RESUMO
Clostridioides difficile infections are a significant threat to our healthcare system, and rapid and accurate diagnostics are crucial to implement the necessary infection prevention and control measurements. Nucleic acid amplification tests are such reliable diagnostic tools for the detection of toxigenic Clostridioides difficile strains directly from stool specimens. In this multicenter evaluation, we determined the performance of the revogene C. difficile assay. The analysis was conducted on prospective stool specimens collected from six different sites in Europe. The performance of the revogene C. difficile assay was compared to the different routine diagnostic methods and, for a subset of the specimens, against toxigenic culture. In total, 2621 valid stool specimens were tested, and the revogene C. difficile assay displayed a sensitivity/specificity of 97.1% [93.3-99.0] and 98.9% [98.5-99.3] for identification of Clostridioides difficile infection. Discrepancy analysis using additional methods improved this performance to 98.8% [95.8-99.9] and 99.6% [99.2-99.8], respectively. In comparison to toxigenic culture, the revogene C. difficile assay displayed a sensitivity/specificity of 93.0% [86.1-97.1] and 99.5% [98.7-99.9], respectively. These results indicate that the revogene C. difficile assay is a robust and reliable aid in the diagnosis of Clostridioides difficile infections.
Assuntos
Clostridioides difficile/isolamento & purificação , Infecções por Clostridium/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Clostridioides difficile/genética , Testes Diagnósticos de Rotina , Europa (Continente) , Fezes/microbiologia , Humanos , Técnicas de Amplificação de Ácido Nucleico , Testes Imediatos , Estudos Prospectivos , Sensibilidade e EspecificidadeRESUMO
Clostridioides difficile is the main causative agent of antibiotic-associated diarrhea. Prompt diagnosis is required for initiation of timely infection control measures and appropriate adjustment of antibiotic treatment. The cobas Cdiff assay for use on the cobas Liat system enables a diagnostic result in 20 minutes. A total of 252 prospective (n = 150) and retrospective (n = 102) stool specimens from The Netherlands, France, and Switzerland were tested on the cobas Cdiff assay using the Xpert C. difficile assay as a reference method. The overall positive and negative percent agreement (PPA and NPA, respectively) of the cobas Cdiff assay compared with the Xpert C. difficile assay was 98.0% (100/102; 95% confidence interval [CI], 93.1% to 99.5%) and 94.0% (141/150; 95% CI, 89.0% to 96.8%), respectively. When comparing the PPAs of cobas Cdiff and Xpert C. difficile with culture, the results were 91.7% (55/60; 95% CI, 81.9% to 96.4%) and 85.0% (51/60; 95% CI, 73.9% to 91.9%), respectively. The difference was not statistically significant. The cobas Cdiff assay offers a very rapid alternative for diagnosing C. difficile infection. The 20-minute turnaround time provides the potential for point-of-care testing so that adequate infection control measures can be initiated promptly.
Assuntos
Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Clostridioides difficile/genética , Infecções por Clostridium/diagnóstico , Infecções por Clostridium/microbiologia , Técnicas de Diagnóstico Molecular , Reação em Cadeia da Polimerase/métodos , Humanos , RibotipagemRESUMO
OBJECTIVES: To determine whether it would be hygienic to evaluate dogs and humans in the same MRI scanner. METHODS: We compared the bacterial load in colony-forming units (CFU) of human-pathogenic microorganisms in specimens taken from 18 men and 30 dogs. In addition, we compared the extent of bacterial contamination of an MRI scanner shared by dogs and humans with two other MRI scanners used exclusively by humans. RESULTS: Our study shows a significantly higher bacterial load in specimens taken from men's beards compared with dogs' fur (p = 0.036). All of the men (18/18) showed high microbial counts, whereas only 23/30 dogs had high microbial counts and 7 dogs moderate microbial counts. Furthermore, human-pathogenic microorganisms were more frequently found in human beards (7/18) than in dog fur (4/30), although this difference did not reach statistical significance (p = 0.074). More microbes were found in human oral cavities than in dog oral cavities (p < 0.001). After MRI of dogs, routine scanner disinfection was undertaken and the CFU found in specimens isolated from the MRI scanning table and receiver coils showed significantly lower bacteria count compared with "human" MRI scanners (p < 0.05). CONCLUSION: Our study shows that bearded men harbour significantly higher burden of microbes and more human-pathogenic strains than dogs. As the MRI scanner used for both dogs and humans was routinely cleaned after animal scanning, there was substantially lower bacterial load compared with scanners used exclusively for humans. KEY POINTS: ⢠Bearded men harbour significantly more microbes than dogs. ⢠Dogs are no risk to humans if they use the same MRI. ⢠Deficits in hospital hygiene are a relevant risk for patients.
Assuntos
Cães/microbiologia , Cabelo/microbiologia , Higiene , Imageamento por Ressonância Magnética/instrumentação , Pelo Animal/microbiologia , Animais , Bactérias/isolamento & purificação , Carga Bacteriana , Contagem de Colônia Microbiana , Infecção Hospitalar/prevenção & controle , Infecção Hospitalar/transmissão , Desinfecção , Contaminação de Equipamentos/prevenção & controle , Humanos , MasculinoRESUMO
Each year in the United States, 500 patients are hospitalized for cat-scratch disease, caused by Bartonella henselae infection. We report a case of rare but serious neurologic B. henselae infection. When typical features of cat-scratch disease occur with neurologic findings, Bartonella infection should be suspected and diagnostic testing should be performed.
Assuntos
Bartonella , Doença da Arranhadura de Gato/complicações , Mielite Transversa/etiologia , Anti-Infecciosos/uso terapêutico , Doença da Arranhadura de Gato/líquido cefalorraquidiano , Doença da Arranhadura de Gato/tratamento farmacológico , Doença da Arranhadura de Gato/patologia , Feminino , Humanos , Pessoa de Meia-Idade , Mielite Transversa/líquido cefalorraquidiano , Mielite Transversa/tratamento farmacológicoRESUMO
HIV is known to spread efficiently both in a cell-free state and from cell to cell, however the relative importance of the cell-cell transmission mode in natural infection has not yet been resolved. Likewise to what extent cell-cell transmission is vulnerable to inhibition by neutralizing antibodies and entry inhibitors remains to be determined. Here we report on neutralizing antibody activity during cell-cell transmission using specifically tailored experimental strategies which enable unambiguous discrimination between the two transmission routes. We demonstrate that the activity of neutralizing monoclonal antibodies (mAbs) and entry inhibitors during cell-cell transmission varies depending on their mode of action. While gp41 directed agents remain active, CD4 binding site (CD4bs) directed inhibitors, including the potent neutralizing mAb VRC01, dramatically lose potency during cell-cell transmission. This implies that CD4bs mAbs act preferentially through blocking free virus transmission, while still allowing HIV to spread through cell-cell contacts. Thus providing a plausible explanation for how HIV maintains infectivity and rapidly escapes potent and broadly active CD4bs directed antibody responses in vivo.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antígenos CD4/imunologia , Infecções por HIV/prevenção & controle , Infecções por HIV/transmissão , HIV-1/imunologia , Anticorpos Monoclonais Murinos , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/farmacologia , Feminino , HIV-1/patogenicidade , Células HeLa , Humanos , MasculinoRESUMO
The Two Weeks in the World research project has resulted in a dataset of 3087 clinically relevant bacterial genomes with pertaining metadata, collected from 59 diagnostic units in 35 countries around the world during 2020. A relational database is available with metadata and summary data from selected bioinformatic analysis, such as species prediction and identification of acquired resistance genes.
Assuntos
Bactérias , Genoma Bacteriano , Bactérias/genética , Biologia Computacional , Bases de Dados Factuais , MetadadosRESUMO
BACKGROUND: Actinobaculum schaalii was first described as a causative agent for human infection in 1997. Since then it has mainly been reported causing urinary tract infections (UTI) in elderly individuals with underlying urological diseases. Isolation and identification is challenging and often needs molecular techniques. A. schaalii is increasingly reported as a cause of infection in humans, however data in children is very limited. CASE PRESENTATION: We present the case of an 8-month-old Caucasian boy suffering from myelomeningocele and neurogenic bladder who presented with a UTI. An ultrasound of the urinary tract was unremarkable. Urinalysis and microscopy showed an elevated leukocyte esterase test, pyuria and a high number of bacteria. Empiric treatment with oral co-trimoxazole was started.Growth of small colonies of Gram-positive rods was observed after 48 h. Sequencing of the 16S rRNA gene confirmed an A. schaalii infection 9 days later. Treatment was changed to oral amoxicillin for 14 days. On follow-up urinalysis was normal and urine cultures were negative. CONCLUSIONS: A.schaalii is an emerging pathogen in adults and children. Colonization and subsequent infection seem to be influenced by the age of the patient. In young children with high suspicion of UTI who use diapers or in children who have known abnormalities of their urogenital tract, infection with A. schaalii should be considered and empiric antimicrobial therapy chosen accordingly.
Assuntos
Actinomycetaceae/isolamento & purificação , Doenças Transmissíveis Emergentes/diagnóstico , Doenças Transmissíveis Emergentes/microbiologia , Infecções por Bactérias Gram-Positivas/diagnóstico , Infecções por Bactérias Gram-Positivas/microbiologia , Infecções Urinárias/diagnóstico , Infecções Urinárias/microbiologia , Amoxicilina/administração & dosagem , Antibacterianos/administração & dosagem , Doenças Transmissíveis Emergentes/tratamento farmacológico , Doenças Transmissíveis Emergentes/patologia , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Infecções por Bactérias Gram-Positivas/patologia , Humanos , Lactente , Masculino , Meningomielocele/complicações , Meningomielocele/diagnóstico , Meningomielocele/patologia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Resultado do Tratamento , Combinação Trimetoprima e Sulfametoxazol/administração & dosagem , Bexiga Urinaria Neurogênica/complicações , Bexiga Urinaria Neurogênica/diagnóstico , Bexiga Urinaria Neurogênica/patologia , Infecções Urinárias/tratamento farmacológico , Infecções Urinárias/patologia , Urina/química , Urina/citologia , População BrancaRESUMO
The rapid spread of the SARS-CoV-2 lineages B.1.1.7 (N501Y.V1) throughout the UK, B.1.351 (N501Y.V2) in South Africa, and P.1 (B.1.1.28.1; N501Y.V3) in Brazil has led to the definition of variants of concern (VoCs) and recommendations for lineage specific surveillance. In Switzerland, during the last weeks of December 2020, we established a nationwide screening protocol across multiple laboratories, focusing first on epidemiological and microbiological definitions. In January 2021, we validated and implemented an N501Y-specific PCR to rapidly screen for VoCs, which are then confirmed using amplicon sequencing or whole genome sequencing (WGS). A total of 13,387 VoCs have been identified since the detection of the first Swiss case in October 2020, with 4194 being B.1.1.7, 172 B.1.351, and 7 P.1. The remaining 9014 cases of VoCs have been described without further lineage specification. Overall, all diagnostic centers reported a rapid increase of the percentage of detected VOCs, with a range of 6 to 46% between 25 to 31 of January 2021 increasing towards 41 to 82% between 22 to 28 of February. A total of 739 N501Y positive genomes were analysed and show a broad range of introduction events to Switzerland. In this paper, we describe the nationwide coordination and implementation process across laboratories, public health institutions, and researchers, the first results of our N501Y-specific variant screening, and the phylogenetic analysis of all available WGS data in Switzerland, that together identified the early introduction events and subsequent community spreading of the VoCs.
RESUMO
Here, we describe the generation of a novel type of HIV entry inhibitor using the recently developed Designed Ankyrin Repeat Protein (DARPin) technology. DARPin proteins specific for human CD4 were selected from a DARPin DNA library using ribosome display. Selected pool members interacted specifically with CD4 and competed with gp120 for binding to CD4. DARPin proteins derived in the initial selection series inhibited HIV in a dose-dependent manner, but showed a relatively high variability in their capacity to block replication of patient isolates on primary CD4 T cells. In consequence, a second series of CD4-specific DARPins with improved affinity for CD4 was generated. These 2nd series DARPins potently inhibit infection of genetically divergent (subtype B and C) HIV isolates in the low nanomolar range, independent of coreceptor usage. Importantly, the actions of the CD4 binding DARPins were highly specific: no effect on cell viability or activation, CD4 memory cell function, or interference with CD4-independent virus entry was observed. These novel CD4 targeting molecules described here combine the unique characteristics of DARPins-high physical stability, specificity and low production costs-with the capacity to potently block HIV entry, rendering them promising candidates for microbicide development.
Assuntos
Repetição de Anquirina/fisiologia , Anquirinas/farmacologia , Fármacos Anti-HIV/farmacologia , Antígenos CD4/imunologia , Linfócitos T CD4-Positivos/virologia , HIV/patogenicidade , Animais , Anquirinas/imunologia , Linfócitos T CD4-Positivos/imunologia , Sobrevivência Celular/efeitos dos fármacos , Reações Cruzadas , Células Dendríticas/imunologia , Relação Dose-Resposta Imunológica , Ensaio de Imunoadsorção Enzimática , Feminino , HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Ativação Linfocitária , Macaca mulatta , Masculino , Camundongos , Testes de Neutralização , Engenharia de Proteínas , Replicação Viral/efeitos dos fármacosRESUMO
Sapovirus (SaV) and astrovirus (AstV) increasingly are recognized as cause of acute viral gastroenteritis (AGE). We evaluated the real-time RT-PCR assays RIDA®GENE SaV and viral stool panel II (RGN RT-PCR) for detection of SaV, AstV, adenovirus (AdV) F40/41 and rotavirus (RoV) in clinical stool samples (nâ¯=â¯69). Results were compared with reference singleplex RT-PCRs. The sensitivity for SaV, AstV and RoV are 100%, the specificity ranges from 98.1% to 100%. In 10 out of 11 AdV (all types) samples, the RGN RT-PCR for AdV F40/41 displayed negative results. Retrospectively, 196 stool specimens from adult patients previously tested negative for norovirus (NoV) were analyzed. In about 10% of NoV-negative stool samples, AdV (nâ¯=â¯9), RoV (nâ¯=â¯6), AstV (nâ¯=â¯3) or SaV (nâ¯=â¯3) were found. The RGN RT-PCR assays are useful for detection of enteric viruses other than NoV. This study emphasizes the need for further testing of NoV-negative stool samples in patients with AGE.
Assuntos
Adenoviridae/genética , Fezes/virologia , Gastroenterite/diagnóstico , Gastroenterite/virologia , Mamastrovirus/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Rotavirus/genética , Sapovirus/genética , Adenoviridae/classificação , Adenoviridae/isolamento & purificação , Adulto , Feminino , Humanos , Masculino , Mamastrovirus/classificação , Mamastrovirus/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/normas , Reprodutibilidade dos Testes , Rotavirus/classificação , Rotavirus/isolamento & purificação , Sapovirus/classificação , Sapovirus/isolamento & purificação , Sensibilidade e Especificidade , SuíçaRESUMO
Interference with virus entry is known to be the principle mechanism of HIV neutralization by antibodies, including 2F5 and 4E10, which bind to the membrane-proximal external region (MPER) of the gp41 envelope protein. However, to date, the precise molecular events underlying neutralization by MPER-specific antibodies remain incompletely understood. In this study, we investigated the capacity of these antibodies to irrevocably sterilize HIV virions. Long-term effects of antibodies on virions can differ, rendering neutralization either reversible or irreversible. MPER-specific antibodies irreversibly neutralize virions, and this capacity is associated with induction of gp120 shedding. Both processes have similar thermodynamic properties and slow kinetics requiring several hours. Antibodies directed to the CD4 binding site, V3 loop, and the MPER can induce gp120 shedding, and shedding activity is detected with high frequency in plasma from patients infected with divergent genetic HIV-1 subtypes. Importantly, as we show in this study, induction of gp120 shedding is closely associated with MPER antibody inhibition, constituting either a primary event leading to virion neutralization or representing an immediate consequence thereof, and thus needs to be factored into the mechanistic processes underlying their activity.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp41 do Envelope de HIV/imunologia , HIV-1/imunologia , Eliminação de Partículas Virais/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/fisiologia , Western Blotting , Proteína gp120 do Envelope de HIV/fisiologia , Proteína gp41 do Envelope de HIV/fisiologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , Humanos , Cinética , Testes de Neutralização , Vírion/imunologia , Eliminação de Partículas Virais/fisiologiaRESUMO
OBJECTIVE: Using clinical isolates from a recent passive immunization trial with antibody 2G12, we probed the capacity of frequently used neutralization formats - the primary peripheral blood mononuclear cell (PBMC)-based and the TZM-bl cell-based assay systems - to predict in-vivo activity of 2G12. DESIGN: Antibodies and entry inhibitors of established efficacy were used to neutralize HIV-1 isolates in different in-vitro assay setups. METHODS: Single round infection with Env-pseudotyped and multiple round infection with replication-competent virus was studied on PBMCs and a variety of engineered cell lines. RESULTS: Six out of 12 isolates with high sensitivity to 2G12 in the replication-competent PBMC assay lacked sensitivity to the monoclonal antibody in the env-pseudotype TZM-bl assay. Outcome of passive immunization with 2G12 corroborated the PBMC-assay in-vitro data, as escape mutations to 2G12 emerged, proving the monoclonal antibody's impact on HIV in vivo. Failure to inhibit pseudotype infection of TZM-bl was not due to sequence differences or the pseudotype infection per se, as infection of PBMCs with the identical pseudotyped viruses was sensitive to 2G12 inhibition. Similar shifts in efficacy, though less extreme, were noted for other neutralizing antibodies and inhibitors. Exploration of causes for the observed differences between assay systems revealed that both target cell and virus producer properties influence sensitivity of virus entry to inhibition. CONCLUSION: Our observation that neutralization assay systems employing engineered reporter cell lines can miss in-vivo relevant neutralizing activities strongly argues that preclinical assessment should not be restricted to a single assay type.