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PubMed1991

Culture vs direct antigen assays for detection of microbial pathogens from lower respiratory tract specimens suspected of containing the respiratory syncytial virus.

Following the introduction of effective antiviral chemotherapy, rapid antigen assays have been utilized increasingly, instead of cell cultures, for detection of the respiratory syncytial virus from lower respiratory tract specimens. Because antigen assays, unlike cell culture, cannot amplify low levels of the virus to a detectable level, assay sensitivity is especially dependent on high-quality specimens. In addition, the assays are unable to detect other viruses or bacteria with which the patient may be infected. This review summarizes results from clinical studies of the performance of cell cultures and the more commonly used antigen assays, describes factors that may lead to false-positive or false-negative test results, and makes recommendations for the selection of procedures for the reliable detection of microbial pathogens from patients suspected of being infected with respiratory syncytial virus.

Antigens, Viral↗

PubMed1981

Enzymic analysis for rapid detection of microbial infection in human body fluids: an overview.

One possible means of rapidly detecting microorganisms in patients with suspected infectious diseases is the direct measurement of microbial enzymes in body fluids. This technique is based on the fact that bacterial, fungal, and viral organisms possess enzymes that are not produced by mammalian cells and are thus not found in uninfected human body fluids. Detection of one of these microbial enzymes in blood, cerebrospinal fluid, or other body fluids would thus be indicative of microbial infection. Potentially useful enzymes for this purpose include bacterial beta-lactamases, fungal adenine deaminases, and viral thymidine kinases. In addition, glycosidases such as neuraminidases and galactosidases can be used as markers for microbial infection, provided that the enzymic activity can be appropriately identified as being of microbial origin. The direct measurement of microbial enzymes offers great potential for the rapid diagnosis of infectious diseases.

Bacteria↗

PubMed1985

The role of sampling in the detection of microbial contamination on cadaveric allograft skin used as a biological wound dressing.

The availability of cryopreservation and low temperature storage techniques for cadaveric allograft skin allows it to be preserved while microbial assessments are made before its use as a temporary biological dressing on burn wounds. In a 300-donor, 5-year prospective study, we tested ten skin samples from defined areas on each donor for microbiological contamination. Although the skin from 52.3 per cent of the donors possessed some detectable residual microbial contamination after surgical body preparation and skin removal, such contamination was limited to an average of 1.4 areas per body, leaving 86 per cent of all skin obtained free from detectable contamination and suitable for use as biological wound dressings. The number of skin samples tested per donor body determined the accuracy of detection of the presence of contamination. Testing one skin sample per donor body yielded a correct skin assessment 92 per cent of the time, while testing five skin samples increased the accuracy to 96 per cent, and testing ten skin samples yielded a 99.9 per cent accuracy in detection of skin contamination. Thus, it is within the ability of a skin bank to set the limits of microbiological risk to patients receiving processed cadaveric allograft skin.

Bacteria↗

PubMed1987

Clinical relevance of culture versus screens for the detection of microbial pathogens in urine specimens.

The use of screens to detect "significant levels" of pathogenic microorganisms in urine specimens offers the advantages of both rapidly reporting results and controlling costs. Many of these screens, however, are insensitive at microbial counts below 10(5) colony-forming units (CFU)/ml of urine. It is increasingly apparent that patients with almost any type of urinary tract infection (except for most patients who are asymptomatic or who have pyelonephritis) may have urine concentrations of pathogens as low as 10(2) to 10(3) CFU/ml. This review documents factors that can contribute to diminished concentrations of microorganisms in urine, lists patient populations in whose urine microorganisms in concentrations well below 10(5) CFU/ml have been associated with infection, and makes recommendations for selection of laboratory tests, including rapid screens, for the diagnosis and management of urinary tract infections.

Age Factors↗

PubMed1971

Chemical detection of microbial prey by bacterial predators.

A motile, predacious bacterium which degraded Pythium debaryanum was strongly attracted to substances released into the medium by the fungus. A nonpredacious bacterium was not attracted to these substances. The predator bacterium was specifically attracted to cellulose and its oligomers which are known to be components of the cell wall of Pythium. Ethanol inhibited chemotaxis of the bacterium without affecting either its motility or its ability to degrade cellulose. A second predacious bacterium was isolated for the alga, Skeletonema costatum. The role of chemoreception in the detection of microbial prey by bacterial predators in natural habitats is discussed.

Agar↗

PubMed2001

Time to positivity of neonatal blood cultures.

AIM: To determine how long it takes neonatal blood cultures to become positive. METHODS: Data were collected retrospectively on 451 positive blood cultures from babies on a tertiary neonatal unit between January 1997 and December 1998. During the study period, the laboratory used the BacT/Alert microbial detection system. RESULTS: Complete information was available on 416 blood cultures. Twelve became positive after 72 hours, none of which were considered to be clinically significant. Of the 404 remaining cultures, 86% were positive at 36 hours, 96% at 48 hours, and 98.5% by 60 hours. If definite bacterial pathogens are considered alone, the time to positivity was 90% by 36 hours, 93% by 48 hours, and 98% by 60 hours. If definite and possible bacterial pathogens are considered (coagulase negative staphylococci taken as possible bacterial pathogens), the time to positivity was 89% at 36 hours and 97% at 48 hours. The negative predictive value, for isolation of any organism before 72 hours, of a negative blood culture was 97% at 36 hours and 99% at 48 hours. The negative predictive value for the isolation of definite bacterial pathogens only was 99.7% at 36 hours and 99.8% at 48 hours. CONCLUSIONS: A period of 36 hours is enough to rule out sepsis in the asymptomatic neonate, and a three day incubation period is sufficient to detect all clinically important infections using the BacT/Alert microbial detection system.

Bacteremia↗

PubMedDate not supplied

Alternative microbial testing: a novel DNA-based detection system for specified microorganisms in pharmaceutical preparations.

Fluorescence-coupled PCR technology was employed to quantify DNA segments specific for Staphylococcus aureus, Pseudomonas aeruginosa, and Enterobacteriaceae. The PCR procedure is put forward as an alternative method for detecting microbial contaminations in pharmaceutical preparations and is compared to the tests for specified microorganisms described in European Pharmacopoeia (EP) 2, 2.6.13 and the USP, chapter 61. Data presented here describe the validation of this analytical method when used for proof of absence of specified microorganisms. The detection systems were specific for the microorganisms analyzed, and led to linear results over a wide range (more than 6-7 log intervals). The correlation coefficients lay above 0.99. The precision of replicate determinations within a single test was observed to be high, the relative standard deviation being between 0.39% and 1.53%. The precision between different tests was also high, with a relative standard deviation between 0.76% and 1.91%. The sensitivity without pre-enrichment amounted to 1-10 CFU. Since determination of the specified bacteria was performed following pre-enrichment, the limit of detection amounted to 1 CFU. Equivalent results were obtained in a study on nine batches of a milky hydrophilic cream (SH-No. M 440 A) with the conventional test for microbial contamination and the PCR procedure. The data presented here strongly indicate that the use of fluorescence-coupled PCR techniques can prove the absence of specified bacteria faster and more efficiently than conventional methods.

DNA, Bacterial↗

PubMed2006

Molecular techniques in wastewater: Understanding microbial communities, detecting pathogens, and real-time process control.

Traditionally, the detection of pathogens in water, wastewater, and other environmental samples is restricted by the ability to culture such organisms from complex environmental samples. During the last decade the use of molecular methods have supplied the means for examining microbial diversity and detecting specific organisms without the need for cultivation. The application of molecular techniques to the study of natural and engineered environmental systems has increased our insight into the vast diversity and interaction of microorganisms present in complex environments. In this paper, we will review the current and emerging molecular approaches for characterizing microbial community composition and structure in wastewater processes. Recent studies show that advances in microarray assays are increasing our capability of detecting hundreds and even thousands of DNA sequences simultaneously and rapidly. With the current progress in microfluidics and optoelectronics, the ability to automate a detection/identification system is now being realized. The status of such a system for wastewater monitoring is discussed.

DNA Fingerprinting↗

PubMedDate not supplied

A new method for in vitro detection of microbially produced mitochondrial toxins.

Sperm motility inhibition assay, earlier shown valuable for the detection of food poisoning non-protein toxins of Bacillus species was developed into an assay useful for specific detection of mitochondria damaging toxins. This was done by assessing the dissipation of the mitochondrial inner membrane transmembrane potential, Deltapsim under conditions where the plasma membrane permeability barrier remained intact. The Deltapsim was estimated as the intensity of orange JC-1 fluorescence in the mitochondrial sheath of the exposed spermatozoa. The plasma membrane integrity of the same cells was assessed by observing the exclusion of propidium iodide from the cytoplasm. Three types of mitochondrial toxic responses to microbially made bioactive substances were recognised. Mitochondrial toxicity by gramicidin (A, B, C, D), nigericin, salinomycin, narasin, monensin, calcimycin and antimycin A was characterised by gradual fading of the JC-1 fluorescence in the mitochondria. Dissipation of the Deltapsim by cereulide, valinomycin and enniatin (A, A1, B, B1) was visible as spotwise quenching of the mitochondrial JC-1 fluorescence. In addition these substances caused hyperpolarisation of the plasma membrane. Oligomycin (A, B, C), ionomycin and staurosporine inhibited the spermatozoan motility, but Deltapsim was fully preserved. Surfactin and lichenysin A caused mitochondrial damage at concentrations where the plasma membrane was also damaged.

Animals↗

PubMed1971

Microbial esterase detection with ultraviolet fluorescence.

A method is presented to identify esterase-synthesizing microbial colonies within mixed culture plates by utilizing induced esterase hydrolysis of nonfluorescent butyryl ester of 7-hydroxy-4-methylcoumarin to the highly fluorescent 7-hydroxy-4 methyl umbelliferone. Microscopy procedures for making esterase loci of fungal mycelia visible with this reaction are described.

Aspergillus↗

PubMed1984

The use of beta-lactamase in enzyme immunoassays for detection of microbial antigens.

The sensitivity and performance characteristics of enzyme immunoassays (EIA) depend to a great extent on the kinetics of the enzyme-substrate system used as indicator. We labeled a variety of polyclonal and monoclonal immunoglobulins with purified beta-lactamase and used them in sensitive EIA systems for the detection of a number of microbial antigens. Polyclonal antibodies to rotavirus, adenovirus, and Haemophilus influenzae type b polyribitol phosphate and monoclonal antibodies to dengue virus were labeled with beta-lactamase and used to provide sensitive direct EIA systems for the detection of the corresponding antigens. In addition, antibodies directed at animal immunoglobulins were labeled with beta-lactamase and used in indirect EIA for the detection of viral antigens with unlabeled anti-viral monoclonal and polyclonal antibodies. Similarly, avidin from Streptomyces was labeled with beta-lactamase and used to detect viral antigens tested for in an avidin-biotin format. Enzyme immunoassay systems with beta-lactamase-labeled antibodies were also used to detect rotaviral and adenoviral antigens in rectal swab specimens from children with acute gastroenteritis. The sensitivity of the beta-lactamase EIA compared favorably with that of analogous EIA systems using alkaline phosphatase or horseradish peroxidase. The results of a beta-lactamase EIA were easily determined by naked eye and a permanent record of the qualitative results obtained by the use of a standard office photocopier, obviating the need for an expensive colorimeter. Enzyme immunoassays using beta-lactamase have potential as practical assay systems for the detection of a wide range of microbial antigens using monoclonal and polyclonal antibodies.

Antigens, Bacterial↗

PubMed2005

Immobilized stem-loop structured probes as conformational switches for enzymatic detection of microbial 16S rRNA.

We have designed and evaluated novel DNA stem-loop structured probes for enzymatic detection of nucleic acid targets. These probes constitute a novel class of conformational switches for enzymatic activity, which in the absence of a target sterically shield an affinity label and upon hybridization of the target to the recognition sequence that forms the loop of the probe restore accessibility of the label for the binding of a reporter enzyme. Analysis of probe characteristics revealed stem stability as the most important parameter governing detection functionality, while other factors such as the length of linker molecules attaching the label to the stem-loop structure and the nature of the solid support proved to be less critical. Apparently, the bulky nature of the reporter enzyme facilitates shielding of the label in the absence of the target, thereby conferring considerable structural tolerance to the conformational switch system. The stem-loop structured probes allow sensitive detection of unlabeled nucleic acid targets. Employing a microtiter assay format, 4 ng of bacterial 16S ribosomal RNA corresponding to 8 fmol could be detected, which can be compared favorably with current immobilized molecular beacon concepts based on fluorescence detection.

Colorimetry↗

PubMed1984

Enzyme immunoassays in which biotinillated beta-lactamase is used for the detection of microbial antigens.

The performance characteristics of enzyme immunoassays are determined to a great extent by the enzyme-substrate system utilized for the immunoassay. Beta-lactamases (penicillin amido-beta-lactamhydrolase EC 3.5.2.6) offer a number of advantages which might make them useful in immunoassay systems. We linked beta-lactamase from Bacillus cereus with biotin and used the biotinillated enzyme to devise immunoassay systems for the detection of a number of microbial antigens. An assay system in which antibodies to the polyribitol phosphate antigen of Haemophilus influenzae type b were used was capable of detecting between 0.4 and 1.6 ng of that antigen. Similarly, an assay in which antibodies to the common antigens of adenoviruses and biotin-linked beta-lactamase were used was capable of detecting between 1 and 10 50% tissue culture infective doses of a strain of enteric-type adenovirus. When applied to the detection of rotavirus, a similar system in which biotinillated beta-lactamase was used was capable of detecting small amounts of antigen in a standard rotavirus preparation. This assay could also detect virus in 36 of 37 stool specimens from children with rotavirus gastroenteritis. The positive specimens could easily be distinguished from negative ones by the naked eye, and a permanent record of the qualitative results could be obtained by the use of a standard office photocopying machine. Beta-lactamases have promise for use in practical enzyme immunoassay systems, especially in situations in which expensive colorimetric instrumentation is not available.

Antigens, Bacterial↗

PubMedDate not supplied

[Development of new plate tests for the detection of microbial hydrolysis of esters and oxidation of 2-hydroxycarboxylic acids].

The application of the thin-agar-layer coated filter culture technique (Z. Naturforsch. 42c, 1082 (1987)) has been extended to the detection of ester hydrolysis and 2-hydroxyacid oxidation by microbial colonies. The former was performed by spraying with bromocresol purple and the latter with a salicylhydrazide reagent. Under the optimized conditions, the hydrolysis activity of more than 20 mumol/h.g-wet cells and the oxidation activity of more than 40 mumol/h.g-wet cells were usually detected directly on the filter-plate cultures of bacteria, yeasts and molds.

Alcaligenes↗

PubMed1992

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↗

PubMed1992

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↗

PubMed1986

[Detection of microbial variants of Salmonella typhi in the bone marrow of typhoid patients and carriers].

The immunofluorescence test with the use of antisera to S. typhi and its L-forms permits the detection of the infective agent in bone marrow smears. This diagnostic method is particularly important in cases of carrier state in the latent phase and in differential diagnosis. As revealed in this study, the microbial variants of S. typhi, reacting with antiserum to its L-forms, are present in the bone marrow of carriers. They are localized, as a rule, on the membranes of lymphoid and erythroid cells, which probably leads to the formation of rosettes.

Bone Marrow↗

PubMed1993

The acute infection-associated hemolytic anemia of childhood: immunofluorescent detection of microbial antigens altering the erythrocyte membrane.

The majority of acute infection-associated hemolytic diseases of infancy and childhood have been suggested to be caused by exogenic alterations of the erythrocyte surface, though laboratory methods for their further evaluation were not yet available. Investigating 96 children, the present study characterizes 72% of cases as corresponding to this type of acute acquired hemolytic anemia, which cannot be clearly related to autoantibodies against unmodified components of the host's own red cells. Using a new immunofluorescence test, the erythrocyte membrane of 80% of these children was found to be altered in vivo by nonspecific adsorption of foreign material released from the infectious micro-organisms. In 24% of cases additive binding of complement was detectable by an antiglobulin test. Thus, the adsorption of microbial antigens to the red cell surface is suggested to be one of the causes for the removal of altered erythrocytes due to phagocytosis or a complement-dependent destruction during the course of infection-associated hemolytic anemia. Especially in childhood, the immunofluorescent detection of an erythrocyte sensitization in vivo provides a further characterization of this type of mostly transient hemolytic disease, which probably can be treated without any immunosuppressive drug, merely by elimination of the underlying infection.

Adolescent↗