A systematic approach to microbial forensics.

The coronavirus disease 2019 pandemic accelerated developments in biotechnology that underpin infection science. These advances present an opportunity to refresh the microbial forensic toolkit. Integration of novel analytical techniques with established forensic methods will speed up acquisition of evidence and better support lines of enquiry. A critical part of any such investigation is demonstration of a robust causal relationship and attribution of responsibility for an incident. In the wider context of a formal investigation into agency, motivation and intent, the quick and efficient assembly of microbiological evidence sets the tone and tempo of the entire investigation. Integration of established and novel analytical techniques from infection science into a systematic approach to microbial forensics will therefore ensure that major perspectives are correctly used to frame and shape the evidence into a clear narrative, while recognizing that forensic hypothesis generation, testing and refinement comprise an iterative process. Development of multidisciplinary training exercises that use this approach will enable translation into practice and efficient implementation when the need arises.

Nucleic acid hybridization-based detection of pathogenic RNA using microscale thermophoresis.

Infectious diseases are one of the world's leading causes of morbidity. Their rapid spread emphasizes the need for accurate and fast diagnostic methods for large-scale screening. Here, we describe a robust method for the detection of pathogens based on microscale thermophoresis (MST). The method involves the hybridization of a fluorescently labeled DNA probe to a target RNA and the assessment of thermophoretic migration of the resulting complex in solution within a 2 to 30-time window. We found that the thermophoretic migration of the nucleic acid-based probes is primarily determined by the fluorescent molecule used, rather than the nucleic acid sequence of the probe. Furthermore, a panel of uniformly labeled probes that bind to the same target RNA yields a more responsive detection pattern than a single probe, and moreover, can be used for the detection of specific pathogen variants. In addition, intercalating agents (ICA) can be used to alter migration directionality to improve detection sensitivity and resolving power by several orders of magnitude. We show that this approach can rapidly diagnose viral SARS-CoV2, influenza H1N1, artificial pathogen targets, and bacterial infections. Furthermore, it can be used for anti-microbial resistance testing within 2 h, demonstrating its diagnostic potential for early pathogen detection.

Characterization of bacterial diversity and capacity to remove lead of a consortium from mining soil / Caracterización de la diversidad bacteriana y capacidad de remoción de plomo de un consorcio en suelo minero

Introduction: At present, the presence of lead (Pb2+) continues to be a problem in water bodies due to its continuous use and high toxicity. The aim of this study was to investigate the bacterial diversity of a potential consortium used as a biosorbent for the removal of lead in an aqueous solution. Methods: The minimum inhibitory concentration and the mean lethal dose of the consortium were determined, and then the optimal variables of pH and temperature for the removal process were obtained. With the optimal conditions, the kinetic behavior was evaluated, and adjustments were made to different mathematical models. A Fourier transform infrared spectroscopy analysis was performed to determine the functional groups of the biomass participating in the removal process, and the diversity of the bacterial consortium was evaluated during Pb2+ removal by an Ion Torrent Personal Genome Machine System. Results: It was found that the intraparticle diffusion model was the one that described the adsorption kinetics showing a higher rate constant with a higher concentration of Pb2+, while the Langmuir model was that explained the isotherm at 35 °C, defining a maximum adsorption load for the consortium of 54 mg/g. In addition, it was found that Pb2+ modified the diversity and abundance of the bacterial consortium, detecting genera such as Pseudomonas, Enterobacter, Citrobacter, among others. Conclusions: Thus, it can be concluded that the bacterial consortium from mining soil was a biosorbent with the ability to tolerate high concentrations of Pb2+ exposure. The population dynamics during adsorption showed enrichment of Proteobacteria phyla, with a wide range of bacterial families and genera capable of resisting and removing Pb2+ in solution.(AU)

Understanding the Non-Equivalency of Bio-Fluorescent Particle Counts versus the Colony-Forming Unit.

Adopting emerging microbiological methods is often desirable because it enables more advantageous, real-time monitoring practices. However, when the newer method measures contamination based on a different detection principle and provides results that are based on different units of measure, a paradigm shift is necessary. That shift can be one of the most difficult challenges in any such project and requires careful consideration. In this article, we explore the challenges presented by the bio-fluorescent particle counting (BFPC) technology, when considering that the traditional colony-forming unit (CFU) is the gold standard that any change is measured against. We examine why attempts to correlate newer units of measure used by biofluorescent particle counters, namely the auto-fluorescent units (AFUs), to the traditional CFUs are not necessarily appropriate. The article explores in depth why there is no consistent correlation factor between the two units of measure, and why that should not be a barrier to fully leveraging, implementing, and using such modern technologies in routine monitoring.

Head-to-head comparison of CAMPYAIR aerobic culture medium versus standard microaerophilic culture for Campylobacter isolation from clinical samples.

Campylobacter spp. are considered the most frequent cause of acute gastroenteritis worldwide. However, outside high-income countries, its burden is poorly understood. Limited published data suggest that Campylobacter prevalence in low- and middle-income countries is high, but their reservoirs and age distribution are different. Culturing Campylobacter is expensive due to laboratory equipment and supplies needed to grow the bacterium (e.g., selective culture media, microaerophilic atmosphere, and a 42°C incubator). These requirements limit the diagnostic capacity of clinical laboratories in many resource-poor regions, leading to significant underdiagnosis and underreporting of isolation of the pathogen. CAMPYAIR, a newly developed selective differential medium, permits Campylobacter isolation without the need for microaerophilic incubation. The medium is supplemented with antibiotics to allow Campylobacter isolation in complex matrices such as human feces. The present study aims to evaluate the ability of the medium to recover Campylobacter from routine clinical samples. A total of 191 human stool samples were used to compare the ability of CAMPYAIR (aerobic incubation) and a commercial Campylobacter medium (CASA, microaerophilic incubation) to recover Campylobacter. All Campylobacter isolates were then identified by MALDI-TOF MS. CAMPYAIR showed sensitivity and specificity values of 87.5% (95% CI 47.4%-99.7%) and 100% (95% CI 98%-100%), respectively. The positive predictive value of CAMPYAIR was 100% and its negative predictive value was 99.5% (95% CI 96.7%-99.9%); Kappa Cohen coefficient was 0.93 (95% CI 0.79-1.0). The high diagnostic performance and low technical requirements of the CAMPYAIR medium could permit Campylobacter culture in countries with limited resources.

Diagnostic Performance of BD Phoenix CPO Detect Panels for Detection and Classification of Carbapenemase-Producing Gram-Negative Bacteria.

BD Phoenix CPO Detect panels can identify and classify carbapenemase-producing organisms (CPOs) simultaneously with antimicrobial susceptibility testing (AST) for Gram-negative bacteria. Detection and classification of carbapenemase producers were performed using the BD Phoenix CPO Detect panels NMIC/ID-441 for Enterobacterales, NMIC/ID-442 for nonfermenting bacteria, and NMIC-440 for both. The results were compared with those obtained using comparator methods. A total of 133 strains (32 Klebsiella pneumoniae, 37 Enterobacter cloacae complex, 33 Pseudomonas aeruginosa, and 31 Acinetobacter baumannii complex strains), including 60 carbapenemase producers (54 imipenemases [IMPs] and 6 OXA type), were analyzed. Using panels NMIC-440 and NMIC/ID-441 or NMIC/ID-442, all 54 IMP producers were accurately identified as CPOs (positive percent agreement [PPA], 100.0%; 54/54). Among the 54 IMP producers identified as CPOs using panels NMIC-440 and NMIC/ID-441, 12 and 14 Enterobacterales were not resistant to carbapenem, respectively. Among all 54 IMP producers, 48 (88.9%; 48/54) were correctly classified as Ambler class B using panel NMIC-440. Using panels NMIC-440 and NMIC/ID-442, all four OXA-23-like carbapenemase-producing A. baumannii complex strains (100.0%, 4/4) were correctly identified as CPOs, and three (75.0%, 3/4) were precisely classified as class D using panel NMIC-440. Both carbapenemase producers harboring the blaISAba1-OXA-51-like gene were incorrectly identified as non-CPOs using panels NMIC-440 and NMIC/ID-442. For detecting carbapenemase producers, the overall PPA and negative percent agreement (NPA) between panel NMIC-440 and the comparator methods were 96.7% (58/60) and 71.2% (52/73), respectively, and the PPA and NPA between panels NMIC/ID-441 or NMIC/ID-442 and the comparator methods were 96.7% (58/60) and 74.0% (54/73), respectively. BD Phoenix CPO Detect panels can successfully screen carbapenemase producers, particularly IMP producers, regardless of the presence of carbapenem resistance and can be beneficial in routine AST workflows. IMPORTANCE Simple and efficient screening methods of detecting carbapenemase producers are required. BD Phoenix CPO Detect panels effectively screened carbapenemase producers, particularly IMP producers, with a high overall PPA. As the panels enable automatic screening for carbapenemase producers simultaneously with AST, the workflow from AST to confirmatory testing for carbapenemase production can be shortened. In addition, because carbapenem resistance varies among carbapenemase producers, the BD Phoenix CPO Detect panels, which can screen carbapenemase producers regardless of carbapenem susceptibility, can contribute to the accurate detection of carbapenemase producers. Our results report that these panels can help streamline the AST workflow before confirmatory testing for carbapenemase production in routine microbiological tests.

MALDI-TOF as a tool for microbiological monitoring in areas considered aseptic.

To maintain asepsis in production environments, contamination must be constantly controlled. To this end, microbiological monitoring is constantly used with the objective of evaluating the incidence of microorganisms prevalent in the sampling of air, surface, and people, in the area of an environment considered aseptic, isolated, and identified using the rapid and automated phenotypic microbiological methodology, highlighting the MALDI-TOF mass spectrometry analysis technique (MS), being identified at the level of genus and/or species. For that purpose, microbiological control of environmental monitoring of environments considered aseptic in a pharmaceutical industry was conducted for 12 months. The isolated microorganisms were identified using the mass spectrometry identification method (MALDI-TOF). In area classification A, the most prevalent microorganisms were bacteria in the sampling person. The microbial population was composed of bacteria of the genus Micrococcus sp. and Staphylococcus sp. Based on the results, it is possible to observe that in an environment where the process requires human operations, possible microbial contamination is inevitable and requires the identification of microorganisms at least at the level of species and/or genus. The microorganisms identified and found in the sampling of the aseptic environment must be evaluated with frequency to ensure that the productive environment guarantees the quality of the product produced.

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.

Performance Equivalence and Validation of a Rapid Microbiological Method for Detection and Quantification of Yeast and Mold in an Antacid Oral Suspension.

Alternative and rapid microbiological methods can be effective replacements for more traditional plating approaches for ensuring quality and safety in the pharmaceutical industry. This article compares the efficacy of the Soleris automated method and the traditional plate-count method for the quantitative detection of yeasts and molds at three different microbial bioburden levels. Validation testing was carried out using an antacid oral suspension (aluminum hydroxide 4% + magnesium hydroxide 4% + simethicone 0.4%). Equivalence of data between detection time and colony-forming units was established for both the alternative and the conventional methodologies. Using probability of detection, linear Poisson regression, Fisher's test, and multifactorial analysis of variance (ANOVA), all results from the rapid method were shown to be in statistical agreement with the those of the reference plating procedures. The limits of detection and quantification were statistically similar for both methods (Fisher's exact test, P > 0.05), showing that the alternative method is not inferior in performance to the reference method. Essential validation parameters such as precision (standard deviation <5, coefficient of variance <35%), accuracy (>70%), linearity (R2 >0.9025), ruggedness (ANOVA, P < 0.05), operative range, and specificity were determined. It was shown that all the test results obtained using the alternative method were in statistical agreement with the those of the standard plate-count method. Thus, this new technology was found to meet all the validation criteria needed to be considered as an alternative method for yeast and mold quantification in the antacid oral suspension tested. However, taking into account that the present validation was carried out utilizing A. brasiliensis and C. albicans as suitable models for yeasts and molds and with an antacid oral suspension as a pharmaceutical matrix, further investigation will be required to qualify Soleris technology for other environmental isolates and recovery of these isolates from production batches.

Análise microbiológica de sushi comercializado em Rio Branco, Acre / Microbiological analysis of sushi marketed in Rio Branco, Acre / Análisis microbiológico de sushi comercializado en Rio Branco, Acre

O Brasil é um dos países mais diversificados no ramo gastronômico oferecendo vários alimentos diferentes aos seus consumidores, com base nos próprios pratos típicos ou provenientes de outras culturas. O pescado trata-se de um alimento perecível que necessita de atenções especiais em seu processamento. Falhas nas condições higiênico-sanitárias, associadas com a não cocção do alimento, podem ocasionar em uma contaminação e proliferação de bactérias, o que leva à uma grande preocupação a nível de saúde pública. O estudo analisou os aspectos microbiológicos de sushi comercializado na cidade de Rio Branco ­ Acre verificando os parâmetros de qualidade e as condições higiênicas sanitárias, comparando os resultados obtidos com a legislação vigente estabelecida pela ANVISA. Foram escolhidos 5 estabelecimentos aleatoriamente, sendo escolhidas 3 amostras de sushis do tipo niguiri de cada. As análises microbiológicas incluíram coliformes totais e coliformes termotolerantes utilizando a técnica dos tubos multiplos e a técnica de semeadura por profundidade para mesófilos e Salmonella. Constatou-se que todas as amostras tiveram um crescimento bacteriano e presença sugestiva de Salmonella, tornando o alimento impróprio para o consumo e mostrando uma falha nas condições higiênico- sanitária ao qual o sushi é processado e armazenado. É necessário maior fiscalização dos órgãos responsáveis e cuidado dos estabelecimentos que vendem sushi na cidade de Rio Branco, para que o produto vendido seja de boa qualidade e não cause malefícios a saúde de quem o consome.(AU)

Brazil is one of the most diversified countries in the gastronomic field, offering several different foods to its consumers, based on typical dishes or from other cultures. Fish is a perishable food that requires special attention in its processing. Failures in hygienic-sanitary conditions, coupled with the consumption of undercooked food, can lead to contamination and the proliferation of bacteria, which raises significant concerns regarding public health. The study analyzed the microbiological aspects of sushi sold in the city of Rio Branco - Acre, verifying the quality parameters and the hygienic sanitary conditions, comparing the obtained results with the current legislation established by ANVISA. Five establishments were randomly selected, and three samples of nigiri sushi were chosen from each establishment. The microbiological analysis included total coliforms and thermotolerant coliforms using the multiple tube technique, as well as depth seeding technique for mesophiles and Salmonella. It was found that all samples exhibited bacterial growth and suggested the presence of Salmonella, rendering the food unsuitable for consumption and indicating a failure in the hygienic-sanitary conditions under which the sushi was processed and stored. Greater inspection by the responsible authorities and improved care by establishments selling sushi in the city of Rio Branco are necessary to ensure that the product sold is of good quality and does not pose harm to the health of consumers.(AU)

Brasil es uno de los países más diversificados en el campo gastronómico, ofreciendo muchos alimentos diferentes a sus consumidores, basados en platos típicos ode otras culturas El pescado es un alimento perecedero que necesita especial atención en su elaboración. Las fallas en las condiciones, higiénico-sanitarias asociadas a la no cocción de los alimentos, pueden conducir a la contaminación y proliferación de bacterias, lo que genera una gran preocupación en términos de salud pública. El estudio analizó los aspectos microbiológicos del sushi comercializado en la ciudad de Rio Branco - Acre, verificando los parámetros de calidad y las condiciones higiénicas sanitarias, comparando los resultados obtenidos con la legislación vigente establecida por la ANVISA. Se eligieron 5 establecimientos al azar, y de cada uno se escogieron 3 muestras de sushi niguiri. Los análisis microbiológicos incluyeron coliformes totales y coliformes termotolerantes mediante la técnica de tubos múltiples y la técnica de siembra profunda para mesófilos y Salmonella. Se encontró que todas las muestras presentaban crecimiento bacteriano y la sugestiva presencia de Salmonella, lo que hace que el alimento no sea apto para el consumo y presenta una falla en las condiciones higiénico-sanitarias en las que se procesa y almacena el sushi. Se necesita mayor fiscalización por parte de los órganos responsables y cuidado de los establecimientos que venden sushi en la ciudad de Rio Branco, para que el producto vendido sea de buena calidad y no cause daño a la salud de quien lo consume.(AU)

CRISPRi-based programmable logic inverter cascade for antibiotic-free selection and maintenance of multiple plasmids.

Antibiotics have been widely used for plasmid-mediated cell engineering. However, continued use of antibiotics increases the metabolic burden, horizontal gene transfer risks, and biomanufacturing costs. There are limited approaches to maintaining multiple plasmids without antibiotics. Herein, we developed an inverter cascade using CRISPRi by building a plasmid containing a single guide RNA (sgRNA) landing pad (pSLiP); this inhibited host cell growth by repressing an essential cellular gene. Anti-sgRNAs on separate plasmids restored cell growth by blocking the expression of growth-inhibitory sgRNAs in pSLiP. We maintained three plasmids in Escherichia coli with a single antibiotic selective marker. To completely avoid antibiotic use and maintain the CRISPRi-based logic inverter cascade, we created a novel d-glutamate auxotrophic E. coli. This enabled the stable maintenance of the plasmid without antibiotics, enhanced the production of the terpenoid, (-)-α-bisabolol, and generation of an antibiotic-resistance gene-free plasmid. CRISPRi is therefore widely applicable in genetic circuits and may allow for antibiotic-free biomanufacturing.

Concordance of the filmarray blood culture identification panel 2 and classical microbiological methods in a bacteriemia diagnostic unit.

Our study evaluates the effectiveness of the FilmArray Blood Culture Identification Panel 2 in a rapid bacteremia diagnostic system, using bacteriological culture as a reference. A total of 2042 positive blood cultures were analyzed, the FilmArray was performed for 175. Concordance was higher in monomicrobial bacteremia (95%) than in polymicrobial bacteremia's (72.2%). For detecting bacterial resistance mechanisms, concordance was very high (100% for Gram-positive bacteria and 98.12% for Gram-negative). This methodology provides significant improvements in response time and is especially useful for the detection of monomicrobial bacteremia.

Adaptation to a Multiplex Bead Assay and Seroprevalence to Rift Valley Fever N Protein: Nampula Province, Mozambique, 2013-2014.

Rift Valley fever virus (RVFV) is endemic in sub-Saharan Africa (SSA), with outbreaks reported in the Arabian Peninsula and throughout SSA. The natural reservoir for RVFV are ruminants, with livestock populations exceeding 50% exposure rates in some areas of SSA. Transmission to humans can occur through exposure to infected livestock products or multiple species of mosquito vectors. In 2013 and 2014, cross-sectional surveys occurred in two districts of Nacala-a-Velha and Mecubúri in northern Mozambique, and participants provided blood samples for later serological assays. IgG against the N protein of RVFV was detected through multiplex bead assay (MBA). Of the 2,278 persons enrolled between the two surveys and study sites, 181 (7.9%, 95% confidence interval (CI): 6.9%-9.1%) were found to be IgG seropositive with increasing seroprevalence with older age and significantly higher seroprevalence in Nacala-a-Velha (10.5%, 8.8%-12.5%) versus Mecubúri (5.7%, 4.5%-7.1%). Seroprevalence estimates were not significantly different between the 2013 and 2014 surveys. Significant spatial clustering of IgG positive persons were consistent among surveys and within the two districts, pointing toward the consistency of serology data for making population-level assumptions regarding RVFV seroprevalence. A subset of persons (n = 539) provided samples for both the 2013 and 2014 surveys, and a low percentage (0.81%) of these were found to seroconvert between these two surveys. Including the RVFV N protein in an MBA antigen panel could assist elucidate RVFV exposure in SSA. IMPORTANCE Due to sporadic transmission, human contact with Rift Valley Fever Virus (RVFV) is difficult to ascertain at a population level. Detection of antibodies against RVFV antigens assist in estimating exposure as antibodies remain in the host long after the virus has been cleared. In this study, we show that antibodies against RVFV N protein can be detected from dried blood spot (DBS) samples being assayed by multiplex bead assay. DBS from two districts in northern Mozambique were tested for IgG against the N protein, and 7.9% of all enrolled persons were seropositive. Older persons, males, and persons residing closer to the coast had higher RVFV N protein seroprevalence. Spatial clustering of IgG positive persons was noted in both districts. These results show low exposure rates to RVFV in these two northern districts in Mozambique, and the ability to perform serology for the RVFV N protein from dried blood samples.

Candida auris Identification and Profiling by MALDI-ToF Mass Spectrometry.

MALDI-ToF MS has become the standard method for routine identification of most medically important yeasts in clinical and public health laboratories and has largely replaced phenotypic identification methods as a first-line identification tool. Fungal identification is based on extensive and well-curated mass spectra libraries usually provided by the manufacturer of the MALDI-ToF MS platform; however, many centers do create specialized or in-house database collections to aid analysis. Most MALDI-ToF MS systems offer simple and standardized workflows for the identification of clinically relevant yeasts to species level with a high throughput, high accuracy, and a low overall cost per test. This makes MALDI-ToF MS an ideal platform for use in routine clinical, diagnostic, and research microbiology laboratories which may lack experience or expertise in the identification of pathogenic fungi.In this chapter we review three standard protocols for the proteomic-based identification of Candida auris isolated from cultures of clinical or environmental surveillance samples in diagnostic and research laboratories.

Accuracy of direct examination and culture as compared to the anatomopathological examination for the diagnosis of chromoblastomycosis: a systematic review.

BACKGROUND: Chromoblastomycosis is a skin infection caused by dematiaceous fungi that take the form of muriform cells in the tissue. It mainly manifests as verrucous plaques on the lower limbs of rural workers in tropical countries. OBJECTIVES: The primary objective of this review is to evaluate the accuracy of diagnostic methods for the identification of chromoblastomycosis, considering the histopathological examination as the reference test. METHODS: MEDLINE, LILACS and Scielo databases were consulted using the terms "chromoblastomycosis" AND "diagnosis". The eligibility criteria were: studies that evaluated the accuracy of tests for the diagnosis of chromoblastomycosis. Eleven studies were selected. Statistical analysis included the calculation of sensitivity and specificity of the diagnostic methods. RESULTS: Considering the histopathological examination as the reference test, the culture showed a sensitivity (S) of 37.5% - 90.9% and a specificity (Sp) of 100%; while direct mycological examination showed S =  50% - 91.6% and Sp of 100% . Considering the culture as the reference test, the serology (precipitation techniques) showed S  of 36% - 99%; and Sp  of 80% - 100%; while the intradermal test showed S  of 83.3% - 100% and Sp  of 99.4% - 100%. STUDY LIMITATIONS: The small number of studies and very discrepant sensitivity results among them do not allow the calculation of summary measures through a meta-analysis. CONCLUSIONS: Direct mycological examination, culture, intradermal test and serology show sensitivity and specificity values ​​for the diagnosis of chromoblastomycosis with no significant difference between the studies.

Review of Liquid Chromatography-Mass Spectrometry-Based Proteomic Analyses of Body Fluids to Diagnose Infectious Diseases.

Rapid and precise diagnostic methods are required to control emerging infectious diseases effectively. Human body fluids are attractive clinical samples for discovering diagnostic targets because they reflect the clinical statuses of patients and most of them can be obtained with minimally invasive sampling processes. Body fluids are good reservoirs for infectious parasites, bacteria, and viruses. Therefore, recent clinical proteomics methods have focused on body fluids when aiming to discover human- or pathogen-originated diagnostic markers. Cutting-edge liquid chromatography-mass spectrometry (LC-MS)-based proteomics has been applied in this regard; it is considered one of the most sensitive and specific proteomics approaches. Here, the clinical characteristics of each body fluid, recent tandem mass spectroscopy (MS/MS) data-acquisition methods, and applications of body fluids for proteomics regarding infectious diseases (including the coronavirus disease of 2019 [COVID-19]), are summarized and discussed.

Advanced Understanding of Prokaryotic Biofilm Formation through Use of a Cost-Effective and Versatile Multipanel Adhesion (mPAD) Mount.

Most microorganisms exist in biofilms, which comprise aggregates of cells surrounded by an extracellular matrix that provides protection from external stresses. Based on the conditions under which they form, biofilm structures vary in significant ways. For instance, biofilms that develop when microbes are incubated under static conditions differ from those formed when microbes encounter the shear forces of a flowing liquid. Moreover, biofilms develop dynamically over time. Here, we describe a cost-effective coverslip holder, printed with a three-dimensional (3D) printer, that facilitates surface adhesion assays under a broad range of standing and shaking culture conditions. This multipanel adhesion (mPAD) mount further allows cultures to be sampled at multiple time points, ensuring consistency and comparability between samples and enabling analyses of the dynamics of biofilm formation. As a proof of principle, using the mPAD mount for shaking, oxic cultures, we confirm previous flow chamber experiments showing that the Pseudomonas aeruginosa wild-type strain and a phenazine deletion mutant (Δphz) strain form biofilms with similar structure but reduced density in the mutant strain. Extending this analysis to anoxic conditions, we reveal that microcolony formation and biofilm formation can only be observed under shaking conditions and are decreased in the Δphz mutant compared to wild-type cultures, indicating that phenazines are crucial for the formation of biofilms if oxygen as an electron acceptor is unavailable. Furthermore, while the model archaeon Haloferax volcanii does not require archaella for surface attachment under static conditions, we demonstrate that an H. volcanii mutant that lacks archaella is impaired in early stages of biofilm formation under shaking conditions. IMPORTANCE Due to the versatility of the mPAD mount, we anticipate that it will aid the analysis of biofilm formation in a broad range of bacteria and archaea. Thereby, it contributes to answering critical biological questions about the regulatory and structural components of biofilm formation and understanding this process in a wide array of environmental, biotechnological, and medical contexts.