RESUMEN
Chronic renal failure (CRF) is an incurable disease with unique challenges. Anemia is a frequent complication affecting dialysis patients. Erythropoietin (EPO) is used to treat anemia, but a poor response may result. We investigated genetic polymorphisms of store-operated calcium channel (SOC) signaling, an important erythropoietin-activated pathway that may induce EPO resistance in patients with renal failure. A total of 108 end stage renal disease (ESRD) patients were selected for this study. Patients were divided into two groups according to their erythropoietin resistance index (ERI): 39 patients with an ERI>10 and 69 patients with an ERI<10. We selected four tagging single nucleotide polymorphisms (tSNPs) in STIM1 and five in ORAI1 in our study. A polymerase chain reaction was performed, and genotyping against EPO resistance was correlated. Patients with the AG genotype of rs1561876 in STIM1, the TC genotype of rs6486795 in ORAI1, and the TG or GG genotypes of rs12320939 in ORAI1 were associated with an increased risk of erythropoietin resistance. Overall, we reported a moderately significant relationship between genetic polymorphisms of STIM1 and EPO resistance. We also reported a highly significant relationship between genetic polymorphisms of ORAI1 and EPO resistance. The (A-A-G) haplotype of STIM1 and the (G-T-G-T-A, G-C-G-C-G, or G-T-T-C-G) haplotypes of ORAI1 were significantly associated with EPO resistance.
Asunto(s)
Eritropoyetina , Fallo Renal Crónico , Proteínas de Neoplasias , Proteína ORAI1 , Polimorfismo de Nucleótido Simple , Molécula de Interacción Estromal 1 , Humanos , Molécula de Interacción Estromal 1/genética , Egipto , Fallo Renal Crónico/genética , Masculino , Eritropoyetina/genética , Femenino , Proteína ORAI1/genética , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Adulto , Resistencia a Medicamentos/genéticaRESUMEN
BACKGROUND: Leishmaniasis is a neglected zoonosis caused by parasites of Leishmania spp. The main drug used to treat cutaneous leishmaniasis (CL) is the antimoniate of meglumine. This drug, which has strong adverse and toxic effects, is usually administered intravenously, further complicating the difficult treatment. Factors such as Leishmania gene expression and genomic mutations appear to play a role in the development of drug resistance. OBJECTIVES: This systematic review summarises the results of the literature evaluating parasite genetic markers possibly associated with resistance to pentavalent antimony in CL. METHODS: This study followed PRISMA guidelines and included articles from PubMed, SciELO, and LILACS databases. Inclusion criteria were studies that (i) investigated mutations in the genome and/or changes in gene expression of Leishmania associated with treatment resistance; (ii) used antimony drugs in the therapy of CL; (iii) used naturally resistant strains isolated from patients. The Joanna Briggs Institute Critical Appraisal Checklist was used to assess article quality and risk of bias. FINDINGS: A total of 23 articles were selected, of which 18 investigated gene expression and nine genomic mutations. Of these 23 articles, four examined gene expression and genomic mutations in the same samples. Regarding gene expression, genes from the ABC transporter protein family, AQP1, MRPA, TDR1 and TRYR were most frequently associated with drug resistance. In one of the articles in which mutations were investigated, a mutation was found in HSP70 (T579A) and in three articles mutations were found in AQP1 (A516C, G562A and G700A). A limitation of this review is that in most of the included studies, parasites were isolated from cultured lesion samples and drug resistance was assessed using in vitro drug susceptibility testing. These approaches may not be ideal for accurate genetic evaluation and detection of treatment failure. MAIN CONCLUSIONS: The development of further studies to evaluate the genetic resistance factors of Leishmania spp. is necessary to elucidate the mechanisms of the parasite and improve patient treatment and infection control.
Asunto(s)
Antimonio , Antiprotozoarios , Resistencia a Medicamentos , Leishmania , Leishmaniasis Cutánea , Resistencia a Medicamentos/genética , Leishmaniasis Cutánea/tratamiento farmacológico , Leishmaniasis Cutánea/parasitología , Antiprotozoarios/farmacología , Humanos , Leishmania/efectos de los fármacos , Leishmania/genética , Antimonio/farmacología , Antimonio/uso terapéutico , Mutación , Antimoniato de Meglumina/uso terapéuticoRESUMEN
Varroa destructor is widely recognized as a significant contributor to colony collapse disorder. Chemical acaricides, such as amitraz, have been extensively used for Varroa control due to their selectivity within beehives. However, the increasing number of cases of amitraz resistance across global V. destructor populations poses a significant challenge. In this study, we conducted a comprehensive molecular screening of the ß-adrenergic-like octopamine receptor (Octß2R), the target-site of amitraz, across 66 Turkish and 63 Belgian V. destructor populations. Although previously reported amitraz resistance mutations were not detected, the screening revealed a novel Y337F mutation located within transmembrane 7 (TM7) of Octß2R in Turkish Varroa populations. Notably, this mutation was identified in the last residue of the highly conserved NPxxY motif associated with the activation of G-protein coupled receptors (GPCR). Among the 66 Varroa samples from Türkiye, twenty harbored the Y337F mutation, with eight samples exhibiting fixation of the mutation. Subsequent bioassays revealed over 8-fold resistance to amitraz in populations that contain the Y337F mutation. Genotyping of mites after exposure to 10 mg a.i./l amitraz demonstrated that all surviving mites were homozygous for the Y337F mutation, whereas dead mites carried susceptible alleles, providing genetic linkage between mutation and phenotype. Further, we used CRISPR-Cas9 editing to introduce the Y337F mutation in the orthologous Octß2R of the model organism Tetranychus urticae. Crispants exhibited over threefold resistance to amitraz. In conclusion, this study identified and validated a novel amitraz resistance mutation. Additional research is required to further evaluate the phenotypic strength of Y337F in the context of operational resistance with current treatment strategies.
Asunto(s)
Mutación , Receptores de Amina Biogénica , Toluidinas , Varroidae , Animales , Toluidinas/farmacología , Receptores de Amina Biogénica/genética , Receptores de Amina Biogénica/metabolismo , Varroidae/genética , Varroidae/efectos de los fármacos , Sistemas CRISPR-Cas , Acaricidas/farmacología , Resistencia a Medicamentos/genéticaRESUMEN
Neonatal intensive care unit (NICU), particularly in treating developmental and epileptic encephalopathy (DEE) and metabolic epilepsy (ME), requires a deep understanding of their complex etiologies and treatment responses. After excluding treatable cases such as infectious or autoimmune encephalitis, our focus shifted to a more challenging subgroup of 59 patients for in-depth genetic analysis using exome sequencing (ES). The ES analysis identified 40 genetic abnormalities, significantly including de novo variants. Notably, we found structural variation as duplications in regions 2q24.3, including SCN1A and SCN2A were observed in 7 cases. These genetic variants, impacting ion channels, glucose transport, transcription regulation, and kinases, play a crucial role in determining medication efficacy. More than one-third (34.2%) of patients with DEE had an unfavorable response to anti-seizure medications (ASMs) in the chronic phase. However, since the ketogenic supplementary diet showed a positive effect, more than three-quarters (80%) of these drug-resistant patients improved during a 3-month follow-up. In contrast, the ME had a lower adverse reaction rate of 9.1% (2/22) to specialized medications, yet there were 5 fatalities and 10 cases with unidentified genetic etiologies. This study suggests the potential of categorizing drug-resistant variants and that a ketogenic diet could be beneficial in managing DEE and ME. It also opens new perspectives on the mechanisms of the ketogenic diet on the discovered genetic variants.
Asunto(s)
Genotipo , Humanos , Femenino , Masculino , Epilepsia/tratamiento farmacológico , Epilepsia/genética , Secuenciación del Exoma , Recién Nacido , Dieta Cetogénica , Resultado del Tratamiento , Lactante , Anticonvulsivantes/uso terapéutico , Canal de Sodio Activado por Voltaje NAV1.1/genética , Canal de Sodio Activado por Voltaje NAV1.2/genética , Canal de Sodio Activado por Voltaje NAV1.2/metabolismo , Resistencia a Medicamentos/genética , Epilepsia Refractaria/tratamiento farmacológico , Epilepsia Refractaria/genéticaRESUMEN
BACKGROUND: The spread of antimalarial drug resistance parasites is a major obstacle in eliminating malaria in endemic areas. This increases the urgency for developing novel antimalarial drugs with improved profiles to eliminate both sensitive and resistant parasites in populations. The invention of the drug candidates needs a model for sensitive and resistant parasites on a laboratory scale. METHODS: Repeated Incomplete Treatment (RIcT) method was followed in raising the rodent malaria parasite, Plasmodium berghei, resistant to sulfadoxine. Plasmodium berghei were exposed to an adequate therapeutic dose of sulfadoxine without finishing the treatment to let the parasite recover. Cycles of drug treatment and parasite recovery were repeated until phenotypic resistance appeared. RESULTS: After undergoing 3-4 cycles, phenotypic resistance was not yet found in mice treated with sulfadoxine. Nevertheless, the molecular biology of dhps gene (the target of sulfadoxine) was analyzed at the end of the RIcT cycle. There was no mutations found in the gene target. Interestingly, the appearance of gametocytes at the end of every cycle of drug treatment and parasite recovery was observed. These gametocytes later on would no longer extend their life in the RBC stage, unless mosquitoes bite the infected host. This phenomenon is similar to the case in human malaria infections treated with sulfadoxine-pyrimethamine (SP). CONCLUSIONS: In this study, the antimalarial drug sulfadoxine induced gametocytogenesis in P. berghei, which could raise the risk factor for malaria transmission.
Asunto(s)
Antimaláricos , Plasmodium berghei , Sulfadoxina , Plasmodium berghei/efectos de los fármacos , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Animales , Sulfadoxina/farmacología , Sulfadoxina/uso terapéutico , Ratones , Resistencia a Medicamentos/genética , Gametogénesis/efectos de los fármacos , Femenino , Malaria/tratamiento farmacológico , Malaria/parasitologíaRESUMEN
Background: Stroke or transient ischaemic attack patients are at increased risk of secondary vascular events. Antiplatelet medications, most commonly clopidogrel, are prescribed to reduce this risk. Factors including CYP2C19 genetic variants can hinder clopidogrel metabolism. Laboratory-based or point-of-care tests can detect these variants, enabling targeted treatment. Objective: To assess the effectiveness of genetic testing to identify clopidogrel resistance in people with ischaemic stroke or transient ischaemic attack. Specific objectives: Do people tested for clopidogrel resistance, and treated accordingly, have a reduced risk of secondary vascular events? Do people with loss-of-function alleles associated with clopidogrel resistance have a reduced risk of secondary vascular events if treated with alternative interventions compared to clopidogrel? Do people with loss-of-function alleles associated with clopidogrel resistance have an increased risk of secondary vascular events when treated with clopidogrel? What is the accuracy of point-of-care tests for detecting variants associated with clopidogrel resistance? What is the technical performance and cost of CYP2C19 genetic tests? Is genetic testing for clopidogrel resistance cost-effective compared with no testing? Design: Systematic review and economic model. Results: Objective 1: Two studies assessed secondary vascular events in patients tested for loss-of-function alleles and treated accordingly. They found a reduced risk, but confidence intervals were wide (hazard ratio 0.50, 95% confidence interval 0.09 to 2.74 and hazard ratio 0.53, 95% confidence interval 0.24 to 1.18). Objective 2: Seven randomised controlled trials compared clopidogrel with alternative treatment in people with genetic variants. Ticagrelor was associated with a lower risk of secondary vascular events than clopidogrel (summary hazard ratio 0.76, 95% confidence interval 0.65 to 0.90; two studies). Objective 3: Twenty-five studies compared outcomes in people with and without genetic variants treated with clopidogrel. People with genetic variants were at an increased risk of secondary vascular events (hazard ratio 1.72, 95% confidence interval 1.43 to 2.08; 18 studies). There was no difference in bleeding risk (hazard ratio 0.98, 95% confidence interval 0.68 to 1.40; five studies). Objective 4: Eleven studies evaluated Genomadix Cube accuracy; no studies evaluated Genedrive. Summary sensitivity and specificity against laboratory reference standards were both 100% (95% confidence interval 94% to 100% and 99% to 100%). Objective 5: Seventeen studies evaluated technical performance of point-of-care tests. Test failure rate ranged from 0.4% to 19% for Genomadix Cube. A survey of 8/10 genomic laboratory hubs revealed variation in preferred technologies for testing, and cost per test ranging from £15 to £250. Most laboratories expected test failure rate to be < 1%. Additional resources could enhance testing capacity and expedite turnaround times. Objective 6: Laboratory and point-of-care CYP2C19 testing strategies were cost-saving and increase quality-adjusted life-years compared with no testing. Both strategies gave similar costs, quality-adjusted life-years and expected net monetary benefit. Conclusions: Our results suggest that CYP2C19 testing followed by tailored treatment is likely to be effective and cost-effective in both populations. Future work: Accuracy and technical performance of Genedrive. Test failure rate of Genomadix Cube in a National Health Service setting. Value of testing additional loss-of-function alleles. Appropriateness of treatment dichotomy based on loss-of-function alleles. Limitations: Lack of data on Genedrive. No randomised 'test-and-treat' studies of dipyramidole plus aspirin. Study registration: This study is registered as PROSPERO CRD42022357661. Funding: This award was funded by the National Institute for Health and Care Research (NIHR) Evidence Synthesis programme (NIHR award ref: NIHR135620) and is published in full in Health Technology Assessment; Vol. 28, No. 57. See the NIHR Funding and Awards website for further award information.
The most common type of stroke occurs when the supply of blood to the brain is cut off. Symptoms of stroke happen suddenly and vary depending on which part of the brain is affected. They usually include problems with movement, speech, vision and the face drooping on one side. A 'transient ischaemic attack' is a milder related condition. There are around 100,000 strokes and 60,000 transient ischaemic attacks every year in the UK. People who have a stroke or transient ischaemic attack are at greater risk of having another stroke. To reduce the chances of this happening, doctors will often prescribe medication. The most common medication used is called 'clopidogrel'. However, clopidogrel does not work for everyone. One reason for this is having specific variations of a gene called the CYP2C19 gene. Around one in three people in the UK have this variation. We wanted to know whether introducing genetic testing to identify variations in the CYP2C19 gene for people who have had a stroke or transient ischaemic attack can help doctors prescribe a treatment that will work for them, reducing the risk of having another stroke. We also wanted to know if doing this test would be a good use of NHS money. Doing a genetic test to identify variations in the CYP2C19 gene, and prescribing an alternative medication for people with these variations, may reduce the chances of having a new stroke. It is likely that a genetic test for variations of the CYP2C19 gene would represent value for money for the NHS.
Asunto(s)
Clopidogrel , Análisis Costo-Beneficio , Citocromo P-450 CYP2C19 , Resistencia a Medicamentos , Ataque Isquémico Transitorio , Inhibidores de Agregación Plaquetaria , Clopidogrel/uso terapéutico , Humanos , Ataque Isquémico Transitorio/tratamiento farmacológico , Inhibidores de Agregación Plaquetaria/uso terapéutico , Citocromo P-450 CYP2C19/genética , Resistencia a Medicamentos/genética , Accidente Cerebrovascular Isquémico/tratamiento farmacológico , Genotipo , Modelos Económicos , Pruebas Genéticas , Años de Vida Ajustados por Calidad de VidaRESUMEN
Steroid-resistant asthma (SRA), resisting glucocorticoids such as dexamethasone (DEX), is a bottleneck in the treatment of asthma. It is characterized by a predominantly neutrophilic inflammatory subtype and is prone to developing into severe refractory asthma and fatal asthma. Currently, there is a lack of universally effective treatments for SRA. Moreover, since cold stimulation does increase the risk of asthma development and exacerbate asthma symptoms, the treatment of cold-stimulated SRA (CSRA) will face greater challenges. To find effective new methods to ameliorate CSRA, this study established a CSRA mouse model of allergic airway inflammation mimicking human asthma for the first time and evaluated the alleviating effects of 80% ethanol extract of mountain-cultivated ginseng (MCG) based on multi-omics analysis. The results indicate that cold stimulation indeed exacerbated the SRA-related symptoms in mice; the DEX individual treatment did not show a satisfactory effect; while the combination treatment of DEX and MCG could dose-dependently significantly enhance the lung function; reduce neutrophil aggregation; decrease the levels of LPS, IFN-γ, IL-1ß, CXCL8, and IL-17; increase the level of IL-10; alleviate the inflammatory infiltration; and decrease the mucus secretion and the expression of MUC5AC. Moreover, the combination of DEX and high-dose (200 mg/kg) MCG could significantly increase the levels of tight junction proteins (TJs), regulate the disordered intestinal flora, increase the content of short-chain fatty acids (SCFAs), and regulate the abnormal gene profile and metabolic profile. Multi-omics integrated analysis showed that 7 gut microbes, 34 genes, 6 metabolites, and the involved 15 metabolic/signaling pathways were closely related to the pharmacological effects of combination therapy. In conclusion, integrated multi-omics profiling highlighted the benefits of MCG for CSRA mice by modulating the interactions of microbiota, genes, and metabolites. MCG shows great potential as a functional food in the adjuvant treatment of CSRA.
Asunto(s)
Asma , Dexametasona , Panax , Extractos Vegetales , Animales , Asma/tratamiento farmacológico , Asma/microbiología , Asma/metabolismo , Panax/química , Ratones , Dexametasona/farmacología , Extractos Vegetales/farmacología , Frío , Modelos Animales de Enfermedad , Microbioma Gastrointestinal/efectos de los fármacos , Metabolómica/métodos , Microbiota/efectos de los fármacos , Ratones Endogámicos BALB C , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/microbiología , Pulmón/patología , Mucina 5AC/metabolismo , Mucina 5AC/genética , Citocinas/metabolismo , Resistencia a Medicamentos/genética , Femenino , MultiómicaRESUMEN
East African countries accounted for ~ 10% of all malaria prevalence worldwide in 2022, with an estimated 23.8 million cases and > 53,000 deaths. Despite recent increases in malaria incidence, high-resolution genome-wide analyses of Plasmodium parasite populations are sparse in Kenya, Tanzania, and Uganda. The Kenyan-Ugandan border region is a particular concern, with Uganda confirming the emergence and spread of artemisinin resistant P. falciparum parasites. To establish genomic surveillance along the Kenyan-Ugandan border and analyse P. falciparum population dynamics within East Africa, we generated whole-genome sequencing (WGS) data for 38 parasites from Bungoma, Western Kenya. These sequences were integrated into a genomic analysis of available East African isolate data (n = 599) and revealed parasite subpopulations with distinct genetic structure and diverse ancestral origins. Ancestral admixture analysis of these subpopulations alongside isolates from across Africa (n = 365) suggested potential independent ancestral populations from other major African populations. Within isolates from Western Kenya, the prevalence of biomarkers associated with chloroquine resistance (e.g. Pfcrt K76T) were significantly reduced compared to wider East African populations and a single isolate contained the PfK13 V568I variant, potentially linked to reduced susceptibility to artemisinin. Overall, our work provides baseline WGS data and analysis for future malaria genomic surveillance in the region.
Asunto(s)
Resistencia a Medicamentos , Malaria Falciparum , Plasmodium falciparum , Plasmodium falciparum/genética , Plasmodium falciparum/efectos de los fármacos , Kenia/epidemiología , Humanos , Uganda/epidemiología , Malaria Falciparum/epidemiología , Malaria Falciparum/parasitología , Resistencia a Medicamentos/genética , Secuenciación Completa del Genoma , Dinámica Poblacional , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Genómica/métodos , África Oriental/epidemiología , Genoma de ProtozoosRESUMEN
Despite the overall decline in malaria cases in Thailand, continuous surveillance in endemic areas remains crucial. This retrospective analysis examined Plasmodium falciparum samples from Tak province, Thailand, collected in 1998, 1999, and 2001, to investigate the prevalence and evolution of antimalarial genotypic drug resistance. The study revealed a high prevalence of drug-resistant P. falciparum, particularly to mefloquine and sulfadoxine/pyrimethamine, with significant mutations in genes associated with resistance. Notably, mutations indicative of artemisinin resistance, such as those in the kelch13 gene, were detected at low frequencies, suggesting an evolving resistance pattern. The underlying cause of these resistance mutations appears to be the historical and widespread use of these antimalarial drugs, which exerted selective pressure on the parasite population. These findings underscore the necessity of ongoing surveillance and adaptive control strategies to manage drug resistance, guide treatment policies, and prevent potential outbreaks, even as malaria cases decrease. Continuous monitoring and research are imperative to sustain malaria elimination efforts and address the dynamic challenges posed by evolving drug-resistant strains.
Asunto(s)
Antimaláricos , Resistencia a Medicamentos , Malaria Falciparum , Mutación , Plasmodium falciparum , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Tailandia/epidemiología , Resistencia a Medicamentos/genética , Malaria Falciparum/parasitología , Malaria Falciparum/epidemiología , Malaria Falciparum/tratamiento farmacológico , Humanos , Estudios Retrospectivos , Prevalencia , Mefloquina/farmacología , Mefloquina/uso terapéutico , Animales , Artemisininas/farmacología , Artemisininas/uso terapéutico , Pirimetamina/farmacología , Pirimetamina/uso terapéutico , Sulfadoxina/farmacología , Sulfadoxina/uso terapéutico , Combinación de MedicamentosRESUMEN
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
Chicken coccidiosis causes retarded growth and low production performance in poultry, resulting in huge economic losses to the poultry industry. In order to prevent and control chicken coccidiosis, great efforts have been made to develop new drugs and vaccines, which require pure isolates of Eimeria spp. In this study, we obtained the Eimeira tenella Xiantao isolate by single oocyst isolation technology and compared its genome with the reference genome GCF_000499545.2_ETH001 of the Houghton strain. The results of the comparative genomic analysis indicated that the genome of this isolate contained 46,888 single nucleotide polymorphisms (SNPs). There were 15,107 small insertion and deletion variations (indels), 1693 structural variations (SV), and 3578 copy number variations (CNV). In addition, 64 broilers were used to determine the resistance profile of Xiantao strain. Drug susceptibility testing revealed that this isolate was completely resistant to monensin, diclazuril, halofuginone, sulfachlorpyrazine sodium, and toltrazuril, but sensitive to decoquinate. These data improve our understanding of drug resistance in avian coccidia.
Asunto(s)
Pollos , Coccidiosis , Resistencia a Medicamentos , Eimeria tenella , Enfermedades de las Aves de Corral , Eimeria tenella/genética , Eimeria tenella/efectos de los fármacos , Eimeria tenella/aislamiento & purificación , Animales , China , Pollos/parasitología , Enfermedades de las Aves de Corral/parasitología , Coccidiosis/veterinaria , Coccidiosis/parasitología , Resistencia a Medicamentos/genética , Coccidiostáticos/farmacología , Polimorfismo de Nucleótido Simple , Genoma de ProtozoosRESUMEN
The emergence of drug-resistant Plasmodium falciparum parasites in sub-Saharan Africa will substantially challenge malaria control. Here, we evaluated the frequency of common drug resistance markers among adolescents from Northern Uganda with asymptomatic infections. We used an established amplicon deep sequencing strategy to screen dried blood spot samples collected from 2016 to 2017 during a reported malaria epidemic within the districts of Kitgum and Pader in Northern Uganda. We screened single-nucleotide polymorphisms within: kelch13 (Pfk13), dihydropteroate synthase (Pfdhps), multidrug resistance-1 (Pfmdr1), dihydrofolate reductase (Pfdhfr), and apical membrane antigen (Pfama1) genes. Within the study population, the median age was 15 years (14.3-15.0, 95% CI), and 54.9% (78/142) were Plasmodium positive by 18S rRNA qPCR, which were subsequently targeted for sequencing analysis. We observed a high frequency of resistance markers particularly for sulfadoxine-pyrimethamine (SP), with no wild-type-only parasites observed for Pfdhfr (N51I, C59R, and S108N) and Pfdhps (A437G and K540E) mutations. Within Pfmdr1, mixed infections were common for NF/NY (98.5%). While for artemisinin resistance, in kelch13, there was a high frequency of C469Y (34%). Using the pattern for Pfama1, we found a high level of polygenomic infections with all individuals presenting with complexity of infection greater than 2 with a median of 6.9. The high frequency of the quintuple SP drug-resistant parasites and the C469Y artemisinin resistance-associated mutation in asymptomatic individuals suggests an earlier high prevalence than previously reported from symptomatic malaria surveillance studies (in 2016/2017). Our data demonstrate the urgency for routine genomic surveillance programs throughout Africa and the value of deep sequencing.
Asunto(s)
Antimaláricos , Infecciones Asintomáticas , Resistencia a Medicamentos , Malaria Falciparum , Plasmodium falciparum , Pirimetamina , Sulfadoxina , Plasmodium falciparum/genética , Plasmodium falciparum/efectos de los fármacos , Humanos , Uganda/epidemiología , Adolescente , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Malaria Falciparum/epidemiología , Malaria Falciparum/parasitología , Malaria Falciparum/tratamiento farmacológico , Pirimetamina/farmacología , Pirimetamina/uso terapéutico , Estudios Retrospectivos , Sulfadoxina/farmacología , Sulfadoxina/uso terapéutico , Resistencia a Medicamentos/genética , Femenino , Infecciones Asintomáticas/epidemiología , Masculino , Mutación , Proteínas Protozoarias/genética , Combinación de Medicamentos , Polimorfismo de Nucleótido Simple/genética , Prevalencia , Artemisininas/farmacología , Artemisininas/uso terapéutico , Tetrahidrofolato Deshidrogenasa/genéticaRESUMEN
BACKGROUND: Giardiasis, caused by the protozoan parasite Giardia intestinalis, often presents a treatment challenge, particularly in terms of resistance to metronidazole. Despite extensive research, markers for metronidazole resistance have not yet been identified. METHODS: This study analysed 28 clinical samples of G. intestinalis from sub-assemblage AII, characterised by varying responses to metronidazole treatment. We focussed on copy number variation (CNV) of the multi-copy flavohemoprotein gene, analysed using digital polymerase chain reaction (dPCR) and next generation sequencing (NGS). Additionally, chromosomal ploidy was tested in 18 of these samples. Flavohemoprotein CNV was also assessed in 17 samples from other sub-assemblages. RESULTS: Analyses revealed variable CNVs of the flavohemoprotein gene among the isolates, with no correlation to clinical metronidazole resistance. Discrepancies in CNVs detected from NGS data were attributed to biases linked to the whole genome amplification. However, dPCR helped to clarify these discrepancies by providing more consistent CNV data. Significant differences in flavohemoprotein CNVs were observed across different G. intestinalis sub-assemblages. Notably, Giardia exhibits a propensity for aneuploidy, contributing to genomic variability within and between sub-assemblages. CONCLUSIONS: The complexity of the clinical metronidazole resistance in Giardia is influenced by multiple genetic factors, including CNVs and aneuploidy. No significant differences in the CNV of the flavohemoprotein gene between isolates from metronidazole-resistant and metronidazole-sensitive cases of giardiasis were found, underscoring the need for further research to identify reliable genetic markers for resistance. We demonstrate that dPCR and NGS are robust methods for analysing CNVs and provide cross-validating results, highlighting their utility in the genetic analyses of this parasite.
Asunto(s)
Antiprotozoarios , Variaciones en el Número de Copia de ADN , Resistencia a Medicamentos , Giardia lamblia , Giardiasis , Metronidazol , Giardia lamblia/genética , Giardia lamblia/efectos de los fármacos , Metronidazol/farmacología , Resistencia a Medicamentos/genética , Humanos , Giardiasis/parasitología , Giardiasis/tratamiento farmacológico , Antiprotozoarios/farmacología , Secuenciación de Nucleótidos de Alto Rendimiento , Proteínas Protozoarias/genéticaRESUMEN
To understand the benzimidazole (BZ) resistance of Haemonchus contortus in Southern Xinjiang, three single nucleotide polymorphisms (SNPs) designated as F167Y, E198A, and F200Y, in the isotype-1 ß-tubulin gene which are associated with BZ resistance, were investigated for H. contortus populations from sheep in Hejing and Minfeng counties of Southern Xinjiang. In brief, a total of 190 H. contortus adults were collected from 52 out of 70 slaughtered sheep in city abattoirs across two regions in Southern Xinjiang. The species identity of each adult worm was confirmed by PCR amplification of ITS-2 using H. contortus-specific primers targeting the ITS-2. The samples were then investigated for BZ-related SNPs at locus 167, 198, and 200, by PCR-sequencing of the isotype-1 ß-tubulin gene. The results showed that only E198A and F200Y mutations were detected in the investigated H. contortus populations. The E198A mutation (homozygous and heterozygote resistant: found in 40% and 30% of sequenced samples from Minfeng and Hejing counties, respectively) was predominant compared with the F200Y mutation (homozygous and heterozygote resistant: found in 14% and 13.3% of sequenced samples from Minfeng and Hejing counties, respectively). The results indicate a high prevalence of BZ resistance in H. contortus populations from certain areas of Southern Xinjiang. Our findings provide valuable information for the prevention and control of H. contortus in areas with similar conditions.
Asunto(s)
Antihelmínticos , Bencimidazoles , Resistencia a Medicamentos , Hemoncosis , Haemonchus , Polimorfismo de Nucleótido Simple , Enfermedades de las Ovejas , Tubulina (Proteína) , Animales , Haemonchus/efectos de los fármacos , Haemonchus/genética , Bencimidazoles/farmacología , Ovinos , Resistencia a Medicamentos/genética , Enfermedades de las Ovejas/parasitología , Enfermedades de las Ovejas/epidemiología , China/epidemiología , Tubulina (Proteína)/genética , Hemoncosis/veterinaria , Hemoncosis/parasitología , Antihelmínticos/farmacología , Análisis de Secuencia de ADN , ADN Espaciador Ribosómico/genética , Reacción en Cadena de la PolimerasaRESUMEN
A new superior bacteria complementation model was achieved for testing antifolate compounds and investigating antifolate resistance in the dihydrofolate reductase (DHFR) enzyme of the malaria parasite. Earlier models depended on the addition of trimethoprim (TMP) to chemically suppress the host Escherichia coli (Ec) DHFR function. However, incomplete suppression of EcDHFR and potential interference of antibiotics needed to maintain plasmids for complementary gene expression can complicate the interpretations. To overcome such limitations, the folA (F) and thyA (T) genes were genetically knocked out (Δ) in E. coli BL21(DE3). The resulting EcΔFΔT cells were thymidine auxotroph where thymidine supplementation or functional complementation with heterologous DHFR-thymidylate synthase (TS) is needed to restore the loss of gene functions. When tested against pyrimethamine (PYR) and its analogs designed to target Plasmodium falciparum (Pf) DHFR-TS, the 50 % inhibitory concentration values obtained from EcΔFΔT surrogates expressing wildtype (PfTM4) or double mutant (PfK1) DHFR-TS showed strong correlations to the results obtained from the standard in vitro P. falciparum growth inhibition assay. Interestingly, while TMP had little effect on the susceptibility to PYR and analogs in EcΔFΔT expressing PfDHFR-TS, it hypersensitized the chemically knockdown E. coli BL21(DE3) expressing PfTM4 DHFR-TS but desensitized the one carrying PfK1 DHFR-TS. The low intrinsic expression level of PfTM4 in E. coli BL21(DE3) by western blot analysis may explain the hypersensitive to antifolates of chemical knockdown bacteria surrogate. These results demonstrated the usefulness of EcΔFΔT surrogate as a new tool for antifolate antimalarial screening with potential application for investigation of antifolate resistance mechanism.
Asunto(s)
Escherichia coli , Antagonistas del Ácido Fólico , Técnicas de Inactivación de Genes , Plasmodium falciparum , Pirimetamina , Tetrahidrofolato Deshidrogenasa , Timidilato Sintasa , Escherichia coli/genética , Escherichia coli/efectos de los fármacos , Antagonistas del Ácido Fólico/farmacología , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismo , Timidilato Sintasa/genética , Timidilato Sintasa/metabolismo , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Pirimetamina/farmacología , Antimaláricos/farmacología , Concentración 50 Inhibidora , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Resistencia a Medicamentos/genética , Prueba de Complementación Genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Complejos MultienzimáticosRESUMEN
Recrudescent infections with the human malaria parasite, Plasmodium falciparum, presented traditionally the major setback of artemisinin-based monotherapies. Although the introduction of artemisinin combination therapies (ACT) largely solved the problem, the ability of artemisinin to induce dormant parasites still poses an obstacle for current as well as future malaria chemotherapeutics. Here, we use a laboratory model for induction of dormant P. falciparum parasites and characterize their transcriptome, drug sensitivity profile, and cellular ultrastructure. We show that P. falciparum dormancy requires a ~ 5-day maturation process during which the genome-wide gene expression pattern gradually transitions from the ring-like state to a unique form. The transcriptome of the mature dormant stage carries hallmarks of both cellular quiescence and senescence, with downregulation of most cellular functions associated with growth and development and upregulation of selected metabolic functions and DNA repair. Moreover, the P. falciparum dormant stage is considerably more resistant to antimalaria drugs compared to the fast-growing asexual stages. Finally, the irregular cellular ultrastructure further suggests unique properties of this developmental stage of the P. falciparum life cycle that should be taken into consideration by malaria control strategies.
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Antimaláricos , Artemisininas , Senescencia Celular , Malaria Falciparum , Plasmodium falciparum , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Plasmodium falciparum/crecimiento & desarrollo , Artemisininas/farmacología , Antimaláricos/farmacología , Senescencia Celular/efectos de los fármacos , Malaria Falciparum/parasitología , Malaria Falciparum/tratamiento farmacológico , Humanos , Transcriptoma/efectos de los fármacos , Estadios del Ciclo de Vida/efectos de los fármacos , Resistencia a Medicamentos/genética , Resistencia a Medicamentos/efectos de los fármacosRESUMEN
BACKGROUND: The burden of malaria persists in sub-Saharan Africa and the emergence of artemisinin resistance has introduced complexity to control efforts. Monitoring the efficacy of artemisinin-based treatment for malaria is crucial to address this challenge. This study assessed treatment efficacy of artemether-lumefantrine (AL) and genetic diversity of Plasmodium falciparum isolates in a Nigerian population. METHODS: Participants presenting with clinical symptoms of uncomplicated malaria at a health centre in Lagos, Nigeria, were screened for P. falciparum. Enrolled participants were treated with AL and monitored through scheduled check-up visits, clinical and laboratory examinations for 28 days. Parasite clearance and genetic diversity were assessed through polymerase chain reaction (PCR) analysis of merozoite surface proteins (msp1 and msp2). The prevalence of drug resistance mutations was assessed by P. falciparum multidrug resistance gene 1 (mdr1) genotyping followed by P. falciparum ubiquitin-specific protease 1 (ubp1) gene sequencing. RESULTS: The PCR-uncorrected treatment outcome revealed 94.4% adequate clinical and parasitological response (ACPR) and 5.6% late parasitological failure (LPF) rates. After PCR correction, no suspected LPF case was detected and ACPR 67/67 (100%) was achieved in all the individuals. Moreover, a high prevalence of wild-type alleles for mdr1 N86Y (93.7%), and mdr1 D1246Y (87.5%) was observed. Genetic diversity analysis revealed predominant K1 allelic family for msp1 (90.2%) and FC27 for msp2 (64.4%). Estimated multiplicity of infection (MOI) was 1.7, with the highest MOI observed in the 5-15 years age group. ubp1 sequence analysis identified one nonsynonymous E1528D polymorphism at a low frequency (1.6%). CONCLUSION: The study demonstrated sustained efficacy of AL for treating uncomplicated P. falciparum malaria. Genetic diversity analysis revealed various allelic types, suggesting occurrences of polyclonal infections. Nonetheless, the detection of a significant ubp1 polymorphism could have future implications for the epidemiology of anti-malarial drug resistance in the population.
Asunto(s)
Antimaláricos , Combinación Arteméter y Lumefantrina , Resistencia a Medicamentos , Malaria Falciparum , Plasmodium falciparum , Combinación Arteméter y Lumefantrina/uso terapéutico , Nigeria , Humanos , Plasmodium falciparum/genética , Plasmodium falciparum/efectos de los fármacos , Antimaláricos/uso terapéutico , Antimaláricos/farmacología , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Femenino , Masculino , Niño , Preescolar , Adolescente , Adulto , Adulto Joven , Resistencia a Medicamentos/genética , Persona de Mediana Edad , Lactante , Resultado del Tratamiento , Artemisininas/uso terapéutico , Artemisininas/farmacología , Variación Genética , Anciano , Proteínas Protozoarias/genética , Combinación de Medicamentos , Proteína 1 de Superficie de Merozoito/genética , Etanolaminas/uso terapéutico , Fluorenos/uso terapéuticoRESUMEN
Malaria, an ancient mosquito-borne illness caused by Plasmodium parasites, is mostly treated with Artemisinin Combination Therapy (ACT). However, Single Nucleotide Polymorphisms (SNPs) mutations in the P. falciparum Kelch 13 (PfK13) protein have been associated with artemisinin resistance (ART-R). Therefore, this study aims to generate PfK13 recombinant proteins incorporating of two specific SNPs mutations, PfK13-V494I and PfK13-N537I, and subsequently analyze their binding interactions with artemisinin (ART). The recombinant proteins of PfK13 mutations and the Wild Type (WT) variant were expressed utilizing a standard protein expression protocol with modifications and subsequently purified via IMAC and confirmed with SDS-PAGE analysis and Orbitrap tandem mass spectrometry. The binding interactions between PfK13-V494I and PfK13-N537I propeller domain proteins ART were assessed through Isothermal Titration Calorimetry (ITC) and subsequently validated using fluorescence spectrometry. The protein concentrations obtained were 0.3 mg/ml for PfK13-WT, 0.18 mg/ml for PfK13-V494I, and 0.28 mg/ml for PfK13-N537I. Results obtained for binding interaction revealed an increased fluorescence intensity in the mutants PfK13-N537I (83 a.u.) and PfK13-V494I (143 a.u.) compared to PfK13-WT (33 a.u.), indicating increased exposure of surface proteins because of the looser binding between PfK13 protein mutants with ART. This shows that the PfK13 mutations may induce alterations in the binding interaction with ART, potentially leading to reduced effectiveness of ART and ultimately contributing to ART-R. However, this study only elucidated one facet of the contributing factors that could serve as potential indicators for ART-R and further investigation should be pursued in the future to comprehensively explore this complex mechanism of ART-R.
Asunto(s)
Artemisininas , Plasmodium falciparum , Unión Proteica , Proteínas Protozoarias , Proteínas Recombinantes , Artemisininas/farmacología , Plasmodium falciparum/genética , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/química , Mutación , Polimorfismo de Nucleótido Simple , Antimaláricos/farmacología , Resistencia a Medicamentos/genéticaRESUMEN
Benzimidazole (BZ) anthelmintics are among the most important treatments for parasitic nematode infections in the developing world. Widespread BZ resistance in veterinary parasites and emerging resistance in human parasites raise major concerns for the continued use of BZs. Knowledge of the mechanisms of resistance is necessary to make informed treatment decisions and circumvent resistance. Benzimidazole resistance has traditionally been associated with mutations and natural variants in the C. elegans beta-tubulin gene ben-1 and orthologs in parasitic species. However, variants in ben-1 alone do not explain the differences in BZ responses across parasite populations. Here, we examined the roles of five C. elegans beta-tubulin genes (tbb-1, mec-7, tbb-4, ben-1, and tbb-6) in the BZ response as well as to determine if another beta-tubulin acts redundantly with ben-1. We generated C. elegans strains with a loss of each beta-tubulin gene, as well as strains with a loss of tbb-1, mec-7, tbb-4, or tbb-6 in a genetic background that also lacks ben-1. We found that the loss of ben-1 conferred the maximum level of resistance following exposure to a single concentration of albendazole, and the loss of a second beta-tubulin gene did not alter the level of resistance. However, additional traits other than larval development could be affected by the loss of additional beta-tubulins, and the roles of other beta-tubulin genes might be revealed at different albendazole concentrations. Therefore, further work is needed to fully define the possible roles of other beta-tubulin genes in the BZ response.
Asunto(s)
Albendazol , Antihelmínticos , Caenorhabditis elegans , Resistencia a Medicamentos , Mutación , Tubulina (Proteína) , Animales , Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/genética , Tubulina (Proteína)/genética , Resistencia a Medicamentos/genética , Antihelmínticos/farmacología , Albendazol/farmacología , Proteínas de Caenorhabditis elegans/genéticaRESUMEN
Aldo-keto reductases (AKRs), a superfamily of NADP(H)-dependent oxidoreductases, catalyze the oxidoreduction of a wide variety of eobiotic and xenobiotic aldehydes and ketones. In mammals, AKRs play essential roles in hormone and xenobiotic metabolism, oxidative stress, and drug resistance, but little is known about these enzymes in the parasitic nematode Haemonchus contortus. In the present study, 22 AKR genes existing in the H. contortus genome were investigated and a phylogenetic analysis with comparison to AKRs in Caenorhabditis elegans, sheep and humans was conducted. The constitutive transcription levels of all AKRs were measured in eggs, larvae, and adults of H. contortus, and their expression was compared in a drug-sensitive strain (ISE) and a benzimidazole-resistant strain (IRE) previously derived from the sensitive strain by imposing benzimidazole selection pressure. In addition, the inducibility of AKRs by exposure of H. contortus adults to benzimidazole anthelmintic flubendazole in vitro was tested. Phylogenetic analysis demonstrated that the majority of AKR genes in H. contortus lack orthologues in the sheep genome, which is a favorable finding for considering AKRs as potential drug targets. Large differences in the expression levels of individual AKRs were observed, with AKR1, AKR3, AKR8, and AKR10 being the most highly expressed at most developmental stages. Significant changes in the expression of AKRs during the life cycle and pronounced sex differences were found. Comparing the IRE and ISE strains, three AKRs were upregulated, and seven AKRs were downregulated in adults. In addition, the expression of three AKRs was induced by flubendazole exposure in adults of the ISE strain. Based on these results, AKR1, AKR2, AKR3, AKR5, AKR10 and AKR19 in particular merit further investigation and functional characterization with respect to their potential involvement in drug biotransformation and anthelmintic resistance in H. contortus.