Aspirin Resistance and Ischemic Stroke.

Objective: Acetylsalicylic acid (ASA) is the most widely used antiplatelet agent in treating ischemic strokes. ASA resistance varies between the populations, from 5% to 60%. We aimed to determine the prevalence of ASA resistance in our population, its association with demographic characteristics, risk factors, and the occurrence of recurrent ischemic stroke (IS). (IST). Methods: One hundred consecutive patients with primary or recurrent IS were prospectively included. Strokes were diagnosed with computed tomography (CT) or magnetic resonance imaging (MRI). In all patients a detailed history of cardiovascular risk factors and fasting blood analyzes (blood count, glycemia, HbA1c, degradation products, lipid profile) were taken, and the ASA resistance was examined with the Innovance PFA 200 system. The ASA resistance was examined after a minimum of 1 month of regular use of ASA 100 mg per day. Results: The prevalence of ASA resistance was 32%. ASA-resistant patients were statistically significantly older (69.9±7.5 vs, 61±1 y., p<0.05), and of male gender (75 vs, 45.6%, p<0.05). Previous cardiovascular diseases were statistically significantly more common in the ASA-resistant patients compared to the ASA-sensitive patients (46.8 vs 26.5%, p<0.05). The presence of recurrent stroke in the ASA-resistant versus ASA-sensitive patients was statistically significant (75% vs. 22.05%, p<0.0001). There was no statistically significant difference in the investigated laboratory parameters and the risk factors between the ASA-resistant and the ASA-sensitive patients. Conclusion. The prevalence of ASA resistance in our population is high. According to our results, ASA resistance testing should be done in the older male individuals with a previous burden of cardiovascular diseases.

Molecular assessment of voltage-gated sodium channel (VGSC) gene mutations in Rhipicephalus microplus from Guangxi, China.

BACKGROUND: Pyrethroid chemicals are one of the main acaricides used against ticks. Resistance to these chemicals has been reported to be associated with mutations in the voltage-gated sodium channel (VGSC) gene of the Rhipicephalus microplus. This study investigates R. microplus resistance to pyrethroids in Guangxi region of China, marking one of the first research efforts in this area. The findings are intended to provide vital baseline for the effective implementation of localized tick control strategies. METHODS: From March to July 2021, 447 R. microplus tick samples were collected from five prefecture-level cities in Guangxi. Allele-specific polymerase chain reaction (AS-PCR) was used to amplify segments C190A and G215T of the domain II S4-5 linker and T2134A of domain III S6 in the VGSC, to detect nucleotide mutations associated with resistance to pyrethroid acaricides. Subsequent analyses were conducted to ascertain the prevalence, types of mutations, and genotypic distributions within the sampled populations. RESULTS: Mutations within VGSC gene were identified across all five studied populations of R. microplus, although the mutation rates remained generally low. Specifically, the most prevalent mutation was C190A, observed in 4.9% of the samples (22/447), followed by G215T at 4.0% (18/447), and T2134A at 1.3% (6/447). The distribution of mutations across three critical sites of the VGSC gene revealed four distinct mutation types: C190A, G215T, C190A + G215T, and T2134A. Notably, the single mutation C190A had the highest mutation frequency, accounting for 4.3%, and the C190A + G215T combination had the lowest, at only 0.7%. The analysis further identified seven genotypic combinations, with the wild-type combination C/C + G/G + T/T predominating at a frequency of 90.4%. Subsequently, the C/A + G/G + T/T combination was observed at a frequency of 4.3%, whereas the C/C + T/T + T/T combination exhibited the lowest frequency (0.2%). Additionally, no instances of simultaneous mutations at all three sites were detected. Geographical differences in mutation types were apparent. Both samples from Hechi to Chongzuo cities exhibited the same three mutation types; however, C190A was the most prevalent in Hechi, while G215T dominated in Chongzuo. In contrast, samples from Beihai to Guilin each exhibited only one mutation type: G215T occurred in 12.5% (4/32) of Beihai samples, and C190A in 7.5% (4/53) of Guilin samples. CONCLUSIONS: These findings underscore the relatively low frequency of VGSC gene mutations in R. microplus associated with pyrethroid resistance in the Guangxi, China. Moreover, the variation in mutation types and genotypic distributions across different locales highlights the need for regionalized strategies in monitoring and managing pyrethroid resistance in tick populations. This molecular surveillance is crucial for informing targeted control measures and mitigating the risk of widespread resistance emergence.

Strain-level variations of Dirofilaria immitis microfilariae in two biochemical assays.

BACKGROUND: The increase in reports of resistance to macrocyclic lactones in the canine heartworm, Dirofilaria immitis is alarming. While DNA based tests have been well-validated, they can be expensive. In a previous study, we showed that two biochemical tests adapted to a 96- well plate format and read in a spectrophotometer could detect differences among lab validated D. immitis isolates. The two tests- Resazurin reduction and Hoechst 33342 efflux-detect metabolism and P-glycoprotein activity respectively in microfilariae isolated from infected dog blood. METHODS: Our objective was to optimize the two assays further by testing various assay parameters in D. immitis isolates not tested previously. We tested microfilarial seeding density, incubation time and the effect of in vitro treatment with ivermectin and doxycycline in five other D. immitis isolates-JYD-34, Big Head, Berkeley, Georgia III and LOL. All assays were performed in 3 technical replicates and 2-4 biological replicates. To understand the molecular basis of the assays, we also performed qPCR for selected drug metabolism and elimination associated genes of the ABC transporter and cytochrome P450 gene families. RESULTS: Metabolism and ABC transporter activity as detected by these assays varied between strains. Anthelmintic status (resistant or susceptible) did not correlate with metabolism or P-gp efflux. Basal transcriptional variations were found between strains in ABC transporter and cytochrome P450 genes. CONCLUSIONS: These assays provide a greater understanding of the biochemical variation among isolates of D. immitis, which can be exploited in the future to develop in vitro diagnostic tests capable of differentiating susceptible and resistant isolates.

Vedolizumab plus basiliximab as second-line therapy for steroid-refractory lower gastrointestinal acute graft-versus-host disease.

Background: Steroid-resistant (SR) lower gastrointestinal (LGI) tract graft-versus-host disease (GVHD) is the predominant cause of morbidity and mortality from GVHD after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The role of vedolizumab in the treatment of SR-LGI acute GVHD (aGVHD) remains uncertain. We aimed to assess the efficacy and safety of vedolizumab combined with basiliximab as second-line therapy for SR-LGI-aGVHD. Methods: This study aimed to explore the efficacy of vedolizumab combined with basiliximab for SR-LGI-aGVHD. The primary endpoint was the overall response (OR) on day 28. Secondary and safety endpoints included durable OR at day 56, overall survival (OS), chronic GVHD (cGVHD), non-relapse mortality (NRM), failure-free survival (FFS), and adverse events. Results: Twenty-eight patients with SR-LGI-aGVHD were included. The median time to start of combination therapy after SR-LGI-aGVHD diagnosis was 7 (range, 4-16) days. The overall response rate (ORR) at 28 days was 75.0% (95% CI: 54.8%-88.6%), and 18 achieved a complete response (CR) (64.3%, 95% CI: 44.1%-80.7%). The durable OR at day 56 was 64.3% (95% CI: 44.1%-80.7%). The 100-day, 6-month, and 12-month OS rates for the entire cohort of patients were 60.7% (95% CI: 45.1%-81.8%), 60.7% (95% CI: 45.1%-81.8%), and 47.6% (95% CI: 31.4%-72.1%), respectively. The median failure-free survival was 276 days; (95% CI: 50-not evaluable) 12-month NRM was 42.9% (95% CI: 24.1%-60.3%). The 1-year cumulative incidence of cGVHD was 35.7%. Within 180 days after study treatments, the most common grade 3 and 4 adverse events were infections. Nine (32.1%) patients developed cytomegalovirus (CMV) reactivation complicated with bacterial infections (25.0%, CMV infection; 7.1%, CMV viremia). Epstein-Barr virus (EBV) reactivation occurred in five patients (17.9%, 95% CI: 6.8%-37.6%). Only three patients (10.7%, 95% CI: 2.8%-29.4%) in our study developed pseudomembranous colitis. Conclusions: Vedolizumab plus basiliximab demonstrated efficacy in severe SR-LGI-aGVHD and was well-tolerated. Vedolizumab plus basiliximab may be considered a potential treatment option for patients with LGI-aGVHD.

Artemisinin-resistant malaria in Africa demands urgent action.

Investment in community health workers is essential.

Mutation Screening of Dictyostelium Restriction Enzyme-Mediated Integration (REMI) Libraries.

Identifying the mechanisms of action of existing and novel drugs is essential for the development of new compounds for therapeutic and commercial use. Here we provide a technique to identify these mechanisms through isolating mutant cell lines that show resistance to drug-induced phenotypes using Dictyostelium discoideum REMI libraries. This approach provides a robust and rapid chemical-genetic screening technique that enables an unbiased approach to identify proteins and molecular pathways that control drug sensitivity. Mutations that result in drug resistance often occur in target proteins thus identifying the specific protein targets for drugs and bioactive natural products. Following the identification of a list of putative molecular targets user selected compound targets can be analyzed to confirm and validate direct inhibitory effects.

Investigation of the Antitrichomonal Activity of Cinnamaldehyde, Carvacrol and Thymol and Synergy with Metronidazole.

Objective: Trichomonas vaginalis is a sexually transmitted protozoan parasite that usually causes infections in women. Metronidazole is used as the first choice in the treatment of this parasitic disease, but there is a need for new drugs since 1980's with increasing numbers of reported resistance. In this study, it was aimed to determine the antitrichomonal activity of the major components of Cinnamomum zeylanicum (cinnamon) and Thymus vulgaris (thyme) essential oils, cinnamaldehyde, carvacrol and thymol against metronidazole resistant and susceptible T. vaginalis strains, and to determine their interaction with metronidazole by checkerboard method. Methods: Cinnamaldehyde, carvacrol, thymol and metronidazole were obtained commercially. Two clinical isolates and one metronidazole resistant T. vaginalis reference strain were used in the study. MIC50 and MLC values of essential oil components and metronidazole were determined by broth microdilution method. The combinations of essential oil components with metronidazole were determined by the checkerboard method. Results: According to in vitro activity tests, cinnamaldehyde was determined to be most effective essential oil component. Clinical isolates were susceptible to metronidazole. In combination study, metronidazole showed synergy with cinnamaldehyde and carvacrol, and partial synergy with thymol. Conclusion: It was determined that cinnamaldehyde, carvacrol and thymol, which are known to have high antimicrobial activity, also have strong activity against T. vaginalis isolates and show a synergistic interaction with metronidazole. The use of metronidazole at lower doses in the synergistic interaction may contribute to the literature in terms of reducing drug side effects, creating a versatile antimicrobial target, and reducing the rate of resistance development.

An overview of artemisinin-resistant malaria and associated Pfk13 gene mutations in Central Africa.

Malaria caused by Plasmodium falciparum is one of the deadliest and most common tropical infectious diseases. However, the emergence of artemisinin drug resistance associated with the parasite's Pfk13 gene, threatens the public health of individual countries as well as current efforts to reduce malaria burdens globally. It is of concern that artemisinin-resistant parasites may be selected or have already emerged in Africa. This narrative review aims to evaluate the published evidence concerning validated, candidate, and novel Pfk13 polymorphisms in ten Central African countries. Results show that four validated non-synonymous polymorphisms (M476I, R539T, P553L, and P574L), directly associated with a delayed therapy response, have been reported in the region. Also, two Pfk13 polymorphisms associated to artemisinin resistance but not validated (C469F and P527H) have been reported. Furthermore, several non-validated mutations have been observed in Central Africa, and one allele A578S, is commonly found in different countries, although additional molecular and biochemical studies are needed to investigate whether those mutations alter artemisinin effects. This information is discussed in the context of biochemical and genetic aspects of Pfk13, and related to the regional malaria epidemiology of Central African countries.

TaqMan-quantitative PCR assays applied in Neospora caninum knock-outs generated through CRISPR-Cas9 allow to determine the copy numbers of integrated dihydrofolate reductase-thymidylate synthase drug selectable markers.

As for many other organisms, CRISPR-Cas9 mediated genetic modification has gained increasing importance for the identification of vaccine candidates and drug targets in Neospora caninum, an apicomplexan parasite causing abortion in cattle and neuromuscular disease in dogs. A widely used approach for generating knock-out (KO) strains devoid of virulence factors is the integration of a drug selectable marker such as mutated dihydrofolate reductase-thymidylate synthase (mdhfr-ts) into the target gene, thus preventing the synthesis of respective protein and mediating resistance to pyrimethamine. However, CRISPR-Cas9 mutagenesis is not free of off-target effects, which can lead to integration of multiple mdhfr-ts copies into other sites of the genome. To determine the number of integrated mdhfr-ts in N. caninum, a duplex quantitative TaqMan PCR was developed. For this purpose, primers were designed that amplifies a 106 bp fragment from wild-type (WT) parasites corresponding to the single copy wtdhfrs-ts gene, as well as the mutated mdhfrs-ts present in KO parasites that confers resistance and were used simultaneously with primers amplifying the diagnostic NC5 gene. Thus, the dhfr-ts to NC5 ratio should be approximately 1 in WT parasites, while in KO parasites with a single integrated mdhrf-ts gene this ratio is doubled, and in case of multiple integration events even higher. This approach was applied to the Neospora KO strains NcΔGRA7 and NcΔROP40. For NcΔGRA7, the number of tachyzoites determined by dhfr-ts quantification was twice the number of tachyzoites determined by NC5 quantification, thus indicating that only one mdhfr-ts copy was integrated. The results obtained with the NcΔROP40 strain, however, showed that the number of dhfr-ts copies per genome was substantially higher, indicating that at least three copies of the selectable mdhfr-ts marker were integrated into the genomic DNA during gene editing by CRISPR-Cas9. This duplex TaqMan-qPCR provides a reliable and easy-to-use tool for assessing CRISPR-Cas9 mediated mutagenesis in WT N. caninum strains.

High rates of benzimidazole-resistance-associated alleles in Haemonchus contortus and detection of resistance against macrocyclic lactones in strongylids from German alpaca herds.

The population of South American camelids (SAC) has been steadily growing in Europe, where they are confronted with the regional endoparasite population of ruminants. As there are no anthelmintic drugs registered for use against nematode infections in SACs, anthelmintics (AH) available for ruminants or horses are usually applied. Reports indicating potential failures in administered AH are increasing. However, the generally low egg counts in SACs complicate the application of resistance tests in the field. The present study reports a follow-up study on SAC farms where anthelmintic resistance (AR) was suspected. The aims were (i) to repeat faecal egg count reduction tests (FECRTs) on potentially affected farms identified in a previous study with larger sample sizes, (ii) to verify suspected AR of Haemonchus contortus against benzimidazoles (BZ) by performing a single-nucleotide polymorphism (SNP) analysis using digital polymerase chain reaction (dPCR), and (iii) to apply the mini-FLOTAC technique for more reliable results at low egg counts in line with current recommendations. Seven farms (9-46 animals each) were examined by coproscopy, larval differentiation and SNP analysis. A FECRT was performed on six of these farms with moxidectin (three farms), monepantel (two farms) and ivermectin (one farm). The FEC was calculated according to the current World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines with the clinical protocol (a newly introduced variant of FECRT which can be used for smaller sample sizes and lower egg counts on the cost of sensitivity) and an expected efficacy of 99%. A high level (> 90%) of BZ-resistance-associated SNPs on codon 200 of H. contortus was observed on all farms. With the FECRT, resistance was demonstrated for ivermectin (74% FECR), while it remained inconclusive for one farm for moxidectin treatment. Sustained efficacy was demonstrated for the remaining treatments. This study showed an advanced level of BZ resistance in H. contortus of SACs and the development of AR against macrocyclic lactones on some farms. Thus, constant monitoring of AH treatment and sustainable worm control methods both need to be applied.

Role of Doxofylline, Low Dose Theophylline, and Dexamethasone in Mice (BALB/C) Model of Corticosteroid Resistant Asthma: A Comparative Study.

OBJECTIVE: This study was designed to determine the comparative efficacy of Doxofylline (DOXO) compared to low-dose theophylline (LDT) in treating corticosteroid-resistant asthma. METHODS: This study was conducted on 56 adult BALB/C mice aged six to eight weeks old with an average weight of 20-25 g. They were divided into seven groups: control group, ovalbumin (OVA)+lipopolysaccharide (LPS) group, OVA+LPS+dexamethasone (DEXA) group, OVA+LPS+LDT group, OVA+LPS+ group, OVA+LPS+DEXA+LDT group, and OVA +LPS+DEXA+DOXO group. All mice were administered IP DOXO+DEXA. All the doses were administrated one day before the first challenge and lasted for five consecutive days after one hour of the OVA challenge until sacrificed. Lung biochemical parameters, including interleukin (IL)-2, IL-4, IL-8, IL-10, and IL-17 levels, were measured using enzyme-linked immunosorbent assay (ELISA). In addition, Histone deacetylase (HDAC) activity and lung histological analysis were also performed. Furthermore, the glucocorticoid receptor was measured by nexttec™. RESULTS: The OVA+LPS group exhibited significantly (p<0.05) elevated levels of interleukin (IL)-2, IL-4, IL-8, IL-10, and IL-17 compared to controls, indicative of airway inflammation. Moreover, OVA+LPS induction significantly (p<0.05) increased the levels of Interferon-gamma (IFN-γ), NF[Formula: see text]B, Tumor Necrosis Factor (TNFα), and Immunoglobulin E (IgE) parameters, indicating severe inflammation and immune response and successfully induced the disease model. Meanwhile, LDT and DOXO in conjunction with DEXA, further augmented HDAC2 activity compared to DEXA alone. Similarly, the administration of LDT increased the expression of GR by 64.5% (23.72±0.34), while DOXO increased the expression of GR by 94.10% (27.99±0.15), which restores it back to control. Furthermore, according to Hematoxylin and eosin (H&E) stained sections, the DOXO group exhibited a slight improvement in these histopathological features, suggesting a modest therapeutic effect. Masson's Trichrome staining showed a slightly improved patchy collagen deposition within alveolar spaces in intra-alveolar and interstitial inflammatory cell accumulation in DOXO group, and the combination of these drugs (DEXA+LDT group) improved collagen deposition moderately within alveolar spaces in intra-alveolar and interstitial inflammatory cell accumulation. Overall, treatment with DOXO, LDT alone, and with DEXA combination led to reductions in cytokine levels, with DOXO and LDT showing significant (p<0.05) efficacy to DEXA used alone, which showed non-significant (p>0.05) efficacy. CONCLUSIONS: Doxofylline and LDT were found to be effective therapeutic agents when used alone or in combination with Dexamethasone. However, randomized controlled trials are required to evaluate its further efficacy.

Evidence on sulfadoxine-pyrimethamine resistance molecular markers from India: interpret with caution.

BACKGROUND: Sulfadoxine-pyrimethamine (SP), as a partner to artesunate as ACT is the treatment of choice for uncomplicated P. falciparum infections in the majority of India and SP-resistance has a potential to lead to ACT failure. In the lack of robust surveillance of therapeutic efficacy of SP, validate molecular markers of SP-resistance offer a hint of failing SP. However, studies reporting these validated markers often suffer from certain pitfalls that warrant a careful interpretation. MAIN BODY: Critical analyses of the results and their reported interpretations from a recent study and other studies conducted on the WHO-validated molecular markers of SP-resistance in India were analysed and the main problems with studying and reporting of these markers are presented here. It was noted that almost all studies analysed flawed either on the usage, estimation and/or interpretation of the standardized classification of the studies SP mutations. These flaws not only impart spatiotemporal incomparability of the published data but also have the potential of being misunderstood and wrongly translated. CONCLUSION: Based on this universal problem in studying, reporting and interpreting the data from the studies on molecular markers of SP-resistance, it is stressed that the future studies should be conducted with utmost caution so that robust evidence may be generated and correctly translated to policy.

Two decades of molecular surveillance in Senegal reveal rapid changes in known drug resistance mutations over time.

BACKGROUND: Drug resistance in Plasmodium falciparum is a major threat to malaria control efforts. Pathogen genomic surveillance could be invaluable for monitoring current and emerging parasite drug resistance. METHODS: Data from two decades (2000-2020) of continuous molecular surveillance of P. falciparum parasites from Senegal were retrospectively examined to assess historical changes in malaria drug resistance mutations. Several known drug resistance markers and their surrounding haplotypes were profiled using a combination of single nucleotide polymorphism (SNP) molecular surveillance and whole genome sequence based population genomics. RESULTS: This dataset was used to track temporal changes in drug resistance markers whose timing correspond to historically significant events such as the withdrawal of chloroquine (CQ) and the introduction of sulfadoxine-pyrimethamine (SP) in 2003. Changes in the mutation frequency at Pfcrt K76T and Pfdhps A437G coinciding with the 2014 introduction of seasonal malaria chemoprevention (SMC) in Senegal were observed. In 2014, the frequency of Pfcrt K76T increased while the frequency of Pfdhps A437G declined. Haplotype-based analyses of Pfcrt K76T showed that this rapid increase was due to a recent selective sweep that started after 2014. DISCUSSION (CONCLUSION): The rapid increase in Pfcrt K76T is troubling and could be a sign of emerging amodiaquine (AQ) resistance in Senegal. Emerging AQ resistance may threaten the future clinical efficacy of artesunate-amodiaquine (ASAQ) and AQ-dependent SMC chemoprevention. These results highlight the potential of molecular surveillance for detecting rapid changes in parasite populations and stress the need to monitor the effectiveness of AQ as a partner drug for artemisinin-based combination therapy (ACT) and for chemoprevention.

Current profile of phenotypic pyrethroid resistance in Rhipicephalus microplus (Acari: Ixodidae) populations sampled from Marathwada region of Maharashtra state, India.

This study examined the pattern of resistance to widely applied synthetic pyrethroids, i.e., cypermethrin and deltamethrin, against larvae of Rhipicephalus microplus ticks sampled from Marathwada region in Maharashtra, India. The study also examined the role of α- and ß-esterases and glutathione-S-transferase (GST) in resistance development. All eight R. microplus isolates tested were resistant to deltamethrin (RL IV), having RR50 values from 6.88 to 131.26. LPT analysis exhibited the resistance level II deltamethrin resistance in Beed and Hingoli, III in Dharashiv, and IV in Sambhajinagar, Parbhani, Latur, Jalna, and Nanded isolates. The LIT analysis showed that Dharashiv field isolates had the lowest LC50 value of 229.09 ppm against cypermethrin, while Sambhajinagar field isolates had the highest at 489.78 ppm. The RR50 ranged from 1145.45 to 2448.9. Seven isolates were level I resistant to cypermethrin while the Jalna isolate was level II resistant. In larvae treated with deltamethrin and cypermethrin, the activity of α- and ß-esterase enzymes increased significantly compared to control groups. The enzyme ratios in treated larvae ranged from 0.7533 to 1.7023 for α-esterase and 0.7434 to 3.2054 for ß-esterase. The Hingoli isolate treated with cypermethrin exhibited the highest α-esterase activity (903.261), whereas Sambhajinagar isolate had the highest GST enzyme ratio (2.8224) after deltamethrin exposure. When exposed to cypermethrin, the Hingoli isolate showed the highest GST enzyme ratio, 2.0832. The present study provides the current resistance status in tick populations from Marathwada region indicating deltamethrin and cypermethrin to be ineffective for tick control. The results also suggest that SP compounds should be regulated in this region and alternative control strategies should be introduced.

Neutrophil percentage-to-albumin ratio is a potential marker of intravenous immunoglobulin resistance in Kawasaki disease.

Intravenous immunoglobulin (IVIG) resistance in Kawasaki disease (KD) was associated with coronary artery lesions. Neutrophil percentage-to-albumin ratio (NPAR) is an index of mortality in several inflammatory diseases. This study focused on the association of NPAR with IVIG- resistance in KD. Clinical and laboratory data of 438 children with KD before IVIG treatment were retrospectively analyzed. Notably, high NPAR was associated with older age, high WBC, NP, ALT, total bilirubin and CRP, as well as with high the incidence of IVIG-resistance, and with low hemoglobin (Hb), PLT, ALB and sodium levels. NPAR (OR: 2.366, 95% CI: 1.46-3.897, p = 0.001) and Hb (OR: 0.967, 95% CI: 0.944-0.989, p = 0.004) were independent risk factors for IVIG-resistance. NPAR showed linear relation with IVIG-resistance (p for nonlinear = 0.711) and the nonlinear correlation was found between IVIG-resistance and Hb (p for nonlinear = 0.002). The predictive performance of NPAR was superior to Beijing model (z = 2.193, p = 0.028), and not inferior to Chongqing model (z = 0.983, p = 0.326) and the combination of NPAR and Hb (z = 1.912, p = 0.056). These findings revealed that NPAR is a reliable predictor of IVIG-resistance.

Sterol 14-alpha demethylase (CYP51) activity in Leishmania donovani is likely dependent upon cytochrome P450 reductase 1.

Liposomal amphotericin B is an important frontline drug for the treatment of visceral leishmaniasis, a neglected disease of poverty. The mechanism of action of amphotericin B (AmB) is thought to involve interaction with ergosterol and other ergostane sterols, resulting in disruption of the integrity and key functions of the plasma membrane. Emergence of clinically refractory isolates of Leishmania donovani and L. infantum is an ongoing issue and knowledge of potential resistance mechanisms can help to alleviate this problem. Here we report the characterisation of four independently selected L. donovani clones that are resistant to AmB. Whole genome sequencing revealed that in three of the moderately resistant clones, resistance was due solely to the deletion of a gene encoding C24-sterol methyltransferase (SMT1). The fourth, hyper-resistant resistant clone (>60-fold) was found to have a 24 bp deletion in both alleles of a gene encoding a putative cytochrome P450 reductase (P450R1). Metabolic profiling indicated these parasites were virtually devoid of ergosterol (0.2% versus 18% of total sterols in wild-type) and had a marked accumulation of 14-methylfecosterol (75% versus 0.1% of total sterols in wild-type) and other 14-alpha methylcholestanes. These are substrates for sterol 14-alpha demethylase (CYP51) suggesting that this enzyme may be a bona fide P450R specifically involved in electron transfer from NADPH to CYP51 during catalysis. Deletion of P450R1 in wild-type cells phenocopied the metabolic changes observed in our AmB hyper-resistant clone as well as in CYP51 nulls. Likewise, addition of a wild type P450R1 gene restored sterol profiles to wild type. Our studies indicate that P450R1 is essential for L. donovani amastigote viability, thus loss of this gene is unlikely to be a driver of clinical resistance. Nevertheless, investigating the mechanisms underpinning AmB resistance in these cells provided insights that refine our understanding of the L. donovani sterol biosynthetic pathway.

Plasmodium vivax genomic surveillance in the Peruvian Amazon with Pv AmpliSeq assay.

BACKGROUND: Plasmodium vivax is the most predominant malaria species in Latin America, constituting 71.5% of malaria cases in 2021. With several countries aiming for malaria elimination, it is crucial to prioritize effectiveness of national control programs by optimizing the utilization of available resources and strategically implementing necessary changes. To support this, there is a need for innovative approaches such as genomic surveillance tools that can investigate changes in transmission intensity, imported cases and sources of reintroduction, and can detect molecular markers associated with drug resistance. METHODOLOGY/PRINCIPAL FINDINGS: Here, we apply a modified highly-multiplexed deep sequencing assay: Pv AmpliSeq v2 Peru. The tool targets a newly developed 41-SNP Peru barcode for parasite population analysis within Peru, the 33-SNP vivaxGEN-geo panel for country-level classification, and 11 putative drug resistance genes. It was applied to 230 samples from the Peruvian Amazon (2007-2020), generating baseline surveillance data. We observed a heterogenous P. vivax population with high diversity and gene flow in peri-urban areas of Maynas province (Loreto region) with a temporal drift using all SNPs detected by the assay (nSNP = 2909). In comparison, in an indigenous isolated area, the parasite population was genetically differentiated (FST = 0.07-0.09) with moderate diversity and high relatedness between isolates in the community. In a remote border community, a clonal P. vivax cluster was identified, with distinct haplotypes in drug resistant genes and ama1, more similar to Brazilian isolates, likely representing an introduction of P. vivax from Brazil at that time. To test its applicability for Latin America, we evaluated the SNP Peru barcode in P. vivax genomes from the region and demonstrated the capacity to capture local population clustering at within-country level. CONCLUSIONS/SIGNIFICANCE: Together this data shows that P. vivax transmission is heterogeneous in different settings within the Peruvian Amazon. Genetic analysis is a key component for regional malaria control, offering valuable insights that should be incorporated into routine surveillance.

Genomic surveillance of malaria parasites in an indigenous community in the Peruvian Amazon.

Hard-to-reach communities represent Peru's main challenge for malaria elimination, but information about transmission in these areas is scarce. Here, we assessed Plasmodium vivax (Pv) and P. falciparum (Pf) transmission dynamics, resistance markers, and Pf hrp2/3 deletions in Nueva Jerusalén (NJ), a remote, indigenous community in the Peruvian Amazon with high population mobility. We collected samples from November 2019 to May 2020 by active (ACD) and passive case detection (PCD) in NJ. Parasites were identified with microscopy and PCR. Then, we analyzed a representative set of positive-PCR samples (Pv = 68, Pf = 58) using highly-multiplexed deep sequencing assays (AmpliSeq) and compared NJ parasites with ones from other remote Peruvian areas using population genetics indexes. The ACD intervention did not reduce malaria cases in the short term, and persistent malaria transmission was observed (at least one Pv infection was detected in 96% of the study days). In Nueva Jerusalen, the Pv population had modest genetic diversity (He = 0.27). Pf population had lower diversity (He = 0.08) and presented temporal clustering, one of these clusters linked to an outbreak in February 2020. Moreover, Pv and Pf parasites from NJ exhibited variable levels of differentiation (Pv Fst = 0.07-0.52 and Pf Fst = 0.11-0.58) with parasites from other remote areas. No artemisin resistance mutations but chloroquine (57%) and sulfadoxine-pyrimethamine (35-67%) were detected in NJ's Pf parasites. Moreover, pfhrp2/3 gene deletions were common (32-50% of parasites with one or both genes deleted). The persistent Pv transmission and the detection of a Pf outbreak with parasites genetically distinct from the local ones highlight the need for tailored interventions focusing on mobility patterns and imported infections in remote areas to eliminate malaria in the Peruvian Amazon.

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

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