Comparison of two automated commercial assays for routine detection of anti-hepatitis E Virus IgM antibodies in clinical samples.

Diagnosis of hepatitis E virus (HEV) infection relies first on detection of IgM antibodies (Ab), sometimes completed with HEV RNA detection. This study aimed to compare the performance of two automated anti-HEV IgM Ab assays. Correlation between Virclia® (Vircell) and Liaison® (Diasorin) assays was carried out on 178 routine clinical samples. Both assays were run on 67 samples from HEV RT-PCR (Altona) screened patients, and 52 Wantai® EIA (Euroimmun) tested samples. An excellent correlation was observed between both assays with an overall agreement of 96.6% (172/178), and a kappa coefficient at 0.93. In HEV RNA positive group (n=43), IgM detection rate was 93.3% (14/15) in immunocompetent patients, with both assays. In immunocompromised patients, detection rate was 75% (21/28) and 71.4% (20/28) using Virclia® and Liaison XL® assays, respectively. Virclia® and Liaison® anti-HEV IgM assays have similar performance for the detection of anti-HEV IgM Ab.

Refractory Microascus Bronchopulmonary Infection Treated with Olorofim, France.

We report 3 cases of successful treatment of Microascus spp. bronchopulmonary infection in a multiple-traumatized patient and 2 lung transplant recipients in France. We emphasize the promising use of olorofim antifungal therapy in a rising context of intrinsically less-susceptible respiratory infections caused by mold.

Antibodies as Models and Tools to Decipher Candida albicans Pathogenic Development: Review about a Unique Monoclonal Antibody Reacting with Immunomodulatory Adhesins.

Candidiasis, caused mainly by Candida albicans, a natural commensal of the human digestive tract and vagina, is the most common opportunistic fungal infection at the mucosal and systemic levels. Its high morbi-mortality rates have led to considerable research to identify the molecular mechanisms associated with the switch to pathogenic development and to diagnose this process as accurately as possible. Since the 1980s, the advent of monoclonal antibody (mAb) technology has led to significant progress in both interrelated fields. This linear review, intended to be didactic, was prompted by considering how, over several decades, a single mAb designated 5B2 contributed to the elucidation of the molecular mechanisms of pathogenesis based on ß-1,2-linked oligomannoside expression in Candida species. These contributions starting from the structural identification of the minimal epitope as a di-mannoside from the ß-1,2 series consisted then in the demonstration that it was shared by a large number of cell wall proteins differently anchored in the cell wall and the discovery of a cell wall glycoplipid shed by the yeast in contact of host cells, the phospholipomannan. Cytological analysis revealed an overall highly complex epitope expression at the cell surface concerning all growth phases and a patchy distribution resulting from the merging of cytoplasmic vesicles to plasmalema and further secretion through cell wall channels. On the host side, the mAb 5B2 led to identification of Galectin-3 as the human receptor dedicated to ß-mannosides and signal transduction pathways leading to cytokine secretion directing host immune responses. Clinical applications concerned in vivo imaging of Candida infectious foci, direct examination of clinical samples and detection of circulating serum antigens that complement the Platelia Ag test for an increased sensitivity of diagnosis. Finally, the most interesting character of mAb 5B2 is probably its ability to reveal C. albicans pathogenic behaviour in reacting specifically with vaginal secretions from women infected versus colonized by this species as well as to display higher reactivity with strains isolated in pathogenic circumstances or even linked to an unfavourable prognosis for systemic candidiasis. Together with a detailed referenced description of these studies, the review provides a complementary reading frame by listing the wide range of technologies involving mAb 5B2 over time, evidencing a practical robustness and versatility unique so far in the Candida field. Finally, the basic and clinical perspectives opened up by these studies are briefly discussed with regard to prospects for future applications of mAb 5B2 in current research challenges.

Parietal composition of Lichtheimia corymbifera: Differences between spore and germ tube stages and host-pathogen interactions.

The molecular composition and structural organization of the cell wall of filamentous fungi underlie the ability of the host to identify them as pathogens. Although the organization of the fungal cell wall, composed of 90% polysaccharides, is similar from one fungus to another, small variations condition their ability to trigger pattern recognition receptors. Because the incidence of mucormycosis, an emerging life-threatening infection caused by the species of the order Mucorales is increasing worldwide, the precise composition of the cell wall of two strains of Lichtheimia corymbifera was investigated in the early growth stages of germination (spores and germ-tubes) using trimethylsilylation and confocal microscopy. This study also characterizes the response of THP-1 cells to Mucorales. The study identified the presence of uncommon monosaccharides (fucose, galactose, and glucuronic acid) whose respective proportions vary according to the germination stage, revealing early parietal reorganization. Immunofluorescence studies confirmed the exposure of ß-glucan on the surface of swollen spores and germ-tubes. Both spores and germ-tubes of L. corymbifera promoted an early and strong pro-inflammatory response, through TLR-2. Our results show the singularity of the cell wall of the order Mucorales, opening perspectives for the development of specific diagnostic biomarkers.

Lichtheimia corymbifera is a causative agent of mucormycosis, an emerging invasive fungal infection. Deciphering cell wall composition can lead to the identification of a polysaccharide epitope, which could be used as a biomarker, useful for the diagnosis of mucormycosis.

The Application of Open Searching-based Approaches for the Identification of Acinetobacter baumannii O-linked Glycopeptides.

Protein glycosylation is increasingly recognized as a common modification within bacterial organisms, contributing to prokaryotic physiology and optimal infectivity of pathogenic species. Due to this, there is increasing interest in characterizing bacterial glycosylation and a need for high-throughput analytical tools to identify these events. Although bottom-up proteomics readily enables the generation of rich glycopeptide data, the breadth and diversity of glycans observed in prokaryotic species make the identification of bacterial glycosylation events extremely challenging. Traditionally, the manual determination of glycan compositions within bacterial proteomic datasets made this a largely bespoke analysis restricted to field-specific experts. Recently, open searching-based approaches have emerged as a powerful alternative for the identification of unknown modifications. By analyzing the frequency of unique modifications observed on peptide sequences, open searching techniques allow the identification of common glycans attached to peptides within complex samples. This article presents a streamlined workflow for the interpretation and analysis of glycoproteomic data, demonstrating how open searching techniques can be used to identify bacterial glycopeptides without prior knowledge of the glycan compositions. Using this approach, glycopeptides within samples can rapidly be identified to understand glycosylation differences. Using Acinetobacter baumannii as a model, these approaches enable the comparison of glycan compositions between strains and the identification of novel glycoproteins. Taken together, this work demonstrates the versatility of open database-searching techniques for the identification of bacterial glycosylation, making the characterization of these highly diverse glycoproteomes easier than ever before.

Presence of the Hmq System and Production of 4-Hydroxy-3-Methyl-2-Alkylquinolines Are Heterogeneously Distributed between Burkholderia cepacia Complex Species and More Prevalent among Environmental than Clinical Isolates.

The Burkholderia cepacia complex (Bcc) comprises several species of closely related, versatile bacteria. Some Bcc strains produce 4-hydroxy-3-methyl-2-alkylquinolines (HMAQs), analogous to the 4-hydroxy-2-alkylquinolines of Pseudomonas aeruginosa. Using in silico analyses, we previously estimated that the hmqABCDEFG operon, which encodes enzymes involved in the biosynthesis of HMAQs, is carried by about one-third of Bcc strains, with considerable inter- and intraspecies variability. In the present study, we investigated by PCR, using consensus primers, the distribution of hmqABCDEFG in a collection of 312 Bcc strains (222 of clinical and 90 of environmental origins) belonging to 18 Bcc species. We confirmed that this operon is not distributed evenly among Bcc species. Among the 30% of strains bearing the hmqABCDEFG operon, we found that 92% of environmental isolates and 82% of clinically isolated Bcc strains produce levels of HMAQs detectable by liquid chromatography-mass spectrometry in at least one of the tested culture conditions. Among the hmqABCDEFG-positive but HMAQ-negative strains, none expressed the hmqA gene under the specified culture conditions. Interestingly, the hmqABCDEFG operon is more prevalent among plant root environment species (e.g., Burkholderia ambifaria and Burkholderia cepacia) and absent in species commonly found in chronically colonized individuals with cystic fibrosis (e.g., Burkholderia cenocepacia and Burkholderia multivorans), suggesting a role for the Hmq system in niche adaptation. We investigated the impact of the Hmq system on plant growth promotion and found that Pisum sativum root development by B. ambifaria required a functional HMAQ system. IMPORTANCE Environmental bacteria belonging to the various closely related species forming the Burkholderia cepacia complex (Bcc) can infect plants and animals, including humans. Their pathogenicity is regulated by intercellular communication, or quorum sensing, allowing them to collaborate instead of acting individually. Bcc organisms generally exploit interacting quorum sensing systems based on N-acyl-homoserine lactones as signaling molecules. Several Bcc strains also carry an hmqABCDEFG operon responsible for the biosynthesis of 4-hydroxy-3-methyl-2-alkylquinolines (HMAQs), molecules analogous to the Pseudomonas quinolone signal (PQS) system of P. aeruginosa. Our finding that the prevalences of the Hmq system and HMAQ production are very different between various Bcc species suggests a key role in niche adaptation or pathogenicity. This is supported by a significant reduction in plant growth promotion in the absence of HMAQ production for a beneficial Bcc strain.

Secondary metabolites from the Burkholderia pseudomallei complex: structure, ecology, and evolution.

Bacterial secondary metabolites play important roles in promoting survival, though few have been carefully studied in their natural context. Numerous gene clusters code for secondary metabolites in the genomes of members of the Bptm group, made up of three closely related species with distinctly different lifestyles: the opportunistic pathogen Burkholderia pseudomallei, the non-pathogenic saprophyte Burkholderia thailandensis, and the host-adapted pathogen Burkholderia mallei. Several biosynthetic gene clusters are conserved across two or all three species, and this provides an opportunity to understand how the corresponding secondary metabolites contribute to survival in different contexts in nature. In this review, we discuss three secondary metabolites from the Bptm group: bactobolin, malleilactone (and malleicyprol), and the 4-hydroxy-3-methyl-2-alkylquinolines, providing an overview of each of their biosynthetic pathways and insight into their potential ecological roles. Results of studies on these secondary metabolites provide a window into how secondary metabolites contribute to bacterial survival in different environments, from host infections to polymicrobial soil communities.

First case of fatal bacteremia due to Nocardia neocaledoniensis.

Nocardia neocaledoniensis is an uncommon cause of human-infections. Few cases are reported in the literature. We describe the first case of bacteremia caused by N. neocaledoniensis. This article underlines the importance of mass spectrometry for easy and rapid identification of such bacterium.

Synthesis and Antimicrobial Activity of Burkholderia-Related 4-Hydroxy-3-methyl-2-alkenylquinolines (HMAQs) and Their N-Oxide Counterparts.

The Burkholderia genus offers a promising potential in medicine because of the diversity of biologically active natural products encoded in its genome. Some pathogenic Burkholderia spp. biosynthesize a specific class of antimicrobial 2-alkyl-4(1H)-quinolones, i.e., 4-hydroxy-3-methyl-2-alkenylquinolines (HMAQs) and their N-oxide derivatives (HMAQNOs). Herein, we report the synthesis of a series of six HMAQs and HMAQNOs featuring a trans-Δ2 double bond at the C2-alkyl chain. The quinolone scaffold was obtained via the Conrad-Limpach approach, while the (E)-2-alkenyl chain was inserted through Suzuki-Miyaura cross-coupling under microwave radiation without noticeable isomerization according to the optimized conditions. Subsequent oxidation of enolate-protected HMAQs cleanly led to the formation of HMAQNOs following cleavage of the ethyl carbonate group. Synthetic HMAQs and HMAQNOs were evaluated in vitro for their antimicrobial activity against different Gram-negative and Gram-positive bacteria as well as against molds and yeasts. The biological results support and extend the potential of HMAQs and HMAQNOs as antimicrobials, especially against Gram-positive bacteria. We also confirm the involvement of HMAQs in the autoregulation of the Hmq system in Burkholderia ambifaria.

Routine drug resistance testing in HIV-1 proviral DNA, using an automated next- generation sequencing assay.

BACKGROUND: HIV-1 DNA genotypic drug resistance testing is increasingly performed to guide treatment switching or simplification in controlled patients. The Sentosa NGS platform is a fully automated system marketed for drug resistance testing on HIV-1 RNA samples. OBJECTIVES: The aim of this study was to evaluate this automated NGS solution for routine resistance genotypic resistance testing in proviral HIV-1 DNA. STUDY DESIGN: Sanger sequencing (SS) of the reverse transcriptase (RT), protease (PR) and integrase (IN) genes was performed using the French ANRS protocol. NGS was performed retrospectively on frozen samples, using the Sentosa platform combined with the Sentosa SQ HIV genotyping Assay. RESULTS: A total of 77 samples were run once using NGS. A successful sequencing of the three HIV-1 genes (RT, PR, IN) was obtained for 45 samples. The number of cumulated RAMs was 179, 185 and 219 with SS, NGS 20% and NGS 10% respectively; however most of them were minor mutations in the PR region. The mutation detection rate was similar between SS and NGS 20%. Several discordances were observed between both methods in the RT and PR regions, mainly due to the use of different DNA extracts, and hypermutation. CONCLUSIONS: HIV-1 DNA genotypic resistance testing can be performed with the Sentosa platform. Few technical optimizations are still needed to include the extraction step and to improve the sequencing efficiency.

Keeping an Eye on Wild Brown Trout (Salmo trutta) Populations: Correlation Between Temperature, Environmental Parameters, and Proliferative Kidney Disease.

Proliferative kidney disease (PKD) is an emerging disease of salmonids caused by the myxozoan parasite Tetracapsuloides bryosalmonae, which plays a major role in the decrease of wild brown trout (Salmo trutta) populations in Switzerland. Strong evidence demonstrated that water temperature modulates parasite infection. However, less knowledge exists on how seasonal water temperature fluctuations influence PKD manifestation under field conditions, how further environmental factors such as water quality may modulate the disease, and whether these factors coalesce with temperatures role possibly giving rise to cumulative effects on PKD. The aims of this study were to (1) determine the correlation between seasonal course of water temperature and PKD prevalence and intensity in wild brown trout populations, (2) assess if other factors such as water quality or ecomorphology correlate with the infection, and (3) quantitatively predict the implication of these factors on PKD prevalence with a statistical model. Young-of-the-year brown trout were sampled in 45 sites through the Canton of Vaud (Switzerland). For each site, longitudinal time series of water temperature, water quality (macroinvertebrate community index, presence of wastewater treatment plant effluent) and ecomorphological data were collected and correlated with PKD prevalence and intensity. 251 T. bryosalmonae-infected trout of 1,118 were found (overall prevalence 22.5%) at 19 of 45 study sites (42.2%). Relation between PKD infection and seasonal water temperature underlined that the mean water temperature for June and the number of days with mean temperature ≥15°C were the most significantly correlated parameters with parasite prevalence and intensity. The presence of a wastewater treatment plant effluent was significantly correlated with the prevalence and infection intensity. In contrast, macroinvertebrate diversity and river ecomorphology were shown to have little impact on disease parameters. Linear and logistic regressions highlighted quantitatively the prediction of PKD prevalence depending on environmental parameters at a given site and its possible increase due to rising temperatures. The model developed within this study could serve as a useful tool for identifying and predicting disease hot spots. These results support the importance of temperature for PKD in salmonids and provides evidence for a modulating influence of additional environmental stress factors.

Polysaccharides Cell Wall Architecture of Mucorales.

Invasive fungal infections are some of the most life-threatening infectious diseases in the hospital setting. In industrialized countries, the most common fungal species isolated from immunocompromised patients are Candida and Aspergillus spp. However, the number of infections due to Mucorales spp. is constantly increasing and little is known about the virulence factors of these fungi. The fungal cell wall is an important structure protecting fungi from the environment. A better knowledge of its composition should improve our understanding of host-pathogen interactions. Cell wall molecules are involved in tissue adherence, immune escape strategies, and stimulation of host defenses including phagocytosis and mediators of humoral immunity. The fungal cell wall is also a target of choice for the development of diagnostic or therapeutic tools. The present review discusses our current knowledge on the cell wall structure of Mucorales in terms of the polysaccharides and glyco-enzymes involved in its biosynthesis and degradation, with an emphasis on the missing gaps in our knowledge.

Potential of the Burkholderia cepacia Complex to Produce 4-Hydroxy-3-Methyl-2-Alkyquinolines.

A few Burkholderia species, especially Burkholderia pseudomallei, Burkholderia thailandensis, Burkholderia ambifaria, and Burkholderia cepacia, are known to produce and release various 4-hydroxy-3-methyl-2-alkylquinolines (HMAQs), a family of molecules analogous to the 4-hydroxy-2-alkylquinolines [aka 2-n-alkyl-4(1H)-quinolones] of Pseudomonas aeruginosa, which include the Pseudomonas quinolone signal (PQS). However, while these exoproducts play several roles in P. aeruginosa virulence and survival, the available literature is very limited on their distribution and function in Burkholderia. In this perspective article, we studied the distribution of the hmqABCDEFG operon, which encodes the enzymes involved in the biosynthesis of HMAQs, in the Burkholderia cepacia complex (Bcc) group. Based on the available sequence data, about one third of Bcc species carry a homolog of the hmqABCDEFG, and not all sequenced strains in a given species possess this operon. Looking at the synteny of genes surrounding the hmqABCDEFG operon, we found that for some species, the operon seems to have been deleted or replaced by other genes. Finally, we review the literature on the possible function of HMAQs. Understanding the Hmq system may provide clues concerning their functions in Bcc.