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 IC50 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 IC50 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 . These observations may reflect a > 30 year history of artemisinin use in this region.
In western Cambodia, a steady increase in GM IC50 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 IC50 values from those recorded when CQ was the first-line treatment for P. falciparum infections [20, 21].
QN showed no remarkable trends in GM IC50 values, paralleling continued effectiveness of this 2nd line agent. For LUM, not widely used in Cambodia, increasing GM IC50 values in western Cambodia from 2006–2010, in parallel with trends in other drugs, possibly mirrored increases in MQ IC50s (discussed below). For PPQ, the clinical relevance of our preliminary in vitro IC50 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 . 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 IC50 values showed a steady increase from 2006 to 2010, and comparably high GM IC50 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 Pfmdr1 copy number, so the association of increased MQ GM IC50s with a recent description of reduced Pfmdr1 copy numbers in western Cambodia from 2005 to 2007 is unclear . The WHO 2010 Global Report suggests this molecular event was due to a switch in treatment policy from MQ-AS to DHA-PPQ ; if so, a continued rise in MQ mean IC50 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 .
For northern Cambodia, little is known about IC50 malaria drug susceptibility . Relatively high GM IC50 values in northern Cambodia in 2009 and 2010 are supported by antecedent GM IC50 values in eastern Thailand and western Cambodia [27–29]. In Thailand, GM IC50 values for CQ, MQ, QN and LUM increased from 2005 to 2007, an increase mirrored in nearby western Cambodia, starting in 2006. QN GM IC50 values remained below the WHO historic cut-off of 315 nM denoting "resistance", whereas CQ and MQ GM IC50 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 IC50 values for CQ, MQ, QN, AS, DHA and LUM were noted. However, GM IC50 values dropped in 2007, versus 2006, but then steadily increased from 2008 to 2010. It is unclear why GM IC50 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 IC50 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 . This perhaps illustrates the importance considering factors such as sampling bias when interpreting antimalarial drug IC50 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 . In this survey about 75% of HRP-2 IEV assays were successful, and W2 clone IC50 values for CQ, MQ, QN and AS were within expected ranges, providing context for falciparum isolate IC50 values. This bodes well for field based HRP-2 IEV assays. Moreover, GM IC50 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 IC50 ranges from 2001 to 2007 , 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 IC50 range reported here could reflect continuing emergence of artemisinin resistance, paralleling GM IC50 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 IC50 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 IC50 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 IC50 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.