Timing of treatment failure and mutations in Plasmodium falciparum dihydrofolate reductase in Uganda

Nalunkuma Kazibwe, Anne Juliet (2003) Timing of treatment failure and mutations in Plasmodium falciparum dihydrofolate reductase in Uganda. MSc(R) thesis, University of Glasgow.

Abstract

The spread of drug resistant strains of malaria parasites has made prophylaxis and treatment of the disease increasingly difficult. The resistance of Plasmodium falciparum to chloroquine and sulfadoxine-pyrimethamine (Fansidar) has been reported in various parts of Uganda. A study was carried out in Tororo, Uganda, in order to investigate by polymerase chain reaction/dot-blot hybridisation the association of mutations in the DHFR gene in Plasmodium falciparum parasite isolates with clinical and parasitological treatment failure following sulfadoxine-pyrimethamine or a combination of sulfadoxine-pyrimethamine and chloroquine treatment in Uganda. The study was also done to determine if the number of mutations in DHFR is correlated with the time to recrudescence following sulfadoxine- pyrimethamine and sulfadoxine-pyrimethamine plus chloroquine treatment. In collaboration with the Ministry of Health, blood samples were collected on filter paper on the first day of treatment-Day 0 (D0) and on any other day symptoms developed after administering Fansidar alone and a combination of chloroquine and Fansidar to children with uncomplicated malaria aged between 6 months and 5 years old who were followed up on days 1, 2, 3, 7, 14, 21 and 28. Forty seven paired chloroquine plus Fansidar D0 and R (day of recrudescence) patient DNA samples in the RI-RIII group, were genotyped at position 76 of the Pfcrt locus. Following genotyping at the MSP1 and MSP2 loci, 22 of these patient samples showing evidence of recrudescence were genotyped at the 3 DHFR codons: 108, 51 and 59. For Pfcrt codon 76, all (100%) of the pre- and post-treatment isolates possessed the Pfcrt T76 allele. Only 6 (13%) pre- and 3 (7%) post-treatment isolates possessed both Pfcrt K76 and T76 alleles simultaneously. No isolate possessed only the Pfcrt K76 allele alone. For codon 108, of the 22 (pre-treatment) patient DNA samples successfully typed, all (100%) had only the DHFR Asnl08 allele. The same trend was observed in the post-treatment isolates. No isolate pre- /or post-treatment possessed the DHFR Ser108 allele alone. No isolate pre- /or post-treatment possessed both DHFR Ser108 and Asn 108 alleles simultaneously. At codon 51, there was a high prevalence (96%) of the 51Ile allele alone in the pre-treatment isolates and of the 51Ile allele alone in the post-treatment isolates (100%). No isolate both pre- and post-treatment possessed the DHFR 51Asn allele alone. Only one pre-treatment isolate possessed both DHFR 51Asn and 51Ile alleles simultaneously. At codon 59 there was a low prevalence (14%) of the 59Arg allele alone in the pre- treatment isolates and 38% of the isolates had the 59Arg allele alone in the post-treatment isolates. Seventeen (77%) patient samples possessed both DHFR 59Cys and 59Arg alleles present simultaneously in their pre-treatment isolates. This dropped to 38% in the post-treatment isolates. Only 2 (9%) patient samples pre-treatment had only the 59Cys allele while this increased to 24% in the post- treatment isolates. Thirty three out of 48 paired Fansidar DO and R (day of recrudescence) patient DNA samples in the RI-RIII group, showing evidence of recrudescence were genotyped for the 3 DHFR codons 108, 51 and 59. For codon 108, of the 33 (pretreatment) patient DNA samples successfully typed, all (100%) had only the DHFR Asnl08 allele. Thirty samples, representing 100% of the successfully typed post-treatment isolates, had only the DHFR Asn108 allele. No isolate pre- or post-treatment possessed the DHFR Serl08 allele alone. No patient pre- or post-treatment possessed both DHFR Seri 08 and Asn 108 alleles simultaneously. For codon 51, 100% of samples had only the DHFR 51Ile allele in the pre-treatment isolates and 85% had only the DHFR 51 He allele in the post-treatment isolates. No patient pre-treatment possessed the DHFR 51 Asn allele alone. Only one patient isolate post-treatment possessed the DHFR 51 Asn allele alone. One patient pre- and post-treatment possessed both DHFR 51 Asn and 51Ile alleles present simultaneously. At codon 59, there was a low prevalence (18%) of the 59Arg allele alone in the pre-treatment isolates and a high prevalence (77%>) for 59Arg allele alone in the post-treatment isolates. Nineteen (58%) patient samples possessed both the DHFR 59Cys and 59Arg alleles present simultaneously in their pre-treatment isolates. This dropped to 17% in the post-treatment isolates. Only 8 (24%>) patient samples pre-treatment had only the 59Cys allele while this reduced to 1% in the post-treatment isolates. In conclusion, the findings showed there was no correlation between the presence of Pfcrt alleles encoding T76 and time to recrudescence following treatment with a combination of chloroquine and Fansidar. There was no correlation between the DHFR alleles encoding Asn108 and/ or Ile51 and time to recrudescence following treatment with chloroquine plus Fansidar as a combination and also with Fansidar treatment alone. There was however a correlation between the presence of the DHFR double mutant allele Asn 108/ Arg 59, and a stronger correlation between the triple mutant allele Asn 108/ He 51/ Arg 59, and time to recrudescence following Fansidar treatment in Tororo, Uganda. There was no correlation of theses alleles in the chloroquine plus Fansidar treatment group.

• Item Type: Thesis (MSc(R))
• Qualification Level: Masters
• Keywords: Parasitology.
• Colleges/Schools: College of Medical Veterinary and Life Sciences
• Supervisor's Name: Ranford-Cartwright, Dr. Lisa and Egwang, Dr. Thomas Gordon
• Date of Award: 2003
• Depositing User: Enlighten Team
• Unique ID: glathesis:2003-71472
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 17 May 2019 09:31
• Last Modified: 08 Jun 2021 15:25
• URI: https://theses.gla.ac.uk/id/eprint/71472