & Arnold, K. (1997). Management of multiple drug-resistant malaria in Vietnam. Annals of Academic Medicine Singapore, 26, 659-663. Inselburg, J. (1985). Induction and isolation of artemisinineresistant mutants of Plasmodium falciparum. American Journal of Troaical Medicine and Hwiene, 34. 417-418. Le Bras, J. -(1999). Mechanismsand- dynamics of drug resistance in Plasmodium falciparum. Bulletin de la SociS de Pathologic Exotique, 92,236-241. Litchfield, J. T. & Wilcoxon, F. (1949). A simplified method for evaluating dose-effect. Journal of Pharmacology and Experimental Therapeutics, 96, 93- 113. Morillon, M., Baudon, D. & Dai, B. (1996). Malaria in Vietnam in 1996: brief synthesis of epidemiological data [in French]. Midecine Tropicale (Mars), 56, 197-200. Philipps, J., Radloff, P. D., Wernsdorfer, W. & Kremsner, P. G. (1998). Follow-up of the susceptibility of Plasmodium falcipancm to antimalarials in Gabon. American Journal of Tropical Medicine and Hygiene, 58, 612-618. Pradines, B., Rogier, C., Fusai, T., Tall, A., Trape, J. F. & Doury, J. C. (1998). In vitro activity of artemether against African isolates (Senegal) of Plasmodium falciparum in comparison with standard antimalarial drugs. American JournaZ of Tropical Medicine and Hygiene, 58, 354-357. Reeder, J. C., Rieckmann, K. H., Genton, B., Lorry, K., Wines, B. & Cowman, A. F. (1996). Point mutations in the dihydrofolate reductase and dihydropteroate synthase genes and in vitro susceptibility to pyrimethamine and cycloguanil of Plasmodium falciparum isolates from Papua New Guinea. American Journal of Tropical Medicine and Hygiene, 55, 209-213. Rieckmann, K. H., Campbell, G. H., Sax, L. J. & Mrema, J. E. (1978). Drug sensitivity to Plasmodium falciparum. An &-vitro microtechnique. La&et, i, 22-23. Ryan, B. P. (1975). Sulfadoxine and pyrimethamine in the treatment of falcioarum malaria in Vietnam. Medical .,7oumal OfAustralia, 1, lOi- 103. Shcherbakov, A. M., Nguyen, T. L., Chin, K. A., Chan, K. T.. Rabinovich, S. A., Nrmven, V. D.. Chan, V. F., Nguyen, T. L., Vu,- T. T. h Pidh;lt, H. (199b). The clinical efficacy of and tolerance for Lariam (mefloquine) in tropical malaria in the south of the Socialist Republic of Vietnam [in Russian]. Medical Parazitology (Mask), 5,57-59. Wernsdorfer, W. H. & Wernsdorfer, M. G. (1995). The valuation of in vitro test for the assessment of drug response in Plasmodium falciparum. Parasitology, 17, 22 l-227. WHO ( 1990). In vitro micro-test (Mark III) for the assessmentof the response of Plasmodium falciparum to chloroquine, me$i’oquine, quinine, suljadoxinelpyrimethamine and amodiaquine. Geneva, Switzerland: World Health Organization. WHO/ MAP/87.1. WHO (1997). In vitro micro-test (Mark III) for the assessmentof the response of Plasmodium falciparum to chloroquine, mefloand artemisinin. q&e, quinine, sulfadoxinelpyrimethamine Geneva, Switzerland: World Health Organization. WHO/ CTD/ti97.20.
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SUSCEPTIBILITY
IN VIET
517
NAM
Wongsrichanalai, C., Nguyen, T. D., Trieu, N. T., Wimonwattrawatee, T., Sookto, I’., Heppner, D. G. & Kawamoto, F. (1997). In vitro susceptibility of Plasmodium falciparum isolates in Vietnam to artemisinin derivatives and other antimalarial% Actu Tropica, 63, 151-l 58. Zindrou S., Nguyen, P. D., Nguyen, D. S., Skold, 0. & Swedenberg, G. (1996). Plasmodiumfalciparxm: mutation pattern in the dihydrofolate reductase-thymidylate synthase
TRANSACTIONS
OF THE ROYAL
1 Short Report
SOCIETY
OF TROPICAL
MEDICINE
1
Acute asymptomatic hepatitis in a healthy normal volunteer exposed to 2 oral doses of amodiaquine and artesunate C. Orrell’, W. R. J. Taylor2’3 and P. Olliar02’ Depanment of Pharmacology, University of Cape Town, Cape Town, South Africa; ’ UNDPIWorld BanklWHO Special Programme for Research and Training in Tropical Diseases (TDR), WHO, Geneva, Switzerland; 3The Centre for Infectious Diseases, Royal Free and University College Medical School, London, UK Abstract Combination antimalarial therapy is being explored to delay development of resistance to falciparum malaria. This report describes an unexpected drug-induced hepatitis in a previously healthy young woman exposed to 2 doses of amodiaquine and artesunate. Use of these combinations should be closely monitored. Keywords: antimalarial agents, amodiaquine, artesunate, adverse events, volunteer, liver function, hepatitis, South Africa
Multidrug-resistant Plasmodium falciparum is a global problem (WERNSDORFER & PAYNE, 1991). The use of antimalarial drug combinations with independent mechanisms of action is a rational approach to delay the onset of drug resistance and safeguard existing drugs (WHITE & OLLIARO, 1996). Current field research is assessing oral artesunate combined with standard antimalarials, e.g., sulfadoxine-pyrimethamine, amodiaquine, for treating acute uncomplicated falciparum malaria. Artesunate has a short half-life and produces a rapid and substantial reduction of malaria parasites; those that remain are then killed by the combinant drug acting over a longer time period. To complement the field studies, an interactive pharmacokinetic study of artesunate and amodiaquine was conducted in normal healthy volunteers at the University of Cape Town. We report the occurrence of asymptomatic hepatitis in a woman during this study. This single-dose 3-phase cross-over study was conducted in 15 volunteers, 10 male and 5 female, aged 19-42 years. All volunteers received 1 dose of oral artesunate (4 mg/kg) on day 0, followed, on day 7, by either 1 dose of oral amodiaquine (10 mg/kg) alone or combined with a single dose of artesunate (4 mg/kg). Subjects were given the alternative regimen on day 28. Blood for routine haematology and biochemistry was taken on days 0, 6,27 and 48.
genes of Vietnamese isolates, a novel mutation, and coexistence of two clones in a Thai patient. Experimental Parasitology,84, 56-57.
Received 13 October 2000; revised 5 April 2001; accepted for publication 9 May 2001
AND HYGIENE
(2001) 95,517-518
Our subject was a healthy 20-year-old South African woman of African-Caucasian descent, with no previous significant illnesses. During the study she did not take any prescribed or over-the-counter drugs, including paracetamol, and had no reported drug allergies. She did not drink alcohol and had no risk factors for hepatitis B. Her family history was unremarkable. Clinically, there were no abnormal physical signs and her baseline blood tests were all normal. She received artesunate (day 0), amodiaquine (day 7), followed by the combination (day 28). Six days after taking artesunate alone, her aspartate aminotransferase (AST) and total bilirubin were normal but she had a slight increase in alanine aminotransferase (ALT) of 31 IU/L (normal 1-25 IU/L) that was not considered clinically significant (Figure). On day 27, all blood tests were normal. On day 48, her transaminases were elevated, rising to peak values (AST 236 IU/L, ALT 483 IU/L) on day 69, decreasing thereafter to normal values over 45 days (day 124). Lactic dehydrogenase (LDH) and gamma glutamyl transpeptidase (yGT) were also elevated. She remained asymptomatic and afebrile throughout her illness. She did not develop any physical signs of either liver dysfunction or the possible aetiology of her hepatitis: in particular no rash, lymphadenopathy, splenomegaly, enlarged tonsils. Pertinent investigations were non-contributory and included a normal urea, creatinine, CPK, total white cell, neutrophil, lymphocyte and eosinophil counts, and prothrombin time. There were no atypical lymphocytes. Serologies for hepatitis A, B and C, and autoantibodies [anti-nuclear factor and smooth-muscle anti@NW, anti-mitochondrial bodies] were negative. A liver ultrasound on day 72 was normal. A liver biopsy was postponed after the first improved transaminase results. This normal, healthy female volunteer developed an asymptomatic rise in liver enzymes following the sequential administration of artesunate and amodiaquine which we believe was most probably caused by the amodiaquine alone. Other causes of asymptomatic hepatitis, e.g., Epstein-Barr virus, cytomegalovirus, toxoplasmosis were considered unlikely in the absence of other physical signs, the lack of lymphocytosis, her immune competent state (HSU et al., 1995). Furthermore toxoplasmosis is rare in Cape Town (Liver Unit, Universitv of Cane Town. nersonal communication). Amodiaquinelinduced -hepatitis during malaria prophylaxis is well described and may be associated with neutropenia (LARREY et al., 1986; NEFTEL et al., 1986; WOODTLI et al., 1986; CHARMOT & GOUJON, 1987; BERNUAU et al., 1988; RAYMOND et al., 1989). These side-effects are thought to be immune mediated but the pathogenesis remains unclear (CLARKE et al., 1991). Hepatitis has occurred from as early as 3 weeks (exposure to 3 weekly doses) to as late as 10 months of prophylaxis (CHARMOT & GOUJON, 1987). Clinically, reported cases have ranged from a mild, transient elevation of liver enzymes With few symptoms (LARREY et al., 1986; CHARMOT & GOUTON. 1987) to fulminant hepatitis with either slow recov&y of fun&ion (LARREY
Address for correspondence: Dr C. Orrell, Clinical Research Unit, Somerset Hospital, P.O. Box 50309, Waterfront, 8005, South Africa; phone +27 21 4026393, fax +27 21 4252021, e-mail [email protected]
Cape Town