A novel diagnostic method for a rare fungus: FcMBL facilitates Wickerhamomyces anomalus identification in an immunocompromised neonate.

Fungemia negatively impacts patient outcomes, current diagnostics lack sensitivity to identify emerging rare mycoses, and fungal infections are increasing in prevalence, variety, and resistance. We report a case of Wickerhamomyces anomalus in an immunocompromised neonate in which FcMBL bead-based matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) resulted in species identification roughly 30 hours before standard pathogen identification methods. Deploying FcMBL bead-based MALDI-TOF MS may improve the speed and accuracy of identification, and therefore treatment, of rare pathogens.

Identification of bacterial pathogens in sudden unexpected death in infancy and childhood using 16S rRNA gene sequencing.

Background: Sudden unexpected death in infancy (SUDI) is the most common cause of post-neonatal death in the developed world. Following an extensive investigation, the cause of ~40% of deaths remains unknown. It is hypothesized that a proportion of deaths are due to an infection that remains undetected due to limitations in routine techniques. This study aimed to apply 16S rRNA gene sequencing to post-mortem (PM) tissues collected from cases of SUDI, as well as those from the childhood equivalent (collectively known as sudden unexpected death in infancy and childhood or SUDIC), to investigate whether this molecular approach could help identify potential infection-causing bacteria to enhance the diagnosis of infection. Methods: In this study, 16S rRNA gene sequencing was applied to de-identified frozen post-mortem (PM) tissues from the diagnostic archive of Great Ormond Street Hospital. The cases were grouped depending on the cause of death: (i) explained non-infectious, (ii) infectious, and (iii) unknown. Results and conclusions: In the cases of known bacterial infection, the likely causative pathogen was identified in 3/5 cases using bacterial culture at PM compared to 5/5 cases using 16S rRNA gene sequencing. Where a bacterial infection was identified at routine investigation, the same organism was identified by 16S rRNA gene sequencing. Using these findings, we defined criteria based on sequencing reads and alpha diversity to identify PM tissues with likely infection. Using these criteria, 4/20 (20%) cases of unexplained SUDIC were identified which may be due to bacterial infection that was previously undetected. This study demonstrates the potential feasibility and effectiveness of 16S rRNA gene sequencing in PM tissue investigation to improve the diagnosis of infection, potentially reducing the number of unexplained deaths and improving the understanding of the mechanisms involved.

FcMBL magnetic bead-based MALDI-TOF MS rapidly identifies paediatric blood stream infections from positive blood cultures.

Rapid identification of potentially life-threatening blood stream infections (BSI) improves clinical outcomes, yet conventional blood culture (BC) identification methods require ~24-72 hours of liquid culture, plus 24-48 hours to generate single colonies on solid media suitable for identification by mass spectrometry (MS). Newer rapid centrifugation techniques, such as the Bruker MBT-Sepsityper® IVD, replace culturing on solid media and expedite the diagnosis of BCs but frequently demonstrate reduced sensitivity for identifying clinically significant Gram-positive bacterial or fungal infections. This study introduces a protocol that utilises the broad-range binding properties of an engineered version of mannose-binding lectin linked to the Fc portion of immunoglobulin (FcMBL) to capture and enrich pathogens combined with matrix-assisted laser desorption-ionisation time-of-flight (MALDI-TOF) MS for enhanced infection identification in BCs. The FcMBL method identified 94.1% (64 of 68) of clinical BCs processed, with a high sensitivity for both Gram-negative and Gram-positive bacteria (94.7 and 93.2%, respectively). The FcMBL method identified more patient positive BCs than the Sepsityper® (25 of 25 vs 17 of 25), notably with 100% (3/3) sensitivity for clinical candidemia, compared to only 33% (1/3) for the Sepsityper®. Additionally, during inoculation experiments, the FcMBL method demonstrated a greater sensitivity, identifying 100% (24/24) of candida to genus level and 9/24 (37.5%) top species level compared to 70.8% (17/24) to genus and 6/24 to species (25%) using the Sepsityper®. This study demonstrates that capture and enrichment of samples using magnetic FcMBL-conjugated beads is superior to rapid centrifugation methods for identification of BCs by MALDI-TOF MS. Deploying the FcMBL method therefore offers potential clinical benefits in sensitivity and reduced turnaround times for BC diagnosis compared to the standard Sepsityper® kit, especially for fungal diagnosis.

IFITM3-specific antibody reveals IFN preferences and slow IFN induction of the antiviral factor IFITM3 in humans.

Using a specific antibody, we found that expression of the viral restriction factor IFITM3 differs across cell types within the immune compartment with higher expression in myeloid rather than lymphoid cells. IFITM3 expression was increased following IFN stimulation, mostly type I, in immune cells, with the exception of T cells.

Lack of Truncated IFITM3 Transcripts in Cells Homozygous for the rs12252-C Variant That is Associated With Severe Influenza Infection.

Interferon-induced transmembrane 3 (IFITM3) is known to restrict the entry of a range of enveloped viruses. The single nucleotide polymorphism rs12252-C within IFITM3 has been shown to be associated with severe influenza A virus infection. It has been suggested that rs12252-C results in expression of a truncated IFITM3 protein lacking the first 21 amino acids. By performing high-throughput RNA sequencing on primary dendritic cells and peripheral blood mononuclear cells isolated from pandemic H1N1 influenza and human immunodeficiency virus-1 (HIV-1) infected patients we show that full-length IFITM3 mRNA is dominantly expressed (>99%) across all rs12252 genotypes. Full-length IFITM3 protein can be detected in all genotypes.

The autoimmune-associated genetic variant PTPN22 R620W enhances neutrophil activation and function in patients with rheumatoid arthritis and healthy individuals.

OBJECTIVES: A genetic variant of the leukocyte phosphatase PTPN22 (R620W) is strongly associated with autoimmune diseases including rheumatoid arthritis (RA). Functional studies on the variant have focussed on lymphocytes, but it is most highly expressed in neutrophils. We have investigated the effects of the variant on neutrophil function in health and in patients with RA. METHODS: Healthy individuals and patients with RA were genotyped for PTPN22 (R620W) and neutrophils isolated from peripheral blood. Neutrophil adhesion and migration across inflamed endothelium were measured. Calcium (Ca(2+)) release and reactive oxygen species (ROS) production in response to fMLP stimulation were also assessed. RESULTS: Expression of R620W enhanced neutrophil migration through cytokine activated endothelium (non-R620W=24%, R620W=45% migrating cells, p<0.001). Following fMLP stimulation, neutrophils that were heterozygous and homozygous for R620W released significantly more Ca(2+) when compared to non-R620W neutrophils, in healthy individuals and patients with RA. fMLP stimulation, after TNF-α priming, provoked more ROS from neutrophils heterozygous for R620W in patients with RA (non-R620W vs R620W=∼1.75-fold increase) and healthy individuals (non-R620W vs R620W=fourfold increase) and this increase was statistically significant in healthy individuals (p<0.001) but not in patients with RA (p<0.25). CONCLUSIONS: Expression of PTPN22 (R620W) enhanced neutrophil effector functions in health and RA, with migration, Ca(2+) release and production of ROS increased. Neutrophils are found in large numbers in the RA joint, and this hyperactivity of R620W cells may directly contribute to the joint damage, as well as to the initiation and perpetuation of the chronic immune-mediated inflammatory processes driving the disease.