Genetic polymorphisms in plasmodium falciparum associated with resistance to artemisnin combination therapy: complexity of infection during short-term culture and characterization of in-vitro sensitivity in Kampala Uganda
Nsobya, Samuel Lubwama
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Background Artemisinin-based combination therapy (ACT) is currently advocated in Africa as a means of improving treatment efficacy and slowing the development of drug resistance. However, the selection of resistant parasites, particularly to artemisinin partner drugs, remains a concern. In addition, evaluations of in vitro drug sensitivity of fresh clinical isolates have been limited for components of artemisinin-based combination therapies, and associations between in vitro measures, clinical outcomes, and genotypes are uncertain. We also know that P.falciparum infections are commonly polyclonal in many areas, and, since not all parasites successfully grow in culture, it is unclear how well in vitro culture represents the complexity of clinical infections. Objectives In this work, the following objectives were addressed: i) assessed changes in occurrence of various polymorphisms of a drug resistant allele’s of pfmdr-1 and pfcrt between baseline and new infections during therapy with artesunate-amodiaquine (AS/AQ). ii) Evaluate changes in complexity of infection (COI) during culture. iii) Assess the impact of various amodiaquine containing regimens on the sensitivity of recurrent P. falciparum isolates. iv) Determine in vitro sensitivity patterns of P. falciparum isolates in Uganda against various antimalarial drugs. v) evaluate associations between parasite genetic polymorphisms, in vitro drug sensitivity, and clinical outcomes after antimalarial combination therapy. Methods The following methods were used to address the above objectives: i) polymorphisms of the pfmdr-1 and pfcrt genes known to play a role in altered drug sensitivity to some antimalarials were analysed by PCR and restriction fragment length polymorphism. We compared results between pretreatment isolates (n=201) and in new infections (n=132) in (AS/AQ) identified by genotyping over 28 days of follow-up from samples collected in a randomized trial for the treatment of uncomplicated malaria in Tororo, Uganda. We also evaluated pfcrt haplotypes at positions 72-76 in 90 randomly selected pretreatment and 90 randomly selected new infections ii) To study COI, we placed into culture 211 samples from children with uncomplicated malaria in Kampala. Uganda. Of these samples, 98(46%) were successfully cultured for at least 9 days. We determined COI daily based on msp-2 polymorphisms. In 25 samples initially mixed which lost some of strains, known gene polymorphisms of dhps, dhfr and pfmdr-1 were analysed using restriction fragment polymorphism. iii) Using P. falciparum isolates obtained from a clinical trial comparing efficacy of (AQ/SP andAQ/AS or AL), the impact of AQ-containing therapies on the sensitivity of 61 P. falciparum isolates causing recurrent infections soon after prior therapy and changes in resistance-mediating polymorphisms of pfmdr-1 gene were assessed. iv) For the in vitro sensitivity studies, IC50s for 241 P. falciparum isolates obtained between 2006 and 2008 were analysed in Kampala using an HRP-2-based ELISA. Results The findings for each objective above were as follows: i) in our study of the selection of pfmdr-1 polymorphisms, AS/AQ arm selected for the mutant alleles 86Y, (91%(182/201) to 97%(128/132) (p=0.03) and 1246Y,83%(167/201) to 91%(120/132)(p=0.05):86Yand1246Y, 81.6%(164/201) to 90.2%(119/132) (p=0.04). In contrast, the prevalence of the wild type allele Y184 increased from pretreatment to new infection isolates (171/201, 85.1% to 122/132, 92.4%, p=0.04). Only wild type alleles were seen at positions 1034 and 1042. For pfcrt, the CVIET haplotype at positions 72-76 was seen in all 180 samples analyzed ii) in studies of in vitro cultures, COI decreased over 9 days of culture from 1.73 to 1.56. New strains appeared after day 0 in 20 (44%) out of 45 cultures. Strains disappeared after day 0 in 25(56%) of 45 cultures that were initially mixed; persisting strains more commonly had wild type dhfr(C59) and dhps(K540), and mutant pfmdr-1(86Y) sequences. iii) for the in vitro susceptibility studies, parasites from subjects previously treated with AQ/SP or AQ/AS within 12 weeks were less sensitive to AQ(n=18; mean IC50 62.9 nM; range 12.7-158.3 nM) than parasites from those not treated within 12 weeks (n=43; mean IC50 37.5 nM; p=0.022; range 6.3-184.7 nM) or only those in the treatment arm that did not contain AQ (n=20; mean IC50 28.8 nM; p=0.017; range 6.3-121.8 nM).The proportion of strains with polymorphisms expected to mediate diminished response to AQ (pfmdr-1 86Y and 1246Y) increased after prior AQ therapy, although differences were not significant. iv) in vitro sensitivities (geometric mean IC50 (nM), range) were assessed for: chloroquine(CQ)(101.1, 15.6-767,n=181); monodesethylamodiaquine(MDAQ)(66.4,6.5-312,n=206);quinine(QN)(94.4,15.4-761,n=196); lumefantrine(LUM)(0.51,0.19-29.4,n=200);piperaquine(PIP)(6.1,5-6.8,n=199); and dihydroartemisinin (DHA)(0.55, 0.13-4.8, n=212). Sensitivities were positively correlated between CQ, MDAQ, and QN (r=0.4-0.6; p<0.001 by Pearson’s correlation), but not for other comparisons. Sensitivities to CQ, MDAQ, and QN, but not to the other drugs, decreased over the course of the study. Considering common Pfmdr-1 polymorphisms, parasites highly sensitive to MDAQ, QN, and LM, but not PQ, were more likely to have wild type sequence at allele N86Y; there were no clear associations at Y184F or D1246Y Conclusion The following conclusions were made from this study: i) we report that although AS/AQ combination therapy is effective in clearing malaria parasites, in re-infections most of strains selected are associated with decreased response to AQ in Uganda. ii) for the first time in Uganda, we have developed a molecular research laboratory to study fresh clinical isolates of P. falciparum by performing culture, in vitro culture and molecular studies. iii) To the best our knowledge, this is the first assessment of complexity of infection during in vitro culture of freshly isolated malaria parasites. In drug efficacy trials, strains missed on day 0 and seen after treatment will be misclassified as new infections, thus understating levels of drug resistance. iv) Loss of strains during culture of freshly isolated malaria parasites may be due to diminished fitness of some drug resistant strains. v) These results suggest diminishing efficacy of AQcontaining combination regimens as they are increasingly used in Uganda. vi) The generated baseline in vitro sensitivity data for the first time in Uganda will be used as yard stick to monitor emerging early resistance to ACTs.