Updates on antimalarial drug efficacy and resistance surveillance in the Americas Document for information REGIONAL MALARIA PROGRAM 2019, PAHO/WHO

This document was prepared as a pre-read for the meeting of the PAHO Malaria Technical Advisory Group and is not an official document of PAHO/WHO

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CONTENTS 1 Background ........................................................................................................................................... 2

1.1 Definitions ..................................................................................................................................... 2

1.2 Artemisinin partial resistance ........................................................................................................ 2

2 Responding to declines in drug efficacy ............................................................................................... 3

2.1 Monitoring the therapeutic efficacy of ACTs (WHO recommendations) ..................................... 3

2.2 Strategic approach for the surveillance of resistance in the Americas .......................................... 3

2.2.1 Monitoring the therapeutic efficacy of the antimalarials in the Americas ............................ 3

2.2.2 Surveillance in the Guiana Shield, PfK13 mutation and its presence in the Region ............ 5

2.2.3 pfcrt 76T polymorphism in the Region (resistance to CQ) ................................................... 6

2.3 Monitoring antimalarial resistance in P. vivax .............................................................................. 7

3 Country updates on antimalarial resistance 2017-2019 ........................................................................ 7

3.1 Updates on ACT efficacy in P. falciparum (TES) ........................................................................ 7

3.1.1 Colombia: .............................................................................................................................. 7

3.1.2 Guyana .................................................................................................................................. 8

3.2 Update on therapeutic efficacy in P. vivax.................................................................................. 10

3.2.1 Brazil ................................................................................................................................... 10

3.3 Updates on surveillance based on molecular markers ................................................................ 10

3.3.1 Central America and Hispaniola Island .............................................................................. 10

3.3.2 South America..................................................................................................................... 11

4 MAIN POINTS ....................................................................................................................................... 12

5 NEXT STEPS .......................................................................................................................................... 12

6 REFERENCES......................................................................................................................................... 13

7 ANNEX 1............................................................................................................................................... 14

Drug efficacy studies for assessing antimalarial drug resistance conducted in RAVREDA/AMI, 2002 – 2018. ....................................................................................................................................................... 14

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1 Background The Plan of Action for the Elimination of Malaria 2016-2020 (1) has five components, one of them to ensure universal access to malaria prevention, diagnosis and treatment, highlighting the importance of timely diagnosis and effective treatment as key element for the reduction of malaria morbidity and mortality. The success of this component is based on the ability of the malaria programs at the ministries of health to provide effective antimalarial drugs. Given the history of the region in emergence and spread of antimalarial resistance and the existence of risk factors related to the use of antimalarial drugs in the endemic areas in the Americas, surveillance of resistance should be a key element of malaria programs in the Region and mainly in South America where resistance emerged in the past and P. falciparum continues to be a challenge.

1.1 Definitions Antimalarial resistance is defined as the ability of a parasite strain to survive and/or multiply despite the administration and absorption of a drug given in doses equal to or higher than those usually recommended but within tolerance of the subject. Multidrug resistance (MDR) is defined as resistance to more than two antimalarial compounds of different chemical classes. Treatment failure is defined as the inability to clear malarial parasitaemia or prevent recrudescence after administration of an antimalarial medicine, regardless of whether clinical symptoms are resolved. Many factors can contribute to treatment failure, including incorrect dosage, poor patient compliance, poor drug quality, and drug interactions and resistance. Most of these factors are addressed by therapeutic efficacy studies (TESs).

1.2 Artemisinin partial resistance Artemisinin resistance is defined as delayed parasite clearance; it represents a partial resistance that has affected only ring-stage parasites thus far. Nevertheless, the majority of patients who have delayed parasite clearance are still able to clear their infections following treatment with an ACT with an effective partner drug or with an artesunate treatment lasting seven days. An analysis of P. falciparum parasites selected for artemisinin resistance in vitro as well as parasites from patients experiencing slow parasite clearance led to the identification of a genetic locus linked to the current artemisinin resistance profile: a kelch domain-containing protein located on chromosome 13 (pfk13) (9). To date, many mis-sense mutations have been described in the BTP/POZ or kelch propeller domain-containing regions this protein in clinical isolates, however only nine of them have been associated with delayed parasite clearance (F446I, N458Y, M476I, Y493H, R539T, I543T, P553L, R561H and C580Y) (8,9,10). The identification of the K13 mutations as markers for artemisinin resistance has allowed for a more refined definition of partial artemisinin resistance that includes information on the genotype. More than 200 non-synonymous mutations in the pfK13 gene have been reported. Distinct alleles originating from multiple independent emergence events have been observed in South-East Asia. The KARMA project has reported frequent C580Y, R539T, Y493H and I543T mutations in the eastern GMS (Cambodia, Lao PDR and Viet Nam) and frequent F446L, N458Y, P574L and R561H mutations in the western GMS (China, Myanmar and Thailand). The P553L allele has been found in the two areas. There is

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evidence that selective sweeps have occurred throughout the GMS. Currently, the K13 C580Y mutation can be found in several genetic backgrounds (haplotypes) throughout the GMS. The prevalence of one specific K13 C580Y haplotype is increasing, replacing other haplotypes in an area that includes sites in western Cambodia, north-eastern Thailand and southern Lao PDR. This indicates a selective sweep in this part of the GMS. However, the frequencies of different K13 C580Y haplotypes vary by region, and no single haplotype is dominant throughout the GMS. Figure1. Distribution of C580Y mutations worldwide

2 Responding to declines in drug efficacy

2.1 Monitoring the therapeutic efficacy of ACTs (WHO recommendations) TES studies are the standard method of determining the efficacy or inefficacy of antimalarial drugs. TES results for ACTs used in the treatment of P. falciparum allow for the determination of:

• the proportion of patients who are parasitemic on day 3, which is currently the indicator of choice for routine monitoring to identify suspected artemisinin partial resistance in P. falciparum;

• the proportion of treatment failure by day 28 or 42 (days of follow-up is determined according to the half-life of the ACT partner drug).

A change in the national malaria treatment policy should be initiated if the total treatment failure rate is ≥10%, as assessed through TESs. NMPs should adopt antimalarial medicines with a parasitological cure rate greater than 95% If artemisinin resistance is suspected due to the observation of slow clearance in a clinical trial or TES, K13 marker analysis should be prioritized, e.g., from filter paper blood spots. If resistance is suspected based on a survey with molecular data only, resistance should be confirmed by obtaining information on both the clinical phenotype (delayed parasite clearance) and the K13 genotype from the same parasite strain.

2.2 Strategic approach for the surveillance of resistance in the Americas

2.2.1 Monitoring the therapeutic efficacy of the antimalarials in the Americas

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In the Americas, the majority of national malaria control programs (NMCPs) used data from in vivo studies to evaluate the efficacy of first- and second-line drugs, and to decide whether malaria treatment policies need to be changed. The protocol most commonly used for monitoring antimalaria resistance, follows the WHO guidelines (2), which were revised and include modifications recommended by PAHO for studies in the Americas (3). The objective of the studies have been to evaluate antimalarial drugs currently being used as the first and second lines of treatment for uncomplicated malaria caused by P. falciparum, as well as for P. vivax treatment, and to compare their efficacy with that of possible alternative treatments. Such data have been fundamental in guiding the development of policy on the use of antimalarial drugs in endemic areas. During 2001-2006 the Amazon countries, supported by PAHO and USAID (Amazon Malaria Initiative) created the RAVREDA network that supported the realization of TES studies that led to consolidate the introduction of ACTs in the region by 2006. Annex 1 summarize the studies done by the Amazon countries on that period. Since the introduction of ACT, PAHO in coordination with WHO, had continued promoting within the Region the surveillance of antimalarial resistance, not only for ACT, but also for CQ in P. falciparum in Central America as well as the surveillance of resistance in P. vivax. To ensure that the treatments recommended in the national treatment policies are efficacious, PAHO/WHO recommends that malaria-endemic countries perform routine monitoring of antimalarial drug efficacy at sentinel sites at least once every 24-36 months in order to detect changes in therapeutic efficacy (for P. falciparum policies). Areas for which there is evidence of resistance, should consider adding more sentinel sites in order to facilitate early detection of new resistance foci. Table 1 summarize results from the TES studies done in the Region from 2010-2014 after the introduction of ACT.

Table 1. Summary of treatment failure rates among patient infected with P. falciparum, grouped by country and treatment (4)*

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With the support from PAHO/WHO Collaborating Centres (CDC, Atlanta, USA, and Institute Pasteur, Cayenne, French Guiana), in coordination with National reference laboratories and research institutions in the countries, the Region has also implemented the use of known and validated molecular markers such as the pfK13, pfcrt, pfmdr1, pfdhfr, and pfdhps to determine the state of antimalarial drug resistance in specific situations. Molecular markers have also served as additional information to that provided by in vivo studies and as an alert system. Table 2 shows how tools are being implemented according to the strategic approach with member states. Table 2: How to use the available tools according to the epidemiological situation

Surveillance with

High or Moderate transmission

Low or no transmission (elimination)

In vivo Up to 8 sentinel sites Every 3 years One arm

One site at least every 3 years Multicentre studies* One arm

Molecular markers Concomitant with in vivo studies Collection in hotspots for possible selection of resistance

Collect samples every 18 months Collection in hotspots for possible selection of resistance

In vitro (ELISA) Concomitant with in vivo studies when requested

*in places within a country or between countries that have similar epidemiological features.

2.2.2 Surveillance in the Guiana Shield, PfK13 mutation and its presence in the Region Guiana Shield are among the most challenging settings for malaria control and elimination in the Americas because of their inaccessibility, highly mobile populations, lack of formal health facilities, and risk for emergence of antimalarial resistance that is associated with risk factors related mainly with gold mining activities. Routine surveillance of ACT efficacy between 2005–2006 and 2011 among gold miners in Suriname showed an increase in the day-3 positivity rate (from 2% to more than 20%), with a high cure rate at day 28. There were however concerns regarding the quality of the microscopy conducted. In 2013–2014, a study using artesunate and mefloquine did not confirm the high day-3 positivity rate, and the sequencing of the K13 strains collected during this study revealed only wildtype K13. First time pfK13 mutation C580Y was identified in our region was in Guyana, during a retrospective analysis of blood samples collected in 2010 for a Pfhrp2 surveillance study by Chenet S.M. et al. 2010 (6). All five pfK13 C580Y mutant samples detected had nearly identical haplotypes, suggesting a common origin distinct from the South-East Asian K13 C580Y haplotype. Actions taken: In 2014 a confirmatory in vivo study conducted in Guyana with 7 days Artesunate (4 mg/kg/day)

+ primaquine single dose. n = 50 (26% from zone 1; 54% zone 7; 16% zone 8). The study showed 100% efficacy at day 28 (Table 1), whereas only 2% of the patients had persistent parasitaemia on day 3 after treatment. The 47 strains collected all showed wild-type K13.

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During end of 2016 to beginning of 2017, samples on paper filter were collected for P. falciparum malaria positive patients as part of an alert system based on PAHO/WHO guidance. A total of 854 samples were analysed in collaboration with the WHO Collaborating Centre (WHO CC) Institute Pasteur in Cayenne, French Guiana, showing an overall of 1.6% of pfK13 mutant C580Y in Guyana [CI95: 0.9-2.9], mutation was found mainly in Region 1: 8.8% [CI95: 3.6-15.2], then Region 8 (0.7%) and Region 7 (0.5%) (Figure 2). It was confirmed that the Pfkelch 13 C580Y variant arose on a single Guyanese haplotypic background, and was not imported from South-East Asia.

In 2017 the TAG was informed about these findings and on the decision of performing a new TES study in Guyana, following PAHO/WHO procedures with samples from regions 1, and Georgetown to confirm efficacy of first line treatment currently in use in the country.

Figure 2. Prevalence of Pfkelch13 C580Y by region in Guyana

N C580Y % mutant Region 1 114 10 8.8 Region 2 2 0 0 Region 3 10 0 0 Region 7 572 3 0.5 Region 8 150 1 0.7 Region 9 4 0 0 Region 10 2 0 0 Total 854 14 1.6

2.2.3 pfcrt 76T polymorphism in the Region (resistance to CQ) Resistance of Plasmodium falciparum to chloroquine (CQ) has never been detected in Central America and the Caribbean (Mesoamerica) where CQ is still used as first-line therapy for uncomplicated infections caused by either P. vivax or P. falciparum. Currently pfcrt 76T polymorphism associated to the position C350, is considered the most critical change that confers resistance to CQ. Therefore, a search was conducted to retrieve available data of molecular studies performed on samples collected in Central America and the Caribbean since 2005. (11) Data were reviewed to estimate the prevalence of mutated allele pfcrt 76T in these regions. TES with chloroquine conducted since 2005 were also identified in order to correlate treatment outcomes with parasite genotypes (4). Based on the available data (11), these findings justify the continuation of chloroquine as first-line therapy for uncomplicated falciparum malaria in Mesoamerica

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2.3 Monitoring antimalarial resistance in P. vivax Table 6. Summary of treatment failure rates among patient infected with P. vivax, grouped by country and treatment (4)*

*all studies have a 28 day follow up.

3 Country updates on antimalarial resistance 2017-2019

3.1 Updates on ACT efficacy in P. falciparum (TES) Since last TAG meeting three new TES were supported in the Region, two to evaluate efficacy of ACT (Artemether + Lumefantrine) in P. falciparum, one in Guyana due to the importance of findings of the presence pfk13 mutation in 2017, and one study in the Pacific Coast in Colombia, the region with higher number of cases of Plasmodium falciparum malaria, outside the current situation in Venezuela. The third study, was a TES on the efficacy of chloroquine and primaquine in P. vivax that was implemented in Brazil. Preliminary results are as follow:

3.1.1 Colombia:

One site: Choco, Quibdó, Colombia Drug: Artemether 20mg + Lumefantrine 120 mg (First line treatment for P. falciparum

uncomplicated infections) Starting date 7/17/19 last patient follow up 2/28/2019 Total patients enrolled 89 patients (ratio male/female: 54/35; Age mean (sd): 30.5 (16.5); Age

range (min – max) 5-64)

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Patients follow up D28: 84 Table 3 summarize the results of this study. Table 3. Colombia results - PCR uncorrected data

Number % lower 95%CI upper 95% CI

ETF 0 0.0 0.0 4.3

LCF 1 1.2 0.0 6.5

LPF 0 0.0 0.0 4.3

ACPR 83 98.8 93.5 100.0

Total patients per protocol 84

WTH 3

LFU 2

Total patients LFU/WTH 5 5.6

Total patients at baseline 89

Note: The patient classified as LCF, with a very low parasitaemia (12 parasites/μl) could not be amplified, hence it could not be established if it was a reinfection or a recrudescence.

3.1.2 Guyana Two sites: #1 Georgetown and #2 Port Kaituma Drug: Artemether 20mg + Lumefantrine 120 mg + primaquine 0.75mg/kg bw (First line

treatment for P. falciparum uncomplicated infections) Starting date site #1: June 2018 and last patient follow up October 2018. Site #2 starting date:

September 2018 and last patient follow up April 2019 Total patients enrolled: Site #1 99 patients (ratio male/female: 79/20; Age mean (sd): 32.4

(13.5); Age range (min – max) 5-69); Site #2 75 patients (ratio male/female: 52/23; Age mean (sd): 28.9 (14.9); Age range (min – max) 5-62)

Patients follow up D28: Site #1 84; site #2 50 Currently molecular analyses underway by Institute Pasteur, Cayenne, French Guiana. Up to now no K13 mutation has been found (n= 99 site #1 Georgetown, analysis completed, and n= 29 site #2 Port Kaituma)

Tables 4 and 5 summarize the results of this study.

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Table 4. Guyana site #1 results - PCR uncorrected data

number % lower 95%CI upper 95% CI

ETF 0 0.0 0.0 4.3

LCF 2 2.4 0.3 8.3

LPF 3 3.6 0.7 10.1

ACPR 79 94.0 86.7 98.0

Total patients per protocol 84

WTH 12

LFU 3

Total patients LFU/WTH 15 15.2

Total patients at baseline 99

Table 5. Guyana site #2 results – PCR uncorrected data

number % lower 95%CI upper 95% CI

ETF 2 4.0 0.5 13.7

LCF 0 0.0 0.0 7.1

LPF 0 0.0 0.0 7.1

ACPR 48 96.0 86.3 99.5

Total patients per protocol 50

WTH 15

LFU 10

Total patients LFU/WTH 25 33.3

Total patients at baseline 75

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3.2 Update on therapeutic efficacy in P. vivax

3.2.1 Brazil Since last TAG meeting in 2017, one additional in vivo study has been implemented to evaluate the efficacy and safety of first line treatment in Brazil for P. vivax. One site: Acre Drug: Chloroquine + Primaquine 3 arm study. Assess the efficacy of 3 different regimens of

chloroquine and primaquine for the treatment of P. vivax infections in Cruzeiro do Sul, Acre, Brazil

Starting date: February 2018 and last patient follow up to November 2018 (6-month follow-up period (168 days))

Total patients included: 257 (63, 98 and 93 groups 1, 2 and 3 correspondingly) Adequate clinical and parasitological response at Day 168 could be estimated at 59.6% (95% confidence interval [CI] =46.3–73.0%), 57.1% (95% CI =46.1–68.2%), and 85.9% (95% CI =78.2–93.6%), for Groups 1, 2, and 3, respectively. CQ and PQ remain efficacious to treat the acute phase of uncomplicated P. vivax malaria, but the higher PQ dose was associated with superior efficacy within 168 days post-treatment

Table 6. Total patient enrolled, follow-up and recurrent infections observed

GROUP

TOTAL

PATIENT

FOLLOW-UP

RECURRENT INFECTION

NUMBER OF PATIENT

D28 D168

1 63 1 21

2 98 2 33

3 93 0 11

TOTAL 257

3.3 Updates on surveillance based on molecular markers

3.3.1 Central America and Hispaniola Island Routine surveillance of P. falciparum positive cases in Central American countries (Honduras, Nicaragua, Dominican Republic, and Haiti) during last years showed absence of mutations for pfcrt demonstrating that chloroquine still is efficacious on that sub region for treating falciparum malaria cases. Recently (2016-2017 samples) Haiti analysed more 741 blood samples on paper filter from P. falciparum positive

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infections and results showed also 100% wild type circulation, so no mutations founded indicating that chloroquine remains efficacious on those countries1.

3.3.2 South America

Guyana:

To understand origin of those mutants presented in data collected from 2016-2017, during 2018 nine microsatellite loci flanking the pfK13 gene, whole genome sequencing (WGS) and selective whole genome amplification (SWGA) were performed. Results confirm that the pfK13 C580Y variant arose on a single Guyanese haplotypic background and was not imported from South-East Asia. Also, the presence of this mutation generates an increase in the parasite survival rate in vitro (7).

Also, new data from samples from the TES (2018-2019) already analysed from Georgetown site #1 (n= 99) and for Port Kaituma site #2 (n=29) no pfK13 C580Y mutation found. The other samples from site #2 are under analyses.

Suriname:

Samples collected from 2013 and 2014 in the country (N= 41) showed only wild type no mutant presence.

French Guiana:

Samples collected between 2009 and 2018 (N=657) only one mutant was founded (M671I) the others were wild type.

Venezuela:

Samples collected as part as routine surveillance in the country in 2017/2018 (N=101) no pfK13 C580Y mutants were detected and two samples were observed of mixed genotype: one sample T367D and another one only wild type.

Brazil:

The country has been collecting samples as part of the TES studies, founding the following mutants in Manaus: 2014: N= 130 (n=1 A481V); 2015: N= 63 (n=1 P475S) and (n=1 P553S). Samples collected from 2016 to February 20182 a total of 751 patients enrolled (346 Pacaraima; 348 Boa Vista; 57 Rorainapolis) all wild type, no pfK13 gene mutation found.

Colombia:

Samples collected in studies done in 2010 (N=523) no mutants were detected only wild type. In samples collected during last in vivo study in 2019 (N= 89) no pfK13 mutations were detected, also previously reported haplotypes for pfmdr1 detected (NFSDD/NFSDY).

Peru:

Samples collected in studies done in 2010 (N=69) no mutants were detected only wild type.

1 Rogier E., et al, unpublished 2 Collaboration between FIOCRUZ, Federal University of Roraima and CDC. Data presented by CDC.

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4 MAIN POINTS

Until now there is not any evidence of resistance to ACT in the Americas. Since 2010 there is evidence of the presence in the Americas of one of K13 mutations that globally

are considered associated with ACT resistance (pfK13 C580Y). Cases with that mutation have been reported only in one country (Guyana), had not been associated with delay in clearance or therapeutic failure and although they have been reported in samples collected in two moments (2010 and 2016-17), they were not registered in 128 samples of 2018.

Presence of mutants for resistance to artemisinin identified in Guyana did not spread from the Greater Mekong Subregion. Results indicate de novo emergence.

K13 mutations associated with resistance (C580Y or others) have not been detected in surveillance activities in other countries, including Venezuela.

Two recent in vivo studies from two different parts of South America confirm the absence of therapeutic failure to artesunate lumefantrine, which the ACT more used in the Region.

There is not evidence of presence of resistance markers to CQ in Central America and the Hispaniola Island.

Therapeutic failure to CQ in P. vivax malaria exists in the Region but remain in very low levels. The priorities in resistance surveillance are maintaining surveillance activity in Guyana and

promote an intensify surveillance in main foci in Venezuela. Efforts for preventing and containing ACT resistance in the Guyana Shield are highly relevant in a

context of a Regional framework to eliminate P. falciparum from the Region.

5 NEXT STEPS

As per PAHO/WHO guidelines key priorities for member states are:

• TES needs to be conducted every 2-3 years (depending on epidemiological situation) o To evaluate the efficacy of 1st. and 2nd. line treatment. Provable next TES to be

implemented in: Bolivar State, Venezuela Loreto, Peru

o Capacities needs to be strength at national level when a TES is planned

• Surveillance with molecular markers should continue to be conducted systematically: o Concomitant with in vivo studies, o As well as collection in hotspots for possible selection of resistance

Guyana and Venezuela: we will continue this surveillance with molecular markers Also, detection and collection in other possible sites with high risk for resistance

development. Some countries in South America (Colombia, Brazil, Guyana, Venezuela) should identify better key situation with presence of risk factors

• Quality: External evaluation of TES needs to be in place, and WHO CC for molecular markers • Implementation of the Framework for artemisinin resistance containment and elimination in

South America –Guyana Shield o With the objective to eliminate Plasmodium falciparum before resistance arise.

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6 REFERENCES

1. Pan American Health Organization. Plan of Action for Malaria Elimination of 2016-2020. 2. World Health Organization. Methods for surveillance of antimalarial drug efficacy, 2003. 3. Pan American Health Organization. 2011. Strategic Orientation Document on Monitoring the

Efficacy of and Resistance to Antimalarials in the Current Epidemiological Context. Produced by the Pan American Health Organization (PAHO/WHO) in collaboration with the US Centres for Disease Control and Prevention (CDC) and Links Media, LLC, for the U.S. Agency for International Development.

4. World Health Organization. Status Report. Artemisinin and artemisinin-based combination therapy resistance. October 2016.

5. WHO. Malaria Policy Advisory Committee meeting report. April 2019. https://apps.who.int/iris/bitstream/handle/10665/312198/WHO-CDS-GMP-2019.04-eng.pdf?ua=1

6. Chenet, S. M. et al. Independent Emergence of the Plasmodium falciparum Kelch Propeller Domain Mutant Allele C580Y in Guyana. J. Infect. Dis. 213, 1472–1475 (2016).

7. Musset L. et al 2018. In vitro artemisinin resistance and the pfk13 C580Y mutation in Guyana: a confirmed link and emergence. Abst n°71. 67th Annual meeting of the American society of tropical medicine and hygiene, October 25th to November 1st, New Orleans (USA).

8. World Health Organization. Status report on Artemisinin resistance and artemisinin-based combination therapy. (2018). Available at: http://apps.who.int/iris/bitstream/handle/10665/274362/WHO-CDS-GMP-2018.18-eng

9. Ariey, F. et al. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature 505, 50–55 (2013)

10. Straimer, J. et al. K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates. Science 347, 428–431 (2015)

11. PAHO/WHO Data collected. Efficacy of chloroquine and prevalence of pfcrt mutations in Mesoamerica.

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7 ANNEX 1

Drug efficacy studies for assessing antimalarial drug resistance conducted in RAVREDA/AMI, 2002 – 2018.

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