Molecular epidemiology and reverse transcript ase (RT) and protease inhibitors (PR) resistance genotyping of human immunodeficiency virus (HIV) in Kelantan

The wide use of antiretroviral (AR V) therapy has greatly reduced the rate of morbidity and mortality among Human immunodeficiency virus type- I (HIV -I) infected patients. However, high mutation and recombination rates of HIV -I lead to the emergence of various subtypes and drug-resistance viruses, rendering AR V -therapy ineffective in many patients. This study was undertaken to determine for the first time the prevailing HIV -I subtypes and the patterns of drug resistance mutations among 53 HIV -!-infected patients in Kelantan, Malaysia. Blood samples from 53 HIV-1 infected patients (naive and treated) were collected from Hospital Raja Perernpuan Zainab II, Kelantan, Malaysia from May 2009 to December 20 I 0. Protease (PR) and reverse transcriptase (RT) genes were amplified from patient plasma, and then sequenced and analysed for mutations. Subtype determination was done using NCB! Genotyping Tool while ARV resistance mutations were analysed using Stanford HIV Drug Resistance Database

Diagnosis of Dengue Infection Using Conventional and Biosensor Based Techniques

Dengue is an arthropod-borne viral disease caused by four antigenically different serotypes of dengue virus. This disease is considered as a major public health concern around the world. Currently, there is no licensed vaccine or antiviral drug available for the prevention and treatment of dengue disease. Moreover, clinical features of dengue are indistinguishable from other infectious diseases such as malaria, chikungunya, rickettsia and leptospira. Therefore, prompt and accurate laboratory diagnostic test is urgently required for disease confirmation and patient triage. The traditional diagnostic techniques for the dengue virus are viral detection in cell culture, serological testing, and RNA amplification using reverse transcriptase PCR. This paper discusses the conventional laboratory methods used for the diagnosis of dengue during the acute and convalescent phase and highlights the advantages and limitations of these routine laboratory tests. Subsequently, the biosensor based assays developed using various transducers for the detection of dengue are also reviewed

Performance Evaluation of Commercial Dengue Diagnostic Tests for Early Detection of Dengue in Clinical Samples

The shattering rise in dengue virus infections globally has created a need for an accurate and validated rapid diagnostic test for this virus. Rapid diagnostic test (RDT) and reverse transcription-polymerase chain reaction (RT-PCR) diagnostic detection are useful tools for diagnosis of early dengue infection. We prospectively evaluated the diagnostic performance of nonstructural 1 (NS1) RDT and real-time RT-PCR diagnostic kits in 86 patient serum samples. Thirty-six samples were positive for dengue NS1 antigen while the remaining 50 were negative when tested with enzyme-linked immunosorbent assay (ELISA). Commercially available RDTs for NS1 detection, RTK ProDetect™, and SD Bioline showed high sensitivity of 94% and 89%, respectively, compared with ELISA. GenoAmp® Trioplex Real-Time RT-PCR and RealStar® Dengue RT-PCR tests presented a comparable kappa agreement with 0.722. The result obtained from GenoAmp® Real-Time RT-PCR Dengue test showed that 14 samples harbored dengue virus type 1 (DENV-1), 8 samples harbored DENV-2, 2 samples harbored DENV-3, and 1 sample harbored DENV-4. 1 sample had a double infection with DENV-1 and DENV-2. The NS1 RDTs and real-time RT-PCR tests were found to be a useful diagnostic for early and rapid diagnosis of acute dengue and an excellent surveillance tool in our battle against dengue

FCGR3B gene copy number variation and host susceptibility to vascular leakage in Dengue Hemorrhagic Fever / Hoh Boon Peng , Sazaly Abu Bakar and Rafidah Hanim Shueb

DENV causes significantly more human disease than any other abovirus. Annually, an estimated of 50-100 million cases of severe dengue require hospitalization in which 500,000 resulted in DHF/DSS, with more than 20,000 death worldwide (WHO DengueNet report, 2005). Hence, it has now been recognized as a major expanding public health problem of the country. Dengue viruses cause a spectrum of illness ranging from asymptomatic infection or mild febrile illness to severe and fatal hemorrhagic disease. While majority experience uncomplicated Dengue Fever (DF), Dengue Hemorrhagic Fever (DHF) can present with severe clinical manifestations including transient vascular permeability resulting in plasma leakage (WHO, 1997). No specific treatment is available to date. Previous studies suggested the involvement of the events in the peripheral blood in association with the DENV disease severity, such as dengue viral replication, cytokine expression, and cellular activation / proliferation and robust host inflammatory immune response (Rothman, 2004). Antibody-dependent enhancement of viral replication is the most widely accepted explanation for the association between DHF and pre-existing antibody. However, it remains considerable uncertainty as to how virus-host interaction triggers the inflammatory response resulting in plasma leakage, the hallmark of DHF/DSS. FcGRII has been reported to play a role in pathogenesis of severe dengue infections (Loke et al., 2002; Littaua et al., 1990). It functions to mediate antibody enhancement in vitro by binding to virus-IgG complexes. A fundamental to any discussion of DENV pathogenesis is the association of secondary infection with heterologous serotypes with DHF/DSS. Antibody Dependent Enhancement (ADE) model during secondary infections, postulates that DENV specific antibodies either cross reactive antibodies, can interact with DENV without neutralizing the virus, and thereby requires FcG receptors to mediate entry of antibody coated DENV into cells (Clyde et al., 2006; Coffey et al, 2009). Therefore, this proposed study investigated and to characterized the CNV of FcGRII gene among the DF and DHF patients. Though association of FcGRII gene SNP polymorphism with dengue has been reported earlier (Loke et al, 2002), none investigated the copy number of this gene and its association to the susceptibility of plasma leakage

Diagnostic accuracy of rapid antigen test kits for detecting SARS-CoV-2:a systematic review and meta-analysis of 17,171 suspected COVID-19 patients

Early diagnosis is still as crucial as the initial stage of the COVID-19 pandemic. As RT-PCR sometimes is not feasible in developing nations or rural areas, health professionals may use a rapid antigen test (RAT) to lessen the load of diagnosis. However, the efficacy of RAT is yet to be investigated thoroughly. Hence, we tried to evaluate the overall performance of RAT in SARS-CoV-2 diagnosis. Based on our PROSPERO registered protocol (CRD42021231432), we searched online databases (i.e., PubMed, Google Scholar, Scopus, and Web of Science) and analysed overall pooled specificity and sensitivity of RAT along with study quality, publication bias, heterogeneity and more. The overall pooled specificity and sensitivity of RAT were detected as 99.4% (95% CI: 99.1–99.8; I2 = 90%) and 68.4% (95% CI: 60.8–75.9; I2 = 98%), respectively. In subgroup analyses, nasopharyngeal specimens and symptomatic patient’s samples were more sensitive in RAT, while cycle threshold (Ct) values were found to have an inverse relationship with sensitivity. In the European and American populations, RAT showed better performance. Although the sensitivity of RAT is yet to be improved, it could still be an alternative in places with poor laboratory set up. Nevertheless, the negative samples of RAT can be re-tested using RT-PCR to reduce false negative results

Facile hydrothermal and solvothermal synthesis and characterization of nitrogen-doped carbon dots from palm kernel shell precursor

Carbon dots (CDs), a nanomaterial synthesized from organic precursors rich in carbon content with excellent fluorescent property, are in high demand for many purposes, including sensing and biosensing applications. This research focused on preparing CDs from natural and abundant waste, palm kernel shells (PKS) obtained from palm oil biomass, aiming for sensing and biosensing applications. Ethylenediamine and L-phenylalanine doped CDs were produced via the hydrothermal and solvothermal methods using one-pot synthesis techniques in an autoclave batch reactor. The as-prepared N-CDs shows excellent photoluminescence (PL) property and a quantum yield (QY) of 13.7% for ethylenediamine (EDA) doped N-CDs (CDs-EDA) and 8.6% for L-phenylalanine (L-Ph) doped N-CDs (CDs-LPh) with an excitation/emission wavelength of 360 nm/450 nm. The transmission electron microscopy (TEM) images show the N-CDs have an average particle size of 2 nm for both CDs. UV-Visible spectrophotometric results showed C=C and C=O transition. FTIR results show and confirm the presence of functional groups, such as -OH, -C=O, -NH2 on the N-CDs, and the X-ray diffraction pattern showed that the N-CDs were crystalline, depicted with sharp peaks. This research work demonstrated that palm kernel shell biomass often thrown away as waste can produce CDs with excellent physicochemical properties

A Comparison of Assays for Accurate Copy Number Measurement of the Low-Affinity Fc Gamma Receptor Genes FCGR3A and FCGR3B

The FCGR3 locus encoding the low affinity activating receptor FcγRIII, plays a vital role in immunity triggered by cellular effector and regulatory functions. Copy number of the genes FCGR3A and FCGR3B has previously been reported to affect susceptibility to several autoimmune diseases and chronic inflammatory conditions. However, such genetic association studies often yield inconsistent results; hence require assays that are robust with low error rate. We investigated the accuracy and efficiency in estimating FCGR3 CNV by comparing Sequenom MassARRAY and paralogue ratio test-restriction enzyme digest variant ratio (RT-REDVR). In addition, since many genetic association studies of FCGR3B CNV were carried out using real-time quantitative PCR, we have also included the evaluation of that method’s performance in estimating the multi-allelic CNV of FCGR3B. The qPCR assay exhibited a considerably broader distribution of signal intensity, potentially introducing error in estimation of copy number and higher false positive rates. Both Sequenom and PRT-REDVR showed lesser systematic bias, but Sequenom skewed towards copy number normal (CN = 2). The discrepancy between Sequenom and PRT-REDVR might be attributed either to batch effects noise in individual measurements. Our study suggests that PRT-REDVR is more robust and accurate in genotyping the CNV of FCGR3, but highlights the needs of multiple independent assays for extensive validation when performing a genetic association study with multi-allelic CNVs

The Effect of Hydrocotyle sibthorpioides Lam. Extracts on in Vitro Dengue Replication

Objective. To investigate the potential effect of Hydrocotyle sibthorpioides Lam. (H. sibthorpioides) extracts against in vitro dengue viral replication. Methods. The cytotoxicity of H. sibthorpioides was evaluated using a cell viability assay. Cells were pre- and posttreated with water and methanol extracts of H. sibthorpioides, and the viral inhibitory effect was investigated by observing the morphological changes, which were further confirmed by plaque assay. Results. The methanolic extract cytotoxicity was higher in Vero and C6/36 cells than the cytotoxicity of the water extract. Preincubation of the cells with H. sibthorpioides extract showed nonexistent to mild prophylactic effects. The posttreatment of Vero cells with H. sibthorpioides methanolic extract presented higher antidengue activities when compared with the water extract. Surprisingly, posttreatment of C6/36 cells resulted in an enhancement of viral replication. Conclusion. H. sibthorpioides had variable effects on dengue viral replication, depending on the treatment, cell lines, and solvent types. This study provides important novel insights on the phytomedicinal properties of H. sibthorpioides extracts on dengue virus

Strategies for the preparation of non-amplified and amplified genomic dengue gene samples for electrochemical DNA biosensing applications

The application of electrochemical DNA biosensors in real genomic sample detection is challenging due to the existence of complex structures and low genomic concentrations, resulting in inconsistent and low current signals. This work highlights strategies for the treatment of non-amplified and amplified genomic dengue virus gene samples based on real samples before they can be used directly in our DNA electrochemical sensing system, using methylene blue (MB) as a redox indicator. The main steps in this study for preparing non-amplified cDNA were cDNA conversion, heat denaturation, and sonication. To prepare amplified cDNA dengue virus genomic samples using an RT-PCR approach, we optimized a few parameters, such as the annealing temperature, sonication time, and reverse to forward (R/F) primer concentration ratio. We discovered that the generated methylene blue (MB) signals during the electrochemical sensing of non-amplified and amplified samples differ due to the different MB binding affinities based on the sequence length and base composition. The findings show that our developed electrochemical DNA biosensor successfully discriminates MB current signals in the presence and absence of the target genomic dengue virus, indicating that both samples were successfully treated. This work also provides interesting information about the critical factors in the preparation of genomic gene samples for developing miniaturized PCR-based electrochemical sensing applications in the future. We also discuss the limitations and provide suggestions related to using redox-indicator-based electrochemical biosensors to detect real genomic nucleic acid genes

Strategies in the optimization of DNA hybridization conditions and its role in electrochemical detection of dengue virus (DENV) using response surface methodology (RSM)

In recent years, limited research has been conducted on enhancing DNA hybridization-based biosensor approaches using statistical models. This study explores the application of response surface methodology (RSM) to improve the performance of a DNA hybridization biosensor for dengue virus (DENV) detection. The biosensor is based on silicon nanowires decorated with gold nanoparticles (SiNWs/AuNPs) and utilizes methylene blue as a redox indicator. The DNA hybridization process between the immobilized DNA probe and the target DENV gene was monitored using differential pulse voltammetry (DPV) based on the reduction of methylene blue. Fourier-transform infrared spectroscopy (FTIR) and electrochemical impedance spectroscopy (EIS) were employed to confirm successful DNA hybridization events on the modified screen-printed gold electrode (SPGE) surface. Several parameters, including pH buffer, NaCl concentration, temperature, and hybridization time, were simultaneously optimized, with NaCl concentration having the most significant impact on DNA hybridization events. This study enhances the understanding of the role of each parameter in influencing DNA hybridization detection in electrochemical biosensors. The optimized biosensor demonstrated the ability to detect complementary oligonucleotide and amplified DENV gene concentrations as low as 0.0891 ng mL−1 (10 pM) and 2.8 ng mL−1 , respectively. The developed biosensor shows promise for rapid clinical diagnosis of dengue virus infection