Development of DNA-Based Lateral Flow Assay for Detection of LDLR Gene Mutation for Familial Hypercholesterolemia.

Background: The techniques for detecting single nucleotide polymorphisms (SNP) require lengthy and complex experimental procedures and expensive instruments that may only be available in some laboratories. Thus, a deoxyribonucleic acid (DNA)-based lateral flow assay (LFA) was developed as a point-of-care test (POCT) diagnostic tool for genotyping. In this study, single nucleotide variation (E101K) in the low-density lipoprotein receptor (LDLR) gene leading to familial hypercholesterolemia (FH) was chosen as a model. Methods: Hypercholesterolemic individuals (n = 103) were selected from the Malaysian Cohort project (UKM Medical Molecular Biology Institute) while the control samples were selected from the Biobank (UKM Medical Molecular Biology Institute). The DNA samples were isolated from whole blood. Polymerase chain reaction (PCR) amplification process was performed using bifunctional labelled primers specifically designed to correspond to the variant that differentiates wild-type and mutant DNA for visual detection on LFA. The variant was confirmed using Sanger sequencing, and the sensitivity and specificity of the LFA detection method were validated using the Agena MassARRAY® technique. Results: Out of 103 hypercholesterolemic individuals, 5 individuals (4.8%) tested positive for E101K, LDLR mutation and the rest, including healthy control individuals, tested negative. This result was concordant with Sanger sequencing and Agena MassARRAY®. These five individuals could be classified as Definite FH, as the DNA diagnosis was confirmed. The sensitivity and specificity of the variant detection by LFA is 100% compared to results using the genotyping method using Agena MassARRAY®. Conclusion: The developed LFA can potentially be used in the POC setting for detecting the E101K variant in the LDLR gene. This LFA can also be used to screen family members with E101K variant in the LDLR gene and is applicable for other SNP's detection.

Kaedah Diagnostik Semasa dan Penggunaan Ujian Titik Penjagaan Pantas (POC) Bagi Mendiagnos Hiperkolesterolemia Famili (FH) (Current Diagnostic Techniques and the Use of Rapid Point-of-Care (POC) Testing to Diagnose Familial Hypercholesterolemia (FH)) / Jurnal Sains Kesihatan Malaysia

@#Familial hypercholesterolemia (FH) is an autosomal dominant inherited genetic disease characterized by increased concentrations of low-density lipoprotein (LDL-C) cholesterol in the blood. The risk of premature coronary heart disease in FH patients may increase without early treatment. Advancement in molecular biology techniques has enable early detection and diagnosis of FH. These techniques are cost-effective and have a shorter turnaround time. The current diagnostic tools available for FH diagnosis involving algorithm-based scoring criteria and various molecular diagnosis methods including next-generation sequencing (NGS), Sanger sequencing, Multiplex ligation-dependent probe amplification (MLPA) and DNA hybridisation assay are discussed in this review. However, molecular genetic testing is not widely available due to time-consuming procedures, high cost and requires trained personnel. Thus, this 36 review highlights the use of point of care (POC) testing as an approach to diagnose FH, particularly in countries lacking infrastructure and expertise in this field. Lateral flow testing (LFA) has gained attention as a POC diagnostic tool due to its simplicity, low cost and involved simple procedure and settings. The advantages of LFA made this technique a potential tool in addressing challenges in diagnosing FH, particularly for early diagnosis of family members.

Comprehensive approaches for the detection of Burkholderia pseudomallei and diagnosis of melioidosis in human and environmental samples.

Melioidosis is endemic in Southeast Asia and northern Australia. The causative agent of melioidosis is a Gram-negative bacterium, Burkholderia pseudomallei. Its invasion can be fatal if melioidosis is not treated promptly. It is intrinsically resistant to a variety of antibiotics. In this paper, we present a comprehensive overview of the current trends on melioidosis cases, treatments, B. pseudomallei virulence factors, and molecular techniques to detect the bacterium from different samples. The clinical and microbial diagnosis methods of identification and detection of B. pseudomallei are commonly used for the rapid diagnosis and typing of strains, such as polymerase chain reaction or multi-locus sequence typing. The genotyping strategies and techniques have been constantly evolving to identify genomic loci linked to or associated with this human disease. More research strategies for detecting and controlling melioidosis should be encouraged and conducted to understand the current situation. In conclusion, we review existing diagnostic methodologies for melioidosis detection and provide insights on prospective diagnostic methods for the bacterium.

Podcast acceptance for pedagogy: the levels and significant influences.

There is limited empirical evidence arguing against accepting and using podcasts for educational purposes. This may in part, explain the recent surge in the acceptance of podcasts for pedagogy, alongside the COVID-19 pandemic. Both students and lecturers have been greatly affected by this pandemic which may explain the uptake in the use of podcasts. However, few studies have explored podcast use for pedagogy and thus, there is limited empirical backing. This study investigated pedagogy and considered podcast performance expectancy, effort expectancy, social influence, facilitating condition, cultural, social, and political beliefs factors in Nigerian universities. This research introduced cross-sectional quantitative methods, which utilised a questionnaire, gathering data from three Federal Universities in Nigeria. The formulated hypothesis was rejected using multiple regression and a total of eight hundred and twenty-nine questionnaires were gathered from Nigerian university lecturers. The data was analysed and the results showed a low-level outcome with regards to podcast acceptance for pedagogy in Nigeria Federal Universities.

Mediating effect of behavioural intention on podcast acceptance.

This research contributes to knowledge in mediating effect of lecturers' behavioural intention towards the acceptance of podcasts technology in universities. A cross-sectional quantitative research design was involved in this study. Also, a total of eight hundred and twenty-nine (829) lecturers participated in this research from three different south-west Nigeria Federal universities. The research instrument was titled lecturer's acceptance questionnaire (LAQ) with a calculated Cronbach's alpha of .919, which confirmed the instrument validity. The gathered data was analysed by using both descriptive and inferential statistics. The formulated hypothesis was tested at .05 level of significance. The result from descriptive analysis of data suggested that behavioural intention was at a low level among lecturers and the results of hypothesis testing revealed the effect of behavioural intention as partial mediator of podcast acceptance. Based on these findings relevant conclusions and recommendations were suggested in this research.

Fabrication and Characterization of Simple Structure Fluidic-Based Memristor for Immunosensing of NS1 Protein Application.

Non-structural protein 1 (NS1 protein) is becoming a commonplace biomarker for the diagnostic of early detection of dengue. In this study, we sought to use a label-free approach of detecting NS1 protein by harnessing fluidic-based memristor sensor. The sensor was fabricated using sol-gel spin coating technique, by which TiO2 thin film is coated on the surface of Indium tin oxide (ITO) and a glass substrate. The sensor was then functionalized with glycidoxypropyl-trimethoxysilane (GPTS), acting as antibody for NS1. The addition of the target NS1 formed an antibody-antigen complex which altered the physical and electrical properties in sensing region. Sensing of the sensor is incumbent upon the measurement of Off-On resistance ratio. Imaging with Field Emission Scanning Electron Microscope (FESEM) evinced the successful immobilization of the antibody and the subsequent capture of the NS1 protein by the immobilized antibody. The detection limit actualized by the developed sensor was 52 nM and the diameter of 2 mm gives the most optimal measurement. The developed sensor demonstrated an immense potential towards the development of label-free diagnostic of early dengue infection.

Diagnostic techniques for COVID-19 and new developments.

COVID-19 pandemic is a serious global health issue today due to the rapid human to human transmission of SARS-CoV-2, a new type of coronavirus that causes fatal pneumonia. SARS -CoV-2 has a faster rate of transmission than other coronaviruses such as SARS and MERS and until now there are no approved specific drugs or vaccines for treatment. Thus, early diagnosis is crucial to prevent the extensive spread of the disease. The reverse transcription-polymerase chain reaction (RT-PCR) is the most routinely used method until now to detect SARS-CoV-2 infections. However, several other faster and accurate assays are being developed for the diagnosis of COVID-19 aiming to control the spread of infection through the identification of patients and immediate isolation. In this review, we will discuss the various detection methods of the SARS-CoV-2 virus including the recent developments in immunological assays, amplification techniques as well as biosensors.

Laboratory Diagnosis of Paratyphoid Fever: Opportunity of Surface Plasmon Resonance.

Paratyphoid fever is caused by the bacterium Salmonella enterica serovar Paratyphi (A, B and C), and contributes significantly to global disease burden. One of the major challenges in the diagnosis of paratyphoid fever is the lack of a proper gold standard. Given the absence of a licensed vaccine against S. Paratyphi, this diagnostic gap leads to inappropriate antibiotics use, thus, enhancing antimicrobial resistance. In addition, the symptoms of paratyphoid overlap with other infections, including the closely related typhoid fever. Since the development and utilization of a standard, sensitive, and accurate diagnostic method is essential in controlling any disease, this review discusses a new promising approach to aid the diagnosis of paratyphoid fever. This advocated approach is based on the use of surface plasmon resonance (SPR) biosensor and DNA probes to detect specific nucleic acid sequences of S. Paratyphi. We believe that this SPR-based genoassay can be a potent alternative to the current conventional diagnostic methods, and could become a rapid diagnostic tool for paratyphoid fever.

Immune response to asymptomatic infections by Entamoeba histolytica and other enteric pathogens in pregnant women and their infants in a high HIV burdened setting in Zimbabwe.

BACKGROUND: Asymptomatic Entamoeba histolytica infections in pregnant women puts infants at risk of infection through vertical transmission or transmission during breastfeeding in high HIV prevalence areas. The study aimed at investigating the immune response to asymptomatic E.histolytica infection in pregnant women and their infants in a high HIV burdened setting in Harare, Zimbabwe. METHODOLOGY: Serum samples from 39 predominantly breastfeeding mother-infant pairs were analyzed for inflammatory cytokine and immunoglobulin profiles using BIOPLEX. The infants' ages ranged from 10 days to 14 weeks. RESULTS: IL-1r, IL-4, IL-9, IL-12p70, IL-17a, G-CSF and PDGF-BB were significantly raised in E. histolytica infected compared to non-infected lactating mothers (p < 0.05). Carriage of any form of enteric infection such as Non-lactose fermenters (NLFs) including E. histolytica significantly increased concentration levels of IL-1r, IL-4, IL-9, IL-10, IL-12p70, IL17a, G-CSF, GM-CSF, IFN-γ, PDGF-BB and TNF-α cytokines (p < 0.05) but no significant differences in immunoglobulin levels among the mothers. Anti-inflammatory cytokines (IL-1r, IL-2, IL-4, IL-5, IL-6), pro-inflammatory cytokines (IL-9, IL-12-p70, IL-15, IL-17a, TNF-α) and growth factors (FGF-ß, G-CSF, GM-CSF, PDGF-bb) were significantly raised in HIV-uninfected mothers and not HIV-infected mothers during E. histolytica infection (p < 0.05). In infants, E. histolytica carriage and HIV exposure had no significant impact on the cytokine and immunoglobulin concentrations. CONCLUSION: Pro-inflammatory cytokines and chemokines are highly raised in lactating mothers with asymptomatic enteric pathogens hence there is need to check cytokine profiles in pregnant women and their infants to assist in decision making linked to treatment and prevention in times of pandemics.

Detection methods of avian influenza - current and novel approaches

@# Avian influenza (AI), caused by the avian strain of influenza A virus (AIV) is one of the significant health concerns globally. Human infections with AI viruses were reported sporadically and often exhibited high mortality and morbidity rate. AI outbreaks also influenced the safety of the food supply and caused significant economic losses. Immediate control measures are required during AI outbreaks in poultry to prevent further viruses spreading. Hence, accurate, sensitive, and rapid detection methods are pivotal for decision making. Traditional methods of detection, such as virus isolation in embryonated chicken eggs, immuno-based methods, and nucleic acid amplification method, pose different limitations. These always grab the attention of researchers to improve existing methods or invent novel diagnostic approaches to compensate for the shortcoming of current methods applied. However, the method of choice is highly dependent on the availability of facilities and resources. Among the detection methods, reverse transcription-polymerase chain reaction (RT-PCR) is the most favourable method used for detecting AIV. However, a constant review of the virus genome is crucial to maintain the assay’s sensitivity. More comprehensive research and evaluation study are needed for new diagnostic approaches.

Influence of Maternal Characteristics during Pregnancy on the Infant Early Life Immune Responses in a High HIV Burdened Setting in Harare, Zimbabwe.

This study aimed at investigating the maternal characteristics that in turn influence the immunological status of infants in asymptomatic enteric pathogen carriers in mother baby pairs (MBPs) in a high HIV burdened population in Harare, Zimbabwe. BIOPLEX immunoassay was used to analyse serum samples from 39 MBPs for 27 cytokines and 6 immunoglobulins. The MBP were purposively selected based on HIV infection and Entamoeba histolytica carriage. Logistic regression was used to identify any link between maternal demographic and clinical data with infant cytokine and immunoglobulin levels. Maternal E. histolytica carriers were more likely to have infants with low levels of IL-12p70, FGF-basic, GM-CSF and TNF-α cytokines (OR: 0.14; 95% CI: 0.03-0.79) and high levels of IgA immunoglobulin (OR: 8.1; 95% CI: 1.45-45.06). HIV infected mothers were more likely to have infants with low levels of IgG2 (OR: 0.24; 95% CI: 0.06-1.00) and IgA (OR: 0.22; 95% CI: 0.05-0.90) immunoglobulins. Notably, it was highly likely to deliver infants with low IgG4 levels (OR: 0.24; 95% CI: 0.06-1.02) for maternal mean age above 30.38 years (Standard deviation 6.09) though not significant (p=0.05). Maternal E. histolytica asymptomatic carriage, and HIV-infection status result in low levels of pro-inflammatory cytokines IL-12p70, FGF-basic, GM-CSF and TNF-α and immunoglobulins IgG2, IgG4 and IgA on their infants.

Cloning, expression, and purification of the hemolysin/cytolysin (HlyE antigen) from Salmonella enterica serovar Typhi: potential application for immunoassay development.

The hemolysin (HlyE) protein of Salmonella enterica serovar Typhi was reported to be antigenic. This work describes the cloning, expression, and purification of a hexahistidine-tagged HlyE protein under native conditions. Immunoblot analysis and a competitive enzyme-linked immunosorbent assay using sera from typhoid patients showed the presence of HlyE-specific antibodies in circulation.