RESUMO
The preparation of extremely thin samples, which are required for high-resolution electron microscopy, poses extreme risk of damaging biological macromolecules due to interactions with the air-water interface. Although the rapid increase in the number of published structures initially gave little indication that this was a problem, the search for methods that substantially mitigate this hazard is now intensifying. The two main approaches under investigation are (a) immobilizing particles onto structure-friendly support films and (b) reducing the length of time during which such interactions may occur. While there is little possibility of outrunning diffusion to the interface, intentional passivation of the interface may slow the process of adsorption and denaturation. In addition, growing attention is being given to gaining more effective control of the thickness of the sample prior to vitrification.
Assuntos
Microscopia Crioeletrônica/instrumentação , Microscopia Crioeletrônica/métodos , Complexos Multiproteicos/química , Ar , Carbono/química , Difusão , Grafite/química , Lipídeos/química , Complexos Multiproteicos/isolamento & purificação , Desnaturação Proteica , Manejo de Espécimes/métodos , Estreptavidina/química , ÁguaRESUMO
Addressing interfacial effects during specimen preparation in cryogenic electron microscopy remains challenging. Here we introduce ESI-cryoPrep, a specimen preparation method based on electrospray ionization in native mass spectrometry, designed to alleviate issues associated with protein denaturation or preferred orientation induced by macromolecule adsorption at interfaces. Through fine-tuning spraying parameters, we optimized protein integrity preservation and achieved the desired ice thickness for analyzing target macromolecules. With ESI-cryoPrep, we prepared high-quality cryo-specimens of five proteins and obtained three-dimensional reconstructions at near-atomic resolution. Our findings demonstrate that ESI-cryoPrep effectively confines macromolecules within the middle of the thin layer of amorphous ice, facilitating the preparation of blotting-free vitreous samples. The protective mechanism, characterized by the uneven distribution of charged biomolecules of varying sizes within charged droplets, prevents the adsorption of target biomolecules at air-water or graphene-water interfaces, thereby avoiding structural damage to the protein particles or the introduction of dominant orientation issues.
Assuntos
Microscopia Crioeletrônica , Manejo de Espécimes , Espectrometria de Massas por Ionização por Electrospray , Microscopia Crioeletrônica/métodos , Manejo de Espécimes/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Proteínas/química , Humanos , Substâncias Macromoleculares/químicaRESUMO
Cryo-electron tomography (cryo-ET) is a revolutionary technique for resolving the structure of subcellular organelles and macromolecular complexes in their cellular context. However, the application of the cryo-ET is hampered by the sample preparation step. Performing cryo-focused ion beam milling at an arbitrary position on the sample is inefficient, and the target of interest is not guaranteed to be preserved when thinning the cell from several micrometers to less than 300 nm thick. Here, we report a cryogenic correlated light, ion and electron microscopy (cryo-CLIEM) technique that is capable of preparing cryo-lamellae under the guidance of three-dimensional confocal imaging. Moreover, we demonstrate a workflow to preselect and preserve nanoscale target regions inside the finished cryo-lamellae. By successfully preparing cryo-lamellae that contain a single centriole or contact sites between subcellular organelles, we show that this approach is generally applicable, and shall help in innovating more applications of cryo-ET.
Assuntos
Tomografia com Microscopia Eletrônica , Manejo de Espécimes , Tomografia com Microscopia Eletrônica/métodos , Substâncias Macromoleculares/química , Manejo de Espécimes/métodos , Elétrons , Imageamento Tridimensional/métodos , Microscopia Crioeletrônica/métodosRESUMO
Nearly seventy percent of diagnostic lab test errors occur due to variability in preanalytical factors. These are the parameters involved with all aspects of tissue processing, starting from the time tissue is collected from the patient in the operating room, until it is received and tested in the laboratory. While there are several protocols for transporting fixed tissue, organs, and liquid biopsies, such protocols are lacking for transport and handling of live solid tumor tissue specimens. There is a critical need to establish preanalytical protocols to reduce variability in biospecimen integrity and improve diagnostics for personalized medicine. Here, we provide a comprehensive protocol for the standard collection, handling, packaging, cold-chain logistics, and receipt of solid tumor tissue biospecimens to preserve tissue viability.
Assuntos
Neoplasias , Manejo de Espécimes , Humanos , Neoplasias/diagnóstico , Manejo de Espécimes/métodos , Medicina de Precisão/métodosRESUMO
Skin microbiome sampling is currently performed with tools such as swabs and tape strips to collect microbes from the skin surface. However, these conventional approaches may be unable to detect microbes deeper in the epidermis or in epidermal invaginations. We describe a sampling tool with a depth component, a transepidermal microprojection array (MPA), which captures microbial biomass from both the epidermal surface and deeper skin layers. We leveraged the rapid customizability of 3D printing to enable systematic optimization of MPA for human skin sampling. Evaluation of sampling efficacy on human scalp revealed the optimized MPA was comparable in sensitivity to swab and superior to tape strip, especially for nonstandard skin surfaces. We observed differences in species diversity, with the MPA detecting clinically relevant fungi more often than other approaches. This work delivers a tool in the complex field of skin microbiome sampling to potentially address gaps in our understanding of its role in health and disease.
Assuntos
Epiderme , Microbiota , Impressão Tridimensional , Manejo de Espécimes , Análise Serial de Tecidos , Epiderme/microbiologia , Humanos , Manejo de Espécimes/métodosRESUMO
The goals of the Earth Biogenome Project-to sequence the genomes of all eukaryotic life on earth-are as daunting as they are ambitious. The Darwin Tree of Life Project was founded to demonstrate the credibility of these goals and to deliver at-scale genome sequences of unprecedented quality for a biogeographic region: the archipelago of islands that constitute Britain and Ireland. The Darwin Tree of Life Project is a collaboration between biodiversity organizations (museums, botanical gardens, and biodiversity institutes) and genomics institutes. Together, we have built a workflow that collects specimens from the field, robustly identifies them, performs sequencing, generates high-quality, curated assemblies, and releases these openly for the global community to use to build future science and conservation efforts.
Assuntos
Eucariotos/genética , Genômica/métodos , Biodiversidade , Evolução Biológica , Evolução Molecular , Genoma/genética , Filogenia , Seleção Genética/genética , Análise de Sequência de DNA/métodos , Manejo de Espécimes/métodos , Manejo de Espécimes/normas , Fluxo de TrabalhoRESUMO
Collagen from paleontological bones is an important organic material for isotopic measurement, radiocarbon analysis, and paleoproteomic analysis to provide information on diet, dating, taxonomy, and phylogeny. Current paleoproteomic methods are destructive and require from a few milligrams to several tens of milligrams of bone for analysis. In many cultures, bones are raw materials for artifacts that are conserved in museums, which hampers damage to these precious objects during sampling. Here, we describe a low-invasive sampling method that identifies collagen, taxonomy, and post-translational modifications from Holocene and Upper Pleistocene bones dated to 130,000 and 150 BC using dermatological skin tape discs for sampling. The sampled bone micropowders were digested following our highly optimized enhanced filter-aided sample preparation protocol and then analyzed by MALDI FTICR MS and LC-MS/MS for identifying the genus taxa of the bones. We show that this low-invasive sampling does not deteriorate the bones and achieves results similar to those obtained by more destructive sampling. Moreover, this sampling method can be carried out at archeological sites or in museums.
Assuntos
Osso e Ossos , Colágeno , Fósseis , Paleontologia , Proteômica , Osso e Ossos/química , Proteômica/métodos , Paleontologia/métodos , Animais , Colágeno/química , Colágeno/análise , Arqueologia/métodos , Manejo de Espécimes/métodos , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Processamento de Proteína Pós-Traducional , HumanosRESUMO
Triacylglycerols and wax esters are two lipid classes that have been linked to diseases, including autism, Alzheimer's disease, dementia, cardiovascular disease, dry eye disease, and diabetes, and thus are molecules worthy of biomarker exploration studies. Since triacylglycerols and wax esters make up the majority of skin-surface lipid secretions, a viable sampling method for these potential biomarkers would be that of groomed latent fingerprints. Currently, however, blood-based sampling protocols predominate in the field. The invasiveness of a blood draw limits its utility to protected populations, including children and the elderly. Herein we describe a noninvasive means for sample collection (from fingerprints) paired with fast MS data-acquisition (MassIVE data set MSV000092742) and efficient data analysis via machine learning. Using both supervised and unsupervised classification, we demonstrate the usefulness of this method in determining whether a variable of interest imparts measurable change within the lipidomic data set. As a proof-of-concept, we show that the method is capable of distinguishing between the fingerprints of different individuals as well as between anatomical sebum collection regions. This noninvasive, high-throughput approach enables future lipidomic biomarker researchers to more easily include underrepresented, protected populations, such as children and the elderly, thus moving the field closer to definitive disease diagnoses that apply to all.
Assuntos
Biomarcadores , Lipidômica , Aprendizado de Máquina , Humanos , Lipidômica/métodos , Biomarcadores/sangue , Biomarcadores/análise , Espectrometria de Massas/métodos , Triglicerídeos/sangue , Triglicerídeos/análise , Dermatoglifia , Idoso , Criança , Masculino , Feminino , Sebo/metabolismo , Sebo/química , Lipídeos/sangue , Lipídeos/análise , Manejo de Espécimes/métodosRESUMO
Cryo-focussed ion beam (FIB)-milling is a powerful technique that opens up thick, cellular specimens to high-resolution structural analysis by electron cryotomography (cryo-ET). FIB-milled lamellae can be produced from cells on grids, or cut from thicker, high-pressure frozen specimens. However, these approaches can put geometrical constraints on the specimen that may be unhelpful, particularly when imaging structures within the cell that have a very defined orientation. For example, plunge frozen rod-shaped bacteria orient parallel to the plane of the grid, yet the Z-ring, a filamentous structure of the tubulin-like protein FtsZ and the key organiser of bacterial division, runs around the circumference of the cell such that it is perpendicular to the imaging plane. It is therefore difficult or impractical to image many complete rings with current technologies. To circumvent this problem, we have fabricated monolithic gold specimen supports with a regular array of cylindrical wells in a honeycomb geometry, which trap bacteria in a vertical orientation. These supports, which we call "honeycomb gold discs", replace standard EM grids and when combined with FIB-milling enable the production of lamellae containing cross-sections through cells. The resulting lamellae are more stable and resistant to breakage and charging than conventional lamellae. The design of the honeycomb discs can be modified according to need and so will also enable cryo-ET and cryo-EM imaging of other specimens in otherwise difficult to obtain orientations.
Assuntos
Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Ouro , Microscopia Crioeletrônica/métodos , Ouro/química , Tomografia com Microscopia Eletrônica/métodos , Escherichia coli/ultraestrutura , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Manejo de Espécimes/métodosRESUMO
BACKGROUND: Sputum-based testing is a barrier to increasing access to molecular diagnostics for tuberculosis (TB). Many people with TB are unable to produce sputum, and sputum processing increases assay complexity and cost. Tongue swabs are emerging as an alternative to sputum, but performance limits are uncertain. METHODS: From June 2022 to July 2023, we enrolled 397 consecutive adults with cough >2 weeks at 2 health centers in Kampala, Uganda. We collected demographic and clinical information, sputum for TB testing (Xpert MTB/RIF Ultra and 2 liquid cultures), and tongue swabs for same-day quantitative polymerase chain reaction (qPCR) testing. We evaluated tongue swab qPCR diagnostic accuracy versus sputum TB test results, quantified TB targets per swab, assessed the impact of serial swabbing, and compared 2 swab types (Copan FLOQSWAB and Steripack spun polyester). RESULTS: Among 397 participants, 43.1% were female, median age was 33 years, 23.5% were diagnosed with human immunodeficiency virus, and 32.0% had confirmed TB. Sputum Xpert Ultra and tongue swab qPCR results were concordant for 98.2% (95% confidence interval [CI]: 96.2-99.1) of participants. Tongue swab qPCR sensitivity was 92.6% (95% CI: 86.5 to 96.0) and specificity was 99.1% (95% CI: 96.9 to 99.8) versus microbiological reference standard. A single tongue swab recovered a 7-log range of TB copies, with a decreasing recovery trend among 4 serial swabs. Swab types performed equivalently. CONCLUSIONS: Tongue swabs are a promising alternative to sputum for molecular diagnosis of TB, with sensitivity approaching sputum-based molecular tests. Our results provide valuable insights for developing successful tongue swab-based TB diagnostics.
Assuntos
Mycobacterium tuberculosis , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Manejo de Espécimes , Escarro , Língua , Humanos , Feminino , Escarro/microbiologia , Masculino , Uganda , Adulto , Língua/microbiologia , Manejo de Espécimes/métodos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Tuberculose/diagnóstico , Tuberculose/microbiologia , Pessoa de Meia-Idade , Adulto Jovem , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/microbiologiaRESUMO
Endometrial cancer (EC) is the most prevalent gynaecological cancer in high-income countries and its incidence is continuing to rise sharply. Simple and objective tools to reliably detect women with EC are urgently needed. We recently developed and validated the DNA methylation (DNAme)-based women's cancer risk identification-quantitative polymerase chain reaction test for endometrial cancer (WID-qEC) test that could address this need. Here, we demonstrate that the stability of the WID-qEC test remains consistent regardless of: (i) the cervicovaginal collection device and sample media used (Cervex brush and PreservCyt or FLOQSwab and eNAT), (ii) the collector of the specimen (gynaecologist- or patient-based), and (iii) the precise sampling site (cervical, cervicovaginal and vaginal). Furthermore, we demonstrate sample stability in eNAT medium for 7 days at room temperature, greatly facilitating the implementation of the test into diagnostic laboratory workflows. When applying FLOQSwabs (Copan) in combination with the eNAT (Copan) sample collection media, the sensitivity and specificity of the WID-qEC test to detect uterine (i.e., endometrial and cervical) cancers in gynaecologist-taken samples was 92.9% (95% confidence interval [CI] = 75.0%-98.8%) and 98.6% (95% CI = 91.7%-99.9%), respectively, whilst the sensitivity and specificity in patient collected self-samples was 75.0% (95% CI = 47.4%-91.7%) and 100.0% (95% CI = 93.9%-100.0%), respectively. Taken together these data confirm the robustness and clinical potential of the WID-qEC test.
Assuntos
Metilação de DNA , Neoplasias do Endométrio , Humanos , Feminino , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/diagnóstico , Manejo de Espécimes/métodos , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Neoplasias Uterinas/genética , Neoplasias Uterinas/diagnóstico , Idoso , Detecção Precoce de Câncer/métodos , Adulto , Biomarcadores Tumorais/genéticaRESUMO
With the objective to investigate associations between sociodemographic characteristics and participation in interventions designed to increase participation in cervical cancer screening among under-screened women, we randomized a random sample of 6000 women in Norway aged 35-69 years who had not attended cervical screening for ≥10 years to receive either (i) a reminder to attend regular screening (control), (ii) an offer to order a self-sampling kit (opt-in), or (iii) a self-sampling kit unsolicited (send-to-all). We analyzed how sociodemographic characteristics were associated with screening participation within and between screening arms. In the send-to-all arm, increased screening participation ranged from 17.1% (95% confidence interval [95% CI] = 10.3% to 23.8%) to 30.0% (95% CI = 21.5% to 38.6%) between sociodemographic groups. In the opt-in arm, we observed smaller, and at times, non-significant increases within the range 0.7% (95% CI = -5.8% to 7.3%) to 19.1% (95% CI = 11.6% to 26.7%). In send-to-all versus control comparisons, there was greater increase in participation for women in the workforce versus not (6.1%, 95% CI = 1.6% to 10.6%), with higher versus lower income (7.6%, 95% CI = 2.2% to 13.1%), and with university versus primary education (8.5%, 95% CI = 2.4% to 14.6%). In opt-in versus control comparisons, there was greater increase in participation for women in the workforce versus not (4.6%, 95% CI = 0.7% to 8.5%), with higher versus lower income (6.3%, 95% CI = 1.5% to 11.1%), but lower increase for Eastern European versus Norwegian background (-12.7%, 95% CI = -19.7% to -5.7%). Self-sampling increased cervical screening participation across all sociodemographic levels, but inequalities in participation should be considered when introducing self-sampling, especially with the goal to reach long-term non-attending women.
Assuntos
Detecção Precoce de Câncer , Infecções por Papillomavirus , Neoplasias do Colo do Útero , Humanos , Feminino , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/virologia , Neoplasias do Colo do Útero/epidemiologia , Pessoa de Meia-Idade , Noruega/epidemiologia , Adulto , Detecção Precoce de Câncer/métodos , Detecção Precoce de Câncer/estatística & dados numéricos , Idoso , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/virologia , Infecções por Papillomavirus/epidemiologia , Esfregaço Vaginal/métodos , Esfregaço Vaginal/estatística & dados numéricos , Manejo de Espécimes/métodos , Fatores Socioeconômicos , Programas de Rastreamento/métodos , Programas de Rastreamento/estatística & dados numéricos , Fatores Sociodemográficos , Aceitação pelo Paciente de Cuidados de Saúde/estatística & dados numéricosRESUMO
Japan is lagging in cervical cancer prevention. The effectiveness of a self-sampling human papillomavirus (HPV) test, a possible measure to overcome this situation, has not yet been evaluated. A randomized controlled trial was performed to evaluate the effectiveness of a self-sampling HPV test on detection of cervical intraepithelial neoplasia grade 2 or worse (CIN2+) and screening uptake. Women between 30 and 58 years old who did not participate in the cervical cancer screening program for ≥3 years were eligible and assigned to the intervention group (cytology or self-sampling HPV test) or control group (cytology). Participants assigned to the intervention group were sent a self-sampling kit according to their ordering (opt-in strategy). A total of 7337 and 7772 women were assigned to the intervention and control groups, respectively. Screening uptake in the intervention group was significantly higher than that in the control group (20.0% vs. 6.4%; risk ratio: 3.10; 95% confidence interval [CI]: 2.82, 3.42). The compliance rate with cytology triage for HPV-positive women was 46.8% (95% CI: 35.5%, 58.4%). CIN2+ was detected in five and four participants in the intervention and control groups, respectively; there was no difference for intention-to-screen analysis (risk ratio: 1.32; 95% CI: 0.36, 4.93). Self-sampling of HPV test increased screening uptake; however, no difference was observed in the detection of CIN2+, probably due to the low compliance rate for cytology triage in HPV-positive women. Efforts to increase cytology triage are essential to maximize precancer detections.
Assuntos
Detecção Precoce de Câncer , Infecções por Papillomavirus , Displasia do Colo do Útero , Neoplasias do Colo do Útero , Humanos , Feminino , Pessoa de Meia-Idade , Adulto , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/virologia , Infecções por Papillomavirus/epidemiologia , Japão/epidemiologia , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/virologia , Detecção Precoce de Câncer/métodos , Displasia do Colo do Útero/diagnóstico , Displasia do Colo do Útero/virologia , Displasia do Colo do Útero/epidemiologia , Papillomaviridae/isolamento & purificação , Esfregaço Vaginal/métodos , Manejo de Espécimes/métodos , Programas de Rastreamento/métodos , Lesões Pré-Cancerosas/diagnóstico , Lesões Pré-Cancerosas/virologia , Papillomavirus HumanoRESUMO
Metabolites from feces provide important insights into the functionality of the gut microbiome. As immediate freezing is not always feasible in gut microbiome studies, there is a need for sampling protocols that provide the stability of the fecal metabolome and microbiome at room temperature (RT). Here, we investigated the stability of various metabolites and the microbiome (16S rRNA) in feces collected in 95% ethanol (EtOH) and commercially available sample collection kits with specific preservatives OMNImetâ¢GUT/OMNIgeneâ¢GUT. To simulate field-collection scenarios, the samples were stored at different temperatures at varying durations (24 h + 4 °C, 24 h RT, 36 h RT, 48 h RT, and 7 days RT) and compared to aliquots immediately frozen at -80 °C. We applied several targeted and untargeted metabolomics platforms to measure lipids, polar metabolites, endocannabinoids, short-chain fatty acids (SCFAs), and bile acids (BAs). We found that SCFAs in the nonstabilized samples increased over time, while a stable profile was recorded in sample aliquots stored in 95% EtOH and OMNImetâ¢GUT. When comparing the metabolite levels between aliquots stored at room temperature and at +4 °C, we detected several changes in microbial metabolites, including multiple BAs and SCFAs. Taken together, we found that storing samples at RT and stabilizing them in 95% EtOH yielded metabolomic results comparable to those from flash freezing. We also found that the overall composition of the microbiome did not vary significantly between different storage types. However, notable differences were observed in the α diversity. Altogether, the stability of the metabolome and microbiome in 95% EtOH provided results similar to those of the validated commercial collection kits OMNImetâ¢GUT and OMNIgeneâ¢GUT, respectively.
Assuntos
Etanol , Fezes , Microbioma Gastrointestinal , Metabolômica , Etanol/metabolismo , Etanol/análise , Fezes/microbiologia , Fezes/química , Humanos , Manejo de Espécimes/métodos , RNA Ribossômico 16S , TemperaturaRESUMO
The COVID-19 pandemic has highlighted the need for rapid and reliable diagnostics that are accessible in resource-limited settings. To address this pressing issue, we have developed a rapid, portable, and electricity-free method for extracting nucleic acids from respiratory swabs (i.e. nasal, nasopharyngeal and buccal swabs), successfully demonstrating its effectiveness for the detection of SARS-CoV-2 in residual clinical specimens. Unlike traditional approaches, our solution eliminates the need for micropipettes or electrical equipment, making it user-friendly and requiring little to no training. Our method builds upon the principles of magnetic bead extraction and revolves around a low-cost plastic magnetic lid, called SmartLid, in combination with a simple disposable kit containing all required reagents conveniently prealiquoted. Here, we clinically validated the SmartLid sample preparation method in comparison to the gold standard QIAamp Viral RNA Mini Kit from QIAGEN, using 406 clinical isolates, including 161 SARS-CoV-2 positives, using the SARS-CoV-2 RT-qPCR assays developed by the US Centers for Disease Control and Prevention (CDC). The SmartLid method showed an overall sensitivity of 95.03% (95% CI: 90.44-97.83%) and a specificity of 99.59% (95% CI: 97.76-99.99%), with a positive agreement of 97.79% (95% CI: 95.84-98.98%) when compared to QIAGEN's column-based extraction method. There are clear benefits to using the SmartLid sample preparation kit: it enables swift extraction of viral nucleic acids, taking less than 5 min, without sacrificing significant accuracy when compared to more expensive and time-consuming alternatives currently available on the market. Moreover, its simplicity makes it particularly well-suited for the point-of-care where rapid results and portability are crucial. By providing an efficient and accessible means of nucleic acid extraction, our approach aims to introduce a step-change in diagnostic capabilities for resource-limited settings.
Assuntos
COVID-19 , RNA Viral , SARS-CoV-2 , Humanos , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/genética , COVID-19/diagnóstico , COVID-19/virologia , RNA Viral/isolamento & purificação , RNA Viral/análise , Teste de Ácido Nucleico para COVID-19/métodos , Teste de Ácido Nucleico para COVID-19/instrumentação , Manejo de Espécimes/métodos , Teste para COVID-19/métodos , Teste para COVID-19/instrumentação , Técnicas de Diagnóstico Molecular/métodos , Região de Recursos LimitadosRESUMO
Many virus lysis/transport buffers used in molecular diagnostics, including the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, contain guanidine-based chaotropic salts, primarily guanidine hydrochloride (GuHCl) or guanidine isothiocyanate (GITC). Although the virucidal effects of GuHCl and GITC alone against some enveloped viruses have been established, standardized data on their optimum virucidal concentrations against SARS-CoV-2 and effects on viral RNA stability are scarce. Thus, we aimed to determine the optimum virucidal concentrations of GuHCl and GITC against SARS-CoV-2 compared to influenza A virus (IAV), another enveloped respiratory virus. We also evaluated the effectiveness of viral RNA stabilization at the determined optimum virucidal concentrations under high-temperature conditions (35°C) using virus-specific real-time reverse transcription polymerase chain reaction. Both viruses were potently inactivated by 1.0 M GITC and 2.5 M GuHCl, but the GuHCl concentration for efficient SARS-CoV-2 inactivation was slightly higher than that for IAV inactivation. GITC showed better viral RNA stability than GuHCl at the optimum virucidal concentrations. An increased concentration of GuHCl or GITC increased viral RNA degradation at 35°C. Our findings highlight the need to standardize GuHCl and GITC concentrations in virus lysis/transport buffers and the potential application of these guanidine-based salts alone as virus inactivation solutions in SARS-CoV-2 and IAV molecular diagnostics.
Assuntos
Guanidina , Vírus da Influenza A , RNA Viral , SARS-CoV-2 , Manejo de Espécimes , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/genética , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/genética , Guanidina/farmacologia , Guanidina/química , RNA Viral/genética , Humanos , Manejo de Espécimes/métodos , Genoma Viral , COVID-19/virologia , COVID-19/diagnóstico , Chlorocebus aethiops , Células Vero , Inativação de Vírus/efeitos dos fármacos , Animais , Estabilidade de RNA/efeitos dos fármacos , Contenção de Riscos Biológicos , Guanidinas/farmacologia , Guanidinas/química , Sais/farmacologia , Sais/químicaRESUMO
BACKGROUND: Modern high-throughput technologies enable the processing of a large number of samples simultaneously, while also providing rapid and accurate procedures. In recent years, automated liquid handling workstations have emerged as an established technology for reproducible sample preparation. They offer flexibility, making them suitable for an expanding range of applications. Commonly, such approaches are well-developed for experimental procedures primarily designed for cell-line processing and xenobiotics testing. Conversely, little attention is focused on the application of automated liquid handlers in the analysis of whole organisms, which often involves time-consuming laboratory procedures. RESULTS: Here, we present a fully automated workflow for all steps, from RNA extraction to real-time PCR processing, for gene expression quantification in the ascidian marine model Ciona robusta. For procedure validation, we compared the results obtained with the liquid handler with those of the classical manual procedure. The outcome revealed comparable results, demonstrating a remarkable time saving particularly in the initial steps of sample processing. CONCLUSIONS: This work expands the possible application fields of this technology to whole-body organisms, mitigating issues that can arise from manual procedures. By minimizing errors, avoiding cross-contamination, decreasing hands-on time and streamlining the procedure, it could be employed for large-scale screening investigations.
Assuntos
Perfilação da Expressão Gênica , Manejo de Espécimes , Automação , Reação em Cadeia da Polimerase em Tempo Real , Análise em Microsséries , Manejo de Espécimes/métodosRESUMO
A prior analysis suggested that wound swab culture (WSC) results were driving unnecessary antibiotic use in patients who were not already receiving treatment. As a quality-improvement initiative, our laboratory introduced an "exception-reporting" protocol on 1 March 2023, whereby typical wound pathogens susceptible to recommended empiric therapy (flucloxacillin/cefalexin) were not reported, and a comment was provided, stating no significant resistant organisms had been detected. Full results were available to clinicians on request. Cultures falling outside protocol criteria were reported in the standard fashion. This analysis sought to assess the effect of exception-reporting on post-report antibiotic initiation (PRAI). All community WSC results were matched to antibiotic dispensing records from October 2021 to December 2023. Sampling without treatment pre-report was termed "test and wait" (TaW). Following TaW, PRAI was identified if antibiotics were started within 5 days post-report. There were 1,819 and 764 WSCs received in the pre-change and post-change periods, respectively, where an initial TaW approach had been taken and an organism eligible for exception-reporting had been isolated. In the post-change period, 407 (53.3%) met the criteria and were exception-reported. PRAI occurred in 901 (49.5%) pre-change samples, compared to 102 (25.1%, P < 0.01) with exception-reporting. There was no detectable increase in hospitalization or repeat WSC collection in the 30 days following exception-reporting. Exception-reporting was associated with a markedly reduced proportion of patients being initiated on antibiotics following WSC where an organism had been isolated. The naming of organisms in reports appears to drive unnecessary antibiotic prescribing in many patients. These results require confirmation in other jurisdictions. IMPORTANCE: Wound swab culture is a high-volume test performed in clinical microbiology laboratories. In this analysis, we have shown that an alternative approach to reporting positive wound swab cultures has resulted in a large reduction in post-report antibiotic initiation, suggesting that the current standard method of reporting generates considerable unnecessary antibiotic use. If these findings are replicated elsewhere, wider adoption of this reporting would represent an opportunity for many clinical microbiology laboratories to have a significant impact on community antimicrobial stewardship.
Assuntos
Antibacterianos , Infecção dos Ferimentos , Humanos , Antibacterianos/uso terapêutico , Infecção dos Ferimentos/microbiologia , Infecção dos Ferimentos/tratamento farmacológico , Infecção dos Ferimentos/diagnóstico , Feminino , Masculino , Idoso , Pessoa de Meia-Idade , Adulto , Gestão de Antimicrobianos , Manejo de Espécimes/métodos , Idoso de 80 Anos ou maisRESUMO
Home sample collection for sexually transmitted infection (STI) screening options can improve access to sexual healthcare across communities. For Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG), genital infections have classically been the focus for remote collection options. However, infections may go undiagnosed if sampling is limited to urogenital sites because some individuals only participate in oral and/or anal intercourse. Here we evaluated samples for CT/NG detection after several pre-analytical collection challenges. A paired provider to self-collection validation was performed on rectal [n = 162; 22 + for CT and 9 + for NG by provider-collected (PC)] and throat (N = 158; 2 + for CT and 11 + for NG by provider-collected) swabs. The positive percent agreement for CT and NG ranged from 90.9% to 100%. The discrepancies were more often positive on self-collected (SC) (n = 9 SC+/PC-; n = 1 PC+/SC-; n = 1 PC+/SC Equiv.; n = 2 PC-/SC Equiv.). An empirical limit of detection (LoD) lower than the manufacturer's claim (0.031 vs 2.5 IFU/mL for CT and 0.063 vs 124.8 CFU/ml for NG, respectively) was used to challenge additional variables. Common hand contaminants, including soap, hand sanitizer, lotion, and sunscreen were added to known positive (3× empirical LoD) or negative samples and did not influence detection. Samples at 2× and 10× the empirical LoD were challenged with extreme temperature cycling and extended room temperature storage. Detection was not affected by these conditions. These results indicate that remote self-collection is an appropriate method of sample acquisition for detecting extragenital CT/NG infections. Additionally, they provide a foundation towards meeting the regulatory standards for commercial testing of home collected extragenital samples. IMPORTANCE: There is a clinical need for expanded extragenital bacterial sexually transmitted infection (STI) testing options, but the current regulatory landscape limits the wide-spread promotion and adoption of such services. Improved access, particularly for the LGBTQ+ community, can be achieved by validating testing for specimens that are self-collected at a remote location and arrive at the laboratory via a postal carrier or other intermediary route. Here we provide valuable data showing that self-collected samples for anal and oropharyngeal STI testing are equally or increasingly sensitive compared with those collected by a provider. We systematically consider the effects of storage time, exposure to temperature extremes, and the addition of common toiletries on results.
Assuntos
Infecções por Chlamydia , Chlamydia trachomatis , Gonorreia , Neisseria gonorrhoeae , Manejo de Espécimes , Humanos , Manejo de Espécimes/métodos , Chlamydia trachomatis/isolamento & purificação , Gonorreia/diagnóstico , Gonorreia/microbiologia , Neisseria gonorrhoeae/isolamento & purificação , Feminino , Infecções por Chlamydia/diagnóstico , Infecções por Chlamydia/microbiologia , Masculino , Adulto , Faringe/microbiologia , Infecções Sexualmente Transmissíveis/diagnóstico , Reto/microbiologia , Adulto Jovem , Sensibilidade e EspecificidadeRESUMO
The COVID-19 pandemic highlighted the necessity of fast, sensitive, and efficient methods to test large populations for respiratory viruses. The "gold standard" molecular assays for detecting respiratory viruses, such as quantitative polymerase chain reaction (qPCR) and reverse transcription qPCR (RT-qPCR), rely on invasive swab samples and require time-consuming and labor-intensive extraction processes. Moreover, the turnaround time for RT-qPCR-based assays is too lengthy for rapid screening. Extraction-free saliva-based methods provide a non-invasive sampling process with a fast turnaround time and are suitable for high-throughput applications. However, when used with a standard RT-qPCR system, the absence of extraction significantly reduces the assays' sensitivity. Here, using a novel optical modulation biosensing (OMB) platform, we developed a rapid and highly sensitive extraction-free saliva-based molecular assay. We blindly tested 364 paired nasopharyngeal swabs and saliva samples from suspected SARS-CoV-2 cases in Israel. Compared with the gold standard swab-based RT-qPCR assay, the sensitivity of the extraction-free saliva-based OMB assay is 90.7%, much higher than the sensitivity of extraction-free saliva-based RT-qPCR assay (77.8%) with similar specificity (95.3% and 97.6%, respectively). Moreover, out of 12 samples identified by the OMB-based assay as positive, 8 samples were collected from hospitalized patients in a COVID-19 ward and were verified to be SARS-CoV-2-positive upon admission, indicating that the actual clinical sensitivity and specificity of the OMB assay are higher. Considering its user-friendly saliva-based protocol, short and cost-effective extraction-free process, and high clinical accuracy, the OMB-based molecular assay is very suitable for high-throughput testing of large populations for respiratory viruses. IMPORTANCE: Three years after the SARS-CoV-2 outbreak, there are no molecular tests that combine low-cost and straightforward sample preparation, effective sample handling, minimal reagent and disposable requirements, high sensitivity, and high throughput required for mass screening. Existing rapid molecular techniques typically sacrifice certain requirements to meet others. Yet, localized outbreaks of novel viral diseases happen daily in different parts of the world. In this context, respiratory diseases are of specific importance, as they are frequently airborne and highly contagious, with the potential for a rapid global spread. The widely accepted opinion is that another pandemic is just a question of time. To ensure that the containment efforts for the upcoming "disease X" are successful, introducing rapid, high-throughput, and highly sensitive diagnostic methods for detecting and identifying pathogens is critical. A few months into the pandemic, saliva was suggested as a diagnostic matrix for SARS-CoV-2 detection. The collection of saliva does not require swabs and is minimally invasive. In particular, extraction-free saliva-based assays require fewer reagents and disposables, and therefore are faster and cheaper, offering an appealing alternative for low-income countries. Unfortunately, current extraction-free saliva-based detection methods, such as direct RT-qPCR or isothermal amplification, have either low sensitivity or low throughput. Therefore, we believe that the presented highly sensitive ht-OMBi platform and the extraction-free saliva-based molecular assay can become an essential tool in the infectious disease monitoring toolbox.