Rapid Diagnostics of Joint Infections Using IS-Pro.

Diagnosis of bone and joint infections (BJI) relies on microbiological culture which has a long turnaround time and is challenging for certain bacterial species. Rapid molecular methods may alleviate these obstacles. Here, we investigate the diagnostic performance of IS-pro, a broad-scope molecular technique that can detect and identify most bacteria to the species level. IS-pro additionally informs on the amount of human DNA present in a sample, as a measure of leukocyte levels. This test can be performed in 4 h with standard laboratory equipment. Residual material of 591 synovial fluid samples derived from native and prosthetic joints from patients suspected of joint infections that were sent for routine diagnostics was collected and subjected to the IS-pro test. Bacterial species identification as well as bacterial load and human DNA load outcomes of IS-pro were compared to those of culture. At sample level, percent positive agreement (PPA) between IS-pro and culture was 90.6% (95% CI 85.7- to 94%) and negative percent agreement (NPA) was 87.7% (95% CI 84.1 to 90.6%). At species level PPA was 80% (95% CI 74.3 to 84.7%). IS-pro yielded 83 extra bacterial detections over culture for which we found supporting evidence for true positivity in 40% of the extra detections. Missed detections by IS-pro were mostly related to common skin species in low abundance. Bacterial and human DNA signals measured by IS-pro were comparable to bacterial loads and leukocyte counts reported by routine diagnostics. We conclude that IS-pro showed an excellent performance for fast diagnostics of bacterial BJI.

MicrobiomeAnalyst 2.0: comprehensive statistical, functional and integrative analysis of microbiome data.

Microbiome studies have become routine in biomedical, agricultural and environmental sciences with diverse aims, including diversity profiling, functional characterization, and translational applications. The resulting complex, often multi-omics datasets demand powerful, yet user-friendly bioinformatics tools to reveal key patterns, important biomarkers, and potential activities. Here we introduce MicrobiomeAnalyst 2.0 to support comprehensive statistics, visualization, functional interpretation, and integrative analysis of data outputs commonly generated from microbiome studies. Compared to the previous version, MicrobiomeAnalyst 2.0 features three new modules: (i) a Raw Data Processing module for amplicon data processing and taxonomy annotation that connects directly with the Marker Data Profiling module for downstream statistical analysis; (ii) a Microbiome Metabolomics Profiling module to help dissect associations between community compositions and metabolic activities through joint analysis of paired microbiome and metabolomics datasets; and (iii) a Statistical Meta-Analysis module to help identify consistent signatures by integrating datasets across multiple studies. Other important improvements include added support for multi-factor differential analysis and interactive visualizations for popular graphical outputs, updated methods for functional prediction and correlation analysis, and expanded taxon set libraries based on the latest literature. These new features are demonstrated using a multi-omics dataset from a recent type 1 diabetes study. MicrobiomeAnalyst 2.0 is freely available at microbiomeanalyst.ca.

A lab-on-a-chip utilizing microwaves for bacterial spore disruption and detection.

Bacterial spores are problematic in agriculture, the food industry, and healthcare, with the fallout costs from spore-related contamination being very high. Spores are difficult to detect since they are resistant to many of the bacterial disruption techniques used to bring out the biomarkers necessary for detection. Because of this, effective and practical spore disruption methods are desirable. In this study, we demonstrate the efficiency of a compact microfluidic lab-on-chip built around a coplanar waveguide (CPW) operating at 2.45 GHz. We show that the CPW generates an electric field hotspot of ∼10 kV/m, comparable to that of a commercial microwave oven, while using only 1.2 W of input power and thus resulting in negligible sample heating. Spores passing through the microfluidic channel are disrupted by the electric field and release calcium dipicolinic acid (CaDPA), a biomarker molecule present alongside DNA in the spore core. We show that it is possible to detect this disruption in a bulk spore suspension using fluorescence spectroscopy. We then use laser tweezers Raman spectroscopy (LTRS) to show the loss of CaDPA on an individual spore level and that the loss increases with irradiation power. Only 22% of the spores contain CaDPA after exposure to 1.2 W input power, compared to 71% of the untreated control spores. Additionally, spores exposed to microwaves appear visibly disrupted when imaged using scanning electron microscopy (SEM). Overall, this study shows the advantages of using a CPW for disrupting spores for biomarker release and detection.

Wooden sticks for plaque streaking and microbiological inoculation might be more cost- effective, but is its large scale use feasible? Quality control methods and proof of concept / Métodos de controle de qualidade e prova de conceito para a utilização de palitos de madeira em microbiologia podem ser mais econômicas, mas seu uso em larga escala é viável? Método de controle de qualidade e prova de conceito

The loop is a material classically used in the laboratory for the purpose of plate streaking and handling biological materials. However, metal loops techniques might be time consuming, considering the amount of time spent to guarantee its cooling process through each inoculation. Furthermore, plastic loops may also represent environmental issues during its production and discard process and can also represent higher costs for the laboratory. Thus, in situations of limited resources, even the simplest materials can be restricted due to logistical and budgetary issues, especially in developing countries. Inspired by demands like these, facing an occasional shortage of supply of laboratory plastic handles, we hereby present a quality control for sterilization methods and cost-effectiveness studies towards the use of wooden sticks in a Latin American country and we discuss the possibility of the large-scale use of this technique.

A alça calibrada é um material usado classicamente em laboratório para fins de inoculação em placas e manuseio de materiais biológicos. No entanto, as técnicas de alças metálicas podem consumir muito tempo, considerando a quantidade de tempo gasto para garantir seu processo de resfriamento a cada inoculação. Além disso, alças de plástico também podem representar questões ambientais durante o processo de produção e descarte e também podem representar custos mais altos para o laboratório. Assim, em situações de recursos limitados, até os materiais mais simples podem ser restringidos devido a questões logísticas e orçamentárias, especialmente nos países em desenvolvimento. Inspirados por demandas como essas, diante de uma escassez ocasional de suprimentos de alças de plástico de laboratório, apresentamos um controle de qualidade para métodos de esterilização e estudos de custo-efetividade para o uso de varas de madeira em um país latino-americano e discutimos a possibilidade de grande uso em escala dessa técnica.

Establishment of novel in vitro culture system with the ability to reproduce oral biofilm formation on dental materials.

Intensive research has been conducted with the aim of developing dental restorative/prosthetic materials with antibacterial and anti-biofilm effects that contribute to controlling bacterial infection in the oral cavity. In situ evaluations were performed to assess the clinical efficacy of these materials by exposing them to oral environments. However, it is difficult to recruit many participants to collect sufficient amount of data for scientific analysis. This study aimed to assemble an original flow-cell type bioreactor equipped with two flow routes and assess its usefulness by evaluating the ability to reproduce in situ oral biofilms formed on restorative materials. A drop of bacterial suspension collected from human saliva and 0.2% sucrose solution was introduced into the assembled bioreactor while maintaining the incubation conditions. The bioreactor was able to mimic the number of bacterial cells, live/dead bacterial volume, and volume fraction of live bacteria in the in situ oral biofilm formed on the surface of restorative materials. The usefulness of the established culture system was further validated by a clear demonstration of the anti-biofilm effects of a glass-ionomer cement incorporating zinc-releasing glasses when evaluated by this system.

Pre-processing tissue specimens with a tissue homogenizer: clinical and microbiological evaluation.

BACKGROUND: Tissues are valuable specimens in diagnostic microbiology because they are often obtained by invasive methods, and effort should thus be taken to maximize microbiological yield. The objective of this study was to evaluate the added value of using tissue pre-processing (tissue homogenizer instrument gentleMACS Dissociator) in detecting microorganisms responsible for infections. METHODS: We included 104 randomly collected tissue samples, 41 (39.4 %) bones and 63 (60.6 %) soft tissues, many of those (42/104 (40.4 %)) were of periprosthetic origins. We compared the agreement between pre-processing tissues using tissue homogenizer with routine microbiology diagnostic procedure, and we calculated the performance of these methods when clinical infections were used as reference standard. RESULTS: There was no significant difference between the two methods (McNemar test, p = 0.3). Among the positive culture using both methods (n = 62), 61 (98.4 %) showed at least one similar microorganism. Exactly similar microorganisms were found in 42/62 (67.7 %) of the samples. From the included tissues, 55/ 104 (52.9 %) were deemed as infected. We found that the sensitivity of homogenized tissue procedure was lower (83.6 %) than when tissue was processed using tissue homogenizer (89.1 %). Sub-analysis on periprosthetic tissues and soft or bone tissues showed comparable results. CONCLUSIONS: The added value of GentleMACS Dissociator tissue homogenizer is limited in comparison to routine tissue processing.

Red fluorescent ultra-small gold nanoclusters functionalized with signal molecules to probe specificity in quorum sensing receptors in gram-negative bacteria.

Ultra-small (size < 2 nm) gold nanoclusters (AuNCs) are used as fluorescent probes which have excellent applications in bioimaging and sensing due to their emission in visible and NIR spectral region. Here, this property is exploited for understanding the quorum sensing phenomenon in bacteria which is regulated by signal molecules which are specific to various species. AuNCs are then functionalized with the signal molecules, Acyl Homoserine Lactones (AHL) of varying carbon chain length, C-6, C-8, and C-12 without 3rd C modification, to sense different strains of gram-negative bacteria i.e., Escherichia coli, Cronobacter sakazakii and Pseudomonas aeruginosa. In the concentration employed, selectivity to a limited extent is observed between the three Gram-negative bacteria tested. E. coli showed emission with all the AHL conjugates and P. aeruginosa did not interact with any of the three conjugates, whereas C. sakazakii showed specificity to C-8AHL. This is probably due to selectivity for cognate AHL molecules of appropriate concentrations.

Contaminação de aparelhos celulares da equipe de enfermagem em unidade de terapia intensiva de um hospital público do noroeste paranaense / Contamination of cell phones from the nursing team in an intensive care unit in a public hospital in the northwest paranaense

O ambiente hospitalar é um dos locais com maiores chances de acontecer quadros de infecções, sendo um dos motivos a utilização irrestrita dos aparelhos celulares tanto por pacientes quanto por profissionais da saúde que não se preocupam com as boas práticas de higienização. O objetivo deste estudo foi determinar a prevalência de micro­organismos em aparelhos celulares da equipe de enfermagem da Unidade de Terapia Intensiva (UTI) de um hospital na região noroeste paranaense. Participaram da pesquisa 22 colaboradores da UTI, sendo colhidos swabs umedecidos em caldo Brain Heart Infusion (BHI) dos aparelhos celulares de cada um dos participantes e, posteriormente, as amostras foram incubadas e realizadas as análises microbiológicas. Além disso, aplicou-se um questionário para se conhecer sobre o manuseio do telefone celular por parte dos colaboradores. Na análise microbiológica, observou-se crescimento em todas as amostras de pelo menos um micro­organismo (100% nos meios Ágar Sal Manitol e Ágar Sangue, e 27,3% em Ágar MacConkey). Posteriormente, realizou-se comparação com as respostas do questionário e com o resultado da amostra, sendo que apenas 13,6% dos colaboradores relataram realizar sempre higienização dos aparelhos, porém também houve crescimento de micro­organismos nos aparelhos desses colaboradores. A partir dos dados obtidos, espera-se o envolvimento da Comissão de Controle de Infecção Hospitalar (CCIH) para desenvolver ações que reduzam a prevalência e a gravidade da contaminação no ambiente hospitalar.(AU)

Hospital environments are one of the most likely places for the development of infections, with one of the reasons being the unrestricted use of cell phones by both patients and health professionals, with disregard to good hygiene practices. The purpose of this study is to determine the prevalence of microorganisms on cell phones of the nursing staff at the Intensive Care Unit (ICU) of a hospital in the northwestern region of Paraná. A total of twenty-four ICU employees participated in the research. Swabs were collected from the cell phones of each participant and were then moistened in Brain Heart Infusion (BHI) broth. Subsequently, the samples were incubated, and a microbiological analysis was carried out. In addition, a questionnaire was applied to learn out about the employee's handling of the cell phone. From the microbiological analysis, growth of at least one microorganism (100% in the Agar Salt Mannitol and Blood Agar, and 27.3% in MacConkey Agar) could be observed in all samples. Subsequently, a comparison was made with the answers to the questionnaire and with the sample result, with only 13.6% of employees reporting that they always clean the devices. However, even the devices that were reported as being cleaned presented microorganisms. With the data obtained, the Hospital Infection Control Commission (HICC) is expected to be involved in order to develop actions to reduce prevalence, incidence, and the severity of contamination in hospital environments.(AU)

Evaluation of a Novel Culture System for Rapid Pathogen Identification and Detection of Cephalosporin Resistance in Neonatal Gram-negative Sepsis at a Tertiary Referral Unit in Harare, Zimbabwe.

BACKGROUND: Neonatal sepsis accounts for a large proportion of neonatal deaths in sub-Saharan Africa. The lack of access to diagnostic testing and excessively long turnaround times to result contributes to delays in sepsis identification and initiation of appropriate treatment. This study aims to evaluate the novel InTrays COLOREX Screen and extended-spectrum beta-lactamase for rapid identification of bacterial pathogens causing sepsis and detection of resistance. METHODS: Neonates with suspected sepsis admitted to the Harare Central Hospital were prospectively enrolled. One blood culture was collected and incubated using the BacT/ALERT automated system. Positive blood cultures with potential pathogens identified by Gram stain were inoculated on the InTray COLOREX Screen and extended-spectrum beta-lactamase culture plates. RESULTS: A total of 216 neonates with suspected sepsis were recruited. Pathogens were isolated from blood cultures in 56 (25.9%) neonates of which 54 were Klebsiella pneumoniae. All K. pneumoniae were resistant to ceftriaxone and 53 (98%) were resistant to gentamicin. Sensitivity and specificity for ceftriaxone-resistant K. pneumoniae detection using InTrays were 100%. InTrays results were interpretable as early as 5-10 hours (median 7 hours, interquartile range 7-7) post blood culture positivity enabling rapid identification and notification of result and leading to a 60% reduction in time to result from blood culture collection. CONCLUSIONS: This study shows that the implementation of a novel culture method was feasible and reduced turnaround times for results by 60% compared with standard microbiologic techniques. An impact on patient outcomes and cost-effectiveness of this method needs to be demonstrated.

A Facile High-Throughput Model of Surface-Independent Staphylococcus aureus Biofilms by Spontaneous Aggregation.

Many microbes in their natural habitats are found in biofilm ecosystems attached to surfaces and not as free-floating (planktonic) organisms. Furthermore, it is estimated that nearly 80% of human infections are associated with biofilms. Biofilms are traditionally defined as three-dimensional, structured microbial communities that are attached to a surface and encased in a matrix of exopolymeric material. While this view of biofilm largely arises from in vitro studies under static or flow conditions, in vivo observations have indicated that this view of biofilms is essentially true only for foreign-body infections on catheters or implants where biofilms are attached to the biomaterial. In mucosal infections such as chronic wounds or cystic fibrosis or joint infections, biofilms can be found unattached to a surface and as three-dimensional aggregates. In this work, we describe a high-throughput model of aggregate biofilms of methicillin-resistant Staphylococcus aureus (MRSA) using 96-well plate hanging-drop technology. We show that MRSA forms surface-independent biofilms, distinct from surface-attached biofilms, that are rich in exopolymeric proteins, polysaccharides, and extracellular DNA (eDNA), express biofilm-related genes, and exhibit heightened antibiotic resistance. We also show that the surface-independent biofilms of clinical isolates of MRSA from cystic fibrosis and central catheter-related infections demonstrate morphological differences. Overall, our results show that biofilms can form by spontaneous aggregation without attachment to a surface, and this new in vitro system can model surface-independent biofilms that may more closely mimic the corresponding physiological niche during infection.IMPORTANCE The canonical model of biofilm formation begins with the attachment and growth of microbial cells on a surface. While these in vitro models reasonably mimic biofilms formed on foreign bodies such as catheters and implants, this is not the case for biofilms formed in cystic fibrosis and chronic wound infections, which appear to present as aggregates not attached to a surface. The hanging-drop model of biofilms of methicillin-resistant Staphylococcus aureus (MRSA), the major causative organism of skin and soft tissue infections, shows that these biofilms display morphological and antibiotic response patterns that are distinct from those of their surface-attached counterparts, and biofilm growth is consistent with their in vivo location. The simplicity and throughput of this model enable adoption to investigate other single or polymicrobial biofilms in a physiologically relevant setting.

Headspace analyses using multi-capillary column-ion mobility spectrometry allow rapid pathogen differentiation in hospital-acquired pneumonia relevant bacteria.

BACKGROUND: Hospital-acquired pneumonia (HAP) is a common problem in intensive care medicine and the patient outcome depends on the fast beginning of adequate antibiotic therapy. Until today pathogen identification is performed using conventional microbiological methods with turnaround times of at least 24 h for the first results. It was the aim of this study to investigate the potential of headspace analyses detecting bacterial species-specific patterns of volatile organic compounds (VOCs) for the rapid differentiation of HAP-relevant bacteria. METHODS: Eleven HAP-relevant bacteria (Acinetobacter baumanii, Acinetobacter pittii, Citrobacter freundii, Enterobacter cloacae, Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Staphylococcus aureus, Serratia marcescens) were each grown for 6 hours in Lysogeny Broth and the headspace over the grown cultures was investigated using multi-capillary column-ion mobility spectrometry (MCC-IMS) to detect differences in the VOC composition between the bacteria in the panel. Peak areas with changing signal intensities were statistically analysed, including significance testing using one-way ANOVA or Kruskal-Wallis test (p < 0.05). RESULTS: 30 VOC signals (23 in the positive ion mode and 7 in the negative ion mode of the MCC-IMS) showed statistically significant differences in at least one of the investigated bacteria. The VOC patterns of the bacteria within the HAP panel differed substantially and allowed species differentiation. CONCLUSIONS: MCC-IMS headspace analyses allow differentiation of bacteria within HAP-relevant panel after 6 h of incubation in a complex fluid growth medium. The method has the potential to be developed towards a feasible point-of-care diagnostic tool for pathogen differentiation on HAP.

Novel Diagnostics for Kaposi Sarcoma and Other Skin Diseases in Resource-Limited Settings.

In resource-limited settings, point-of-care diagnostic devices have the potential to reduce diagnostic delays and improve epidemiologic surveillance of dermatologic conditions. We outline novel-point-of care diagnostics that have recently been developed for dermatologic conditions that primarily affect patients living in resource-limited settings, namely, Kaposi sarcoma, cutaneous leishmaniasis, leprosy, Buruli ulcer, yaws, onchocerciasis, and lymphatic filariasis. All of the technologies described in this article are prototypes, and some have undergone field testing. These devices still require validation in real-world settings and effective pricing to have a major impact on dermatologic care in resource-limited settings.

Adesão, internalização e sobrevivência de Salmonella spp. e Listeria monocytogenes em mangas (Mangifera indica), variedade 'Tommy Atkins' / Adhesion, internalization and survival of Salmonella spp. and Listeria monocytogenes in mangoes (Mangifera indica), variety 'Tommy Atkins'

Surtos de salmonelose e listeriose associados ao consumo de frutas inteiras ou minimamente processadas ocorrem com frequência. O objetivo deste estudo foi investigar a capacidade de adesão e internalização de Salmonella spp. e Listeria monocytogenes em mangas (Mangifera indica) variedade Tommy Atkins, em diferentes condições de contaminação experimental e tratamento hidrotérmico, bem como avaliar a multiplicação dos patógenos internalizados na polpa das frutas durante armazenamento em refrigeração (8oC ) e temperatura ambiente (25oC). O estudo foi conduzido com as cepas S. Enteritidis ATCC 13076, S. Thyphimurium ATCC 14028, L. monocytogenes ATCC 7644 e L. monocytogenes Scott A. Inicialmente as cepas foram avaliadas segundo o índice de hidrofobicidade e capacidade de formação de biofilme em poliestireno. A adesão à superfície da manga foi avaliada por técnicas microbiológicas e também pela técnica de microscopia eletrônica de varredura. A internalização foi avaliada a partir de inoculação na cicatriz do pedúnculo (6 log UFC/mL) e após tratamento hidrotérmico e imersão em solução contaminada (6 log UFC/mL), mantidas a 8 °C e a 25 °C por 24h, 5 e 10 dias. A sobrevivência foi avaliada através da inoculação em região demarcada, em cenário de baixo (2 log UFC/mL) e alto nível de contaminação (6 log UFC/mL), a 8 °C e 25 °C, nos tempos 0, 1, 2, 3, 5 e 10 dias. A adesão foi observada nos dois patógenos, mesmo após sucessivas lavagens, com diferença significativa (p<0,05) após 1h de exposição e observou-se presença de estruturas exopolissacarídicas em diferentes tempos e condições de temperatura. A internalização ocorreu em todas as amostras avaliadas e a região do pedúnculo foi a mais afetada pela contaminação, diferindo significativamente na comparação com a região blossom end (p<0,05) a 8 °C e 25 °C. A sobrevivência foi observada nas duas temperaturas até o décimo dia. A multiplicação a 8°C foi significativamente mais baixa (p<0,05). Os resultados demonstraram que a Salmonella spp e L. monocytogenes são capazes de aderir à superfície, de internalizar e se alastrar pela polpa e ainda sobreviverem por períodos consideráveis, em 8 °C e 25 °C. Esses dados poderão auxiliar produtores e órgãos de saúde no desenvolvimento de avaliações quantitativas de risco e no estabelecimento de medidas adequadas para evitar surtos

Outbreaks of salmonellosis and listeriosis associated with the consumption of whole or minimally processed fruits occur frequently. The aim of this study was to investigate the ability of spp. and Listeria monocytogenes to adhere and internalize in mangoes (Mangifera indica) variety Tommy Atkins, under different conditions of experimental contamination and hydrothermal treatment, as well as evaluate the multiplication of the internalized pathogens in the fruit pulp during storage under refrigeration (8oC) and room temperature (25oC). The study was conducted with the strains S. Enteritidis ATCC 13076, S. Thyphimurium ATCC 14028, L. monocytogenes ATCC 7644 and L. monocytogenes Scott A. Initially the strains were evaluated according to the hydrophobicity index and capability to form biofilms on polystyrene surface. Adhesion to the mango surface was evaluated by microbiological techniques and also by scanning electron microscopy. Internalization was evaluated by inoculating the peduncle scar (6 log CFU / mL) and immersion of the fruits in contaminated solution (6 log CFU / mL) after hydrothermal treatment, during storage at 8 °C and 25 °C for 24h, 5 and 10 days. Survival was assessed by inoculation in a demarcated region, using low (2 log CFU / mL) and high level of contamination (6 log CFU / mL), and storage at 8 °C and 25 °C during 0, 1, 2, 3, 5 and 10 days. Adhesion was observed for both pathogens, even after successive washes, with a significant difference (p <0.05) after 1 h of exposure. Adhesion was mediated by exopolysaccharide structures, observed at different times and temperature conditions. Internalization occurred in all samples and the peduncle region was the most affected by the contamination, differing significantly in comparison with the blossom end region (p <0.05) at 8 °C and 25 oC. Survival was observed at both temperatures until the tenth day. The multiplication at 8 °C was significantly lower than at 25 oC (p <0.05). The results showed that Salmonella spp and L. monocytogenes were able to adhere to the surface, to internalize and spread through the pulp and still survive for considerable periods, at 8 °C and 25 °C. This data may help producers and health agencies to develop quantitative risk assessments and to establish appropriate measures to prevent outbreaks

Evaluation of physicochemical and microbiological stability of liquid preparation from tizanidine hydrochloride tablets - a Hospital concern

Tizanidine hydrochloride is a centrally acting skeletal muscle relaxant, used in the management of spasticity. This drug is commercially available only as tablets, which highlights the need to develop oral liquid formulations. In the hospital environment, this aspect is circumvented by the preparation of suspensions, to allow administration to children and adults with impaired swallowing, but there are no data regarding their stability. The purpose of this study was to evaluate the physicochemical andmicrobiological stability of liquid dosage forms prepared in the hospital environment from tizanidine hydrochloride tablets, applying high performance liquid chromatography (HPLC) and microbiological analysis. A simple and stability-indicating HPLC method was developed and validated for specificity, linearity, limits of detection and quantification, precision, accuracy and robustness. The liquid formulations were placed in amber PET and glass bottles, which were stored under three different conditions: at room temperature, under refrigeration and at 40 ºC. The liquid formulations were analyzed and demonstrated chemical stability for 56 days, allowing their use for long periods. However, the determination of microbiological stability showed that these formulations are prone to microbial contamination, which has dramatically reduced its stability to 7 days, in both bottles and at all evaluated temperatures

Flow Cytometric Measurement of Efflux in Candida Species.

A technique to assess the ability of distinct Candida strains to efflux substrates, as well as to compare the effectiveness of efflux inhibitors, is important for analysis of antifungal drug resistance mechanisms and the mode of action of antifungals. We describe a method that measures the ability of Candida species to extrude the fluorescent dye Nile red as an output for efflux activity. This involves exposing cells to Nile red and using flow cytometry to quantify cellular fluorescence, enabling numerous samples to be processed in a limited time frame. This protocol provides a simple, yet effective method for quantifying efflux in drug-resistant Candida species. © 2020 Wiley Periodicals LLC Basic Protocol 1: Growth and sample preparation of stained Candida Basic Protocol 2: Quantitative measurement of fluorescence by flow cytometry Alternate Protocol: Qualitative determination of fluorescence using microscopy.

Evaluation of the Risk of Laboratory Microbial Contamination during Routine Testing in Automated Clinical Chemistry and Microbiology Laboratories.

BACKGROUND: Every clinical specimen is potentially infectious, but data regarding risk for contamination of the laboratory environment during routine testing are scarce. We assessed contamination during routine sample analysis in automated clinical chemistry and microbiology laboratories. METHODS: A fluorescent marker was applied to specimen container exteriors to assess the impact of gross contamination. Nonpathogenic MS2 virus was added to remnant blood, urine, and ESwab matrices as a biomarker of cross-contamination. Samples were processed and analyzed using Roche Cobas 8100 and ISE, c502, e602, and c702 modules (blood) and BD Kiestra total laboratory automation (blood, urine, ESwabs) over 3 experiments. Fluorescence transfer to laboratory surfaces and personnel was visualized using ultraviolet light. Surfaces were swabbed and assessed for MS2 cross-contamination by RT-PCR. Adherence to standard precautions by laboratory staff was assessed by observation. RESULTS: Fluorescence was observed on 49 of 165 (30%) laboratory surfaces and personnel and 21 of 93 (23%) total laboratory automation instruments. Fluorescence transferred most frequently to gloves (31/40), computer accessories (9/18), and specimen loading racks (12/12). None of 123 areas swabbed were positive for MS2. Improper personal protective equipment use occurred at a rate of 0.36 and 0.15 events per staff per hour in the chemistry and microbiology laboratories, respectively. Hand-washing compliance was observed for 61 of 132 (46%) staff members evaluated. CONCLUSIONS: Analysis of grossly contaminated specimens on automated chemistry and microbiology equipment elicits a low likelihood of instrument contamination. However, handling contaminated specimen containers can result in contamination of environmental laboratory surfaces, representing a source of risk that is heightened by low adherence to appropriate personal protective equipment.

Electrochemical biosensors for pathogen detection.

Recent advances in electrochemical biosensors for pathogen detection are reviewed. Electrochemical biosensors for pathogen detection are broadly reviewed in terms of transduction elements, biorecognition elements, electrochemical techniques, and biosensor performance. Transduction elements are discussed in terms of electrode material and form factor. Biorecognition elements for pathogen detection, including antibodies, aptamers, and imprinted polymers, are discussed in terms of availability, production, and immobilization approach. Emerging areas of electrochemical biosensor design are reviewed, including electrode modification and transducer integration. Measurement formats for pathogen detection are classified in terms of sample preparation and secondary binding steps. Applications of electrochemical biosensors for the detection of pathogens in food and water safety, medical diagnostics, environmental monitoring, and bio-threat applications are highlighted. Future directions and challenges of electrochemical biosensors for pathogen detection are discussed, including wearable and conformal biosensors, detection of plant pathogens, multiplexed detection, reusable biosensors for process monitoring applications, and low-cost, disposable biosensors.

Antimicrobial susceptibility testing: currently used methods and devices and the near future in clinical practice.

Rapid identification of pathogen and its resistance to antimicrobial drugs, and subsequent appropriate antimicrobial treatment are essential for correct patient outcomes. Conventional detection methods of bacterial resistance, such as disc diffusion, broth microdilution and automated instruments, are constantly widely used and primarily standardized. Nevertheless, the results cannot be obtained earlier than 48 h after receiving a sample, which may lead to prolonged use or overuse of broad-spectrum antibiotics. Hence, there is a drive to develop and introduce novel, faster, standardized, sensitive and specific methods with reliable results into routine microbiological laboratory practice. Recently developed matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been introduced in recent years into laboratory practice, and methods based on microfluidics and microdroplets might be introduced in the near future. This review is focused on the methods and instruments in use both currently and in the foreseeable future, applicable to determine antimicrobial efficacy in clinical microbiology laboratories.