RESUMEN
Retinal pigment epithelial (RPE) cells are exclusive to the retina, critically multifunctional in maintaining the visual functions and health of photoreceptors and the retina. Despite their vital functions throughout lifetime, RPE cells lack regenerative capacity, rendering them vulnerable which can lead to degenerative retinal diseases. With advancements in stem cell technology enabling the differentiation of functional cells from pluripotent stem cells and leveraging the robust autocrine and paracrine functions of RPE cells, extracellular vesicles (EVs) secreted by RPE cells hold significant therapeutic potential in supplementing RPE cell activity. While previous research has primarily focused on the trophic factors secreted by RPE cells, there is a lack of studies investigating miRNA, which serves as a master regulator of gene expression. Profiling and defining the functional role of miRNA contained within RPE-secreted EVs is critical as it constitutes a necessary step in identifying the optimal phenotype of the EV-secreting cell and understanding the biological cargo of EVs to develop EV-based therapeutics. In this study, we present a comprehensive profile of miRNA in small extracellular vesicles (sEVs) secreted during RPE maturation following differentiation from human embryonic stem cells (hESCs); early-stage hESC-RPE (20-21 days in culture), mid-stage hESC-RPE (30-31 days in culture) and late-stage hESC-RPE (60-61 days in culture). This exploration is essential for ongoing efforts to develop and optimize EV-based intraocular therapeutics utilizing RPE-secreted EVs, which may significantly impact the function of dysfunctional RPE cells in retinal diseases.
RESUMEN
The introduction of artemisinin combination therapies (ACTs) has significantly reduced the burden of Plasmodium falciparum malaria, yet the emergence of artemisinin partial resistance (ART-R) as well as partner drug resistance threatens these gains. Recent confirmations of prevalent de novo ART-R mutations in Africa, in particular in Rwanda, Uganda and Ethiopia, underscore the urgency of addressing this issue in Africa. Our objective is to characterise this evolving resistance landscape in Africa and understand the speed with which ART-R will continue to spread. We produce estimates of both ART-R and partner drug resistance by bringing together WHO, WWARN and MalariaGen Pf7k data on antimalarial resistance in combination with a literature review. We integrate these estimates within a mathematical modelling approach, aincorporating to estimate parameters known to impact the selection of ART-R for each malaria-endemic country and explore scenarios of ART-R spread and establishment. We identify 16 malaria-endemic countries in Africa to prioritise for surveillance and future deployment of alternative antimalarial strategies, based on ART-R reaching greater than 10% prevalence by 2040 under current malaria burden and effective-treatment coverage. If resistance continues to spread at current rates with no change in drug policy, we predict that partner drug resistance will emerge and the mean percentage of treatment failure across Africa will reach 30.74% by 2060 (parameter uncertainty range: 24.98% - 34.54%). This translates to an alarming number of treatment failures, with 52,980,600 absolute cases of treatment failure predicted in 2060 in Africa (parameter uncertainty range: 26,374,200 - 93,672,400) based on current effective treatment coverage. Our results provide a refined and updated prediction model for the emergence of ART-R to help guide antimalarial policy and prioritise future surveillance efforts and innovation in Africa. These results put into stark context the speed with which antimalarial resistance may spread in Africa if left unchecked, confirming the need for swift and decisive action in formulating antimalarial treatment policies focused on furthering malaria control and containing antimalarial resistance in Africa. The rise of artemisinin partial resistance (ART-R) and increasing partner drug tolerance by Plasmodium falciparum malaria in Africa threatens to undo malaria control efforts. Recent confirmations of de novo ART-R markers in Rwanda, Uganda, and Ethiopia highlight the urgent need to address this threat in Africa, where the vast majority of cases and deaths occur. This study characterises the resistance landscape and predicts the spread of antimalarial resistance across Africa. We estimate and map the current levels of resistance markers related to artemisinin and its partner drugs using WHO, WWARN, and MalariaGen Pf7k data. We combine these estimates with current malaria transmission and treatment data and use an established individual-based model of malaria resistance to simulate future resistance spread. We identify 16 African countries at highest risk of ART-R for prioritisation of enhanced surveillance and alternative antimalarial strategies. We project that, without policy changes, ART-R will exceed 10% in these regions by 2040. By 2060, if resistance spreads unchecked, we predict mean treatment failure rates will reach 30.74% (parameter uncertainty range: 24.98% - 34.54%) across Africa. This alarming spread of resistance is predicted to cause 52.98 million treatment failures (uncertainty range: 26.37 million - 93.67 million) in 2060. The impact of antimalarial resistance in Africa, if left unchecked, would hugely damage efforts to reduce malaria burden. Our results underscore the critical need for swift policy action to contain resistance and guide future surveillance and intervention efforts.
RESUMEN
BACKGROUND: In 2021, nationwide malaria molecular surveillance revealed a high prevalence of a validated artemisinin resistance marker, the kelch13 (k13) Arg561His mutation, in the Kagera region of northwestern Tanzania. We aimed to investigate the efficacy of artemether-lumefantrine and artesunate-amodiaquine and to confirm the presence of artemisinin partial resistance (ART-R) in the Karagwe district of this region. METHODS: This single-arm, therapeutic efficacy study was carried out at the Bukangara dispensary in the Karagwe district of the Kagera region in northwestern Tanzania. Eligible participants were aged between 6 months and 120 months, had confirmed Plasmodium falciparum asexual parasitaemia, and met other inclusion criteria according to WHO's standard protocol. Participants were enrolled, treated sequentially with either artemether-lumefantrine or artesunate-amodiaquine, and assessed clinically and parasitologically for 28 days as per WHO protocol. Parasitaemia was measured every 8 h until day 3, on day 7, and then during weekly follow-up visits until day 28. Mutations in the k13 gene and extended haplotypes with the mutations were analysed, and comparisons were made with previous samples collected in the same region of Kagera and in Rwanda and southeast Asia. The primary endpoint was PCR-corrected cure rate. FINDINGS: Between April 29 and Sept 1, 2022, 343 patients were screened, of whom 176 were enrolled: 88 in each treatment group. The PCR-corrected cure rate was 98% (95% CI 91-100) in the artemether-lumefantrine group and 100% (96-100) in the artesunate-amodiaquine group. Persistent parasitaemia on day 3 occurred in 11 (13%) of 88 patients in the artemether-lumefantrine group and 17 (19%) of 88 patients in the artesunate-amodiaquine group. Arg561His mutations on day 0 and parasitaemia on day 3 were reported in eight (9%) of 87 patients in the artemether-lumefantrine group and ten (12%) of 86 patients in the artesunate-amodiaquine group. The median parasite clearance half-life in patients harbouring parasites with Arg561His mutation was 6·1 h in the artemether-lumefantrine group and 6·0 h in the artesunate-amodiaquine group. Parasites with the Arg561His mutation were not similar to those from southeast Asia and Rwanda but had similar haplotypes to parasites reported in the same Tanzanian region of Kagera in 2021. INTERPRETATION: This study confirms the presence of ART-R in Tanzania, although artemether-lumefantrine and artesunate-amodiaquine showed high efficacy. A context-specific response strategy and vigilance to detect the reduced efficacy of current antimalarial treatments and ART-R in other parts of the country are urgently needed. FUNDING: The Bill & Melinda Gates Foundation and the US National Institutes of Health.
RESUMEN
BACKGROUND: The emergence of the artemisinin partial resistance (ART-R) mutation in the Plasmodium falciparum kelch13 gene (k13), Arg561His, in Rwanda and the regional presence of polymorphisms affecting sulfadoxine-pyrimethamine have raised concern in neighbouring Tanzania. The goal of this study was to assess the status of antimalarial resistance in Tanzania, with a focus on the border with Rwanda, to understand the distribution of the Arg561His mutation, partner drug resistance, and resistance to chemoprevention drugs. METHODS: In this cross-sectional survey, capillary dried blood spots were collected from malaria positive asymptomatic individuals in the community and symptomatic individuals in health facilities aged 6 months and older, in 13 regions of mainland Tanzania from Jan 31 to June 26, 2021. Exclusion criteria included residence of the areas other than the target sites, presenting to the health facility for care and treatment of conditions other than malaria, and not providing informed consent. Samples were assessed for antimalarial resistance polymorphisms and genetic relatedness using molecular inversion probes targeting P falciparum and short-read whole-genome sequencing. The primary outcome was the prevalence of molecular markers of antimalarial resistance at the region level, as well as at the district level in Kagera, a region in the northwest of the country at the border with Rwanda. FINDINGS: 6855 (88·1%) of 7782 capillary dried blood spot samples collected were successfully genotyped. The overall prevalence of k13 Arg561His in Kagera was 7·7% (90% CI 6·0-9·4; 50 of 649), with the highest prevalence in the districts near the Rwandan border (22·8% [31 of 136] in Karagwe, 14·4% [17 of 118]) in Kyerwa, and 1·4% [two of 144] in Ngara). k13 Arg561His was uncommon in the other regions. Haplotype analysis suggested that some of these parasites are related to isolates collected in Rwanda in 2015, supporting regional spread of Arg561His. However, a novel k13 Arg561His haplotype was observed, potentially indicating a second origin in the region. Other validated k13 resistance polymorphisms (one Arg622Ile and two Ala675Val isolates) were also identified. A region of prevalent dihydrofolate reductase Ile164Leu mutation, associated with sulfadoxine-pyrimethamine resistance, was also identified in Kagera (15·2% [12·6-17·8%]; 80 of 526). The mutant crt Lys76Thr mutation, associated with chloroquine and amodiaquine resistance, was uncommon, occurring only in 75 of 2861 genotyped isolates, whereases the wild-type mdr1 Asn86Tyr allele, associated with reduced sensitivity to lumefantrine, was found in 99·7% (3819 of 3830) of samples countrywide. INTERPRETATION: These findings show that the k13 Arg561His mutation is common in northwest Tanzania and that multiple emergences of ART-R, similar as to what was seen in southeast Asia, have occurred. Mutations associated with high levels of sulfadoxine-pyrimethamine resistance are common. These results raise concerns about the long-term efficacy of artemisinin and chemoprevention antimalarials in the region. Understanding how multiple emergences interact with drivers of regional spread is essential for combating ART-R in Africa. FUNDING: This study was funded by the Bill & Melinda Gates Foundation and the National Institutes of Health.
RESUMEN
The emergence of antimalarial drug resistance is a major threat to malaria control and elimination. Using whole genome sequencing of 282 P. falciparum samples collected during the 2018 Zambia National Malaria Indicator Survey, we determined the prevalence and spatial distribution of known and candidate antimalarial drug resistance mutations. High levels of genotypic resistance were found across Zambia to pyrimethamine, with over 94% (n=266) of samples having the Pfdhfr triple mutant (N51I, C59R, and S108N), and sulfadoxine, with over 84% (n=238) having the Pfdhps double mutant (A437G and K540E). In northern Zambia, 5.3% (n=15) of samples also harbored the Pfdhps A581G mutation. Although 29 mutations were identified in Pfkelch13, these mutations were present at low frequency (<2.5%), and only three were WHO-validated artemisinin partial resistance mutations: P441L (n=1, 0.35%), V568M (n=2, 0.7%) and R622T (n=1, 0.35%). Notably, 91 (32%) of samples carried the E431K mutation in the Pfatpase6 gene, which is associated with artemisinin resistance. No specimens carried any known mutations associated with chloroquine resistance in the Pfcrt gene (codons 72-76). P. falciparum strains circulating in Zambia were highly resistant to sulfadoxine and pyrimethamine but remained susceptible to chloroquine and artemisinin. Despite this encouraging finding, early genetic signs of developing artemisinin resistance highlight the urgent need for continued vigilance and expanded routine genomic surveillance to monitor these changes.
RESUMEN
Novel antimalarials are urgently needed to combat rising resistance to available drugs. The imidazolopiperazine ganaplacide is a promising drug candidate, but decreased susceptibility of laboratory strains has been linked to polymorphisms in the Plasmodium falciparum cyclic amine resistance locus (PfCARL), acetyl-CoA transporter (PfACT), and UDP-galactose transporter (PfUGT). To characterize parasites causing disease in Africa, we assessed ex vivo drug susceptibilities to ganaplacide in 750 P. falciparum isolates collected in Uganda from 2017 to 2023. Drug susceptibilities were assessed using a 72-hour SYBR Green growth inhibition assay. The median IC50 for ganaplacide was 13.8 nM, but some isolates had up to 31-fold higher IC50s (31/750 with IC50 > 100 nM). To assess genotype-phenotype associations, we sequenced genes potentially mediating altered ganaplacide susceptibility in the isolates using molecular inversion probe and dideoxy sequencing methods. PfCARL was highly polymorphic, with eight mutations present in >5% of isolates. None of these eight mutations had previously been selected in laboratory strains with in vitro drug pressure and none were found to be significantly associated with decreased ganaplacide susceptibility. Mutations in PfACT and PfUGT were found in ≤5% of isolates, except for two frequent (>20%) mutations in PfACT; one mutation in PfACT (I140V) was associated with a modest decrease in susceptibility. Overall, Ugandan P. falciparum isolates were mostly highly susceptible to ganaplacide. Known resistance mediators were polymorphic, but mutations previously selected with in vitro drug pressure were not seen, and mutations identified in the Ugandan isolates were generally not associated with decreased ganaplacide susceptibility.
Asunto(s)
Antimaláricos , Resistencia a Medicamentos , Plasmodium falciparum , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Antimaláricos/farmacología , Uganda , Humanos , Resistencia a Medicamentos/genética , Malaria Falciparum/parasitología , Malaria Falciparum/tratamiento farmacológico , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Concentración 50 Inhibidora , Piperazinas/farmacología , Pruebas de Sensibilidad ParasitariaRESUMEN
Background: Given the altered responses to both artemisinins and lumefantrine in Eastern Africa, monitoring antimalarial drug resistance in all African countries is paramount. Methods: We measured the susceptibility to six antimalarials using ex vivo growth inhibition assays (IC50) for a total of 805 Plasmodium falciparum isolates obtained from travelers returning to France (2016-2023), mainly from West and Central Africa. Isolates were sequenced using molecular inversion probes (MIPs) targeting fourteen drug resistance genes across the parasite genome. Findings: Ex vivo susceptibility to several drugs has significantly decreased in 2019-2023 versus 2016-2018 parasite samples: lumefantrine (median IC50: 23·0 nM [IQR: 14·4-35·1] in 2019-2023 versus 13·9 nM [8·42-21·7] in 2016-2018, p<0·0001), monodesethylamodiaquine (35·4 [21·2-51·1] versus 20·3 nM [15·4-33·1], p<0·0001), and marginally piperaquine (20·5 [16·5-26·2] versus 18.0 [14·2-22·4] nM, p<0·0001). Only four isolates carried a validated pfkelch13 mutation. Multiple mutations in pfcrt and one in pfmdr1 (N86Y) were significantly associated with altered susceptibility to multiple drugs. The susceptibility to lumefantrine was altered by pfcrt and pfmdr1 mutations in an additive manner, with the wild-type haplotype (pfcrt K76-pfmdr1 N86) exhibiting the least susceptibility. Interpretation: Our study on P. falciparum isolates from West and Central Africa indicates a low prevalence of molecular markers of artemisinin resistance but a significant decrease in susceptibility to the partner drugs that have been the most widely used since a decade -lumefantrine and amodiaquine. These phenotypic changes likely mark parasite adaptation to sustained drug pressure and call for intensifying the monitoring of antimalarial drug resistance in Africa. Funding: This work was supported by the French Ministry of Health (grant to the French National Malaria Reference Center) and by the Agence Nationale de la Recherche (ANR-17-CE15-0013-03 to JC). JAB was supported by NIH R01AI139520. JR postdoctoral fellowship was funded by Institut de Recherche pour le Développement.
RESUMEN
The chromatin-associated protein WDR5 has been widely studied due to its role in histone modification and its potential as a pharmacological target for the treatment of cancer. In humans, the protein with highest sequence homology to WDR5 is encoded by the retrogene WDR5B, which remains unexplored. Here, we used CRISPR-Cas9 genome editing to generate WDR5B knockout and WDR5B-FLAG knock-in cell lines for further characterization. In contrast to WDR5, WDR5B exhibits low expression in pluripotent cells and is upregulated upon neural differentiation. Loss or shRNA depletion of WDR5B impairs cell growth and increases the fraction of non-viable cells in proliferating retinal pigment epithelial (RPE) cultures. CUT&RUN chromatin profiling in RPE and neural progenitors indicates minimal WDR5B enrichment at established WDR5 binding sites. These results suggest that WDR5 and WDR5B exhibit several divergent biological properties despite sharing a high degree of sequence homology.
Asunto(s)
Proliferación Celular , Epitelio Pigmentado de la Retina , Animales , Humanos , Diferenciación Celular , Línea Celular , Sistemas CRISPR-Cas/genética , Células Epiteliales/metabolismo , Edición Génica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/citologíaRESUMEN
Background: Given the altered responses to both artemisinins and lumefantrine in Eastern Africa, monitoring antimalarial drug resistance in all African countries is paramount. Methods: We measured the susceptibility to six antimalarials using ex vivo growth inhibition assays (IC 50 ) for a total of 805 Plasmodium falciparum isolates obtained from travelers returning to France (2016-2023), mainly from West and Central Africa. Isolates were sequenced using molecular inversion probes (MIPs) targeting fourteen drug resistance genes across the parasite genome. Findings: Ex vivo susceptibility to several drugs has significantly decreased in 2019-2023 versus 2016-2018 parasite samples: lumefantrine (median IC 50 : 23·0 nM [IQR: 14·4-35·1] in 2019-2023 versus 13·9 nM [8·42-21·7] in 2016-2018, p<0·0001), monodesethylamodiaquine (35·4 [21·2-51·1] versus 20·3 nM [15·4-33·1], p<0·0001), and marginally piperaquine (20·5 [16·5-26·2] versus 18.0 [14·2-22·4] nM, p<0·0001). Only four isolates carried a validated pfkelch13 mutation. Multiple mutations in pfcrt and one in pfmdr1 (N86Y) were significantly associated with altered susceptibility to multiple drugs. The susceptibility to lumefantrine was altered by pfcrt and pfmdr1 mutations in an additive manner, with the wild-type haplotype ( pfcrt K76- pfmdr1 N86) exhibiting the least susceptibility. Interpretation: Our study on P. falciparum isolates from West and Central Africa indicates a low prevalence of molecular markers of artemisinin resistance but a significant decrease in susceptibility to the partner drugs that have been the most widely used since a decade -lumefantrine and amodiaquine. These phenotypic changes likely mark parasite adaptation to sustained drug pressure and call for intensifying the monitoring of antimalarial drug resistance in Africa.
RESUMEN
Plasmodium vivax transmission occurs throughout the tropics and is an emerging threat in areas of Plasmodium falciparum decline, causing relapse infections that complicate treatment and control. Targeted sequencing for P. falciparum has been widely deployed to detect population structure and the geographic spread of antimalarial and diagnostic resistance. However, there are fewer such tools for P. vivax . Leveraging global variation data, we designed four molecular inversion probe (MIP) genotyping panels targeting geographically differentiating SNPs, neutral SNPs, putative antimalarial resistance genes, and vaccine candidate genes. We deployed these MIP panels on 866 infections from the Peruvian Amazon and identified transmission networks with clonality (IBD>0.99), copy number variation in Pvdbp and multiple Pvrbps , fixation of putative antimalarial resistance, and balancing selection in 13 vaccine candidate genes. Our MIP panels are the broadest genotyping panel currently available and are poised for successful deployment in other regions of P. vivax transmission.
RESUMEN
The emergence of antimalarial drug resistance is an impediment to malaria control and elimination in Africa. Analysis of temporal trends in molecular markers of resistance is critical to inform policy makers and guide malaria treatment guidelines. In a low and seasonal transmission region of southern Zambia, we successfully genotyped 85.5% (389/455) of Plasmodium falciparum samples collected between 2013-2018 from 8 spatially clustered health centres using molecular inversion probes (MIPs) targeting key drug resistance genes. Aside from one sample carrying K13 R622I, none of the isolates carried other World Health Organization-validated or candidate artemisinin partial resistance (ART-R) mutations in K13. However, 13% (CI, 9.6-17.2) of isolates had the AP2MU S160N mutation, which has been associated with delayed clearance following artemisinin combination therapy in Africa. This mutation increased in prevalence between 2015-2018 and bears a genomic signature of selection. During this time period, there was an increase in the MDR1 NFD haplotype that is associated with reduced susceptibility to lumefantrine. Sulfadoxine-pyrimethamine polymorphisms were near fixation. While validated ART-R mutations are rare, a mutation associated with slow parasite clearance in Africa appears to be under selection in southern Zambia.
RESUMEN
Background: The Zanzibar archipelago of Tanzania has become a low-transmission area for Plasmodium falciparum. Despite being considered an area of pre-elimination for years, achieving elimination has been difficult, likely due to a combination of imported infections from mainland Tanzania and continued local transmission. Methods: To shed light on these sources of transmission, we applied highly multiplexed genotyping utilizing molecular inversion probes to characterize the genetic relatedness of 282 P. falciparum isolates collected across Zanzibar and in Bagamoyo district on the coastal mainland from 2016 to 2018. Results: Overall, parasite populations on the coastal mainland and Zanzibar archipelago remain highly related. However, parasite isolates from Zanzibar exhibit population microstructure due to the rapid decay of parasite relatedness over very short distances. This, along with highly related pairs within shehias, suggests ongoing low-level local transmission. We also identified highly related parasites across shehias that reflect human mobility on the main island of Unguja and identified a cluster of highly related parasites, suggestive of an outbreak, in the Micheweni district on Pemba island. Parasites in asymptomatic infections demonstrated higher complexity of infection than those in symptomatic infections, but have similar core genomes. Conclusions: Our data support importation as a main source of genetic diversity and contribution to the parasite population in Zanzibar, but they also show local outbreak clusters where targeted interventions are essential to block local transmission. These results highlight the need for preventive measures against imported malaria and enhanced control measures in areas that remain receptive to malaria reemergence due to susceptible hosts and competent vectors. Funding: This research was funded by the National Institutes of Health, grants R01AI121558, R01AI137395, R01AI155730, F30AI143172, and K24AI134990. Funding was also contributed from the Swedish Research Council, Erling-Persson Family Foundation, and the Yang Fund. RV acknowledges funding from the MRC Centre for Global Infectious Disease Analysis (reference MR/R015600/1), jointly funded by the UK Medical Research Council (MRC) and the UK Foreign, Commonwealth & Development Office (FCDO), under the MRC/FCDO Concordat agreement and is also part of the EDCTP2 program supported by the European Union. RV also acknowledges funding by Community Jameel.
Asunto(s)
Malaria Falciparum , Plasmodium falciparum , Tanzanía/epidemiología , Plasmodium falciparum/genética , Plasmodium falciparum/aislamiento & purificación , Malaria Falciparum/transmisión , Malaria Falciparum/parasitología , Malaria Falciparum/epidemiología , Humanos , GenotipoRESUMEN
Newly arrived refugees offer insights into malaria epidemiology in their countries of origin. We evaluated asymptomatic refugee children within 7 days of arrival in Uganda from South Sudan and the Democratic Republic of Congo (DRC) in 2022 for parasitemia, parasite species, and Plasmodium falciparum drug resistance markers. Asymptomatic P. falciparum infections were common in both populations. Coinfection with P. malariae was more common in DRC refugees. Prevalences of markers of aminoquinoline resistance (PfCRT K76T, PfMDR1 N86Y) were much higher in South Sudan refugees, of antifolate resistance (PfDHFR C59R and I164L, PfDHPS A437G, K540E, and A581G) much higher in DRC refugees, and of artemisinin partial resistance (ART-R; PfK13 C469Y and A675V) moderate in both populations. Prevalences of most mutations differed from those seen in Ugandans attending health centers near the refugee centers. Refugee evaluations yielded insights into varied malaria epidemiology and identified markers of ART-R in 2 previously little-studied countries.
Asunto(s)
Antimaláricos , Resistencia a Medicamentos , Malaria Falciparum , Plasmodium falciparum , Proteínas Protozoarias , Refugiados , Humanos , Uganda/epidemiología , Antimaláricos/uso terapéutico , Antimaláricos/farmacología , Resistencia a Medicamentos/genética , Prevalencia , Preescolar , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Malaria Falciparum/epidemiología , Malaria Falciparum/parasitología , Malaria Falciparum/tratamiento farmacológico , Femenino , Masculino , Niño , Proteínas Protozoarias/genética , Lactante , Proteínas de Transporte de Membrana/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Sudán/epidemiología , Biomarcadores/sangre , Artemisininas/uso terapéutico , Artemisininas/farmacología , Parasitemia/epidemiología , Parasitemia/tratamiento farmacológico , Plasmodium malariae/genética , Plasmodium malariae/efectos de los fármacosRESUMEN
Increasing sulfadoxine-pyrimethamine (SP) resistance in the Democratic Republic of the Congo (DRC) has threatened its use for prevention of malaria in one of the most malarious countries in the world. Using geographic information on mining operations in the DRC and genetic data on SP drug resistance markers from the 2013-2014 Demographic and Health Surveys, we evaluated associations between close residence to mining and the presence of mutations conferring resistance to sulfadoxine. Close residential proximity to mining was associated with increased prevalence odds ratio (POR) of the dhps540E mutation (POR: 2.11, 95% uncertainty interval: 1.15-3.96) with adjustments for confounding variables and space. Our findings indicate that exposure to mining is associated with increased presence of an antimalarial drug resistance haplotype that threatens effective use of SP for vulnerable populations. Areas actively engaged in mining could be considered for interventions to reduce the spread of emerging drug resistance in the DRC.
Asunto(s)
Antimaláricos , Resistencia a Medicamentos , Minería , Mutación , Pirimetamina , Sulfadoxina , República Democrática del Congo/epidemiología , Humanos , Antimaláricos/uso terapéutico , Antimaláricos/farmacología , Resistencia a Medicamentos/genética , Pirimetamina/farmacología , Pirimetamina/uso terapéutico , Sulfadoxina/uso terapéutico , Sulfadoxina/farmacología , Prevalencia , Plasmodium falciparum/genética , Plasmodium falciparum/efectos de los fármacos , Dihidropteroato Sintasa/genética , Combinación de Medicamentos , Malaria Falciparum/epidemiología , Malaria Falciparum/parasitología , FemeninoRESUMEN
BACKGROUND: Emerging artemisinin partial resistance and diagnostic resistance are a threat to malaria control in Africa. Plasmodium falciparum kelch13 (k13) propeller-domain mutations that confer artemisinin partial resistance have emerged in Africa. k13-561H was initially described at a frequency of 7.4% from Masaka in 2014-2015, but not present in nearby Rukara. By 2018, 19.6% of isolates in Masaka and 22% of isolates in Rukara contained the mutation. Longitudinal monitoring is essential to inform control efforts. In Rukara, an assessment was conducted to evaluate recent k13-561H prevalence changes, as well as other key mutations. Prevalence of hrp2/3 deletions was also assessed. METHODS: Samples collected in Rukara in 2021 were genotyped for key artemisinin and partner drug resistance mutations using molecular inversion probe assays and for hrp2/3 deletions using qPCR. RESULTS: Clinically validated k13 artemisinin partial resistance mutations continue to increase in prevalence with the overall level of mutant infections reaching 32% in Rwanda. The increase appears to be due to the rapid emergence of k13-675V (6.4%, 6/94 infections), previously not observed, rather than continued expansion of 561H (23.5% 20/85). Mutations to partner drugs and other anti-malarials were variable, with high levels of multidrug resistance 1 (mdr1) N86 (95.5%) associated with lumefantrine decreased susceptibility and dihydrofolate reductase (dhfr) 164L (24.7%) associated with a high level of antifolate resistance, but low levels of amodiaquine resistance polymorphisms with chloroquine resistance transporter (crt) 76T: at 6.1% prevalence. No hrp2 or hrp3 gene deletions associated with diagnostic resistance were found. CONCLUSIONS: Increasing prevalence of artemisinin partial resistance due to k13-561H and the rapid expansion of k13-675V is concerning for the longevity of artemisinin effectiveness in the region. False negative RDT results do not appear to be an issue with no hrp2 or hpr3 deletions detected. Continued molecular surveillance in this region and surrounding areas is needed to follow artemisinin partial resistance and provide early detection of partner drug resistance, which would likely compromise control and increase malaria morbidity and mortality in East Africa.
Asunto(s)
Antimaláricos , Artemisininas , Resistencia a Medicamentos , Malaria Falciparum , Mutación , Plasmodium falciparum , Proteínas Protozoarias , Plasmodium falciparum/genética , Plasmodium falciparum/efectos de los fármacos , Artemisininas/farmacología , Antimaláricos/farmacología , Proteínas Protozoarias/genética , Resistencia a Medicamentos/genética , Rwanda , Malaria Falciparum/parasitología , Malaria Falciparum/epidemiología , Humanos , Antígenos de Protozoos/genética , Prevalencia , Niño , Adulto Joven , Adolescente , Adulto , PreescolarAsunto(s)
Antimaláricos , Artemisininas , Resistencia a Medicamentos , Plasmodium falciparum , Artemisininas/uso terapéutico , Artemisininas/farmacología , Humanos , Antimaláricos/uso terapéutico , Antimaláricos/farmacología , Resistencia a Medicamentos/genética , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Malaria/tratamiento farmacológico , África/epidemiología , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitologíaRESUMEN
BACKGROUND: In 2021 and 2023, the World Health Organization approved RTS,S/AS01 and R21/Matrix M malaria vaccines, respectively, for routine immunization of children in African countries with moderate to high transmission. These vaccines are made of Plasmodium falciparum circumsporozoite protein (PfCSP), but polymorphisms in the gene raise concerns regarding strain-specific responses and the long-term efficacy of these vaccines. This study assessed the Pfcsp genetic diversity, population structure and signatures of selection among parasites from areas of different malaria transmission intensities in Mainland Tanzania, to generate baseline data before the introduction of the malaria vaccines in the country. METHODS: The analysis involved 589 whole genome sequences generated by and as part of the MalariaGEN Community Project. The samples were collected between 2013 and January 2015 from five regions of Mainland Tanzania: Morogoro and Tanga (Muheza) (moderate transmission areas), and Kagera (Muleba), Lindi (Nachingwea), and Kigoma (Ujiji) (high transmission areas). Wright's inbreeding coefficient (Fws), Wright's fixation index (FST), principal component analysis, nucleotide diversity, and Tajima's D were used to assess within-host parasite diversity, population structure and natural selection. RESULTS: Based on Fws (< 0.95), there was high polyclonality (ranging from 69.23% in Nachingwea to 56.9% in Muheza). No population structure was detected in the Pfcsp gene in the five regions (mean FST = 0.0068). The average nucleotide diversity (π), nucleotide differentiation (K) and haplotype diversity (Hd) in the five regions were 4.19, 0.973 and 0.0035, respectively. The C-terminal region of Pfcsp showed high nucleotide diversity at Th2R and Th3R regions. Positive values for the Tajima's D were observed in the Th2R and Th3R regions consistent with balancing selection. The Pfcsp C-terminal sequences revealed 50 different haplotypes (H_1 to H_50), with only 2% of sequences matching the 3D7 strain haplotype (H_50). Conversely, with the NF54 strain, the Pfcsp C-terminal sequences revealed 49 different haplotypes (H_1 to H_49), with only 0.4% of the sequences matching the NF54 strain (Hap_49). CONCLUSIONS: The findings demonstrate high diversity of the Pfcsp gene with limited population differentiation. The Pfcsp gene showed positive Tajima's D values, consistent with balancing selection for variants within Th2R and Th3R regions. The study observed differences between the intended haplotypes incorporated into the design of RTS,S and R21 vaccines and those present in natural parasite populations. Therefore, additional research is warranted, incorporating other regions and more recent data to comprehensively assess trends in genetic diversity within this important gene. Such insights will inform the choice of alleles to be included in the future vaccines.
Asunto(s)
Plasmodium falciparum , Polimorfismo Genético , Proteínas Protozoarias , Selección Genética , Humanos , Enfermedades Endémicas , Malaria Falciparum/parasitología , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , TanzaníaRESUMEN
In Africa, the first Plasmodium falciparum Kelch13 (K13) artemisinin partial resistance mutation 561H was first detected and validated in Rwanda. Surveillance to better define the extent of the emergence in Rwanda and neighboring countries as other mutations arise in East Africa is critical. We employ a novel scheme of liquid blood drop preservation combined with pooled sequencing to provide a cost-effective rapid assessment of resistance mutation frequencies at multiple collection sites across Rwanda and neighboring countries. Malaria-positive samples (n=5,465) were collected from 39 health facilities in Rwanda, Uganda, Tanzania, and the Democratic Republic of the Congo (DRC) between May 2022 and March 2023 and sequenced in 199 pools. In Rwanda, K13 561H and 675V were detected in 90% and 65% of sites with an average frequency of 19.0% (0-54.5%) and 5.0% (0-35.5%), respectively. In Tanzania, 561H had high frequency in multiple sites while it was absent from the DRC although 675V was seen at low frequency. Conceringly candidate mutations were observed: 441L, 449A, and 469F co-occurred with validated mutations suggesting they are arising under the same pressures. Other resistance markers associated with artemether-lumefantrine are common: P. falciparum multidrug resistance protein 1 N86 at 98.0% and 184F at 47.0% (0-94.3%) and P. falciparum chloroquine resistance transporter 76T at 14.7% (0-58.6%). Additionally, sulfadoxine-pyrimethamine-associated mutations show high frequencies. Overall, K13 mutations are rapidly expanding in the region further endangering control efforts with the potential of engendering partner drug resistance.
RESUMEN
Plasmodium falciparum with the histidine rich protein 2 gene (pfhrp2) deleted from its genome can escape diagnosis by HRP2-based rapid diagnostic tests (HRP2-RDTs). The World Health Organization (WHO) recommends switching to a non-HRP2 RDT for P. falciparum clinical case diagnosis when pfhrp2 deletion prevalence causes ≥ 5% of RDTs to return false negative results. Tanzania is a country of heterogenous P. falciparum transmission, with some regions approaching elimination and others at varying levels of control. In concordance with the current recommended WHO pfhrp2 deletion surveillance strategy, 100 health facilities encompassing 10 regions of Tanzania enrolled malaria-suspected patients between February and July 2021. Of 7863 persons of all ages enrolled and providing RDT result and blood sample, 3777 (48.0%) were positive by the national RDT testing for Plasmodium lactate dehydrogenase (pLDH) and/or HRP2. A second RDT testing specifically for the P. falciparum LDH (Pf-pLDH) antigen found 95 persons (2.5% of all RDT positives) were positive, though negative by the national RDT for HRP2, and were selected for pfhrp2 and pfhrp3 (pfhrp2/3) genotyping. Multiplex antigen detection by laboratory bead assay found 135/7847 (1.7%) of all blood samples positive for Plasmodium antigens but very low or no HRP2, and these were selected for genotyping as well. Of the samples selected for genotyping based on RDT or laboratory multiplex result, 158 were P. falciparum DNA positive, and 140 had sufficient DNA to be genotyped for pfhrp2/3. Most of these (125/140) were found to be pfhrp2+/pfhrp3+, with smaller numbers deleted for only pfhrp2 (n = 9) or only pfhrp3 (n = 6). No dual pfhrp2/3 deleted parasites were observed. This survey found that parasites with these gene deletions are rare in Tanzania, and estimated that 0.24% (95% confidence interval: 0.08% to 0.39%) of false-negative HRP2-RDTs for symptomatic persons were due to pfhrp2 deletions in this 2021 Tanzania survey. These data provide evidence for HRP2-based diagnostics as currently accurate for P. falciparum diagnosis in Tanzania.
Asunto(s)
Antígenos de Grupos Sanguíneos , Malaria Falciparum , Humanos , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Eliminación de Gen , Tanzanía/epidemiología , Pruebas Diagnósticas de Rutina/métodos , Antígenos de Protozoos/genética , Malaria Falciparum/diagnóstico , Malaria Falciparum/epidemiología , Malaria Falciparum/genética , Instituciones de Salud , ADNRESUMEN
Recent developments in cardiac macrophage biology have broadened our understanding of the critical functions of macrophages in the heart. As a result, there is further interest in understanding the independent contributions of distinct subsets of macrophage to cardiac development and function. Here, we demonstrate that genetic loss of interferon regulatory factor 8 (Irf8)-positive embryonic-derived macrophages significantly disrupts cardiac conduction, chamber function, and innervation in adult zebrafish. At 4 months post-fertilization (mpf), homozygous irf8st96/st96 mutants have significantly shortened atrial action potential duration and significant differential expression of genes involved in cardiac contraction. Functional in vivo assessments via electro- and echocardiograms at 12 mpf reveal that irf8 mutants are arrhythmogenic and exhibit diastolic dysfunction and ventricular stiffening. To identify the molecular drivers of the functional disturbances in irf8 null zebrafish, we perform single cell RNA sequencing and immunohistochemistry, which reveal increased leukocyte infiltration, epicardial activation, mesenchymal gene expression, and fibrosis. Irf8 null hearts are also hyperinnervated and have aberrant axonal patterning, a phenotype not previously assessed in the context of cardiac macrophage loss. Gene ontology analysis supports a novel role for activated epicardial-derived cells (EPDCs) in promoting neurogenesis and neuronal remodeling in vivo. Together, these data uncover significant cardiac abnormalities following embryonic macrophage loss and expand our knowledge of critical macrophage functions in heart physiology and governing homeostatic heart health.