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INTRODUCTION: Here we tested the correlation between minimum inhibitory concentrations (MICs) of major antifungal agents and sequence types (STs) within Cryptococcus neoformans VNI isolates, and explored the ERG11 gene of included strains. MATERIALS AND METHODS: We analysed 23 C. neoformans strains categorised into two groups according to the distribution of the ST profile in Kinshasa clinics (Democratic Republic of Congo): major ST [ST93 (n = 15)], and less common STs [ST659 (n = 2), ST5 (n = 2), ST4 (n = 1), ST 53 (n = 1), ST31 (n = 1), and ST69 (n = 1)]. The MICs of the major antifungal agents [amphotericin B (AMB), 5-fluorocytosine (5FC) and fluconazole (FCZ)] were determined following EUCAST guidelines. ERG11 gene sequences were extracted from whole genome sequence of the isolates and compared with the wild-type gene sequence of the C. neoformans VNI. RESULTS: Although major ST isolates appeared to have lower median MICs for AMB and 5FU than less common ST isolates (0.50 vs. 0.75 mg/L for AMB, 2 vs. 4 mg/L for 5FU, respectively), FCZ susceptibility was similar in both groups (4 mg/L) (p-value >0.05). The susceptibility profile of C. neoformans strains separately considered did not significantly affect the patients' clinical outcomes (p-value >0.05). Furthermore, two structural modalities of the ERG11 gene were observed: (1) that of the reference gene, and (2) that containing two exonic silent point substitutions, and one intronic point substitution located in a sequence potentially involved in pre-mRNA splicing (c.337-22C > T); with no association with the MICs of the isolates (p-value >0.05). CONCLUSIONS: The lack of association/correlation found in this study calls for further investigations to better understand the mechanisms of C. neoformans resistance to antifungal agents.

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Mathematical modelling studies have shown that repetitive screening can be used to mitigate SARS-CoV-2 transmission in primary schools while keeping schools open. However, not much is known about how transmission progresses within schools and whether there is a risk of importation to households. During the academic year 2020-2021, a prospective surveillance study using repetitive screening was conducted in a primary school and associated households in Liège (Belgium). SARS-CoV-2 screening was performed via throat washing either once or twice a week. We used genomic and epidemiological data to reconstruct the observed school outbreaks using two different models. The outbreaker2 model combines information on the generation time and contact patterns with a model of sequence evolution. For comparison we also used SCOTTI, a phylogenetic model based on the structured coalescent. In addition, we performed a simulation study to investigate how the accuracy of estimated positivity rates in a school depends on the proportion of a school that is sampled in a repetitive screening strategy. We found no difference in SARS-CoV-2 positivity between children and adults and children were not more often asymptomatic compared to adults. Both models for outbreak reconstruction revealed that transmission occurred mainly within the school environment. Uncertainty in outbreak reconstruction was lowest when including genomic as well as epidemiological data. We found that observed weekly positivity rates are a good approximation to the true weekly positivity rate, especially in children, even when only 25% of the school population is sampled. These results indicate that, in addition to reducing infections as shown in modelling studies, repetitive screening in school settings can lead to a better understanding of the extent of transmission in schools during a pandemic and importation risk at the community level.

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Healthcare workers (HCWs) are known to be at higher risk of developing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections although whether these risks are equal across all occupational roles is uncertain. Identifying these risk factors and understand SARS-CoV-2 transmission pathways in healthcare settings are of high importance to achieve optimal protection measures. We aimed to investigate the implementation of a voluntary screening program for SARS-CoV-2 infections among hospital HCWs and to elucidate potential transmission pathways though phylogenetic analysis before the vaccination era. HCWs of the University Hospital of Liège, Belgium, were invited to participate in voluntary reverse transcriptase-polymerase chain reaction (RT-PCR) assays performed every week from April to December 2020. Phylogenetic analysis of SARS-CoV-2 genomes were performed for a subgroup of 45 HCWs. 5095 samples were collected from 703 HCWs. 212 test results were positive, 15 were indeterminate, and 4868 returned negative. 156 HCWs (22.2%) tested positive at least once during the study period. All SARS-CoV-2 test results returned negative for 547 HCWs (77.8%). Nurses (p < 0.05), paramedics (p < 0.05), and laboratory staff handling respiratory samples (p < 0.01) were at higher risk for being infected compared to the control non-patient facing group. Our phylogenetic analysis revealed that most positive samples corresponded to independent introduction events into the hospital. Our findings add to the growing evidence of differential risks of being infected among HCWs and support the need to implement appropriate protection measures based on each individual's risk profile to guarantee the protection of both HCWs and patients. Furthermore, our phylogenetic investigations highlight that most positive samples correspond to distinct introduction events into the hospital.

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Prevention of perinatal Group B Streptococcus (GBS) transmission is crucial in our effort to prevent Early-onset GBS disease. Here, we established the performance of the Revogene GBS DS assay for the detection of group B streptococcus on intrapartum vaginal samples in a laboratory environment using a prospective noninterventional study design. Intrapartum vaginal swabs were enriched using a selective culture method which served as study reference method. Overall, 119 patients were enrolled with an antenatal and intrapartum Group B Streptococcus colonization prevalence of 12.9% and 11.8%, respectively. Compared to intrapartum culture, the Revogene GBS DS assay had a sensitivity of 92.9% and a specificity of 99.1%, while the antenatal culture displayed a sensitivity 78.6% of and specificity of 96.2%. The Revogene GBS DS assay displayed an acceptable performance according to the European Group B Streptococcus consensus recommendations. Complementary studies in clinical practice are needed to confirm these findings.

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The testing and isolation of patients with coronavirus disease 2019 (COVID-19) are indispensable tools to control the ongoing COVID-19 pandemic. PCR tests are considered the "gold standard" of COVID-19 testing and mostly involve testing nasopharyngeal swab specimens. Our study aimed to compare the sensitivity of tests for various sample specimens. Seventy-five participants with confirmed COVID-19 were included in the study. Nasopharyngeal swabs, oropharyngeal swabs, Oracol-collected saliva, throat washes and rectal specimens were collected along with pooled swabs. Participants were asked to complete a questionnaire to correlate specific clinical symptoms and the symptom duration with the sensitivity of detecting COVID-19 in various sample specimens. Sampling was repeated after 7 to 10 days (T2), then after 14 to 20 days (T3) to perform a longitudinal analysis of sample specimen sensitivity. At the first time point, the highest percentages of SARS-CoV-2-positive samples were observed for nasopharyngeal samples (84.3%), while 74%, 68.2%, 58.8% and 3.5% of throat washing, Oracol-collected saliva, oropharyngeal and rectal samples tested positive, respectively. The sensitivity of all sampling methods except throat wash samples decreased rapidly at later time points compared to the first collection. The throat washing method exhibited better performance than the gold standard nasopharyngeal swab at the second and third time points after the first positive test date. Nasopharyngeal swabs were the most sensitive specimens for early detection after symptom onset. Throat washing is a sensitive alternative method. It was found that SARS-CoV-2 persists longer in the throat and saliva than in the nasopharynx.

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COVID-19 transmission rates are often linked to locally circulating strains of SARS-CoV-2. Here we describe 203 SARS-CoV-2 whole genome sequences analyzed from strains circulating in Rwanda from May 2020 to February 2021. In particular, we report a shift in variant distribution towards the emerging sub-lineage A.23.1 that is currently dominating. Furthermore, we report the detection of the first Rwandan cases of the B.1.1.7 and B.1.351 variants of concern among incoming travelers tested at Kigali International Airport. To assess the importance of viral introductions from neighboring countries and local transmission, we exploit available individual travel history metadata to inform spatio-temporal phylogeographic inference, enabling us to take into account infections from unsampled locations. We uncover an important role of neighboring countries in seeding introductions into Rwanda, including those from which no genomic sequences were available. Our results highlight the importance of systematic genomic surveillance and regional collaborations for a durable response towards combating COVID-19.

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At the end of 2020, several new variants of SARS-CoV-2-designated variants of concern-were detected and quickly suspected to be associated with a higher transmissibility and possible escape of vaccine-induced immunity. In Belgium, this discovery has motivated the initiation of a more ambitious genomic surveillance program, which is drastically increasing the number of SARS-CoV-2 genomes to analyse for monitoring the circulation of viral lineages and variants of concern. In order to efficiently analyse the massive collection of genomic data that are the result of such increased sequencing efforts, streamlined analytical strategies are crucial. In this study, we illustrate how to efficiently map the spatio-temporal dispersal of target mutations at a regional level. As a proof of concept, we focus on the Belgian province of Liège that has been consistently sampled throughout 2020, but was also one of the main epicenters of the second European epidemic wave. Specifically, we employ a recently developed phylogeographic workflow to infer the regional dispersal history of viral lineages associated with three specific mutations on the spike protein (S98F, A222V and S477N) and to quantify their relative importance through time. Our analytical pipeline enables analysing large data sets and has the potential to be quickly applied and updated to track target mutations in space and time throughout the course of an epidemic.

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BACKGROUND: The coronavirus infectious disease 19 (COVID-19) pandemic has resulted in significant morbidities, severe acute respiratory failures and subsequently emergency departments' (EDs) overcrowding in a context of insufficient laboratory testing capacities. The development of decision support tools for real-time clinical diagnosis of COVID-19 is of prime importance to assist patients' triage and allocate resources for patients at risk. METHODS AND PRINCIPAL FINDINGS: From March 2 to June 15, 2020, clinical patterns of COVID-19 suspected patients at admission to the EDs of Liège University Hospital, consisting in the recording of eleven symptoms (i.e. dyspnoea, chest pain, rhinorrhoea, sore throat, dry cough, wet cough, diarrhoea, headache, myalgia, fever and anosmia) plus age and gender, were investigated during the first COVID-19 pandemic wave. Indeed, 573 SARS-CoV-2 cases confirmed by qRT-PCR before mid-June 2020, and 1579 suspected cases that were subsequently determined to be qRT-PCR negative for the detection of SARS-CoV-2 were enrolled in this study. Using multivariate binary logistic regression, two most relevant symptoms of COVID-19 were identified in addition of the age of the patient, i.e. fever (odds ratio [OR] = 3.66; 95% CI: 2.97-4.50), dry cough (OR = 1.71; 95% CI: 1.39-2.12), and patients older than 56.5 y (OR = 2.07; 95% CI: 1.67-2.58). Two additional symptoms (chest pain and sore throat) appeared significantly less associated to the confirmed COVID-19 cases with the same OR = 0.73 (95% CI: 0.56-0.94). An overall pondered (by OR) score (OPS) was calculated using all significant predictors. A receiver operating characteristic (ROC) curve was generated and the area under the ROC curve was 0.71 (95% CI: 0.68-0.73) rendering the use of the OPS to discriminate COVID-19 confirmed and unconfirmed patients. The main predictors were confirmed using both sensitivity analysis and classification tree analysis. Interestingly, a significant negative correlation was observed between the OPS and the cycle threshold (Ct values) of the qRT-PCR. CONCLUSION AND MAIN SIGNIFICANCE: The proposed approach allows for the use of an interactive and adaptive clinical decision support tool. Using the clinical algorithm developed, a web-based user-interface was created to help nurses and clinicians from EDs with the triage of patients during the second COVID-19 wave.

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Since the start of the COVID-19 pandemic, an unprecedented number of genomic sequences of SARS-CoV-2 have been generated and shared with the scientific community. The unparalleled volume of available genetic data presents a unique opportunity to gain real-time insights into the virus transmission during the pandemic, but also a daunting computational hurdle if analyzed with gold-standard phylogeographic approaches. To tackle this practical limitation, we here describe and apply a rapid analytical pipeline to analyze the spatiotemporal dispersal history and dynamics of SARS-CoV-2 lineages. As a proof of concept, we focus on the Belgian epidemic, which has had one of the highest spatial densities of available SARS-CoV-2 genomes. Our pipeline has the potential to be quickly applied to other countries or regions, with key benefits in complementing epidemiological analyses in assessing the impact of intervention measures or their progressive easement.

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Artemisinin-based combination therapies (ACTs) have been recommended by the World Health Organization (WHO) as first-line treatment of uncomplicated Plasmodium falciparum (P. falciparum) malaria since 2005 in Democratic Republic of Congo (DRC) and a regular surveillance of the ACT efficacy is required to ensure the treatment effectiveness. Mutations in the propeller domain of the pfk13 gene were identified as molecular markers of artemisinin resistance (ART-R). This study investigated the pfk13-propeller gene polymorphism in clinical isolates of P. falciparum collected in the DRC. In 2017, ten geographical sites across DRC were selected for a cross-sectional study that was conducted first in Kinshasa from January to March, then in the nine other sites from September to December. Dried blood samples were collected from patients attending health centers for fever where diagnosis of Malaria was first made by rapid diagnostic test (RDT) available on site (SD Bioline malaria Ag Pf or CareStart Malaria Pf) or by thick blood smear and then confirmed by a P. falciparum real-time PCR assay. A pfk13-propeller segment containing a fragment that codes for amino acids at positions 427-595 was amplified by conventional PCR before sequencing. In total, 1070 patients were enrolled in the study. Real-time PCR performed confirmed the initial diagnosis of P. falciparum infection in 806 samples (75.3%; 95% CI: 72.6%- 77.9%). Of the 717 successfully sequenced P. falciparum isolates, 710 (99.0%; 95% CI: 97.9% - 99.6) were wild-type genotypes and 7 (1.0%; 95% CI: 0.4% - 2.1%) carried non-synonymous (NS) mutations in pfk13-propeller including 2 mutations (A578S and V534A) previously detected and 2 other (M472I and A569T) not yet detected in the DRC. Mutations associated with ART-R in Southeast Asia were not observed in DRC. However, the presence of other mutations in pfk13-propeller gene calls for further investigations to assess their implication in drug resistance.

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Control of the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic requires accurate laboratory testing to identify infected individuals while also clearing essential staff to continue to work. At the current time, a number of quantitative real-time PCR (qRT-PCR) assays have been developed to identify SARS-CoV-2, targeting multiple positions in the viral genome. While the mutation rate of SARS-CoV-2 is moderate, given the large number of transmission chains, it is prudent to monitor circulating viruses for variants that might compromise these assays. Here, we report the identification of a C-to-U transition at position 26340 of the SARS-CoV-2 genome that is associated with failure of the cobas SARS-CoV-2 E gene qRT-PCR in eight patients. As the cobas SARS-CoV-2 assay targets two positions in the genome, the individuals carrying this variant were still called SARS-CoV-2 positive. Whole-genome sequencing of SARS-CoV-2 showed all to carry closely related viruses. Examination of viral genomes deposited on GISAID showed this mutation has arisen independently at least four times. This work highlights the necessity of monitoring SARS-CoV-2 for the emergence of single-nucleotide polymorphisms that might adversely affect RT-PCRs used in diagnostics. Additionally, it argues that two regions in SARS-CoV-2 should be targeted to avoid false negatives.

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Real-time PCR are often used for the diagnosis and monitoring of Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) infections in susceptible populations. In this context, we evaluated the analytical performances of the Abbott RealTime CMV/EBV maxCycle protocol automated on the m2000 platform (Abbott). It was compared to our routinely-used procedure consisting of a NucleoMag® DNA extraction automated on a STARlet platform followed by manually processed CMV and EBV quantitative real-time PCR (Diagenode). In this study, we showed that both EBV assays exhibited a similar sensitivity but with a better precision for the EBV Abbott RealTime assay. For the CMV performances, the Abbott assay was more sensitive and more precise than our routine method. The use of WHO International Standards also indicated a slight underestimation of the viral loads (-0.25 log10 IU/mL and -0.21 log10 IU/mL for CMV and EBV assays respectively) while these were rather overestimated with the Starlet/Diagenode method (0.48 log10 IU/mL and 0.19 log10 IU/mL for CMV and EBV assays respectively). These trends were confirmed using relevant whole-blood clinical samples and external quality controls. The workflows were also compared and we highlighted a significant technician hands-on time reduction (-63%) using the Abbott CMV/EBV maxCycle automated protocol.

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We report a case of louse-borne relapsing fever (LBRF) in a refugee from Somalia who had arrived in Belgium a few days earlier. He complained of myalgia and secondarily presented fever. Blood smears revealed spirochetes later identified as Borrelia recurrentis. LBRF should be considered in countries hosting refugees, particularly those who transit through endemic regions.

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BACKGROUND: Herpes zoster (HZ) in patients receiving tumor necrosis factor (TNF) antagonists may be more severe and the incidence seems increased. The influence of TNF antagonists on varicella zoster virus (VZV) reactivation is unknown. OBJECTIVE: To prospectively search in a pilot study for VZV DNA in sequential blood samples before and after infliximab administration. SETTING: University medical center. SUBJECTS AND METHODS: Blood samples of six patients with longstanding and severe plaque psoriasis were taken on day 1 (before infliximab administration) and on days 2, 7, 21 and 42 for the determination of VZV viremia by ORF21 real-time polymerase chain reaction. Patients with varicella, HZ and normal subjects were included as controls. RESULTS: None of the six patients presented VZV viremia at any of the time points. High-load viremia was present during varicella, low-load viremia in some HZ patients and no viremia in the control patients. LIMITATIONS: Small number of patients. CONCLUSIONS: In this pilot study, infliximab did not reactivate VZV and did not induce subclinical VZV viremia.

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The first step of herpesviruses virion assembly occurs in the nucleus. However, the exact site where nucleocapsids are assembled, where the genome and the inner tegument are acquired, remains controversial. We created a recombinant VZV expressing ORF23 (homologous to HSV-1 VP26) fused to the eGFP and dually fluorescent viruses with a tegument protein additionally fused to a red tag (ORF9, ORF21 and ORF22 corresponding to HSV-1 UL49, UL37 and UL36). We identified nuclear dense structures containing the major capsid protein, the scaffold protein and maturing protease, as well as ORF21 and ORF22. Correlative microscopy demonstrated that the structures correspond to capsid aggregates and time-lapse video imaging showed that they appear prior to the accumulation of cytoplasmic capsids, presumably undergoing the secondary egress, and are highly dynamic. Our observations suggest that these structures might represent a nuclear area important for capsid assembly and/or maturation before the budding at the inner nuclear membrane.

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AIM: To document the epidemiologic patterns and risk factors of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections in Mali in order to develop prevention means for both diseases. METHODS: Two prospective studies were conducted in Bamako in 2009 among 1000 pregnant women (i.e., young women) who consulted six reference health centers, and in 2010, among 231 older women who attended general practice in two hospitals. Antibody tests and molecular analysis (performed only for HCV) were used to quantify the frequencies of both infections. The data were collected from patients recruited through a questionnaire. Transmission risk factors of both diseases were identified by univariate and multivariate analysis. RESULTS: HCV seroprevalence was 0.2% for young and 6.5% for older women. HIV prevalence was similar in both populations (4.1% vs 6.1%). In older women, the analysis of risk factors highlighted an association between HCV infection and episodes of hospitalization (P < 0.01). The study did not show an association between HIV infection and the variables such as hospitalization, transfusion, tattoo, dental care, and endoscopy. A significant decrease of HIV seroprevalence was detected in young women who used condoms for contraception more than for other purposes (P < 0.01). By contrast, HIV seroprevalence was significantly increased in young women using condoms mainly to prevent sexual infections rather than for contraception (P < 0.01). No HCV/HIV coinfection was detected in our study. CONCLUSION: Risk factors and epidemiologic data of HIV and HCV as well as the absence of co-infection strongly suggest epidemiological disparities between these diseases.

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The role of the tegument during the herpesvirus lytic cycle is still not clearly established, particularly at the late phase of infection, when the newly produced viral particles need to be fully assembled before being released from the infected cell. The varicella-zoster virus (VZV) protein coded by open reading frame (ORF) 9 (ORF9p) is an essential tegument protein, and, even though its mRNA is the most expressed during the productive infection, little is known about its functions. Using a GalK positive/negative selection technique, we modified a bacterial artificial chromosome (BAC) containing the complete VZV genome to create viruses expressing mutant versions of ORF9p. We showed that ORF9p is hyperphosphorylated during the infection, especially through its interaction with the viral Ser/Thr kinase ORF47p; we identified a consensus site within ORF9p recognized by ORF47p and demonstrated its importance for ORF9p phosphorylation. Strikingly, an ultrastructural analysis revealed that the mutation of this consensus site (glutamate 85 to arginine) strongly affects viral assembly and release, reproducing the ORF47 kinase-dead VZV phenotype. It also slightly diminishes the infectivity toward immature dendritic cells. Taken together, our results identify ORF9p as a new viral substrate of ORF47p and suggest a determinant role of this phosphorylation for viral infectivity, especially during the process of viral particle formation and egress.

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AIM: To estimate the prevalence of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections in women in Mali and to evaluate the performance of serological assays. METHODS: Two prospective studies were conducted in 2009 and 2010 in Mali. They concerned first, 1000 pregnant women attending six reference health centers in Bamako (Malian capital) between May 26 and June 16, 2009; and secondly, 231 women over 50 years who consulted general practitioners of two hospitals in Bamako between October 25 and December 24, 2010. Blood samples were collected and kept frozen in good condition before analysis. All samples depicted as positive using HIV/HCV enzyme immuno-assay screening assays were submitted to confirmation analysis. Molecular markers of HCV were characterized. RESULTS: The seroprevalence of HIV and HCV in the population of pregnant women was 4.1% and 0.2% respectively. Among older women the seroprevalence was higher and similar for HIV and HCV (6.1% vs 6.5%). The anti-HIV prevalence was not different in young and older women (4.1% vs 6.1%). In contrast, the anti-HCV prevalence was higher in older compared to younger women (6.5% vs 0.2%, P < 0.01). Of 2 pregnant women who were HCV seropositive, only one was polymerase chain reaction (PCR) reactive and infected by genotype 2, with a viral load of 1600 IU/mL. Regarding older women who were HCV seropositive, 13 out of 15 were PCR reactive, infected by genotype 1 or 2. Globally HCV genotype 2 was predominant. The positive predictive value (PPV) measured with VIKIA HIV test in young women was 100% therefore significantly higher than the 87.5% measured in older women (P < 0.05). Conversely, the PPV measured with Monolisa HCV assay in older women was 88.2% and higher than the 14.3% measured in younger women (P < 0.01). CONCLUSION: Whereas HIV prevalence was similar in both subpopulations HCV was more frequent among older women (P < 0.01). The PPV of screening assays varied with the age of the subjects.

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Skin carcinoma are among the most spread diagnosed tumours in the world. In this study, we investigated the transfection of immortalized keratinocytes, used as an in vitro model for skin carcinoma, using antisense technology and poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA)-based polymers, with original architecture and functionalities. We tested PDMAEMA polymers with different structures: linear, with two (DEA-PDMAEMA) or three (TEA-PDMAEMA) arms. The cytotoxicity of these polymers was assessed over a wide range of apparent M n (from 7600 to 64 600). At a N/P ratio of 7.38, cytotoxicity increases with the M n. Keratinocytes were transfected with a fluorescent oligonucleotide and then analyzed by flow cytometry. For the three architectures tested, the percentage of transfected cells and abundance of internalized oligonucleotide were closely related to the M n of the polymer. Confocal microscopy and FACS analyses showed a wide spread fine granular distribution of the oligonucleotide up to 3 days post-transfection. Then, we assessed the silencing efficiency of the polymers, targeting GFP in GFP expressing keratinocytes. The maximal silencing effect (±40%) was obtained using a DEA-PDMAEMA polymer (M n = 30 300). These results suggest that PDMAEMA-based polymers can be efficiently used to transfect immortalized keratinocytes and, thus, open new perspectives in the therapy of skin carcinoma.

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