Comparison of different genetic testing modalities applied in paediatric patients with steroid-resistant nephrotic syndrome.

BACKGROUND: Steroid-resistant nephrotic syndrome (SRNS) are monogenic in some cases, however, there are still no clear guidelines on genetic testing in the clinical practice of SRNS in children. METHODS: Three hundred thirty-two children were diagnosed with SRNS, and all children underwent genetic testing, including gene panels and/or whole-exome/genome sequencing (WES/WGS), during treatment. We analysed the relationship between clinical manifestation and genotype, and compared different genetic testing methods' detection rates and prices. RESULTS: In this study, 30.12% (100/332) of children diagnosed with SRNS had monogenic causes of the disease. With 33.7% (122/332) of children achieving complete remission, 88.5% (108/122) received steroids combined with tacrolimus (TAC). In detectability, WES increased by 8.69% (4/46) on gene panel testing, while WGS increased by 4.27% (5/117) on WES, and WES was approximately 1/7 of the price of WGS for every further 1% increase in pathogenicity. CONCLUSIONS: We verified that steroids combined with TAC were the most effective option in paediatric SRNS. In detection efficiency, we found that WGS was the highest, followed by WES. The panel was the lowest, but the most cost-effective method when considering the economic-benefit ratio, and thus it should be recommended first in SRNS.

A Biotinylated cpFIT-PNA Platform for the Facile Detection of Drug Resistance to Artemisinin in Plasmodium falciparum.

The evolution of drug resistance to many antimalarial drugs in the lethal strain of malaria (Plasmodium falciparum) has been a great concern over the past 50 years. Among these drugs, artemisinin has become less effective for treating malaria. Indeed, several P. falciparum variants have become resistant to this drug, as elucidated by specific mutations in the pfK13 gene. This study presents the development of a diagnostic kit for the detection of a common point mutation in the pfK13 gene of P. falciparum, namely, the C580Y point mutation. FIT-PNAs (forced-intercalation peptide nucleic acid) are DNA mimics that serve as RNA sensors that fluoresce upon hybridization to their complementary RNA. Herein, FIT-PNAs were designed to sense the C580Y single nucleotide polymorphism (SNP) and were conjugated to biotin in order to bind these molecules to streptavidin-coated plates. Initial studies with synthetic RNA were conducted to optimize the sensing system. In addition, cyclopentane-modified PNA monomers (cpPNAs) were introduced to improve FIT-PNA sensing. Lastly, total RNA was isolated from red blood cells infected with P. falciparum (WT strain - NF54-WT or mutant strain - NF54-C580Y). Streptavidin plates loaded with either FIT-PNA or cpFIT-PNA were incubated with the total RNA. A significant difference in fluorescence for mutant vs WT total RNA was found only for the cpFIT-PNA probe. In summary, this study paves the way for a simple diagnostic kit for monitoring artemisinin drug resistance that may be easily adapted to malaria endemic regions.

Absence of association between Pfnfs1 mutation and in vitro susceptibility to lumefantrine in Plasmodium falciparum.

Artemether-lumefantrine (AL) is the most widely used antimalarial drug for treating uncomplicated falciparum malaria. This study evaluated whether the K65Q mutation in the Plasmodium falciparum cysteine desulfurase IscS (Pfnfs1) gene was associated with alternated susceptibility to lumefantrine using clinical parasite samples from Ghana and the China-Myanmar border area. Parasite isolates from the China-Myanmar border had significantly higher IC50 values to lumefantrine than parasites from Ghana. In addition, the K65 allele was significantly more prevalent in the Ghanaian parasites (34.5%) than in the China-Myanmar border samples (6.8%). However, no difference was observed in the lumefantrine IC50 value between the Pfnfs1 reference K65 allele and the non reference 65Q allele in parasites from the two regions. These data suggest that the Pfnfs1 K65Q mutation may not be a reliable marker for reduced susceptibility to lumefantrine.

Distribution patterns of molecular markers of antimalarial drug resistance in Plasmodium falciparum isolates on the Thai-Myanmar border during the periods of 1993-1998 and 2002-2008.

BACKGROUND: Polymorphisms of Plasmodium falciparum chloroquine resistance transporter (pfcrt), Plasmodium falciparum multi-drug resistance 1 (pfmdr1) and Plasmodium falciparum kelch 13-propeller (pfk13) genes are accepted as valid molecular markers of quinoline antimalarials and artemisinins. This study investigated the distribution patterns of these genes in P. falciparum isolates from the areas along the Thai-Myanmar border during the two different periods of antimalarial usage in Thailand. RESULTS: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were used to detect pfcrt mutations at codons 76, 220, 271, 326, 356, and 371 as well as pfmdr1 mutation at codon 86. The prevalence of pfcrt mutations was markedly high (96.4-99.7%) in samples collected during both periods. The proportions of mutant genotypes (number of mutant/total isolate) at codons 76, 220, 271, 326, 356 and 371 in the isolates collected during 1993-1998 (period 1) compared with 2002-2008 (period 2) were 97.9% (137/140) vs. 97.1% (401/413), 97.9% (140/143) vs. 98.8% (171/173), 97.2% (139/143) vs. 97.1% (333/343), 98.6% (140/142) vs. 99.7% (385/386), 96.4% (134/139) vs. 98.2% (378/385) and 97.8% (136/139) vs. 98.9% (375/379), respectively. Most isolates carried pfmdr1 wild-type at codon 86, with a significant difference in proportions genotypes (number of wild type/total sample) in samples collected during period 1 [92.9% (130/140)] compared with period 2 [96.9% (379/391)]. Investigation of pfmdr1 copy number was performed by real-time PCR. The proportions of isolates carried 1, 2, 3 and 4 or more than 4 copies of pfmdr1 (number of isolates carried correspondent copy number/total isolate) were significantly different between the two sample collecting periods (65.7% (90/137) vs. 87.8% (390/444), 18.2% (25/137) vs. 6.3%(28/444), 5.1% (7/137) vs. 1.4% (6/444) and 11.0% (15/137) vs. 4.5% (20/444), for period 1 vs. period 2, respectively). No pfk13 mutation was detected by nested PCR and nucleotide sequencing in all samples with successful analysis (n = 68). CONCLUSIONS: The persistence of pfcrt mutations and pfmdr1 wild-types at codon 86, along with gene amplification in P. falciparum, contributes to the continued resistance of chloroquine and mefloquine in P. falciparum isolates in the study area. Regular surveillance of antimalarial drug resistance in P. falciparum, incorporating relevant molecular markers and treatment efficacy assessments, should be conducted.

Trends of Plasmodium falciparum molecular markers associated with resistance to artemisinins and reduced susceptibility to lumefantrine in Mainland Tanzania from 2016 to 2021.

BACKGROUND: Therapeutic efficacy studies (TESs) and detection of molecular markers of drug resistance are recommended by the World Health Organization (WHO) to monitor the efficacy of artemisinin-based combination therapy (ACT). This study assessed the trends of molecular markers of artemisinin resistance and/or reduced susceptibility to lumefantrine using samples collected in TES conducted in Mainland Tanzania from 2016 to 2021. METHODS: A total of 2,015 samples were collected during TES of artemether-lumefantrine at eight sentinel sites (in Kigoma, Mbeya, Morogoro, Mtwara, Mwanza, Pwani, Tabora, and Tanga regions) between 2016 and 2021. Photo-induced electron transfer polymerase chain reaction (PET-PCR) was used to confirm presence of malaria parasites before capillary sequencing, which targeted two genes: Plasmodium falciparum kelch 13 propeller domain (k13) and P. falciparum multidrug resistance 1 (pfmdr1). RESULTS: Sequencing success was ≥ 87.8%, and 1,724/1,769 (97.5%) k13 wild-type samples were detected. Thirty-seven (2.1%) samples had synonymous mutations and only eight (0.4%) had non-synonymous mutations in the k13 gene; seven of these were not validated by the WHO as molecular markers of resistance. One sample from Morogoro in 2020 had a k13 R622I mutation, which is a validated marker of artemisinin partial resistance. For pfmdr1, all except two samples carried N86 (wild-type), while mutations at Y184F increased from 33.9% in 2016 to about 60.5% in 2021, and only four samples (0.2%) had D1246Y mutations. pfmdr1 haplotypes were reported in 1,711 samples, with 985 (57.6%) NYD, 720 (42.1%) NFD, and six (0.4%) carrying minor haplotypes (three with NYY, 0.2%; YFD in two, 0.1%; and NFY in one sample, 0.1%). Between 2016 and 2021, NYD decreased from 66.1% to 45.2%, while NFD increased from 38.5% to 54.7%. CONCLUSION: This is the first report of the R622I (k13 validated mutation) in Tanzania. N86 and D1246 were nearly fixed, while increases in Y184F mutations and NFD haplotype were observed between 2016 and 2021. Despite the reports of artemisinin partial resistance in Rwanda and Uganda, this study did not report any other validated mutations in these study sites in Tanzania apart from R622I suggesting that intensified surveillance is urgently needed to monitor trends of drug resistance markers and their impact on the performance of ACT.

Antigenic strain diversity predicts different biogeographic patterns of maintenance and decline of antimalarial drug resistance.

The establishment and spread of antimalarial drug resistance vary drastically across different biogeographic regions. Though most infections occur in sub-Saharan Africa, resistant strains often emerge in low-transmission regions. Existing models on resistance evolution lack consensus on the relationship between transmission intensity and drug resistance, possibly due to overlooking the feedback between antigenic diversity, host immunity, and selection for resistance. To address this, we developed a novel compartmental model that tracks sensitive and resistant parasite strains, as well as the host dynamics of generalized and antigen-specific immunity. Our results show a negative correlation between parasite prevalence and resistance frequency, regardless of resistance cost or efficacy. Validation using chloroquine-resistant marker data supports this trend. Post discontinuation of drugs, resistance remains high in low-diversity, low-transmission regions, while it steadily decreases in high-diversity, high-transmission regions. Our study underscores the critical role of malaria strain diversity in the biogeographic patterns of resistance evolution.

Drug resistance among strains of the parasites that cause malaria is a growing problem for people relying on antimalarial drugs to protect them from the disease. This phenomenon is global yet exactly how resistance emerges, spreads and persists in a population often differs greatly between regions, which can complicate malaria control projects. For example, discontinuing the use of antimalarials can lead to the frequency of resistant strains declining in an area, such as Africa, but persisting at high levels in others, including Asia and South America. Gaining resistance often leads to parasites becoming less transmissible than other strains. When antimalarials are not used, sensitive strains usually outcompete their resistant counterparts. However, prolonged use of antimalarial drugs tends to eliminate susceptible strains, allowing the previously outcompeted resistant strains to dominate. The local dynamics of antimalarial resistance are also shaped by multiple other factors such as transmission levels (how common the disease is in the region), the type of antimalarial measures used (such as drugs and mosquito nets), or previous immunity the population may have developed to specific strains. While many computational models have been developed to capture these dynamics, they usually fail to include strain diversity ­ a parameter reflecting the number of malaria strains the immune system is exposed to. This parameter is important as parasites need to escape both host immunity and drugs in order to be successful. To address this gap, He, Chaillet, and Labbé created a computational model to investigate how strain diversity, transmission levels and other related factors influence antimalarial resistance. The model was used to explore how the frequency of resistant and susceptible strains changes over time once antimalarial drugs are rolled out and then halted. These analyses show that in areas with both low strain diversity and low transmission levels, susceptible parasites are more likely to be wiped out from the population, leading to a high frequency of resistant strains that persist after drugs are discontinued. However, in high diversity and high transmission regions, susceptible strains can remain in the population. Therefore, when drug treatments are stopped, resistance levels are more likely to drop due to these parasites outcompeting the drug-resistant ones. Overall, this work demonstrates how modelling approaches that include strain diversity can help inform public health decisions aimed at reducing antimalarial resistance. In particular, they can provide important insights into the control strategies that are best suited for a specific region, suggesting that in low transmission areas intensive drug treatment may contribute to resistance. Instead, preventative strategies such as eliminating mosquitos and preventing bites with bed nets may prove more beneficial at reducing transmission rates in such areas.

[Single - nucleotide polymorphisms of artemisinin resistance - related Pfubp1 and Pfap2mu genes in Bioko Island, Equatorial Guinea from 2018 to 2020].

OBJECTIVE: To investigate the prevalence of single nucleotide polymorphisms (SNPs) of artemisinin resistance-related Pfubp1 and Pfap2mu genes in Plasmodium falciparum isolates from Bioko Island, Equatorial Guinea, so as to to provide baseline data for the formulation of malaria control strategies in Bioko Island. METHODS: A total of 184 clinical blood samples were collected from patients with P. falciparum malaria in Bioko Island, Equatorial Guinea from 2018 to 2020, and genomic DNA was extracted. The Pfubp1 and Pfap2mu gene SNPs of P. falciparum were determined using a nested PCR assay and Sanger sequencing, and the gene sequences were aligned. RESULTS: There were 159 wild-type P. falciparum isolates (88.83%) from Bioko Island, Equatorial Guinea, and 6 SNPs were identified in 20 Pfubp1-mutant P. falciparum isolates (11.17%), in which 4 non-synonymous mutations were detected, including E1516G, K1520E, D1525E, E1528D. There was only one Pfubp1gene mutation site in 19 Pfubp1-mutant P. falciparum isolates (95.00%), in which non-synonymous mutations accounted for 68.42% (13/19). D1525E and E1528D were identified as major known epidemic mutation sites in the Pfubp1 gene associated with resistance to artemisinin-based combination therapies (ACTs). At amino acid position 1525, there were 178 wild-type P. falciparum isolates (99.44%) and 1 mutant isolate (0.56%), with such a mutation site identified in blood samples in 2018, and at amino acid position 1528, there were 167 wild-type P. falciparum isolates (93.30%) and 12 mutant isolates (6.70%). The proportions of wild-type P. falciparum isolates were 95.72% (134/140), 79.25% (126/159) and 95.83% (161/168) in the target amplification fragments of the three regions in the Pfap2mu gene (Pfap2mu-inner1, Pfap2mu-inner2, Pfap2mu-inner3), respectively. There were 16 different SNPs identified in all successfully sequenced P. falciparum isolates, in which 7 non-synonymous mutations were detected, including S160N, K199T, A475V, S508G, I511M, L595F, and Y603H. There were 7 out of 43 Pfap2mu-mutant P. falciparum isolates (16.28%) that harbored only one gene mutation site, in which non-synonymous mutations accounted for 28.57% (2/7). For the known delayed clearance locus S160N associated with ACTs, there were 143 wild-type (89.94%) and 16 Pfap2mu-mutant P. falciparum isolates (10.06%). CONCLUSIONS: Both Pfubp1 and Pfap2mu gene mutations were detected in P. falciparum isolates from Bioko Island, Equatorial Guinea from 2018 to 2020, with a low prevalence rate of Pfubp1 gene mutation and a high prevalence rate of Pfap2mu gene mutation. In addition, new mutation sites were identified in the Pfubp1 (E1504E and K1520E) and Pfap2mu genes (A475V and S508G).

Assessing emergence risk of double-resistant and triple-resistant genotypes of Plasmodium falciparum.

Delaying and slowing antimalarial drug resistance evolution is a priority for malaria-endemic countries. Until novel therapies become available, the mainstay of antimalarial treatment will continue to be artemisinin-based combination therapy (ACT). Deployment of different ACTs can be optimized to minimize evolutionary pressure for drug resistance by deploying them as a set of co-equal multiple first-line therapies (MFT) rather than rotating therapies in and out of use. Here, we consider one potential detriment of MFT policies, namely, that the simultaneous deployment of multiple ACTs could drive the evolution of different resistance alleles concurrently and that these resistance alleles could then be brought together by recombination into double-resistant or triple-resistant parasites. Using an individual-based model, we compare MFT and cycling policies in malaria transmission settings ranging from 0.1% to 50% prevalence. We define a total risk measure for multi-drug resistance (MDR) by summing the area under the genotype-frequency curves (AUC) of double- and triple-resistant genotypes. When prevalence ≥ 1%, total MDR risk ranges from statistically similar to 80% lower under MFT policies than under cycling policies, irrespective of whether resistance is imported or emerges de novo. At 0.1% prevalence, there is little statistical difference in MDR risk between MFT and cycling.

Detection of Artemisinin Resistance Marker Kelch-13 469Y in Plasmodium falciparum, South Kivu, Democratic Republic of the Congo, 2022.

Partial artemisinin resistance has emerged in East Africa, posing a threat to malaria control across the continent. The Democratic Republic of the Congo carries one of the heaviest malaria burdens globally, and the South Kivu province directly borders current artemisinin resistance hot spots, but indications of such resistance have not been observed so far. We assessed molecular markers of antimalarial drug resistance in 256 Plasmodium falciparum isolates collected in 2022 in South Kivu, Democratic Republic of the Congo. One isolate carried the P. falciparum Kelch-13 469Y variant, a marker associated with partial artemisinin resistance and decreased lumefantrine susceptibility in Uganda. In addition, the multidrug resistance-1 mutation pattern suggested increased lumefantrine tolerance.

Ex vivo drug susceptibility and resistance mediating genetic polymorphisms of Plasmodium falciparum in Bobo-Dioulasso, Burkina Faso.

Malaria remains a leading cause of morbidity and mortality in Burkina Faso, which utilizes artemether-lumefantrine as the principal therapy to treat uncomplicated malaria and seasonal malaria chemoprevention with monthly sulfadoxine-pyrimethamine plus amodiaquine in children during the transmission season. Monitoring the activities of available antimalarial drugs is a high priority. We assessed the ex vivo susceptibility of Plasmodium falciparum to 11 drugs in isolates from patients presenting with uncomplicated malaria in Bobo-Dioulasso in 2021 and 2022. IC50 values were derived using a standard 72 h growth inhibition assay. Parasite DNA was sequenced to characterize known drug resistance-mediating polymorphisms. Isolates were generally susceptible, with IC50 values in the low-nM range, to chloroquine (median IC5010 nM, IQR 7.9-24), monodesethylamodiaquine (22, 14-46) piperaquine (6.1, 3.6-9.2), pyronaridine (3.0, 1.3-5.5), quinine (50, 30-75), mefloquine (7.1, 3.7-10), lumefantrine (7.1, 4.5-12), dihydroartemisinin (3.7, 2.2-5.5), and atovaquone (0.2, 0.1-0.3) and mostly resistant to cycloguanil (850, 543-1,290) and pyrimethamine (33,200, 18,400-54,200), although a small number of outliers were seen. Considering genetic markers of resistance to aminoquinolines, most samples had wild-type PfCRT K76T (87%) and PfMDR1 N86Y (95%) sequences. For markers of resistance to antifolates, established PfDHFR and PfDHPS mutations were highly prevalent, the PfDHPS A613S mutation was seen in 19% of samples, and key markers of high-level resistance (PfDHFR I164L; PfDHPS K540E) were absent or rare (A581G). Mutations in the PfK13 propeller domain known to mediate artemisinin partial resistance were not detected. Overall, our results suggest excellent susceptibilities to drugs now used to treat malaria and moderate, but stable, resistance to antifolates used to prevent malaria.

Gene co-expression network analysis reveal core responsive genes in Parascaris univalens tissues following ivermectin exposure.

Anthelmintic resistance in equine parasite Parascaris univalens, compromises ivermectin (IVM) effectiveness and necessitates an in-depth understanding of its resistance mechanisms. Most research, primarily focused on holistic gene expression analyses, may overlook vital tissue-specific responses and often limit the scope of novel genes. This study leveraged gene co-expression network analysis to elucidate tissue-specific transcriptional responses and to identify core genes implicated in the IVM response in P. univalens. Adult worms (n = 28) were exposed to 10-11 M and 10-9 M IVM in vitro for 24 hours. RNA-sequencing examined transcriptional changes in the anterior end and intestine. Differential expression analysis revealed pronounced tissue differences, with the intestine exhibiting substantially more IVM-induced transcriptional activity. Gene co-expression network analysis identified seven modules significantly associated with the response to IVM. Within these, 219 core genes were detected, largely expressed in the intestinal tissue and spanning diverse biological processes with unspecific patterns. After 10-11 M IVM, intestinal tissue core genes showed transcriptional suppression, cell cycle inhibition, and ribosomal alterations. Interestingly, genes PgR028_g047 (sorb-1), PgB01_g200 (gmap-1) and PgR046_g017 (col-37 & col-102) switched from downregulation at 10-11 M to upregulation at 10-9 M IVM. The 10-9 M concentration induced expression of cuticle and membrane integrity core genes in the intestinal tissue. No clear core gene patterns were visible in the anterior end after 10-11 M IVM. However, after 10-9 M IVM, the anterior end mostly displayed downregulation, indicating disrupted transcriptional regulation. One interesting finding was the non-modular calcium-signaling gene, PgR047_g066 (gegf-1), which uniquely connected 71 genes across four modules. These genes were enriched for transmembrane signaling activity, suggesting that PgR047_g066 (gegf-1) could have a key signaling role. By unveiling tissue-specific expression patterns and highlighting biological processes through unbiased core gene detection, this study reveals intricate IVM responses in P. univalens. These findings suggest alternative drug uptake of IVM and can guide functional validations to further IVM resistance mechanism understanding.

Analysis of P-gp genes relative expression associated to ivermectin resistance in Haemonchus contortus larval stages from in vitro cultures (L3 and xL3) and from gerbils (Meriones unguiculatus) (L4) as models of study.

The aim of the study was to compare the relative gene expression of Haemonchus contortus P-glycoprotein genes (Hco-pgp) between fourth (L4), infective (L3), and transitory infective (xL3) larval stages as laboratory models to study ivermectin (IVM) resistance. The H. contortus resistant to IVM (IVMr) and susceptible to IVM (IVMs) strains were used to develop xL3in vitro culture and to infect Meriones unguiculatus (gerbils) to collect L4 stages. Morphometric differences were evaluated from 25 individuals of H. contortus from each strain. Relative gene expression from xL3 and L4 was determined between comparison of IVMr stages and from IVMr vs IVMs stages. Seven Hco-pgp genes (1, 2, 3, 4, 9, 10, and 16) were analysed by RT-qPCR using L3 stage as control group, per strain, and GAPDH and ß-tubulin as constitutive genes. Morphological changes were confirmed between xL3 and L4 developing oral shape, oesophagus, and intestinal tube. In addition, the body length and width showed statistical differences (p < 0.05). The Hco-pgp1, 2, 3, and 4 genes (p < 0.05) were upregulated from 7.1- to 463.82-fold changes between IVMr stages, and Hco-pgp9 (13.12-fold) and Hco-pgp10 (13.56-fold) genes showed differences between L4 and xL3, respectively. The comparative study between IVMr vs IVMs strains associated to xL3 and L4 displayed significant upregulation for most of the Hco-pgp genes among 4.89-188.71 fold-change. In conclusion, these results suggest the use of H. contortus xL3 and L4 as suitable laboratory models to study IVMr associated with Hco-pgp genes to contribute to the understanding of anthelmintic resistance.

Geographical distribution of pyrethroid resistance mutations in Varroa destructor across Türkiye and a European overview.

Varroa destructor Anderson & Trueman (Acari: Varroidae) is of paramount significance in modern beekeeping, with infestations presenting a primary challenge that directly influences colony health, productivity, and overall apicultural sustainability. In order to control this mite, many beekeepers rely on a limited number of approved synthetic acaricides, including the pyrethroids tau-fluvalinate, flumethrin and organophosphate coumaphos. However, the excessive use of these substances has led to the widespread development of resistance in various beekeeping areas globally. In the present study, the occurrence of resistance mutations in the voltage-gated sodium channel (VGSC) and acetylcholinesterase (AChE), the target-site of pyrethroids and coumaphos, respectively, was examined in Varroa populations collected throughout the southeastern and eastern Anatolia regions of Türkiye. All Varroa samples belonged to the Korean haplotype, and a very low genetic distance was observed based on cytochrome c oxidase subunit I (COI) gene sequences. No amino acid substitutions were determined at the key residues of AChE. On the other hand, three amino acid substitutions, (L925V/I/M), previously associated with pyrethroid resistance, were identified in nearly 80% of the Turkish populations. Importantly, L925M, the dominant mutation in the USA, was detected in Turkish Varroa populations for the first time. To gain a more comprehensive perspective, we conducted a systematic analysis of the distribution of pyrethroid resistance mutations across Europe, based on the previously reported data. Varroa populations from Mediterranean countries such as Türkiye, Spain, and Greece exhibited the highest frequency of resistance mutation. Revealing the occurrence and geographical distribution of pyrethroid resistance mutations in V. destructor populations across the country will enhance the development of more efficient strategies for mite management.

Functional validation of novel levamisole resistance marker S168T in Haemonchus contortus.

Recently, a S168T variant in the acetylcholine receptor subunit ACR-8 was associated with levamisole resistance in the parasitic helminth Haemonchus contortus. Here, we used the Xenopus laevis oocyte expression system and two-electrode voltage-clamp electrophysiology to measure the functional impact of this S168T variant on the H. contortus levamisole-sensitive acetylcholine receptor, L-AChR-1.1. Expression of the ACR-8 S168T variant significantly reduced the current amplitude elicited by levamisole compared to acetylcholine, with levamisole changing from a full to partial agonist on the recombinant L-AChR. Functional validation of the S168T mutation on modulating levamisole activity at the receptor level highlights its critical importance as both a mechanism and a marker of levamisole resistance.

Relationship between acaricide resistance and acetylcholinesterase gene polymorphisms in the cattle tick Rhipicephalus microplus.

In this study, we aimed to develop a comprehensive methodology for identifying amino acid polymorphisms in acetylcholinesterase transcript 2 (AChE2) in acaricide-resistant Rhipicephalus microplus ticks. This included assessing AChE2 expression levels through qPCR and conducting 3D modeling to evaluate the interaction between acaricides and AChE2 using docking techniques. The study produced significant results, demonstrating that acaricide-resistant R. microplus ticks exhibit significantly higher levels of AChE expression than susceptible reference ticks. In terms of amino acid sequence, we identified 9 radical amino acid substitutions in AChE2 from acaricide-resistant ticks, when compared to the gene sequence of the susceptible reference strain. To further understand the implications of these substitutions, we utilized 3D acaricide-AChE2 docking modeling to examine the interaction between the acaricide and the AChE2 catalytic site. Our models suggest that these amino acid polymorphisms alter the configuration of the binding pocket, thereby contributing to differences in acaricide interactions and ultimately providing insights into the acaricide-resistance phenomenon in R. microplus.

Title: Relations entre la résistance aux acaricides et les polymorphismes du gène de l'acétylcholinestérase chez la tique du bétail Rhipicephalus microplus. Abstract: Notre étude vise à développer une méthodologie complète pour identifier les polymorphismes d'acides aminés dans le transcrit 2 de l'acétylcholinestérase (AChE2) chez les tiques Rhipicephalus microplus résistantes aux acaricides. Cela comprend l'évaluation des niveaux d'expression d'AChE2 via qPCR et la réalisation d'une modélisation 3D pour évaluer l'interaction entre les acaricides et l'AChE2 à l'aide de techniques d'amarrage moléculaire. L'étude a produit des résultats significatifs, démontrant que les tiques R. microplus résistantes aux acaricides présentent des niveaux d'expression d'AChE significativement plus élevés que les tiques sensibles de référence. En termes de séquence d'acides aminés, nous avons identifié 9 substitutions d'acides aminés dans AChE2 provenant de tiques résistantes aux acaricides par rapport à la séquence génétique de la souche sensible de référence. Pour mieux comprendre les implications de ces substitutions, nous avons utilisé la modélisation de l'amarrage acaricide-AChE2 pour examiner l'interaction entre l'acaricide et le site catalytique AChE2. Nos modèles suggèrent que ces polymorphismes d'acides aminés modifient la configuration de la poche de liaison, contribuant ainsi aux différences dans les interactions acaricides et fournissant finalement un aperçu du phénomène de résistance aux acaricides chez R. microplus.

Epoxidase inhibitor-aspirin resistance and the relationship with genetic polymorphisms: a review.

Strokes are the leading cause of death in most regions of the world. Epoxidase inhibitors include the drug aspirin (acetylsalicylic acid). Aspirin is widely used as first-line treatment for the prevention of cardiovascular and cerebrovascular diseases in at-risk patients. However, patients using conventional doses of aspirin can still develop ischaemic cardiovascular and cerebrovascular diseases, a phenomenon known as aspirin resistance. The occurrence of aspirin resistance hinders the prevention and treatment of ischaemic cardiovascular and cerebrovascular diseases. There are many factors affecting aspirin resistance, such as sex, drug dose, metabolic disease, genetic polymorphisms, drug interactions and pharmacokinetics. Genetic polymorphism refers to the simultaneous and frequent presence of two or more discontinuous variants or genotypes or alleles in a population of organisms. Platelets contain a large number of highly polymorphic transmembrane glycoprotein receptors encoded by two or more isomeric alleles. Changes in gene polymorphisms in various pathways during platelet aggregation can lead to aspirin resistance. This narrative review describes the gene polymorphisms that have been demonstrated to be significantly associated with aspirin resistance. Research on the mechanisms of aspirin resistance and increased knowledge should provide accurate drug guidance in individuals that require first-line antiplatelet therapy.

Deaggregation of mutant Plasmodium yoelii de-ubiquitinase UBP1 alters MDR1 localization to confer multidrug resistance.

Mutations in a Plasmodium de-ubiquitinase UBP1 have been linked to antimalarial drug resistance. However, the UBP1-mediated drug-resistant mechanism remains unknown. Through drug selection, genetic mapping, allelic exchange, and functional characterization, here we show that simultaneous mutations of two amino acids (I1560N and P2874T) in the Plasmodium yoelii UBP1 can mediate high-level resistance to mefloquine, lumefantrine, and piperaquine. Mechanistically, the double mutations are shown to impair UBP1 cytoplasmic aggregation and de-ubiquitinating activity, leading to increased ubiquitination levels and altered protein localization, from the parasite digestive vacuole to the plasma membrane, of the P. yoelii multidrug resistance transporter 1 (MDR1). The MDR1 on the plasma membrane enhances the efflux of substrates/drugs out of the parasite cytoplasm to confer multidrug resistance, which can be reversed by inhibition of MDR1 transport. This study reveals a previously unknown drug-resistant mechanism mediated by UBP1 through altered MDR1 localization and substrate transport direction in a mouse model, providing a new malaria treatment strategy.

The proof is in the poo-ding: Benefits of the longitudinal molecular surveillance of drug resistance demonstrated in a New South Wales cattle herd.

Our understanding of anthelmintic resistance in the gastrointestinal nematodes of Australian cattle relies exclusively on small-scale phenotypic reports utilising traditional faecal egg count reduction tests. This approach is not readily scalable to establish the national prevalence of resistance, nor is it conducive of routine longitudinal surveillance for the emergence of resistance in its early stages. This study introduces the benefits of applying mixed amplicon metabarcoding longitudinally for timely and cost-efficient molecular surveillance of multiple anthelmintic resistance mutations, as they emerge on farms. Using opportunistically collected faecal samples from a cattle herd in central west New South Wales (2019-2023), we detected the early emergence of Haemonchus spp. levamisole-resistant S168T shortly after levamisole introduction, while benzimidazole-resistant allele frequencies remained constant. Additionally, we observed the possible spill-over of resistant Haemonchus contortus from sheep, along with variations in faecal burdens and species diversity influenced by climate stochasticity and host immunity. This study emphasises the power of molecular diagnostics for farm-level anthelmintic resistance management, providing essential evidence to support its integration into routine surveillance programmes.

Oxidative stress changes the effectiveness of artemisinin in Plasmodium falciparum.

Malaria parasites have adaptive mechanisms to modulate their intracellular redox status to tolerate the enhanced oxidizing effects created by malaria fever, hemoglobinopathies and other stress conditions, including antimalaria drugs. Emerging artemisinin (ART) resistance in Plasmodium falciparum is a complex phenotype linked to the parasite's tolerance of the activated drug's oxidative damage along with changes in vesicular transport, lipid metabolism, DNA repair, and exported proteins. In an earlier study, we discovered that many of these metabolic processes are induced in P. falciparum to respond to the oxidative damage caused by artemisinin, which exhibited a highly significant overlap with the parasite's adaptive response mechanisms to survive febrile temperatures. In addition, there was a significant overlap with the parasite's survival responses to oxidative stress. In this study, we investigated these relationships further using an in vitro model to evaluate if oxidative stress and heat-shock conditions could alter the parasite's response to artemisinin. The results revealed that compared to ideal culture conditions, the antimalarial efficacy of artemisinin was significantly reduced in parasites growing in intraerythrocytic oxidative stress but not in heat-shock condition. In contrast, heat shock significantly reduced the efficacy of lumefantrine that is an important ART combination therapy partner drug. We propose that prolonged exposure to intraerythrocytic microenvironmental oxidative stress, as would occur in endemic regions with high prevalence for sickle trait and other hemoglobinopathies, can predispose malaria parasites to develop tolerance to the oxidative damage caused by antimalarial drugs like artemisinin. IMPORTANCE: Emerging resistance to the frontline antimalarial drug artemisinin represents a significant threat to worldwide malaria control and elimination. The patterns of parasite changes associated with emerging resistance represent a complex array of metabolic processes evident in various genetic mutations and altered transcription profiles. Genetic factors identified in regulating P. falciparum sensitivity to artemisinin overlap with the parasite's responses to malarial fever, sickle trait, and other types of oxidative stresses, suggesting conserved inducible survival responses. In this study we show that intraerythrocytic stress conditions, oxidative stress and heat shock, can significantly decrease the sensitivity of the parasite to artemisinin and lumefantrine, respectively. These results indicate that an intraerythrocytic oxidative stress microenvironment and heat-shock condition can alter antimalarial drug efficacy. Evaluating efficacy of antimalarial drugs under ideal in vitro culture conditions may not accurately predict drug efficacy in all malaria patients.