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 Allplex^TM 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.

Influenza and coronaviruses cause highly contagious respiratory diseases that cause millions of deaths worldwide. Public health measures implemented during the current coronavirus disease (COVID-19) pandemic have gradually reduced influenza circulation worldwide. As COVID-19 measures have relaxed, it is necessary to monitor and control seasonal influenza during this COVID-19 pandemic. In particular, the development of rapid and accurate diagnostic methods for influenza and COVID-19 is of paramount importance because both diseases have significant public health and economic impacts. To address this, we developed a multi-loop-mediated isothermal amplification (LAMP) kit capable of simultaneously detecting influenza A/B and SARS-CoV-2. The kit was optimized by testing various ratios of primer sets for influenza A/B (FluA/FluB) and SARS-CoV-2 and internal control (IC). The FluA/FluB/SARS-CoV-2 multiplex LAMP assay showed 100% specificity for uninfected clinical samples and sensitivities of 90.6%, 86.89%, and 98.96% for LAMP kits against influenza A, influenza B, and SARS-CoV-2 clinical samples, respectively. Finally, the attribute agreement analysis for clinical tests indicated substantial agreement between the multiplex FluA/FluB/SARS-CoV-2/IC LAMP and commercial Allplex^TM SARS-CoV-2/FluA/FluB/RSV assays.

After three years of the SARS-CoV-2 pandemic, the demand for developing field-deployable point-of-care (PoC) molecular diagnostic tests has increased. Although RT-qPCR is the molecular diagnostic gold standard and is accurate, it is not readily applied to point-of-care testing (POCT). Meanwhile, rapid diagnostic kits have the disadvantage of low sensitivity. Recently, rapid isothermal nucleic acid amplification technology has emerged as an alternative for rapid diagnosis. Here, we developed a rapid SARS-CoV-2 reverse transcription loop-mediated isothermal amplification (RT-LAMP)-lateral flow assay (LFA) kit. This kit includes a Chelex-100/boiling nucleic acid extraction device and a one-step amplification detection apparatus capable of performing the entire process, from RNA extraction to detection, and diagnosing SARS-CoV-2 infection within 40 min without contamination. The detection limits of the rapid SARS-CoV-2 RT-LAMP-LFA kit were 100 plaque-forming units (PFUs) mL⁻¹ and 10⁻¹ PFU mL⁻¹ for RNA samples extracted using the Chelex-100/boiling nucleic acid extraction device and commercial AdvansureTM E3 system, respectively. The sensitivity and specificity of the rapid SARS-CoV-2 RT-LAMP-LFA kit were 97.8% and 100%, respectively. Our SARS-CoV-2 RT-LAMP-LFA kit exhibited high sensitivity and specificity within 40 min without requiring laboratory instruments, suggesting that the kit could be used as a rapid POC molecular diagnostic test for SARS-CoV-2.

Tuberculosis (TB) is one of the leading causes of infectious mortality from a single infectious agent, Mycobacterium tuberculosis (MTB). This study evaluated the performance of the newly developed BZ TB/NTM NALF assay, which integrated loop-mediated isothermal amplification and lateral flow immunochromatographic assay technologies, for the detection of MTB. A total of 80 MTB-positive samples and 115 MTB-negative samples were collected, all of which were confirmed by TB real-time PCR (RT-PCR) using either AdvanSure^TM TB/NTM RT-PCR Kit or Xpert^® MTB/RIF Assay. The performance of the BZ TB/NTM NALF assay was evaluated by calculating its sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) in comparison to those of the RT-PCR methods. Compared to the RT-PCR, the sensitivity, specificity, PPV, and NPV of BZ TB/NTM NALF assay were 98.7%, 99.1%, 98.7%, and 99.1%, respectively. The concordance rate between BZ TB/NTM NALF and RT-PCR was 99.0%. Rapid and simple detection of MTB is essential for global case detection and further elimination of TB. The performance of the BZ TB/NTM NALF Assay is acceptable with a high concordance with RT-PCR, indicating that it is reliable for use in a low-resource environment.

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.

Background: Tuberculosis (TB) remains a significant global health challenge, necessitating rapid and reliable diagnostic solutions. Current methods such as microscopy, culture, and PCR have limitations in resource-limited settings due to high costs and infrastructure requirements. Methods: In this study, we developed the MTB/IC LAMP-MS assay, a novel molecular diagnostic platform that integrates loop-mediated isothermal amplification (LAMP) technology with microscope (MS) detection. The assay enables TB diagnosis within 40 min, from nucleic acid extraction to result, requiring minimal equipment and simple operation for rapid and accurate detection. Results: Compared to the commercial AdvanSure TB/NTM real-time PCR kit, the MTB/IC LAMP-MS assay had a lower analytical sensitivity, with a limit of detection of 10³ CFU/mL versus 10² CFU/mL for the commercial PCR kit, but showed a sensitivity of 93.3% and specificity of 100% in clinical tests. Additionally, the assay demonstrated minimal cross-reactivity with other respiratory pathogens, underscoring its robust specificity. In addition, the preloaded microchips demonstrated stable performance for up to 12 weeks at room temperature, supporting their field applicability. Conclusions: With its lower cost, simplified operation, and minimal infrastructure requirements, the MTB/IC LAMP-MS assay provides a practical and efficient solution for TB diagnosis in resource-limited settings.