Molecular Markers and Gene Flow in Plasmodium falciparum in Southern Africa

 

The in vivo drug resistance trials to be carried out at sentinel sites as a central part of the SEACAT evaluation will provide an important opportunity to study molecular markers and gene flow in Plasmodium falciparum populations. As part of these studies, blood samples in the form of dried spots will be taken from patients for PCR identification of Plasmodium species and distinction between recrudescence and reinfection. The samples can further be used to study molecular markers of drug resistance. A protocol for the study of geneflow between the geographic areas has been submitted for separate funding by Dr C Roper, London School for Hygiene and Tropical Medicine.  Microsatellite genotypes which are known to be selectively neutral in parasite populations under drug pressure will be used to establish whether the parasite populations of the study areas (which are geographically contiguous, except for Northern Province) form a single panmictic unit.

     
 

The particular combination of a number of distinct geographical zones with a varied history of SP use raises several questions relating to gene flow which will be addressed in this study and include:
  Are the point mutations correlated with drug resistance in vivo in the various study areas?
  How does combination therapy impact on the rate of accumulation of mutations?
  Does Plasmodium falciparum in the study area form a single panmictic population?
 Will the presence of a gene pool in KZN containing a relatively high frequency of mutations to SP cause a more rapid build-up in parasites of neighbouring areas?
 
     

It is widely accepted that stepwise accumulations of point mutations in the dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) enzyme systems of Plasmodium falciparum are well (but not completely) correlated with clinical resistance to sulphadoxine-pyrimethamine (SP)(1). These point mutations are easily detected by polymerase chain reactions (PCR) using DNA extracted from dried blood spots.

A workshop was recently held by WHO/TDR to standardise technical aspects of detection of point mutations in the DHFR/DHPS enzyme systems of Plasmodium falciparum (P Olliaro, personal communication.) The concensus opinion  recommends detection of mutations at least codons 51 and 59 (DHFR) and 437 and 581 (DHPS) as optimal for the prediction of resistance to SP. Relevant other resistance markers will also be evaluated in this study, as the mutations required for prediction of resistance remains controversial.

Given this background, the following questions arise regarding the spread/ appearance by selective pressure of SP resistance associated point mutations in the study areas. The particular combination of a number of distinct geographical zones with a varied history of SP use ranging from 11 years as the first line drug (KZN), through 2 years (Mpumalanga) to no use (Swaziland,Mozambique) raises several questions relating to gene flow which will be addressed in this study and include:

      Are the point mutations correlated with drug resistance in vivo in the various study areas?
      How does combination therapy impact on the rate of accumulation of mutations?
     Does Plasmodium falciparum in the study area form a single panmictic population?
     Will the presence of a gene pool in KZN containing a relatively high frequency of mutations to SP cause a more rapid build-up in parasites of neighbouring areas?

A parallel, separately funded study of cross sectional samples of resistance markers in 1200 patients will be made available in a shared repository and will strengthen our understanding of the above.  These cross sectional studies will be repeated annually as part of the SDI.

Another parallel, separately funded study of in vitro resistance (nested in the in vivo studies in the 5 study sites) will be correlated with resistance mutations and in vivo resistance.

Objectives:

Within the population at each sentinel site:

      Establish base-line gene frequency for mutations related to SP resistance and correlate with in vivo resistance in all the study areas.
       Monitor changes in these gene frequencies at all sentinel sites over time.

Within each individual:

      Confirmation of Plasmodium species and detection of low parasitaemias.
      Use of GLURP and MSP I & II markers to distinguish recrudescence from reinfection. 

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