COVID-19 has become a major global public health burden, currently causing a rapidly growing number of infections and significant morbidity and mortality around the world. Early detection with fast and sensitive assays and timely intervention are crucial for interrupting the spread of the COVID-19 virus (SARS-CoV-2). Using a mismatch-tolerant amplification technique, we developed a simple, rapid, sensitive and visual reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for SARS-CoV-2 detection based on its N gene. The assay has a high specificity and sensitivity, and robust reproducibility, and its results can be monitored using a real-time PCR machine or visualized via colorimetric change from red to yellow. The limit of detection (LOD) of the assay is 118.6 copies of SARS-CoV-2 RNA per 25 μL reaction. The reaction can be completed within 30 min for real-time fluorescence monitoring, or 40 min for visual detection when the template input is more than 200 copies per 25 μL reaction. To evaluate the viability of the assay, a comparison between the RT-LAMP and a commercial RT-qPCR assay was made using 56 clinical samples. The SARS-CoV-2 RT-LAMP assay showed perfect agreement in detection with the RT-qPCR assay. The newly-developed SARS-CoV-2 RT-LAMP assay is a simple and rapid method for COVID-19 surveillance.

Human coronaviruses HCoV-OC43, HCoV-229E, HCoV-NL63 and HCoV-HKU1 are common respiratory viruses associated with acute respiratory infection. They have a global distribution. Rapid and accurate diagnosis of HCoV infection is important for the management and treatment of hospitalized patients with HCoV infection. Here, we developed a melting curve-based multiplex RT-qPCR assay for simultaneous detection of the four HCoVs. In the assay, SYTO 9 was used to replace SYBR Green I as the fluorescent dye, and GC-modified primers were designed to improve the melting temperature (Tm) of the specific amplicon. The four HCoVs were clearly distinguished by characteristic melting peaks in melting curve analysis. The detection sensitivity of the assay was 3 × 10² copies for HCoV-OC43, and 3 × 10¹ copies for HCoV-NL63, HCoV-229E and HCoV-HKU1 per 30 μL reaction. Clinical evaluation and sequencing confirmation demonstrated that the assay was specific and reliable. The assay represents a sensitive and reliable method for diagnosis of HCoV infection in clinical samples.

Staphylococcus aureus ( S. aureus ) is a leading cause of bacteremia and blood stream infections. Methicillin-resistant S. aureus (MRSA) that first appeared in 1961 often caused hospital-acquired infections (HAIs) and community-acquired infections (CAIs) and was associated with high mortality rate. Accurate and rapid point-of-care testing (POCT) of MRSA is crucial for clinical management and treatment of MRSA infections, as well as the prevention and control of HAIs and CAIs. Here, we reported a novel extraction-free dual HiFi-LAMP assay for discriminative detection of methicillin-susceptible S. aureus and MRSA. The dual HiFi-LAMP assay can detect 30 copies/reaction of nuc and mecA genes with detection limits of 147 and 158 copies per 25 µL reaction, respectively. A retrospective clinical evaluation with 107 clinical S. aureus isolates showed both sensitivity and specificity of 100%. A prospective clinical evaluation with 35 clinical samples revealed a specificity of 100% and a sensitivity of 92.3%. The dual HiFi-LAMP assay can detect almost all S. aureus samples (141/142; 99.3%) within 20 min, implying that the entire HiFi-LAMP assay (including sample process) can be completed within 40 min, extremely significantly shorter than 3–5 days by the traditional clinical microbial culture and antibiotic susceptibility testing. The novel extraction-free dual HiFi-LAMP assay can be used as a robust POCT tool to promote precise diagnosis and treatment of MRSA infections in hospitals and to facilitate surveillance of MRSA at hospital and community settings. IMPORTANCE: Methicillin-resistant Staphylococcus aureus (MRSA) was associated with high mortality rate and listed as a “priority pathogen” by the World Health Organization. Accurate and rapid point-of-care testing (POCT) of MRSA is critically required for clinical management and treatment of MRSA infections. Some previous LAMP-based POCT assays for MRSA might be questionable due to their low specificity and the lack of appropriate evaluation directly using clinical samples. Furthermore, they are relatively tedious and time-consuming because they require DNA extraction and lack multiplex detection capacity. Here, we reported a novel extraction-free dual HiFi-LAMP assay for discriminative detection of MRSA and methicillin-susceptible S. aureus . The assay has high specificity and sensitivity and can be completed within 40 min. Clinical evaluation with real clinical samples and clinical isolates showed excellent performance with 100% specificity and 92.3%–100% sensitivity. The novel extraction-free assay may be a robust POCT tool to promote precise diagnosis of MRSA infections and facilitate surveillance of MRSA at hospital and community settings.

Hand, foot and mouth disease (HFMD) is usually caused by EVA71 and CVA16 except for a few cases that are caused by non-EVA71 non-CAV16 enteroviruses. Coxsackievirus B3 (CVB3) is mostly associated with myocarditis, occasionally with HFMD. The partial VP1 gene of enteroviruses were amplified and sequenced from 610 throat swabs from clinically confirmed HFMD children. All available CVB3 near full-length genomic and VP1 sequences were downloaded from GenBank. Phylogenetic and distance analyses were performed using MEGA 7.0. A total of 238 partial VP1 sequences were obtained, including 93 EVA71 (39%), 79 CAV16 (33%), 29 CVB3 (12%), 24 CVA6 (10%), and 13 other enterovirus serotypes (5.5%). CVB3 is classified into seven genotypes A-G according to phylogenetic and distance analyses. All CVB3 strains from Zhenjiang belonged to genotype A. In contrast to other genotypes that are prevalent in Europe and other regions of China, and often associated with aseptic meningitis and myocarditis, CVB3 genotype A strains identified in Zhenjiang were only detected among HFMD patients. This high prevalence of CVB3 genotype A among HFMD children has never been reported. This phenomenon has revealed a new epidemic trend of CVB3 among HFMD in China, and it has epidemiological implications for monitoring the epidemic risk of CVB3.

The on-going generation of HIV-1 inter-subtype recombination has led to new circulating recombinant forms (CRFs) and unique recombinant forms (URFs) in Asia. Here, we evaluated whether previously reported URFs were actually CRFs. All available complete or near full-length HIV-1 URFs sequences from Asia were retrieved from the HIV Los Alamos National Laboratory Sequence database, and phylogenetic, transmission cluster and bootscan analyses were performed using MEGA 6.0, Cluster Picker 1.2.1, and SimPlot3.5.1. According to the criterion of new CRF, two new HIV-1 CRFs (CRF87_cpx and CRF88_BC) were identified from these available URFs. CRF87_cpx was comprised of HIV-1 subtypes B, C and CRF01_AE, and CRF88_BC was comprised of subtypes B and C. HIV Blast and bootscan analysis revealed that besides the three representative strains, there were two additional CRF87_cpx strains. Furthermore, we defined 7 dominant URFs (dURF01-dURF07), each of which contained two strains sharing same recombination map and can be used as sequence references to facilitate the finding of new potential CRFs in future. These results will benefit the molecular epidemiological investigation of HIV-1 in Asia.

The human plasma virome is dominated by anelloviruses which are increasingly associated with several clinical conditions including among others HIV-1, COVID-19, autoimmune diseases, and cardiovascular and metabolic diseases. Due to their high genetic divergence, most studies investigated human anellome at broad family or genus level. These approaches obscure the contributions of specific anellovirus species to clinical conditions. We conducted plasma metagenomics in 218 individuals from young (0–16 years) and old (63–100 years) cohorts to resolve the anellome at the species level and examine its patterns across age, sex, and associations with cytokines and cardiometabolic outcomes. Older adults exhibited near-universal anellovirus detection and significantly higher abundance compared with youth. Species-specific analysis revealed that Alphatorquevirus_homin1 and Alphatorquevirus_homin13 were markedly enriched in diseased older adults. Predictive modeling based on machine learning algorithms distinguished disease status in the young cohort with high accuracy (AUC = 0.86), but performance was limited in the elderly (AUC = 0.58), suggesting a lack of diagnostic value in advanced age. Specific species abundances and diversity were associated with stroke and coronary heart disease, while cytokine correlations revealed module-specific immune signatures: Gammatorquevirus-dominated modules associated positively with pro-inflammatory cytokines and growth factors (e.g., IL-1β, IL-15, VEGF), whereas Beta- and some Alphatorquevirus-dominated modules showed predominantly negative correlations with several inflammatory and regulatory mediators (e.g., IL-6, TNF- α , IL-10). These findings demonstrate that the anellome is influenced by age and immune status and shows associations with cardiometabolic health, although these relationships do not guarentee diagnostic or causal significance. Additonally, we found no significant differences of Human endogenous retrovirus K Env expression between disease and healthy controls. This work underscores the importance of resolving human anollome to species level in future longitudinal studies to strengthen their clinical significance and biomarker potential.