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PubMed1991

Sterility testing of a total nutrient admixture with a biphasic blood-culture system.

The ability of a biphasic blood-culture system to detect microbial contamination of a total nutrient admixture (TNA) was studied. A TNA consisting of amino acids, dextrose, lipid emulsion, electrolytes, and trace elements was prepared under aseptic conditions. Septi-Chek blood-culture bottles were then injected with a TNA sample and with one of various dilutions of five bacterial or fungal suspensions, and an agar slide unit was attached to each bottle. One bottle was injected only with a TNA sample. The bottles were incubated and examined once daily. Each bottle was inverted immediately after inoculation and at each observation time to allow the broth to wash over the agar slide unit. Because the lipid contained in the TNA caused the broth in all bottles to become turbid, it was impossible to determine whether microbial growth was present in the broth. Microbial growth, however, became evident in 24-72 hours on all slide units except the one attached to the bottle that had been injected only with TNA. The Septi-Chek biphasic blood-culture system appears to be useful in evaluating the sterility of TNAs.

Bacteria↗
PubMed1989

Molecular analysis of nosocomial epidemics.

Newer developments in molecular biology offer the hospital epidemiologist and clinical microbiologist powerful tools for analysis of nosocomial epidemics. Plasmid fingerprinting and genomic REA are rapid and often more definitive alternatives to serotyping and biotyping. Isolation of specific plasmids for special study involves more complicated manipulations. Polypeptide patterns, though at times diffuse in appearance, can be easily produced with SDS-PAGE of whole cells or outer membranes. Specific monoclonal antibodies as probes for individual proteins can generate more definitive answers about the exact type of protein(s) present. Multilocus enzyme electrophoresis (MLE) has become an ingenious tool for characterizing closely related nosocomial bacterial strains. The newest molecular methods using ribotyping, DNA probes, and the PCR will open many doors into the microbial genomes that were previously closed to the hospital microbial detective. These advances will compel hospitals to plan for their funding and implementation. Yet, like other scientific progress, the new biology in the nosocomial setting will raise as many questions as it will answer.

Bacteria↗
PubMed1987

Diagnosis of pneumonia: techniques and problems.

All diagnostic strategies in patients suspected of having pneumonia have considerable limitations. While the diagnostic strategy varies from patient to patient, an overview of one possible approach is summarized in Figures 2 and 3. The diagnosis and management of pneumonia usually poses little difficulty in the community setting, but the value of various diagnostic approaches in patients with hospital-acquired pneumonia is a subject of considerable controversy. Clinical criteria of pneumonia, including fever, purulent tracheobronchial secretions, leukocytosis, and a new infiltrate on chest radiograph are hampered by the high frequency with which these findings are observed in patients without pneumonia. Since tracheobronchial secretions are commonly contaminated by microorganisms colonizing the upper airways, routine culture of expectorated sputum, with the inevitable recovery of a potpourri of potential pathogens, can hardly be regarded as a meaningful exercise for the physician. Such cultures with subsequent extensive susceptibility testing form one of the largest workloads and expenses in microbiology laboratories. Clinical decisions based on such information may result in serious patient mismanagement with antibiotics, with the potential of superinfection and drug complication, which further add to hospital expenditures. Blood cultures are valuable when positive, but negative results are more common even in severe pneumonia. Transtracheal aspiration of tracheobronchial secretions is satisfactory in the diagnosis of community-acquired pneumonia in patients without pre-existing lung disease, but its value in the diagnosis of hospital-acquired pneumonia needs further evaluation. Transthoracic aspiration, especially with the newer finer needles, holds considerable promise but the significant risk of barotrauma deters most physicians from employing this procedure in patients requiring mechanical ventilation. Immunologic techniques of detecting microbial antigens, like countercurrentimmunoelectrophoresis and ELISA, are promising but presently inadequate to screen for a wide variety of organisms. Although it also has its limitations, fiberoptic bronchoscopy appears to be the most satisfactory technique if an invasive approach is being considered in a patient suspected of pneumonia. Samples taken with the plugged telescoping catheter technique, when properly performed, combined with quantitative cultures (and possibly antibody coating of bacteria) probably provide the least misleading information when a bacterial pathogen is being considered.(ABSTRACT TRUNCATED AT 400 WORDS)

Biopsy, Needle↗
PubMedDate not supplied

Immunodiagnosis of sexually transmitted disease.

Methods for detecting microbial antigens in clinical specimens offer an alternative to culture in the diagnosis of some sexually transmitted diseases. Developers of the immunologic methods are faced with a number of problems in evaluating the new tests. Traditionally, these tests are compared to culture as the "gold standard." Unfortunately, culture for Neisseria gonorrhoeae or Chlamydia trachomatis--the two agents most commonly sought--is considerably less sensitive than 100 percent. Immunologic methods may appear to produce false positives when the paired specimens are actually false-negative cultures. Another source of discordant results is sampling variation. These considerations, however, will not account for all false-positive results. Even the best non-culture methods have a low rate of false-positive results. If a new test has a specificity of 97 percent, it, by definition, yields approximately 3 percent false-positive reactions. In low-prevalence settings this false-positive rate will create problems in interpreting the results. For example, in a population with 3 percent prevalence of infection, a positive result in a 97 percent specificity test could only have a predictive value of 50 percent. Most testing for STD agents is performed in low-prevalence settings. None of the currently available immunodiagnostic procedures has a performance profile that suggests it will be satisfactory for diagnostic use in the low-prevalence setting.

Antigens, Bacterial↗
PubMed1985

[Cardiac abscess in infectious endocarditis. Apropos of 25 anatomo-clinical cases].

Although rarely suspected clinically, cardiac abscesses are a serious and not uncommon complication of infective endocarditis (IE). Twenty-five cardiac abscesses were found at autopsy in 125 cases of IE on native valves. The anatomoclinical features of these cases are described. Cardiac abscesses usually occur in patients with previous valvular heart disease (21/25) and more commonly in aortic valve endocarditis (15/25) especially when complicating calcific aortic stenosis (9/25). The predominant infecting organism was staphylococcus aureus (9 cases). An iatrogenic portal on entry was confirmed in 9/25 cases. Conduction defects (14/25) commonly led on to sudden death (9 cases) despite attempts at temporary pacing (14/25) commonly led on to sudden death (9 cases) despite attempts at temporary pacing (2 cases%. The clinical history was shorter than 2 months in 4/5 cases. The average age of the patients was 53.3 years. The commonest site of infection was the aortic valve (16/25), affecting the posterior cusp in all cases. It is difficult to summarise the localisation of the abscesses as collection of pus extended in all directions. Involvement of the valve rings (aortic 10 cases--mitral 5 cas), of the interventricular septum (15 cases), of the LV free wall (17 cases), was common, sometimes in association, and fistula formation between two different cavities was observed in 4 cases. The abscess may communicate with a cardiac chamber or remain enclosed in the parietal structures (17 cas) reaching an average size of 2 cm diameter. Histological examination distinguished collected forms with an identifiable pyogenic membrane from the extensive, gangrenous, necrotic form with detectable microbial colonies in 9 cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Abscess↗
PubMed1981

Mycoplasma detection-an obligation to scientific accuracy.

Mycoplasma contamination of cell cultures is, unfortunately, a common occurrence. Because of their extremely small size the contamination is not readily apparent and the presence of mycoplasmas is confirmed by culture on agar. Certain mycoplasmas, most notably Mycoplasma hyorhinis, often do not form colonies on agar due to the presence of toxic components. Other noncultural methods have been devised to detect these "noncultivable" mycoplasmas. A brief overview of these methods will be presented and an attempt made to compare their relative efficiency in detecting microbial contamination in cell cultures.

Cells, Cultured↗
PubMed1985

Novel microbial screen for detection of 1,4-butanediol, ethylene glycol, and adipic acid.

A novel microbial-screening procedure was developed for separate detection of 1,4-butanediol, ethylene glycol, and adipic acid, three commercially important oxychemicals potentially derivable from bacterial omega-oxidation of n-butanol, ethanol, and hexanoic acid, respectively. The screening method involved postproduction addition of one of several specific Pseudomonas strains which produce a soluble fluorescent pigment during growth on the product of interest. A mutation and selection procedure was developed for isolation of specific strains with phenotypes for growth and pigment production on the desired product (e.g., 1,4-butanediol), but not on its bioconversion substrate (e.g., n-butanol), common by-products (e.g., n-butyrate), or product isomers. Pigment production was growth associated and required cultivation of the screening strains under limiting Fe3+ concentrations. The pigments resembled well-characterized, iron-chelating siderophores produced by other fluorescent pseudomonads. The sensitivity of the assay for product accumulation was enhanced by (i) conducting the screening in microtiter dishes to permit examination of individual isolates of putative producers and to control product diffusion, (ii) using a wavelength cutoff filter to reduce background source light, and (iii) using adapted screening strains which grew at lower (0.3 mM) concentrations of test compounds. The potential utility of the method for detecting a variety of oxidative catabolic products is discussed.

1-Butanol↗
PubMed2005

An electrokinetically-controlled immunoassay for simultaneous detection of multiple microbial antigens.

An electrokinetically-controlled heterogeneous immunoassay microchip for multiple analyte detection was developed in this study. Numerical simulation was employed to study the transport process in a microfluidic network (microFN). The operation parameters obtained from numerical simulation was then applied to immunoassay experiment. The effectiveness of the automatic electrokinetic control was demonstrated in a separate experiment using fluorescein dye. The immunoassay microchip was made of poly(dimethylsiloxane)(PDMS)/PDMS-coated glass using soft lithography and replica molding. Multi-antigen immobilization was accomplished by adsorbing the antigen molecules onto a PDMS-coated glass slide and by using a microFN. Immobilized lysate antigen of Escherichia coli O157: H7 at different concentrations was assayed and the lower detect limit was 3 microg/mL. The assay also displayed very good specificity, when different microbial lysate antigens were immobilized, including Escherichia coli and Helicobacter pylori, and the primary and secondary antibodies were mixtures of different species. The time required for the immunoassay, from antigen coating to signal detection, was only one hour. While still an un-optimized prototype, this automatic-operating, high-throughput immunoassay microchip shows a great potential in detecting multiple pathogenic infections efficiently for clinical applications.

Antigens, Bacterial↗
PubMed2004

Specificity and performance of PCR detection assays for microbial pathogens.

PCR has become a widely used tool for detection, identification and differentiation of pathogenic microorganisms in diagnosis of animal and human diseases. However, quite a number of currently used protocols can be further optimized to exclude nonspecific reactions. On the one hand, target sequences as defined by primer binding sites should be checked carefully for the absence of significant homologies to other organisms in order to insure high specificity of detection. A major part of PCR assays is still based on target sequences in the ribosomal RNA operon, but, as the differentiating potential of this region is limited, genes encoding cellular proteins, such as toxins, surface antigens or enzymes, have been shown to be a viable alternative in many instances. On the other hand, various approaches are available to improve the performance of the amplification reaction itself. The kinetics of amplification is known to be heavily dependent on primer-to-template ratio, efficiency of primer annealing and enzyme-to-template ratio. In the present paper, recently published PCR detection assays for microorganisms, particularly bacterial pathogens, are reviewed and optimization strategies are explained. The practical implications and epidemiological consequences of routine use of PCR in the diagnostic laboratory are also discussed.

Bacterial Typing Techniques↗
PubMed2002

Rapid and quantitative detection of the microbial spoilage of meat by fourier transform infrared spectroscopy and machine learning.

Fourier transform infrared (FT-IR) spectroscopy is a rapid, noninvasive technique with considerable potential for application in the food and related industries. We show here that this technique can be used directly on the surface of food to produce biochemically interpretable "fingerprints." Spoilage in meat is the result of decomposition and the formation of metabolites caused by the growth and enzymatic activity of microorganisms. FT-IR was exploited to measure biochemical changes within the meat substrate, enhancing and accelerating the detection of microbial spoilage. Chicken breasts were purchased from a national retailer, comminuted for 10 s, and left to spoil at room temperature for 24 h. Every hour, FT-IR measurements were taken directly from the meat surface using attenuated total reflectance, and the total viable counts were obtained by classical plating methods. Quantitative interpretation of FT-IR spectra was possible using partial least-squares regression and allowed accurate estimates of bacterial loads to be calculated directly from the meat surface in 60 s. Genetic programming was used to derive rules showing that at levels of 10(7) bacteria.g(-1) the main biochemical indicator of spoilage was the onset of proteolysis. Thus, using FT-IR we were able to acquire a metabolic snapshot and quantify, noninvasively, the microbial loads of food samples accurately and rapidly in 60 s, directly from the sample surface. We believe this approach will aid in the Hazard Analysis Critical Control Point process for the assessment of the microbiological safety of food at the production, processing, manufacturing, packaging, and storage levels.

Artificial Intelligence↗
PubMed2006

Detection of individual microbial pathogens by proximity ligation.

BACKGROUND: Nucleic acid amplification allows the detection of single infectious agents. Protein-based assays, although they provide information on ongoing infections, have substantially less detection sensitivity. METHODS: We used proximity ligation reactions to detect proteins on bacteria and virus particles via nucleic acid amplification. Antibodies recognizing viral or bacterial surface proteins were equipped with DNA strands that could be joined by ligation when several antibodies were bound in proximity to surface proteins of individual infectious agents. RESULTS: Detection sensitivities similar to those of nucleic acid-based detection reactions were achieved directly in infected samples for a parvovirus and an intracellular bacterium. CONCLUSIONS: This method enables detection of ligated DNA strands with good sensitivity by real-time PCR and could be of value for early diagnosis of infectious disease and in biodefense.

Animals↗
PubMed2003

Application of the real-time PCR for the detection of airborne microbial pathogens in reference to the anthrax spores.

To establish the rapid detection method of airborne bacterial spores, we examined Bacillus anthracis spores by real-time PCR. One hundred liters of air were trapped on a filter of an air monitor device. After it was suspended in PBS, spores of B. anthracis were artificially added. The suspension was also heated at 95 degrees C for 15 min and used for real-time PCR using anthrax-specific primers. A single cell of B. anthracis was detected by real-time PCR within 1 h. Our results provide evidence that anthrax spores from the atmosphere can be detected rapidly, suggesting that real-time PCR provides a flexible and powerful tool to prevent epidemics.

Air Microbiology↗
PubMed2004

Microbial screening of UC blood units by an automated culture system: effect of delayed testing on bacterial detection.

BACKGROUND: Microbial screening is a mandatory test for banked UC blood (UCB) to comply with the code of good manufacturing practice (GMP). Cord blood banks (CBBs) are not always closely located to a GMP-licensed microbiology laboratory, resulting in time delays for transport of specimens prior to microbiological testing. This study investigated the influence of >/=24 h delays in initiating automated microbial screening on the detection of bacteria in UCB, by analysis of specimens deliberately spiked with bacteria and the recovery of bacteria from cryopreserved spiked-UCB. MATERIALS AND METHODS: UCB was processed according to standard CBB procedures and spiked with Staphylococcus epidermidis or Escherichia coli [2-2000 colony forming units (CFU)/mL]. Spiked-UCB (0.5 mL) was (1) held at room temperature (RT) and inoculated into pediatric BacT/Alert bottles (bioMérieux) at Days 1, 4 and 7 (delayed inoculation); and (2) inoculated directly (Day 0) into replicate BacT/Alert bottles and held at RT for 1, 4 or 7 days before loading onto the BacT/ALERT system (delayed loading). Spiked-UCB samples were cryopreserved. Bacterial counts were quantitated on horse blood agar plates. RESULTS: Bacterial growth in UCB spiked with a single bacterium was capable of detection by the BacT/ALERT system. S. epidermidis grew in all conditions of delayed testing (ie. delayed inoculation and delayed loading). E. coli failed to grow under conditions of delayed inoculation but grew at all time points of delayed loading. S. epidermidis and E. coli were recovered from cryopreserved spiked-UCB. DISCUSSION: Inoculation of culture bottles as soon as possible after sample preparation is preferable. Bacteria can maintain viability in BacT/ALERT bottles inoculated and held at RT for up to 7 days prior to automated culture testing. Bacteria can be successfully recovered from cryopreserved UCB.

Bacteria↗
PubMed2025

Rapid pan-microbial metagenomics for pathogen detection and personalised therapy in the intensive care unit: a single-centre prospective observational study.

BACKGROUND: Most clinical metagenomic studies do not provide rapid results, detect pathogens from all microbial kingdoms, or measure clinical impacts. We aimed to evaluate the feasibility, performance, and clinical impacts of a rapid pan-microbial respiratory metagenomic service for patients admitted to intensive care units (ICUs). METHODS: This was a single-centre observational study of a rapid metagenomics service that tests respiratory samples from ICU patients at Guy's and St Thomas' hospitals, London, UK, between Dec 5, 2023, and April 12, 2024. Testing used a previously published pan-microbial metagenomics workflow, which simultaneously detects bacteria, fungi, and DNA and RNA viruses; provides same-day preliminary results after 2 h; and provides final results after 24 h. Patients were included if they were aged 18 years or older, admitted to the ICU, had confirmed respiratory failure requiring supplemental oxygen or advanced airway support, and had at least one of the following: (1) clinical suspicion of lower respiratory tract infection based on clinical, biochemical, or radiological findings, (2) sepsis of unknown origin, and (3) concern from an intensive care physician regarding inflammatory pathology. Patients with a suspected or confirmed containment level three organism were excluded. The outcome was performance characteristics of the metagenomic test compared with routine diagnostic testing, detection of additional pathogens by metagenomics, change in antimicrobial prescribing within 24 h of testing, and initiation of immunomodulation. FINDINGS: We processed 114 samples (1-5 per day) from 74 patients (39 [53%] female and 35 [47%] male). 107 (94%) of 114 samples passed quality control, of which 101 (94%) provided same-day preliminary results. Bacteria were detected in 45 (43%) of 104 tested specimens, fungal organisms in 17 (16%) of 104 tested specimens, and viruses in 28 (34%) of 83 tested specimens. Sensitivity in lower respiratory tract samples after 24 h was 97% (95% CI 87-100) for bacteria, 89% (65-99) for fungi, and 89% (71-98) for viruses, with only one false positive for bacteria. Metagenomics identified 42 pathogens not detected by other tests in 32 (30%) of 107 samples. Antimicrobial therapy was changed after metagenomic results from 30 (28%) of 107 samples: 22 (21%) were de-escalated and eight (7%) were escalated. Metagenomics contributed to the initiation of immunomodulation in 15 (20%) of 74 patients for a range of inflammatory conditions. Pathogens with clinical significance to local infection control or national public health were found in ten (14%) of 74 patients, including three invasive Group A streptococci, two parvovirus B19, and one each of HIV-1, measles virus, Mycobacterium tuberculosis, Neisseria meningitidis, and Mycoplasma pneumoniae. INTERPRETATION: Respiratory metagenomics for ICU patients showed good performance and turnaround time, and diverse clinical and public health benefits. This ability to inform both personalised patient therapy and infectious disease surveillance needs evaluation in multicentre studies. FUNDING: None.

Humans↗
PubMed1998

COVASIAM: an image analysis method that allows detection of confluent microbial colonies and colonies of various sizes for automated counting.

In this work we introduce the confluent and various sizes image analysis method (COVASIAM), an automated colony count technique that uses digital imaging technology for detection and separation of confluent microbial colonies and colonies of various sizes growing on petri dishes. The proposed method takes advantage of the optical properties of the surfaces of most microbial colonies. Colonies in the petri dish are epi-illuminated in order to direct the reflection of concentrated light coming from a halogen lamp towards an image-sensing device. In conjunction, a multilevel threshold algorithm is proposed for colony separation and counting. These procedures improved the quantification of colonies showing confluence or differences in size. We tested COVASIAM with a sample set of microorganisms that form colonies with contrasting physical properties: Saccharomyces cerevisiae, Aspergillus nidulans, Escherichia coli, Azotobacter vinelandii, Pseudomonas aeruginosa, and Rhizobium etli. These physical properties range from smooth to hairy, from bright to opaque, and from high to low convexities. COVASIAM estimated an average of 95.47% (sigma = 8.55%) of the manually counted colonies, while an automated method based on a single-threshold segmentation procedure estimated an average of 76% (sigma = 16.27) of the manually counted colonies. This method can be easily transposed to almost every image-processing analyzer since the procedures to compile it are generically standard.

Aspergillus nidulans↗
PubMed2004

Rapid and quantitative detection of the microbial spoilage in chicken meat by diffuse reflectance spectroscopy (600-1100 nm).

AIMS: To evaluate the feasibility of visible and short-wavelength near-infrared (SW-NIR) diffuse reflectance spectroscopy (600-1100 nm) to quantify the microbial loads in chicken meat and to develop a rapid methodology for monitoring the onset of spoilage. METHODS AND RESULTS: Twenty-four prepackaged fresh chicken breast muscle samples were prepared and stored at 21 degrees C for 24 h. Visible and SW-NIR was used to detect and quantify the microbial loads in chicken breast muscle at time intervals of 0, 2, 4, 6, 8, 10, 12 and 24 h. Spectra were collected in the diffuse reflectance mode (600-1100 nm). Total aerobic plate count (APC) of each sample was determined by the spread plate method at 32 degrees C for 48 h. Principal component analysis (PCA) and partial least squares (PLS) based prediction models were developed. PCA analysis showed clear segregation of samples held 8 h or longer compared with 0-h control. An optimum PLS model required eight latent variables for chicken muscle (R = 0.91, SEP = 0.48 log CFU g(-1)). CONCLUSIONS: Visible and SW-NIR combined with PCA is capable of perceiving the change of the microbial loads in chicken muscle once the APC increases slightly above 1 log cycle. Accurate quantification of the bacterial loads in chicken muscle can be calculated from the PLS-based prediction method. SIGNIFICANCE AND THE IMPACT OF THE STUDY: Visible and SW-NIR spectroscopy is a technique with a considerable potential for monitoring food safety and food spoilage. Visible and SW-NIR can acquire a metabolic snapshot and quantify the microbial loads of food samples rapidly, accurately, and noninvasively. This method would allow for more expeditious applications of quality control in food industries.

Food Microbiology↗