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‐
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‐
11], Indonesia [
12], Irian Jaya [
13‐
16], Guyana South America [
17], Peru [
18], Colombia [
19], India [
20], Myanmar [
21‐
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.
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‐
7,
32] all indicated full sensitivity of
P. vivax to standard dose of chloroquine. In a recent multicenter randomized, double-blind, 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 first-line 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.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
PM performed all the laboratory analysis. RR participated in patients' recruitment and sample collection. WC performed data analysis. KR participated in in vitro sensitivity analysis. KN participated in the design of the study, manage the study and finalize the manuscript. All authors read and approved the final manuscript.