RESUMEN
Crimean-Congo hemorrhagic fever virus (CCHFV) is an important human pathogen. In cell culture, CCHFV is sensed by the cytoplasmic RNA sensor retinoic acid-inducible gene I (RIG-I) molecule and its adaptor molecule mitochondrial antiviral signaling (MAVS) protein. MAVS initiates both type I interferon (IFN-I) and proinflammatory responses. Here, we studied the role MAVS plays in CCHFV infection in mice in both the presence and absence of IFN-I activity. MAVS-deficient mice were not susceptible to CCHFV infection when IFN-I signaling was active and showed no signs of disease. When IFN-I signaling was blocked by antibody, MAVS-deficient mice lost significant weight, but were uniformly protected from lethal disease, whereas all control mice succumbed to infection. Cytokine activity in the infected MAVS-deficient mice was markedly blunted. Subsequent investigation revealed that CCHFV infected mice lacking TNF-α receptor signaling (TNFA-R-deficient), but not IL-6 or IL-1 activity, had more limited liver injury and were largely protected from lethal outcomes. Treatment of mice with an anti-TNF-α neutralizing antibody also conferred partial protection in a post-virus exposure setting. Additionally, we found that a disease causing, but non-lethal strain of CCHFV produced more blunted inflammatory cytokine responses compared to a lethal strain in mice. Our work reveals that MAVS activation and cytokine production both contribute to CCHFV pathogenesis, potentially identifying new therapeutic targets to treat this disease.
Asunto(s)
Virus de la Fiebre Hemorrágica de Crimea-Congo , Fiebre Hemorrágica de Crimea , Animales , Citocinas , Modelos Animales de Enfermedad , Virus de la Fiebre Hemorrágica de Crimea-Congo/genética , Ratones , Ratones Noqueados , Índice de Severidad de la Enfermedad , Inhibidores del Factor de Necrosis TumoralRESUMEN
Adintrevimab is a human immunoglobulin G1 monoclonal antibody engineered to have broad neutralization against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants and other SARS-like coronaviruses with pandemic potential. In both Syrian golden hamster and rhesus macaque models, prophylactic administration of a single dose of adintrevimab provided protection against SARS-CoV-2/WA1/2020 infection in a dose-dependent manner, as measured by significant reductions in lung viral load and virus-induced lung pathology, and by inhibition of viral replication in the upper and lower respiratory tract.
Asunto(s)
COVID-19 , SARS-CoV-2 , Cricetinae , Animales , Humanos , COVID-19/prevención & control , Anticuerpos Monoclonales/uso terapéutico , Macaca mulatta , Pulmón/patología , Mesocricetus , Anticuerpos Antivirales/uso terapéutico , Glicoproteína de la Espiga del CoronavirusRESUMEN
BACKGROUND: Next-generation sequencing (NGS) is revolutionizing a variety of molecular biology fields including bioforensics, biosurveillance, and infectious disease diagnostics. For pathogen detection, the ability to sequence all nucleic acids in a sample allows near limitless multiplexability, free from a priori knowledge regarding an etiologic agent as is typically required for targeted molecular assays such as real-time PCR. Furthermore, sequencing capabilities can generate in depth genomic information, allowing detailed molecular epidemiological studies and bioforensics analysis, which is critical for source agent identification in a biothreat outbreak. However, lack of analytical specificity, inherent to NGS, presents challenges for regulated applications such as clinical diagnostics and molecular attribution. CONTENT: Here, we discuss NGS applications in the context of preparedness and biothreat readiness. Specifically, we investigate current and future applications of NGS technologies to affect the fields of biosurveillance, bioforensics, and clinical diagnostics with specific focus on biodefense. SUMMARY: Overall, there are many advantages to the implementation of NGS for preparedness and readiness against biowarfare agents, from forensics to diagnostics. However, appropriate caveats must be associated with any technology. This includes NGS. While NGS is not the panacea replacing all molecular techniques, it will greatly enhance the ability to detect, characterize, and diagnose biowarfare agents, thus providing an excellent addition to the biodefense toolbox of biosurveillance, bioforensics, and biothreat diagnosis.
Asunto(s)
Armas Biológicas , Bioterrorismo/prevención & control , Enfermedades Transmisibles/diagnóstico , Ciencias Forenses/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Biovigilancia/métodos , Biología Computacional , Secuenciación de Nucleótidos de Alto Rendimiento/estadística & datos numéricos , HumanosRESUMEN
BACKGROUND: Research with high biocontainment pathogens such as Rift Valley fever virus (RVFV) and Lassa virus (LASV) is expensive, potentially hazardous, and limited to select institutions. Surrogate pathogens such as Punta Toro virus (PTV) for RVFV infection and Pichinde virus (PICV) for LASV infection allow research to be performed under more permissive BSL-2 conditions. Although used as infection models, PTV and PICV have no standard real-time RT-qPCR assays to detect and quantify pathogenesis. PTV is also a human pathogen, making a standardized detection assay essential for biosurveillance. Here, we developed and characterized two real-time RT-qPCR assays for PICV and PTV by optimizing assay conditions and measuring the limit of detection (LOD) and performance in multiple clinical matrices. METHODS: Total nucleic acid from virus-infected Vero E6 cells was used to optimize TaqMan-minor groove binder (MGB) real-time RT-qPCR assays. A 10-fold dilution series of nucleic acid was used to perform analytical experiments with 60 replicates used to confirm assay LODs. Serum and whole blood spiked with 10-fold dilutions of PTV and PICV virus were assessed as matrices in a mock clinical context. The Cq, or cycle at which the fluoresce of each sample first crosses a threshold line, was determined using the second derivative method using Roche LightCycler 480 software version 1.5.1. Digital droplet PCR (ddPCR) was utilized to quantitatively determine RNA target counts/µl for PTV and PICV. RESULTS: Optimized PTV and PICV assays had LODs of 1000 PFU/ml and 100 PFU/ml, respectively, and this LOD was confirmed in 60/60 (PTV) and 58/60 (PICV) positive replicates. Preliminary mock clinical LODs remained consistent in serum and whole blood for PTV and PICV at 1000 PFU/ml and 100 PFU/ml. An exclusivity panel showed no cross reaction with near neighbors. CONCLUSIONS: PTV and PICV Taq-man MGB based real-time RT-qPCR assays developed here showed relevant sensitivity and reproducibility in samples extracted from a variety of clinical matrices. These assays will be useful as a standard by researchers for future experiments utilizing PTV and PICV as infection models, offering the ability to track infection and viral replication kinetics during research studies.
Asunto(s)
Infecciones por Arenaviridae/diagnóstico , Infecciones por Bunyaviridae/diagnóstico , Phlebovirus/aislamiento & purificación , Virus Pichinde/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Virología/métodos , Humanos , Técnicas de Diagnóstico Molecular/métodos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
The activation of a high affinity Ca(2+) influx system (HACS) in the plasma membrane is required for survival of yeast cells exposed to natural or synthetic inhibitors of essential processes (secretory protein folding or sterol biosynthesis) in the endoplasmic reticulum (ER). The mechanisms linking ER stress to HACS activation are not known. Here we show that Kch1, a recently identified low affinity K(+) transporter in the plasma membrane of Saccharomyces cerevisiae, is up-regulated in response to several ER stressors and necessary for HACS activation. The activation of HACS required extracellular K(+) and was also dependent on the high affinity K(+) transporters Trk1 and Trk2. However, a paralog of Kch1 termed Kch2 was not expressed and not necessary for HACS activation in these conditions. The pathogenic yeast Candida albicans carries only one homolog of Kch1/Kch2, and homozygous knock-out mutants were similarly deficient in the activation of HACS during the responses to tunicamycin. However, the Kch1 homolog was not necessary for HACS activation or cell survival in response to several clinical antifungals (azoles, allylamines, echinocandins) that target the ER or cell wall. Thus, Kch1 family proteins represent a conserved linkage between HACS and only certain classes of ER stress in these yeasts.
Asunto(s)
Candida albicans/genética , Proteínas de Transporte de Catión/genética , Estrés del Retículo Endoplásmico , Saccharomyces cerevisiae/genética , Calcio/metabolismo , Canales de Calcio/metabolismo , Señalización del Calcio/genética , Candida albicans/fisiología , Proteínas de Transporte de Catión/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Potasio/metabolismo , Saccharomyces cerevisiae/fisiología , Transducción de Señal/genéticaRESUMEN
In the budding yeast Saccharomyces cerevisiae, mating pheromones activate a high-affinity Ca(2+) influx system (HACS) that activates calcineurin and is essential for cell survival. Here we identify extracellular K(+) and a homologous pair of transmembrane proteins, Kch1 and Kch2 (Prm6), as necessary components of the HACS activation mechanism. Expression of Kch1 and especially Kch2 was strongly induced during the response to mating pheromones. When forcibly overexpressed, Kch1 and Kch2 localized to the plasma membrane and activated HACS in a fashion that depended on extracellular K(+) but not pheromones. They also promoted growth of trk1 trk2 mutant cells in low K(+) environments, suggesting they promote K(+) uptake. Voltage-clamp recordings of protoplasts revealed diminished inward K(+) currents in kch1 kch2 double-mutant cells relative to the wild type. Conversely, heterologous expression of Kch1 in HEK293T cells caused the appearance of inwardly rectifying K(+) currents. Collectively, these findings suggest that Kch1 and Kch2 directly promote K(+) influx and that HACS may electrochemically respond to K(+) influx in much the same way as the homologous voltage-gated Ca(2+) channels in most animal cell types.
Asunto(s)
Señalización del Calcio , Proteínas de Transporte de Catión/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Canales de Calcio/metabolismo , Cloruro de Calcio/metabolismo , Proteínas de Transporte de Catión/genética , Expresión Génica , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Factor de Apareamiento , Potenciales de la Membrana , Técnicas de Placa-Clamp , Péptidos/fisiología , Potasio/metabolismo , Transporte de Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Antimicrobial resistance (AR) is one of the greatest threats to global health and is associated with higher treatment costs, longer hospital stays, and increased mortality. Current gold standard antimicrobial susceptibility tests (AST) rely on organism growth rates that result in prolonged time-to-answer for slow growing organisms. Changes in the cellular transcriptome can be rapid in the presence of stressors such as antibiotic pressure, providing the opportunity to develop AST towards transcriptomic signatures. Here, we show that relative quantification of the recA gene is an indicator of pathogen susceptibly when select species are challenged with relevant concentrations of ciprofloxacin. We demonstrate that ciprofloxacin susceptible strains of Y. pestis and B. anthracis have significant increases in relative recA gene expression after 15 min of exposure while resistant strains show no significant differences. Building upon this data, we designed and optimized seven duplex RT-qPCR assays targeting the recA and 16S rRNA gene, response and housekeeping genes, respectively, for multiple biothreat and ESKAPE pathogens. Final evaluation of all seven duplex assays tested against 124 ciprofloxacin susceptible and resistant strains, including Tier 1 pathogens, demonstrated an overall categorical agreement compared to microbroth dilution of 97% using a defined cutoff. Testing pathogen strains commonly associated with urinary tract infections in contrived mock sample sets demonstrated an overall categorical agreement of 96%. These data indicate relative quantification of a single highly conserved gene accurately determines susceptibility for multiple bacterial species in response to ciprofloxacin.
Asunto(s)
Bacillus anthracis , Infecciones Urinarias , Yersinia pestis , Humanos , Ciprofloxacina/farmacología , ARN Ribosómico 16S , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Infecciones Urinarias/tratamiento farmacológico , Pruebas de Sensibilidad MicrobianaRESUMEN
Rapid pathogen identification is a critical first step in patient isolation, treatment, and controlling an outbreak. Real-time PCR is a highly sensitive and specific approach commonly used for infectious disease diagnostics. However, mismatches in the primer or probe sequence and the target organism can cause decreased sensitivity, assay failure, and false negative results. Limited genomic sequences for rare pathogens such as Ebola virus (EBOV) can negatively impact assay performance due to undiscovered genetic diversity. We previously developed and validated several EBOV assays prior to the 2013-2016 EBOV outbreak in West Africa, and sequencing EBOV Makona identified sequence variants that could impact assay performance. Here, we assessed the impact sequence mismatches have on EBOV assay performance, finding one or two primer or probe mismatches resulted in a range of impact from minimal to almost two log sensitivity reduction. Redesigning this assay improved detection of all EBOV variants tested. Comparing the performance of the new assay with the previous assays across a panel of human EBOV samples confirmed increased assay sensitivity as reflected in decreased Cq values with detection of three positive that tested negative with the original assay.
Asunto(s)
Ebolavirus , Fiebre Hemorrágica Ebola , Humanos , Ebolavirus/genética , África Occidental , Brotes de Enfermedades , GenómicaRESUMEN
Human monkeypox (mpox) has recently become a global public health emergency; however, assays that detect mpox infection are not widely available, largely due to cross-reactivity within the Orthopoxvirus genus. Immunoassay development was largely confined to researchers who focus on biothreats and endemic areas (Central and West Africa) until the 2022 outbreak. As was noted in the COVID-19 pandemic, antigen detection assays, integrated with molecular assays, are necessary to help curb the spread of disease. Antigen-detecting immunoassays offer the advantage of providing results ranging from within min to h and in lateral flow formats; they can be deployed for point-of-care, home, or field use. This study reports the development of an mpox-specific antigen detection immunoassay developed on a multiplexed, magnetic-bead-based platform utilizing reagents from all research sectors (commercial, academic, and governmental). Two semi-quantitative assays were developed in parallel and standardized with infectious mpox virus (MPXV) cell culture fluid and MPXV-positive non-human primate (NHP) sera samples. These assays could detect viral antigens in serum, were highly specific toward MPXV as compared to other infectious orthopoxviruses (vaccinia virus, cowpox virus, and camelpox virus), and exhibited a correlation with quantitative PCR results from an NHP study. Access to a toolbox of assays for mpox detection will be key for identifying cases and ensuring proper treatment, as MPXV is currently a global traveler.
RESUMEN
Next-generation sequencing is rapidly finding footholds in numerous microbiological fields, including infectious disease diagnostics. Here, we describe a molecular inversion probe panel for the identification of bacterial, viral, and parasitic pathogens. We describe the ability of Illumina and Oxford Nanopore Technologies (ONT) to sequence small amplicons originating from this panel for the identification of pathogens in complex matrices. The panel correctly classified 31 bacterial pathogens directly from positive blood culture bottles with a genus-level concordance of 96.7% and 90.3% on the Illumina and ONT platforms, respectively. Both sequencing platforms detected 18 viral and parasitic organisms directly from mock clinical samples of plasma and whole blood at concentrations of 104 PFU/mL with few exceptions. In general, Illumina sequencing exhibited greater read counts with lower percent mapped reads; however, this resulted in no effect on limits of detection compared with ONT sequencing. Mock clinical evaluation of the probe panel on the Illumina and ONT platforms resulted in positive predictive values of 0.91 and 0.88 and negative predictive values of 1 and 1 from de-identified human chikungunya virus samples compared with gold standard quantitative RT-PCR. Overall, these data show that molecular inversion probes are an adaptable technology capable of pathogen detection from complex sample matrices on current next-generation sequencing platforms.
Asunto(s)
Secuenciación de Nanoporos , Nanoporos , Bacterias/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Sondas MolecularesRESUMEN
The emergence of SARS-CoV-2 and the subsequent pandemic has highlighted the need for animal models that faithfully replicate the salient features of COVID-19 disease in humans. These models are necessary for the rapid selection, testing, and evaluation of potential medical countermeasures. Here, we performed a direct comparison of two distinct routes of SARS-CoV-2 exposure-combined intratracheal/intranasal and small particle aerosol-in two nonhuman primate species, rhesus and cynomolgus macaques. While all four experimental groups displayed very few outward clinical signs, evidence of mild to moderate respiratory disease was present on radiographs and at necropsy. Cynomolgus macaques exposed via the aerosol route also developed the most consistent fever responses and had the most severe respiratory disease and pathology. This study demonstrates that while all four models produced suitable representations of mild COVID-like illness, aerosol exposure of cynomolgus macaques to SARS-CoV-2 produced the most severe disease, which may provide additional clinical endpoints for evaluating therapeutics and vaccines.
Asunto(s)
COVID-19 , Aerosoles , Animales , Modelos Animales de Enfermedad , Macaca fascicularis , SARS-CoV-2 , Índice de Severidad de la EnfermedadRESUMEN
PURPOSE OF REVIEW: This review is aimed at highlighting recent research and articles on the complicated relationship between virus, vector, and host and how biosurveillance at each level informs disease spread and risk. RECENT FINDINGS: While human cases of CCHFV and tick identification in non-endemic areas in 2019-2020 were reported to sites such as ProMed, there is a gap in recent published literature on these and broader CCHFV surveillance efforts from the late 2010s. SUMMARY: A review of the complex aspects of CCHFV maintenance in the environment coupled with high fatality rate and lack of vaccines and therapeutics warrants the need for a One-Health approach toward detection and increased biosurveillance programs for CCHFV.
RESUMEN
Ebola virus is a continuing threat to human populations, causing a virulent hemorrhagic fever disease characterized by dysregulation of both the innate and adaptive host immune responses. Severe cases are distinguished by an early, elevated pro-inflammatory response followed by a pronounced lymphopenia with B and T cells unable to mount an effective anti-viral response. The precise mechanisms underlying the dysregulation of the host immune system are poorly understood. In recent years, focus on host-derived miRNAs showed these molecules to play an important role in the host gene regulation arsenal. Here, we describe an investigation of RNA biomarkers in the fatal Ebola virus disease (EVD) cynomolgus macaque model. We monitored both host mRNA and miRNA responses in whole blood longitudinally over the disease course in these non-human primates (NHPs). Analysis of the interactions between these classes of RNAs revealed several miRNA markers significantly correlated with downregulation of genes; specifically, the analysis revealed those involved in dysregulated immune pathways associated with EVD. In particular, we noted strong interactions between the miRNAs hsa-miR-122-5p and hsa-miR-125b-5p with immunological genes regulating both B and T-cell activation. This promising set of biomarkers will be useful in future studies of severe EVD pathogenesis in both NHPs and humans and may serve as potential prognostic targets.
RESUMEN
Next-generation sequencing (NGS) for infectious disease diagnostics is a relatively new and underdeveloped concept. If this technology is to become a regulatory-grade clinical diagnostic, standardization in the form of locked-down assays and firmly established underlying processes is necessary. Targeted sequencing, specifically by amplification of genomic signatures, has the potential to bridge the gap between PCR- and NGS-based diagnostics; however, existing NGS assay panels lack validated analytical techniques to adjudicate high background and error-prone NGS data. Herein, we present the Diagnostic targETEd seQuencing adjudicaTion (DETEQT) software, consisting of an intuitive bioinformatics pipeline entailing a set of algorithms to translate raw sequencing data into positive, negative, and indeterminate diagnostic determinations. After basic read filtering and mapping, the software compares abundance and quality metrics against heuristic and fixed thresholds. A novel generalized quality function provides an amalgamated quality score for the match between sequence reads of an assay and panel targets, rather than considering each component factor independently. When evaluated against numerous assay samples and parameters (mock clinical, human, and nonhuman primate clinical data sets; diverse amplification strategies; downstream applications; and sequence platforms), DETEQT demonstrated improved rejection of false positives and accuracies >95%. Finally, DETEQT was implemented in the user-friendly Empowering the Development of Genomics Expertise (EDGE) bioinformatics platform, providing a complete, end-to-end solution that can be operated by nonexperts in a clinical laboratory setting.
Asunto(s)
Enfermedades Transmisibles/diagnóstico , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Programas Informáticos , Algoritmos , Biblioteca de Genes , Genómica/métodos , HumanosRESUMEN
Targeted sequencing promises to bring next-generation sequencing (NGS) into routine clinical use for infectious disease diagnostics. In this context, upfront processing techniques, including pathogen signature enrichment, must amplify multiple targets of interest for NGS to be relevant when applied to patient samples with limited volumes. Here, we demonstrate an optimized molecular inversion probe (MIP) assay targeting multiple variable regions within the 16S ribosomal gene for the identification of biothreat and ESKAPE pathogens in a process that significantly reduces complexity, labor, and processing time. Probes targeting the Klebsiella pneumoniae carbapenemase (KPC) antibiotic resistance (AR) gene were also included to demonstrate the ability to concurrently identify etiologic agent and ascertain valuable secondary genetic information. Our assay captured gene sequences in 100% of mock clinical samples prepared from flagged positive blood culture bottles. Using a simplified processing and adjudication method for mapped sequencing reads, genus and species level concordance was 100% and 80%, respectively. In addition, sensitivity and specificity for KPC gene detection was 100%. Our MIP assay produced sequenceable amplicons for the identification of etiologic agents and the detection of AR genes directly from blood culture bottles in a simplified single tube assay.
Asunto(s)
Proteínas Bacterianas/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Técnicas de Diagnóstico Molecular/métodos , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN/métodos , beta-Lactamasas/genética , Sangre/microbiología , Secuenciación de Nucleótidos de Alto Rendimiento/normas , Humanos , Klebsiella/genética , Klebsiella/patogenicidad , Técnicas de Diagnóstico Molecular/normas , Análisis de Secuencia de ADN/normasRESUMEN
Development and implementation of rapid antimicrobial susceptibility testing is critical for guiding patient care and improving clinical outcomes, especially in cases of sepsis. One approach to reduce the time-to-answer for antimicrobial susceptibility is monitoring the inhibition of DNA production, as differences in DNA concentrations are more quickly impacted compared to optical density changes in traditional antimicrobial susceptibility testing. Here, we use real-time PCR to rapidly determine antimicrobial susceptibility after short incubations with antibiotic. Application of this assay to a collection of 144 isolates in mock blood culture, covering medically relevant pathogens displaying high rates of resistance, provided susceptibility data in under 4 hours. This assay provided categorical agreement with a reference method in 96.3% of cases across all species. Sequencing of a subset of PCR amplicons showed accurate genus level identification. Overall, implementation of this method could provide accurate susceptibility results with a reduced time-to-answer for a number of medically relevant bacteria commonly isolated from blood culture.
Asunto(s)
Antibacterianos/farmacología , Cultivo de Sangre , Pruebas de Sensibilidad Microbiana/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa , Bacterias/efectos de los fármacos , Bacterias/genética , Análisis de Secuencia , Factores de TiempoRESUMEN
Glycosylation reactions in the Golgi complex and the endoplasmic reticulum utilize nucleotide sugars as donors and produce inorganic phosphate (Pi) and acid (H+) as byproducts. Here we show that homologs of mammalian XPR1 and TMEM165 (termed Erd1 and Gdt1) recycle luminal Pi and exchange luminal H+ for cytoplasmic Ca2+, respectively, thereby promoting growth of yeast cells in low Pi and low Ca2+ environments. As expected for reversible H+/Ca2+ exchangers, Gdt1 also promoted growth in high Ca2+ environments when the Golgi-localized V-ATPase was operational but had the opposite effect when the V-ATPase was eliminated. Gdt1 activities were negatively regulated by calcineurin signaling and by Erd1, which recycled the Pi byproduct of glycosylation reactions and prevented the loss of this nutrient to the environment via exocytosis. Thus, Erd1 transports Pi in the opposite direction from XPR1 and other EXS family proteins and facilitates byproduct removal from the Golgi complex together with Gdt1.
Asunto(s)
Canales de Calcio/genética , Retículo Endoplásmico/genética , Aparato de Golgi/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos/genética , Antiportadores , Calcineurina/genética , Calcineurina/metabolismo , Calcio/metabolismo , Proteínas de Transporte de Catión , Retículo Endoplásmico/metabolismo , Glicosilación , Aparato de Golgi/genética , Humanos , Proteínas de la Membrana/genética , Nucleótidos/genética , Nucleótidos/metabolismo , Fosfatos/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Virales/genética , Transducción de Señal/genética , Receptor de Retrovirus Xenotrópico y PolitrópicoRESUMEN
Antibiotic resistance (AR) is an epidemic of increasing magnitude requiring rapid identification and profiling for appropriate and timely therapeutic measures and containment strategies. In this context, ciprofloxacin is part of the first-line of countermeasures against numerous high consequence bacteria. Significant resistance can occur via single nucleotide polymorphisms (SNP) and deletions within ciprofloxacin targeted genes. Ideally, use of ciprofloxacin would be prefaced with AR determination to avoid overuse or misuse of the antibiotic. Here, we describe the development and evaluation of a panel of 44 single-stranded molecular inversion probes (MIPs) coupled to next-generation sequencing (NGS) for the detection of genetic variants known to confer ciprofloxacin resistance in Bacillus anthracis, Yersinia pestis, and Francisella tularensis. Sequencing results demonstrate MIPs capture and amplify targeted regions of interest at significant levels of coverage. Depending on the genetic variant, limits of detection (LOD) for high-throughput pooled sequencing ranged from approximately 300-1800 input genome copies. LODs increased 10-fold in the presence of contaminating human genome DNA. In addition, we show that MIPs can be used as an enrichment step with high resolution melt (HRM) real-time PCR which is a sensitive assay with a rapid time-to-answer. Overall, this technology is a multiplexable upfront enrichment applicable with multiple downstream molecular assays for the detection of targeted genetic regions.