Portable Paper-Based Immunoassay Combined with Smartphone Application for Colorimetric and Quantitative Detection of Dengue NS1 Antigen.

Dengue virus (DENV) infection, which is transmitted by Aedes mosquitoes, is a major public health concern in tropical and subtropical countries. With an annual incidence of approximately 10 million cases and 20,000-25,000 deaths, particularly among children, there is an urgent need for practical diagnostic tools. The presence of dengue non-structural protein 1 (NS1) during early infection has been linked to cytokine release, vascular leakage, and endothelial dysfunction, making it a potential marker for severe dengue. Paper-based immunoassays such as lateral flow assays (LFAs) and microfluidic paper-based analytical devices (PADs) have gained popularity as diagnostic tests due to their simplicity, rapidity, inexpensiveness, specificity, and ease of interpretation. However, conventional paper-based immunoassays for dengue NS1 detection typically rely on visual inspection, yielding only qualitative results. To address this limitation and enhance sensitivity, we proposed a highly portable NS1 dengue detection assay on a Paper-based Analytical Device (PAD), namely, DEN-NS1-PAD, that integrates a smartphone application as a colorimetric and quantitative reader. The development system enables direct quantification of NS1 concentrations in clinical samples. Serum and blood samples obtained from patients were utilized to demonstrate the system prototype performance. The results were obtained immediately and can be employed for clinical assessment, both in well-equipped healthcare facilities and resource-limited settings. This innovative combination of a paper-based immunoassay with a smartphone application offers a promising approach for enhanced detection and quantification of dengue NS1 antigen. By augmenting sensitivity beyond the capabilities of the naked eye, this system holds great potential for improving clinical decision-making in dengue management, particularly in remote or underserved areas.

Target amplification-free detection of urinary microRNA for diabetic nephropathy diagnosis with electrocatalytic reaction.

Diabetic nephropathy (DN) is a serious diabetic complication, usually developed from type II diabetes mellitus (T2DM) and known as type II DN (T2DN). New emerging biomarkers for T2DN are microRNAs (miRNAs) which have been studied for the noninvasive early-stage detection of the disease. In this work, a nucleic acid amplification-free miRNA-124 sensor based on target-induced strand displacement on magnetic beads, and by using methylene blue-loaded silica particles as a label was developed. Measurement methods can be either visual observation, spectrophotometry, or electrochemistry. After incubation and separation of the magnetic particles, a blue-violet solution (564 nm) appeared, depending on the concentration of miRNA displaced. For electrochemical detection, methylene blue on the silica served as a redox mediator for the coupled reaction with ferricyanide in the solution phase. At the electrode surface, ferricyanide was re-reduced to ferrocyanide, and was thus available for further reaction with methylene blue, forming an amplification cycle. After optimization, the total assay time was 60 min, and limits of detection were 1 pM, 6 fM, and 0.65 fM, by the naked eye, spectrophotometry and electrochemistry, respectively. The miRNAs in 42 suspected urine samples from patients suffering from either diabetic nephropathy, diabetes mellitus, or chronic kidney disease were validated by comparing with the droplet digital polymerase chain reaction (ddPCR).

Clinical evaluation of a developed paper-based Dengue NS1 rapid diagnostic test for febrile illness patients.

OBJECTIVES: This study aimed to evaluate a microfluidic paper-based analytical device (DEN-NS1-PAD) based on a rapid NS1 antigen test for diagnosing dengue at the point of care. METHODS: 219 serum samples from suspected dengue cases were tested with the developed DEN-NS1-PAD and commercial RDT by SD BIOLINE. The results were compared with the nested-PCR results. RESULTS: The limit of detection of DEN-NS1-PAD was 0.78 ng mL-1. It showed 88.89% sensitivity, 86.67% specificity, and a substantial agreement correlation (κ = 0.7522) compared with nested-PCR. In contrast, SD BIOLINE for NS1 (SD-NS1) detection showed 87.88% sensitivity, 90.00% specificity, and had a substantial agreement correlation with nested-PCR (κ = 0.7788). CONCLUSIONS: DEN-NS1-PAD is a valuable tool for diagnosing DENV infections, especially for diagnosed patients with early acute phase samples with high viral load. DEN-NS1-PAD has better sensitivity than SD-NS1 but less specificity.

Aptamer-functionalised magnetic particles for highly selective detection of urinary albumin in clinical samples of diabetic nephropathy and other kidney tract disease.

We report a new highly selective detection platform for human albumin (HA) in urine based on aptamer-functionalised magnetic particles. Magnetic separation and re-dispersion was utilised to expose the HA-bound particles to a methylene blue solution. A second magnetic collection step was then used to allow the methylene blue supernatant to be reduced at an unmodified screen-printed electrode. Since methylene blue adsorbs to HA, the reduction current fell in proportion to HA concentration. There was no interference from compounds such as dopamine, epinephrine, vanillylmandelic acid, normetanephrine, metanephrine and creatinine in artificial urine at the concentrations at which they would be expected to appear. A calibration equation was derived to allow for the effect of pH on the response. This enabled measurement to be made directly in clinical urine samples of varying pH. After optimisation of experimental parameters, the total assay time was 40 min and the limit of detection was between 0.93 and 1.16 µg mL-1, depending on the pH used. HA could be detected up to 400 µg mL-1, covering the range from normoalbuminuria to macroalbuminuria. Analysis of urine samples of patients, with diabatic nephropathy, type I & II diabetes mellitus and chronic kidney disease, from a local hospital showed good agreement with the standard urinary human albumin detection method.

Chronic rhinosinusitis with nasal polyps is characterized by dysbacteriosis of the nasal microbiota.

Chronic rhinosinusitis with nasal polyp (CRSwNP) patients are often characterized by asthma comorbidity and a type-2 inflammation of the sinonasal mucosa. The mucosal microbiota has been suggested to be implicated in the persistence of inflammation, but associations have not been well defined. To compare the bacterial communities of healthy subjects with CRSwNP patients, we collected nasal swabs from 17 healthy subjects, 21 CRSwNP patients without asthma (CRSwNP-A), and 20 CRSwNP patients with co-morbid asthma (CRSwNP+A). We analysed the microbiota using high-throughput sequencing of the bacterial 16S rRNA. Bacterial communities were different between the three groups. Haemophilus influenzae was significantly enriched in CRSwNP patients, Propionibacterium acnes in the healthy group; Staphylococcus aureus was abundant in the CRSwNP-A group, even though present in 57% of patients. Escherichia coli was found in high amounts in CRSwNP+A patients. Nasal tissues of CRSwNP+A patients expressed significantly higher concentrations of IgE, SE-IgE, and IL-5 compared to those of CRSwNP-A patients. Co-cultivation demonstrated that P. acnes growth was inhibited by H. influenzae, E. coli and S. aureus. The nasal microbiota of healthy subjects are different from those of CRSwNP-A and CRSwNP+A patients. However, the most abundant species in healthy status could not inhibit those in CRSwNP disease.

Association of Mucosal Organisms with Patterns of Inflammation in Chronic Rhinosinusitis.

BACKGROUND: Chronic rhinosinusitis is a multifactorial process disease in which bacterial infection or colonization may play an important role in the initiation or persistence of inflammatory response. The association between mucosal bacteria presence and inflammatory patterns has only been partially explored. OBJECTIVE: To demonstrate specific mucosal microorganisms possible association with inflammatory patterns. METHODS: We collected nasal polyps or sinus tissues from a clinical selection of six patient groups with defined sinus disease using tissue biomarkers. In the tissues, we detected bacteria using peptide nucleic acid fluorescence in situ hybridization (PNA-FISH). RESULTS: After reviewing a total of 115 samples (15-20 samples per group), the mucosal presence of Staphylococcus aureus was correlated with IL-5 and SE-IgE positive chronic rhinosinusitis with nasal polyps and nasal polyps from cystic fibrosis patients. Chronic rhinosinusitis without nasal polyps with TNFα >20 pg/ml was associated with the mucosal presence of Pseudomonas aeruginosa. CONCLUSION: This study identifies the relationship between intramucosal microbes and inflammatory patterns, suggesting that bacteria may affect the type of inflammation in chronic rhinosinusitis. Additional investigation is needed to further identify the nature of the relationship.

The microbiome of the upper airways: focus on chronic rhinosinusitis.

Upper airway diseases including allergic rhinitis, chronic rhinosinusitis with or without polyps, and cystic fibrosis are characterized by substantially different inflammatory profiles. Traditionally, studies on the association of specific bacterial patterns with inflammatory profiles of diseases had been dependent on bacterial culturing. In the past 30 years, molecular biology methods have allowed bacterial culture free studies of microbial communities, revealing microbiota much more diverse than previously recognized including those found in the upper airway. At presence, the study of the pathophysiology of upper airway diseases is necessary to establish the relationship between the microbiome and inflammatory patterns to find their clinical reflections and also their possible causal relationships. Such investigations may elucidate the path to therapeutic approaches in correcting an imbalanced microbiome. In the review we summarized techniques used and the current knowledge on the microbiome of upper airway diseases, the limitations and pitfalls, and identified areas of interest for further research.

Efficacy of the Precise Climate Controller on the reduction of indoor microorganisms.

BACKGROUND: Nowadays, there are many methods to reduce microorganisms in the air, such as dehumidifier, air purifier or humidity and temperature controller. The Precise Climate Controller is an instrument for controlling humidity and temperature, a concept that is demonstrated. OBJECTIVE: To determine the efficacy of this device, in order to reduce the quantity of the fungi and bacteria in the closed system. METHODS: This study is a perspective experimental study and is conducted as follows - the air sample in the closed system, a 42-cubic-meter room, is collected before the installation of the Precise Climate Controller. Next, the room is fumed with Aspergillus flavus and closed for 2 days. Then the instrument is in use in order to keep the relative humidity (RH) and the temperature constant at 55% RH and 25 degrees Celsius (℃). The air samples are collected every 3 days for 5 times during the period of 15 days to identify the type and calculate the quantity of the microorganisms. RESULTS: Before the Precise Climate Controller has been installed. Three species of bacteria are found in the air samples, but none of the fungus exists in the testing room. Once the room has been fumed with a large amount of A. flavus and the instrument is in use for 3 days, nine colonies of A. flavus are identified, but later on when the instrument is in use for 6, 9, 12, and 15 days, the air samples contain neither fungus nor bacteria. CONCLUSION: After keeping the RH and temperature of the closed system constant at 55% RH and 25℃ by using the Precise Climate Controller, it is found that the efficaciousness in controlling the quantity and species of fungi and bacteria is clinically significant.