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Throughout the COVID-19 pandemic, individuals potentially infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were forcibly recalled to local or central hospitals, where the diagnostic results were obtained a couple of days after the liquid biopsies were subjected to conventional polymerase chain reaction (PCR). This slow output of such a complex and time-consuming laboratory procedure hindered its widespread application. To overcome the limitations associated with such a centralized diagnostic system, we developed a hand-held and all-in-one type test kit in which the analytical results can be obtained in only 30 min. The test kit consists of three major steps for on-site SARS-CoV-2 RNA detection: 1) virus lysis by heat, 2) RNA enrichment by membrane, and 3) real-time detection by colorimetric loop-mediated isothermal amplification (c-LAMP). The proposed device operates in a sample-to-answer format, is fully automated, and reduces dependence on traditional laboratory settings, facilitating large-scale population screening. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic has highlighted the urgent need for rapid and accurate diagnostic methods for various infectious diseases, including SARS-CoV-2. Traditional RT-PCR methods, while highly sensitive and specific, require complex equipment and skilled personnel. In response, we developed an integrated RT-LAMP-MS assay, which combines rapid reverse transcription loop-mediated isothermal amplification (RT-LAMP) with microscanning (MS) technology for detecting SARS-CoV-2. The assay uses magnesium pyrophosphate formed during LAMP amplification as a visual marker, allowing direct observation via microscopy without the need for additional chemical indicators or probes. For the SARS-CoV-2/IC RT-LAMP-MS assay, the sample-LAMP reagent mixture was added to a microchip with SARS-CoV-2 primers and internal controls, then incubated at 62 °C for 30 min in a heat block, followed by amplification analysis using a microscanner. In clinical tests, the RT-LAMP-MS assay showed 99% sensitivity and 100% specificity, which is identical to the RT-LAMP results and comparable to the commercial AllplexTM SARS-CoV-2 assay results. Additionally, the limit of detection (LOD) was determined to be 10−1 PFU mL−1 (dynamic range: 103~10−1 PFU mL−1). The assay delivers results in 30 min, uses low-cost equipment, and demonstrates 100% reproducibility in repeated tests, making it suitable for point-of-care use in resource-limited settings. [ABSTRACT FROM AUTHOR]

A pre-transfusion crossmatch test is crucial for ensuring safe blood transfusions by identifying the compatibility between donor and recipient blood samples. Conventional tube methods for crossmatching have limitations, including subjectivity in result interpretation and the potential for human error. In this study, we evaluated the diagnostic performance of a new crossmatch test using Microscanner C3, which can overcome these shortcomings. The crossmatch test results using the method were obtained in 323 clinical samples. The sensitivity, specificity, positive predictive value, negative predictive value, and concordance rate of the crossmatch test using Microscanner C3 were 98.20%, 100.00%, 100.00%, 98.11%, and 99.07%, respectively. The diagnostic performance of the new system offers a promising alternative to conventional tube methods for pre-transfusion crossmatch testing. Microscanner C3 could also increase the automation, standardization, and accuracy of crossmatch tests. The crossmatch test using Microscanner C3 is thought to increase the efficiency and reliability in identifying blood samples suitable for transfusion, thereby improving patient safety and optimizing the use of blood products in clinical settings. [ABSTRACT FROM AUTHOR]

Background: Periprosthetic joint infection (PJI) is one of the most serious and debilitating complications that can occur after total joint arthroplasty. Therefore, early diagnosis and appropriate treatment are important for a good prognosis. Recently, molecular diagnostic methods have been widely used to detect the causative microorganisms of PJI sensitively and rapidly. The Multiplex Loop-Mediated Isothermal Amplification (LAMP) method eliminates the complex temperature cycling and delays caused by temperature transitions seen in polymerase chain reaction (PCR) methods, making it faster and easier to perform compared to PCR-based assays. Therefore, this study developed a multiplex LAMP assay for diagnosing bacterial PJI using LAMP technology and evaluated its analytical and clinical performance. Methods: We developed a multiplex LAMP assay for the detection of five bacteria: Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae, Pseudomonas aeruginosa, and Escherichia coli, frequently observed to be the causative agents of PJI. The method of analytical sensitivity and cross-reactivity were determined by spiking standard strains into the joint synovial fluid. The analytical sensitivity of the multiplex LAMP assay was compared with that of a quantitative real-time PCR (qPCR) assay. Clinical performance was evaluated using 20 joint synovial fluid samples collected from patients suspected of having bacterial PJI. Results: The analytical sensitivity of the gram-positive bacterial multiplex LAMP assay and qPCR were 105/104 CFU/mL, 103/103 CFU/mL, and 105/104 CFU/mL against S. agalactiae, S. epidermidis, and S. aureus, respectively. For P. aeruginosa and E. coli, the analytical sensitivity of the multiplex LAMP and qPCR assays were 105/104 and 106/104 CFU/mL, respectively. The multiplex LAMP assay detects target bacteria without cross-reacting with other bacteria, and exhibited 100% sensitivity and specificity in clinical performance evaluation. Conclusions: This multiplex LAMP assay can rapidly detect five high-prevalence bacterial species causing bacterial PJI, with excellent sensitivity and specificity, in less than 1 h, and it may be useful for the early diagnosis of PJI. [ABSTRACT FROM AUTHOR]

Influenza viruses cause highly contagious respiratory diseases that cause millions of deaths worldwide. Rapid detection of influenza viruses is essential for accurate diagnosis and the initiation of appropriate treatment. We developed a loop-mediated isothermal amplification and lateral flow assay (LAMP-LFA) capable of simultaneously detecting influenza A and influenza B. Primer sets for influenza A and influenza B were designed to target conserved regions of segment 7 and the nucleoprotein gene, respectively. Optimized through various primer set ratios, the assay operated at 62 °C for 30 min. For a total of 243 (85 influenza A positive, 58 influenza B positive and 100 negative) nasopharyngeal swab samples, the performance of the influenza A/B multiplex LAMP-LFA was compared with that of the commercial AllplexTM Respiratory Panel 1 assay (Seegene, Seoul, Korea). The influenza A/B multiplex LAMP-LFA demonstrated a specificity of 98% for the non-infected clinical samples, along with sensitivities of 94.1% for the influenza A clinical samples and 96.6% for the influenza B clinical samples, respectively. The influenza A/B multiplex LAMP-LFA showed high sensitivity and specificity, indicating that it is reliable for use in a low-resource environment. [ABSTRACT FROM AUTHOR]

Counting CD4+ T lymphocytes using flow cytometry is a standard method for monitoring patients with HIV infections. Simpler and cheaper alternatives to flow cytometry are in high demand because getting access to flow cytometers is difficult or impossible in resource-limited settings. We evaluated the performance of the Microscanner Plus, a simple and automated image-based cell counter, in determining CD4 counts against a flow cytometer. CD4 count results of the Microscanner Plus and flow cytometer were compared using samples from 47 HIV-infected patients and 87 healthy individuals. All CV% for precision and reproducibility tests were less than 10%. The Microscanner Plus's lowest detectable CD4 count was determined to be 15.27 cells/µL of whole blood samples. The correlation coefficient (R) between Microscanner Plus and flow cytometry for CD4 counting in 134 clinical samples was very high, at 0.9906 (p < 0.0001). The automated Microscanner Plus showed acceptable analytical performance for counting CD4+ T lymphocytes and may be particularly useful for monitoring HIV patients in resource-limited settings. [ABSTRACT FROM AUTHOR]

Plasmodium vivax causes the most geographically widespread human malaria, accounting annually for 70-80 million clinical cases throughout the tropical and subtropical regions of the world's continents. We have analyzed the DNA sequences of the Csp (circumsporozoite protein) gene in 24 geographically representative strains of P. vivax and 2 of P. simium, which parasitizes several species of New World monkeys. The Csp sequences are of two types, VK210 and VK247, which differ by three diagnostic amino acid replacements, one in each of the 5' and 3' terminal regions [5' nonrepeat (NR) and 3' NR] of the gene and in an insertion sequence that precedes the 3' NR region. The central region of the gene consists of approximately 38 repetitive "motifs," which are alternatively four and five amino acids long, which also are diagnostically different between the VK210 and VK247 types. There are very few synonymous substitutions within and between the two types of strains, which we hypothesize reflects that the worldwide spread of P. vivax is very recent. The two P. simium Csp sequences belong one to each of the two VK types and are genetically indistinguishable from the corresponding P. vivax strains, suggesting that at least two host transfers have occurred between humans and New World monkeys. We exclude as unlikely the possibility that the two types of sequences could have independently arisen in humans and platyrrhines by natural selection. There are reasons favoring each of the two possible directions of host transfer between humans and monkeys.