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Lodderomyces elongisporus, first isolated in 1952, has increasingly been recognized as a significant pathogen, with a notable rise in human infections since the 1970s. Initially misidentified as Candida parapsilosis due to morphological similarities, L. elongisporus has now been conclusively established as a distinct species, largely due to advancements in molecular biology, particularly DNA sequencing. This review traces the detection history of L. elongisporus, from the earliest documented cases to the most recent reports, underscoring its role as a causative agent in human infections. It also explores therapeutic strategies that have demonstrated efficacy, alongside instances of environmental contamination reported in international literature. A critical evaluation of diagnostic methodologies essential for precise identification is provided, including culture-based techniques such as colony morphology on Sabouraud Dextrose Agar (SDA) and chromogenic media, coupled with microscopic assessments using Lactophenol Cotton Blue (LPCB) and Gram staining. The ultrastructure of L. elongisporus, as observed under Scanning Electron Microscopy (SEM), is also discussed. Furthermore, non-culture-based diagnostics, such as sugar utilization tests (API 20C AUX and the innovative in-house arabinose-based "Loddy" test) and antifungal susceptibility profiling, are reviewed, with a particular focus on molecular tools like ITS-DNA sequencing and MALDI-TOF MS, which, despite their higher costs, offer unparalleled specificity. The accurate distinction and characterization of L. elongisporus are paramount, particularly in vulnerable and immunocompromised patients, where misdiagnosis can lead to severe consequences. This review advocates for intensified research efforts to develop more accessible diagnostic tools and deepen our understanding of this emerging pathogen, ultimately aiming to improve patient outcomes., (© 2024. The Author(s).)

Diagnostic stewardship (DxS) has gained traction in recent years as a cross-disciplinary method to improve the quality of patient care while appropriately managing resources within the healthcare system. Clinical microbiology laboratorians have been highly engaged in DxS efforts to guide best practices with conventional microbiology tests and more recently with molecular infectious disease diagnostics. Laboratories can experience resistance to their role in DxS, especially when the clinical benefits, motivations for interventions, and underlying regulatory requirements are not clearly conveyed to stakeholders. Clinical laboratories must not only ensure ethical practices but also meet obligatory requirements to steward tests responsibly. In this review, we aim to support clinical microbiology laboratorians by providing the background and resources that demonstrate the laboratory's essential role in DxS. The heart of this review is to collate regulatory and accreditation requirements that, in essence, mandate DxS practices as a long-standing, core element of high-quality laboratory testing to deliver the best possible patient care. While examples of the clinical impact of DxS are plentiful in the literature, here, we focus on the operational and regulatory justification for the laboratory's role in stewardship activities., Competing Interests: E.M.M. is an employee of Quest Diagnostics, a commercial clinical reference laboratory, and owns stock in the company.

Competing Interests: I declare no competing interests. I thank the Directorate of Research of Universidad Técnica de Ambato for funding project PFCIAB 39.

Objectives: This study aims to identify infection etiology in febrile neutropenia (FN) is vital. This study explores different microbiological approaches and their impact on diagnosing infections in patients with hematologic malignancies and FN., Methods: This is a retrospective analysis conducted at the Hospital Clinic of Barcelona details microbiological testing strategies used to diagnose infections at FN onset between January 2020 and July 2022., Results: A total of 4520 microbiological tests were ordered in 462 FN episodes, achieving a 10% test positivity rate, with 200 (43.3%) episodes showing microbiological documentation of infection. Blood cultures (40.4%), non-culture blood tests (21.2%), and respiratory tract samples (16.2%) were the most requested. Blood cultures exhibited the highest (16.9%) test positivity rates, whereas non-culture blood tests showed the lowest (3.3%). Bacterial infections were present in 149 of 462 (32.3%) FN episodes. Viral infections (66 of 462, 14.3%)-notably, respiratory viruses-were also frequent. Mortality rate at 60 days was 9.1%; documented infections were associated with a higher risk (15%)., Conclusions: In the current landscape of antimicrobial diagnostics, our findings revealed the highest reported rate of microbiologically documented infections at FN onset. Bacterial infections are common; however, our data reiterate the significance of viral infections in causing fever. Optimizing FN management during respiratory viral infections remains a challenge for antimicrobial de-escalation. The low positivity rates observed in certain diagnostic tests emphasize the need for cost-effective diagnostic stewardship., Competing Interests: Declarations of competing interest CG-V has received honoraria for talks on behalf of Gilead Science, MSD, Pfizer, Jannsen, Novartis, Basilea, GSK, Shionogi, AbbVie, and Advanz Pharma and grant support from Gilead Science, Pfizer, GSK, MSD, and PharmaMar. PP-A has received honoraria for talks on behalf of Merck Sharp and Dohme, Lilly, ViiV Healthcare, and Gilead Science., (Copyright © 2024. Published by Elsevier Ltd.)

Background: Before a new test can be routinely used in your laboratory, its reliability must be established in the laboratory where it will be used. International standards demand validation and verification procedures for new tests. The International Organization for Standardization (ISO) 15189 was recently updated, and the European Commission's In Vitro Diagnostic Regulation (IVDR) came into effect. These events will likely increase the need for validation and verification procedures., Objectives: This paper aims to provide practical guidance in validating or verifying microbiology tests, including antimicrobial susceptibility tests in a clinical microbiology laboratory., Sources: It summarizes and interprets certain parts of standards such as ISO 15189:2022, and regulations, such as IVDR 2017/746 regarding validation or verification of a new test in a routine clinical microbiology laboratory., Content: The reasons for choosing a new test and the outline of the validation and verification plan are discussed. Furthermore, the following topics are touched upon: the choice of reference standard, number of samples, testing procedures, how to solve the discrepancies between results from new test and reference standard, and acceptance criteria. Arguments for selecting certain parameters (such as reference standard and sample size) and examples are given., Implications: With the expected increase in validation and verification procedures because of the implementation of IVDR, this paper may aid in planning and executing these procedures., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Detection and identification of microorganisms are the first steps to guide susceptibility testing and enable clinicians to confirm diseases and guide therapy. The faster the pathogen identification is determined, the quicker the appropriate treatment can be started. In the clinical microbiology laboratory, multiple methodologies can be used to identify organisms, such as traditional biochemical testing or more recent methods like MALDI TOF MS and nucleic acid detection/identification assays. Each of these techniques has advantages and limitations, and clinical laboratories need to determine which methodology is best suited to their particular setting in terms of clinical needs, availability of technical expertise and cost. This article presents a concise review of the history, utilization, advantages and limitations of the main methods used for identifying microorganisms in microbiology laboratories., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

SUMMARYThis guidance presents recommendations for clinical microbiology laboratories for processing respiratory samples from people with cystic fibrosis (pwCF). Appropriate processing of respiratory samples is crucial to detect bacterial and fungal pathogens, guide treatment, monitor the epidemiology of cystic fibrosis (CF) pathogens, and assess therapeutic interventions. Thanks to CF transmembrane conductance regulator modulator therapy, the health of pwCF has improved, but as a result, fewer pwCF spontaneously expectorate sputum. Thus, the collection of sputum samples has decreased, while the collection of other types of respiratory samples such as oropharyngeal and bronchoalveolar lavage samples has increased. To optimize the detection of microorganisms, including Pseudomonas aeruginosa , Staphylococcus aureus , Haemophilus influenzae , and Burkholderia cepacia complex; other less common non-lactose fermenting Gram-negative bacilli, e.g., Stenotrophomonas maltophilia , Inquilinus , Achromobacter , Ralstonia , and Pandoraea species; and yeasts and filamentous fungi, non-selective and selective culture media are recommended for all types of respiratory samples, including samples obtained from pwCF after lung transplantation. There are no consensus recommendations for laboratory practices to detect, characterize, and report small colony variants (SCVs) of S. aureus , although studies are ongoing to address the potential clinical impact of SCVs. Accurate identification of less common Gram-negative bacilli, e.g., S. maltophilia , Inquilinus , Achromobacter , Ralstonia , and Pandoraea species, as well as yeasts and filamentous fungi, is recommended to understand their epidemiology and clinical importance in pwCF. However, conventional biochemical tests and automated platforms may not accurately identify CF pathogens. MALDI-TOF MS provides excellent genus-level identification, but databases may lack representation of CF pathogens to the species-level. Thus, DNA sequence analysis should be routinely available to laboratories for selected clinical circumstances. Antimicrobial susceptibility testing (AST) is not recommended for every routine surveillance culture obtained from pwCF, although selective AST may be helpful, e.g., for unusual pathogens or exacerbations unresponsive to initial therapy. While this guidance reflects current care paradigms for pwCF, recommendations will continue to evolve as CF research expands the evidence base for laboratory practices., Competing Interests: Lisa Saiman receives grant funding (provided to institution) from the U.S. CF Foundation and serves as a consultant to the CF Foundation. Valerie Waters receives grant funding from the U.S. CF Foundation and CF Canada. John J. LiPuma receives grant funding from the U.S. CF Foundation. Lucas R. Hoffman receives grant funding from the U.S. CF Foundation. Kevin Alby has no conflicts of interest to report. Sean X. Zhang receives grant funding from the U.S. CF Foundation. Yvonne C. Yau receives grant funding from the U.S. CF Foundation and CF Canada. Damian G. Downey receives grants from the CF Trust and U.S. CF Foundation. He receives support from the European CF Society and U.S. CF Foundation for travel to meetings. He is the Director of the European CF Society Clinical Trials Network. Isabelle Sermet-Gaudelus receives funding from the European Cystic Fibrosis Society and the UK Cystic Fibrosis Trust. Jean-Philippe Bouchara receives funding from Vaincre la Mucoviscidose, La Pierre Le Bigaut, Association Grégory Lemarchal, and Anjou Muco. Timothy J Kidd has no conflicts of interest to report. Scott C. Bell receives grant funding from the U.S. CF Foundation, the Australian National Health and Medical Research Council, and the Medical Research Future's Fund. A. Whitney Brown is an employee of the U.S. CF Foundation.

Background: The detection of causative pathogens plays a crucial role in the diagnosis and targeted treatment of periprosthetic joint infections (PJI). While there have been improvements in analytic methods in the past, pre-analytical procedures have not yet been sufficiently investigated. The objective of this study was to compare the culture yield of four different pre-analytical procedures., Methods: Patients with perioperative diagnosis of PJI were included in a single center cross-sectional study (2021-2022). Tissue samples (n = 20) of each patient were randomly and equally distributed to each of the four study arms. Tissue samples were either send to the laboratory without culture medium (group A) or were transported in thioglycolate medium immediately after sampling at three different temperatures (room temperature, 4 °C, 37° for 24 h; group B-D). Culture media were investigated for growth on days 1, 3, 7, 12, 14. All organisms, the number of positive samples and the time to positivity were recorded and compared between the study arms. Single positive cultures were considered as contamination., Results: In total, 71 patients were included. The proportions of culture negative samples (10-15%) and polymicrobial infections (51-54%) were comparable between the four arms. Seven patients (10%) were culture-negative in group A, but showed growth in thioglycolate media (group B-D). Furthermore, 13% of patients showed growth in all groups, but additional organisms were cultured in thioglycolate. There was growth beyond day 7 of culturing only in thioglycolate, but not in group A. A storage temperature of 4 °C showed a longer time to positivity compared to the other groups (p < 0.001)., Conclusions: Pre-analytical storage of tissue samples in thioglycolate broth did not improve the culture yield and did not detect additional cases of infection compared to the standard (pre-analytical storage in sterile containers). However, including a thioglycolate medium to the sampling algorithm reduced the rate of culture-negative infections and helped to identify additional organisms., (© 2024. The Author(s).)

Background: Several clinical signs in dermatoscopy are very characteristic of onychomycosis and can be a quick complement for the diagnosis of onychomycosis., Objectives: The aim of this study was to evaluate the diagnostic accuracy of dermatoscopy compared to microbiological culture and polymerase chain reaction (PCR), as well as the clinical signs associated with onychomycosis., Methods: The clinical signs of 125 patients were assessed cross-sectionally using dermatoscopy, and a positive or negative result was assigned. A sample was then taken for PCR and microbiological culture., Results: Of the 125 patients, 69.6% (87/125) had positive results when both laboratory tests were combined. When they were not combined, the prevalence was lower at 48% (60/125) with PCR and at 43.2% (54/125) with culture. Furthermore, 76.8% (96/125) were classified as positive with dermatoscopy with a sensitivity of 1, a specificity of 0.76, positive predictive value of 0.91 and negative predictive value of 1 (with 95% confidence intervals). Of the 96 dermatoscopy-positive samples, 36 were negative with PCR (p < 0.001), 42 were negative with culture (p < 0.001) and nine were negative when both tests were combined (p < 0.001). Clinical signs that were significantly associated with the presence of onychomycosis were subungual hyperkeratosis (dermatoscopy: p = 0.004, odds ratio (OR) = 2.438; PCR + microbiological culture: p = 0.004, OR = 3.221), subungual detritus (p = 0.033, OR = 3.01, only with dermatoscopy) and dermatophytoma (dermatoscopy: p = 0.049, OR = 3.02; PCR + microbiological culture: p = 0.022, OR = 2.40)., Conclusions: The results suggest that dermatoscopy is a good tool for the diagnosis of onychomycosis but should be used as a complementary test or for screening patients to be sampled for laboratory testing. The combination of the three tests can lead to a reduction of false-positive and false-negative clinical and laboratory results. This allows for early diagnosis and specific treatment based on test results., (© 2024 The Author(s). Mycoses published by Wiley‐VCH GmbH.)

In a tour de force, Hart and colleagues recently used a technique known as BASEHIT (bacterial selection to elucidate host-microbe interactions in high throughput) to screen a yeast display library containing 3324 curated human exoproteins with 82 pathogen samples, focusing on vector-borne pathogens, to identify 1303 putative interactions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

In the last decade, MALDI-TOF Mass Spectrometry (MALDI-TOF MS) has been introduced and broadly accepted by clinical laboratory laboratories throughout the world as a powerful and efficient tool for rapid microbial identification. During the MALDI-TOF MS process, microbes are identified using either intact cells or cell extracts. The process is rapid, sensitive, and economical in terms of both labor and costs involved. Whilst MALDI-TOF MS is currently the gold-standard, it suffers from several shortcomings such as lack of direct information on antibiotic resistance, poor depth of analysis and insufficient discriminatory power for the distinction of closely related bacterial species or for reliably sub-differentiating isolates to the level of clones or strains. Thus, new approaches targeting proteins and allowing a better characterization of bacterial strains are strongly needed, if possible, on a very short time scale after sample collection in the hospital. Bottom-up proteomics (BUP) is a nice alternative to MALDI-TOF MS, offering the possibility for in-depth proteome analysis. Top-down proteomics (TDP) provides the highest molecular precision in proteomics, allowing the characterization of proteins at the proteoform level. A number of studies have already demonstrated the potential of these techniques in clinical microbiology. In this review, we will discuss the current state-of-the-art of MALDI-TOF MS for the rapid microbial identification and detection of resistance to antibiotics and describe emerging approaches, including bottom-up and top-down proteomics as well as ambient MS technologies., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

A method for separating M. oryzae from rice samples infected with multiple pathogens using basic laboratory equipment is described. We conducted a series of experiments to obtain a single spore of M. oryzae. This method can also be used to isolate spores from other fungal species., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

To have an impact on the mortality of bloodstream infections, microbiological diagnostics of blood cultures (BC) should provide first results within 12 h. Here, we show how a decentralized BC incubation connected to the central BC incubators via a browser-based application significantly reduces turnaround times., Competing Interests: Declaration of Competing Interest All authors report no conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

The detection and identification of microorganisms are crucial in microbiology laboratories. Traditionally, detecting and identifying microbes require extended periods of incubation, significant manual effort, skilled personnel, and advanced laboratory facilities. Recent progress in nanotechnology has provided novel opportunities for detecting and identifying bacteria, viruses, and microbial metabolites using customized nanoparticles. These improvements are thought to have the ability to surpass the constraints of existing procedures and make a substantial contribution to the development of rapid microbiological diagnosis. This review article examines the customizability of nanoparticles for detecting bacteria, viruses, and microbial metabolites and discusses recent cutting-edge studies demonstrating the use of nanotechnology in biomedical research.

Background: Contamination rates reported in the literature for patient-ready flexible endoscopes vary from 0.4% to 49%. Unfortunately, the comparison and interpretation of these results is almost impossible since several factors including sampling and culturing methods, target levels for contamination, or definition of indicator micro-organisms vary widely from one study to the other., Aim: To compare the efficacy of six duodenoscope sampling and culturing methods by means of extraction efficacy comparison, while at the same time identifying key parameters that provide optimal microbial recovery., Methods: The duodenoscope sample extraction efficacy of each method was assessed using the repetitive recovery method described in ISO 11737-1: 2018., Findings: Mean overall bioburden extraction efficacy varied from 1% for the Australian method to 39% for the French one. The lowest endoscope sample extraction efficacy was associated with the absence of any neutralizer, friction, or tensioactive agent, and when only a small portion of the sampling solution collected was inoculated on to culture media. The efficacy of the sampling and culturing methods also varied according to the nature of micro-organisms present in the endoscope, and the time between sampling and culturing., Conclusion: This study supports the need for a harmonized and standardized sampling and culturing method for flexible endoscopes., (Copyright © 2024 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.)

In the execution of its legislated responsibilities, the United States Food and Drug Administration commonly refers to standard test methods detailed in the United States Pharmacopeia (USP). Microbiological test methods (contained in general chapters) are listed in chapters <51> to <80> with details regarded as enforceable where referenced as a test method. USP <61> "Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests" is a globally harmonized chapter that has been successfully employed for the enumeration of microorganisms recoverable from nonsterile finished drug products. The content of USP <61> is not always scientifically principled nor emphatically understood by all pharmaceutical microbiologists. Consequently, misunderstanding and misapplication of USP <61> may result in analyses and assessments of microbiological quality that are flawed or erroneous. In this article, clarification is provided to assist the pharmaceutical microbiologist in the appropriate and intended use of USP <61>, including provision of details not always commonly known or understood., (© PDA, Inc. 2024.)

SUMMARYImplant-associated infections (IAIs) pose serious threats to patients and can be associated with significant morbidity and mortality. These infections may be difficult to diagnose due, in part, to biofilm formation on device surfaces, and because even when microbes are found, their clinical significance may be unclear. Despite recent advances in laboratory testing, IAIs remain a diagnostic challenge. From a therapeutic standpoint, many IAIs currently require device removal and prolonged courses of antimicrobial therapy to effect a cure. Therefore, making an accurate diagnosis, defining both the presence of infection and the involved microorganisms, is paramount. The sensitivity of standard microbial culture for IAI diagnosis varies depending on the type of IAI, the specimen analyzed, and the culture technique(s) used. Although IAI-specific culture-based diagnostics have been described, the challenge of culture-negative IAIs remains. Given this, molecular assays, including both nucleic acid amplification tests and next-generation sequencing-based assays, have been used. In this review, an overview of these challenging infections is presented, as well as an approach to their diagnosis from a microbiologic perspective., Competing Interests: M.A.A. is a consultant for Cytophage Technologies Ltd., as well as BioFire USA and bioMérieux Canada. R.P. reports grants from MicuRx Pharmaceuticals and BioFire USA. R.P. is a consultant to PhAST, Day Zero Diagnostics, Abbott Laboratories, Sysmex, DEEPULL DIAGNOSTICS, S.L., Netflix and CARB-X. In addition, R.P. has a patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued. R.P. receives honoraria from Up-to-Date and the Infectious Diseases Board Review Course.

MALDI-TOF MS identifications of microorganisms in a clinical laboratory were investigated, comparing steel targets with MBT Biotargets. By using MBT Biotargets, the score values of yeast identifications increased, whereas the score values of Gram-negative bacteria decreased. Switching to MBT Biotargets did not negatively impact overall frequencies of high confidence identifications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Addressing the existing problem in the microbiological diagnosis of infections associated with implants and the current debate about the real power of precision of sonicated fluid culture (SFC), the objective of this review is to describe the methodology and analyze and compare the results obtained in current studies on the subject. Furthermore, the present study also discusses and suggests the best parameters for performing sonication. A search was carried out for recent studies in the literature (2019-2023) that addressed this research topic. As a result, different sonication protocols were adopted in the studies analyzed, as expected, and consequently, there was significant variability between the results obtained regarding the sensitivity and specificity of the technique in relation to the traditional culture method (periprosthetic tissue culture - PTC). Coagulase-negative Staphylococcus (CoNS) and Staphylococcus aureus were identified as the main etiological agents by SFC and PTC, with SFC being important for the identification of pathogens of low virulence that are difficult to detect. Compared to chemical biofilm displacement methods, EDTA and DTT, SFC also produced variable results. In this context, this review provided an overview of the most current scenarios on the topic and theoretical support to improve sonication performance, especially with regard to sensitivity and specificity, by scoring the best parameters from various aspects, including sample collection, storage conditions, cultivation methods, microorganism identification techniques (both phenotypic and molecular) and the cutoff point for colony forming unit (CFU) counts. This study demonstrated the need for standardization of the technique and provided a theoretical basis for a sonication protocol that aims to achieve the highest levels of sensitivity and specificity for the reliable microbiological diagnosis of infections associated with implants and prosthetic devices, such as prosthetic joint infections (PJIs). However, practical application and additional complementary studies are still needed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Silva, De Melo, Oliva and de Araújo.)

We evaluated the performance of a new rapid phenotypic antimicrobial susceptibility test (ASTar; Q-linea AB) on Gram-negative bacilli, directly from positive blood cultures bottles. MIC values obtained by the routine reference method (Microscan, Beckman Coulter) were compared to the ones provided by the tested method (ASTar). ASTar demonstrated an overall essential agreement of 98% and a category agreement of 96.1%. The overall rate of major errors and very major errors was 2.5% and 3.3%, respectively. ASTar can represent a rapid, simple, and reliable method to speed up information about antimicrobial susceptibility of Gram-negative pathogens from positive blood culture bottles.

Background: Early, rapid, and accurate pathogen diagnosis can help clinicians select targeted treatment options, thus improving prognosis and reducing mortality rates of severe pneumonia. Metagenomic next-generation sequencing (mNGS) has a higher sensitivity and broader pathogen spectrum than traditional microbiological tests. However, the effects of mNGS-based antimicrobial treatment procedures on clinical outcomes and cost-effectiveness in patients with severe pneumonia have not been evaluated., Methods: This is a regional, multi-center, open, prospective, randomized controlled trial to evaluate that whether the combination of mNGS and traditional testing methods could decrease 28-day call-cause mortality with moderate cost-effectiveness. A total of 192 patients with severe pneumonia will be recruited from four large tertiary hospitals in China. Bronchoalveolar lavage fluid will be obtained in all patients and randomly assigned to the study group (mNGS combined with traditional microbiological tests) or the control group (traditional microbiological tests only) in a 1:1 ratio. Individualized antimicrobial treatment and strategy will be selected according to the analysis results. The primary outcome is 28-day all-cause mortality. The secondary outcomes are ICU and hospital length of stay (LOS), ventilator-free days and ICU-free days, consistency between mNGS and traditional microbiological tests, detective rate of mNGS and traditional microbiological tests, turn-out time, time from group allocation to start of treatment, duration of vasopressor support, types and duration of anti-infective regimens, source of drug-resistant bacteria or fungi, and ICU cost., Discussion: The clinical benefits of mNGS are potentially significant, but its limitations should also be considered., Trial Registration: ChineseClinicalTrialRegistry.org, ChiCTR2300076853. Registered on 22 October 2023., (© 2024. The Author(s).)

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., Competing Interests: Conflict of interests The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

The study objective was to describe the formation of forensic microbiological examination as an analysis of a new type, defined as the detection and registration of reliably measured environmental and physiological changes within the microbial community of corpse in order to substantiate the possibility of using microbiological parameters to establish the prescription of death coming. It has been determined that the knowledge of the patterns of interaction of a human and his corpse with endogenous and exogenous flora provides the basis for solving a number of traditional and new application-oriented expert tasks and the allocation of such a variety of forensic examination as forensic microbiological examination. Endogenous and exogenous human flora and its interaction with living and dead biological tissues are the objects of this kind of examination, and the dynamic patterns of such interaction are the subject of study. One of the initial relevant tasks of forensic microbiological examination consists in development of methods, adequate for the expert task to be solved, choice of the research «target», «models» for comparative analysis and medium, adequate for task in hand, as well as certification of these methods and standardization of assessment criteria for the obtained results.

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., (© PDA, Inc. 2023.)

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., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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 (R 2 >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., (© PDA, Inc. 2023.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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 bla ISAba1-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., Competing Interests: The authors declare a conflict of interest. Reagents, instrumentation, and funding were provided by the Nippon Becton Dickinson Company, Ltd. M.M., K.K., and K.Y. received honoraria from the Nippon Becton Dickinson Company, Ltd. Y.M., M.O., and A.W. are employees of the Nippon Becton Dickinson Company, Ltd. N.A., K.I., and H.M. declare no other conflicts of interest.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Levican, Varela, Porte, Weitzel, Briceño, Guerra, Mena and Hinton.)

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., (© 2023. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., Competing Interests: Declaration of competing interests The authors declare that they have no conflict of interest in relation to this article., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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.

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., (Copyright © 2022 Sociedade Brasileira de Dermatologia. Published by Elsevier España, S.L.U. All rights reserved.)

Downstream processing is an expensive step for industrial production of recombinant proteins. Cell immobilization is known as one of the ideal solutions in regard to process intensification. In recent years, magnetic immobilization was introduced as a new technique for cell immobilization. This technique was successfully employed to harvest many bacterial and eukaryotic cells. But there are no data about the influence of magnetic immobilization on the eukaryotic inducted recombinant cells. In this study, impacts of magnetic immobilization on the growth and metabolic status of induced recombinant Pichia pastoris as a valuable eukaryotic model cells were investigated. Results based on colony-forming unit, OD600, and trypan blue assay indicated that magnetic immobilization had no adverse effect on the growth and viability of P. pastoris cells. Also, about 20-40% increase in metabolic activity was recorded in immobilized cells that were decorated with 0.5-2 mg/mL nanoparticles. Total protein and carbohydrate of the cells were also measured as main indicatives for cell function and no significant changes were observed in the immobilized cells. Current data show magnetic immobilization as a biocompatible technique for application in eukaryotic expression systems. Results can be considered for further developments in P. pastoris-based expression systems., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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 m ulti p anel ad hesion (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 (Δ phz ) 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 Δ phz 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.

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.

BACKGROUND The human microbiota modulates the immune system and forms the surface flora. Antibiotic administration causes dysbiosis in the intestinal flora. It is not clear if antibiotic administration in the community effects the upper airway flora in the mid-term or long-term. This study aims to define long-term influence of antibiotics on upper airway flora. MATERIAL AND METHODS In this prospective study, aerobic microbiological analysis of nasal and nasopharyngeal surfaces was performed. Antibiotic administration history of the last 6 months was retrieved using the social insurance database. Culture results of antibiotic-treated and antibiotic-naïve subjects were compared by Pearson's chi-square test or Fisher's exact test. RESULTS A total of 210 subjects were included in the study. Normal flora were documented in 86 nasal swabs and 99 nasopharyngeal swabs. Most of the remaining cases demonstrated gram-positive bacterial overgrowth. There were 113 subjects who did not receive any antibiotic, and 93% of the remaining 97 patients received broad-spectrum antibiotics. Statistical analysis showed that nasal and nasopharyngeal flora did not change upon antibiotic administration, but antibiotic administration during the last month caused increased methicillin resistance development of coagulase-negative Staphylococcus and Staphylococcus aureus microorganisms. CONCLUSIONS Antibiotic exposure did not lead to perturbations in general composition of upper airway flora within 6 months, although the incidence of methicillin resistance in coagulase-positive and -negative Staphylococci demonstrated significant increases when patients received antibiotic during the last month. This should be considered in case of broad-spectrum antibiotic administration, since methicillin resistance increases the morbidity and mortality of nosocomial Staphylococcus infections.

Toxicological batch assays are essential to assess a compound's acute effect on microorganisms. This methodology is frequently employed to evaluate the effect of contaminants in sensitive microbial communities from wastewater treatment plants (WWTPs), such as autotrophic nitrifying populations. However, despite nitrifying batch assays being commonly mentioned in the literature, their experimental design criteria are rarely reported or overlooked. Here, we found that slight deviations in culture preparations and conditions impacted bacterial community performance and could skew assay results. From pre-experimental trials and experience, we determined how mishandling and treatment of cultures could affect nitrification activity. While media and biomass preparations are needed to establish baseline conditions (e.g., biomass washing), we found extensive centrifugation selectively destabilised nitrification activities. Further, it is paramount that the air supply is adjusted to minimise nitrite build-up in the culture and maintain suitable aeration levels without sparging ammonia. DMSO and acetone up to 0.03% (v/v) were suitable organic solvents with minimal impact on nitrification activity. In the nitrification assays with allylthiourea (ATU), dilute cultures exhibited more significant inhibition than concentrated cultures. So there were biomass-related effects; however, these differences minimally impacted the EC 50 values. Using different nutrient-media compositions had a minimal effect; however, switching mineral media for the toxicity test from the original cultivation media is not recommended because it reduced the original biomass nitrification capacity. Our results demonstrated that these factors substantially impact the performance of the nitrifying inoculum used in acute bioassays, and consequently, affect the response of AOB-NOB populations during the toxicant exposure. These are not highlighted in operation standards, and unfortunately, they can have significant consequential impacts on the determinations of toxicological endpoints. Moreover, the practical procedures tested here could support other authors in developing testing methodologies, adding quality checks in the experimental framework with minimal waste of time and resources., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Infectious diarrhea is caused by a variety of pathogens, including viruses, bacteria, and parasitic organisms. Though the causative agent of diarrhea has historically been evaluated via stool cultures, recently, culture-independent diagnostic tests (CIDT) have been developed and utilized with increasing frequency. Current practice guidelines recommend their use as adjuncts to stool cultures for diagnosing acute and chronic diarrhea. The three principal CIDT are microscopy, enzyme-based immunoassays (EIAs), and molecular based polymerase chain reaction (PCR). This review explores the common causes of infectious diarrhea, the basics of stool culture, the diagnostic utility of these three culture-independent modalities, and the strengths and weaknesses of all currently available clinical techniques. It also outlines considerations for specific populations including returning travelers and those with inflammatory bowel disease., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Fusarium graminearum is an important fungus causing a variety of maize diseases, including stalk rot, ear rot, and sheath rot. However, conidia of F . graminearum are not easily obtained under normal culture conditions, which seriously affects the identification and pathogenicity assessment of the isolates and screening of resistance sources. This study was undertaken to develop and utilize a rapid sporulation technique of F . graminearum using liquid cultivation, which could meet the needs of various tests. The results show that the optimum conditions for sporulation of F . graminearum were as follows: culture medium, 0.154 mol/liter of saline; temperature, 28 to 30°C; incubation time, 96 h; initial pH, 9 to 10; illumination, continuous ultraviolet light; and shaking speed, 150 rpm. Using this culture method, conidial concentration of tested F . graminearum strains can reach >1.5 × 10 5 conidia/ml. Compared with the existing methods using mung bean and carboxylmethyl cellulose as matrix, saline is relatively inexpensive, and the culture process, relatively quick. Overall, this study provided a systematic, rapid, and simple method to obtain a large number of conidia of F . graminearum .

L-asparaginase from endophytic Fusarium proliferatum (isolate CCH, GenBank accession no. MK685139) isolated from the medicinal plant Cymbopogon citratus (Lemon grass), was optimized for its L-asparaginase production and its subsequent cytotoxicity towards Jurkat E6 cell line. The following factors were optimized; carbon source and concentration, nitrogen source and concentration, incubation period, temperature, pH and agitation rate. Optimization of L-asparaginase production was performed using One-Factor-At-A-Time (OFAT) and Response surface methodology (RSM) model. The cytotoxicity of the crude enzyme from isolate CCH was tested on leukemic Jurkat E6 cell line. The optimization exercise revealed that glucose concentration, nitrogen source, L-asparagine concentration and temperature influenced the L-asparaginase production of CCH. The optimum condition suggested using OFAT and RSM results were consistent. As such, the recommended conditions were 0.20% of glucose, 0.99% of L-asparagine and 5.34 days incubation at 30.50 °C. The L-asparaginase production of CCH increased from 16.75 ± 0.76 IU/mL to 22.42 ± 0.20 IU/mL after optimization. The cytotoxicity of the crude enzyme on leukemic Jurkat cell line recorded IC 50 value at 33.89 ± 2.63% v/v. To conclude, the enzyme extract produced from Fusarium proliferatum under optimized conditions is a potential alternative resource for L-asparaginase., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The rapidity, accuracy, and detection abilities of different laboratory methods (tube agglutination test (SAT), indirect ELISA, fluorescence polarization test (FPA), and blood culture methods) to detect Brucella in the laboratory. The study included 95 patients with documented and 42 patients with suspected brucellosis and 56 healthy control subjects. For the tests, the positive rates of Brucella infection detection in the confirmed group were significantly higher than in group with suspected infection (p<0.01) and in healthy controls (p<0.01). There was no significant difference between indirect ELISA and FPA in detecting antibodies to Brucella in acute (χ 2 =0.335), subacute (χ 2 =0.660), and chronic cases (χ 2 =5.332). Among the detection methods, indirect ELISA showed the highest sensitivity (98.9%), specificity (100%), and Youden index (0.989). The sensitivity and specificity of FPA were 96.8 and 96.4%, respectively. In order to easily and rapidly diagnose brucellosis in clinical practice, a combination of detection methods is recommended, in which Brucella antibodies are screened by FPA and then confirmed by indirect ELISA., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Objectives: The rapid Carbapenem Inactivation Method (rCIM) was evaluated with a strain collection of 164 and 69 carbapenem-resistant Enterobacterales and Pseudomonas aeruginosa, respectively, that produced various carbapenemases. For an improved carbapenemase detection in Enterobacterales, an optimized variant of the rCIM named TSBrCIM was developed., Methods: Bacterial isolates were incubated with two meropenem disks in distilled water (rCIM) or tryptic soy broth (TSBrCIM). After centrifugation, the supernatant was incubated with a susceptible E. coli indicator strain in tryptic soy broth. Growth of the indicator strain implied carbapenemase activity in the test strain., Results: The rCIM detected 100/113 carbapenemase-producing Enterobacterales, resulting in a sensitivity of 88.5% and a specificity of 94.1%. For P. aeruginosa, sensitivity and specificity were 96.0% and 100%, respectively. The TSBrCIM was able to detect 105/113 carbapenemase-producing Enterobacterales, resulting in a sensitivity of 92.9% and a specificity of 96.1%., Conclusion: This study shows that the TSBrCIM can be valuable tool for detection of carbapenemases in Enterobacterales in the clinical laboratory, while the rCIM showed the best results for carbapenemase detection in P. aeruginosa., (Copyright © 2021 Elsevier B.V. All rights reserved.)