Evaluation of parasite subpopulations and genetic diversity of the msp1, msp2 and glurp genes during and following artesunate monotherapy treatment of Plasmodium falciparum malaria in Western Cambodia

Abstract

Background: Despite widespread coverage of the emergence of artemisinin resistance, relatively little is known about the parasite populations responsible. The use of PCR genotyping around the highly polymorphic Plasmodium falciparum msp1, msp2 and glurp genes has become well established both to describe variability in alleles within a population of parasites, as well as classify treatment outcome in cases of recurrent disease. The primary objective was to assess the emergence of minority parasite clones during seven days of artesunate (AS) treatment in a location with established artemisinin resistance. An additional objective was to investigate whether the classification of clinical outcomes remained valid when additional genotyping was performed.

Methods: Blood for parasite genotyping was collected from 143 adult patients presenting with uncomplicated falciparum malaria during a clinical trial of AS monotherapy in Western Cambodia. Nested allelic type-specific amplification of the genes encoding the merozoite surface proteins 1 and 2 (msp1 and msp2) and the glutamate-rich protein (glurp) was performed at baseline, daily during seven days of treatment, and again at failure. Allelic variants were analysed with respect to the size of polymorphisms using Quantity One software to enable identification of polyclonal infections.

Results: Considerable variation of msp2 alleles but well-conserved msp1 and glurp were identified. At baseline, 31% of infections were polyclonal for one or more genes. Patients with recurrent malaria were significantly more likely to have polyclonal infections than patients without recurrence (seven of nine versus 36 of 127, p = 0.004). Emergence of minority alleles during treatment was detected in only one of twenty-three cases defined as being artemisinin resistant. Moreover, daily genotyping did not alter the final outcome classification in any recurrent cases.

Conclusions: The parasites responsible for artemisinin-resistant malaria in a clinical trial in Western Cambodia comprise the dominant clones of acute malaria infections rather than minority clones emerging during treatment. Additional genotyping during therapy was not beneficial. Disproportionately high rates of polyclonal infections in cases of recurrence suggest complex infections lead to poor treatment outcomes. Current research objectives should be broadened to include identification and follow-up of recurrent polyclonal infections so as to define their role as potential agents of emerging resistance.

Figure 1

Parasite genetic diversity detected by msp1, msp2 and glurp. Polymerase chain reaction (PCR) products were categorized into molecular weight groups differing by 10 bp. (A)msp1. Orange bars represent K1; brown bars, MAD-20; and yellow bars, RO33 allelic families. The 18 K1 alleles ranged in size from 100 to 330 bp; the 5 MAD-20 alleles; 150 to 320 bp; and the 1 RO33 alleles, 220-230 bp. (B)msp2. Red bars represent FC27 and black bars represent IC3D7 allelic families. The 18 FC27 alleles ranged in size from 170 to 820 bp, and the 33 IC3D7 alleles ranged in size from 290 to 870 bp. (C)glurp. The glurp alleles ranged in size from 660 to 1,090 bp. Dashed purple lines represent fragment size thresholds for each gene [10].

Figure 2

Pie charts showing genotype frequencies of msp1 and msp2 determined at baseline for fast parasite clearers (artemisinin sensitive) (n = 14), slow parasite clearers (n = 18), recurrences (n = 9), and for remaining (n = 102) and overall (n = 143) study populations. Key: orange, brown, yellow = msp1; dark green = no msp1; black, red = msp2; lime green = no msp2.

Figure 3

Parasite genotype determined daily in individual patients with malaria recurrence (Group C). Allele typing displaying size variation of 18 s alleles for msp1, msp2 and glurp in P. falciparum parasites isolated from nine patients showing the patterns of alleles present during seven days of AS monotherapy and on the day of falciparum malaria recurrence. Key: Bp = base pairs; PCT100 = duration of parasite clearance by microscopy; bands = separate clones for the same allele; dayR = day of malaria recurrence. Dashed lines represent asexual parasitaemia determined by microscopy. (A) Recrudescence cases. Case 66 received only three days of initial AS treatment due to development of neutropenia and completed anti-malarial therapy with an alternative short-acting drug. All remaining cases completed seven days of AS monotherapy. Gametocytes were seen by microscopy for cases 33 (Day 0-21), 80 (Days 35 and 42), and 147 (Days 21 and 42). Case 88 had two shared alleles on D_f plus additional msp1 and msp2 loci. Case 140 had one new allele of msp2 detected on Days 3 and 5 but not on Day 0 or D_f. One shared msp1 allele detected at D_f plus 1 new allele of glurp.(B) New infection. Case 112 new alleles of msp1, msp2 and glurp detected on D_f in addition to Plasmodium vivax.