Drug Resistance Alleles Research Articles

DISTRIBUTION OF PLASMODIUM FALCIPARUM CHLOROQUINE RESISTANCE TRANSPORTER (PFCRT) AND PLASMODIUM FALCIPARUM MULTIDRUG RESISTANCE 1(PFMDR-1) GENES IN ILORIN NORTH-CENTRAL NIGERIA

Globally, malaria is a peculiar health challenge particularly in continent of Africa. The ease of developing resistance to anti-malaria drugs by Plasmodium falciparum is a serious impediment towards programs aim at control and elimination of malaria. Consequently, drug efficacy surveillance is desirable to institute adequate and effective treatment policies. In this study, distribution of these genes in Plasmodium falciparum isolates in Ilorin metropolis were investigated. One hundred and three samples collected randomly from 5 hospitals in Ilorin metropolis. The samples were amplified at codon 76 and 86 for Pfcrt and Pfmdr-1 respectively using PCR / Restricted Fragment Length Polymorphism (RFLP). The most prevalent alleles were drug susceptible alleles in the study area (K76 and N86) with 31% and 40% respectively, while drug resistance alleles (86Y) have 13% and the least prevalence 2% was the mixed alleles K76T. UITH has the highest number of susceptible alleles followed by CSC 38% and 46% for K76 and N86 respectively. There are significant differences in the distribution of these alleles and the study sites (P< 0.05). The prevalence of resistance alleles in the area is a welcome development that can be employed for possibility of re-introduction of CQ for the treatment of malaria in the study area which will serve as an advantage over expensive ACT due to fact that it is safe cheap and readily affordable.

Distribution of Plasmodium falciparum Chloroquine Resistance Transporter (Pfcrt) and Plasmodium falciparum Multidrug Resistance 1(Pfmdr-1) Genes in Ilorin North-central Nigeria

Malaria remains a major health problem globally, especially in Sub-Saharan Africa. The ease of developing resistance to anti-malaria drugs by Plasmodium falciparum is a serious challenge to malaria control and elimination programs. Consequently, continuous monitoring of drug efficacy is necessary to ascertain effective treatment policies. The study investigated the distribution pattern of these genes in Plasmodium falciparum isolates in Ilorin metropolis. One hundred and three samples were collected randomly from 5 hospitals in Ilorin metropolis. The samples were amplified at codon 76 and 86 for Pfcrt and Pfmdr-1 respectively using PCR/Restricted Fragment Length Polymorphism (RFLP). Drug susceptible alleles were most prevalent in the study area (K76 and N86) with 31% and 40% respectively, followed by drug resistance alleles (86Y) with 13%, and the least prevalent (2%) was the mixed alleles K76T. UITH has the highest number of susceptible alleles followed by CSC 38% and 46% for K76 and N86 respectively. There are significant differences in the distribution of these alleles and the study sites (P55< 0.05). The prevalence of resistance alleles in the area is a welcome development that can be employed for the possible re-introduction of CQ to treat malaria in the study area. It will be an advantage over expensive ACT because it is safe, cheap, and readily affordable.

C646 degrades Exportin-1 to modulate p300 chromatin occupancy and function

Molecular glues can induce proximity between a target protein and ubiquitin ligases to induce target degradation, but strategies for their discovery remain limited. We screened 3,200 bioactive small molecules and identified that C646 requires neddylation-dependent protein degradation to induce cytotoxicity. Although the histone acetyltransferase p300 is the canonical target of C646, we provide extensive evidence that C646 directly targets and degrades Exportin-1 (XPO1). Multiple cellular phenotypes induced by C646 were abrogated in cells expressing the known XPO1C528S drug-resistance allele. While XPO1 catalyzes nuclear-to-cytoplasmic transport of many cargo proteins, it also directly binds chromatin. We demonstrate that p300 and XPO1 co-occupy hundreds of chromatin loci. Degrading XPO1 using C646 or the known XPO1 modulator S109 diminishes the chromatin occupancy of both XPO1 and p300, enabling direct targeting of XPO1 to phenocopy p300 inhibition. This work highlights the utility of drug-resistant alleles and further validates XPO1 as a targetable regulator of chromatin state.

Sub-microscopic Plasmodium falciparum infections and multiple drug resistant single nucleotide polymorphic alleles in pregnant women from southwestern Nigeria

ObjectivesThe study evaluated sub-microscopic malaria infections in pregnancy using two malaria Rapid Diagnostic Tests (mRDTs), microscopy and RT-PCR and characterized Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and Plasmodium falciparum dihydropteroate synthase (Pfdhps) drug resistant markers in positive samples.MethodsThis was a cross sectional survey of 121 pregnant women. Participants were finger pricked, blood drops were collected for rapid diagnosis with P. falciparum histidine-rich protein 11 rapid diagnostic test kit and the ultra-sensitive Alere Pf malaria RDT, Blood smears for microscopy and dried blood spots on Whatman filter paper for molecular analysis were made. Real time PCR targeting the var acidic terminal sequence (varATS) gene of P. falciparum was carried out on a CFX 96 real time system thermocycler (BioRad) in discriminating malaria infections. For each run, laboratory strain of P. falciparum 3D7 and nuclease free water were used as positive and negative controls respectively. Additionally, High resolution melt analyses was employed for genotyping of the different drug resistance markers.ResultsOut of one hundred and twenty-one pregnant women sampled, the SD Bioline™ Malaria Ag P.f HRP2-based malaria rapid diagnostic test (mRDT) detected eight (0.06%) cases, the ultra-sensitive Alere™ malaria Ag P.f rapid diagnostic test mRDT had similar outcome in the same samples as detected by the HRP2-based mRDT. Microscopy and RT-PCR confirmed four out of the eight infections detected by both rapid diagnostic tests as true positive and RT-PCR further detected three false negative samples by the two mRDTs providing a sub-microscopic malaria prevalence of 3.3%. Single nucleotide polymorphism in Pfdhps gene associated with sulphadoxine resistance revealed the presence of S613 mutant genotypes in three of the seven positive isolates and isolates with mixed wild/mutant genotype at codon A613S. Furthermore, four mixed genotypes at the A581G codon were also recorded while the other Pfdhps codons (A436G, A437G and K540E) showed the presence of wild type alleles. In the Pfdhfr gene, there were mutations in 28.6%, 28.6%, and 85.7% at the I51, R59 and N108 codons respectively. Mixed wild and mutant type genotypes were also observed in 28.6% each of the N51I, and C59R codons. For the Pfcrt, two haplotypes CVMNK and CVIET were observed. The SVMNT was altogether absent. Triple mutant CVIET 1(14.3%) and triple mutant + wild genotype CVIET + CVMNK 1(14.3%) were observed. The Pfmdr1 haplotypes were single mutants YYND 1(14.3%); NFND 1(14.3%) and double mutants YFND 4(57.1%); YYDD 1(14.3%).

Antimalarial drug resistance profiling of Plasmodium falciparum infections in India using Ion Torrent deep sequencing

IntroductionTracking the emergence and spread of antimalarial drug resistance is critical for supporting progress towards the control and eventual elimination of malaria in South Asia, especially India. Plasmodium falciparum has evolved resistance to virtually every antimalarial drug, and significant progress has been made to identify the molecular genetic mechanisms involved in the most common types of resistance.MethodsAn amplicon sequencing protocol was used for molecular surveillance of antimalarial drug resistance in a total of 158 patient isolates collected from December 2012 to September 2015 from three sites in south, west and east India: Tamil Nadu, Gujarat, and Odisha respectively. Five full length Plasmodium falciparum genes whose mutant proteins are implicated in antimalarial drug resistance were investigated: Pfcrt for chloroquine, Pfdhfr for pyrimethamine, Pfdhps for sulfadoxine, Pfk13 for artemisinin and Pfmdr1 for resistance to multiple antimalarial drugs.ResultsWe observed a high proportion of wild-type Pfcrt and Pfdhfr haplotypes from the P. falciparum-dominant site Rourkela, while mutant Pfcrt and Pfdhfr haplotypes were fixed at the P. vivax dominant sites Chennai and Nadiad. The wild-type Pfdhps haplotype was predominant across all study sites. We identified mutations in the propeller domain of Pfk13, although they are not associated with resistance to artemisinin. Finally, using samples taken from the same patient on day 2, day 7, and day 14 after artemisinin combination treatment, we were able to observe changes in allele frequency of drug resistance genes during the course of an infection.DiscussionThis is the first high-throughput deep sequencing study of five full-length P. falciparum genes in clinical isolates from three different study sites in India with varying transmission ecologies. Amplicon sequencing based on ion-torrent has the potential to track changes in the frequency of drug resistant alleles as a patient is undergoing drug therapy, and to identify new resistance mutations as they increase in frequency in the patient. This study showed possibility of whole gene sequencing, leads to in-depth molecular surveillance of multiple antimalarial resistant candidates and furthermore suggests investigations on reversal of resistant genotypes due to usage of artemisinin combination therapy in P. falciparum prevalent areas of the country.

Drug resistance markers in Plasmodium vivax isolates from a Kanchanaburi province, Thailand between January to May 2023.

Plasmodium vivax has become the predominant species in the border regions of Thailand. The emergence and spread of antimalarial drug resistance in P. vivax is one of the significant challenges for malaria control. Continuous surveillance of drug resistance is therefore necessary for monitoring the development of drug resistance in the region. This study aims to investigate the prevalence of the mutation in the P. vivax multidrug resistant 1 (Pvmdr1), dihydrofolate reductase (Pvdhfr), and dihydropteroate synthetase (Pvdhps) genes conferred resistance to chloroquine (CQ), pyrimethamine (P) and sulfadoxine (S), respectively. 100 P. vivax isolates were obtained between January to May 2023 from a Kanchanaburi province, western Thailand. Nucleotide sequences of Pvmdr1, Pvdhfr, and Pvdhps genes were amplified and sequenced. The frequency of single nucleotide polymorphisms (SNPs)-haplotypes of drug-resistant alleles was assessed. The linkage disequilibrium (LD) tests were also analyzed. In Pvmdr1, T958M, Y976F, and F1076L, mutations were detected in 100%, 21%, and 23% of the isolates, respectively. In Pvdhfr, the quadruple mutant allele (I57R58M61T117) prevailed in 84% of the samples, followed by (L57R58M61T117) in 11%. For Pvdhps, the double mutant allele (G383G553) was detected (48%), followed by the triple mutant allele (G383M512G553) (47%) of the isolates. The most prevalent combination of Pvdhfr (I57R58M61T117) and Pvdhps (G383G553) alleles was sextuple mutated haplotypes (48%). For LD analysis, the association in the SNPs pairs was found between the intragenic and intergenic regions of the Pvdhfr and Pvdhps genes. The study has recently updated the high prevalence of three gene mutations associated with CQ and SP resistance. Genetic monitoring is therefore important to intensify in the regions to further assess the spread of drug resistant. Our data also provide evidence on the distribution of drug resistance for the early warning system, thereby threatening P. vivax malaria treatment policy decisions at the national level.

Geographical emergence of sulfadoxine-pyrimethamine drug resistance-associated P. falciparum and P. malariae alleles in co-existing Anopheles mosquito and asymptomatic human populations across Cameroon.

Malaria molecular surveillance remains critical in detecting and tracking emerging parasite resistance to anti-malarial drugs. The current study employed molecular techniques to determine Plasmodium species prevalence and characterize the genetic diversity of Plasmodium falciparum and Plasmodium malariae molecular markers of sulfadoxine-pyrimethamine resistance in humans and wild Anopheles mosquito populations in Cameroon. Anopheles mosquito collections and parasitological survey were conducted in villages to determine Plasmodium species infection, and genomic phenotyping of anti-folate resistance was accomplished by sequencing the dihydrofolate-reductase (dhfr) and dihydropteroate-synthase (dhps) genes of naturally circulating P. falciparum and P. malariae isolates. The malaria prevalence in Elende was 73.5% with the 5-15 years age group harboring significant P. falciparum (27%) and P. falciparum + P. malariae (19%) infections. The polymorphism breadth of the pyrimethamine-associated Pfdhfr marker revealed a near fixation (94%) of the triple-mutant -A16I51R59N108I164. The Pfdhps backbone mediating sulfadoxine resistance reveals a high frequency of the V431A436G437K540A581A613 alleles (20.8%). Similarly, the Pmdhfr N50K55L57R58S59S114F168I170 haplotype (78.4%) was predominantly detected in the asexual blood stage. In contrast, the Pmdhps- S436A437occured at 37.2% frequency. The combined quadruple N50K55L57R58S59S114F168I170_ S436G437K540A581A613 (31.9%) was the major circulating haplotype with similar frequency in humans and mosquitoes. This study highlights the increasing frequency of the P. malariae parasite mostly common in asymptomatic individuals with apparent P. falciparum infection. Interventions directed at reducing malaria transmission such as the scaling-up of SP are favoring the emergence and spread of multiple drug-resistant alleles between the human and mosquito host systems.

Comparison of CX-4945 and SGC-CK2-1 as inhibitors of CSNK2 using quantitative phosphoproteomics: Triple SILAC in combination with inhibitor-resistant CSNK2

Specificity is a limiting factor when using small-molecule inhibitors to study protein kinase signalling. Since inhibitor-resistant kinase mutants (i.e., drug-resistant alleles) remain active in the presence of inhibitor, they facilitate validation of on-target effects. By combining an inhibitor-resistant kinase mutant with mass spectrometry-based phosphoproteomics, we previously devised a systematic strategy for reliable identification and validation of CSNK2 substrates. In this study, we use the same strategy to evaluate the selectivity of CX-4945, a clinical stage CSNK2 inhibitor, and SGC-CK2-1, a chemical probe selectively targeting CSNK2. Human osteosarcoma (U2OS) cells expressing exogenous wild-type CSNK2A1 (WT) or an inhibitor-resistant triple mutant (TM, V66A/H160D/I174A) were treated with CX-4945 or SGC-CK2-1 prior to analysis using triple SILAC (phospho)proteomics. The minority of phosphosites, 15% at 4 ​h and 5% at 24 ​h, that were significantly downregulated in response to CX-4945 treatment were determined to be CSNK2A1-dependent. By comparison, the majority of phosphosites, >55% at both 4 and 24 ​h, that were significantly downregulated in response to SGC-CK2-1 were identified as CSNK2A1-dependent. This indicates that SGC-CK2-1 exhibits significantly greater selectivity towards CSNK2A1 than CX-4945. Notably, utilization of SGC-CK2-1 in cells expressing CSNK2A1-TM enabled the identification of >300 CSNK2A1-dependent phosphosites. Overall, this study highlights the utility of exploiting highly selective chemical probes together with inhibitor-resistant kinase mutants to facilitate identification of bona fide kinase substrates.

Efficient generation of mNeonGreen Plasmodium falciparum reporter lines enables quantitative fitness analysis.

CRISPR editing has enabled the rapid creation of fluorescent Plasmodium transgenic lines, facilitating a deeper understanding of parasite biology. The impact of genetic perturbations such as gene disruption or the introduction of drug resistance alleles on parasite fitness is typically quantified in competitive growth assays between the query line and a wild type reference. Although fluorescent reporter lines offer a facile and frequently used method to measure relative growth, this approach is limited by the strain background of the existing reporter, which may not match the growth characteristics of the query strains, particularly if these are slower-growing field isolates. Here, we demonstrate an efficient CRISPR-based approach to generate fluorescently labelled parasite lines using mNeonGreen derived from the LanYFP protein in Branchiostoma lanceolatum, which is one of the brightest monomeric green fluorescent proteins identified. Using a positive-selection approach by insertion of an in-frame blasticidin S deaminase marker, we generated a Dd2 reporter line expressing mNeonGreen under the control of the pfpare (P. falciparum Prodrug Activation and Resistance Esterase) locus. We selected the pfpare locus as an integration site because it is highly conserved across P. falciparum strains, expressed throughout the intraerythrocytic cycle, not essential, and offers the potential for negative selection to further enrich for integrants. The mNeonGreen@pare line demonstrates strong fluorescence with a negligible fitness defect. In addition, the construct developed can serve as a tool to fluorescently tag other P. falciparum strains for in vitro experimentation.

Discovering new biology with drug-resistance alleles.

Small molecule drugs form the backbone of modern medicine's therapeutic arsenal. Often less appreciated is the role that small molecules have had in advancing basic biology. In this Review, we highlight how resistance mutations have unlocked the potential of small molecule chemical probes to discover new biology. We describe key instances in which resistance mutations and related genetic variants yielded foundational biological insight and categorize these examples on the basis of their role in the discovery of novel molecular mechanisms, protein allostery, physiology and cell signaling. Next, we suggest ways in which emerging technologies can be leveraged to systematically introduce and characterize resistance mutations to catalyze basic biology research and drug discovery. By recognizing how resistance mutations have propelled biological discovery, we can better harness new technologies and maximize the potential of small molecules to advance our understanding of biology and improve human health.

The Rare, the Best: Spread of Antimalarial-Resistant Plasmodium falciparum Parasites by Anopheles Mosquito Vectors.

ABSTRACTThe emergence of resistance to antimalarials has prompted the steady switch to novel therapies for decades. Withdrawal of antimalarials, such as chloroquine in sub-Saharan Africa in the late 1990s, led to rapid declines in the prevalence of resistance markers after a few years, raising the possibility of reintroducing them for malaria treatment. Here, we provide evidence that the mosquito vector plays a crucial role in maintaining parasite genetic diversity. We followed the transmission dynamics of Plasmodium falciparum parasites through its vector in natural infections from gametocytes contained in the blood of asymptomatic volunteers until sporozoites subsequently developed in the mosquito salivary glands. We did not find any selection of the mutant or wild-type pfcrt 76 allele during development in the Anopheles mosquito vector. However, microsatellite genotyping indicated that minority genotypes were favored during transmission through the mosquito. The analysis of changes in the proportions of mutant and wild-type pfcrt 76 alleles showed that, regardless of the genotype, the less-represented allele in the gametocyte population was more abundant in mosquito salivary glands, indicating a selective advantage of the minority allele in the vector. Selection of minority genotypes in the vector would explain the persistence of drug-resistant alleles in the absence of drug pressure in areas with high malaria endemicity and high genetic diversity. Our results may have important epidemiological implications, as they predict the rapid re-emergence and spread of resistant genotypes if antimalarials that had previously selected resistant parasites are reintroduced for malaria prevention or treatment.IMPORTANCE Drug selection pressure in malaria patients is the cause of the emergence of resistant parasites. Resistance imposes a fitness cost for parasites in untreated infections, so withdrawal of the drug leads to the return of susceptible parasites. Little is known about the role of the malaria vector in this phenomenon. In an experimental study conducted in Cameroon, an area of high malaria transmission, we showed that the vector did not favor the parasites based on sensitivity or resistance criteria, but it did favor the selection of minority clones. This finding shows that the vector increases the diversity of plasmodial populations and could play an important role in falciparum malaria epidemiology by maintaining resistant clones despite the absence of therapeutic pressure.

Genomic analysis reveals independent evolution of Plasmodium falciparum populations in Ethiopia

BackgroundPlasmodium falciparum parasite populations in Ethiopia have been experiencing local selective pressures from drugs and immunity, leading to evolutionary adaptation. However, there was a paucity of data on genomic characterization and evolutionary adaptations of P. falciparum isolates from the central area of Ethiopia.MethodsWhole-genome analysis of 25 P. falciparum isolates from central Ethiopia, specifically from West Arsi, were studied to determine their genetic diversity, population structures, and signatures of selection in known drug resistance alleles against global isolates from Cambodia, Thailand, DR Congo, and Malawi.ResultsA total of 18,517 high-quality single-nucleotide polymorphisms (SNPs) were identified in Ethiopian P. falciparum isolates. About 84% of the Ethiopian P. falciparum isolates had a FWS value > 0.95 showing a dominant single genotype infection in most isolates at the time of collection with little potential for out-crossing as expected in areas with low transmission intensity. Within-host diversity of Ethiopian infections was significantly different from East African (p < 0.001), but not Southeast Asian infections (P > 0.05). A significant population structure has been observed by PCA and population differentiation between Ethiopian parasites and East African (Fst ~ 10%) and Southeast Asian populations (Fst ~ 18%), suggesting limited gene flow and the independent evolution of the Ethiopian parasite population. Moreover, a total of 125 genes under balancing selection was found that include ama1, trap, eba175, and lsa3, previously identified as targets of human host immunity. Recent directional selection analysis using integrated standardized haplotype score (IHS) did not detect any selection signatures in the Pfcrt, Pfdhfr, Pfdhps, Pfmdr1, and PfK13 genes. However, known drug resistance-conferring mutations analysis showed that at least one SNP marker was fixed in these genes, but not in Pfdhps and PfK13.ConclusionPlasmodium falciparum populations in the central region of Ethiopia was structurally diverged from both Southeast Asian and other East African populations. Malaria infections in Ethiopia had low within-host diversity, and parasites carry fixed chloroquine resistance markers despite the withdrawal of this drug for the treatment of P. falciparum.

691. Results of Repeat HIV-1 DNA Resistance Tests Are Highly Concordant

BackgroundDHHS guidelines recommend caution when interpreting HIV-1 DNA resistance testing because not all previously identified drug resistance mutations (DRMs) may be captured. Comparison of multiple reports from the same patient was performed to assess the ability of HIV-1 DNA testing to consistently identify wild-type and drug resistance alleles.MethodsPatients with 3 HIV-1 DNA resistance tests (trios) and corresponding HIV-1 viral load (VL) measurements within ~3 months of each resistance test were identified in a commercial database. Concordance among trio test results was assessed for each patient. VL and trio timespan were evaluated for impact on concordance using one-way ANOVA with post-hoc analyses.ResultsFifty-five patients with test trios were identified for analysis. Average patient age was 53 years, and 88% were male. All 3 tests within 26/55 trios were associated with VL< 200 copies/mL. The average testing timespan was 100 weeks (range 17-228 weeks). Wild-type virus was identified on 3/3 reports for 11/55 (20%) patients. Among resistant viruses, DRMs were identified on average 70%, 20% and 10% on 3/3, 2/3 and 1/3 of HIV-1 DNA reports, respectively. M184V was identified on 3/3, 2/3 and 1/3 reports among 14/17, 2/17, and 1/17 test trios, respectively. K103N was identified on 3/3 and 2/3 reports among 11/12 and 1/12 test trios, respectively. The redetection rate following an initial HIV-1 DNA test was high for M184V (30/34, 88%) and K103N (23/24, 96%).Of 178 DRMs detected across all initial HIV-1 DNA tests, 17 (9.6%) were not detected on the second, but redetected on the third test, including M184V and K103N in one trio each. The average concordance among test trios across all drug classes was 97%. No correlation between VL at time of testing and DRM redetection rates was observed. Significantly fewer DRMs were recaptured when repeat testing was performed > 24 months after the initial test.ConclusionRepeat HIV-1 DNA drug resistance testing reliably detected archived DRMs. DRM decay related to turnover of the viral reservoir may explain some discordance between repeat HIV-1 DNA tests.DisclosuresDusica Curanovic, PhD, Monogram Biosciences (Employee) Suqin Cai, n/a, Monogram Biosciences (Employee) Jonathan Toma, n/a, Monogram Biosciences (Employee) Christos J. Petropoulos, PhD, Monogram Biosciences (Employee) Charles M. Walworth, MD, Monogram Biosciences (Employee)

Multi-locus genotyping reveals established endemicity of a geographically distinct Plasmodium vivax population in Mauritania, West Africa.

Plasmodium vivax has been recently discovered as a significant cause of malaria in Mauritania, although very rare elsewhere in West Africa. It has not been known if this is a recently introduced or locally remnant parasite population, nor whether the genetic structure reflects epidemic or endemic transmission. To investigate the P. vivax population genetic structure in Mauritania and compare with populations previously analysed elsewhere, multi-locus genotyping was undertaken on 100 clinical isolates, using a genome-wide panel of 38 single nucleotide polymorphisms (SNPs), plus seven SNPs in drug resistance genes. The Mauritanian P. vivax population is shown to be genetically diverse and divergent from populations elsewhere, indicated consistently by genetic distance matrix analysis, principal components analyses, and fixation indices. Only one isolate had a genotype clearly indicating recent importation, from a southeast Asian source. There was no linkage disequilibrium in the local parasite population, and only a small number of infections appeared to be closely genetically related, indicating that there is ongoing genetic recombination consistent with endemic transmission. The P. vivax diversity in a remote mining town was similar to that in the capital Nouakchott, with no indication of local substructure or of epidemic population structure. Drug resistance alleles were virtually absent in Mauritania, in contrast with P. vivax in other areas of the world. The molecular epidemiology indicates that there is long-standing endemic transmission that will be very challenging to eliminate. The virtual absence of drug resistance alleles suggests that most infections have been untreated, and that this endemic infection has been more neglected in comparison to P. vivax elsewhere.

Assessment of antimalarial drug resistant markers in asymptomatic Plasmodium falciparum infections after 4 years of indoor residual spraying in Northern Ghana.

BackgroundDrug resistance remains a concern for malaria control and elimination. The effect of interventions on its prevalence needs to be monitored to pre-empt further selection. We assessed the prevalence of Plasmodium falciparum gene mutations associated with resistance to the antimalarial drugs: sulfadoxine-pyrimethamine (SP), chloroquine (CQ) and artemisinin combination therapy (ACTs) after the scale-up of a vector control activity that reduced transmission.MethodsA total of 400 P. falciparum isolates from children under five years were genotyped for seventeen single nucleotide polymorphisms (SNPs) in pfcrt, pfmdr1, pfdhfr, pfdhps and pfk13 genes using polymerase chain reaction (PCR) and high resolution melting (HRM) analysis. These included 80 isolates, each randomly selected from cross-sectional surveys of asymptomatic infections across 2010 (baseline), 2011, 2012, 2013 (midline: post-IRS) and 2014 (endline: post-IRS) during the peak transmission season, when IRS intervention was rolled out in Bunkpurugu Yunyoo (BY) District, Ghana. The proportions of isolates with drug resistant alleles were assessed over this period.ResultsThere were significant decreases in the prevalence of pfdhfr- I51R59N108 haplotype from 2010 to 2014, while the decline in pfdhfr/pfdhps- I51R59N108G437 during the same period was not significant. The prevalence of lumefantrine (LM), mefloquine (MQ) and amodiaquine (AQ) resistance-associated haplotypes pfmdr1-N86F184D1246 and pfmdr1-Y86Y184Y1246 showed decreasing trends (z = -2.86, P = 0.004 and z = -2.71, P = 0.007, respectively). Each of pfcrt-T76 and pfmdr1-Y86 mutant alleles also showed a declining trend in the asymptomatic reservoir, after the IRS rollout in 2014 (z = -2.87, P = 0.004 and z = -2.65, P = 0.008, respectively). Similarly, Pyrimethamine resistance mediating polymorphisms pfdhfr-N108, pfdhfr-I51 and pfdhfr-R59 also declined (z = -2.03, P = 0.042, z = -3.54, P<0.001 and z = -4.63, P<0.001, respectively), but not the sulphadoxine resistance mediating pfdhps-G437 and pfdhps-F436 (z = -0.36, P = 0.715 and z = 0.41, P = 0.684, respectively). No mutant pfk13-Y580 were detected during the study period.ConclusionThe study demonstrated declining trends in the prevalence of drug resistant mutations in asymptomatic P. falciparum infections following transmission reduction after an enhanced IRS intervention in Northern Ghana.

Genetic Polymorphisms of Pfcrt K76T and Pfmdr1 N86Y among Asymptomatic School Children in Forest Communities of Ekondo Titi Subdivision along the Cameroon-Nigeria Border Area

Aims: The study sought to quantify Plasmodium infection and molecular markers for chloroquine resistance among asymptomatic school children.&#x0D; Study Design: The study was cross-sectional.&#x0D; Place and Duration of Study: The study was carried out in Ekondo Titi Subdivision near Cameroon's south-western border with Nigeria from March to May and from September to October 2014.&#x0D; Methodology: The prevalence of human Plasmodium species was determined by nested PCR (Polymerase Chain Reaction) using DNA from dried blood spot in six primary schools. A PCR/RFLP analysis (Restriction Fragment Length Polymorphism) was used to determine the prevalence of chloroquine resistance (CQR) associated pfcrt 76T and pfmdr 1 56Y point mutations in Plasmodium falciparum asymptomatic school children.&#x0D; Results: A nested PCR amplifying the 18S small-subunit ribosomal RNA (SSU rRNA) gene of Plasmodium in 205 samples confirmed 76.1% of the isolates as asymptomatic P. falciparum infections, with a substantial proportion 22% of P. malariae infection. Among these, 3.6% were single P. malariae infections and 15.1% were P. falciparum and P. malariae mixed infections. Mixed P. falciparum and P. ovale infections were 2.0%. Of the 156 Plasmodium falciparum, positive samples by species-specific PCR, 107 samples with P. falciparum mono-infection were analyzed for the presence of drug resistant alleles pfcrt 76T and pfmdr1- Y 86. The prevalence of pfcrt 76T mutation (74.6%) was higher than that of the pfmdr1-Y86 mutation (25.4%). Logistic regression analysis of socio-demographic factors predicted no significant association between pfcrt 76T mutation with gender and communities.&#x0D; Conclusions: The results indicated a high prevalence of P. malariae and mixed infection in the area under study. The high-level distribution of the pfcrtT76 observed in the study could be possibly attributed to the fact that CQ remained widely used at the community level more than 14 years after withdrawal.

High-Complexity Plasmodium falciparum Infections, North Central Nigeria, 2015-2018.

The mass migration that occurred during 2009–2013 and after the insurgency in northeastern Nigeria could have increased malaria incidence and Plasmodium falciparum genetic diversity in North Central Nigeria. To determine P. falciparum sequence diversity in this region, we screened 282 samples collected in regional clinics during 2015–2018 for Plasmodium spp. and, with positive samples, determined P. falciparum infection complexity and allele diversity using PCR. Of 34 P. falciparum–positive samples, 39 msp1, 31 msp2, and 13 glurp alleles were detected, and 88% of infections were polyclonal. We identified trimorphic and dimorphic allele combinations in a high percentage of samples, indicative of a high infection complexity in the study population. High genetic diversity is a catalyst for the evolution of drug-resistant alleles. Improved measures (e.g., better drug quality, diagnostics) are needed to control P. falciparum transmission and reduce the potential for the emergence of drug resistance in Nigeria.

Prevalence of chloroquine and antifolate drug resistance alleles in Plasmodium falciparum clinical isolates from three areas in Ghana.

Background: The emergence and spread of resistance in Plasmodium falciparum to chloroquine (CQ) necessitated the change from CQ to artemisinin-based combination therapies (ACTs) as first-line drug for the management of uncomplicated malaria in Ghana in 2005. Sulphadoxine-pyrimethamine (SP) which was the second line antimalarial drug in Ghana, was now adopted for intermittent preventive treatment of malaria in pregnancy (IPTp). Methods: To examine the prevalence of molecular markers associated with CQ and antifolate drug resistance in Ghana, we employed restriction fragment length polymorphism polymerase chain reaction to genotype and compare single nucleotide polymorphisms (SNPs) in the P. falciparum chloroquine resistance transporter ( pfcrt, PF3D7_0709000), multidrug resistance ( pfmdr1, PF3D7_0523000), bifunctional dihydrofolate reductase-thymidylate synthase ( pfdhfr, PF3D7_0417200) and dihydropteroate synthase ( pfdhps, PF3D7_0810800) genes. Parasites were collected from children with malaria reporting to hospitals in three different epidemiological areas of Ghana (Accra, Kintampo and Navrongo) in 2012-2013 and 2016-2017. Results: The overall prevalence of the CQ resistance-associated pfcrt 76T allele was 8%, whereas pfmdr1 86Y and 184F alleles were present in 10.2% and 65.1% of infections, respectively. The majority of the isolates harboured the antifolate resistance-associated pfdhfr alleles 51I (83.4%), 59R (85.9 %) and 108N (90.5%). Pfdhps 437G and 540E were detected in 90.6% and 0.7% of infections, respectively. We observed no significant difference across the three study sites for all the polymorphisms except for pfdhps 437G , which was more common in Accra compared to Kintampo for the 2016-2017 isolates. Across both pfdhfr and pfdhps genes, a large proportion (61%) of the isolates harboured the quadruple mutant combination ( I 51 R 59 N 108/ G 437). CQ resistance alleles decreased during the 12 years after CQ withdrawal, but an mediate SP resistance alleles increased. Conclusion: Surveillance of the prevalence of resistance alleles is necessary in monitoring the efficacy of antimalarial drugs.

High-Density CRISPR Scan Identifies Functional Regions of DOT1L That Mediate Therapeutic Response in MLL-r Leukemia

Leukemias containing Mixed Lineage Leukemiagene rearrangement (MLL-r) account for 5-10% of human acute leukemia cases and are associated with a poor prognosis. The unmet clinical need and the lack of an effective targeted therapy emphasizes the need for novel approaches for these malignancies.Recent studies have discovered an essential role for the histone H3 lysine 79 (H3K79) methyltransferase DOT1L in the maintenance of MLL-r leukemias. Phase-I clinical trials (NCT01684150 and NCT02141828) have demonstrated the safety and clinical activity of the DOT1L-specific small molecule inhibitor EPZ5676 (Pinometostat, Epizyme Inc.). However, the variable response of the MLL-r patients in these clinical trials indicates the need to further understand the mechanism of action and resistance to DOT1L inhibitors.To address this issue, we designed a high-density CRISPR screen approach (Fig 1) to dissect the function of Dot1l coding regions in mouse MLL-AF9 leukemic cells, a well-established mouse model that mimics human MLL-r acute myeloid leukemia (AML). We constructed a library of 602 sgRNA that targets most of the “NGG” protospacer adjacent motifs (PAM) within Dot1l exons. The average targeting density is 7.7 bp per sgRNA (i.e. 2.5 a.a. per sgRNA). We delivered this sgRNA library into Cas9-expressing MLL-AF9 AML cells using lentiviral transduction, and then compared the frequencies of each integrated sgRNA sequence before vs. after 12 days of culture using high-throughput sequencing (NextSeq, Illumina Inc.). The results revealed a significant depletion of clusters of sgRNA targeting the known functional regions including the lysine methyltransferase (KMT) core and the AF9-binding domains of DOT1L. We mapped the CRISPR scan score to the previously solved three-dimensional structures of DOT1L and found the high-density CRISPR scan clearly distinguishes the substrate binding pocket from the less important regions within the KMT domain. Furthermore, we identified CRISPR hotspots that coincide with amino acid residues directly contacting the enzymatic substrate S-adenosylmethionine (SAM). Similarly, CRISPR scan identifies amino acid hot spots in DOT1L that establish a direct interaction with the cofactor AF9. These results implicate the utility of the high-density CRISPR scan to pinpoint the functional elements within a protein to a sub-domain resolution.To identify regions in DOT1L that mediate response of MLL-r leukemia to DOT1L-inhibitors, we conducted parallel CRISPR scans by culturing the Dot1l sgRNA library transduced MLL-AF9-Cas9+cells in either DMSO (vehicle) or the DOT1L inhibitor (1 uM EPZ5676) for 12 days. These paired screens revealed distinct patterns of the CRISPR scan scores between the two culture conditions. Surprisingly, a cluster of 31 sgRNA targeting the amino acid residues T560 - S661 of DOT1L was significantly enriched only in the presence of the DOT1L inhibitor.Expression of individual sgRNA targeting this region confirmed the EPZ5676-resistant phenotype observed in the CRISPR scans. Computational modeling of the structure of DOT1L suggests this newly identified region overlaps with two alpha-helixes motifs in a predicted coiled-coil domain. Finally, we examined eight clinically observed DOT1L missense mutations in this region (cBioPortal database; 54,510 patients) and foundthree DOT1L-mutant constructs (A591V, G594S and L626P) when expressed in the MLL-AF9 leukemia cells can confer resistance to EPZ5676. These findings suggest the utility of the high-density CRISPR scan for de novoidentification of drug-resistant mutant alleles in the human population, which maydirect future clinical decisions and benefit patients with specific genomic backgrounds. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Fitness Costs and the Rapid Spread of kelch13-C580Y Substitutions Conferring Artemisinin Resistance.

Fitness costs are key determinants of whether drug resistance alleles establish and how fast they spread within populations. More than 125 different kelch13 alleles, each containing a different amino acid substitution, have arisen in Southeast Asian malaria parasite (Plasmodium falciparum) populations under artemisinin selection over the past 15 years in a dramatic example of a soft selective event. However, just one of these alleles (C580Y) is now outcompeting other alleles in multiple different countries and is spreading toward fixation. Here we examine the fitness consequences of C580Y, relative to another less successful kelch13 mutation (R561H), to try to explain the distinctive dynamics of C580Y. We hypothesized that C580Y will show lower fitness costs than other kelch13 substitutions in the absence of artemisinin treatment. We used CRISPR/Cas9 methods to introduce single mutations (C580Y or R561H) or synonymous control edits into a wild-type parasite isolated on the Thailand-Myanmar border, conducted replicated head-to-head competition assays, and determined the outcome of competition using deep sequencing of kelch13 amplicons. Contrary to our predictions, these experiments reveal that C580Y carries higher fitness costs (s [selection coefficient] = 0.15 ± 0.008 [1 standard error {SE}]) than R561H (s = 0.084 ± 0.005). Furthermore, R561H outcompetes C580Y in direct competition (s = 0.065 ± 0.004). We conclude that fitness costs of C580Y in isolation are unlikely to explain the rapid spread of this substitution.