A simplified laboratory approach for isolating M. oryzae spores from rice samples infected with multiple pathogens.

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.

Breaking boundaries in microbiology: customizable nanoparticles transforming microbial detection.

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.

BASEHIT scores home run: elucidates pathogen-host interactions.

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.

How to verify and validate a clinical microbiology test before it can be used in routine diagnostics: a practical guide.

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.

Integrated decentralized blood culture incubation: A step towards a 24/7 microbiology service?

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.

Clarifications on the Intended Use of USP <61> Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests.

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.

Evaluation of an automated rapid phenotypic antimicrobial susceptibility testing (ASTar, Q-linea AB) applied directly on blood cultures bottles positive for Gram-negative pathogens.

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.

Sonication protocols and their contributions to the microbiological diagnosis of implant-associated infections: a review of the current scenario.

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.

Endoscope sampling and culturing methods.

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.

The clinical value of mNGS of bronchoalveolar lavage fluid versus traditional microbiological tests for pathogen identification and prognosis of severe pneumonia (NT-BALF):study protocol for a prospective multi-center randomized clinical trial.

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.

Practical Guidance for Clinical Microbiology Laboratories: Microbiologic diagnosis of implant-associated infections.

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.

Comparison of MBT biotargets with MBT steel targets for matrix-assisted laser desorption/ionization time of flight mass spectrometry identifications of microorganisms in a clinical microbiology laboratory.

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.

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.

Present and future perspectives on mass spectrometry for clinical microbiology.

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.

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.