Can pre-analytical procedures improve microbiological culture yield in patients with periprosthetic infections?

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.

Evolving strategies in microbe identification-a comprehensive review of biochemical, MALDI-TOF MS and molecular testing methods.

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.

Diagnostic Accuracy of Dermatoscopy Versus Microbiological Culture and Polymerase Chain Reaction in the Diagnosis of Onychomycosis: A Cross-Sectional Study.

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.

Protocol for examining the T3SS-mediated cytotoxicity of Pseudomonas aeruginosa using the A549 cell line.

Pseudomonas aeruginosa utilizes a type III secretion system (T3SS) to directly inject effector proteins into host cells, leading to severe acute infections. Here, we present a protocol for detecting the T3SS-mediated cytotoxicity of P. aeruginosa using the A549 cell line. We describe the steps for the preparation of the A549 cell line and P. aeruginosa strains, cell seeding, bacterial culture, infection, and cytotoxicity assay. Additionally, we provide detailed procedures for data analysis. For complete details on the use and execution of this protocol, please refer to Huang et al.1.

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.

Practical Guidance for Clinical Microbiology Laboratories: Updated guidance for processing respiratory tract samples from people with cystic fibrosis.

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.

Identificación de microorganismos entéricos en áreas de preparación y consumo de alimentos / Identification of enteric microorganisms in food preparation and consumption areas / Identificação de microrganismos entéricos em áreas de preparação e consumo de alimentos

La contaminación de las áreas de preparación al entrar en contacto con los alimentos crudos o cocinados, es por esto que una de las principales causas de la contaminación de las superficies inertes es la inadecuada manipulación de los alimentos a la hora de ser preparados. Con el objetivo de controlar la aplicación de normas de higiene en las áreas de preparación y consumo de alimentos mediante análisis microbiológicos para disminuir los riesgos de contaminación alimentaria. Esta investigación es de carácter descriptivo, en la cual se realizó una inspección visual del establecimiento con el propósito de evaluar las condiciones higiénicas sanitarias, mediante la aplicación de la Guía Técnica para el Análisis Microbiológico de Superficies en contacto con Alimentos y Bebidas. Para el análisis microbiológico de las muestras se emplearon las técnicas de inoculación, método de estriado, aislamiento bacteriano, tinción diferencial y utilización de las pruebas bioquímicas como: TSI, SIM, Citrato de Simmons, Urea, Lisina, Catalasa y Oxidasa, además de la utilización de medios de cultivo selectivo y diferencial como agar EMB y agar MacConkey para la identificación de bacterias entéricas como: E. coli, Salmonella, Klebsiella pneumoniae, Shigella, Pseudomona aeruginosa. Los resultados arrojaron que la frecuencia bacteriana de las superficies inertes de los restaurantes en el área de preparación de alimentos (mesón y tabla de picar) tienen presencia de bacterias: Salmonella con mayor frecuencia; E. coli, Klebsiella pneumoniae y Pseudomonas aeruginosa de mediana frecuencia y de baja para Shigella, y en el área de consumo de alimentos (mesas) la bacteria de mayor frecuencia es la E. coli y Shigella, la Klebsiella pneumoniae de mediana y Pseudomona aeruginosa se encuentra en baja frecuencia. Se llegó a la conclusión que las superficies inertes tanto en el área de preparación como en el área de consumo de alimentos se encuentran contaminados por lo que hay un riesgo de infección alimentaria para los comensales de la Universidad Técnica de Machala.

Contamination of preparation areas when coming into contact with raw or cooked foods, which is why one of the main causes of contamination of inert surfaces is inadequate handling of food when it is being prepared. With the aim of controlling the application of hygiene standards in the areas of food preparation and consumption through microbiological analysis to reduce the risks of food contamination. This research is descriptive in nature, in which a visual inspection of the establishment was carried out with the purpose of evaluating the sanitary and hygienic conditions, through the application of the Technical Guide for the Microbiological Analysis of Surfaces in Contact with Food and Beverages. For the microbiological analysis of the samples, inoculation techniques, streaking method, bacterial isolation, differential staining and use of biochemical tests such as: TSI, SIM, Simmons Citrate, Urea, Lysine, Catalase and Oxidase, in addition to use of selective and differential culture media such as EMB agar and MacConkey agar for the identification of enteric bacteria such as: E. coli, Salmonella, Klebsiella pneumoniae, Shigella, Pseudomona aeruginosa. The results showed that the bacterial frequency of the inert surfaces of the restaurants in the food preparation area (counter and cutting board) have the presence of bacteria: Salmonella more frequently; E. coli, Klebsiella pneumoniae and Pseudomonas aeruginosa of medium frequency and low frequency for Shigella, and in the food consumption area (tables) the most frequent bacteria are E. coli and Shigella, Klebsiella pneumoniae of medium and Pseudomona aeruginosa It is at low frequency. It was concluded that the inert surfaces in both the preparation area and the food consumption area are contaminated, so there is a risk of food infection for diners at the Technical University of Machala

Contaminação das áreas de preparo ao entrar em contato com alimentos crus ou cozidos, por isso uma das principais causas de contaminação de superfícies inertes é o manuseio inadequado dos alimentos no momento do preparo. Com o objetivo de controlar a aplicação de padrões de higiene nas áreas de preparação e consumo de alimentos através de análises microbiológicas para reduzir os riscos de contaminação alimentar. Esta pesquisa é de natureza descritiva, na qual foi realizada uma inspeção visual do estabelecimento com a finalidade de avaliar as condições sanitárias e higiênicas, por meio da aplicação do Guia Técnico para Análise Microbiológica de Superfícies em Contato com Alimentos e Bebidas. Para a análise microbiológica das amostras foram utilizadas técnicas de inoculação, método de estrias, isolamento bacteriano, coloração diferencial e utilização de testes bioquímicos como: TSI, SIM, Citrato de Simmons, Ureia, Lisina, Catalase e Oxidase, além de utilização de testes seletivos e diferenciais. meios de cultura como ágar EMB e ágar MacConkey para identificação de bactérias entéricas como: E. coli, Salmonella, Klebsiella pneumoniae, Shigella, Pseudomona aeruginosa. Os resultados mostraram que a frequência bacteriana das superfícies inertes dos restaurantes na área de preparo de alimentos (balcão e tábua de corte) apresentam com maior frequência a presença de bactérias: Salmonella; E. coli, Klebsiella pneumoniae e Pseudomonas aeruginosa de média frequência e baixa frequência para Shigella, e na área de consumo alimentar (tabelas) as bactérias mais frequentes são E. coli e Shigella, Klebsiella pneumoniae de média e Pseudomona aeruginosa Está em baixa frequência. Concluiu-se que as superfícies inertes tanto na área de preparação como na área de consumo de alimentos estão contaminadas, pelo que existe risco de infecção alimentar para os comensais da Universidade Técnica de Machala

Current microbiological testing approaches and documented infections at febrile neutropenia onset in patients with hematologic malignancies.

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.

When less is more: the art of communicating clinical microbiology results.

The clinical microbiology laboratory is capable of identifying microorganisms in clinical specimens faster and more accurately than ever before. At face value, this should enable patient care providers to make better-informed decisions and target antimicrobial therapies to deliver individualized care. Ironically, more complete and specific reporting of microorganisms isolated from specimens may result in overtreatment based on the presence of a pathogen, even in the absence of clear signs of clinical infection. This conundrum calls into question the role of the laboratory in contributing to care through selective or "exception" reporting whereby some results are selectively withheld when there is a low probability that laboratory findings correlate with the clinical infection. In a recent article published in the Journal of Clinical Microbiology, Bloomfield et al. (J Clin Microbiol 62:e00342-24, 2024, https://doi.org/10.1128/jcm.00342-24) examine the impact and safety of an exception reporting strategy applied to wound swab specimens. Canonical pathogens associated with skin and soft tissue infections including S. aureus and beta-hemolytic streptococci are withheld from the laboratory report if certain patient criteria are met that would put them at low risk of adverse outcomes if untreated, or if treated with guideline-recommended empiric therapy. Their central finding was an approximately 50% reduction in post-laboratory report antibiotic initiation without adverse events or increased 30-day admission rate (indicative of infection-related complications, e.g., disseminated disease). While effectively achieving their goal, the premise of exception reporting and other modified reporting strategies raises questions about the potential risk of underreporting and how to ensure that the message is being interpreted, and acted upon, by care providers as was intended by the laboratory.

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.

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.

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.

The Rabbit Model of Cryptococcal Meningitis.

Cryptococcal meningitis (CM) is a fungal disease caused by the invasion of Cryptococcus yeast cells into the central nervous system. The organism is thought to enter the body through the lungs and then escape due to dysregulation of the immune response. Multiple animal species have been used to model the infection and characterize CM including mice, rats, dogs, guinea pigs, and rabbits. The rabbit model has over 40 years of data and has been used to study host-pathogen interactions and the efficacy of antifungal therapeutics. The model begins with immune suppression to eliminate the lymphocytic cell population followed by direct infection of the central nervous system via an injection of a suspension of yeast cells into the cisterna magna. The organism remains in the CNS during the course of infection, and cerebrospinal fluid can be repeatedly sampled to quantify the burden of organism, measure drug levels in the CSF, profile the immune response in the CSF, and/or characterize the yeast cells. The rabbit model of infection is a robust experimental model for better understanding CM and Cryptococcus cellular behavior.

Kodamaea ohmeri: A rare yeast causing invasive infections in immunocompromised patients.

INTRODUCTION: Kodamaea ohmeri is a rare, recognized pathogen that has previously been isolated from environmental sources. The patients commonly affected by this yeast include immunocompromised as well as immunocompetent patients having several associated risk factors. METHODOLOGY: We report three cases in which K. ohmeri was isolated from blood using Bact T/ALERT. Identification was carried out by MALDI-TOF MS (Vitek-MS, BioMérieux, Marcy-l'Etoile, France) in addition to color characteristics on chromogenic media. The patients had diminished immune response on account of a multitude of comorbidities. RESULTS: K. ohmeri can be misidentified as Candida tropicalis, Candida albicans, or Candida hemolounii by conventional methods; correct and timely identification can be achieved by MALDI-TOF MS. Antifungal susceptibility breakpoints for K. ohmeri are currently not defined. An Echinocandin was added to the treatment regimen of all three of the cases. CONCLUSIONS: Identification of K. ohmeri using conventional methods is difficult and unusual yeasts should be carefully observed, especially upon prolonged incubation.

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.