Linezolid is one of the most effective treatments against Gram-positive pathogens. However, linezolid-resistant/intermediate strains have recently emerged in worldwide. The purpose of this study was to analyse the prevalence and resistance mechanisms of linezolid-resistant/intermediate staphylococci and enterococci in Shanghai, China. Thirty-two linezolid-resistant/intermediate strains, including 14Staphylococcus capitis, three Staphylococcus aureus, 14 Enterococcus faecalis and one Enterococcus faecium clinical isolates, were collected in this study which displayed linezolid MICs of 8 to 512 μg/ml, 8-32 μg/ml, 4-8 μg/ml and 4 μg/ml, respectively. All linezolid-resistant S. capitis isolates had a novel C2131T mutation and a G2603T mutation in the 23S rRNA region, and some had a C316T (Arg106Cys) substitution in protein L4 and/or harboured cfr. Linezolid-resistant S. aureus isolates carried a C389G (Ala130Gly) substitution in protein L3, and/or harboured cfr. The cfr gene was flanked by two copies of the IS256-like element, with a downstream orf1 gene. Linezolid-resistant/intermediate enterococci lacked major resistance mechanisms. The semi-quantitative biofilm assay showed that 14 linezolid-resistant E. faecalis isolates produced a larger biofilm than linezolid-susceptible E. faecalis strains. Transmission electron microscopy showed the cell walls of linezolid-resistant/intermediate strains were thicker than linezolid-susceptible strains. Our data indicated that major resistance mechanisms, such as mutations in 23S rRNA and ribosomal proteins L3 and L4, along with cfr acquisition, played an important role in linezolid resistance. Secondary resistance mechanisms, such as biofilm formation and cell wall thickness, should also be taken into account.

Epstein-Barr virus (EBV) infection is a well-known for its association with lymphoproliferative disorders and various lymphomas, causing significant global morbidity and mortality. EBV-positive vitreoretinal lymphoma (VRL) is exceedingly rare. As a result, the pathogenic role and genomic characteristics of EBV in VRL remain poorly understood. In this study, we employed droplet digital PCR (ddPCR) combined with EBV-specific immunofluorescence assay to detect EBV in the vitreous fluid of fifty-three VRL patients. We found that approximately 28% (15/53) of the patients were EBV positive. Analysis of clinical data showed that EBV-positive VRL patients had shorter progression-free survival (PFS) compared to EBV-negative patients (P = 0.004). Additionally, through integration of EBV-targeted sequencing and PCR-based deep sequencing, we found that all five VRL-derived EBV genomes formed a distinct cluster within one phylogenetic branch. Meanwhile, several non-synonymous mutations were exclusively detected in the VRL group, including S229T in latent membrane protein 1 (LMP1) and G2248R in the Epstein-Barr virus BamHI-PraL fragment 1 (BPLF1). In conclusion, our findings suggest that EBV as a risk factor associated with poor prognosis in VRL, and we provide a genome-wide view of EBV sequence variations from VRL patients. This may offer insights into the pathogenic role of EBV in VRL and could potentially assist in the diagnosis and treatment of this disease.

Cryptococcal meningitis (CM) is a global threat with significant attributable morbidity and mortality. Information on integrated detection for CM diagnosis is still limited. This is mainly due to the presence of a large polysaccharide capsule and the tough cell wall of Cryptococcus , which makes it difficult to extract nucleic acids on the chip. In this study, we developed a LAMP-based microfluidic chip for rapid detection of pathogen in CM. We adopted 4 duplicate filtration membrane structures to improve target capture and simplify the enrichment process, and combined lyticase digestion and thermal alkaline lysisto optimize the nucleic acid extraction of Cryptococcus on the chip, and selected a portable UVA flashlight to shine the LAMP products to obtain the visual detection results which could be observed by the naked eye. This microfluidic chip, integrating sample Cryptococcus enrichment, nucleic acid extraction and LAMP detection unit, streamlined the operation process and reduced the exposure risk of directly handling cryptococcal samples. It did not require any additional instruments and demonstrated a rapid, reliable, as well as high-efficiency approach. It truly realized the “sample-to-answer” application and could be easily used for clinical cryptococcal prediagnosis.