抗疟药物耐药性分子标记简编中使用的方法和阈值

Methods andthresholds(version 1.0, 8 December 2025) Introduction TheCompendium of molecular markers for antimalarial drug resistancewas developed to address theneed for an evidence-based, up-to-date resource presenting genetic alterations associated withreduced susceptibility to antimalarial drugs. The compendium summarizes and classifies molecular markers using evidence from three domains:laboratory, clinical and genetic epidemiology. These domains represent distinct but complementarylines of evidence that support the association between specific genetic alterations and drug resistance. The approach builds on earlier classification efforts, including those applied toPfKelch13mutationsandis designed to evolve as new evidence emerges. The compendium will be updated annually following areview of new evidence and, where applicable, revisions to molecular markers and methodologies. Developmentprocess The compendium was developed through a multi-step process: 1.Development of predefined criteria and thresholds for evidence evaluation.Thresholds wereestablished to assess the quality and relevance of evidence within each domain. These weredeveloped inconsultation with independent experts convened by WHO. Heterogeneity inmeasurement and reporting across the published literature presented a significant challenge tostandardization. The criteria were designed to balance scientific rigor with practical applicabilityacross different sources of evidence and study designs of varying quality. 2.Literature review.Published laboratory, clinical, and genetic epidemiology data were reviewedand evaluated against these thresholds by a group of primary reviewers.3.Expert consultations.Two rounds of expert review were conducted to refine and validatemarker classifications, assess study quality, and consider contextual factors affectinginterpretation. The experts also served as secondary reviewers for the initialassignment ofmarkers. Methods To support a transparent and consistent evaluation, all genetic markers were classified according topredefined thresholds established for each of the three evidence domains: laboratory, clinical, andgenetic epidemiology. Within each domain, different types of evidence were considered to assesscausal relationships between genetic alterations and antimalarial drug resistance, as describedin thesection on Thresholds. Markers that met the domain-specific thresholdswere considered for inclusion in the compendium.Based on the combined assessment of evidence across domains, each genetic alteration was classifiedas a potential, candidate, or validated marker of antimalarial drug resistance (Figure 1 and Table 1). Figure 1.Markerclassification framework Thresholds A.Laboratory evidence Three types of evidence are considered in the assessment of laboratory data. These include studiesdemonstrating that a genetic alteration reduces parasite susceptibility to a drugin vitro, usingtransfected strains,fieldisolates orculture-adaptedlaboratorystrains, or progeny derived from geneticcrosses. Among these, data from transfected strains are regarded as stronger evidence, as they providedirect evidence of causality. Consequently, priority is given to studies using transfection and gene-editing techniques. When thresholds are met using these methods, additionalin vitrodata are notreviewed. In-vitro drug-pressure selection studies, where parasites acquire mutations followingprolonged exposure to antimalarials, can provide supportive evidence of adaptivepotential. While notproving direct causality, such findings may inform prioritisation of studies on candidategenesforfurther validation. A1.Transfection-based confirmation Threshold •When comparing a culture-adapted, recombinant isogenic parasite line incorporating thegenetic alteration–produced through transfection and gene-editing techniques–with acontrol isogenic line of the same strain, the threshold is met under the following conditions: •For markers associated with artemisinin partial resistance:a significant difference (p <0.05) inthe Ring-Stage Assay (RSA0–3h),1with a minimum survival of >1% in the gene-edited linecompared to the control isogenic line (same strain).•For markers associated with piperaquine (PPQ) resistance:a significant difference (p <0.05) inthe Piperaquine Survival Assay (PSA),2with≥10% survival at 200 nM PPQ in the gene-edited linecompared to thewild-typeisogenic line.•For markers associated with resistance to other drugs:oFor single point mutations, or alleles where multiple mutations are introduced: astatistically significant (p<0.05) increase in half-maximal inhibitory concentration (IC50)or 90% inhibitory concentration (IC90)3in the gene-edited line compared with thewild-typeisogenic line.oFor copy number variations where transfection is used to overexpress thegene:astatistically significant (p<0.05) increase in IC50or IC90in the gene-edited line comparedwith thewild-typeisogenic line.•ForP. vivax(and rarely forP. falci