Resistance of plasmodium falciparum to a

TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE (2001) 95,325-329 Resistance of Plasmodium falciparum to antimalarial drugs in a highly area of southern Viet Nam: a study in viva and in vitro endemic Nguyen Mai Huong’, Sean Hewitt*, Timothy M E Davis2’3, Le Due Dao’, Tran Quoc Toan’, ‘Nutianal Tran Bach Kim’, Nguyen Thi Hanh’, Vo Nhu Phuong’ , Doan Hanh Nhan’ and Le Dinh Gong’ Institute of Malariology, Parasitology and Entomology, Luong The Virzh Road, Hanoi, Viet Nam; ‘Vie, Nam-Australia Malaria Control Project, Ministy of Health, 138A Giang Vo, Hanoi, Viet Nam; University of Western Australia, Department of Medicine, Fremantle Hospital, Fremantle, Australia Abstract To assess the antimalarial sensitivity of Plasmodium falciparum in vivo and in vitro in a highly endemic area of southern Viet Nam a field studv was conducted (in 1999) at a rubber plantation in Binh Phuoc Province north of Ho Chi r&h City F&y patients were ireated with either a;esunate (4 mg/kg on day 0, then mg/kg on day to 4) or mefloquine (10 mg/kg at h, then mg/kg at h), and their progress was followed for 28 days under standard WHO protocols Blood spots were taken at baseline from all patients, as well as from those who redeveloped parasitaemia during follow-up, for polymerase chain reaction (PCR) determination of parasite genotypes to assist differentiation of re-infection from recrudescence Both treatments cleared parasites within days, Ofthe 25 mefloquine-treated patients, (8%) re-presented with probable re-infections For artesunate, patients (16%) had re-infections and (20%) had recrudescences Sensitivity tests in vitro of pre-treatment I? fulciparum isolates showed geometric mean IC,, values of 29,38, 209 and 15 nmol/L for chloroquine (n = 32), mefloquine (n = 33), quinine (n = 31) and artemisinin (n = 1), respectively There were significant correlations between IC+ for artemisinin and mefloquine (r = 0.72, P = 0.004), and chloroquine and quinine (r = 0.44, P = 0.05) These data show that, although mefloquine has been used for 10 years in Binh Phuoc Province, it remains fully effective, perhaps because an artemisinin derivative is commonly given at the same time The recrudescence rate for artesunate is similar to those reported in other epidemiological contexts The present in-vitro data imply that quinine remains effective and that reduced drug pressure has been associated with increased sensitivity of local strains of l? falciparum to chloroquine Although from one hyperendemic area, these results may have implications for antimalarial prophylaxis and treatment strategies for residents and travellers to southern Viet Nam malaria, Plasmodium falciparum, artesunate, artemisinin, Viet Nam Keywords: drug sensitivity, in viva, in vitro, chloroquine, mefloquine, quinine, Introduction The threat of resistance of Plasmodium falciparum to available antimalarial drugs remains of great concern, especially in south-east Asian countries such as Viet Nam Although formal reports in the literature from Indochina have been understandably few, clinical evidence of chloroquine-resistant I? falciparum was first found in southern Viet Nam in 1961 and similar reports from the central provinces of Quang Binh and Nghe An followed years later (PHAN, 1998) As a result, the National Institute of Malariology, Parasitology and Entomology (NIMPE) in Hanoi established routine monitoring of drug resistance in I? falciparum in 1968 The standard 7-day test recommended by the World Health Organization (WHO) was used initially The extended 28-day in-vivo test was introduced in 1980 together with the WHO macro and micro in-vitro tests Drug-resistant I? falciparum is now widespread, particularlv in central and southern Viet Nam Chloroauine resistance in vivo is currently thought to range from*30% to 85% while sulfadoxine-pyrimethamine resistance is encountered in 30-80% of patients (N D Sy, personal communication) In response to the need for alternative treatments, mefloquine and artemisinin derivatives were introduced in 1990 Mefloquine use was limited initially to combination therapy with artemisinin derivatives in drug-resistant cases However, mefloquine-artemisinin combinations have had wider annlication since 1995 and the use of chloroquine, sulfadokine-pyrimethamine and quinine has declined Although an attenuated in-vivo response to artemisinin has been described (LUXEMBERGER et al., 1998), evidence from Thailand suggests that mefloquine and artemisinin may be murually protective when used in combination (NOSTEN et al., 1998; WHITE, 1998) In Address for correspondence: Professor T M E Davis, University ofwestern Australia, Department of Medicine, Fremantle Hospital, P.O Box 480, Fremantle, Western Australia 6959, Australia; phone +618 9431 3229, fax +618 9431 2977, e-mail tdavis@cyllene.uwa.edu.au the light of these reports, and given the history of drug use and parasite resistance in Viet Nam, we aimed to establish the sensitivity of I? fulciparum to (i) artesunate and mefloquine in vivo and in vitro, and (ii) chloroquine and quinine in vitro in an endemic rural area in the south of the country Methods Study site The study was approved by the Ethics and Scientific Committee of NIMPE and nerformed at Phu Riena rubber plantation, Phuoc Ling district, Binh Phuo: province Phuoc Long district is in a highly endemic area in southern Viet Nam well known for multidrug-resistant I? fulciparum Malaria transmission occurs all year round, with peaks reported between May and June and betweenNovember and December Between August and October 1998, cross-sectional surveys revealed a-malaria orevalence of23% 170% I? falciaarum 27% I? vivax and 3% mixed infectiois) In &e early 1lOOO/pL whole blood Exclusion criteria included (i) pregnancy, (ii) concomitant illness, and (iii) prior antimalarial therapy, specifically quinine or an artemisinin drug within the previous days, a 4-aminoquinoline within the previous 14 days, or 326 NGUYEN MA1 HUONG ETA,!, pyrimethamine and/or sulphonamide within the previous 28 days Dill-Glazko urine tests (reagents preDared bv NIMPE) were nerformed to confirm that all recruits had taken neither chloroquine nor sulfadoxinepyrimethamine All patients were admitted to hospital and duplicate thick and thin blood films prepared Twenty-five patients were randomized to receive days of artesunate (50 mg salt, National Pharmaceutical Company No 1, Hanoi, Viet Nam), consisting of mg/kg single dose on day and then mg/kg morning doses on days to inclusive A further 25 patients were randomized to a l-day course of MephaquineTM (250 mg base, Mepha, Switzerland), 10 mg/kg at h followed by mg/kg at h This group received antiemetic (atropine 0.1 mg/kg bodyweight) and antipyretic (paracetamol 10 mg/kg) medication 30 before treatment was given All treatments were supervised and patients were observed for at least 60 post administration Any patients vomiting during this period were retreated but excluded from the study Oral temperatures were measured daily As well as those scheduled on davs and additional thick and thin blood films were taken daily until parasite clearance was confirmed by consecutive negative results (no asexual forms in 100 fields of view at X 1000 magnification) Patients were kept in hospital for at least days and were discharged when asymptomatic and aparasitaemic All were asked to return for follow-up on days 14,2 and 28 or if they became symptomatic A blood film was taken and a clinical assessment (including oral temperature) was performed on each of these occasions Those who failed to attend were contacted by local health workers in an attempt to provide as complete a followup as possible A sensitive (S) response was recorded if no asexual stages were found on day and parasites had not reappeared by day 28 If asexual parasites disappeared by day but reappeared before day 28 the isolate was considered to have probable RI resistance If the asexual parasitaemia had dropped by at least 75% at 48 h but not cleared, and if parasites were still present on day 7, the parasites were considered resistant at the RI1 level If the asexual parasitaemia had dropped by less than 75% at 48 h and the patient remained slide-positive on day 7, the parasites were considered resistant at the RI11 level Cytogenetic studies were carried out to distinguish recrudescence from re-infection Parasite DNA was extracted from Whatman filter-paper using a modification of the method described bv KAIN & LANAR C199 1) The blood-soaked portion of the papers was cut into fine strips, placed in 1*5-mL polyethylene tubes with sodium dodecyl sulphate (0.5% final concentration) and proteinase K (500 pg/mL final concentration) and incubated at 37°C for h The DNA was then extracted with phenol, precipitated with alcohol and amplified using nested polymerase chain reaction (PCR) A set of primers was chosen to identify 16 distinct association types These consisted of merozoite surface protein (MSPl, block 2) allelic families (MAD20, Kl and R033), MSP2 allelic families (FC27 and Indochina), and glutamate-rich protein (GLURP) PCR products were analysed by gel electrophoresis In-vitro test The WHO standard Mark III micro-test was applied for in-vitro assessment of the drug sensitivity of parasites from the patients recruited to the in-vivo study Patients with an asexual parasitaemia between 1000 and 80 000 parasites/pL were considered suitable for testing In all, parasite cultures from 33 patients were studied These cultures were tested against artemisinin, mefloquine, chloroquine and quinine The artemisinin plates used were produced by NIMPE under guidance from WHO All others were supplied directly by WHO For mefloquine, chloroquine, and quinine, parasites were considered sensitive if complete inhibition of schizonts occurred at 3200,800 and 2560 nmol/L or less, respectively There is currently no sensitivity threshold set for artemisinin Data analysis Drug concentrations inhibiting parasite growth by 50% (ICsOs) were calculated using nonlinear regression based on software developed by WERNSDORFER & WERNSDORFER (1995) and are reported as geometric means and 95% confidence intervals Two sample comparisons were by Student’s t-test or, in the case of nonnormally distributed or discontinuous data, by Wilcoxon-Mann-Whitney tests The x2 test was used to assess differences in proportions between groups Associations between variables were assessed using Pearson’s product-moment correlation co-efficient A 2-tailed level of significance was used throughout Results Details of the patients are summarized in Table There were no significant differences in age, sex distribution, bodyweight or admission oral temperature between the groups (P>O.5) Although the day-0 median parasite density in the artesunate group was significantly greater than in the mefloquine group (P = 0,024), the median parasite clearance time was shorter (P = 0,004) Parasite clearance curves are shown in the Figure All patients were afebrile by day The majority of patients (85%) in the mefloquine group reported suffering side-effects including dizziness (80%), headache (64%), nausea (32%), vomiting (16%), tremor (5%) and abdominal pain (5%) Symptoms were usually mild and patients recovered without medical intervention No side-effects were reported in the artesunate group Data from in-vitro tests are summarized in Table Sensitivity in vitro to mefloquine was 100% amongst the isolates tested, with complete inhibition of schizont formation occurring in the wells containing 320 nmol/L (the WHO discriminating concentration) Table Details of the Vietnamese malaria patients at the time of admission to the study (July-November 1999), classified by allocated treatment Characteristic Mefloquine Artesunate Number of patients Age (yea& Sex (% males) Bodyweight (kg) Oral temperature (“C) Parasitaemia (/@J Parasite clearance time (days) Fever clearance time (days) 25 26.5 + 13.3 26.2: 40.3 g-62) 38.8 It 0.8 7400 (1040-114000) (l-5) (O-2) 13.5 43.9 (qo5-68) 38.8 f 0.7 18 200 (1560-75 300)* (l-4)* (l-2) Data are mean f SD or median (range) *P < 0.05 in comparison with the mefloquine group PLASMODIUM FALCIPARUM 327 DRUG RESISTANCE IN VIET NAM 120000 m E s 60000 r m" 40000 z 16 18 20 22 24 26 28 20000 Days after admission Figure Median (circles) and range (vertical bars) for I? fulciparum parasite clearance curves for artesunate (0) and mefloquine (0) The upper right insert shows the patients who redeveloped parasitaemia during follow-up: mefloquine reinfection (grey bars), artesunate reinfection (white bars) and artesunate recrudescence (black bars) concentrations for the four antimalarial drugs evaluated Table Inhibitory during in-vitro testing of Vietnamese l? falciparum isolates (1999) Concentration Dw Chloroquine Mefloquine Quinine Artemisinin n 32 33 31 31 IC,, IC,, 29 38 209 15.0 (22-39) (28-50) (155-282) (9.4-24.1) (nmol/L) 110 80 628 84.0 (72-169) (53-121) (406-973) (43.4-162.5) G9 323 148 1540 341 (170-612) (79-276) (805-2944) (130-892) Values are geometric means (95% confidence intervals in parentheses) Among the 32 isolates tested successfully against chloroquine in vitro, (16%) were resistant (discriminating concentration 80 nmol/L) All 31 isolates tested against quinine and artemisinin were sensitive to quinine (discriminating concentration 2560 nmol/L) and showed complete schizont inhibition at artemisinin concentrations >300 nmol/L Further analysis of the in-vitro results for individual isolates revealed significant correlations between I&s for artemisinin and mefloquine (r = 0.72, P = 0.004), and chloroquine and quinine (r = 0.44, P = 0.05) Parasitaemia re-appeared in patients from the mefloquine group (8%) and in patients from the artesunate group (36%) between 14 and 28 days (P = 0.03) Cytogenetic studies indicated that both the mefloquinetreated patients had re-infections while, in the artesunate group, patients (16%) had re-infections and (20%) recrudescences (see Figure) Cytogenetic studies that were done on 49 of the 50 patients at the time of admission to the study using the full primer set showed that no patients had the same PCR profile It is very unlikely, therefore, that the patients whose PCR results were interpreted as recrudescences were, in reality, reinfected with the same strain No patients exhibited resistance at the RI1 or RI11 level All 11 patients who redeveloped a parasitaemia during follow-up were retreated with the alternative drug regimen and prompt parasite clearance was observed in each case Of the day-0 isolates taken from patients subsequently exhibiting recrudescence after artesunate therapy, failed to grow in vitro, displayed complete schizont inhibition at 30 nmol/L artemisinin and at 100 nmol/L artemisinin One of these latter isolates also demonstrated relatively high tolerance to mefloquine with complete inhibition of schizont formation occurring only in the well containing 320 nmol/L In patients who did not recrudesce after artesunate, inhibition of schizont formation occurred only at 1000 nmol/L artemisinin Discussion Our in-vivo data, supported by PCR analysis, provide evidence that mefloquine remains a fully effective antimalarial drug despite being in use for almost a decade in the endemic area of southern Viet Nam in which the study was performed The reason may be that mefloquine has been protected by the concomitant use of an artemisinin derivative during this time Artesunate cleared parasites more rapidly than mefloquine but was associated with recrudescence in 20% of patients as assessed using PCR in paired blood spots Nevertheless, this recrudescence rate is similar to that reported by other investigators who have also used a 5-day artesunate regimen (BARRADELL & FI~TON, 1995) These in-vivo findings are supported by the present in-vitro data Mefloquine sensitivity was 100% based on current WHO criteria Isolates taken from the artesunate-treated patients showed complete schizont inhibition in the presence of artemisinin concentrations at or below pmol/L Our in-vitro data also provide evidence that quinine is still an effective first-line agent against P falciparum in Viet Nam, and suggest that reduced drug pressure has had a significant positive impact on parasite sensitivity to chloroquine 328 NGUYENMAIHUONGETAL In Viet Nam, antimalarials are the only drugs supplied by the health services to all groups free of charge There has, therefore, been the potential for overuse Antimalarial treatment may be given to patients with fever irrespective of other symptoms or given out in exchange for blood smears during mass surveys These practices would favour the development of parasite resistance The dispensing of antimalarial drugs is, however, increasingly restricted, and in many areas they are now provided only for the treatment of clinically diagnosed malaria or as single dose ‘prophylactic treatment’ for non-immune forest workers Mefloquine has, however, been a special case From 1984 to 1990, mefloquine was in widespread and often indiscriminate use along the western border of Thailand As a result its antimalarial efficacy fell dramatically LOOAREESUWAN et al (1992) reported that the cure rate for a 15-mg/kg dose decreased from 98% in 1983-86 to 1% in 1930.IMefloquine was first introduced to Viet Nam in 1990 but, based on the reports from Thailand and because of its relatively high cost, its use was limited to combination therapy with artemisinin derivatives for patients from areas with drug-resistant parasites Only over the past 4-5 years has such combination therapy become more commonplace Despite this trend, there does not appear to have-been a decline in the efficacy of either drug In Thailand the introduction of artesunate in 1994 for use in combination with mefloquine prevented further reductions in the efficacy of mefloquine (NOSTEN et al., 1998) This might also explain the 100% cure rate observed in the present study, which is comparable to that when mefloquine was first introduced to Viet Nam as well as to rates recorded in 1993 and 1998 (N D Sy & D X Huong, unpublished observations) Furthermore, sensitivity in vitro to mefloquine in the present study was 100% and the geometric mean I&, was 38 nmol/L These results were similar to those from previous’ unpublished studies carried out in a variety of locations in Viet Nam since 1986 in which 392% sensitivity and an IC,, G83 nmol/L were found (V T Tuyet, N T Tien, T T Tinh, N V Thanh, unpublished observations) Despite the fact that the admission parasitaemia was significantly higher in the artesunate than the mefloquine group, parasite clearance occurred earlier in the former group There were no RI1 or RI11 cases, but a 5-day artesunate regimen was associated with a recrudescence rate of 20% In many parts of Viet Nam, artemisinin and artesunate have been used as a first-line treatment for falciparum malaria for nearly 10 years Up until now Table A summary showing fluctuations of unpublished in recrudescence studies carried rates (%R) there have been no documented reports of resistance in viva but high recrudescence rates were reported from the outset (see Table 3; NGUYEN, 1993) Longer regimens give better cure rates (BUNNAG et al 1991) but patient compliance falls off rapidly after the symptoms of malaria subside These considerations also argue for the combination of an artemisinin drug such as artesunate with a second longer half-life drug such as mefloquine The geometric mean IC,, of 15.0 nmol/L for artemisinin observed during this study was very similar to that of 14.5 nmol/L from a study conducted at a nearby hospital (15 km distant) in 1995 (N V Huong, unpublished report) However, the short shelf-life for artemisinin plates has caused problems in the past (F&E et al., 1999) They must be kept at or below 4°C during transport and storage, and should be used within months While refrigeration and storage during the present study were optimized, the results of previous similar in-vitro studies of artemisinin in Viet Nam may not be valid In view of the range of sensitivities we encountered, especially in recrudescent cases, there is a definite need for in-vitro surveillance of artemisinin drugs to be maintained on a regular basis in areas in which they are used Chloroquine remains first-line treatment for l? vivax in Viet Nam, but in highly endemic southern and central regions it has not officially been used to treat falciparum malaria for more than 10 years Among the 32 isolates we tested against chloroquine in vitro, 84% were sensitive A comparison of these results with those from previous unpublished studies in the area confirms that sensitivity in vitro has risen dramatically, presumably owing to reduction in drug pressure In 1986 V T Tuyet reported that 83% of 496 isolates he tested were resistant with an IC,, of 900 nmol/L In 1998 N V Thanh reported that 17% of 30 isolates tested were resistant with an IC,, of 359 nmol/L Tuyet’s study was carried out in a hospital and some of the patients recruited are likely to have had a history of treatment failure This sample would, therefore, not have been a representative one Nevertheless, the magnitude of the difference between the results from 1986 and the 1990s suggests that resistance was very much greater then than now Consistent with the results of a recent large-scale comparative trial in which quinine was as effective as artemether in southern Vietnamese patients with severe malaria (HIEN et al, 1996), all isolates investigated in the present study were sensitive to quinine in vitro This result is also similar to those from a number of recent unpublished studies conducted in Viet Nam In 1998 N out in Phu Rieng Commune, Date Drug Regimen 1990 1991 Mar-May 199 May- Jun 199 Sep-Nov 1992 Ott-Dee 1993 Ott-Dee 1993 Mar-May 1994 Ott-Dee 1995 May-Jun 1995 Aug-Ott 1996 Ott-Dee Artemisinin Artesunate (C) Artemisinin Artemisinin Artemisinin Artemisinin Artemisinin Artemisinin Artemisinin Artemisinin Artesunate’” 1998 May-Jun 1997 Ott-Dee 1998 Aug-Ott 1998 Ott-Dee Artemisinin Artesunate Artesunate” 10 mg/k/day Do4 D14 mg/k/day Do & mg/k/day 15 mg/kg/day Do4 20 mg/kg/day Do & 10 mg/kg/day D,-, 20 mg/kg/day Do & 10 mg/kg/day DI-4 10 mglklday DOA 10 mg/k/dv DO4 10 mg/k/day Ds6 10 mg/k/day h6 10 mg/kg/dv Do4 2.4 mg/kg/day Do & 1.2 mg/kg/day DIA 10 mg/k/day Do4 20 mg/kg/day Do & 10 mg/kg/day Dim4 20 mg/kg/day Do & 10 mg/kg/day D,, 2.4 mg/kg/day Do & 1.2 mg/kg/day D,-, Binh Phuoc Province, n %R 102 18 86 50 32 22 15 22 22 30 52 15 12 12 28 34 *26 9 *26 14 65 28 *E 48 40 14 The possibility that some of these were re-infections cannot be ruled out Im, intramuscular; iv, intravenous; (C), made in China; *, 21day rather than 28-day follow-up (N D Sy, D H Nham, N M Huong, N V Huong, T T Thin, N V Thanh, D X Huong & T N Hai, unpublished results) l’LASMODIUM FALCPARUM DRUG RESISTANCE IN VIET NAM V Thanh reported 100% sensitivity and an IC50 of 340 nmol/L in a study of isolates exposed to quinine and, in 1999, T T Tinh reported 100% sensitivity and an I&, of 142 nmol/L in 19 isolates A number of researchers have observed a positive correlation between responses in vitro to artemisinin (or its derivatives) and mefloauine (DOURY et al 1992: BASCO & LE BRAS, 1993; -BUST& et al., 1994; RING: WALD et al., 1999; WONGSRICHANALAI et al., 1999) Where this pattern is apparent at low ICsos it might be attributed to the general biological fitness of the isolates, but when the pattern holds at higher concentrations, cross-resistance seems more likely This explanation may have been applicable to the isolate in our study for which schizont inhibition in vitro required high concentrations of both mefloquine and artemisinin Although, as discussed above, such combination therapy appears mutually protective, there is also a need to sustain regular in-vitro testing where such a regimen is used so that true cross-resistance can be identified at an early stage Our results have implications for antimalarial prophylaxis and treatment strategies for residents and travellers to Viet Nam In addition, they may contribute to our understanding of the epidemiology of parasite drug resistance We have confirmed that artesunate clears malaria parasites quickly and that it is well tolerated Unfortunately the use of artesunate alone is not viable because of the high recrudescence rates seen with short treatment regimens Mefloquine is a drug with a significant side-effect profile and it is also relatively expensive Our findings suggest that it may be possible to take advantage of increased sensitivity of l? falciparum to chloroquine by using this drug in combination with artesunate as an alternative to mefloquine Although antagonism in vitro has been reported for chloroquine plus artemisinin (STAHEL et al., 1988), the effects were weak and therefore not likely to present a clinical problem Unless the antagonism was extreme and resulted in a significant drop in parasite clearance time, the combination would be likely to be beneficial (WHITE, 1998) Chloroquine-based prophylactic regimens might also currently afford protection in some endemic areas of Viet Nam for groups such as migrant workers, pregnant women and travellers Nevertheless, further studies are needed to determine the effectiveness of chloroquine in viva as part of combination therapy where parasite sensitivity appears to have been re-established Acknowledgements We thank MS Cath Barker, Dr Le Thi Nga and Dr Phillip Passmore of VAMCP and Dr Allan Schapira of WHO for their valuable assistance This study received financial support from AusAID (the Australian Agency for International Developmerit) References Barradell, L B & Fitton, A (1995) Artesunate A review of its pharmacology and therapeutic efftcacy in the treatment of malaria Drugs, 50, 714-741 Basco, L K & Le Bras, J (1993) In vitro activity of 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