Background
Since the 1980s, measuring
in vitro drug 50% inhibitory concentrations (IC
50) against
P. falciparum field isolates has been useful in tracking clinical drug susceptibility patterns [
1‐
3]. In Southeast Asia, especially along the Thailand-Cambodia border, changes in drug susceptibility often emerge first, with worldwide implications, underscoring the region’s importance as a sentinel site for anti-malarial drug resistance [
3].
In 2003, artesunate (AS) + mefloquine (MQ) was implemented as the first-line artemisinin-combination therapy (ACT) for falciparum malaria in Cambodia. By 2005, subjects with falciparum malaria along Thai-Cambodia border treated with oral artesunate were showing longer parasite clearance times, suggesting the emergence of reduced susceptibility to artemisinins, as well as to the partner drug [
4].
The non-radioisotope histidine-rich protein-2 (HRP-2) ELISA, a relatively sensitive assay, which reliably depicts drug IC
50 values for
P. falciparum isolates, reduces obstacles for conducting assays in remote settings [
5,
6]. HRP-2 assays are expedient, safer and less costly than radioisotope assays. HRP-2 assay, when field deployed proximal to
P. falciparum collection sites, allows for “immediate
ex vivo” (IEV) field isolate processing [
7]. IEV, by avoiding cryopreservation and culture-adaptation before IC
50 determination, may reduce clonal selection, better preserving parasite subpopulations with variable drug susceptibility profiles, the latter likely present in a smaller proportion than wild-type drug susceptible parasites.
Here, to characterize recent geographical and temporal trends in western and northern Cambodia, and eastern Thailand, including years when reduced artemisinin susceptibility was first described, an HRP-2 assay, with IEV field isolate processing was used to determine IC50 values of P. falciparum field isolates obtained from 2005 to 2010.
Discussion
Among P. falciparum field isolates obtained in western Cambodia from 2005 to 2010, steady increases were observed for GM IC50 values measured by a HRP-2 in vitro assay against a range of anti-malarial drugs, including AS and DHA. Moreover, in northern Cambodia, assessed in 2009 and 2010, most GM IC50 values approximated those in western Cambodia, during the same period. This supports the notion that western Cambodia is associated with sustained and likely progressive reductions in anti-malarial drug susceptibility, with possible spread to northern Cambodia.
As the same HRP-2 IEV assay was used for all
P. falciparum field isolates, increasing GM IC
50 values for AS and DHA may reflect possible emerging artemisinin resistance, first described in subjects in western Cambodia in 2005 [
7,
17,
18]. Statistically significant increases in GM IC
50 values for DHA in Trat Province, Thailand, adjacent to the Cambodian border, from 2005 to 2007, coincided with declining efficacy of AS + MQ in Thailand [
19]. These observations may reflect a > 30 year history of artemisinin use in this region.
In western Cambodia, a steady increase in GM IC
50 values for CQ from 2006 to 2010 was observed. This may reflect continued drug pressure due to its use for
P. vivax blood stage treatment, as well as widespread unregulated availability. This is in contrast to the situation in parts of Africa, where the introduction of ACTs and withdrawal of CQ use has led to a reduction in mean IC
50 values from those recorded when CQ was the first-line treatment for
P. falciparum infections [
20,
21].
QN showed no remarkable trends in GM IC
50 values, paralleling continued effectiveness of this 2nd line agent. For LUM, not widely used in Cambodia, increasing GM IC
50 values in western Cambodia from 2006–2010, in parallel with trends in other drugs, possibly mirrored increases in MQ IC
50s (discussed below). For PPQ, the clinical relevance of our preliminary
in vitro IC
50 data remains unclear. Of note, 15% of subjects in two recent malaria treatment trials in Cambodia had detectable pre-treatment PPQ levels, suggesting increasing drug pressure [
22]. Continued
in vivo and
ex vivo monitoring of PPQ, the recently adopted first-line partner drug for DHA in Cambodia, is of paramount importance.
In western Cambodia, MQ GM IC
50 values showed a steady increase from 2006 to 2010, and comparably high GM IC
50 values were observed in northern Cambodia in 2009–2010; all were well above the proposed WHO historical cut-off indicative of clinical MQ monotherapy “resistance”, paralleling reduced clinical efficacy of MQ in this region [
23,
24]. The cause of reduced MQ sensitivity (and possibly LUM) is generally accepted to be related to increased
Pfmdr 1 copy number, so the association of increased MQ GM IC
50s with a recent description of reduced Pfmdr1 copy numbers in western Cambodia from 2005 to 2007 is unclear [
25]. The WHO 2010 Global Report suggests this molecular event was due to a switch in treatment policy from MQ-AS to DHA-PPQ [
4]; if so, a continued rise in MQ mean IC
50 values to 2010 is potentially concerning. As before, unregulated availability of antimalarial medications during that period may have been a contributing factor. Notably, MQ-AS still appears to retain high efficacy despite reduced
in vitro sensitivity to both drugs, shown most recently in a trial comparing mefloquine-artesunate with pyronaridine-artesunate [
26].
For northern Cambodia, little is known about IC
50 malaria drug susceptibility [
27]. Relatively high GM IC
50 values in northern Cambodia in 2009 and 2010 are supported by antecedent GM IC
50 values in eastern Thailand and western Cambodia [
27‐
29]. In Thailand, GM IC
50 values for CQ, MQ, QN and LUM increased from 2005 to 2007, an increase mirrored in nearby western Cambodia, starting in 2006. QN GM IC
50 values remained below the WHO historic cut-off of 315 nM denoting "resistance", whereas CQ and MQ GM IC
50 values rose above WHO historic cut-off values, and DHA showed marked increases in both countries. These trends followed drug policies during the survey period, with AS-MQ the first-line regimen for
P. falciparum, and CQ as the first-line agent for blood stage
P. vivax in both Thailand and Cambodia.
In western Cambodia, measured annually 2006–2010, statistically significant, relatively steady increases in GM IC
50 values for CQ, MQ, QN, AS, DHA and LUM were noted. However, GM IC
50 values dropped in 2007, versus 2006, but then steadily increased from 2008 to 2010. It is unclear why GM IC
50 decreases occurred in 2007 only. Review of data from 2007 showed a smaller sample size (n = 26) compared to other years, although the location of sampled sites was unchanged. Interestingly Lim
et al. observed a similar dip in IC
50 values for a range of antimalarial drugs in 2006, and attributed the observation to sampling bias since most samples that year were collected from a single, new field site; the data was included in the report for completeness and so that future comparisons could be made [
27]. This perhaps illustrates the importance considering factors such as sampling bias when interpreting antimalarial drug IC
50 surveys.
HRP-2 assays with IEV isolate processing conducted at field laboratories, which are simpler techniques than radioisotopic assays and culture-adaptation, may also reduce clonal selection and better preserve sub-populations of susceptible and potentially drug resistant parasites [
11]. In this survey about 75% of HRP-2 IEV assays were successful, and W2 clone IC
50 values for CQ, MQ, QN and AS were within expected ranges, providing context for falciparum isolate IC
50 values. This bodes well for field based HRP-2 IEV assays. Moreover, GM IC
50 values reported here generally paralleled other published data from eastern Thailand and Cambodia, which assayed
P. falciparum isolates by
3 H-hypoxanthine uptake [
28,
30]. For example, a comparison of western Cambodia GM IC
50 ranges from 2001 to 2007 [
27], with these results were, respectively (nM): CQ (131–237 vs. 96–242), MQ (12–57 vs. 7–58), QN (94–302 vs. 53–163), and AS (0.6-1.8 vs.5.1-6.8). The higher AS GM IC
50 range reported here could reflect continuing emergence of artemisinin resistance, paralleling GM IC
50 increases for AS and DHA from 2008 to 2010. Alternatively, it might reflect methodological differences, which affect drug or parasite behaviour, illustrating the importance of inter-assay consistency.
Trends in IC
50 values, coupled with molecular marker assays, help track emergence and spread of anti-malarial drug resistance. For the artemisinins, although associations between validated resistance markers, treatment outcome and IC
50 susceptibility remain elusive [
18,
31]., in part because the clinical definition of artemisinin “resistance” is unclear; the converse is not true for partner drugs, such as MQ and CQ, which have established thresholds of resistance and validated molecular markers. Nonetheless, determining AS and DHA GM IC
50 values in a substantial number of
P. falciparum isolates from 2005 to 2010 establishes a baseline which may be useful for future comparisons if evidence of reduced artemisinin susceptibility grows, and especially if the methodology remains consistent.
Acknowledgments
We thank AFRIMS and Cambodian clinical and laboratory field teams for their support. This work was supported by numerous organizations. Training of medical staff, capacity building and facility upgrades were provided by Global Emerging Infections Surveillance (GEIS) Program, US Department of Defense. Additional funding for some of the work was received from US Department of Defense Global Emerging Infections System, Silver Spring, Maryland, USA, Bill & Melinda Gates Foundation World Health Organization (via World Health Organization), Medicines for Malaria Venture (MMV), and US Army Military Infectious Disease Research Program (MIDRP) and US Army Medical Materiel Development Activity (USAMMDA), Fort Detrick, Maryland, USA. We thank Ms. Tippa Wongstitwilairoong for making the map.
The opinions and assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the U.S. Department of the Army.
Presented in part at 2010 Annual Meeting of the American Society of Tropical Medicine and Hygiene, Atlanta, GA USA.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
Study designs, oversight, data interpretation, manuscript preparation: CL, ST, DS (Dr Socheat), YS, HN, DB, KS, WR, WS, MF, SC (Dr. Suwanna), AT, DS (Dr Saunders), DW Subject recruitment, interactions: DS (Dr Sea), YS, SC (Ms. Chann), NB, SS (Ms Sabaithip) Conducted experiments, summarized data: ST, KS, WR, SC, KY, SS (Ms Siratchana), PC. All authors read and approved the final manuscript.