ICTV Virus Taxonomy Profile: Hantaviridae 2024.

Hantaviridae is a family for negative-sense RNA viruses with genomes of about 10.5-14.6 kb. These viruses are maintained in and/or transmitted by fish, reptiles, and mammals. Several orthohantaviruses can infect humans, causing mild, severe, and sometimes-fatal diseases. Hantavirids produce enveloped virions containing three single-stranded RNA segments with open reading frames that encode a nucleoprotein (N), a glycoprotein precursor (GPC), and a large (L) protein containing an RNA-directed RNA polymerase (RdRP) domain. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Hantaviridae, which is available at ictv.global/report/hantaviridae.

Multi-center evaluation of the Research Use Only NeuMoDx monkeypox virus (MPXV) fully automated real-time PCR assay.

The mpox outbreak, caused by monkeypox virus (MPXV), accelerated the development of molecular diagnostics. In this study, we detail the evaluation of the Research Use Only (RUO) NeuMoDx MPXV assay by multiple European and US sites. The assay was designed and developed by Qiagen for the NeuMoDx Molecular Systems. Primers and probes were tested for specificity and inclusivity in silico. The analytical sensitivity of the assay was determined by testing dilutions of synthetic and genomic MPXV DNA. A total of 296 clinical samples were tested by three sites; the Johns Hopkins University (US), UZ Gent (Belgium, Europe), and Hospital Universitario San Cecilio (Spain, Europe). The analytical sensitivity of the assay was 50 copies/mL for both clades I and II. The assay showed 100% in silico identity for 80 clade I and 99.98% in silico identity for 5,162 clade II genomes. Clade II primers and probes showed 100% in silico specificity; however, identity of at least one of the two sets of clade I primers and probes with variola, cowpox, camelpox, and vaccinia viruses was noticed. The clinical validation showed sensitivity of 99.21% [95% confidence interval (CI): 95.66-99.98%] and specificity of 96.64% (95% CI: 91.62-99.08%) for lesion swab samples. The NeuMoDx MPXV Test shows acceptable analytical and clinical performance. The assay improves the laboratory's workflow as it consolidates nucleic acid extraction, PCR, data analysis, and interpretation and can be interfaced. The Test Strip can differentiate clades I and II, which has important laboratory safety implications. IMPORTANCE: In this manuscript, we provide detailed in silico analysis and clinical evaluation of the assay using a large cohort of clinical samples across three academic centers in Europe and the United States. Because the assay differentiates MPXV clades I and II, this manuscript is timely due to the current need to rule out the regulated clade I by diagnostic clinical laboratories. In December 2023, and due to first report of cases of sexually transmitted clade I infections in the Democratic Republic of the Congo, when generic assays that do not differentiate the clades are used, samples are considered regulated. The assay meets the need of full automation and has a marked positive impact on the laboratory workflow.

Nationwide quality assurance of high-throughput diagnostic molecular testing during the SARS-CoV-2 pandemic: role of the Belgian National Reference Centre.

Since the onset of the coronavirus disease (COVID-19) pandemic in Belgium, UZ/KU Leuven has played a crucial role as the National Reference Centre (NRC) for respiratory pathogens, to be the first Belgian laboratory to develop and implement laboratory developed diagnostic assays for SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) and later to assess the quality of commercial kits. To meet the growing demand for decentralised testing, both clinical laboratories and government-supported high-throughput platforms were gradually deployed across Belgium. Consequently, the role of the NRC transitioned from a specialised testing laboratory to strengthening capacity and coordinating quality assurance. Here, we outline the measures taken by the NRC, the national public health institute Sciensano and the executing clinical laboratories to ensure effective quality management of molecular testing throughout the initial two years of the pandemic (March 2020 to March 2022).

Assessment of the clinical and analytical performance of three Seegene Allplex SARS-CoV-2 assays within the VALCOR framework.

The coronavirus disease 2019 (COVID-19) pandemic demonstrated the need for accurate diagnostic testing for the early detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although the pandemic has ended, accurate assays are still needed to monitor viral spread at national levels and beyond through population and wastewater surveillance. To enhance early detection, SARS-CoV-2 assays should have high diagnostic accuracy and should be validated to assure accurate results. Three distinct SARS-CoV-2 assays were evaluated with clinical samples using the VALCOR (VALidation of SARS-CORona Virus-2 assays) framework, with the TaqPath COVID-19 assay (ThermoFisher Scientific, USA) as a comparator. We evaluated clinical sensitivity, specificity, limit of detection (LOD), and overall concordance between comparator and three index Allplex SARS-CoV-2 assays (Seegene, South Korea): Allplex-SC2, Allplex-SC2Fast (Fast PCR), and Allplex-SC2FabR (SARS-CoV-2/FluA/FluB/respiratory syncytial virus). Analytical performance and LOD of index assays were assessed using a dilution series of three synthetic SARS-CoV-2 sequence reference materials (RMs). Ninety SARS-CoV-2 positives and 90 SARS-CoV-2 negatives were tested. All Allplex assays had 100.0% sensitivity (95%CI = 95.9%-100.0%). Allplex-SC2 and Allplex-SC2Fast assays had 97.8% specificity (95%CI = 92.3%-99.7%) and 98.9% overall concordance [κ = 0.978 (95%CI = 0.947-1.000)]. Allplex-SC2FabR assay showed 100.0% specificity (95%CI = 95.9%-100.0%) and 100.0% overall concordance [κ = 1.000 (95%CI = 1.000-1.000)]. LOD assessment of index assays revealed detection down to 2.61 × 102 copies/mL in clinical samples, while the analytical LOD was 9.00 × 102 copies/mL. In conclusion, the evaluation of the three Seegene Allplex SARS-CoV-2 assays showed high sensitivity and specificity and an overall good assay concordance with the comparator. The assays showed low analytical LOD using RM and even a slightly lower LOD in clinical samples. Non-overlapping target gene sequences between SARS-CoV-2 assays and RMs emphasize the need for aligning targeted sequences of diagnostic assays and RMs.IMPORTANCEThe coronavirus disease 2019 pandemic has a significant impact on global public health, economies, and societies. As shown through the first phases of the pandemic, accurate and timely diagnosis is crucial for disease control, prevention, and monitoring. Though the pandemic phase of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has concluded, diagnostic assays remain in demand to monitor SARS-CoV-2 at the individual patient level, regionally, and nationally, as well as to remain an infectious disease preparedness instrument to monitor any new SARS-CoV-2 dissemination across borders using population and wastewater surveillance. The anticipation by WHO and central health care policy entities such as the Center for Disease Control, EMA, and multiple national health authorities is that SARS-CoV-2 will reside as an endemic respiratory disease for years to come. The key strategic consideration is hence shifting from combating a pandemic situation with a high number of patients to instead allowing precise diagnostics of suspected patients with the intention of correct management in a low-prevalence setting.

The value of point-of-care tests for the detection of SARS-CoV-2 RNA or antigen in bronchoalveolar lavage fluid.

BACKGROUND: Transmission of SARS-CoV-2 from donor to recipient is a clinically relevant risk for developing severe COVID-19 after lung transplantation (LTx). This risk of iatrogenic transmission can be reduced by timely detection of viral RNA or antigen in samples of bronchoalveolar lavage (BAL) fluid obtained at the time of lung procurement. We aimed to retrospectively evaluate the detection of SARS-CoV-2 RNA or antigen in BAL fluid samples using three point-of-care tests (POCTs). METHODS: BAL fluid samples came from patients hospitalized in an intensive care unit during the COVID-19 pandemic. These pandemic samples were scored as positive or negative for SARS-CoV-2 by a RT-qPCR comparator assay for orf1ab. Three commercially available POCTs were then evaluated: cobas SARS-CoV-2 & Influenza A/B assay with the cobas Liat RT-qPCR system (Roche Diagnostics), ID NOW COVID-19 and COVID-19 2.0 (Abbott), and SARS-CoV-2 Rapid Antigen Test (RAT) (Roche Diagnostics). Samples from the pre-pandemic era served as negative controls. RESULTS: We analyzed a total of 98 BAL fluid samples, each from a different patient: 58 positive pandemic samples (orf1ab Ct<38), 20 putatively negative pandemic samples (orf1ab Ct≥38), and 20 pre-pandemic samples. Univariate logistic regression shows that the probability of detection was highest for cobas Liat, followed by ID NOW, and then RAT. Of clinical relevance, cobas Liat detected SARS-CoV-2 RNA in 30 of the 31 positive pandemic samples that were collected within 10 days after RT-qPCR diagnosis of SARS-CoV-2 infection. None of the 20 pre-pandemic samples had a false-positive result for any POCT. CONCLUSIONS: POCTs enable the detection of SARS-CoV-2 RNA or antigen in BAL fluid samples and may provide additional information to decide if donor lungs are suitable for transplantation. Detection of respiratory pathogens with POCTs at the time of donor lung procurement is a potential strategy to increase safety in LTx by preventing iatrogenic transmission and severe postoperative infections.

Unravelling the effect of New Year's Eve celebrations on SARS-CoV-2 transmission.

Public holidays have been associated with SARS-CoV-2 incidence surges, although a firm link remains to be established. This association is sometimes attributed to events where transmissions occur at a disproportionately high rate, known as superspreading events. Here, we describe a sudden surge in new cases with the Omicron BA.1 strain amongst higher education students in Belgium. Contact tracers classed most of these cases as likely or possibly infected on New Year's Eve, indicating a direct trigger by New Year celebrations. Using a combination of contact tracing and phylogenetic data, we show the limited role of superspreading events in this surge. Finally, the numerous simultaneous transmissions allowed a unique opportunity to determine the distribution of incubation periods of the Omicron strain. Overall, our results indicate that, even under social restrictions, a surge in transmissibility of SARS-CoV-2 can occur when holiday celebrations result in small social gatherings attended simultaneously and communitywide.

Genomic monitoring of SARS-CoV-2 variants using sentinel SARI hospital surveillance.

Background: To support the COVID-19 pandemic response, many countries, including Belgium, implemented baseline genomic surveillance (BGS) programs aiming to early detect and characterize new SARS-CoV-2 variants. In parallel, Belgium maintained a sentinel network of six hospitals that samples patients with severe acute respiratory infections (SARI) and integrated SARS-CoV-2 detection within a broader range of respiratory pathogens. We evaluate the ability of the SARI surveillance to monitor general trends and early signals of viral genetic evolution of SARS-CoV-2 and compare it with the BGS as a reference model. Methods: Nine-hundred twenty-five SARS-CoV-2 positive samples from patients fulfilling the Belgian SARI definition between January 2020 and December 2022 were sequenced using the ARTIC Network amplicon tiling approach on a MinION platform. Weekly variant of concern (VOC) proportions and types were compared to those that were circulating between 2021 and 2022, using 96,251 sequences of the BGS. Results: SARI surveillance allowed timely detection of the Omicron (BA.1, BA.2, BA.4, and BA.5) and Delta (B.1.617.2) VOCs, with no to 2 weeks delay according to the start of their epidemic growth in the Belgian population. First detection of VOCs B.1.351 and P.1 took longer, but these remained minor in Belgium. Omicron BA.3 was never detected in SARI surveillance. Timeliness could not be evaluated for B.1.1.7, being already major at the start of the study period. Conclusions: Genomic surveillance of SARS-CoV-2 using SARI sentinel surveillance has proven to accurately reflect VOCs detected in the population and provides a cost-effective solution for long-term genomic monitoring of circulating respiratory viruses.

Design and validation of a laboratory-developed diagnostic assay for monkeypox virus.

Mpox is a viral zoonosis with endemic circulation in animals and humans in some West and Central African countries. The disease was imported a few times in the past to countries outside the African continent through infected animals or travelers, one of which resulted in an unprecedented global outbreak sustained by human-to-human transmission in 2022. Although timely and reliable diagnosis is a cornerstone of any disease control, availability of accurate diagnostic assays and comparative performance studies of diagnostic assays remains limited despite of the long-known identification of monkeypox virus (MPXV) as a human pathogen since 1970. We laboratory-developed a real-time PCR test (LDT) and evaluated its performance against the commercial TaqMan™ Monkeypox Virus Microbe Detection Assay (Applied Biosystems, Cat A50137). The limit of detection of the LDT was established at 1.2 genome copies/ml. The sensitivity and specificity of both assays were 99.14% and 100%, respectively, and both are capable of detecting both clade I and clade II of MPXV. Our results demonstrate the validity and accuracy of the LDT for confirmation of MPXV infection from lesion swabs samples.

Indoor air surveillance and factors associated with respiratory pathogen detection in community settings in Belgium.

Currently, the real-life impact of indoor climate, human behaviour, ventilation and air filtration on respiratory pathogen detection and concentration are poorly understood. This hinders the interpretability of bioaerosol quantification in indoor air to surveil respiratory pathogens and transmission risk. We tested 341 indoor air samples from 21 community settings in Belgium for 29 respiratory pathogens using qPCR. On average, 3.9 pathogens were positive per sample and 85.3% of samples tested positive for at least one. Pathogen detection and concentration varied significantly by pathogen, month, and age group in generalised linear (mixed) models and generalised estimating equations. High CO2 and low natural ventilation were independent risk factors for detection. The odds ratio for detection was 1.09 (95% CI 1.03-1.15) per 100 parts per million (ppm) increase in CO2, and 0.88 (95% CI 0.80-0.97) per stepwise increase in natural ventilation (on a Likert scale). CO2 concentration and portable air filtration were independently associated with pathogen concentration. Each 100ppm increase in CO2 was associated with a qPCR Ct value decrease of 0.08 (95% CI -0.12 to -0.04), and portable air filtration with a 0.58 (95% CI 0.25-0.91) increase. The effects of occupancy, sampling duration, mask wearing, vocalisation, temperature, humidity and mechanical ventilation were not significant. Our results support the importance of ventilation and air filtration to reduce transmission.

Pending Reorganization of Hantaviridae to Include Only Completely Sequenced Viruses: A Call to Action.

The official classification of newly discovered or long-known unassigned viruses by the International Committee on Taxonomy of Viruses (ICTV) requires the deposition of coding-complete or -near-complete virus genome sequences in GenBank to fulfill a requirement of the taxonomic proposal (TaxoProp) process. However, this requirement is fairly new; thus, genomic sequence information is fragmented or absent for many already-classified viruses. As a result, taxon-wide modern phylogenetic analyses are often challenging, if not impossible. This problem is particularly eminent among viruses with segmented genomes, such as bunyavirals, which were frequently classified solely based on single-segment sequence information. To solve this issue for one bunyaviral family, Hantaviridae, we call on the community to provide additional sequence information for incompletely sequenced classified viruses by mid-June 2023. Such sequence information may be sufficient to prevent their possible declassification during the ongoing efforts to establish a coherent, consistent, and evolution-based hantavirid taxonomy.

Severity of COVID-19 among Hospitalized Patients: Omicron Remains a Severe Threat for Immunocompromised Hosts.

The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in the general population in the context of a relatively high immunity gained through the early waves of coronavirus disease 19 (COVID-19), and vaccination campaigns. Despite this context, a significant number of patients were hospitalized, and identifying the risk factors associated with severe disease in the Omicron era is critical for targeting further preventive, and curative interventions. We retrospectively analyzed the individual medical records of 1501 SARS-CoV-2 positive hospitalized patients between 13 December 2021, and 13 February 2022, in Belgium, of which 187 (12.5%) were infected with Delta, and 1036 (69.0%) with Omicron. Unvaccinated adults showed an increased risk of moderate/severe/critical/fatal COVID-19 (crude OR 1.54; 95% CI 1.09-2.16) compared to vaccinated patients, whether infected with Omicron or Delta. In adults infected with Omicron and moderate/severe/critical/fatal COVID-19 (n = 323), immunocompromised patients showed an increased risk of in-hospital mortality related to COVID-19 (adjusted OR 2.42; 95% CI 1.39-4.22), compared to non-immunocompromised patients. The upcoming impact of the pandemic will be defined by evolving viral variants, and the immune system status of the population. The observations support that, in the context of an intrinsically less virulent variant, vaccination and underlying patient immunity remain the main drivers of severe disease.

Anatomical barriers against SARS-CoV-2 neuroinvasion at vulnerable interfaces visualized in deceased COVID-19 patients.

Can SARS-CoV-2 hitchhike on the olfactory projection and take a direct and short route from the nose into the brain? We reasoned that the neurotropic or neuroinvasive capacity of the virus, if it exists, should be most easily detectable in individuals who died in an acute phase of the infection. Here, we applied a postmortem bedside surgical procedure for the rapid procurement of tissue, blood, and cerebrospinal fluid samples from deceased COVID-19 patients infected with the Delta, Omicron BA.1, or Omicron BA.2 variants. Confocal imaging of sections stained with fluorescence RNAscope and immunohistochemistry afforded the light-microscopic visualization of extracellular SARS-CoV-2 virions in tissues. We failed to find evidence for viral invasion of the parenchyma of the olfactory bulb and the frontal lobe of the brain. Instead, we identified anatomical barriers at vulnerable interfaces, exemplified by perineurial olfactory nerve fibroblasts enwrapping olfactory axon fascicles in the lamina propria of the olfactory mucosa.

The characterization of multiple novel paramyxoviruses highlights the diverse nature of the subfamily Orthoparamyxovirinae.

The subfamily Orthoparamyxovirinae is a group of single-stranded, negative-sense RNA viruses that contains many human, animal, and zoonotic pathogens. While there are currently only forty-two recognized species in this subfamily, recent research has revealed that much of its diversity remains to be characterized. Using a newly developed nested PCR-based screening assay, we report here the discovery of fifteen orthoparamyxoviruses in rodents and shrews from Belgium and Guinea, thirteen of which are believed to represent new species. Using a combination of nanopore and sanger sequencing, complete genomes could be determined for almost all these viruses, enabling a detailed evaluation of their genome characteristics. While most viruses are thought to belong to the rapidly expanding genus Jeilongvirus, we also identify novel members of the genera Narmovirus, Henipavirus, and Morbillivirus. Together with other recently discovered orthoparamyxoviruses, both henipaviruses and the morbillivirus discovered here appear to form distinct rodent-/shrew-borne clades within their respective genera, clustering separately from all currently classified viruses. In the case of the henipaviruses, a comparison of the different members of this clade revealed the presence of a secondary conserved open reading frame, encoding for a transmembrane protein, within the F gene, the biological relevance of which remains to be established. While the characteristics of the viruses described here shed further light on the complex evolutionary origin of paramyxoviruses, they also illustrate that the diversity of this group of viruses in terms of genome organization appears to be much larger than previously assumed.

Characterization of Emerging Serotype 19A Pneumococcal Strains in Invasive Disease and Carriage, Belgium.

After switching from 13-valent to 10-valent pneumococcal conjugate vaccine (PCV10) (2015-2016) for children in Belgium, we observed rapid reemergence of serotype 19A invasive pneumococcal disease (IPD). Whole-genome sequencing of 166 serotype 19A IPD isolates from children (n = 54) and older adults (n = 56) and carriage isolates from healthy children (n = 56) collected after the vaccine switch (2017-2018) showed 24 sequence types (STs). ST416 (global pneumococcal sequence cluster [GPSC] 4) and ST994 (GPSC146) accounted for 75.9% of IPD strains from children and 65.7% of IPD (children and older adults) and carriage isolates in the PCV10 period (2017-2018). These STs differed from predominant 19A IPD STs after introduction of PCV7 (2011) in Belgium (ST193 [GPSC11] and ST276 [GPSC10]), which indicates that prediction of emerging strains cannot be based solely on historical emerging strains. Despite their susceptible antimicrobial drug profiles, these clones spread in carriage and IPD during PCV10 use.

Antiviral treatment with fluoxetine for rituximab-associated chronic echovirus 13 meningoencephalitis and myofasciitis.

BACKGROUND AND PURPOSE: Enterovirus infections pose a serious threat for patients with humoral deficiencies and may be lethal, whilst the efficacy of proposed treatment options such as corticosteroids, intravenous immunoglobulins and fluoxetine remains debated. METHODS: Viral clearance was investigated in a patient with rituximab-induced B-cell depletion and chronic echovirus 13 (E13) meningoencephalitis/myofasciitis in response to intravenous immunoglobulins and fluoxetine using sequential semi-quantitative E13 viral load measurements by real-time reverse transcription polymerase chain reaction. Fluoxetine concentrations in plasma and cerebrospinal fluid were determined by liquid chromatography mass spectrometry. RESULTS: Intravenous immunoglobulins appeared ineffective in this case of E13 infection, whereas virus clearance in cerebrospinal fluid was obtained after 167 days of oral fluoxetine. Since treatment with corticosteroids resulted in a flare of symptoms, rechallenge with viral load measurements was not attempted. CONCLUSION: In this report of a patient with rituximab-associated chronic echovirus 13 meningoencephalitis, viral clearance in response to single treatment options is assessed for the first time. Our observations further support the in vivo efficacy of fluoxetine against enteroviral infections. More research is needed to establish its efficacy in different enterovirus strains.

Early SARS-CoV-2 Reinfections within 60 Days and Implications for Retesting Policies.

Illustrated by a clinical case supplemented by epidemiologic data, early reinfections with SARS-CoV-2 Omicron BA.1 after infection with Delta variant, and reinfection with Omicron BA.2 after Omicron BA.1 infection, can occur within 60 days, especially in young, unvaccinated persons. The case definition of reinfection, which influences retesting policies, should be reconsidered.

Nationwide Harmonization Effort for Semi-Quantitative Reporting of SARS-CoV-2 PCR Test Results in Belgium.

From early 2020, a high demand for SARS-CoV-2 tests was driven by several testing indications, including asymptomatic cases, resulting in the massive roll-out of PCR assays to combat the pandemic. Considering the dynamic of viral shedding during the course of infection, the demand to report cycle threshold (Ct) values rapidly emerged. As Ct values can be affected by a number of factors, we considered that harmonization of semi-quantitative PCR results across laboratories would avoid potential divergent interpretations, particularly in the absence of clinical or serological information. A proposal to harmonize reporting of test results was drafted by the National Reference Centre (NRC) UZ/KU Leuven, distinguishing four categories of positivity based on RNA copies/mL. Pre-quantified control material was shipped to 124 laboratories with instructions to setup a standard curve to define thresholds per assay. For each assay, the mean Ct value and corresponding standard deviation was calculated per target gene, for the three concentrations (107, 105 and 103 copies/mL) that determine the classification. The results of 17 assays are summarized. This harmonization effort allowed to ensure that all Belgian laboratories would report positive PCR results in the same semi-quantitative manner to clinicians and to the national database which feeds contact tracing interventions.

Reliable and Scalable SARS-CoV-2 qPCR Testing at a High Sample Throughput: Lessons Learned from the Belgian Initiative.

We present our approach to rapidly establishing a standardized, multi-site, nation-wide COVID-19 screening program in Belgium. Under auspices of a federal government Task Force responsible for upscaling the country's testing capacity, we were able to set up a national testing initiative with readily available resources, putting in place a robust, validated, high-throughput, and decentralized qPCR molecular testing platform with embedded proficiency testing. We demonstrate how during an acute scarcity of equipment, kits, reagents, personnel, protective equipment, and sterile plastic supplies, we introduced an approach to rapidly build a reliable, validated, high-volume, high-confidence workflow based on heterogeneous instrumentation and diverse assays, assay components, and protocols. The workflow was set up with continuous quality control monitoring, tied together through a clinical-grade information management platform for automated data analysis, real-time result reporting across different participating sites, qc monitoring, and making result data available to the requesting physician and the patient. In this overview, we address challenges in optimizing high-throughput cross-laboratory workflows with minimal manual intervention through software, instrument and assay validation and standardization, and a process for harmonized result reporting and nation-level infection statistics monitoring across the disparate testing methodologies and workflows, necessitated by a rapid scale-up as a response to the pandemic.

Multidrug-resistant tuberculosis control in Rwanda overcomes a successful clone that causes most disease over a quarter century.

SUMMARY BACKGROUND: Multidrug-resistant (MDR) tuberculosis (TB) poses an important challenge in TB management and control. Rifampicin resistance (RR) is a solid surrogate marker of MDR-TB. We investigated the RR-TB clustering rates, bacterial population dynamics to infer transmission dynamics, and the impact of changes to patient management on these dynamics over 27 years in Rwanda. METHODS: We analysed whole genome sequences of a longitudinal collection of nationwide RR-TB isolates. The collection covered three important periods: before programmatic management of MDR-TB (PMDT; 1991-2005), the early PMDT phase (2006-2013), in which rifampicin drug-susceptibility testing (DST) was offered to retreatment patients only, and the consolidated phase (2014-2018), in which all bacteriologically confirmed TB patients had rifampicin DST done mostly via Xpert MTB/RIF assay. We constructed clusters based on a 5 SNP cut-off and resistance conferring SNPs. We used Bayesian modelling for dating and population size estimations, TransPhylo to estimate the number of secondary cases infected by each patient, and multivariable logistic regression to assess predictors of being infected by the dominant clone. RESULTS: Of 308 baseline RR-TB isolates considered for transmission analysis, the clustering analysis grouped 259 (84.1%) isolates into 13 clusters. Within these clusters, a single dominant clone was discovered containing 213 isolates (82.2% of clustered and 69.1% of all RR-TB), which we named the "Rwanda Rifampicin-Resistant clone" (R3clone). R3clone isolates belonged to Ugandan sub-lineage 4.6.1.2 and its rifampicin and isoniazid resistance were conferred by the Ser450Leu mutation in rpoB and Ser315Thr in katG genes, respectively. All R3clone isolates had Pro481Thr, a putative compensatory mutation in the rpoC gene that likely restored its fitness. The R3clone was estimated to first arise in 1987 and its population size increased exponentially through the 1990s', reaching maximum size (∼84%) in early 2000 s', with a declining trend since 2014. Indeed, the highest proportion of R3clone (129/157; 82·2%, 95%CI: 75·3-87·8%) occurred between 2000 and 13, declining to 64·4% (95%CI: 55·1-73·0%) from 2014 onward. We showed that patients with R3clone detected after an unsuccessful category 2 treatment were more likely to generate secondary cases than patients with R3clone detected after an unsuccessful category 1 treatment regimen. CONCLUSIONS: RR-TB in Rwanda is largely transmitted. Xpert MTB/RIF assay as first diagnostic test avoids unnecessary rounds of rifampicin-based TB treatment, thus preventing ongoing transmission of the dominant R3clone. As PMDT was intensified and all TB patients accessed rifampicin-resistance testing, the nationwide R3clone burden declined. To our knowledge, our findings provide the first evidence supporting the impact of universal DST on the transmission of RR-TB.

Exploration of the Ixodes ricinus virosphere unveils an extensive virus diversity including novel coltiviruses and other reoviruses.

Recent metagenomics studies have revealed several tick species to host a variety of previously undiscovered RNA viruses. Ixodes ricinus, which is known to be a vector for many viral, bacterial, and protozoan pathogens, is the most prevalent tick species in Europe. For this study, we decided to investigate the virosphere of Belgian I. ricinus ticks. High-throughput sequencing of tick pools collected from six different sampling sites revealed the presence of viruses belonging to many different viral orders and families, including Mononegavirales, Bunyavirales, Partitiviridae, and Reoviridae. Of particular interest was the detection of several new reoviruses, two of which cluster together with members of the genus Coltivirus. This includes a new strain of Eyach virus, a known causative agent of tick-borne encephalitis. All genome segments of this new strain are highly similar to those of previously published Eyach virus genomes, except for the fourth segment, encoding VP4, which is markedly more dissimilar, potentially indicating the occurrence of a genetic reassortment. Further polymerase chain reaction-based screening of over 230 tick pools for 14 selected viruses showed that most viruses could be found in all six sampling sites, indicating the wide spread of these viruses throughout the Belgian tick population. Taken together, these results illustrate the role of ticks as important virus reservoirs, highlighting the need for adequate tick control measures.