Field-applicable simultaneous multiplex LAMP assay for screening HBV and HCV co-infection in a single tube.

BACKGROUND: Globally, around 7 to 20 million people are believed to be suffering from coinfection with both hepatitis B virus (HBV) and hepatitis C virus (HCV). The loop-mediated isothermal amplification (LAMP) approach, introduced by Notomi and colleagues, has undergone substantial advancements as an effective molecular tool that enables the simultaneous analysis of multiple samples in a single tube. METHODS: The present study examined the simultaneous detection of HBV and HCV in a single tube using melt curve analysis multiplex LAMP (mLAMP), which is based on the identification of unique melting peak temperatures. Selected regions for primer design including the S gene of HBV and the UTR gene of HCV. Primer optimization is initially performed through individual HBV and HCV LAMP analysis. Following the optimization process, the mLAMP assay was evaluated by optimizing the multiplex reaction mixture, determining the reaction time, and analyzing the limit of detection (LOD). The results are also analyzed using lateral flow dipsticks (LFD), which enable the visual detection of HBV and HCV by adding 20 pmol FITC-labeled LF primers into the reaction mixture prior the mLAMP. RESULTS: The LOD for the mLAMP assay was determined as 10 copies/µl, and no cross-reactivity with other microorganisms was detected. The detection results obtained from patient plasma were also visually demonstrated using LFD, and displayed significant concordance with those obtained from Real-Time Polymerase Chain Assay. The mLAMP assay revealed a diagnostic sensitivity of 95% for detecting the HBV, and LOD is 90% for HCV. The overall diagnostic sensitivity of the mLAMP assay for both viruses was 85%. The assay confirmed a specificity of 100%. CONCLUSION: The mLAMP assay displays significant promise for analyzing coinfected samples by simultaneously detecting the dual targets HBV and HCV within a set temperature of 62 °C, all within a time frame of 1 h. Additionally, when paired with disposable LFD, the mLAMP assay enables rapid visual detection of assay results in a matter of minutes. The result contributes to the mLAMP assay being highly suitable for coinfection screening, particularly in field conditions.

Development of Molecular-Based Screening Test for Hepatitis B Virus in Human Plasma Samples.

Despite regular administration of hepatitis B virus (HBV) vaccine in several countries, the mortality rate associated with HBV remains significant. The antiviral medications available for the treatment of HBV infection do not suffice for the serious complications related to chronic HBV infection. Additionally, the serological tests fail to detect early viral replication preventing early treatment response. Recently, many studies have demonstrated the significant advantages of loop-mediated isothermal amplification (LAMP) over serological testing and polymerase chain reaction (PCR), for the rapid detection of microbial pathogens. Here we developed a rapid, sensitive, and portable system-integrative LAMP assay for the detection of HBV DNA in plasma samples. The final optimized assay was achieved with an amplification time of less than 45 min at 62°C. The assay showed 100% specificity, 92.20% sensitivity, and a detection limit of 10 copies/µL in 77 HBV-positive plasma samples with known Cq values. Our results showed that the colorimetric LAMP assay is sensitive, efficient, and supremely reliable for rapid detection of HBV, and may be potentially used as a screening test in areas with poor laboratory facilities and limited resource availability.

Rapid Molecular Diagnosis of Group A Streptococcus with a Novel Loop Mediated Isothermal Amplification Method.

BACKGROUND: Group A Streptococcus (GAS) is the most common bacterial cause of acute tonsillopharyngitis. In this study, it was aimed to evaluate the performance of a novel Loop Mediated Isothermal Amplification (LAMP) method in the rapid diagnosis of GAS in samples taken from children with a prediagnosis of acute bacterial tonsillopharyngitis by comparing it with culture and rapid antigen test (RAT) methods. METHODS: A total of 100 throat swab samples taken from children at the pediatrics outpatient clinic with suspected tonsillopharyngitis were included in the study. Throat swab samples were analyzed by RAT, throat culture, and LAMP method. GAS suspected colonies were identified with MALDI-TOF MS system. The isothermal amplification reaction for LAMP was conducted by a novel LAMP instrument. RESULTS: According to the results of throat cultures; 53 of them were positive and 47 were negative in terms of GAS. Six (11.32%) of the culture positive samples were found to be negative by the RAT (sensitivity; 88.68%, specificity 100%). While the antigen test was positive, no culture negative sample was detected. One of the culture positive samples was found negative by LAMP. In two samples, while throat culture was negative, it was observed that LAMP was positive (sensitivity; 98.11%, specificity; 95.74%). In one of these samples, the bacteria grown in the culture were identified as Streptococcus dysgalactiae by mass spectrophotometry. CONCLUSIONS: In this study, it was determined that the LAMP method used in the diagnosis of throat infections caused by GAS has high sensitivity and specificity. We believe that the instrument is easy to use, low cost, portable, and adaptable to point of care tests. There are very few studies in the literature regarding the use of the instrument in this field, and it should be evaluated in terms of its usability in daily practice with new studies.