Effective Rapid Diagnosis of Bacterial and Fungal Bloodstream Infections by T2 Magnetic Resonance Technology in the Pediatric Population.

Children are prone to bloodstream infections (BSIs), the rapid and accurate diagnosis of which is an unmet clinical need. The T2MR technology is a direct molecular assay for identification of BSI pathogens, which can help to overcome the limits of blood culture (BC) such as diagnostic accuracy, blood volumes required, and turnaround time. We analyzed results obtained with the T2Bacteria (648) and T2Candida (106) panels in pediatric patients of the Bambino Gesù Children's Hospital between May 2018 and September 2020 in order to evaluate the performance of the T2Dx instrument with respect to BC. T2Bacteria and T2Candida panels showed 84.2% and 100% sensitivity with 85.9% and 94.1% specificity, respectively. The sensitivity and specificity of the T2Bacteria panel increased to 94.9% and 98.7%, respectively, when BC was negative but other laboratory data supported the molecular result. T2Bacteria sensitivity was 100% with blood volumes <2 mL in neonates and infants. T2Bacteria and T2Candida provided definitive microorganism identification in a mean time of 4.4 and 3.7 h, respectively, versus 65.7 and 125.5 h for BCs (P < 0.001). T2 panels rapidly and accurately enable a diagnosis of a pediatric BSI, even in children under 1 year of age and for very small blood volumes. These findings support their clinical use in life-threatening pediatric infections, where the time to diagnosis is of utmost importance, in order to improve survival and minimize the long-term sequalae of sepsis. The T2 technology could be further developed to include more bacteria and fungi species that are involved in the etiology of sepsis.

Gut Microbiota Ecology and Inferred Functions in Children With ASD Compared to Neurotypical Subjects.

Autism spectrum disorders (ASDs) is a multifactorial neurodevelopmental disorder. The communication between the gastrointestinal (GI) tract and the central nervous system seems driven by gut microbiota (GM). Herein, we provide GM profiling, considering GI functional symptoms, neurological impairment, and dietary habits. Forty-one and 35 fecal samples collected from ASD and neurotypical children (CTRLs), respectively, (age range, 3-15 years) were analyzed by 16S targeted-metagenomics (the V3-V4 region) and inflammation and permeability markers (i.e., sIgA, zonulin lysozyme), and then correlated with subjects' metadata. Our ASD cohort was characterized as follows: 30/41 (73%) with GI functional symptoms; 24/41 (58%) picky eaters (PEs), with one or more dietary needs, including 10/41 (24%) with food selectivity (FS); 36/41 (88%) presenting high and medium autism severity symptoms (HMASSs). Among the cohort with GI symptoms, 28/30 (93%) showed HMASSs, 17/30 (57%) were picky eaters and only 8/30 (27%) with food selectivity. The remaining 11/41 (27%) ASDs without GI symptoms that were characterized by HMASS for 8/11 (72%) and 7/11 (63%) were picky eaters. GM ecology was investigated for the overall ASD cohort versus CTRLs; ASDs with GI and without GI, respectively, versus CTRLs; ASD with GI versus ASD without GI; ASDs with HMASS versus low ASSs; PEs versus no-PEs; and FS versus absence of FS. In particular, the GM of ASDs, compared to CTRLs, was characterized by the increase of Proteobacteria, Bacteroidetes, Rikenellaceae, Pasteurellaceae, Klebsiella, Bacteroides, Roseburia, Lactobacillus, Prevotella, Sutterella, Staphylococcus, and Haemophilus. Moreover, Sutterella, Roseburia and Fusobacterium were associated to ASD with GI symptoms compared to CTRLs. Interestingly, ASD with GI symptoms showed higher value of zonulin and lower levels of lysozyme, which were also characterized by differentially expressed predicted functional pathways. Multiple machine learning models classified correctly 80% overall ASDs, compared with CTRLs, based on Bacteroides, Lactobacillus, Prevotella, Staphylococcus, Sutterella, and Haemophilus features. In conclusion, in our patient cohort, regardless of the evaluation of many factors potentially modulating the GM profile, the major phenotypic determinant affecting the GM was represented by GI hallmarks and patients' age.

Case Report: Trichosporon japonicum Fungemia in a Pediatric Patient With Refractory Acute B Cell Lymphoblastic Leukemia.

Trichosporon japonicum is a very rare opportunistic yeast causing fungal disease in humans, especially in immunocompromised hosts. Here, we describe a new case of T. japonicum isolated from the blood of a pyrexial pediatric patient with refractory acute B cell lymphoblastic leukemia and acute respiratory distress. Prompt diagnosis through early clinical suspicion and appropriate molecular microbiology analysis allowed the yeast to be accurately identified at species level. Subsequent drug susceptibility testing and focused antifungal treatment with voriconazole and amphotericin B led to a complete clinical and mycological resolution of the infection, which represents the second successful case of T. japonicum bloodstream infection described in literature to date.

Harmonization of six quantitative SARS-CoV-2 serological assays using sera of vaccinated subjects.

BACKGROUND AND AIMS: Vaccines, to limit SARS-CoV-2 infection, were produced and reliable assays are needed for their evaluation. The WHO produced an International-Standard (WHO-IS) to facilitate the standardization/comparison of serological methods. The WHO-IS, produced in limited amount, was never tested for reproducibility. This study aims at developing a reproducible and accessible working standard (WS) to complement the WHO-IS. MATERIALS AND METHODS: Sera from vaccinated individuals were used to produce the WSs. The WHO-IS, the WSs and single serum samples (n = 48) were tested on 6 quantitative serological devices. Neutralization assays were performed for the 48 samples and compared with their antibody titers. RESULTS: The WS carry an antibody titer 20-fold higher than the WHO-IS. It was reproducible, showed both good linearity and insignificant intra- and inter-laboratory variability. However, the WSs behave differently from the WHO-IS. Analysis of the 48 samples showed that single correlation factors are not sufficient to harmonize results from different assays. Yet, all the devices predict neutralization activity based on the antibody titer. CONCLUSIONS: A reproducible and highly concentrated WS, specific for IgG against SARS-CoV-2 Spike-glycoprotein was produced. Such characteristics make it particularly suited for the harmonization of commercially available assays and the consequent evaluation of post-vaccinated individuals.

Bordetella holmesii: Lipid A Structures and Corresponding Genomic Sequences Comparison in Three Clinical Isolates and the Reference Strain ATCC 51541.

Bordetella holmesii can cause invasive infections but can also be isolated from the respiratory tract of patients with whooping-cough like symptoms. For the first time, we describe the lipid A structure of B. holmesii reference strain ATCC 51541 (alias NCTC12912 or CIP104394) and those of three French B. holmesii clinical isolates originating from blood (Bho1) or from respiratory samples (FR4020 and FR4101). They were investigated using chemical analyses, gas chromatography-mass spectrometry (GC-MS), and matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). The analyses revealed a common bisphosphorylated ß-(1→6)-linked d-glucosamine disaccharide with hydroxytetradecanoic acid in amide linkages. Similar to B. avium, B. hinzii and B. trematum lipids A, the hydroxytetradecanoic acid at the C-2' position are carrying in secondary linkage a 2-hydroxytetradecanoic acid residue resulting of post-traductional biosynthesis modifications. The three clinical isolates displayed characteristic structural traits compared to the ATCC 51541 reference strain: the lipid A phosphate groups are more or less modified with glucosamine in the isolates and reference strain, but the presence of 10:0(3-OH) is only observed in the isolates. This trait was only described in B. pertussis and B. parapertussis strains, as well as in B. petrii isolates by the past. The genetic bases for most of the key structural elements of lipid A were analyzed and supported the structural data.

Complete Bordetella avium, Bordetella hinzii and Bordetella trematum lipid A structures and genomic sequence analyses of the loci involved in their modifications.

Endotoxin is recognized as one of the virulence factors of the Bordetella avium bird pathogen, and characterization of its structure and corresponding genomic features are important for an understanding of its role in pathogenicity and for an improved general knowledge of Bordetella spp virulence factors. The structure of the biologically active part of B. avium LPS, lipid A, is described and compared to those of another bird pathogen, opportunistic in humans, Bordetella hinzii, and to that of Bordetella trematum, a human pathogen. Sequence analyses showed that the three strains have homologues of acyl-chain modifying enzymes PagL, PagP and LpxO, of the 1-phosphatase LpxE, in addition to LgmA, LgmB and LgmC, which are required for the glucosamine modification. MALDI mass spectrometry identified a high amount of glucosamine substituting the phosphate groups of B. avium lipid A; this modification was absent from B. hinzii and B. trematum. The acylation patterns of the three lipid As were similar, but they differed from those of Bordetella pertussis and Bordetella parapertussis. They were also found to be close to the lipid A structure of Bordetella bronchiseptica, a mammalian pathogen, only differing from the latter by the degree of hydroxylation of the branched fatty acid.

Comparison of lipopolysaccharide structures of Bordetella pertussis clinical isolates from pre- and post-vaccine era.

Endotoxins are lipopolysaccharides (LPS), and major constituents of the outer membrane of Gram-negative bacteria. Bordetella pertussis LPS were the only major antigens, of this agent of whooping-cough, that were not yet analyzed on isolates from the pre- and post-vaccination era. We compared here the LPS structures of four clinical isolates with that of the vaccine strain BP 1414. All physico-chemical analyses, including SDS-PAGE, TLC, and different MALDI mass spectrometry approaches were convergent. They helped demonstrating that, on the contrary to some other B. pertussis major antigens, no modification occurred in the dodecasaccharide core structure, as well as in the whole LPS molecules. These results are rendering these major antigens good potential vaccine components. Molecular modeling of this conserved LPS structure also confirmed the conclusions of previous experiments leading to the production of anti-LPS monoclonal antibodies and defining the main epitopes of these major antigens.

Minor modifications to the phosphate groups and the C3' acyl chain length of lipid A in two Bordetella pertussis strains, BP338 and 18-323, independently affect Toll-like receptor 4 protein activation.

Lipopolysaccharides (LPS) of Bordetella pertussis are important modulators of the immune system. Interaction of the lipid A region of LPS with the Toll-like receptor 4 (TLR4) complex causes dimerization of TLR4 and activation of downstream nuclear factor κB (NFκB), which can lead to inflammation. We have previously shown that two strains of B. pertussis, BP338 (a Tohama I-derivative) and 18-323, display two differences in lipid A structure. 1) BP338 can modify the 1- and 4'-phosphates by the addition of glucosamine (GlcN), whereas 18-323 cannot, and 2) the C3' acyl chain in BP338 is 14 carbons long, but only 10 or 12 carbons long in 18-323. In addition, BP338 lipid A can activate TLR4 to a greater extent than 18-323 lipid A. Here we set out to determine the genetic reasons for the differences in these lipid A structures and the contribution of each structural difference to the ability of lipid A to activate TLR4. We show that three genes of the lipid A GlcN modification (Lgm) locus, lgmA, lgmB, and lgmC (previously locus tags BP0399-BP0397), are required for GlcN modification and a single amino acid difference in LpxA is responsible for the difference in C3' acyl chain length. Furthermore, by introducing lipid A-modifying genes into 18-323 to generate isogenic strains with varying penta-acyl lipid A structures, we determined that both modifications increase TLR4 activation, although the GlcN modification plays a dominant role. These results shed light on how TLR4 may interact with penta-acyl lipid A species.