Date Published: September 20, 2018
Publisher: Public Library of Science
Author(s): Christian Nsanzabana, Frederic Ariey, Hans-Peter Beck, Xavier C. Ding, Edwin Kamau, Sanjeev Krishna, Eric Legrand, Naomi Lucchi, Olivo Miotto, Sidsel Nag, Harald Noedl, Cally Roper, Philip J. Rosenthal, Henk D. F. H. Schallig, Steve M. Taylor, Sarah K. Volkman, Iveth J. Gonzalez, Silvie Huijben.
Antimalarial drug resistance is a major constraint for malaria control and elimination efforts. Artemisinin-based combination therapy is now the mainstay for malaria treatment. However, delayed parasite clearance following treatment with artemisinin derivatives has now spread in the Greater Mekong Sub region and may emerge or spread to other malaria endemic regions. This spread is of great concern for malaria control programmes, as no alternatives to artemisinin-based combination therapies are expected to be available in the near future. There is a need to strengthen surveillance systems for early detection and response to the antimalarial drug resistance threat. Current surveillance is mainly done through therapeutic efficacy studies; however these studies are complex and both time- and resource-intensive. For multiple common antimalarials, parasite drug resistance has been correlated with specific genetic mutations, and the molecular markers associated with antimalarial drug resistance offer a simple and powerful tool to monitor the emergence and spread of resistant parasites. Different techniques to analyse molecular markers associated with antimalarial drug resistance are available, each with advantages and disadvantages. However, procedures are not adequately harmonized to facilitate comparisons between sites. Here we describe the target product profiles for tests to analyse molecular markers associated with antimalarial drug resistance, discuss how use of current techniques can be standardised, and identify the requirements for an ideal product that would allow malaria endemic countries to provide useful spatial and temporal information on the spread of resistance.
Antimalarial drug resistance is a major concern for malaria control and elimination programmes. Indeed, Plasmodium falciparum parasites have consistently developed resistance to the most widely used antimalarials, pushing national malaria control programmes to regular changes in antimalarial drug policy . Artemisinin-based combination therapy (ACT) is now the mainstay for malaria treatment in endemic regions, following recommendations from the World Health Organization (WHO) . However parasites with decreased susceptibility to artemisinin derivatives have emerged over the last ten years in different parts of the Greater Mekong Sub region (GMS) [3–7]. ACTs are failing due to both decreased susceptibility to artemisinin compounds and resistance to their partner drugs in Southeast Asia [8–14]. Strengthening of existing surveillance systems is needed to detect drug resistance in malaria endemic countries as it emerges or spreads to other regions. Antimalarial drug resistance surveillance is currently done through three different strategies: in vivo studies such as therapeutic efficacy studies (TESs), in vitro/ex vivo studies of cultured malaria parasites, and molecular studies assessing known markers of antimalarial drug resistance. These three techniques are complementary, but each has advantages and disadvantages . TES remains the gold standard for informing antimalarial drug policy change, as outcomes have direct clinical relevance , but these studies are challenging to conduct due to heavy financial and logistical constraints , and they cannot always confirm resistance, especially for combination therapies . Indeed, only monotherapy studies allow for the accurate differentiation of the drug component causing apparent ACT treatment failure . In vivo/ex vivo studies, such as measurement of IC50 (50% inhibitory concentration of a drug) or ring stage survival assays, can provide useful information about parasite susceptibility to antimalarial drugs, but require heavy infrastructure for parasite culture. Performance of these assays is generally restricted to well-equipped laboratories to validate new molecular markers of antimalarial drug resistance , or to link a resistance phenotype to a genotype . Molecular studies of antimalarial drug resistance markers provide information about the parasite genetics associated with resistance, i.e. single nucleotide polymorphisms (SNPs) or gene copy number variations (CNVs) that are associated with decreased susceptibility of parasites to antimalarial drugs. After markers of resistance have been identified by genotype-phenotype discovery studies, detection of these molecular markers provides a feasible means of tracking emergence and/or spread of antimalarial drug resistance, as easy-to-collect dried blood spot (DBS) samples can be used [22,23]. While numerous methodologies for blood spot collection, DNA extraction, PCR amplification, and analysis of molecular markers have been described, standardisation of these approaches is lacking . Given the potential role of molecular surveillance of drug resistance markers, a standardised approach is important to allow for comparability across the globe.
A draft TPP was developed based on a landscape analysis of antimalarial drug resistance surveillance methods performed by FIND . The listed properties were defined according to FIND’s standard procedures (https://www.finddx.org/target-product-profiles/), with characteristics described as either “minimal” or “optimal”. The experts were selected based on their experience and expertise in the field of molecular markers of antimalarial drug resistance. The participants selection was based on a review previously conducted by FIND on the methods used for surveillance of antimalarial drug resistance . Identified experts were contacted by email, invited to participate in the meeting and provided with a brief summary of the meeting’s objectives (S1 Table). Those who confirmed their attendance were provided with the draft TPP prior to the meeting including a questionnaire (S2 Table). The meeting was organised by FIND and held in Geneva on 21 and 22 September 2017 to reach consensus on the TPP. The experts were asked to present the different molecular techniques that are used in their laboratories and discussed their advantages and disadvantages (Table 1).
Molecular markers of antimalarial drug resistance have proved to be useful for detection of early resistance emergence [5,7,60], spread of resistance , or absence of resistance , and are easy to interpret . Although TESs provide valuable resistance measurements that are easiest to directly translate to policy, they are confounded by many factors, including clinical immunity and varied pharmacokinetics, and they require extensive time for completion, so resistance may only be apparent once parasites resistant to both components of a drug combination have spread widely . Molecular techniques have the advantage of providing information in real time about the prevalence and ideally the frequency of resistant parasite strains circulating in the population using easily collected DBS or RDT samples [42,58,65], and this information is not typically confounded by clinical immunity. Even though, the presence of resistant parasites does not necessarily predict treatment failure , increasing prevalence of validated molecular markers of antimalarial drug resistance is associated with increasing treatment failure, and thus molecular markers offer a valuable early indicator of resistance emergence , and a practicable means of determining thresholds for policy makers. As an example, the WHO policy on Intermittent preventive treatment for infants (IPTi) with SP recommends ≤50% prevalence of Pfdhps 540 mutation as the threshold for implementation of SP-IPTi . A variety of different techniques to assess molecular markers associated with antimalarial drug resistance are already available (Table 1), however standardisation is needed to improve the quality of generated data .
In summary, techniques already exist with most of the required characteristics in this TPP for assays to analyse molecular markers associated with antimalarial drug resistance, and could be rapidly implemented in reference laboratories. Other techniques in development fulfil most of the criteria specified by the TPP and could potentially improve data analysis standardisation. However, the implementation of different techniques for routine surveillance of antimalarial drug resistance would need a consensus from policy makers to define implementation procedures, optimise their use, and implement good EQA practices. This TPP can also be used by assay manufacturers to guide development of new technologies to facilitate efficient surveillance of molecular markers associated with antimalarial drug resistance in endemic settings.