Xiao-Ping Chen

Due to the severe consequences of viral infection in allogeneic hematopoietic stem cell transplantation (allo-HSCT) patients, the routine use of polymerase chain reaction (PCR) screening of viruses is common in clinic, while the sensitivities of molecular methods are not always sufficient for blood samples. We aimed to increase the detection efficiency of viremia in allo-HSCT patients with M1 bead enrichment. Blood samples of allo-HSCT patients with fever were collected. Simultaneously with analyses of real-time PCR without enrichment, M1 bead enrichment followed by real-time PCR was applied to detect possible viruses in these samples, and metagenomic next-generation sequencing analyses were also applied in 10 samples negative with real-time PCR without enrichment. Various species of viruses were detected with M1 enrichment method. Significantly, some viruses that had not been found by real-time PCR without M1 enrichment were also detected by those with M1 enrichment and verified by metagenomic next-generation sequencing analyses. Furthermore, blood samples enriched with M1 beads had lower Ct values of real-time PCR assay than those that had not been treated. In conclusion, M1 bead enrichment increased the detection efficiency of most viruses in hematological malignancy patients.

Blood culture-based sepsis diagnostic methods usually cannot obtain positive results in a timely manner. Molecular diagnostic methods, such as real-time PCR without blood culture, would be more time-saving and suitable for pathogenic diagnosis of sepsis, while their sensitivities have always been unsatisfactory for the usually low concentration of pathogens in the blood of sepsis patients. In this study, we established a fast diagnostic method using magnetic beads coated with human recombined mannose-binding lectin that makes it possible to concentrate pathogens from human plasma that have low concentrations of pathogens. With subsequent microculture (MC) and real-time PCR, this method allowed the detection of 1–10 CFUs/ml of Staphylococcus aureus, Group A Streptococcus, Escherichia coli, Pseudomonas aeruginosa, Candida tropicalis, or C. albicans from human plasma within 9.5 h, which was 21–80 h earlier than blood culture. The combination of pathogen enrichment and MC made the detection of sepsis-causing pathogens more time-saving and more sensitive than blood culture or real-time PCR alone.

Epstein-Barr virus (EBV), a common human γ-herpesvirus, infects more than 90% of adults worldwide. The purpose of this study was to establish a novel EBV detection method by combining the recombinase aided amplification (RAA) assay with an initial enrichment step that utilizes magnetic beads coated with a recombinant human mannan-binding lectin (rhMBL, M1 protein). An M1 protein–protein A magnetic bead complex (M1 beads) was prepared and used to achieve separation and enrichment of EBV from blood. After nucleic acid extraction, DNA was amplified by RAA. Using 388 whole blood samples and 1 serum sample, we explored the specificity, sensitivity and applicability of the newly developed detection method and compared it with commercial quantitative real-time polymerase chain reaction (qPCR) following M1 bead enrichment, traditional qPCR and traditional RAA. After enrichment, the positivity rate of EBV was increased from 15.94% to 17.74% by RAA (P < 0.05) and from 7.20% to 15.17% by qPCR (P < 0.05). The viral loads after enrichment were increased by 1.13 to 23.19-fold (P < 0.05). Our data demonstrates that an RAA assay incorporating M1 bead enrichment is a promising tool for detecting low EBV viral loads in blood samples that will facilitate an early response to EBV infection. Graphical abstract

A rapid and accurate method to identify the species and antibiotic resistance of Candida spp. in blood is vital to increase the survival rates of patients with bloodstream infections. However, the extremely low levels of Candida spp. in blood make rapid diagnosis by standard blood culture difficult. In this study, we constructed a direct blood culturing method (i.e., the M1 method) by a rapid enrichment method with magnetic beads coated with a recombined human mannan-binding lectin (rhMBL; i.e., M1 protein), which demonstrated much higher Candida sp.-binding capacity than that of full-length MBL expressed in vitro (i.e., M2). With the M1 method, individual colonies were obtained before the standard blood culture method for each species of Candida spp. tested at <1 CFU/ml (an average of 29 h earlier). Additionally, the clinical sensitivity of the M1 method was 90.5% compared with that of the standard blood culture method when detecting frozen plasma from patients. More significantly, the turnaround time of the M1 method for blood culture could be reduced by approximately 37 to 43 h compared with that of the standard blood culture method in clinical sample identification.

Citrobacter spp. are opportunistic human pathogens which can cause nosocomial infections, sporadic infections and outbreaks. In order to determine the genetic diversity, in vitro virulence properties and antimicrobial resistance profiles of Citrobacter spp., 128 Citrobacter isolates obtained from human diarrheal patients, foods and environment were assessed by multilocus sequence typing (MLST), antimicrobial susceptibility testing and adhesion and cytotoxicity testing to HEp-2 cells. The 128 Citrobacter isolates were typed into 123 sequence types (STs) of which 101 were novel STs, and these STs were divided into five lineages. Lineages I and II contained C. freundii isolates; Lineage III contained all C. braakii isolates, while Lineage IV and V contained C. youngae isolates. Lineages II and V contained more adhesive and cytotoxic isolates than Lineages I, III, and IV. Fifty-one of the 128 isolates were found to be multidrug-resistant (MDR, ≥3) and mainly distributed in Lineages I, II, and III. The prevalence of quinolone resistance varied with Lineage III (C. braakii) having the highest proportion of resistant isolates (52.6%), followed by Lineage I (C. freundii) with 23.7%. Seven qnrB variants, including two new alleles (qnrB93 and qnrB94) were found with Lineage I being the main reservoir. In summary, highly cytotoxic MDR isolates from diarrheal patients may increase the risk of severe disease.

Systemic bacterial infections are prone to secondary Candida albicans superinfection. However, the molecular mechanisms involved remain poorly understood. In this study, a model comprising sub‐lethal cecal ligation and puncture (CLP) plus C. albicans intravenous injection (i.v.) was applied to mimic the situation in superinfection. Compared with mice without systemic bacterial infection, mice with systemic bacterial infection had lower anti‐fungal gene expression (including Il1b, Tnf, Il6, Ifnb, Ifng, Cxcl1, and Ccr2) in monocytes, and less inflammatory monocytes and neutrophils infiltrating into the kidney when challenged with C. albicans. Further, lentivirus‐mediated Setdb2‐knockout and overexpression experiments verified that Setdb2 levels in monocytes correlated negatively with anti‐fungal gene expression and survival rates. Transcriptional repression was probably achieved by Setdb2 through H3 methylation at lysine 9 (H3K9me) in promoter regions of these anti‐fungal genes.

Coagulase-negative staphylococci (CoNS) are recognized as a large reservoir of staphylococcal cassette chromosome mec (SCCmec) harboured by Staphylococcus aureus. However, data of SCCmec in CoNS are relatively absent particularly in China. Seventy-eight CoNS clinical and 47 community isolates were collected in Beijing. PCR was performed to classify SCCmec types. Under oxacillin treatment, quantitative real-time reverse transcription PCR (qRT-PCR) was performed to compare mecA mRNA levels and mRNA half-life between isolates with single SCCmec element and those with multiple one. Their growth curves were analysed. Their bacterial cell wall integrity was also compared by performing a Gram stain. All ccr complex segments were sequenced and obtained ccr segments were analysed by phylogenetic analyses. All 78 clinical isolates had mecA segments compared with 38% in community isolates (total 47). Only 29% clinical isolates and 33% community isolates (among mecA positive isolates) harboured a single previously identified SCCmec type; notably, 17% clinical isolates and 28% community isolates had multiple SCCmec types. Further studies indicated that isolates with multiple SCCmec elements had more stable mecA mRNA expression compared with isolates with single SCCmec elements. CoNS with multiple SCCmec elements demonstrated superior cell wall integrity. Interestingly, phylogenetic analyses of obtained 70 ccr segments indicated that horizontal gene transfer of the ccr complex might exist among various species of clinical CoNS, community CoNS and S. aureus. CoNS recovered from patients carried extremely diverse but distinctive SCCmec elements compared with isolates from the community. More attention should be given to CoNS with multiple SCCmec not only because they had superior cell wall integrity, but also because CoNS and S. aureus might acquire multiple SCCmec through the ccr complex.