A pinboard by
Angela Omondi

PhD Student, University of New Mexico


Phthalimide inhibition of Plasmodium falciparum in culture and P. berghei infection in mouse mode

We synthesized a series of phthalimide analogues and evaluated them as noteworthy growth inhibitors of Plasmodium falciparum (3D7 and W2) in culture. Analogues 6a, 6h and 6 u showed highest activity to inhibit the growth of the parasite with IC50 values in submicromolar range. Structure-activity correlation indicated the necessity of unsubstituted triazoles and leucine linker to obtain maximal growth inhibition of the parasite. Notably, phthalimide 6a and 6u selectively inhibited the ring-stage growth and parasite maturation. On other hand, phthalimide 6h displayed selective schizonticidal activity. Besides, they displayed synergistic interactions with chloroquine and dihydroartemisinin against parasite. Additional in vivo experiments using P. berghei infected mice showed that administration of 6h and 6u alone, as well as in combination with dihydroartemisinin, substantially reduced the parasite load. The high antimalarial activity of 6h and 6u, coupled with low toxicity advocate their potential role as novel antimalarial agents, either as standalone or combination therapies.


Five-year tracking of Plasmodium falciparum allele frequencies in a holoendemic area with indistinct seasonal transitions.

Abstract: The renewed malaria eradication efforts require an understanding of the seasonal patterns of frequency of polymorphic variants in order to focus limited funds productively. Although cross-sectional studies in holoendemic areas spanning a single year could be useful in describing parasite genotype status at a given point, such information is inadequate in describing temporal trends in genotype polymorphisms. For Plasmodium falciparum isolates from Kisumu District Hospital, Plasmodium falciparum chloroquine-resistance transporter gene (Pfcrt-K76T) and P. falciparum multidrug resistance gene 1 (PfMDR1-N86Y), were analyzed for polymorphisms and parasitemia changes in the 53 months from March 2008 to August 2012. Observations were compared with prevailing climatic factors, including humidity, rainfall, and temperature.Parasitemia (the percentage of infected red blood cells per total red blood cells) was established by microscopy for P. falciparum malaria-positive samples. P. falciparum DNA was extracted from whole blood using a Qiagen DNA Blood Mini Kit. Single nucleotide polymorphism identification at positions Pfcrt-K76T and PfMDR1-N86Y was performed using real-time polymerase chain reaction and/or sequencing. Data on climatic variables were obtained from http://www.tutiempo.net/en/.A total of 895 field isolates from 2008 (n=169), 2009 (n=161), 2010 (n=216), 2011 (n=223), and 2012 (n=126) showed large variations in monthly frequency of PfMDR1-N86Y and Pfcrt-K76T as the mutant genotypes decreased from 68.4%±15% and 38.1%±13% to 29.8%±18% and 13.3%±9%, respectively. The mean percentage of parasitemia was 2.61%±1.01% (coefficient of variation 115.86%; n=895). There was no correlation between genotype or parasitemia and climatic factors.This study shows variability in the frequency of Pfcrt-K76T and PfMDR1-N86Y polymorphisms during the study period, bringing into focus the role of cross-sectional studies in describing temporal genotype trends. The lack of correlation between genotypes and climatic changes, especially precipitation, emphasizes the cost of investment in genotype change.

Pub.: 15 Nov '14, Pinned: 18 Aug '17

Reduced in vitro doxycycline susceptibility in plasmodium falciparum field isolates from Kenya is associated with PfTetQ KYNNNN sequence polymorphism.

Abstract: Doxycycline is widely used for malaria prophylaxis by international travelers. However, there is limited information on doxycycline efficacy in Kenya, and genetic polymorphisms associated with reduced efficacy are not well defined. In vitro doxycycline susceptibility profiles for 96 Plasmodium falciparum field isolates from Kenya were determined. Genetic polymorphisms were assessed in P. falciparum metabolite drug transporter (Pfmdt) and P. falciparum GTPase tetQ (PftetQ) genes. Copy number variation of the gene and the number of KYNNNN amino acid motif repeats within the protein encoded by PftetQ were determined. Reduced in vitro susceptibility to doxycycline was defined by 50% inhibitory concentrations (IC50s) of ≥35,000 nM. The odds ratio (OR) of having 2 PfTetQ KYNNNN amino acid repeats in isolates with IC50s of >35,000 nM relative to those with IC50s of <35,000 nM is 15 (95% confidence interval [CI], 3.0 to 74.3; P value of <0.0002). Isolates with 1 copy of the Pfmdt gene had a median IC50 of 6,971 nM, whereas those with a Pfmdt copy number of >1 had a median IC50 of 9,912 nM (P = 0.0245). Isolates with 1 copy of PftetQ had a median IC50 of 6,370 nM, whereas isolates with a PftetQ copy number of >1 had a median IC50 of 3,422 nM (P < 0.0007). Isolates with 2 PfTetQ KYNNNN motif repeats had a median IC50 of 26,165 nM, whereas isolates with 3 PfTetQ KYNNNN repeats had a median IC50 of 3,352 nM (P = 0.0023). PfTetQ sequence polymorphism is associated with a reduced doxycycline susceptibility phenotype in Kenyan isolates and is a potential marker for susceptibility testing.

Pub.: 30 Jul '14, Pinned: 18 Aug '17

Analysis of major genome loci underlying artemisinin resistance and pfmdr1 copy number in pre- and post-ACTs in western Kenya.

Abstract: Genetic analysis of molecular markers is critical in tracking the emergence and/or spread of artemisinin resistant parasites. Clinical isolates collected in western Kenya pre- and post- introduction of artemisinin combination therapies (ACTs) were genotyped at SNP positions in regions of strong selection signatures on chromosome 13 and 14, as described in Southeast Asia (SEA). Twenty five SNPs were genotyped using Sequenom MassArray and pfmdr1 gene copy number by real-time PCR. Parasite clearance half-life and in vitro drug sensitivity testing were performed using standard methods. One hundred twenty nine isolates were successfully analyzed. Fifteen SNPs were present in pre-ACTs isolates and six in post-ACTs. None of the SNPs showed association with parasite clearance half-life. Post-ACTs parasites had significantly higher pfmdr1 copy number compared to pre-ACTs. Seven of eight parasites with multiple pfmdr1 were post-ACTs. When in vitro IC50s were compared for parasites with single vs. multiple gene copies, only amodiaquine and piperaquine reached statistical significance. Data showed SNPs on chromosome 13 and 14 had different frequency and trend in western Kenya parasites compared SEA. Increase in pfmdr1 gene copy is consistent with recent studies in African parasites. Data suggests genetic signature of artemisinin resistance in Africa might be different from SEA.

Pub.: 07 Feb '15, Pinned: 18 Aug '17

Temporal trends in prevalence of Plasmodium falciparum molecular markers selected for by artemether-lumefantrine treatment in pre-ACT and post-ACT parasites in western Kenya.

Abstract: Artemether-lumefantrine (AL) became the first-line treatment for uncomplicated malaria in Kenya in 2006. Studies have shown AL selects for SNPs in pfcrt and pfmdr1 genes in recurring parasites compared to the baseline infections. The genotypes associated with AL selection are K76 in pfcrt and N86, 184F and D1246 in pfmdr1. To assess the temporal change of these genotypes in western Kenya, 47 parasite isolates collected before (pre-ACT; 1995-2003) and 745 after (post-ACT; 2008-2014) introduction of AL were analyzed. In addition, the associations of parasite haplotype against the IC50 of artemether and lumefantrine, and clearance rates were determined. Parasite genomic DNA collected between 1995 and 2014 was analyzed by sequencing or PCR-based single-base extension on Sequenom MassARRAY. IC50s were determined for a subset of the samples. One hundred eighteen samples from 2013 to 2014 were from an efficacy trial of which 68 had clearance half-lives. Data revealed there were significant differences between pre-ACT and post-ACT genotypes at the four codons (chi-square analysis; p < 0.0001). The prevalence of pfcrt K76 and N86 increased from 6.4% in 1995-1996 to 93.2% in 2014 and 0.0% in 2002-2003 to 92.4% in 2014 respectively. Analysis of parasites carrying pure alleles of K + NFD or T + YYY haplotypes revealed that 100.0% of the pre-ACT parasites carried T + YYY and 99.3% of post-ACT parasites carried K + NFD. There was significant correlation (p = 0.04) between lumefantrine IC50 and polymorphism at pfmdr1 codon 184. There was no difference in parasite clearance half-lives based on genetic haplotype profiles. This study shows there is a significant change in parasite genotype, with key molecular determinants of AL selection almost reaching saturation. The implications of these findings are not clear since AL remains highly efficacious. However, there is need to closely monitor parasite genotypic, phenotypic and clinical dynamics in response to continued use of AL in western Kenya.

Pub.: 04 Aug '15, Pinned: 18 Aug '17