One-shot high-resolution melting curve analysis for KRAS point-mutation discrimination on a digital microfluidics platform

Proposed high-accuracy one-shot melting curve analysis on a digital microfluidics platform, enabling KRAS gene discrimination with single-nucleotide mutations in 3 seconds.

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.

Interferon-λ3 Gene Polymorphic Variants, rs4803217 and rs12980275, Responsiveness to HBV Vaccine and Outcome of HBV and HCV Exposure in Hemodialyzed Patients

Background: In non-uremic populations, rs4803217 in the IFNL3 messenger RNA 3’ untranslated region or rs12980275 downstream of IFNL3 is connected with the spontaneous or therapeutic clearance of HCV and HBV, and rs12980275 is correlated with plasma IFN-λ3 levels. Moreover, rs12980275 is associated with the sustained virological response following antiviral therapy of chronic hepatitis C in hemodialysis patients. Objectives: We investigated IFNL3 polymorphisms, rs4803217 and rs12980275, for association with responsiveness to HBV vaccine and natural consequences of HBV and HCV exposure among hemodialyzed individuals. Methods: The capacity to produce protective anti-HBs titers was recognized if they were ≥ 10 IU/L after vaccination or natural exposure. The IFNL3 rs4803217 (G > T) and rs12980275 (A > G) genetic variants were analyzed using a high-resolution melting curve method in 1,337 hemodialysis subjects. Plasma IFN-λ3 was determined in 188 individuals using ELISA. The Kaplan-Meier method was applied for the analysis of survival probability. Results: The tested polymorphisms did not show associations with the capacity to generate protective anti-HBs titers after HBV vaccination or exposition and self-limitation of HBV exposure. Natural HCV clearance was connected with the IFNL3 rs4803217 GG genotype (OR: 3.036, 95% CI: 1.544 - 5.969, P = 0.001) and haplotypes comprising at least two more frequent alleles but without any variant allele of IFNL3/IFNL4 genetic variants (P < 0.05). Plasma IFN-λ3 levels were not directly influenced by IFNL3 rs4803217 and rs12980275, but differed concerning HBV/HCV serum markers (P = 0.00005) and firmly correlated with anti-HBs titers (r = 0.537, P = 4.15E-16). Both tested polymorphisms were not significantly associated with the survival of hemodialysis patients. Conclusions: Genotyping IFNL3 rs4803217 may be advantageous in the prognosis of natural HCV clearance but does not predict the self-limitation of HBV exposure, responsiveness to HBV vaccine, or hemodialysis patients’ mortality.

Comparison of High-resolution Melting Curve Analysis with Specific Target Gene Sequencing to Identify the Most Common Species of Aspergillus and Fusarium

Background: Currently, it appears that new molecular-based methods could substitute microscopic and culture assessment for the first-line detection of microorganisms isolated from clinical specimens. However, it will remain the "continual strategy" until this technology is attuned to identifying all fungi that can be isolated from biological specimens. Objectives: The present study aimed to validate a high-resolution melting (HRM) technique to identify clinical filamentous fungi. Moreover, it was attempted to compare the results with those of the target gene’s polymerase chain reaction (PCR) sequencing. Methods: A total of 54 specimens of bronchoalveolar lavage (BAL), nail, ear discharge, blood culture, and cornea were collected from patients suspected of fungal infection. All Fusarium spp. and Aspergillus spp. were recognized based on Tef-α and beta-tubulin region sequencing, as well as PCR-HRM analysis. Results: The Tef-α sequence analysis revealed the most frequent spp. to be Fusarium solani followed by F. oxysporum (n = 3), F. caucasicum (n = 3), F. coeruleum (n = 3), F. falciforme (n = 1), F. proliferatum (n = 1), F. brevicatenulatum (n = 1), F. globosom (n = 1), and F. verticillioides (n = 1). Based on the beta-tubulin sequences, Aspergillus flavus (n = 10), A. fumigatus (n = 7), A. niger (n = 2), A. terreus (n = 1), and A. orezea (n = 1) were identified in this study. Furthermore, the dataset analysis of PCR-HRM revealed that 33 isolates belonging to Fusarium spp. were F. solani (n = 24), F. oxysporum (n = 3), F. proliferatum (n = 3), F. falciforme (n = 1), F. verticillioides (n = 1), and F. brevicatenulatum (n = 1). Moreover, isolates (n = 21) belonging to Aspergillus spp. included A. flavus (n = 11), A. fumigatus (n = 7), A. niger (n = 2), and A. terreus (n = 1). Conclusions: The sequencing method has a time-consuming and costly nature, and there exists conformity between the sequence results of the Tef-α/beta-tubulin regions and PCR-HRM. The PCR-HRM method is a reliable approach in the clinical laboratory to identify Aspergillus and Fusarium spp. However, some closely related spp. show no curve algorithm differences in PCR-HRM.