HISTORICAL IMPACT OF ARTEMISININ
A precarious situation is looming in the ongoing battle against malaria: decreasing efficacy of what is presently our last clinically approved and universally effective antimalarial drug therapy. The current first-line treatments for uncomplicated Plasmodium falciparum malaria recommended by the WHO are artemisinin (ART)-based combination therapies (ACTs). The most common ACT, known by the trade name “CoArtem,” is composed of artemether (the ACT “parent drug,” Figure 1A), an ether form of the natural product drug ART, and lumefantrine (LF; the ACT “partner” drug, Figure 1B). Other commonly used ACTs include artesunate/mefloquine (AS/MQ), artesunate/amodiaquine (AS/AQ), and dihydroartemisinin/piperaquine (DHA/PPQ) (cf. Figure 1).
(A) Artemisinin-based drugs. Dihydroartemisinin, artemether, and artesunate are the most commonly used artemisinin combination therapy (ACT) “parent” drugs. (B) Partner drugs commonly used in current ACTs.
Citation: The American Journal of Tropical Medicine and Hygiene 101, 6; 10.4269/ajtmh.19-0379
(A) Artemisinin-based drugs. Dihydroartemisinin, artemether, and artesunate are the most commonly used artemisinin combination therapy (ACT) “parent” drugs. (B) Partner drugs commonly used in current ACTs.
Citation: The American Journal of Tropical Medicine and Hygiene 101, 6; 10.4269/ajtmh.19-0379
(A) Artemisinin-based drugs. Dihydroartemisinin, artemether, and artesunate are the most commonly used artemisinin combination therapy (ACT) “parent” drugs. (B) Partner drugs commonly used in current ACTs.
Citation: The American Journal of Tropical Medicine and Hygiene 101, 6; 10.4269/ajtmh.19-0379
The effectiveness of ART and its derivatives has been well documented since the late 1970s/early 1980s,1 and during the 1990s as resistance to quinoline and antifolate antimalarial drugs spread, ART-based drugs (Figure 1A) were introduced in Southeast Asia (SEA). Early reports on the use of ART monotherapy and combination therapy documented rapid parasite clearance by the drugs2,3; however, it was not until the late 1980s/early 1990s that formal ACT clinical trials were first completed in SEA4–8 and Africa (Table 1).9–12 In 2006, the WHO officially recommended ACTs as first-line therapy for falciparum malaria.13
Selected early clinical trials of artemisinin-based drug monotherapy and artemisinin combination therapies in Southeast Asia and Africa (from over 200 beginning 1982)
| Author | Publication year | Study site | Drugs tested | Funding | Reference |
|---|---|---|---|---|---|
| Jiang et al. | 1982 | Hainan Island, China | ART, MQ | Roche Far East Research Foundation (K. A.) and the Kevin Hsu Research Fund (Y. C. K.) | 2 |
| Li et al. | 1984 | Hainan Island, China | ART, ART/MQ,MQ/SP,ART/MQ/SP | Roche Far East Research Foundation | 3 |
| Myint et al. | 1987 | Myanmar | ATM, QN | – | 4 |
| Shwe et al. | 1988 | Myanmar | ATM/MQ, QN | – | 5 |
| Arnold et al. | 1990 | Vietnam | ART, QN | – | 6 |
| Bunnag et al. | 1991 | Thailand | AS | Atlantic Laboratories Corp., Ltd., Bangkok, Thailand | 7 |
| Looareesuwan et al. | 1992 | Thailand | AS/MQ | Mahidol University research grant and the Roche Research Foundation, Hong Kong. | 8 |
| White et al. | 1992 | Gambia | ATM, CQ | Wellcome Trust of Great Britain | 9 |
| Alin et al. | 1995 | Tanzania | AS | Swedish Agency for Research Cooperation with Developing Countries (SAREC) | 10 |
| von Seidlein et al. | 2000 | Gambia | AS/SP | UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases | 11 |
| Adjuik et al. | 2002 | Kenya, Senegal, Gabon | AS/AQ, AQ | WHO and Special Programme for Research and Training in Tropical Diseases | 12 |
AQ = amodiaquine; ART = artemisinin; AS = artesunate; ATM = artemether; MQ = mefloquine; SP = sulfadoxine-pyrimethamine.
Southeast Asia, and particularly the Greater Mekong Subregion (GMS), has historically been the epicenter for development of antimalarial drug resistance phenomena. Unsurprisingly then, because of widespread chloroquine resistance, this area was the first to adopt ACTs. Building from the success of clinical trials (see Table 1), Thailand adopted AS/MQ in the mid-1990s. Cambodia quickly followed suit in 2000, Laos adopted CoArtem (ATM/LF) in 2001, Myanmar began using several ACTs (ATM/LF, AS/MQ, and DHA/PPQ) in 2002, and Vietnam adopted DHA/PPQ in 2003 (Figure 2). This varied GMS use of ACTs follows a history of varied use of quinoline-based antimalarial drugs and others in previous decades. Troublingly, this region now witnesses increasing numbers of malaria patients infected with delayed clearance phenotype (DCP) parasites that show various levels of evolving artemisinin resistance (ARTR). Delayed clearance phenotype is defined via clinical criteria; non-DCP infections show profound (3–4 logarithmic) drop in parasitemia within ∼3 hours of ACT treatment, whereas DCP infections take a longer time (≥ 5–6 hours) to achieve a similar response. “Artemisinin resistance” is quantified in the laboratory typically via the “ring-stage survival assay” (RSA), as described in the following text.
Year of artemisinin combination therapy (ACT) use introduction vs. Greater Mekong Subregion countries. If multiple ACTs are listed for a particular year, they were all recommended country-wide. Current treatment regimens are artesunate/mefloquine (AS/MQ) (square), dihydroartemisinin/piperaquine (DHA/PPQ) (triangle), or artemether/lumefantrine (ATM/LF) (circle) in Myanmar; DHA/PPQ in Thailand; DHA/PPQ or ATM/LF in Laos; AS/MQ in Cambodia; and DHA/PPQ or AS/MQ in Vietnam.
Citation: The American Journal of Tropical Medicine and Hygiene 101, 6; 10.4269/ajtmh.19-0379
Year of artemisinin combination therapy (ACT) use introduction vs. Greater Mekong Subregion countries. If multiple ACTs are listed for a particular year, they were all recommended country-wide. Current treatment regimens are artesunate/mefloquine (AS/MQ) (square), dihydroartemisinin/piperaquine (DHA/PPQ) (triangle), or artemether/lumefantrine (ATM/LF) (circle) in Myanmar; DHA/PPQ in Thailand; DHA/PPQ or ATM/LF in Laos; AS/MQ in Cambodia; and DHA/PPQ or AS/MQ in Vietnam.
Citation: The American Journal of Tropical Medicine and Hygiene 101, 6; 10.4269/ajtmh.19-0379
Year of artemisinin combination therapy (ACT) use introduction vs. Greater Mekong Subregion countries. If multiple ACTs are listed for a particular year, they were all recommended country-wide. Current treatment regimens are artesunate/mefloquine (AS/MQ) (square), dihydroartemisinin/piperaquine (DHA/PPQ) (triangle), or artemether/lumefantrine (ATM/LF) (circle) in Myanmar; DHA/PPQ in Thailand; DHA/PPQ or ATM/LF in Laos; AS/MQ in Cambodia; and DHA/PPQ or AS/MQ in Vietnam.
Citation: The American Journal of Tropical Medicine and Hygiene 101, 6; 10.4269/ajtmh.19-0379
Delayed clearance phenotype initially observed in SEA does not represent “drug resistance” in the traditional use of the term. Antimicrobial drug resistance is typically defined by quantifiable shifts in the cytostatic or cytocidal potency of the antimicrobial drug; however, although there are exceptions, most studies since 2009 have shown that no significant and stable shifts in ART-based drug half-maximal inhibitory concentration (IC50) (cytostatic activity) or half-maximal lethal concentration (LD50) (cytocidal activity) occur for DCP parasites.14–17 However, in some cases, ARTR phenotypes that show IC50 or LD50 shifts have been derived in the laboratory.17–21 The most commonly used quantification of ARTR is the RSA, which relies on the outgrowth of young ring-stage parasites after a bolus dose of ART-based drug monotherapy.22 The use of the assay is still evolving. We use the term “evolving ARTR” to emphasize the aforementioned points, as also noted in the recent WHO definition of DCP.23
Full understanding of the emergence and spread of what are now known to be multiple examples of DCP (meaning resistances to ART-based drugs +/− resistance to various partner drugs used in ART-based ACTs), which have evolved from parasites exhibiting a variety of other drug resistance phenomena, requires analysis of the history of the use of ACT drugs within the GMS.
MECHANISMS OF EVOLVING ARTR
Non-synonymous mutations in the pfk13 gene that clearly cause reduced susceptibility to ART-based drugs in the laboratory14,24,25 have been associated with evolving ARTR within the GMS,24–27 making them a valuable marker for tracking the emergence and spread of ARTR/DCP. However, DCP parasites that do not harbor pfk13 mutations have also been observed,28–34 and, in Africa, correlation between the small number of DCP cases reported to date and pfk13 mutations is yet to be established.28–33 In vitro, pfk13 mutations are not essential for the development of ARTR.15,18–21,26,35–37 Also, in the field, not all PfK13 substitutions observed to date are associated with DCP; to date, a subset of about 20 have been defined as associated or confirmed resistance mutations,23,38–41 with C580Y being the most common. Taken together, these data paint a somewhat complicated picture for the molecular mechanism of evolving ARTR, with factors beyond PfK13 likely involved. Other mechanisms of ARTR, including non-PfK13, are discussed in more detail in other recent reviews.42–45 Nonetheless, within the GMS, PfK13-mediated evolving ARTR dominates in DCP parasites, facilitating an analysis of ARTR/DCP emergence and spread within the region.
MOLECULAR MARKERS PROVIDE IMPROVED SURVEILLANCE FOR DCP/ARTR
Clearly, the world is witnessing the development of new antimalarial drug resistance in real time, yet has the opportunity to effectively slow and perhaps even reverse its spread. With the identification of DCP in 2008, cost-effective and rapid whole-genome sequencing made rapid progress in isolating DCP molecular markers. A genome-wide association study (GWAS) identified a 35-kb region on chr13 as correlated with DCP in 91 samples from Cambodia, Thailand, and Laos in 2012,46 and a subsequent GWAS quickly identified specific single nucleotide polymorphisms (SNPs) positively associated with DCP.47
In 2014, Ariey et al.24 identified non-synonymous SNPs in a gene on chr13 that were candidates for causing DCP. In this work, an ARTR strain was created in the laboratory via stepwise drug selection over the course of 5 years. A point mutation in what was named “pfk13,” causing M476I substitution in the encoded PfK13 protein, was found and associated with increased parasite survival. Pfk13 is a single exon gene well conserved among Plasmodia spp. In P. falciparum, the gene encodes a 726–amino acid protein with three distinct domains, one being a Kelch propeller domain. Subsequent sequencing of DCP parasite field isolates did not find the same M476I mutation but found a number of other mutations (Y493H, R539T, I543T, and C580Y) in the PfK13 propeller region to be associated with ARTR as defined by the RSA.22 Some PfK13 mutations may not necessarily yield a clinically defined DCP27,33,38,48–50; however, in the large majority of cases to date, a subset of specific Kelch propeller mutations correlate with DCP.23,26,27,29,33,41 The endogenous function of PfK13 is unknown, but sequence similarity to the human Kelch-like ECH-associated protein 1 (KEAP1; a negative regulator of antioxidant response) suggests it could have a role in regulation of gene expression and intracellular transport.24 Alternatively, the PfK13 propeller domain shows some homology to KLHL2 and KLHL12 proteins, suggesting a role in ubiquitination and proteosomal degradation.24
Some GWASs and population genetics studies have compared the emergence and spread of different DCP populations. Not surprisingly, as with most genes associated with a drug resistance phenotype, additional minor pfk13 allele frequencies have been found in these studies. To date, there have been more than 200 non-synonymous SNPs identified in pfk13.51 Most are rare and may have arisen independently of ART drug pressure. When analyzing GMS pfk13 alleles, it is clear that a select few are dominant (see in the following text and Table 2). To elucidate the evolution of drug resistance, it is important to distinguish between mutation(s) that arose once and then spread throughout a region versus multiple unique examples of emergence of the mutation(s) within a region. Alleles that appear to have arisen multiple times in different regions of the GMS are C580Y (presently the most dominant mutation found in the field), R539T, and Y493H.26,27,37–39,49,52 Several GWASs have identified multiple different “founder populations.”26,27,37–39,49,52 Data suggest that there are ≥ 9 distinct founder populations for PfK13 C580Y: two originating in western Cambodia/eastern Thailand, one from northern Cambodia, one from Vietnam near the Cambodian border, and five originating along the Thai-Myanmar border.26,27,37–39,49,52 There are at least two distinct R539T populations: one that predominates in western Cambodia/eastern Thailand and one near the China–Myanmar border,26,37,38,49,52 and there are three Y493H subpopulations found in western Cambodia.26,27,37,38,52
Origins of common, WHO validated, resistance conferring pfk13 mutations
| Propeller mutation | First identification | Reference |
|---|---|---|
| F446I | Southeast Asia | 33 |
| N458Y | Cambodia | 24 |
| M476I | Laboratory | 24 |
| Y493H | Cambodia | 24 |
| R539T | Cambodia | 24 |
| I543T | Cambodia | 24 |
| P553L | Cambodia | 24 |
| R561H | Cambodia | 24 |
| C580Y | Cambodia | 24 |
Reverse genetic experiments have solidified the link between specific pfk13 mutations and DCP.15 PfK13 substitutions introduced into ARTS parasites not pressured with any ART-based drug caused various degrees of ARTR as measured by the RSA.14 These effects varied depending on the recipient strain of the parasite, suggesting that pfk13 mutations confer ARTR, but that other genetic factors modulate ARTR/DCP.14
HISTORY AND DECLINING EFFICACY OF ARTEMISININS
Tracing temporal and geographic patterns in the use of various ACTs provides insight into presently emerging patterns of ARTR/DCP. Since the early 1990s, ART monotherapy demonstrated unsatisfactory efficacy for long-term clearance of infection; parasite burden was reduced by orders of magnitude within a few hours; however, the short half-life of ART drugs often resulted in recrudescence of infections within a few weeks.7 Also, use of monotherapy was widely viewed as suboptimal6,53–56 because it would be predicted to promote ART-based drug resistance more quickly. It was hoped that use of ACTs would keep resistance at bay; unfortunately, despite early success and a widespread reduction in worldwide deaths due to infections, a harbinger of ARTR known as the DCP was identified in western Cambodia in 2006–2007.57 This study characterized prolonged parasite clearance times after AS monotherapy, and additional studies a year later suggested that DCP correlated with slightly elevated DHA IC50 (cytostatic activity) values,58 prolonged parasite clearance, and increased recrudescence.16
Consistent with infrequent changes in IC50 or LD50, most patients infected with DCP parasites still clear infections, particularly when treated with a combination regimen.28,31,33,48,59–61 The WHO suggests switching between ACTs if the proportion of patients who are parasitemic on day 3 is greater than 10% or if the proportion of treatment failure by day 28 or 42 is greater than 10%.23 Presently, these parameters only apply to five GMS countries: Cambodia, Thailand, Vietnam, Myanmar, and Laos (Figure 3). There have been a few reports of treatment failure rates > 10% in Africa; however, no African country is close to an overall 10% failure rate,28–32 as is also the case in India.61–63
Temporal spread of ARTR/DCP within the Greater Mekong Subregion (GMS). Delayed clearance phenotype originated in 2001–2002 in western Cambodia (solid white) and subsequently spread or emerged in different areas of the GMS. Delayed clearance was identified in Thailand and the Myanmar–China border in 2006–2007 (dashed white); Myanmar in 2009 (dotted white); northwest Thailand and Binh Phuoc, Vietnam, in 2010–2011 (double white); Preah Vihear, Cambodia, in 2011–2013 (dashed gray); North Tra My, Vietnam, and Chey Saen, Cambodia, in 2012–2013 (dotted gray); Thuan Bac, Vietnam, in 2012–2016 (dashed double white); central Myanmar in 2013–2014 (double gray); Gia Lai, Vietnam, and Campasak, Laos, in 2014 (solid gray); and the Savannakhet, Salavan, Sekong, and Attapeu provinces in Laos in 2015–2016 (dotted double white).
Citation: The American Journal of Tropical Medicine and Hygiene 101, 6; 10.4269/ajtmh.19-0379
Temporal spread of ARTR/DCP within the Greater Mekong Subregion (GMS). Delayed clearance phenotype originated in 2001–2002 in western Cambodia (solid white) and subsequently spread or emerged in different areas of the GMS. Delayed clearance was identified in Thailand and the Myanmar–China border in 2006–2007 (dashed white); Myanmar in 2009 (dotted white); northwest Thailand and Binh Phuoc, Vietnam, in 2010–2011 (double white); Preah Vihear, Cambodia, in 2011–2013 (dashed gray); North Tra My, Vietnam, and Chey Saen, Cambodia, in 2012–2013 (dotted gray); Thuan Bac, Vietnam, in 2012–2016 (dashed double white); central Myanmar in 2013–2014 (double gray); Gia Lai, Vietnam, and Campasak, Laos, in 2014 (solid gray); and the Savannakhet, Salavan, Sekong, and Attapeu provinces in Laos in 2015–2016 (dotted double white).
Citation: The American Journal of Tropical Medicine and Hygiene 101, 6; 10.4269/ajtmh.19-0379
Temporal spread of ARTR/DCP within the Greater Mekong Subregion (GMS). Delayed clearance phenotype originated in 2001–2002 in western Cambodia (solid white) and subsequently spread or emerged in different areas of the GMS. Delayed clearance was identified in Thailand and the Myanmar–China border in 2006–2007 (dashed white); Myanmar in 2009 (dotted white); northwest Thailand and Binh Phuoc, Vietnam, in 2010–2011 (double white); Preah Vihear, Cambodia, in 2011–2013 (dashed gray); North Tra My, Vietnam, and Chey Saen, Cambodia, in 2012–2013 (dotted gray); Thuan Bac, Vietnam, in 2012–2016 (dashed double white); central Myanmar in 2013–2014 (double gray); Gia Lai, Vietnam, and Campasak, Laos, in 2014 (solid gray); and the Savannakhet, Salavan, Sekong, and Attapeu provinces in Laos in 2015–2016 (dotted double white).
Citation: The American Journal of Tropical Medicine and Hygiene 101, 6; 10.4269/ajtmh.19-0379
Regardless, complacency is not a luxury that the infectious disease community can entertain; the fact remains that within the GMS, each country has experienced declining efficacy of one or more ACTs, and past history shows that, left unaddressed, the spread of antimalarial drug resistance from the GMS is inevitable. Evolving ARTR must be understood, and new combination therapies that halt the spread of DCP infections must be deployed.
Similar to the case for emergence of other resistance phenomena,64,65 Cambodia has been at the forefront in the emergence of ARTR/DCP and now appears to be in the most perilous situation of any GMS country. Artesunate/mefloquine was first replaced in western Cambodia (Pailin) in 2008 and then nationwide in 2010. Dihydroartemisinin/piperaquine not only filled the void temporarily but also was replaced in 2014 because of concerns over DCP and increasing PPQ resistance (PPQR).23,59,66,67 A national plan of action reintroduced AS/MQ as first-line treatment, despite high failure rates in some parts of the country. A major contributing factor in this decision was that five ACTs in Cambodia have been identified as failing, leaving a limited number of options.51 Coordinated use of new ACTs in this region is desperately needed.
Thailand used a 2-day regimen of AS/MQ until 2009 when the country then switched to a 3-day regimen.23 Around the same time, reports of AS/MQ treatment failures emerged.60,68,69 Mefloquine resistance in the area combined with evolving ARTR led to treatment failure rates > 10% for much of Thailand,69–71 and failure rates for ATM/LF, which was not a first-line therapy, rose to > 10% in some areas.23,72–75 Dihydroartemisinin/piperaquine was adopted as first-line treatment in 2015,23,72 despite a handful of treatment failures near the Cambodian border.66
Reports of failing efficacy for all three ACTs used in Myanmar have appeared since 2009.68,76–78 Whereas formal ACT failure rates in Myanmar are still well less than 10%, available data reveal DCP in a large portion of the country. Despite these warning signs, ATM/LF, AS/MQ, and DHA/PPQ remain recommended in Myanmar because few other options are presently available.
In the past few years, Laos has seen decreasing efficacy of ATM/LF.79 In 2013, towns bordering Cambodia and Thailand showed 22.2% of patients treated with ATM/LF were parasitemic on day 3.23 The overall rate for treatment failure in these bordering areas was 10%, with more interior areas of Laos showing lower treatment failure rates.23 In 2015, similar trends for ATM/LF were identified; areas that bordered Thailand and Cambodia showed loss of efficacy (defined either by day 3 parasitemia or 28-day recrudescence) of greater than 10%.23,29,38 In response, DHA/PPQ is presently being used in areas with documented ATM/LF failure. Unfortunately, some loss of DHA/PPQ efficacy has also recently been reported in southern Laos near Cambodia,23,38,80 which may correlate with increasing PPQR.38,59,80 An additional observation from this work is the key concept that some PfK13 mutations may have occurred multiple times, but only very few lineages with that mutation have survived.80 This perhaps suggests a yet to be determined connection among parasite genetic background, DCP-associated PfK13 mutations, and parasite fitness.81
Delayed clearance phenotype versus DHA/PPQ treatment in Vietnam was first noted in 2009.23 Binh Phuoc, which is in the southern part of the country and borders eastern Cambodia, showed greater than 10% loss of efficacy by 2015.23 Possible PPQR in this area has recently led Vietnam to introduce the use of AS/MQ.82,83
EVOLUTION OF DCP IN SOUTHEAST ASIA
Although DCP was identified clinically as early as 2006–2007,57 it is possible that ART-tolerant (or even ARTR) parasites existed even earlier. For example, in 2001–2002, 70% of the parasites in Pailin (western Cambodia) already harbored mutant pfk13, with greater than 40% of these encoding C580Y PfK13.24 Because Pailin is on the western border of Cambodia, it is likely that these parasites had infiltrated eastern Thailand at this time. Indeed, in 2006, parasite clearance half-lives were found to be increasing in western Thailand.70 Between 2006 and 2007, pfk13 mutations were also increasing in frequency in the same area,39 with > 25% of Kanchanaburi parasite samples, ∼20% of Prachuap, ∼20% of Ranong, and > 30% of Chumporn region samples harboring mutant pfk13.39 Another study using samples from Kanchanaburi and Ranong collected at a similar time identified an even higher frequency (∼75%) of pfk13 mutations.49 Eastern Thailand (Trat, Chanthaburi, and Sisaket) samples from the same studies showed > 50% of pfk13 mutations.39,49 Mutations were found 5 years earlier in neighboring Pailin24; however, pfk13 mutations were not common in northern Thailand at this time (Figure 3). Between 2006 and 2007, pfk13 mutations were also identified on the China–Myanmar border49; 50% of isolates from the Nabang-Lazan valley carried mutant pfk13, with ∼30% in neighboring Tengchong, China.49
Because DCP was first identified in western Cambodia, it is perhaps surprising that spread took several years to reach northeast Cambodia. Neighboring areas examined in 2011–2013 showed low frequency of mutant pfk13 (Figure 3)33; however, in 2013, 63% of isolates examined from Chey Saen in northeast Cambodia were positive for mutant pfk13.84 During this period, Laotian towns bordering Cambodia and Thailand reported that 22.2% of patients treated with ACTs were parasitemic 3 days after treatment.23 In 2014, pfk13 mutations were found in southern Laos (Champasak) at ∼90% frequency.38 Again, based on these data, it is likely that mutant pfk13 parasites were present in the area before 2014. Champasak is ∼200 km northeast of Preah Vihear and had identified low mutant pfk13 frequency in 2011–2013. At the same time, Attapeu, ∼100 km east of Champasak, showed low frequency (< 5%). Therefore, it appears that mutant pfk13 frequency steadily increased in this region during 2011–2014 (Figure 3).
Western Thailand showed increased pfk13 mutations in 2006–2007; therefore, it is possible that such parasites had invaded southern Myanmar at the same time. Indeed, in 2009, 33% of isolates collected from Kawthaung, Myanmar, were mutant pfk13.76 That same year, the WHO reported DCP in Vietnam.85 A closer examination of this time period shows that only ∼10% of Binh Phuoc (Vietnam) isolates were mutant pfk13; however, by 2010–2011, this had doubled to > 20%.50 Binh Phuoc is on the eastern Cambodia border (Figure 3), suggesting that pfk13 mutations had invaded eastern Cambodia near this time. Between 2010 and 2011, an increase in mutant pfk13 isolates was found in northwest Thailand.40 Previously, pfk13 mutations were only seen in western/southwestern Thailand.39,49 Tak and Mae Hong Son, which are more northern relative to the regions studied earlier,39,49 witnessed pfk13 mutation frequencies increasing to > 33% in 2010 for Tak and to ∼90% in Mae Hong Son by 2011.40
From 2011 to 2013, available data suggest that the spread of pfk13 mutations was confined as earlier. Other than areas bordering Thailand and China, most of Myanmar showed < 10% mutant pfk13 isolates.33,76 This correlates with reports that within Myanmar no ACTs were considered failing via the WHO definition, other than Artesunate/Sulfadoxine-pyrimethamine (SP) (likely due to SP resistance).64,86–90 Northeastern Cambodia also showed few mutant pfk13 isolates. Preah Vihear (west of the southern border with Laos) harbored ∼20% mutant pfk13 isolates, but Ratanakiri (northeastern Cambodia) and Attapeu (southeastern Laos) both showed < 5%.33 Additional clinical studies suggested that there was no DCP for Savannakhet (southern Laos), about 400 km northwest of Attapeu in 2010.91
Between 2012 and 2013, > 80% of isolates were identified as mutant pfk13 in the North Tra My district within central Vietnam (∼600 km north of Binh Phuoc, where mutant pfk13 had been present in 2009–2011),92 suggesting mutations were probably present earlier (this was the only time point during which isolates were collected). By 2014–2015, Gia Lai, just south of North Tra My, was confirmed to harbor parasites with pfk13.50 The period 2014–2015 was the earliest time point in this particular study as well, so it is likely that pfk13 mutations spread to this area before the beginning of the study (pfk13 mutations likely migrated north from Binh Phuoc and had already been identified in North Tra My by 2012–2013). A concurrent study (between 2012 and 2016) also identified high frequencies of pfk13 mutations in Gia Lai (76%) as well as Thuan Bac (15%), which is on the eastern shore of Vietnam.93 In addition, between 2013 and 2014, DCP parasites were identified throughout much of Myanmar.78,94 The Myanmar sites are located between those already confirmed to contain DCP parasites (bordering China and western Thailand, Figure 3) and show frequencies of pfk13 mutations between 24% and 67%.94
Unfortunately, between 2015 and 2016, Laos, which until this point had been relatively devoid of pfk13 mutations outside of Champasak, witnessed a northern migration of the DCP through the Attapeu, Sekong, Salavan, and Savannakhet provinces.95 This encompasses all areas 400–500 km north from the southern border. This same study also identified C580Y mutant parasites in the Phongsaly Province, on the Chinese border.95 Most recently,96,97 two studies have shown that ARTR/DCP is spreading more rapidly within Laos (as well as the rest of the GMS) than was initially perceived to be the case, with one of these studies97 also finding rapidly evolving dominance or “fixation” of parasite subgroups with specific genetic characteristics including pfk13 mutations conferring C580Y substitution within PfK13 protein and (for 3/6 subgroups) novel pfcrt mutations. These pfcrt mutations may be more relevant for PPQR than ARTR, suggesting that resistance to the popular DHA/PPQ ACT may now be profound within sections of the GMS as shown by van der Pluijm et al.96
Finally, presently, regions of GMS remain where mutant pfk13 frequency is at or near zero. Yala, which is the southernmost point in Thailand, is one example; however, a recent study suggests that parasites harboring pfk13 mutations may now be in the area.40,98 Ninh Thuan, on the eastern shore of Vietnam, showed ∼5% frequency as of 2015–2016,50 and, as previously mentioned, much of western Myanmar and northern Laos appear to have low frequency.33,91
FUTURE OUTLOOK
Proper distribution and usage of various ACTs is particularly urgent because of the aforementioned patterns in the spread of evolving ARTR/DCP malarial parasites. Presently, patients who show DCP are still able to clear infections with additional available treatment.3,28,33,48,55,56 Other ACTs show promise.99–107 Eliminating reservoirs of DCP parasites before additional spread would slow or halt the spread and additional evolution of ARTR.23,45 Spread of pfk13-mediated DCP to Africa would be catastrophic, as most deaths due to P. falciparum infections occur in sub-Saharan Africa. Understanding drug use histories as well as genetic, cellular, and molecular mechanisms of DCP will help shape future drug policies and the development of additional treatments, such as newer ACTs with new partner drugs and “triple combination” ACTs.99–101,103,105–116 With respect to the former, a number of next-generation antimalarials are presently being examined, including longer lasting synthetic ART-based drugs that have shown promise in the laboratory99,100,108 and the clinic,105–107 novel ACT partner drugs that are synergistic with presently used ARTs,101 and new classes of drugs with novel targets.109–115 With respect to the later, two promising clinical trials are presently ongoing that are examining the efficacy of triple ACTs in SEA, specifically DHA/PPQ/MQ103,116 and ATM/LF/AQ.116
All of these avenues must be pursued, rapidly, in coming years to contain and eliminate evolving ARTR/DCP. Two very recent reports suggest an alarmingly increased spread of resistance to both components of at least one presently popular ACT.96,97 The world must act to contain ARTR/DCP immediately.
Acknowledgments:
We thank our laboratory colleagues Laura Heller, Kalpana Iyengar, Bryce Riegel, and Anna Sternberg for their helpful conversations; the USP (United States Pharmacopeia, Rockville, MD) for financial support; the reviewers of the manuscript for their helpful suggestions; and the editor for additional helpful suggestions.
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