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The Next Step: The Role of Metagenomic Next-Generation Sequencing in Microbial Detection of Culture-Negative Cardiovascular Infections.

Cardiovascular infections, including those that involve native and prosthetic heart valves, implantable cardiac devices, mechanical circulatory assist devices, and vascular grafts, are associated with significant morbidity and mortality risks. Optimal management of these complex infections requires pathogen-directed antimicrobial therapy. However, standard culture-based methods often fail to identify causative organisms due to prior antimicrobial use, infections due to fastidious organisms, or biofilm-associated infections. Emerging evidence suggests that microbial cell-free DNA (mcfDNA) and metagenomic testing can enhance pathogen detection, particularly in culture-negative cases. However, their results require careful clinical interpretation, often necessitating input from infectious diseases specialists. In this review, we examine published evidence regarding metagenomic testing for cardiovascular infections and its impact on patient care. We propose a framework for microbiological adjudication of mcfDNA results, introduce standardized definitions for clinical impact assessment, and provide guidance on integrating mcfDNA testing into diagnostic evaluation of patients with culture-negative cardiovascular infections.

Humans

Multichannel electrochemical microbial detection unit.

A compact multichannel unit for electrochemical detection of microorganisms that automatically displays detection time length is described. This unit was successfully tested with various members of the Enterobacteriaceae group.

Bacteriological Techniques

Preprototype of an automated microbial detection and identification system: a developmental investigation.

The AutoMicrobic System is an automated, computerized instrument that uses highly selective media and an optical system for detection, enumeration, and identification of bacteria and some yeasts in 13 h. A preprototype instrument (AutoMicrobic System-1) and its urine culture kit (Identi-Pak), developed for the detection, enumeration, and identification of eight species or groups of bacteria and of Candida species and Torulopsis glabrata in urine specimens, was evaluated during its development. An overall agreement of approximately 90% between the preprototype instrument and conventional (manual) culture methods has been obtained both with 1,473 seeded (simulated) and 1,688 clinical (mono- or polymicrobial) specimens containing 70,000 (or more) colony-forming units per ml of Escherichia coli, Klebsiella-Enterobacter species, Proteus species, Citrobacter freundii, Serratia species, group D enterococci, or yeasts (Candida species and T. glabrata). Lower agreements in identification were obtained with Pseudomonas aeruginosa-containing (average of 75% in clinical specimens) and Staphylococcus aureus-containing (76%) specimens. Comparison of specimens tested simultaneously in two preprototype systems resulted in =/<4% disagreement; true negativity agreements in all specimen groups tested were at least 94%. Among problems remaining are adaptation of system for specimens other than urine, improvement of sensitivity for P. aeruginosa and S. aureus, and standardization of manual methods used for comparison and validation.

Bacteria

Evaluation of rapid diagnostic tests in the detection of microbial invasion of the amniotic cavity.

OBJECTIVE: We sought to determine and compare the value of several rapid diagnostic tests in the detection of intraamniotic infection. STUDY DESIGN: Gram stain, intraamniotic glucose level determination, leukocyte esterase assay, and the Limulus amebocyte lysate assay were performed on 144 amniotic fluid specimens retrieved by transabdominal amniocentesis in 136 patients with preterm premature rupture of the membranes or preterm labor. Diagnostic indices for a positive amniotic fluid culture and the development of clinical infection were calculated for each rapid test. Receiver-operator characteristic curves were generated to help select the optimal glucose level and combination of tests to detect intraamniotic infection. RESULTS: The greatest sensitivity for predicting either a positive culture or subsequent clinical infection in preterm labor patients and in predicting clinical infection in patients with preterm premature rupture of the membranes was demonstrated by a low glucose level. The Gram stain provided the greatest positive predictive value in patients with preterm labor. Combining the Gram stain and measurement of intraamniotic glucose levels did not improve sensitivity above glucose alone or positive predictive value above Gram stain alone. CONCLUSION: Leukocyte esterase determination and Limulus amebocyte lysate assay are insensitive indicators of intraamniotic infection. Selection of Gram stain or glucose level measurement alone or in combination as an appropriate screen for intraamniotic infection will depend on the clinicians' false-positive rate threshold.

Adult

Detection of microbial nucleic acids for diagnostic purposes.

The last decade has seen an explosion of interest in the use of molecular genetic techniques to diagnose infectious diseases. Specific molecular probes have been developed for nearly all of the significant, known microbial pathogens. The evolution of incrementally more powerful technologies, particularly gene amplification, has made it possible to detect pathogens with exquisite sensitivity, as well as specificity, based on their nucleic acids. As increasingly rapid and automated methods have become incorporated into successive generations of probe tests, these diagnostics have gained increasing acceptance for routine clinical use.

Cloning, Molecular

Lecithin agar for detection of microbial phospholipases.

Lecithin agar was developed on which phospholipase C produced turbid zones and phospholipase A produced clear zones. Reactions on lecithin agar agreed 74% of the time with reactions in egg yolk broth. On lecithin agar, interpretation was easier, phospholipase A was detectable, and opaque zones were visible 1 or 2 days earlier than on egg yolk agar. All constituents of the medium can be autoclaved.

Agar

Rapid detection of microbial contamination in frozen vegetables by automated impedance measurements.

Automated impedance measurements can be used to rapidly assess whether a sample of frozen vegetables contains greater or less than 10(5) organisms per g. Microorganisms growing pureed food samples cause a change in the impedance of the medium when the organisms reach a threshold concentration of between 10(6) and 10(7) organisms per ml. Estimates of the concentration of microorganisms initially present in the food sample can be made by recording the time required for the organisms in the sample to replicate to threshold levels. In this study, the detection times for 357 samples of frozen vegetables were compared with standard plate counts for each sample. The agreement between the two methods in distinguishing samples containing more than 10(5) organisms per g was 92.6% for 257 assorted frozen vegetables and somewhat higher (93 to 96%) when separate cutoff times were used for each type of vegetable. The time required for analysis was about 5 h, compared to the 48 to 72 h required for standard plate counts.

Bacteria

[Detection of microbial protein synthesis in the small intestine of sheep using an intraduodenal 15N-urea infusion].

Sheep (3 animals, 50 kg LW) with reentrant cannulas in duodenum and at the end of the ileum received 700 g hay and 800 g alfalfa pellets per animal and day. In a previous 1st period of three days duodenal digesta and in a 2nd period of four days ileal digesta were collected and stored deep frozen. In the main period the digesta flow was interrupted for 28 hours. The duodenal and ileal digesta were collected quantitatively. The previously collected duodenal and ileal digesta portions were introduced hourly. The duodenal digesta was supplemented with 15N-labelled urea for a 24 hour period. 4.5% of the introduced 15N-excess were detected at the end of the ileum in the 24 hour period. 5.6% of the 15N-excess at the end of the ileum were incorporated in bacterial protein. It was measured that the ileal digesta contained 4.62 g N in the TCE precipitable fraction and 24.4% of the TCE precipitable N-fraction was bacterial nitrogen.

Animals

Comparative performance of sponge versus flocked swabs for culture-based and metagenomic detection of microbial contamination in the healthcare environment.

BACKGROUND: Identifying optimal methods for sampling surfaces in the healthcare environment is critical for future research requiring the identification of multidrug-resistant organisms (MDROs) on surfaces. METHODS: We compared 2 swabbing methods, use of a flocked swab versus a sponge-stick, for recovery of MDROs by both culture and recovery of bacterial DNA via quantitative 16S polymerase chain reaction (PCR). This comparison was conducted by assessing swab performance in a longitudinal survey of MDRO contamination in hospital rooms. Additionally, a laboratory-prepared surface was also used to compare the recovery of each swab type with a matching surface area. RESULTS: Sponge-sticks were superior to flocked swabs for culture-based recovery of MDROs, with a sensitivity of 80% compared to 58%. Similarly, sponge-sticks demonstrated greater recovery of Staphylococcus aureus from laboratory-prepared surfaces, although the performance of flocked swabs improved when premoistened. In contrast, recovery of bacterial DNA via quantitative 16S PCR was greater with flocked swabs by an average of 3 log copies per specimen. CONCLUSIONS: The optimal swabbing method of environmental surfaces differs by method of analysis. Sponge-sticks were superior to flocked swabs for culture-based detection of bacteria but inferior for recovery of bacterial DNA.

Humans

Interleukin 6 determination in the detection of microbial invasion of the amniotic cavity.

A growing body of evidence suggests a role for cytokines in the mechanisms responsible for preterm parturition associated with intrauterine infection. Interleukin 6, a polyfunctional cytokine that is secreted by tissues in the feto-maternal interface in response to microbial products, has been implicated in the host response to intrauterine infection. The purpose of this study was to establish whether measurement of amniotic fluid concentrations of interleukin 6 could be of value in the diagnosis of microbial invasion of the amniotic cavity. Fluid was obtained by transabdominal amniocentesis from patients with preterm labour and intact chorioamniotic membranes and cultured for aerobic and anaerobic bacteria and mycoplasmas. Interleukin 6 concentrations were determined by an ELISA validated for human amniotic fluid. An interleukin 6 concentration above 11.2 ng/ml had a 93.7% sensitivity and a 92.3% specificity in the diagnosis of intra-amniotic infection. Moreover, patients with an amniotic fluid interleukin 6 level above 11.2 ng/ml and a negative amniotic fluid culture failed to respond to tocolysis, delivered a preterm infant and showed histological evidence of chorioamnionitis, and their neonates were at risk for congenital infections.

Amniotic Fluid

Particle size distribution analysis for the rapid detection of microbial infection of urine.

The accuracy and practicality of particle size distribution analysis for rapid screening of urine specimens are assessed. Six hundred urines were subjected simultaneously to routine bacteriological examinations and particle size distribution analysis using a Coulter Counter (ZBI) linked to a C1000 Channelyzer. There was complete agreement in the results of 593 (98.8%) specimens. Characteristic profiles of various bacterial species in infected specimens were consistently obtained. This system can easily be linked to any existing computer reporting in a district hospital laboratory, and the results of negative specimens (70--80%) can be obtained within 5--10 minutes.

Bacteriological Techniques

Blood cultures with the BACTEC 225 radiometric microbial growth detection system.

The BACTEC 225 automated radiometric blood culture system was compared with a conventional blood culture bottle method for its ability to improve the rapid laboratory diagnosis of bacteremia in cancer patients. The BACTEC 225, in combination with routine blind subcultures and smears of radiometrically negative culture vials, detected two thirds, of all positive cultures within 24 h and shortered the detection time generally by 24-48 h. With the recommended growth index setting of 30, radiometrically false-positive findings are rare and are usually due to leukocytosis resulting from infection or leukemia.

Bacteriological Techniques

[Microbial colonization and succession in the large intestine of newborn infants during their stay with mothers at the maternity hospital].

Formation of microflora in the large intestine of 5-day old infants was studied in one of the Moscow maternity homes. The up-to-date procedures for isolation and identification of aerobic and anaerobic organisms were used in the study and the findings were processed on a computer. In the newborns of the maternity home of the "mother-infant" type there was observed colonization of the large intestine with aerobic and anaerobic organisms. A wave-like dynamics in the formation of the symbiotic microflora was revealed. It reflected the phenomenon of the microbial succession in the infants. The attempts to detect microbial interference between the species colonizing the large intestine showed that it was extremely rare in the 5-day old infants. This was likely the reason of the low intestine resistance to the colonization in the newborns which in its turn defined the frequent colonization of the intestine mucosa with S. aureus and the organisms of the Klebsiella, Enterobacter and Citrobacter group.

Adult

Microbial DNA analysis of paired blood-bronchoalveolar lavage fluid in post-HSCT patients with pneumonia implying application conditions of blood as a surrogate in pathogen detection.

BACKGROUND: Blood testing aids pneumonia diagnosis, but its effectiveness varies. Given the invasiveness of bronchoalveolar lavage fluid (BALF) sampling versus blood testing's simplicity, this study investigates when blood can reliably substitute for BALF in detecting microbial presence, especially for pathogens. RESULTS: Metagenomic sequencing was performed on paired BALF-blood samples from 21 post-HSCT immunocompromised (ICP) and 21 immunocompetent (ICT) patients. The ICP cohort was expanded to 62 for biomarker validation. Host responses were profiled via metatranscriptomics (30 BALF samples). Microbial alpha and beta diversity differed significantly between blood and BALF in ICP, but not ICT, patients. ICP patients' BALF contained a greater diversity and abundance of microbes. A higher proportion of microbial DNA sequences in ICP patients' blood was also present in their BALF, suggesting a potentially more permeable alveolar-capillary barrier. Related genes (e.g., NABA CORE MATRISOME, extracellular matrix organization, cell-cell adhesion) were downregulated. Upregulated pathways like VEGFA-VEGFR2 signaling and Rho GTPases suggested increased vascular permeability. In ICP patients, 419 microbial sequences in blood indicated their presence in the lower respiratory tract with >&#x2009;70% certainty. CONCLUSION: Host immune status significantly influences blood-BALF microbial diversity differences. Shared blood-BALF microbial DNA sequences show potential for aiding pneumonia pathogen diagnosis, offering a novel biomarker identification approach.

Humans