Alignment of DNA sequences

AS-AQ/AL Molecular Marker Study Group

Investigating the role of candidate molecular markers of lumefantrine and amodiaquine resistance in clinical outcomes of ACTs

Update and overview

The Artesunate-Amodiaquine/Artemether Lumefantrine (AS-AQ/AL) Molecular Marker Study Group demonstrated that if patients are infected with malaria parasites that carry particular mutations in genes pfcrt and pfmdr1, they are at higher risk of treatment failure after artemether-lumefantrine (AL). The group showed that artesunate-amodiaquine (ASAQ) and AL exerted opposing selective effects on mutations known as, single-nucleotide polymorphisms, in pfcrt and pfmdr1.

The Study Group recommends that prevalence of molecular markers, at least pfcrt K76T and pfmdr1 N86Y, should be determined routinely to track any changes in their prevalence, as an indicator of changes in efficacy of the lumefantrine and amodiaquine partners of the artemisinin combination therapies, artemether-lumefantrine and artesunate-amodiaquine.  The study was published in the American Journal of Tropical Medicine and Hygiene in October 2014.

The Study Group pooled individual patient and linked parasite genotype data from 31 studies. Data from 7,249 patients who were treated with AL (5,003) or ASAQ (2,246) were included in the analysis. 27 studies were published, representing 91 per cent of all published clinical data on AL and ASAQ in which pfcrt or pfmdr1 genotypes were determined.

The Study Group formed in October 2011, with an open invitation to potential participants. Known research groups with relevant data sets were contacted in October-November 2011. Potential participants met at the ASTMH Annual Meeting in December 2011 to discuss governance and publication policy. The study group closed in April 2012,  the analyseswere completed in 2013 and the paper was published in October 2014.

Publication details:

Venkatesan M, Gadalla NB, Stepniewska K, et al. Polymorphisms in Plasmodium falciparum Chloroquine Resistance Transporter and Multidrug Resistance 1 Genes: Parasite Risk Factors that Affect Treatment Outcomes for P. falciparum Malaria after Artemether-Lumefantrine and Artesunate-Amodiaquine.  Am J Trop Med Hyg. 2014 Oct;91(4):833-43. doi: 10.4269/ajtmh.14-0031. PubMedID:  25048375

Rationale

The initial reduction in peripheral Plasmodium falciparum parasitemia following artemisinin-based combination therapy (ACT) is driven predominantly by the potency and rapid action of the artemisinin component, but overall efficacy requires sustained therapeutic concentrations of the longer-acting partner drug. Candidate molecular markers associated with resistance in the pfcrt and pfmdr1 genes ofP falciparum have been reported to be involved in decreased sensitivity to amodiaquine and lumefantrine. However, the utility of these markers for predicting therapeutic responses to artesunate-amodiaquine (AS-AQ) and artemether-lumefantrine (AL) remains unclear. Correlation studies are confounded by the overall high PCR-corrected parasitological cure rates associated with these ACTs and by regional variation in immunity and parasite genetic background.

Objectives

  • Establish whether known molecular polymorphisms in P falciparum can predict clinical outcome following treatment with AL and AS-AQ
  • Determine whether resistance genotypes are selected in recurrent parasites

To address these objectives, we are conducting a pooled analysis of retrospective data on clinical outcomes and candidate resistance markers from studies conducted in Africa and Asia.

Essential inclusion criteria

  • Prospective clinical efficacy studies of P falciparum;
  • Treatment with Artemether-Lumefantrine (6 dose regimen), Artesunate-Amodiaquine (3 day regimen fixed dose or co-blistered) or  DHA-Piperaquine with a minimum of 28 days of follow up;
  • Individual patient data including baseline characteristics, parasitemia, and temperature;
  • PCR correction to determine true P falciparum recrudescence;
  • Molecular data defining pfcrt and/or pfmdr1 polymorphisms (point mutations and copy number variation) in parasites isolated from patients on day 0, prior to treatment; and
  • Ethical clearance of study protocol.

Desirable criteria

  • Molecular resistance marker data from day of recurrent parasitemia
  • Multiplicity of infection

Primary end point

  • PCR adjusted risk of P falciparum recrudescence

Secondary end points

  • Risk of P falciparum reinfection
  • Unadjusted risk of P falciparum recurrence
  • Time to P falciparum recrudescence, recurrence and reinfection
  • Early parasite clearance
  • Risk of P vivax recurrence
  • Post-treatment and early (day 2 and 3) selection of resistance genotypes

Data standardisation and analysis

After upload to the WWARN Data Repository, WWARN will standardise the data sets according to the WWARN Clinical and Molecular Data Management and Statistical Analysis Plans, and pool the data into a single database of quality-assured individual patient data. Analyses (subject to Study Group approval) may include: 

  • Proportion of patients remaining parasitaemic at days 1, 2 and 3
  • Clinical outcome (Pf PCR adjusted and unadjusted treatment failure at day 28) and identification of significant baseline risk factors, for example age and parasitaemia
  • The effect of molecular markers on day 3 positivity and clinical outcome after controlling for other known confounding factors
  • Comparison of pre- and post-treatment resistance genotypes

Kaplan Meier survival analysis will be used to define treatment efficacy for both univariate and multivariate analyses.

Outcome

This analysis will clarify the roles of molecular markers for partner-drug resistance in monitoring ACT efficacy and will help to guide the selection of informative genetic markers in future studies.

Study Group governance

The Study Group comprises participating investigators who contribute relevant data sets to the pooled analysis. Data sets remain the property of the investigator. The Study Group collectively makes decisions with respect to including additional studies, data analysis and plans for publication, in line with the WWARN Publication Policy. Two Study Group Leaders, Meera Venkatesan and Nahla Gadalla, were identified to coordinate activities including data analysis, and drafting of publications and reports for group review. The data management procedures will be in line with WWARN procedures. The Study Group leaders will be responsible for statistical analyses with support from WWARN statisticians.

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