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Between 2018 and 2024, we conducted systematic whole-genome sequencing and phylogenomic analysis on 263 V. cholerae O1 isolates from cholera patients across four provinces in the Democratic Republic of Congo (North-Kivu, South-Kivu, Tanganyika, and Kasai Oriental). These isolates were classified into the AFR10d and AFR10e sublineages of AFR10 lineage, originating from the third wave of the seventh El Tor cholera pandemic (7PET). Compared to the strains analysed between 2014 and 2017, both sublineages had few genetic changes in the core genome but recent isolates (2022-2024) had significant CTX prophage rearrangement. AFR10e spread across all four provinces, while AFR10d appeared to be extinct by the end of 2020. Since 2022, most V. cholerae O1 isolates exhibited significant CTX prophage rearrangements, including a tandem repeat of an environmental satellite phage RS1 downstream the ctxB toxin gene of the CTX-Φ-3 prophage on the large chromosome, as well as two or more arrayed copies of an environmental pre-CTX-Φ prophage precursor on the small chromosome. We used Illumina data for mapping and coverage estimation to identify isolates with unique CTX-Φ genomic features. Gene localization was then determined on MinION-derived assemblies, revealing an organization similar to that of non-O1 V. cholerae isolates found in Asia (O139 VC1374, and environmental O4 VCE232), but never described in V. cholerae O1 El Tor from the third wave. In conclusion, while the core genome of AFR10d and AFR10e showed minimal changes, significant alterations in the CTX-Φ and pre-CTX-Φ prophage content and organization were identified in AFR10e from 2022 onwards.

Abstract Background Pulsed ultraviolet (UV)-C light sources, such as excimer lasers, are used in emerging non-thermal food-decontamination methods and also have high potential for use in a wide range of microbial decontamination applications. The acaricidal effect of an experimental UV-C irradiation device was assessed using female adults and eggs of a model organism, the two-spotted spider mite Tetranychus urticae. Methods UV-C light was generated by a pulsed krypton fluoride excimer laser operating at 248-nm emission wavelength. The pulse energy and pulse repetition rate were 5 mJ and up to 100 Hz, respectively. The distance from the light source to the target was 150 mm; the target surface area was 2.16 cm2. The exposure time for the mites and fresh eggs varied from 1 to 4 min at 5–300 mW, which corresponded to UV doses of 5–80 kJ/m2. Post-irradiation acaricidal effects (mite mortality) were assessed immediately and also measured at 24 h. The effects of UV-C irradiation on the hatchability of eggs were observed daily for up to 12 days post-irradiation. Results The mortality of mites at 5 and 40 kJ/m2 was 26% and 92%, respectively. Mite mortality reached 98% at 80 kJ/m2. The effect of exposure duration on mortality was minimal. The effect of irradiation on egg hatchability was even more significant than that on adult mite mortality, i.e. about 100% egg mortality at an accumulated dose of as little as 5 kJ/m2 for each exposure time. Conclusions A high rate of mite mortality and lethal egg damage were observed after less than 1 min of exposure to 5 mJ UV-C pulsed irradiation at 60 Hz. Pending further developments (such as beam steering, beam shaping and miniaturisation) and feasibility studies (such as testing with mites in real-life situations), the reported results and characteristics of the UV-C generator (modulation of energy output and adaptability to varying spot sizes) open up the use of this technology for a vast field of acaricidal applications that require long-range radiation. Graphical Abstract

ABSTRACT: Objectives: Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli are an increasingly significant cause of hospital- and community-acquired infections worldwide. Whereas several reports have highlighted their increased prevalence also in North African countries, genomic data on isolates associated with these infections are still scarce. This study aimed to provide data on ESBL-producing E. coli isolates from patients with extraintestinal infections at the Military Teaching Hospital Mohamed V of Rabat, Morocco. Methods: Whole-genome sequencing was carried out on 18 ESBL-producing extraintestinal pathogenic E. coli (ExPEC) isolates for analysis of phylogenomic evolution, virulence factors and antimicrobial resistance genes. Data were compared with ExPEC lineages from several surrounding countries using multilocus sequence typing (MLST) and single nucleotide polymorphism-based phylogenetic approaches. Results: The majority of E. coli isolates were ST131 (n = 15), followed by ST617 (n = 2) and a novel sequence type (ST10703) that is closely related to the pandemic ST405 clone. All ST131 isolates belonged to the O25b-ST131 pandemic clone. They harboured more virulence genes than their non-ST131 counterparts. IncF plasmid replicons and the blaCTX-M-15 β-lactamase gene were identified in all isolates. No ESBL-producing E. coli isolates carried any known carbapenemase gene. Conclusion: Our findings underscore the pre-eminence of ST131 as the major factor driving the expansion of ExPEC in the Rabat region while highlighting the potential links with isolates circulating in other neighbouring countries.

Abstract Background Toxocara canis and Toxocara cati are globally distributed roundworms and causative agents of human toxocariasis, via ingestion of Toxocara eggs. Control of Toxocara infections is constrained by a lack of sensitive methods for screening of animal faeces and environmental samples potentially contaminated by Toxocara eggs. In this work, a pre-analytical method for efficient extraction of DNA from Toxocara eggs in environmental samples was set up using our previously validated T. canis- and T. cati-specific quantitative real-time polymerase chain reaction (qPCR). For this purpose, the influence of different methods for egg lysis, DNA extraction and purification for removal of PCR inhibitors were assessed on environmental samples. Methods To select the best egg disruption method, six protocols were compared on pure T. canis egg suspensions, including enzymatic lysis and thermal or mechanical disruption. Based on the selected best method, an analytical workflow was set up to compare two DNA extraction methods (FastDNA™ SPIN Kit for Soil versus DNeasy® PowerMax® Soil Kit) with an optional dilution and/or clean-up (Agencourt® AMPure®) step. This workflow was evaluated on 10-g soil and 10-g sand samples spiked with egg suspensions of T. canis (tenfold dilutions of 104 eggs in triplicate). The capacity of the different methods, used alone or in combination, to increase the ratio of positive tests was assessed. The resulting optimal workflow for processing spiked soil samples was then tested on environmental soil samples and compared with the conventional flotation-centrifugation and microscopic examination of Toxocara eggs. Results The most effective DNA extraction method for Toxocara eggs in soil samples consisted in the combination of mechanical lysis of eggs using beads, followed by DNA extraction with the DNeasy® PowerMax® Soil Kit, and completed with an additional DNA clean-up step with AMPure® beads and a sample DNA dilution (1:10). This workflow exhibited a limit of detection of 4 and 46 T. canis eggs in 10-g sand and 10-g soil samples, respectively. Conclusions The pre-analytical flow process developed here combined with qPCR represents an improved, potentially automatable, and cost-effective method for the surveillance of Toxocara contamination in the environment. Graphical Abstract

Abstract The coronavirus pandemic, which appeared in Wuhan, China, in December 2019, rapidly spread all over the world in only a few weeks. Faster testing techniques requiring less resources are key in managing the pandemic, either to enable larger scale testing or even just provide developing countries with limited resources, particularly in Africa, means to perform tests to manage the crisis. Here, we report an unprecedented, rapid, reagent-free and easy-to-use screening spectroscopic method for the detection of SARS-CoV-2 on RNA extracts. This method, validated on clinical samples collected from 280 patients with quantitative predictive scores on both positive and negative samples, is based on a multivariate analysis of FTIR spectra of RNA extracts. This technique, in agreement with RT-PCR, achieves 97.8% accuracy, 97% sensitivity and 98.3% specificity while reducing the testing time post RNA extraction from hours to minutes. Furthermore, this technique can be used in several laboratories with limited resources.

Multidrug-resistant (MDR) and extended spectrum β-lactamase (ESBL)-producing extra-intestinal K. pneumoniae are associated with increased morbidity and mortality. This study aimed to characterize the resistance and virulence profiles of extra-intestinal MDR ESBL-producing K. pneumoniae associated with infections at a tertiary hospital in South-Kivu province, DRC. Whole-genome sequencing (WGS) was carried out on 37 K. pneumoniae isolates displaying MDR and ESBL-producing phenotype. The assembled genomes were analysed for phylogeny, virulence factors and antimicrobial resistance genes (ARG) determinants. These isolates were compared to sub-Saharan counterparts. K. pneumoniae isolates displayed a high genetic variability with up to 16 sequence types (ST). AMR was widespread against β-lactamases (including third and fourth-generation cephalosporins, but not carbapenems), aminoglycosides, ciprofloxacin, tetracycline, erythromycin, nitrofurantoin, and cotrimoxazole. The blaCTX-M-15 gene was the most common β-lactamase gene among K. pneumoniae isolates. No carbapenemase gene was found. ARG for aminoglycosides, quinolones, phenicols, tetracyclines, sulfonamides, nitrofurantoin were widely distributed among the isolates. Nine isolates had the colistin-resistant R256G substitution in the pmrB efflux pump gene without displaying reduced susceptibility to colistin. Despite carrying virulence genes, none had hypervirulence genes. Our results highlight the genetic diversity of MDR ESBL-producing K. pneumoniae isolates and underscore the importance of monitoring simultaneously the evolution of phenotypic and genotypic AMR in Bukavu and DRC, while calling for caution in administering colistin and carbapenem to patients.

Dependence on multiple nutritional bacterial symbionts forming a metabolic unit has repeatedly evolved in many insect species that feed on nutritionally unbalanced diets such as plant sap. This is the case for aphids of the subfamilies Lachninae and Chaitophorinae, which have evolved di-symbiotic systems in which the ancient obligate nutritional symbiont Buchnera aphidicola is metabolically complemented by an additional nutritional symbiont acquired more recently. Deciphering how different symbionts integrate both metabolically and anatomically in such systems is crucial to understanding how complex nutritional symbiotic systems function and evolve. In this study, we sequenced and analyzed the genomes of the symbionts B. aphidicola and Serratia symbiotica associated with the Chaitophorinae aphids Sipha maydis and Periphyllus lyropictus. Our results show that, in these two species, B. aphidicola and S. symbiotica complement each other metabolically (and their hosts) for the biosynthesis of essential amino acids and vitamins, but with distinct metabolic reactions supported by each symbiont depending on the host species. Furthermore, the S. symbiotica symbiont associated with S. maydis appears to be strictly compartmentalized into the specialized host cells housing symbionts in aphids, the bacteriocytes, whereas the S. symbiotica symbiont associated with P. lyropictus exhibits a highly invasive phenotype, presumably because it is capable of expressing a larger set of virulence factors, including a complete flagellum for bacterial motility. Such contrasting levels of metabolic and anatomical integration for two S. symbiotica symbionts that were recently acquired as nutritional co-obligate partners reflect distinct coevolutionary processes specific to each association.

Macrophages are not only derived from circulating blood monocytes or embryonic precursors but also expand by proliferation. The origin determines macrophage fate and functions in steady state and pathological conditions. Macrophages predominantly infiltrate fibre-induced mesothelioma tumors and contribute to cancer development. Here, we revealed their ontogeny by comparing the response to needle-like mesotheliomagenic carbon nanotubes (CNT-7) with tangled-like non-mesotheliomagenic CNT-T. In a rat peritoneal cavity model of mesothelioma, both CNT induced a rapid macrophage disappearance reaction (MDR) of MHCIIlow resident macrophages generating an empty niche available for macrophage repopulation. Macrophage depletion after mesotheliomagenic CNT-7 was followed by a substantial inflammatory reaction, and macrophage replenishment completed after 7 days. Thirty days after non-mesotheliomagenic CNT-T, macrophage repopulation was still incomplete and accompanied by a limited inflammatory reaction. Cell depletion experiments, flow cytometry and RNA-seq analysis demonstrated that, after mesotheliomagenic CNT-7 exposure, resident macrophages were mainly replaced by an influx of monocytes, which differentiated locally into MHCIIhigh inflammatory macrophages. In contrast, the low inflammatory response induced by CNT-T was associated by the accumulation of self-renewing MHCIIlow macrophages that initially derive from monocytes. In conclusion, the mesotheliomagenic response to CNT specifically relies on macrophage niche recolonization by monocyte-derived inflammatory macrophages. In contrast, the apparent homeostasis after non-mesotheliomagenic CNT treatment involves a macrophage regeneration by proliferation. Macrophage depletion and repopulation are thus decisive events characterizing the carcinogenic activity of particles and fibres.

Mutualistic associations between insects and heritable bacterial symbionts are ubiquitous in nature. The aphid symbiont Serratia symbiotica is a valuable candidate for studying the evolution of bacterial symbiosis in insects because it includes a wide diversity of strains that reflect the diverse relationships in which bacteria can be engaged with insects, from pathogenic interactions to obligate intracellular mutualism. The recent discovery of culturable strains, which are hypothesized to resemble the ancestors of intracellular strains, provide an opportunity to study the mechanisms underlying bacterial symbiosis in its early stages. In this study, we analyzed the genomes of three of these culturable strains that are pathogenic to aphid hosts, and performed comparative genomic analyses including mutualistic host-dependent strains. All three genomes are larger than those of the host-restricted S. symbiotica strains described so far, and show significant enrichment in pseudogenes and mobile elements, suggesting that these three pathogenic strains are in the early stages of the adaptation to their host. Compared to their intracellular mutualistic relatives, the three strains harbor a greater diversity of genes coding for virulence factors and metabolic pathways, suggesting that they are likely adapted to infect new hosts and are a potential source of metabolic innovation for insects. The presence in their genomes of secondary metabolism gene clusters associated with the production of antimicrobial compounds and phytotoxins supports the hypothesis that S. symbiotia symbionts evolved from plant-associated strains and that plants may serve as intermediate hosts. Mutualistic associations between insects and bacteria are the result of independent transitions to endosymbiosis initiated by the acquisition of environmental progenitors. In this context, the genomes of free-living S. symbiotica strains provide a rare opportunity to study the inventory of genes held by bacterial associates of insects that are at the gateway to a host-dependent lifestyle.

Abstract Background Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli are responsible for severe infections worldwide. Whereas their genotypic and pathogenic characteristics are not documented in Democratic Republic of Congo (DRC), recent studies conducted at the Bukavu General Hospital in the South Kivu province highlighted their high prevalence in extra-intestinal infections. Here we provide data on molecular characterization of ESBL producing-Escherichia coli isolates from patients with extra-intestinal infections at this provincial hospital. Methods Whole-genome sequencing was carried out on 21 of these ESBL-producing Extra-intestinal Pathogenic Escherichia coli (ExPEC) for analysis of phylogenomic evolution, virulence factor and antimicrobial resistance (AMR) genes. Data were compared to phylogenetically close genomes using Multi-Locus Sequence Typing and Single Nucleotide Polymorphism-based phylogenetic approaches. Results The distribution of E. coli sequence types (ST) was as follows: ST 131 (n = 7), ST405 (n = 4), ST410 (n = 2), and other STs (ST10, ST58, ST95, ST393, ST443, S617, ST648, and ST2450). All ST131 belonged to the O25b-ST131 pandemic clone. Unexpectedly, they harbored more virulence genes than their GenBank counterparts. IncF plasmid replicons included novel FIB 69, FII 105 and FII 107 alleles. ESBL-genes included the plasmid-mediated CTX-M-15 in all isolates, and the SHV-12 allele. Other AMR genes included blaOXA-1, blaTEM-1, as well as genes encoding resistance against aminoglycosides, quinolones, chloramphenicol, rifampicin, tetracyclines, sulfonamides and trimethoprim. Conclusion Current data confirm the clonal spread of ESBL-producing ST131 and ST405 clones in patients from South Kivu, and the acquisition of resistance and virulence genes. A closer survey of AMR and virulence should therefore be prompted in this high-risk area.

Many insect species are associated with bacterial partners that can significantly influence their evolutionary ecology. Compared to other insect groups, aphids harbor a bacterial microbiota that has the reputation of being poorly diversified, generally limited to the presence of the obligate nutritional symbiont Buchnera aphidicola and some facultative symbionts. In this study, we analyzed the bacterial diversity associated with the dogwood-grass aphid Anoecia corni, an aphid species that spends much of its life cycle in a subterranean environment. Little is known about the bacterial diversity associated with aphids displaying such a lifestyle, and one hypothesis is that close contact with the vast microbial community of the rhizosphere could promote the acquisition of a richer bacterial diversity compared to other aphid species. Using 16S rRNA amplicon Illumina sequencing on specimens collected on wheat roots in Morocco, we identified 10 bacterial operational taxonomic units (OTUs) corresponding to five bacterial genera. In addition to the obligate symbiont Buchnera, we identified the facultative symbionts Serratia symbiotica and Wolbachia in certain aphid colonies. The detection of Wolbachia is unexpected as it is considered rare in aphids. Moreover, its biological significance remains unknown in these insects. Besides, we also detected Arsenophonus and Dactylopiibacterium carminicum. These results suggest that, despite its subterranean lifestyle, A. corni shelter a bacterial diversity mainly limited to bacterial endosymbionts.

Background: Chronic obstructive pulmonary disease (COPD) is a devastating lung disease, mainly due to cigarette smoking, which represents the third cause of mortality worldwide. The mechanisms driving its epithelial salient features remain largely elusive. We aimed to evaluate the activation and the role of the canonical, β-catenin-dependant WNT pathway in the airway epithelium from COPD patients. Methods: The WNT/β-catenin pathway was first assessed by WNT-targeted RNA sequencing of the air/liquid interface-reconstituted bronchial epithelium from COPD and control patients. Airway expression of total and active β-catenin was assessed in lung sections, as well as WNT components in laser-microdissected airway epithelium. Finally, we evaluated the role of WNT at the bronchial epithelial level by modulating the pathway in the reconstituted COPD epithelium. Findings: We show that the WNT/β-catenin pathway is upregulated in the COPD airway epithelium as compared with that of non-smokers and control smokers, in targeted RNA-sequencing of in vitro reconstituted airway epithelium, and in situ in lung tissue and laser-microdissected epithelium. Extrinsic activation of this pathway in COPD-derived airway epithelium inhibited epithelial differentiation, polarity and barrier function, and induced TGF-β-related epithelial-to-mesenchymal transition (EMT). Conversely, canonical WNT inhibition increased ciliated cell numbers, epithelial polarity and barrier function, whilst inhibiting EMT, thus reversing COPD features. Interpretation: In conclusion, the aberrant reactivation of the canonical WNT pathway in the adult airway epithelium recapitulates the diseased phenotype observed in COPD patients, suggesting that this pathway or its downstream effectors could represent a future therapeutic target. Funding: This study was supported by the Fondation Mont-Godinne, the FNRS and the WELBIO.

BackgroundOver the past recent years, Vibrio cholerae has been associated with outbreaks in sub-Saharan Africa, notably in Democratic Republic of the Congo (DRC). This study aimed to determine the genetic relatedness of isolates responsible for cholera outbreaks in eastern DRC between 2014 and 2017, and their potential spread to bordering countries.Methods/principal findingsPhenotypic analysis and whole genome sequencing (WGS) were carried out on 78 clinical isolates of V. cholerae associated with cholera in eastern provinces of DRC between 2014 and 2017. SNP-based phylogenomic data show that most isolates (73/78) were V. cholerae O1 biotype El Tor with CTX-3 type prophage. They fell within the third transmission wave of the current seventh pandemic El Tor (7PET) lineage and were contained in the introduction event (T)10 in East Africa. These isolates clustered in two sub-clades corresponding to Multiple Locus Sequence Types (MLST) profiles ST69 and the newly assigned ST515, the latter displaying a higher genetic diversity. Both sub-clades showed a distinct geographic clustering, with ST69 isolates mostly restricted to Lake Tanganyika basin and phylogenetically related to V. cholerae isolates associated with cholera outbreaks in western Tanzania, whereas ST515 isolates were disseminated along the Albertine Rift and closely related to isolates in South Sudan, Uganda, Tanzania and Zambia. Other V. cholerae isolates (5/78) were non-O1/non-O139 without any CTX prophage and no phylogenetic relationship with already characterized non-O1/non-O139 isolates.Conclusions/significanceCurrent data confirm the association of both DRC O1 7PET (T)10 sub-clades ST69 and ST515 with recurrent outbreaks in eastern DRC and at regional level over the past 10 years. Interestingly, while ST69 is predominantly a locally endemic sequence type, ST515 became adaptable enough to expand across DRC neighboring countries.

BACKGROUND:Multiple-Locus Variable Number of Tandem Repeats (VNTR) Analysis (MLVA) is widely used by laboratory-based surveillance networks for subtyping pathogens causing foodborne and water-borne disease outbreaks. However, Whole Genome Sequencing (WGS) has recently emerged as the new more powerful reference for pathogen subtyping, making a data conversion method necessary which enables the users to compare the MLVA identified by either method. The MLVAType shiny application was designed to extract MLVA profiles of Vibrio cholerae isolates from WGS data while ensuring backward compatibility with traditional MLVA typing methods. METHODS:To test and validate the MLVAType algorithm, WGS-derived MLVA profiles of nineteen Vibrio cholerae isolates from Democratic Republic of the Congo (n = 9) and Uganda (n = 10) were compared to MLVA profiles generated by an in silico PCR approach and Sanger sequencing, the latter being used as the reference method. RESULTS:Results obtained by Sanger sequencing and MLVAType were totally concordant. However, the latter were affected by censored estimations whose percentage was inversely proportional to the k-mer parameter used during genome assembly. With a k-mer of 127, less than 15% estimation of V. cholerae VNTR was censored. Preventing censored estimation was only achievable when using a longer k-mer size (i.e. 175), which is not proposed in the SPAdes v.3.13.0 software. CONCLUSION:As NGS read lengths and qualities tend to increase with time, one may expect the increase of k-mer size in a near future. Using MLVAType application with a longer k-mer size will then efficiently retrieve MLVA profiles from WGS data while avoiding censored estimation.

Here, we present first draft genome sequence of the trypanosomatid Herpetomonas muscarum ingenoplastis. This parasite was isolated repeatedly in the black blowfly, Phormia regina, and it forms a phylogenetically distinct clade in the Trypanosomatidae family.

BACKGROUND:In 2014-2015, we assessed favipiravir tolerance and efficacy in patients with Ebola virus (EBOV) disease (EVD) in Guinea (JIKI trial). Because the drug had never been used before for this indication and that high concentrations of the drugs were needed to achieve antiviral efficacy against EBOV, a pharmacokinetic model had been used to propose relevant dosing regimen. Here we report the favipiravir plasma concentrations that were achieved in participants in the JIKI trial and put them in perspective with the model-based targeted concentrations. METHODS AND FINDINGS:Pre-dose drug concentrations were collected at Day-2 and Day-4 of treatment in 66 patients of the JIKI trial and compared to those predicted by the model taking into account patient's individual characteristics. At Day-2, the observed concentrations were slightly lower than the model predictions adjusted for patient's characteristics (median value of 46.1 versus 54.3 μg/mL for observed and predicted concentrations, respectively, p = 0.012). However, the concentrations dropped at Day-4, which was not anticipated by the model (median values of 25.9 and 64.4 μg/mL for observed and predicted concentrations, respectively, p

Health care workers (HCWs) in contact with patients with Ebola virus disease (EVD) are exposed to a risk of viral contamination. Fomites contaminated with the patient's blood or body fluids represents this risk. Our study aims to detect Ebola virus (EBOV) RNA within the high- and low-risk areas of an Ebola treatment unit (ETU) located in inland Guinea during the 2014-2015 West African Ebola epidemics. For samples from patients' immediate vicinity, we aim to seek an association between viral RNA detectability and level of plasma viral load of patients (intermediate to high, or very high).Swabbing was performed on immediate vicinity of Ebola patients, on surfaces of an ETU, and on personal protective equipment (PPE) of HCWs after patient care and prior to doffing. All samples were assessed by quantitative reverse-transcribed PCR (RT-qPCR).32% (22/68) of swabs from high-risk areas were tested positive for EBOV RNA, including 42% (18/43) from patients' immediate vicinity, and 16% (4/25) from HCWs PPE. None of specimens from low-risk areas were tested positive (0/19). Swabs were much more often viral RNA positive in the vicinity of patients with a very high plasma viral load (OR 6.7, 95% CI [1.7-23.4]).Our findings show the persistence of EBOV RNA in the environment of Ebola patients and of HCWs, in a Guinean ETU, despite strict infection prevention and control measures. This detection raises the possibility that patients' environment could be a potential source of contamination with the virus.

[This corrects the article DOI: 10.1371/journal.pmed.1001967.].

BackgroundEbola virus disease (EVD) is a highly lethal condition for which no specific treatment has proven efficacy. In September 2014, while the Ebola outbreak was at its peak, the World Health Organization released a short list of drugs suitable for EVD research. Favipiravir, an antiviral developed for the treatment of severe influenza, was one of these. In late 2014, the conditions for starting a randomized Ebola trial were not fulfilled for two reasons. One was the perception that, given the high number of patients presenting simultaneously and the very high mortality rate of the disease, it was ethically unacceptable to allocate patients from within the same family or village to receive or not receive an experimental drug, using a randomization process impossible to understand by very sick patients. The other was that, in the context of rumors and distrust of Ebola treatment centers, using a randomized design at the outset might lead even more patients to refuse to seek care. Therefore, we chose to conduct a multicenter non-randomized trial, in which all patients would receive favipiravir along with standardized care. The objectives of the trial were to test the feasibility and acceptability of an emergency trial in the context of a large Ebola outbreak, and to collect data on the safety and effectiveness of favipiravir in reducing mortality and viral load in patients with EVD. The trial was not aimed at directly informing future guidelines on Ebola treatment but at quickly gathering standardized preliminary data to optimize the design of future studies.Methods and findingsInclusion criteria were positive Ebola virus reverse transcription PCR (RT-PCR) test, age ≥ 1 y, weight ≥ 10 kg, ability to take oral drugs, and informed consent. All participants received oral favipiravir (day 0: 6,000 mg; day 1 to day 9: 2,400 mg/d). Semi-quantitative Ebola virus RT-PCR (results expressed in "cycle threshold" [Ct]) and biochemistry tests were performed at day 0, day 2, day 4, end of symptoms, day 14, and day 30. Frozen samples were shipped to a reference biosafety level 4 laboratory for RNA viral load measurement using a quantitative reference technique (genome copies/milliliter). Outcomes were mortality, viral load evolution, and adverse events. The analysis was stratified by age and Ct value. A "target value" of mortality was defined a priori for each stratum, to guide the interpretation of interim and final analysis. Between 17 December 2014 and 8 April 2015, 126 patients were included, of whom 111 were analyzed (adults and adolescents, ≥13 y, n = 99; young children, ≤6 y, n = 12). Here we present the results obtained in the 99 adults and adolescents. Of these, 55 had a baseline Ct value ≥ 20 (Group A Ct ≥ 20), and 44 had a baseline Ct value < 20 (Group A Ct < 20). Ct values and RNA viral loads were well correlated, with Ct = 20 corresponding to RNA viral load = 7.7 log10 genome copies/ml. Mortality was 20% (95% CI 11.6%-32.4%) in Group A Ct ≥ 20 and 91% (95% CI 78.8%-91.1%) in Group A Ct < 20. Both mortality 95% CIs included the predefined target value (30% and 85%, respectively). Baseline serum creatinine was ≥110 μmol/l in 48% of patients in Group A Ct ≥ 20 (≥300 μmol/l in 14%) and in 90% of patients in Group A Ct < 20 (≥300 μmol/l in 44%). In Group A Ct ≥ 20, 17% of patients with baseline creatinine ≥110 μmol/l died, versus 97% in Group A Ct < 20. In patients who survived, the mean decrease in viral load was 0.33 log10 copies/ml per day of follow-up. RNA viral load values and mortality were not significantly different between adults starting favipiravir within ConclusionsIn the context of an outbreak at its peak, with crowded care centers, randomizing patients to receive either standard care or standard care plus an experimental drug was not felt to be appropriate. We did a non-randomized trial. This trial reaches nuanced conclusions. On the one hand, we do not conclude on the efficacy of the drug, and our conclusions on tolerance, although encouraging, are not as firm as they could have been if we had used randomization. On the other hand, we learned about how to quickly set up and run an Ebola trial, in close relationship with the community and non-governmental organizations; we integrated research into care so that it improved care; and we generated knowledge on EVD that is useful to further research. Our data illustrate the frequency of renal dysfunction and the powerful prognostic value of low Ct values. They suggest that drug trials in EVD should systematically stratify analyses by baseline Ct value, as a surrogate of viral load. They also suggest that favipiravir monotherapy merits further study in patients with medium to high viremia, but not in those with very high viremia.Trial registrationClinicalTrials.gov NCT02329054.

Early diagnosis of rheumatoid arthritis (RA) is an unmet medical need in the field of rheumatology. Previously, we performed high-density transcriptomic studies on synovial biopsies from patients with arthritis, and found that synovial gene expression profiles were significantly different according to the underlying disorder. Here, we wanted to further explore the consistency of the gene expression signals in synovial biopsies of patients with arthritis, using low-density platforms.Low-density assays (cDNA microarray and microfluidics qPCR) were designed, based on the results of the high-density microarray data. Knee synovial biopsies were obtained from patients with RA, spondyloarthropathies (SA) or osteoarthritis (OA) (n = 39), and also from patients with initial undifferentiated arthritis (UA) (n = 49).According to high-density microarray data, several molecular pathways are differentially expressed in patients with RA, SA and OA: T and B cell activation, chromatin remodelling, RAS GTPase activation and extracellular matrix regulation. Strikingly, disease activity (DAS28-CRP) has a significant influence on gene expression patterns in RA samples. Using the low-density assays, samples from patients with OA are easily discriminated from RA and SA samples. However, overlapping molecular patterns are found, in particular between RA and SA biopsies. Therefore, prediction of the clinical diagnosis based on gene expression data results in a diagnostic accuracy of 56.8%, which is increased up to 98.6% by the addition of specific clinical symptoms in the prediction algorithm. Similar observations are made in initial UA samples, in which overlapping molecular patterns also impact the accuracy of the diagnostic algorithm. When clinical symptoms are added, the diagnostic accuracy is strongly improved.Gene expression signatures are overall different in patients with OA, RA and SA, but overlapping molecular signatures are found in patients with these conditions. Therefore, an accurate diagnosis in patients with UA requires a combination of gene expression and clinical data.

Separating CBRN mixed samples that contain both chemical and biological warfare agents (CB mixed sample) in liquid and solid matrices remains a very challenging issue. Parameters were set up to assess the performance of a simple filtration-based method first optimized on separate C- and B-agents, and then assessed on a model of CB mixed sample. In this model, MS2 bacteriophage, Autographa californica nuclear polyhedrosis baculovirus (AcNPV), Bacillus atrophaeus and Bacillus subtilis spores were used as biological agent simulants whereas ethyl methylphosphonic acid (EMPA) and pinacolyl methylphophonic acid (PMPA) were used as VX and soman (GD) nerve agent surrogates, respectively. Nanoseparation centrifugal devices with various pore size cut-off (30 kD up to 0.45 µm) and three RNA extraction methods (Invisorb, EZ1 and Nuclisens) were compared. RNA (MS2) and DNA (AcNPV) quantification was carried out by means of specific and sensitive quantitative real-time PCRs (qPCR). Liquid chromatography coupled to time-of-flight mass spectrometry (LC/TOFMS) methods was used for quantifying EMPA and PMPA. Culture methods and qPCR demonstrated that membranes with a 30 kD cut-off retain more than 99.99% of biological agents (MS2, AcNPV, Bacillus Atrophaeus and Bacillus subtilis spores) tested separately. A rapid and reliable separation of CB mixed sample models (MS2/PEG-400 and MS2/EMPA/PMPA) contained in simple liquid or complex matrices such as sand and soil was also successfully achieved on a 30 kD filter with more than 99.99% retention of MS2 on the filter membrane, and up to 99% of PEG-400, EMPA and PMPA recovery in the filtrate. The whole separation process turnaround-time (TAT) was less than 10 minutes. The filtration method appears to be rapid, versatile and extremely efficient. The separation method developed in this work constitutes therefore a useful model for further evaluating and comparing additional separation alternative procedures for a safe handling and preparation of CB mixed samples.

BACKGROUND: In case of outbreak of rash illness in remote areas, clinically discriminating monkeypox (MPX) from severe form of chickenpox and from smallpox remains a concern for first responders. OBJECTIVE: The goal of the study was therefore to use MPX and chickenpox outbreaks in Democratic Republic of Congo (DRC) as a test case for establishing a rapid and specific diagnosis in affected remote areas. METHODS: In 2008 and 2009, successive outbreaks of presumed MPX skin rash were reported in Bena Tshiadi, Yangala and Ndesha healthcare districts of the West Kasai province (DRC). Specimens consisting of liquid vesicle dried on filter papers or crusted scabs from healing patients were sampled by first responders. A field analytical facility was deployed nearby in order to carry out a real-time PCR (qPCR) assay using genus consensus primers, consensus orthopoxvirus (OPV) and smallpox-specific probes spanning over the 14 kD fusion protein encoding gene. A PCR-restriction fragment length polymorphism was used on-site as backup method to confirm the presence of monkeypox virus (MPXV) in samples. To complete the differential diagnosis of skin rash, chickenpox was tested in parallel using a commercial qPCR assay. In a post-deployment step, a MPXV-specific pyrosequencing was carried out on all biotinylated amplicons generated on-site in order to confirm the on-site results. RESULTS: Whereas MPXV proved to be the agent causing the rash illness outbreak in the Bena Tshiadi, VZV was the causative agent of the disease in Yangala and Ndesha districts. In addition, each on-site result was later confirmed by MPXV-specific pyrosequencing analysis without any discrepancy. CONCLUSION: This experience of rapid on-site dual use DNA-based differential diagnosis of rash illnesses demonstrates the potential of combining tests specifically identifying bioterrorism agents and agents causing natural outbreaks. This opens the way to rapid on-site DNA-based identification of a broad spectrum of causative agents in remote areas.

In the Balkan and Taiwan, the relationship between exposure to aristolochic acid and risk of urothelial neoplasms was inferred from the A>T genetic hallmark in TP53 gene from malignant cells. This study aimed to characterize the TP53 mutational spectrum in urothelial cancers consecutive to Aristolochic Acid Nephropathy in Belgium. Serial frozen tumor sections from female patients (n=5) exposed to aristolochic acid during weight-loss regimen were alternatively used either for p53 immunostaining or laser microdissection. Tissue areas with at least 60% p53-positive nuclei were selected for microdissecting sections according to p53-positive matching areas. All areas appeared to be carcinoma in situ. After DNA extraction, mutations in the TP53 hot spot region (exons 5-8) were identified using nested-PCR and sequencing. False-negative controls consisted in microdissecting fresh-frozen tumor tissues both from a patient with a Li-Fraumeni syndrome who carried a p53 constitutional mutation, and from KRas mutated adenocarcinomas. To rule out false-positive results potentially generated by microdissection and nested-PCR, a phenacetin-associated urothelial carcinoma and normal fresh ureteral tissues (n=4) were processed with high laser power. No unexpected results being identified, molecular analysis was pursued on malignant tissues, showing at least one mutation in all (six different mutations in two) patients, with 13/16 exonic (nonsense, 2; missense, 11) and 3/16 intronic (one splice site) mutations. They were distributed as transitions (n=7) or transversions (n=9), with an equal prevalence of A>T and G>T (3/16 each). While current results are in line with A>T prevalence previously reported in Balkan and Taiwan studies, they also demonstrate that multiple mutations in the TP53 hot spot region and a high frequency of G>T transversion appear as a complementary signature reflecting the toxicity of a cumulative dose of aristolochic acid ingested over a short period of time.

BackgroundAngiotensin-converting enzyme (ACE) (EC 4.15.1) metabolizes many biologically active peptides and plays a key role in blood pressure regulation and vascular remodeling. Elevated ACE levels are associated with different cardiovascular and respiratory diseases.Methods and resultsTwo Belgian families with a 8-16-fold increase in blood ACE level were incidentally identified. A novel heterozygous splice site mutation of intron 25 - IVS25+1G>A (c.3691+1G>A) - cosegregating with elevated plasma ACE was identified in both pedigrees. Messenger RNA analysis revealed that the mutation led to the retention of intron 25 and Premature Termination Codon generation. Subjects harboring the mutation were mostly normotensive, had no left ventricular hypertrophy or cardiovascular disease. The levels of renin-angiotensin-aldosterone system components in the mutated cases and wild-type controls were similar, both at baseline and after 50 mg captopril. Compared with non-affected members, quantification of ACE surface expression and shedding using flow cytometry assay of dendritic cells derived from peripheral blood monocytes of affected members, demonstrated a 50% decrease and 3-fold increase, respectively. Together with a dramatic increase in circulating ACE levels, these findings argue in favor of deletion of transmembrane anchor, leading to direct secretion of ACE out of cells.ConclusionsWe describe a novel mutation of the ACE gene associated with a major familial elevation of circulating ACE, without evidence of activation of the renin-angiotensin system, target organ damage or cardiovascular complications. These data are consistent with the hypothesis that membrane-bound ACE, rather than circulating ACE, is responsible for Angiotensin II generation and its cardiovascular consequences.

Umbilical cord matrix stem cells (UCMSC) have generated great interest in various therapeutic approaches, including liver regeneration. This article aims to analyze the specific characteristics and the potential occurrence of premalignant alterations of UCMSC during long-term expansion, which are important issues for clinical applications. UCMSC were isolated from the umbilical cord of 14 full-term newborns and expanded in vitro until senescence. We examined the long-term growth potential, senescence characteristics, immunophenotype and multilineage differentiation capacity of these cells. In addition, their genetic stability was assessed through karyotyping, telomerase maintenance mechanisms and analysis of expression and functionality of cell cycle regulation genes. The tumorigenic potential was also studied in immunocompromised mice. In vitro, UCMSC reached up to 33.7 ± 2.1 cumulative population doublings before entering replicative senescence. Their immunophenotype and differentiation potential, notably into hepatocyte-like cells, remained stable over time. Cytogenetic analyses did not reveal any chromosomal abnormality and the expression of oncogenes was not induced. Telomere maintenance mechanisms were not activated. Just as UCMSC lacked transformed features in vitro, they could not give rise to tumors in vivo. UCMSC could be expanded in long-term cultures while maintaining stable genetic features and endodermal differentiation potential. UCMSC therefore represent safe candidates for liver regenerative medicine.