Monitoring of clinical efficacy and in vitro sensitivity of Plasmodium vivax to chloroquine in area along Thai Myanmar border during 2009-2010

Background In Thailand, the proportion of Plasmodium vivax infection has become equal to Plasmodium falciparum. Reports of a trend of gradual decline of in vitro sensitivity of P. vivax to chloroquine in some areas of the country, together with accumulating evidences of chloroquine resistance P. vivax in other parts of the world, emphasize the need for closely and continuously monitoring clinical efficacy in conjunction with in vitro sensitivity of P. vivax isolates. Methods The study was conducted at Mae Tao clinic for migrant workers, Tak Province during March 2008 - August 2009. A total of 130 patients (17 Thais and 113 Burmeses; 64 males and 66 females) with mono-infection of P. vivax malaria, aged between 15-60 years and weighing more than 40 kg, were included in the study. Patients received treatment with chloroquine (2,000 mg chloroquine phosphate over three days) and the anti-relapse drug primaquine (15 mg for 14 days). In vitro sensitivity of P. vivax isolates was evaluated by schizont maturation inhibition assay. Results All patients showed satisfactory response to treatment. The cure rate was virtually 100% within the follow-up period of 42 days. Neither recurrence of P. vivax parasitaemia nor appearance of P. falciparum occurred during the investigation period. In vitro data showed a stable sensitivity of chloroquine in this area since 2006. Geometric mean and median (95% CI) values of IC50 for chloroquine were 100.1 and 134.7 (1.1-264.9) nM, respectively. Conclusion In vivo results suggest that the standard regimen of chloroquine was still very effective for the treatment of blood infections with P. vivax in the Thai-Myanmar border area. In vitro sensitivity data however, raise the possibility of potential advent of resistance in the future. Regular monitoring of the chloroquine sensitivity of P. vivax is essential to facilitate the early recognition of treatment failures and to expedite the formulation of appropriate changes to the drug policy.


Background
Plasmodium vivax is responsible for approximately 70 to 80 million cases of malaria worldwide annually, and is the major cause of human malaria in parts of Pacific region and Central and South America [1]. The disease is rarely life-threatening, but morbidity from prolonged illness and the possibility of relapses from a persistent hepatic form (hypnozoite) are of major concern. Plasmodium vivax can only infect reticulocytes, which limits parasitaemia, usually to densities lower than 100,000/ml blood. The relapses can occurs weeks, months or years after initial exposure [2]. In Thailand, the proportion of P. vivax infection has now been increasing and has become equal to Plasmodium falciparum since 1998. On the western border of Thailand, the incidence of P. vivax has recently been reported as 20 per 1,000 population per year, similarly to that of P. falciparum [3]. The blood schizontocide chloroquine and tissue schizontocide primaquine have remained the mainstay chemotherapeutics for the treatment of P. vivax infection in Thailand for more than 60 years, with conserved clinical efficacy of virtually 100% [3][4][5]. To date, there has been no clinical-parasitological evidence of chloroquine-resistant P. vivax in Thailand. Nevertheless, a trend of gradual decline of in vitro sensitivity to chloroquine has been documented in some areas of the country, particularly Thai-Myanmar border [6,7]. Furthermore, the accumulating reports of chloroquine resistance P. vivax in other parts of the world during the past three decades including Papua New Guinea [8][9][10][11], Indonesia [12], Irian Jaya [13][14][15][16], Guyana South America [17], Peru [18], Colombia [19], India [20], Myanmar [21][22][23], Vietnam [24], Turkey [25], and Ethiopia [26] emphasize the need for closely and continuously monitoring clinical efficacy in conjunction with in vitro sensitivity of P. vivax isolates.
The objectives of the present study were to assess in vivo efficacy of first-line regimen of chloroquine given with primaquine, and in vitro susceptibility of P. vivax isolates in areas along the Thai-Myanmar border to chloroquine and the new antifolate WR99120.

Study site
The study was conducted at Mae Tao clinic for migrant workers, Tak Province during March 2008 -August 2009. Malaria is a serious imported medical problem in this area with a low and stable disease transmission with two seasonal peaks and forest-related during May-August and November-January of each year. Anopheles minimus and Anopheles dirus are the principal vectors. P. falciparum and P. vivax are the two predominant species with an incidence of 1:1, Plasmodium malariae is occasionally found and Plasmodium ovale is rare [3]. All age groups are affected and nearly all the P. vivax infections are symptomatic. The study was approved by the Ethics Committee of the Ministry of Public Health of Thailand.
Assessment of in vivo efficacy of chloroquine/primaquine Patient recruitment and follow-up A total of 130 patients (17 Thais and 113 Burmeses; 64 males and 66 females) with mono-infection with P. vivax malaria, aged between 15-60 years (target population) and weighing more than 40 kg were included in the study. Inclusion criteria included a parasitaemia of 1,000-100,000 parasites/μl blood, no signs of severe disease, no anti-malarial treatment during the preceding four weeks, no history of hepatic or kidney diseases. Written informed consent for study participation was obtained from all patients. All were admitted to the clinic during the three-day course of chloroquine and were requested to return for follow-up on days 7, 14, 21, 28, 35 and 42 after treatment initiation. Patients who developed fever or signs/symptoms of malaria were asked to return to the clinic for malaria blood examination.
On enrolment, each patient underwent a physical examination. A symptom questionnaire was also completed. Parasite counts, body temperature and blood for in vitro sensitivity test (1 ml blood collected into sodium heparinized plastic tube).

Evaluation of clinical efficacy
The efficacy of chloroquine when given with primaquine for the treatment of P. vivax malaria were assessed by (i) the proportion of patients with cure (cure rate), i.e., those who showed clearance of parasitaemia in the peripheral blood and absence of reappearance of parasitaemia within 42 days after treatment initiation, (ii) the parasite clearance time (PCT: the time taken for the parasite count to fall below the level of microscopic detection), and (iii) the fever clearance time (FCT: the time taken for the temperature to return to normal, i.e., < 37.3°C and remain so for at least 24 hours).

Treatment
Patients received treatment with chloroquine given concurrently with primaquine for eradication of hepatic stages of P. vivax parasites. Chloroquine (Government Pharmaceutical Organization of Thailand, 250 mg chloroquine phosphate per tablet) at a total dose of 2,000 mg given over 3 days (500 mg, four times at 0 and 6-12 hours on day-0, followed by 500 mg daily for two days) and primaquine (Government Pharmaceutical Organization of Thailand, 15 mg base given daily for 14 days starting from the second day of chloroquine treatment and then once daily until day-14). All drugs were administered with a glass of 250 ml drinking water under the supervision of medical staff. Subjects were closely observed for at least 30 minutes after each drug ingestion.
Patients whose parasitaemia was not cleared or those who had a reappearance of P. vivax were to be treated with the same regimen of chloroquine and primaquine but the dose of primaquine was to be increased to 20 mg. Patients who developed parasitaemia with P. falciparum during the investigation period were to be given a three-day treatment with oral artesunate (600 mg given in doses of 200 mg at 0, 6 and 24 hours) in combination with mefloquine (1,250 mg given as 750 and 500 mg at 6-8 hours apart).

Laboratory investigations
Following the first dose of drug administration, parasite counts (from finger-prick blood samples) were performed in all patients at six hourly intervals until two consecutive slides yielded negative results, and then during the follow-up period until day 42. Blood films were stained with Giemsa and examined by light microscope. Asexual stages of P. vivax were counted against 1,000 erythrocytes in thin blood films or against 200 white blood cells in thick films. The examination was reported as negative only after at least 200 fields of the thick film had been examined without encountering a parasite. Parasite species, morphology, and parasitaemia were assessed by microscopic examination.
In vitro drug sensitivity assay The schizont maturation assay was performed with P. vivax field isolates collected from all patients using a modified method of Russell and colleagues [27]. The concentration of serum added to the culture medium was increased from 10 to 30%. Plasmodium vivax field isolates were tested for their sensitivities against chloroquine and WR99210 at the concentration ranges of 0-10,000 nM for chloroquine (chloroquine phosphate: Liverpool School of Tropical Medicine, University of Liverpool, UK) and 0-2,560 nM for WR99210 (Jacobus Pharmaceutical Inc, Princeton, NJ, USA). The doseresponse curve was analysed by nonlinear regression analysis using CalcuSyn™software (Biosoft™, Cambridge, UK). The results were expressed as inhibitory concentrations (IC) 10, 50 and 90, which are defined as the concentrations of chloroquine or WR99120 producing 10, 50 and 90% inhibition of parasite development as compared to the control. Quality control of the assay was implemented by parallel determination of the IC 50 of chloroquine against chloroquine-sensitive and chloroquine-resistant P. falciparum clones.

In vitro sensitivity
In vitro sensitivity assay was performed in all of the 130 fresh P. vivax isolates collected; 32 (24.6%) with initial parasitaemia between 2,120 and 64,360 parasites/μl were successfully assessed for in vitro sensitivity to chloroquine and WR99120. All isolates completely developed to mature schizont within 20-40 h depending on dominant stage prior to testing. Despite the variation of the started parasite stages, abnormality in parasite morphology was observed in all wells exposed to the test drugs. Table 2 summarizes the IC 10 , IC 50 and IC 90 values of chloroquine and WR99120. Marked variation in IC 50 values were observed for both chloroquine and WR99120. Geometric mean and median (95% CI) values of IC 50 for chloroquine were 100.1 and 134.7 (1.1-264.9) nM, respectively. Based on the cut-off IC 50 of 100 nM used to classify chloroquine resistance in P. falciparum [28], 20 and 12 isolates, respectively exhibited IC 50 values of < and > 100 nM, respectively. Geometric mean and median (95%CI) values of IC 50 for WR99120 were 112.7 and 139.9 (0.2-523.0) nM, respectively.

Discussion
Difficulty in controlling vivax malaria is due to its biological characteristics of repeated pre-erythrocytic development due to relapse, with the maximum survival span of hypnozoites of five years [3]. Radical treatment of the infection, therefore, normally consists of a blood schizontocidal course of chloroquine and a course of primaquine for the elimination of the hypnozoites as antirelapse therapy [29]. In most parts of the world, chloroquine remains the first-line treatment for P. vivax infection in pregnant and non-pregnant individuals [1]. In the present study, all of the P. vivax patients enrolled in the study were satisfactorily treated with the standard regimen of chloroquine with primaquine. All responded well to treatment, with no reappearance of P. vivax parasitaemia (recrudescence or relapse) or appearance of P. falciparum in peripheral blood during the 42 days follow-up. There was no sign of delayed parasitological and clinical response. The PCT (30 h) and FCT (24 h) were similar to our previous observation in 2006 [5] and a recent observation [30] in the same area. Double infection with P. falciparum and P. vivax is common in certain malaria-endemic areas of Thailand. The occurrence of subsequent P. falciparum following treatment of P. vivax malaria has been reported to be less frequent than that of P. vivax after treatment of P. falciparum [31]. One possible explanation is the action of primaquine on pre-erythrocytic and eryhtrocytic forms of P. falciparum [32]. In addition, all patients stayed in malaria non-endemic area during the follow up period, which excluded the possibility of re-infection. So far, there has been no reported case of chloroquine resistant P. vivax in Thailand. Data from previous studies in different endemic areas including Thai-Cambodian and Thai-Myanmar borders during 1989 to 2011 [4][5][6][7]32] all indicated full sensitivity of P. vivax to standard dose of chloroquine. In a recent multicenter randomized, doubleblind, non-inferiority trial conducted in Cambodia, India, Indonesia and Thailand (Mae Sot and Mae Ramat), a 42-day cure rate of chloroquine given with primaquine was 100% [32]. Nevertheless, a recent clinical trial in Ethiopia confirmed resistance of P. vivax to chloroquine with a 28-day cure rate of 7.5% [33]. Reappearance of P. vivax parasitaemia beyond day 28 which is suggested to be due to true relapse due to primaquine failure [29], and the relapse rates within 1-6 months were reported to be 5-18% in adult patients both in Thailand and in other tropical areas [14,29]. Clinical effectiveness of primaquine as an anti-relapse and patients' compliance could not be evaluated in this short follow-up investigation period.
The in vitro sensitivity data based on schizont maturation inhibition test [27] demonstrated more or less the stability of sensitivity of P. vivax isolates in this area of Thailand to chloroquine [geometric mean IC 50 of 100.1, median (95%CI) of 134.7 (1.17-264.9) nM] since 2002 [27,34]. Sensitivity of P. vivax to chloroquine was shown to be increased by about 2-fold (IC 50 : from 131 to 71 nM) in the presence of primaquine, which also possesses direct blood schizontocidal activity [5]. It was noted that the IC 50 of chloroquine in P. vivax was about 2-fold of that of P. falciparum, but the variation is probably higher with P. vivax [35]. A previous study conducted in the same area for monitoring of in vitro susceptibilities and molecular markers of resistance of P. falicparum isolates to chloroquine, quinine, mefloquine and artesunate [35] showed the reversed sensitivity of chloroquine after a period of about 40 years withdrawal from first-line treatment for falciparum malaria, with median (95% CI) IC 50 of chloroquine of 73 (10-164) nM. One (4%), 19 (73%) and 6 (23%) isolates were classified as chloroquine-sensitive, moderately resistant and highly resistant P. falciparum, respectively. In other studies, about 3-4 fold higher IC 50 of chloroquine was observed in P. vivax compared with P. falciparum [35,36]. This may imply intrinsic characteristic (innate resistance) of P. vivax in response to chloroquine. The in vitro cut-off value defining clinically relevant chloroquine resistance in P. vivax malaria has yet to be clearly defined. For P. falciparum, cut-off IC 50 of 100 nM was used to define chloroquine resistance. Suwanarusk and colleagues [37] defined the cut-off IC 50 of 220 nM based on the 35 th percentile of the clinical failure rate of 65% observed in Indonesian patients with P. vivax malaria. In the same study [37], the IC 50 of chloroquine in Thai isolates collected from Mae Sot District, Thai-Myanmar border (same area as the present study) was also found to be significantly lower than that from Indonesian isolates (geometric mean IC 50 of 312 vs 46.8 nM). Eleven out of 81 Thai isolates (13.6%) exhibited IC 50 of chloroquine over 220 nM. Based on this criteria, six out of 32 isolates (18.8%) observed in the present study showed IC 50 of greater than 220 nM. The current in vivo and in vitro results suggest that chloroquine is still an effective firstline treatment for P. vivax in Thailand. Resistance level may remain obviously below the threshold of detectability by the in vivo method. It is noted that definitive  conclusion on the efficacy of chloroquine is not appropriate since chloroquine was given with primaquine, and the fact that the study design did not include control arm with chloroquine monotherapy due to ethical reason. Regular monitoring of the chloroquine sensitivity of P. vivax is essential as to facilitate the early recognition of treatment failures and to expedite the formulation of appropriate changes to the drug policy. Alternative treatment options for P. vivax infection in case of chloroquine resistance may include a three-day course of quinine given concurrently with primaquine [5] or artemisinin combination therapy [38]. Various in vitro assay systems with different endpoint criteria have also been applied for monitoring of sensitivity of P. vivax isolates to anti-malarial drugs. Direct comparison of in vitro sensitivity data using different methods should however, be interpreted with caution. Since P. vivax infection is predominantly asynchronous, the microscopic method based on inhibition of parasite's growth previously developed by our group [39] is considered the best-suited method for assessing sensitivity of P. vivax to anti-malarial drugs [40]. The test method based on schizont maturation inhibition used in the present study, although may be less accurate, but the method is extremely less labour-intensive, more applicable for field studies and does not require expensive or dangerous reagents (monoclonal antibodies or radioisotopes). Unluckily, the success rate of in vitro sensitivity test observed in the current study was relatively low (24.6%), which is possibly due to variation in asynchronicity of parasite isolates in this area. Russel et al [41] demonstrated the marked stage-specific activity of chloroquine with variable growth rates. Isolates initially at the trophozoite stage had significantly higher chloroquine IC 50 values than those initially at the ring stage. Synchronous isolates which reached the target of 40% schizonts in the control wells within 30 hours had significantly higher geometic mean IC 50 of chloroquine. In vitro susceptibility was found to be correlated with initial stage of the parasite, with isolates predominantly at the trophozoite stage having a 2-fold increase in IC 50 values compared to those of parasites predominantly at the ring stage [41].
The spread of chloroquine resistance in P. falciparum has led to the use of the antifolate combination sulphadoxine-pyrimethamine (SP) as the first-line drug for malaria treatment in several countries including Thailand, where P. vivax and P. falciparum often co-exist and occur at approximately equal frequencies [3]. In vitro sensitivity of WR99210, a novel inhibitor of enzyme dihydrofolate reductase (DHFR) was assessed in our study in view of previous reports showing its promise as a possible treatment of P. vivax malaria. The drug shows activity against the most pyrimethamine-resistant P. falciparum strains and extremely effective inhibitor of the P. vivax DHFR including mutations that confer high-level resistance to pyrimethamine [42,43]. Median (95% CI) IC 50 of WR99120 in P. vivax isolates collected in the present study was 139.9 (0.21-523.0) nM. The relatively poor in vitro susceptibility of P. vivax to WR99120 observed was similar to our previous observation in the same area [44], could be explained by the slow action of this drug and/or the innate resistance as well as the presence of p-aminobenzoic acid and folate in the media used which acted as competitive antagonists of antifolate activity [45].
The combination of new antifolates, like WR99210, that are effective against SP-resistant parasites, with appropriate partners, may also play an important role in a rational drug treatment strategy.

Conclusions
Chloroquine is still sufficiently effective for blood schizontocidal therapy in areas along the Thai-Myanmar border. In vitro sensitivity data however raise the possibility of potential advent of resistance in the future. Regular monitoring of chloroquine sensitivity of P. vivax is essential.