Amplification of GTP-cyclohydrolase 1 gene in Plasmodium falciparum isolates with the quadruple mutant of dihydrofolate reductase and dihydropteroate synthase genes in Ghana

Abstract

Sulfadoxine-pyrimethamine (SP) is used as malaria chemoprophylaxis for pregnant women and children in Ghana. Plasmodium falciparum resistance to SP is linked to mutations in the dihydropteroate synthase gene (pfdhps), dihydrofolate reductase gene (pfdhfr) and amplification of GTP cyclohydrolase 1 (pfgch1) gene. The pfgch1 duplication is associated with pfdhfr L164, a crucial mutant for high level pyrimethamine resistance which is rare in Ghana. The presence of amplified pfgch1 in Ghanaian isolates could be an indicator of the evolution of the L164 mutant. This study therefore determined the pfgch1 copy number variations and SP resistance mutations in clinical isolates from Ghana. One hundred and ninety-two (192) blood samples collected from children aged ≤14 years with uncomplicated malaria in 2013–14 and 2015–16 were used. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the pfgch1 copy number and nested PCR-Sanger sequencing used to detect mutations in pfdhps and pfdhfr genes. Twelve parasites (6.3%) harbored double copies of the pfgch1 gene out of the 192 samples. Of the 12, 75% had the pfdhfr I51-R59-N108, 92% had the pfdhps G437 mutant, 8% had the pfdhps E540 and 67% had the SP resistance haplotype IRNG. No L164 was detected in samples with amplified pfgch1. The rare T108 mutant associated with cycloguanil resistance showed predominance (60%) over N108 in the 2015–16 isolates. The observation of parasites with increased copy number of pfgch1 gene is indicative of the future evolution of the rare quadruple pfdhfr mutant, I51-R59-N108-L164, in Ghanaian parasites. Mutant pfdhps isolates also had increased gch1 copy number suggestive that it may also facilitate sulphadoxine resistance. The selection of parasites with pfgch1 gene amplification will enhance the sustenance and persistence of parasites with SP resistance in the country. Policy makers need to begin the search for a replacement chemoprophylaxis drug for malaria vulnerable groups in Ghana.

Molecular characterization of Plasmodium falciparum antifolate resistance markers in Thailand between 2008 and 2016

“…In this study, an association between pfgch1 gene multiple copy number and the pfdhfr-I164L mutation was also observed. However, in Ghana with low level of pfgch1 gene amplification (6%; 12/192), no dhfr-I164L was detected in samples with amplified pfgch1 [30]. The positive correlation between pfdhps (540E) and pfgch1 multiple copies observed in this study was not observed before.…”

The prevalence of molecular markers of resistance to sulfadoxine-pyrimethamine among pregnant women at first antenatal clinic attendance and delivery in the forest-savannah area of Ghana

“…There is no published data on the prevalence of resistance markers to SP among pregnant in the studied area. However, compared to results from samples previously collected from children in the study area, the current study showed a higher prevalence in Pfdhfr SNPs at codons 51, 59 and 108 than in samples collected in 2004 (51-66%) [27] but similar to results of samples collected in 2013-2014 (92-95%) [26], whiles the Pfdhfr triple mutant IRN also showed a marked increase from 31% in 2004 [27] to 81% in the current study. Similar to samples collected in 2013-2014, Pfdhps K540E which was not detected in 2004 was present at low levels, but no A581G was detected in the current study.…”

“…In Ghana, a few studies on SP resistance markers among pregnant women from different locations attending ANC showed IRN, IRN-SGKAA and IRN-SGEAA mutations covering a range from 71% to 80%, 12-41%, and 0-1% [23][24][25]. There are data for SP resistance markers in children resident in the forest-savannah zone of Ghana [26,27] but there are no published data on SP resistance markers in P. falciparum isolates obtained from pregnant women in this region.…”

Genetic diversity of the Plasmodium falciparum GTP-cyclohydrolase 1, dihydrofolate reductase and dihydropteroate synthetase genes reveals new insights into sulfadoxine-pyrimethamine antimalarial drug resistance

“…Furthermore, the location of breakpoints in areas of mono- or di-nucleotide repeats, encourage even more distinctive variations due to an AT-rich genome of P. falciparum . Nonetheless, there were minor exceptions, including in Cameroon, DRC, Kenya and Ghana [ 10 ], where both promoter and gene amplifications were present. Further, the extent of amplification copy number appears to be linked to geography.…”

“…Increased copy number of pfgch1 has been linked to SP resistance in Southeast Asia [ 8 ], with a direct association to pfdhfr and pfdhps alleles [ 9 ]. Similarly, a pfgch1 promoter copy number variation (amplification) in Malawian parasites with quintuple mutations (I51-R59-N108-G437-E540) has been identified, which differs from the whole gene amplification found in Southeast Asia [ 1 , 10 ]. The multiple copies of pfgch1 are thought to compensate for the putatively fitness-reducing mutations in pfdhfr and pfdhps by providing higher concentrations of upstream substrates in the folate-biosynthetic pathway [ 10 ].…”