Identification of inositol monophosphatase as a broad-spectrum antiviral target of ivermectin.

Ivermectin has broad-spectrum antiviral activities. Despite the failure in clinical application of COVID-19, it can serve as a lead compound for the development of more effective broad-spectrum antivirals, for which a better understanding of its antiviral mechanisms is essential. We thus searched for potential novel targets of ivermectin in host cells by label-free thermal proteomic profiling using Huh-7 cells. Inositol monophosphatase (IMPase) was found among the proteins with shifted thermal stability by ivermectin. Ivermectin could inhibit IMPase activity and reduce cellular myo-inositol and phosphatidylinositol-4-phosphate levels. On the other hand, inositol could impair the antiviral activity of ivermectin and lithium, an IMPase inhibitor with known antiviral activity. As phosphatidylinositol phosphate is crucial for the replication of many RNA viruses, inhibition of cellular myo-inositol biosynthesis may be an important antiviral mechanism of ivermectin. Hence, inhibition of IMPase could serve as a potential target for broad-spectrum antiviral development.

Genetic analysis and molecular basis of G6PD deficiency among malaria patients in Thailand: implications for safe use of 8-aminoquinolines.

BACKGROUND: It was hypothesized that glucose-6-phosphate dehydrogenase (G6PD) deficiency confers a protective effect against malaria infection, however, safety concerns have been raised regarding haemolytic toxicity caused by radical cure with 8-aminoquinolines in G6PD-deficient individuals. Malaria elimination and control are also complicated by the high prevalence of G6PD deficiency in malaria-endemic areas. Hence, accurate identification of G6PD deficiency is required to identify those who are eligible for malaria treatment using 8-aminoquinolines. METHODS: The prevalence of G6PD deficiency among 408 Thai participants diagnosed with malaria by microscopy (71), and malaria-negative controls (337), was assessed using a phenotypic test based on water-soluble tetrazolium salts. High-resolution melting (HRM) curve analysis was developed from a previous study to enable the detection of 15 common missense, synonymous and intronic G6PD mutations in Asian populations. The identified mutations were subjected to biochemical and structural characterisation to understand the molecular mechanisms underlying enzyme deficiency. RESULTS: Based on phenotypic testing, the prevalence of G6PD deficiency (< 30% activity) was 6.13% (25/408) and intermediate deficiency (30-70% activity) was found in 15.20% (62/408) of participants. Several G6PD genotypes with newly discovered double missense variants were identified by HRM assays, including G6PD Gaohe + Viangchan, G6PD Valladolid + Viangchan and G6PD Canton + Viangchan. A significantly high frequency of synonymous (c.1311C>T) and intronic (c.1365-13T>C and c.486-34delT) mutations was detected with intermediate to normal enzyme activity. The double missense mutations were less catalytically active than their corresponding single missense mutations, resulting in severe enzyme deficiency. While the mutations had a minor effect on binding affinity, structural instability was a key contributor to the enzyme deficiency observed in G6PD-deficient individuals. CONCLUSIONS: With varying degrees of enzyme deficiency, G6PD genotyping can be used as a complement to phenotypic screening to identify those who are eligible for 8-aminoquinolines. The information gained from this study could be useful for management and treatment of malaria, as well as for the prevention of unanticipated reactions to certain medications and foods in the studied population.

Substitution of arginine 219 by glycine compromises stability, dimerization, and catalytic activity in a G6PD mutant.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most common enzymopathies in humans, present in approximately half a billion people worldwide. More than 230 clinically relevant G6PD mutations of different classes have been reported to date. We hereby describe a patient with chronic hemolysis who presents a substitution of arginine by glycine at position 219 in G6PD protein. The variant was never described in an original publication or characterized on a molecular level. In the present study, we provide structural and biochemical evidence for the molecular basis of its pathogenicity. When compared to the wild-type enzyme, the Arg219Gly mutation markedly reduces the catalytic activity by 50-fold while having a negligible effect on substrate binding affinity. The mutation preserves secondary protein structure, but greatly decreases stability at higher temperatures and to trypsin digestion. Size exclusion chromatography elution profiles show monomeric and dimeric forms for the mutant, but only the latter for the wild-type form, suggesting a critical role of arginine 219 in G6PD dimer formation. Our findings have implications in the development of small molecule activators, with the goal of rescuing the phenotype observed in this and possibly other related mutants.

Anthelmintic efficacy evaluation and mechanism of N-methylbenzo[d]oxazol-2-amine.

Parasitic roundworms cause significant sickness and mortality in animals and humans. In livestock, these nematodes have severe economic impact and result in losses in food production on a global scale. None of the currently available drugs ideally suit all treatment circumstances, and the development of drug-resistant nematode strains has become a challenge to control the infection. There is an urgent need to develop novel anthelmintic compounds. According to our previous report, N-methylbenzo[d]oxazol-2-amine (1) showed anthelmintic activity and lowest cytotoxicity. In this study, in vivo anthelmintic properties were evaluated using Trichinella spiralis infected mice. Toxicity was evaluated using the rats and mode of action using molecular docking and metabolomics approaches. The in vivo results demonstrate that a dose of 250 mg/kg reduced the T. spiralis abundance in the digestive tract by 49%. The 250 mg/kg Albendazole was served as control. The relatively low acute toxicity was categorized into chemical category 5, with an LD50 greater than 2000 mg/kg body. Molecular docking analysis showed the T. spiralis tubulin beta chain and glutamate-gated channels might not be the main targets of compound 1. Metabolomics analysis was used to explain the effects of compound 1 on the T. spiralis adult worm. The results demonstrated that compound 1 significantly up-regulated the metabolism of purine, pyrimidine and down-regulated sphingolipid metabolism. In conclusion, compound 1 could be a potential molecule for anthelmintic development. The bioavailability, pharmacokinetics, and absorption of this compound should be studied further to provide information for its future efficacy improvement.

Molecular characterization of G6PD mutations identifies new mutations and a high frequency of intronic variants in Thai females.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked enzymopathy caused by mutations in the G6PD gene. A medical concern associated with G6PD deficiency is acute hemolytic anemia induced by certain foods, drugs, and infections. Although phenotypic tests can correctly identify hemizygous males, as well as homozygous and compound heterozygous females, heterozygous females with a wide range of G6PD activity may be misclassified as normal. This study aimed to develop multiplex high-resolution melting (HRM) analyses to enable the accurate detection of G6PD mutations, especially among females with heterozygous deficiency. Multiplex HRM assays were developed to detect six G6PD variants, i.e., G6PD Gaohe (c.95A>G), G6PD Chinese-4 (c.392G>T), G6PD Mahidol (c.487G>A), G6PD Viangchan (c.871G>A), G6PD Chinese-5 (c.1024C>T), and G6PD Union (c.1360C>T) in two reactions. The assays were validated and then applied to genotype G6PD mutations in 248 Thai females. The sensitivity of the HRM assays developed was 100% [95% confidence interval (CI): 94.40%-100%] with a specificity of 100% (95% CI: 88.78%-100%) for detecting these six mutations. The prevalence of G6PD deficiency was estimated as 3.63% (9/248) for G6PD deficiency and 31.05% (77/248) for intermediate deficiency by phenotypic assay. The developed HRM assays identified three participants with normal enzyme activity as heterozygous for G6PD Viangchan. Interestingly, a deletion in intron 5 nucleotide position 637/638 (c.486-34delT) was also detected by the developed HRM assays. G6PD genotyping revealed a total of 12 G6PD genotypes, with a high prevalence of intronic variants. Our results suggested that HRM analysis-based genotyping is a simple and reliable approach for detecting G6PD mutations, and could be used to prevent the misdiagnosis of heterozygous females by phenotypic assay. This study also sheds light on the possibility of overlooking intronic variants, which could affect G6PD expression and contribute to enzyme deficiency.

Cocultures of Enterococcus faecium and Aeromonas veronii Induce the Secretion of Bacteriocin-like Substances against Aeromonas.

Lactic acid bacteria (LAB) were screened from Lutjanus russellii (red sea bass), and their antimicrobial activities were evaluated against two Aeromonas species isolated from the Nile tilapia, namely, Aeromonas veronii (AV) and Aeromonas jandaei (AJ). Three LAB isolates, Enterococcus faecium MU8 (EF_8), Enterococcus faecalis MU2 (EFL_2), and E. faecalis MU9 (EFL_9), were found to inhibit both AV and AJ; however, their cell-free supernatant (CFS) did not do so. Interestingly, bacteriocin-like substances (BLS) induced by cocultures of EF_8 with AV exhibited the highest antimicrobial activity against both Aeromonas sp. The size of BLS was less than 1.0 kDa; the purified BLS were susceptible to proteinase K digestion, indicating that they are peptides. BLS contained 13 identified peptides derived from E. faecium, as determined by liquid chromatography-tandem mass spectrometry. Cocultures of Gram-positive-producing and -inducing LAB strains have been used to increase bacteriocin yields. To our knowledge, this is the first report describing inducible BLS produced by cocultures of Gram-positive-producing and Gram-negative-inducing strains.

Regulation of immune response against third-stage Gnathostoma spinigerum larvae by human genes.

Background: Gnathostomiasis is an important zoonosis in tropical areas that is mainly caused by third-stage Gnathostoma spinigerum larvae (G. spinigerum L3). Objectives: This study aimed to prove whether G. spinigerum L3 produces extracellular vesicles (EVs) and investigate human gene profiles related to the immune response against the larvae. Methods: We created an immune cell model using normal human peripheral blood mononuclear cells (PBMCs) co-cultured with the larvae for 1 and 3 days, respectively. The PBMCs were harvested for transcriptome sequencing analysis. The EV ultrastructure was examined in the larvae and the cultured medium. Results: Extracellular vesicle-like particles were observed under the larval teguments and in the pellets in the medium. RNA-seq analysis revealed that 2,847 and 3,118 genes were significantly expressed on days 1 and 3 after culture, respectively. The downregulated genes on day 1 after culture were involved in pro-inflammatory cytokines, the complement system and apoptosis, whereas those on day 3 were involved in T cell-dependent B cell activation and wound healing. Significantly upregulated genes related to cell proliferation, activation and development, as well as cytotoxicity, were observed on day 1, and genes regulating T cell maturation, granulocyte function, nuclear factor-κB and toll-like receptor pathways were predominantly observed on day 3 after culture. Conclusion: G. spinigerum L3 produces EV-like particles and releases them into the excretory-secretory products. Overall, genotypic findings during our 3-day observation revealed that most significant gene expressions were related to T and B cell signalling, driving T helper 2 cells related to chronic infection, immune evasion of the larvae, and the pathogenesis of gnathostomiasis. Further in-depth studies are necessary to clarify gene functions in the pathogenesis and immune evasion mechanisms of the infective larvae.

Cytochrome P450 2D6 (CYP2D6) and glucose-6-phosphate dehydrogenase (G6PD) genetic variations in Thai vivax malaria patients: Implications for 8-aminoquinoline radical cure.

BACKGROUND: Primaquine and tafenoquine are the only licensed drugs that effectively kill the hypnozoite stage and are used to prevent Plasmodium vivax malaria relapse. However, both primaquine and tafenoquine can cause acute hemolysis in glucose-6-phosphate dehydrogenase (G6PD)-deficient people with varying degrees of severity depending on G6PD variants. Additionally, primaquine efficacy against malaria parasites was decreased in individuals with impaired cytochrome P450 2D6 (CYP2D6) activity due to genetic polymorphisms. This study aimed to characterize G6PD and CYP2D6 genetic variations in vivax malaria patients from Yala province, a malaria-endemic area along the Thai-Malaysian border, and determine the biochemical properties of identified G6PD variants. METHODOLOGY/PRINCIPLE FINDINGS: Multiplexed high-resolution melting assay and DNA sequencing detected five G6PD variants, including G6PD Kaiping, G6PD Vanua Lava, G6PD Coimbra, G6PD Mahidol, and G6PD Kerala-Kalyan. Biochemical and structural characterization revealed that G6PD Coimbra markedly reduced catalytic activity and structural stability, indicating a high susceptibility to drug-induced hemolysis. While Kerala-Kalyan had minor effects, it is possible to develop mild adverse effects when receiving radical treatment. CYP2D6 genotyping was performed using long-range PCR and DNA sequencing, and the phenotypes were predicted using the combination of allelic variants. Decreased and no-function alleles were detected at frequencies of 53.4% and 14.2%, respectively. The most common alleles were CYP2D6*36+*10 (25.6%), *10 (23.9%), and *1 (22.2%). Additionally, 51.1% of the intermediate metabolizers showed CYP2D6*10/*36+*10 as the predominant genotype (15.9%). CONCLUSIONS/SIGNIFICANCE: Our findings provide insights about genetic variations of G6PD and CYP2D6 in 88 vivax malaria patients from Yala, which may influence the safety and effectiveness of radical treatment. Optimization of 8-aminoquinoline administration may be required for safe and effective treatment in the studied population, which could be a significant challenge in achieving the goal of eliminating malaria.

Genotype-phenotype association and biochemical analyses of glucose-6-phosphate dehydrogenase variants: Implications for the hemolytic risk of using 8-aminoquinolines for radical cure.

Background: Plasmodium vivax remains the malaria species posing a major threat to human health worldwide owing to its relapse mechanism. Currently, the only drugs of choice for radical cure are the 8-aminoquinolines (primaquine and tafenoquine), which are capable of killing hypnozoites and thus preventing P. vivax relapse. However, the therapeutic use of primaquine and tafenoquine is restricted because these drugs can cause hemolysis in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency. This study aimed to assess and understand the hemolytic risk of using 8-aminoquinolines for radical treatment in a malaria endemic area of Thailand. Methods: The prevalence of G6PD deficiency was determined using a quantitative test in 1,125 individuals. Multiplexed high-resolution meltinging (HRM) assays were developed and applied to detect 12 G6PD mutations. Furthermore, biochemical and structural characterization of G6PD variants was carried out to understand the molecular basis of enzyme deficiency. Results: The prevalence of G6PD deficiency was 6.76% (76/1,125), as assessed by a phenotypic test. Multiplexed HRM assays revealed G6PD Mahidol in 15.04% (77/512) of males and 28.38% (174/613) of females, as well as G6PD Aures in one female. G6PD activity above the 30% cut-off was detected in those carrying G6PD Mahidol, even in hemizygous male individuals. Two variants, G6PD Murcia Oristano and G6PD Songklanagarind + Viangchan, were identified for the first time in Thailand. Biochemical characterization revealed that structural instability is the primary cause of enzyme deficiency in G6PD Aures, G6PD Murcia Oristano, G6PD Songklanagarind + Viangchan, and G6PD Chinese 4 + Viangchan, with double G6PD mutations causing more severe enzyme deficiency. Conclusion: In western Thailand, up to 22% of people may be ineligible for radical cure. Routine qualitative tests may be insufficient for G6PD testing, so quantitative tests should be implemented. G6PD genotyping should also be used to confirm G6PD status, especially in female individuals suspected of having G6PD deficiency. People with double G6PD mutations are more likely to have hemolysis than are those with single G6PD mutations because the double mutations significantly reduce the catalytic activity as well as the structural stability of the protein.

Combined effects of double mutations on catalytic activity and structural stability contribute to clinical manifestations of glucose-6-phosphate dehydrogenase deficiency.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy in humans, affecting ~ 500 million worldwide. A detailed study of the structural stability and catalytic activity of G6PD variants is required to understand how different mutations cause varying degrees of enzyme deficiency, reflecting the response of G6PD variants to oxidative stress. Furthermore, for G6PD double variants, investigating how two mutations jointly cause severe enzyme deficiency is important. Here, we characterized the functional and structural properties of nine G6PD variants: G6PD Gaohe, G6PD Mahidol, G6PD Shoklo, G6PD Canton, G6PD Kaiping, G6PD Gaohe + Kaiping, G6PD Mahidol + Canton, G6PD Mahidol + Kaiping and G6PD Canton + Kaiping. All variants were less catalytically active and structurally stable than the wild type enzyme, with G6PD double mutations having a greater impact than single mutations. G6PD Shoklo and G6PD Canton + Kaiping were the least catalytically active single and double variants, respectively. The combined effects of two mutations were observed, with the Canton mutation reducing structural stability and the Kaiping mutation increasing it in the double mutations. Severe enzyme deficiency in the double mutants was mainly determined by the trade-off between protein stability and catalytic activity. Additionally, it was demonstrated that AG1, a G6PD activator, only marginally increased G6PD enzymatic activity and stability.

Genetic population of Plasmodium knowlesi during pre-malaria elimination in Thailand.

BACKGROUND: Thailand is committed to eliminating malaria by 2024. From 2013 to 2020, the total number of malaria cases have decreased, from 37,741 to 4474 (an 88.1% reduction). However, infections with Plasmodium knowlesi, a monkey malarial pathogen that can also infect humans, have been increasingly observed. This study focused on the molecular analysis of P. knowlesi parasites causing malaria in Thailand. METHODS: Under Thailand's integrated Drug Efficacy Surveillance (iDES), which includes drug-resistance monitoring as part of routine case-based surveillance and responses, specimens were collected from malaria patients (n = 966) between 2018 and 2020. Thirty-one mono P. knowlesi infections (3.1%), most of which were from eastern and southern Thailand, were observed and confirmed by nested PCR assay and DNA sequencing. To evaluate whether these pathogens were from different lineages, cluster analysis based on seven microsatellite genotyping markers and the merozoite surface protein 1 (pkmsp1) gene was carried out. The P. knowlesi pyrimethamine resistance gene dihydrofolate reductase (pkdhfr) was sequenced and homology modelling was constructed. RESULTS: The results of analysing the seven microsatellite markers and pkmsp1 sequence demonstrated that P. knowlesi parasites from eastern Thailand were of the same lineage as those isolated in Cambodia, while the parasites causing malaria in southern Thailand were the same lineage as those isolated from Malaysia. The sequencing results for the pkdhfr genes indicated the presence of two mutations, Arg34Leu and a deletion at position 105. On analysis with homology modelling, the two mutations were not associated with anti-malarial drug resistance. CONCLUSIONS: This report compared the genetic populations of P. knowlesi parasites in Thailand from 2018 to 2020 and have shown similar lineages as those isolated in Cambodia and Malaysia of P. knowlesi infection in Thailand and demonstrated that the P. knowlesi parasites were of the same lineages as those isolated in Cambodia and Malaysia. The parasites were also shown to be sensitive to pyrimethamine.

Human Single-Chain Antibodies That Neutralize Elastolytic Activity of Pseudomonas aeruginosa LasB.

LasB (elastase/pseudolysin) is an injurious zinc-metalloprotease secreted by the infecting Pseudomonas aeruginosa. LasB is recognized as the bacterial key virulence factor for establishment of successful infection, acquisition of nutrients, dissemination, tissue invasion, and immune modulation and evasion. LasB digests a variety of the host tissue proteins, extracellular matrices, as well as components of both innate and adaptive immune systems, including immunoglobulins, complement proteins, and cytokines. Thus, this enzyme is an attractive target for disarming the P. aeruginosa. This study generated human single-chain antibodies (HuscFvs) that can neutralize the elastolytic activity of native LasB by using phage display technology. Gene sequences coding HuscFvs (huscfvs) isolated from HuscFv-displaying phage clones that bound to enzymatically active LasB were sub-cloned to expression plasmids for large scale production of the recombinant HuscFvs by the huscfv-plasmid transformed Escherichia coli. HuscFvs of two transformed E. coli clones, i.e., HuscFv-N42 and HuscFv-N45, neutralized the LasB elastolytic activities in vitro. Computer simulation by homology modeling and molecular docking demonstrated that antibodies presumptively formed contact interfaces with the LasB residues critical for the catalytic activity. Although the LasB neutralizing mechanisms await elucidation by laboratory experiments, the HuscFvs should be tested further towards the clinical application as a novel adjunctive therapeutics to mitigate severity of the diseases caused by P. aeruginosa.

Molecular Characterization of Seasonal Influenza A and B from Hospitalized Patients in Thailand in 2018-2019.

Influenza viruses continue to be a major public health threat due to the possible emergence of more virulent influenza virus strains resulting from dynamic changes in virus adaptability, consequent of functional mutations and antigenic drift in surface proteins, especially hemagglutinin (HA) and neuraminidase (NA). In this study, we describe the genetic and evolutionary characteristics of H1N1, H3N2, and influenza B strains detected in severe cases of seasonal influenza in Thailand from 2018 to 2019. We genetically characterized seven A/H1N1 isolates, seven A/H3N2 isolates, and six influenza B isolates. Five of the seven A/H1N1 viruses were found to belong to clade 6B.1 and were antigenically similar to A/Switzerland/3330/2017 (H1N1), whereas two isolates belonged to clade 6B.1A1 and clustered with A/Brisbane/02/2018 (H1N1). Interestingly, we observed additional mutations at antigenic sites (S91R, S181T, T202I) as well as a unique mutation at a receptor binding site (S200P). Three-dimensional (3D) protein structure analysis of hemagglutinin protein reveals that this unique mutation may lead to the altered binding of the HA protein to a sialic acid receptor. A/H3N2 isolates were found to belong to clade 3C.2a2 and 3C.2a1b, clustering with A/Switzerland/8060/2017 (H3N2) and A/South Australia/34/2019 (H3N2), respectively. Amino acid sequence analysis revealed 10 mutations at antigenic sites including T144A/I, T151K, Q213R, S214P, T176K, D69N, Q277R, N137K, N187K, and E78K/G. All influenza B isolates in this study belong to the Victoria lineage. Five out of six isolates belong to clade 1A3-DEL, which relate closely to B/Washington/02/2009, with one isolate lacking the three amino acid deletion on the HA segment at position K162, N163, and D164. In comparison to the B/Colorado/06/2017, which is the representative of influenza B Victoria lineage vaccine strain, these substitutions include G129D, G133R, K136E, and V180R for HA protein. Importantly, the susceptibility to oseltamivir of influenza B isolates, but not A/H1N1 and A/H3N2 isolates, were reduced as assessed by the phenotypic assay. This study demonstrates the importance of monitoring genetic variation in influenza viruses regarding how acquired mutations could be associated with an improved adaptability for efficient transmission.

Glucose-6-phosphate dehydrogenase mutations in malaria endemic area of Thailand by multiplexed high-resolution melting curve analysis.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency, the most common enzymopathy in humans, is prevalent in tropical and subtropical areas where malaria is endemic. Anti-malarial drugs, such as primaquine and tafenoquine, can cause haemolysis in G6PD-deficient individuals. Hence, G6PD testing is recommended before radical treatment against vivax malaria. Phenotypic assays have been widely used for screening G6PD deficiency, but in heterozygous females, the random lyonization causes difficulty in interpreting the results. Over 200 G6PD variants have been identified, which form genotypes associated with differences in the degree of G6PD deficiency and vulnerability to haemolysis. This study aimed to assess the frequency of G6PD mutations using a newly developed molecular genotyping test. METHODS: A multiplexed high-resolution melting (HRM) assay was developed to detect eight G6PD mutations, in which four mutations can be tested simultaneously. Validation of the method was performed using 70 G6PD-deficient samples. The test was then applied to screen 725 blood samples from people living along the Thai-Myanmar border. The enzyme activity of these samples was also determined using water-soluble tetrazolium salts (WST-8) assay. Then, the correlation between genotype and enzyme activity was analysed. RESULTS: The sensitivity of the multiplexed HRM assay for detecting G6PD mutations was 100 % [95 % confidence interval (CI): 94.87-100 %] with specificity of 100 % (95 % CI: 87.66-100 %). The overall prevalence of G6PD deficiency in the studied population as revealed by phenotypic WST-8 assay was 20.55 % (149/725). In contrast, by the multiplexed HRM assay, 27.17 % (197/725) of subjects were shown to have G6PD mutations. The mutations detected in this study included four single variants, G6PD Mahidol (187/197), G6PD Canton (4/197), G6PD Viangchan (3/197) and G6PD Chinese-5 (1/197), and two double mutations, G6PD Mahidol + Canton (1/197) and G6PD Chinese-4 + Viangchan (1/197). A broad range of G6PD enzyme activities were observed in individuals carrying G6PD Mahidol, especially in females. CONCLUSIONS: The multiplexed HRM-based assay is sensitive and reliable for detecting G6PD mutations. This genotyping assay can facilitate the detection of heterozygotes, which could be useful as a supplementary approach for high-throughput screening of G6PD deficiency in malaria endemic areas before the administration of primaquine and tafenoquine.

The integrity and stability of specimens under different storage conditions for glucose-6-phosphate dehydrogenase deficiency screening using WST-8.

Accurate measurement of glucose-6-phosphate dehydrogenase (G6PD) activity is critical for malaria treatment as misclassification of G6PD deficiency could cause serious harm to patients. G6PD activity should be assessed in blood samples on the day of collection. Otherwise, specimens should be stored under suitable conditions to prevent loss of G6PD activity. Here, we assessed stability and integrity of G6PD testing in samples from normal controls, heterozygous females, and G6PD deficient individuals using water-soluble tetrazolium salts (WST-8) assay. Specimens were stored as ethylenediaminetetraacetic acid (EDTA) whole blood and dried blood spots (DBS) at various temperatures (37 °C, room temperature, 4 °C and -20 °C) and under different humidity conditions (with and without desiccant). G6PD normal samples were stable for up to 1 year when stored at -20 °C under controlled conditions, with 85% and 91% G6PD activity in EDTA whole blood and DBS in the presence of desiccant, respectively. Specimens from heterozygous females showed greater G6PD activity when stored as DBS, with 85% enzyme activity after 1 year of storage at -20 °C under controlled conditions in the presence of desiccant. G6PD deficient samples rapidly lost enzyme activity in all storage conditions tested. However, the reduction in G6PD enzyme activity in G6PD deficient samples did not interfere with G6PD classification. Samples stored under suitable conditions for G6PD testing will allow accurate measurement of enzyme activity, prevent misclassification of G6PD deficiency and enable safe and effective use of antimalarial drugs such as primaquine and tafenoquine.

Genetic analysis of the orthologous crt and mdr1 genes in Plasmodium malariae from Thailand and Myanmar.

BACKGROUND: Plasmodium malariae is a widely spread but neglected human malaria parasite, which causes chronic infections. Studies on genetic polymorphisms of anti-malarial drug target genes in P. malariae are limited. Previous reports have shown polymorphisms in the P. malariae dihydrofolate reductase gene associated with pyrimethamine resistance and linked to pyrimethamine drug pressure. This study investigated polymorphisms of the P. malariae homologous genes, chloroquine resistant transporter and multidrug resistant 1, associated with chloroquine and mefloquine resistance in Plasmodium falciparum. METHODS: The orthologous P. malariae crt and mdr1 genes were studied in 95 patients with P. malariae infection between 2002 and 2016 from Thailand (N = 51) and Myanmar (N = 44). Gene sequences were analysed using BioEdit, MEGA7, and DnaSP programs. Mutations and gene amplifications were compared with P. falciparum and Plasmodium vivax orthologous genes. Protein topology models derived from the observed pmcrt and pmmdr1 haplotypes were constructed and analysed using Phyre2, SWISS MODEL and Discovery Studio Visualization V 17.2. RESULTS: Two non-synonymous mutations were observed in exon 2 (H53P, 40%) and exon 8 (E278D, 44%) of pmcrt. The topology model indicated that H53P and E278D were located outside of the transmembrane domain and were unlikely to affect protein function. Pmmdr1 was more diverse than pmcrt, with 10 non-synonymous and 3 synonymous mutations observed. Non-synonymous mutations were located in the parasite cytoplasmic site, transmembrane 11 and nucleotide binding domains 1 and 2. Polymorphisms conferring amino acid changes in the transmembrane and nucleotide binding domains were predicted to have some effect on PmMDR1 conformation, but were unlikely to affect protein function. All P. malariae parasites in this study contained a single copy of the mdr1 gene. CONCLUSIONS: The observed polymorphisms in pmcrt and pmmdr1 genes are unlikely to affect protein function and unlikely related to chloroquine drug pressure. Similarly, the absence of pmmdr1 copy number variation suggests limited mefloquine drug pressure on the P. malariae parasite population, despite its long time use in Thailand for the treatment of falciparum malaria.

Polymorphisms in Plasmodium vivax antifolate resistance markers in Afghanistan between 2007 and 2017.

BACKGROUND: Plasmodium vivax is the predominant Plasmodium species in Afghanistan. National guidelines recommend the combination of chloroquine and primaquine (CQ-PQ) for radical treatment of P. vivax malaria. Artesunate in combination with the antifolates sulfadoxine-pyrimethamine (SP) has been first-line treatment for uncomplicated falciparum malaria until 2016. Although SP has been the recommended treatment for falciparum and not vivax malaria, exposure of the P. vivax parasite population to SP might still have been quite extensive because of community based management of malaria. The change in the P. vivax antifolate resistance markers between 2007 and 2017 were investigated. METHODS: Dried blood spots were collected (n = 185) from confirmed P. vivax patients in five malaria-endemic areas of Afghanistan bordering Tajikistan, Turkmenistan and Pakistan, including Takhar, Faryab, Laghman, Nangarhar, and Kunar, in 2007, 2010 and 2017. Semi-nested PCR, RFLP and nucleotide sequencing were used to assess the pyrimethamine resistant related mutations in P. vivax dihydrofolate reductase (pvdhfr I13L, P33L, N50I, F57L, S58R, T61I, S93H, S117N, I173L) and the sulfonamide resistance related mutations in P. vivax dihydropteroate synthase (pvdhps A383G, A553G). RESULTS: In the 185 samples genotyped for pvdhfr and pvdhps mutations, 11 distinct haplotypes were observed, which evolved over time. In 2007, wild type pvdhfr and pvdhps were the most frequent haplotype in all study sites (81%, 80/99). However, in 2017, the frequency of the wild-type was reduced to 36%, (21/58; p value ≤ 0.001), with an increase in frequency of the double mutant pvdhfr and pvdhps haplotype S58RS117N (21%, 12/58), and the single pvdhfr mutant haplotype S117N (14%, 8/58). Triple and quadruple mutations were not found. In addition, pvdhfr mutations at position N50I (7%, 13/185) and the novel mutation S93H (6%, 11/185) were observed. Based on in silico protein modelling and molecular docking, the pvdhfr N50I mutation is expected to affect only moderately pyrimethamine binding, whereas the S93H mutation does not. CONCLUSIONS: In the course of ten years, there has been a strong increase in the frequency pyrimethamine resistance related mutations in pvdhfr in the P. vivax population in Afghanistan, although triple and quadruple mutations conferring high grade resistance were not observed. This suggests relatively low drug pressure from SP on the P. vivax parasite population in the study areas. The impact of two newly identified mutations in the pvdhfr gene on pyrimethamine resistance needs further investigation.

Functional analysis of BPSS2242 reveals its detoxification role in Burkholderia pseudomallei under salt stress.

A bpss2242 gene, encoding a putative short-chain dehydrogenase/oxidoreductase (SDR) in Burkholderia pseudomallei, was identified and its expression was up-regulated by ten-fold when B. pseudomallei was cultured under high salt concentration. Previous study suggested that BPSS2242 plays important roles in adaptation to salt stress and pathogenesis; however, its biological functions are still unknown. Herein, we report the biochemical properties and functional characterization of BPSS2242 from B. pseudomallei. BPSS2242 exhibited NADPH-dependent reductase activity toward diacetyl and methylglyoxal, toxic electrophilic dicarbonyls. The conserved catalytic triad was identified and found to play critical roles in catalysis and cofactor binding. Tyr162 and Lys166 are involved in NADPH binding and mutation of Lys166 causes a conformational change, altering protein structure. Overexpression of BPSS2242 in Escherichia coli increased bacterial survival upon exposure to diacetyl and methylglyoxal. Importantly, the viability of B. pseudomallei encountered dicarbonyl toxicity was enhanced when cultured under high salt concentration as a result of BPSS2242 overexpression. This is the first study demonstrating that BPSS2242 is responsible for detoxification of toxic metabolites, constituting a protective system against reactive carbonyl compounds in B. pseudomallei..

Controlled reversible assembly of gold nanoparticles as a new colorimetric and sensitive detection of glucose-6-phosphate dehydrogenase deficiency.

Recently, several studies have examined possible applications of nanoparticles for the development of electronic and optical sensors. The plasmon absorbance of gold nanoparticles has been used extensively to study biomolecular processes, including nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate-dependent enzymatic reactions. In this report, we describe the development of gold nanoparticles as a new colorimetric and sensitive detection method of glucose-6-phosphate dehydrogenase deficiency by means of controlled reversible assembly of gold nanoparticles. 3-nm polyvinylpyrrolidone/N,N'-dimethylaminopyridine-stabilized gold nanoparticles were synthesized, characterized and applied for an in vitro activity assay of 11 recombinant human glucose-6-phosphate dehydrogenase variants. Differences in the activity of the glucose-6-phosphate dehydrogenase variants from different deficiency classes were readily detected using the synthesized gold nanoparticles. The developed method can be easily distinguished with color change by naked eye for the detection of glucose-6-phosphate dehydrogenase deficiency. Moreover, we are the first to propose the segregation mechanism of polyvinylpyrrolidone/N,N'-dimethylaminopyridine-stabilized gold nanoparticles by reduced nicotinamide adenine dinucleotide phosphate. The method enables visual detection of glucose-6-phosphate dehydrogenase deficiency, which could be further developed for diagnostic testing of glucose-6-phosphate dehydrogenase deficiency.

In vitro passage alters virulence, immune activation and proteomic profiles of Burkholderia pseudomallei.

Serial passage is a problem among many bacterial species, especially those where strains have been stored (banked) for several decades. Prior to banking with an organization such as ATCC, many bacterial strains were passaged for many years, so the characteristics of each strain may be extremely different. This is in addition to any differences in the original host environment. For Burkholderia pseudomallei, the number of serial passages should be carefully defined for each experiment because it undergoes adaptation during the course of serial passages. In the present study, we found that passaged B. pseudomallei fresh clinical isolates and reference strain in Luria-Bertani broth exhibited increased plaque formation, invasion, intracellular replication, Galleria mellonella killing abilities, and cytokine production of host cells. These bacteria also modulated proteomic profiles during in vitro passage. We presume that the modulation of protein expression during in vitro passage caused changes in virulence and immunogenicity phenotypes. Therefore, we emphasize the need for caution regarding the use of data from passaged B. pseudomallei. These findings of phenotypic adaptation during in vitro serial passage can help researchers working on B. pseudomallei and on other species to better understand disparate findings among strains that have been reported for many years.