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PubMed1993

A comparative study of the diagnostic performance of amniotic fluid glucose, white blood cell count, interleukin-6, and gram stain in the detection of microbial invasion in patients with preterm premature rupture of membranes.

OBJECTIVE: Our aim was to compare the value of amniotic fluid tests for the detection of microbial invasion of the amniotic cavity and in the prediction of the amniocentesis-to-delivery interval and neonatal complications in patients with preterm premature rupture of membranes. STUDY DESIGN: Amniotic fluid was obtained by transabdominal amniocentesis from 110 consecutive patients with preterm premature rupture of membranes. Fluid was cultured for aerobic and anaerobic bacteria, as well as mycoplasmas. Amniotic fluid analysis included a Gram stain examination, white blood cell count, and glucose and interleukin-6 determinations. Logistic regression and survival techniques (proportional hazards model) were used for statistical analysis. RESULTS: (1) The prevalence of positive amniotic fluid cultures in patients with preterm premature rupture of membranes was 38% (42/110); (2) patients with microbial invasion had a shorter amniocentesis-to-delivery interval and a higher neonatal complication rate than patients with negative cultures; (3) the most sensitive test for the detection of microbial invasion of the amniotic cavity was amniotic fluid interleukin-6 determinations (cutoff 7.9 ng/ml) (sensitivity: for IL-6 80.9%; for white blood cell count 57.1%; for glucose 57.1%; for Gram stain 23.8%; p < 0.05 for all comparisons); (4) the most specific test for the detection of microbial invasion was the Gram stain of amniotic fluid (specificity: for Gram stain 98.5%; for white blood cell count 77.9%; for interleukin-6 75%; for glucose 73.5%; p < 0.01 for all); (5) of all amniotic fluid tests, interleukin-6 determination was the only test that had significant clinical value in the prediction of the amniocentesis-to-delivery interval and neonatal complications. CONCLUSION: Interleukin-6 concentrations in amniotic fluid are a better predictor of microbial invasion of the amniotic cavity, amniocentesis-to-delivery interval and neonatal complications than the amniotic fluid Gram stain, glucose, or white blood cell count in patients with preterm premature rupture of membranes.

Amniocentesis↗
PubMed1986

Enhancement of fluorescence development of end products by use of a fluorescence developer solution in a rapid and sensitive fluorescent spot test for specific detection of microbial beta-lactamases.

A fluorescent spot test method for specific detection of microbial beta-lactamases as previously published (K. C. S. Chen, J. S. Knapp, and K. K. Holmes, J. Clin. Microbiol. 19:818-825, 1984) was improved by the use of a fluorescence developer solution. The fluorescence developer solution used in this study consisted of 0.78 M sodium tartrate buffer containing 12% formaldehyde at a final pH of 4.5. An addition of 1 volume of fluorescence developer solution to 5 volumes of ampicillin or cephalex substrate solution incubated with beta-lactamase-producing organisms, followed by heating the mixture at 45 degrees C for 10 min resulted in enhancement of fluorescence of the end products of beta-lactamase activity. This provides a more sensitive assay for microbial beta-lactamases and offers the potential for direct detection of beta-lactamases in clinical specimens.

Amidohydrolases↗
PubMed2002

Evaluation of the 3D BacT/ALERT automated culture system for the detection of microbial contamination of platelet concentrates.

Bacterial transmission remains the major component of morbidity and mortality associated with transfusion-transmitted infections. Platelet concentrates are the most common cause of bacterial transmission. The BacT/ALERT 3D automated blood culture system has the potential to screen platelet concentrates for the presence of bacteria. Evaluation of this system was performed by spiking day 2 apheresis platelet units with individual bacterial isolates at final concentrations of 10 and 100 colony-forming units (cfu) mL-1. Fifteen organisms were used which had been cited in platelet transmission and monitoring studies. BacT/ALERT times to detection were compared with thioglycollate broth cultures, and the performance of five types of BacT/ALERT culture bottles was evaluated. Sampling was performed immediately after the inoculation of the units, and 10 replicates were performed per organism concentration for each of the five types of BacT/ALERT bottles. The mean times for the detection of these 15 organisms by BacT/ALERT, with the exception of Propionibacterium acnes, ranged from 9.1 to 48.1 h (all 10 replicates were positive). In comparison, the time range found using thioglycollate was 12.0-32.3 h (all 10 replicates were positive). P. acnes' BacT/ALERT mean detection times ranged from 89.0 to 177.6 h compared with 75.6-86.4 h for the thioglycollate broth. BacT/ALERT, with the exception of P. acnes, which has dubious clinical significance, gave equivalent or shorter detection times when compared with the thioglycollate broth system. The BacT/ALERT system detected a range of organisms at levels of 10 and 100 cfu mL-1. This study validates the BacT/ALERT microbial detection system for screening platelets. Currently, the system is the only practically viable option available for routinely screening platelet concentrates to prevent bacterial transmission.

Aerobiosis↗
PubMed1997

Rapid molecular detection of microbial pathogens: breakthroughs and challenges.

Microbiological contamination of foods and drinking water is a global problem, and a significant amount of expense is being incurred as a result of such contamination. The microorganisms associated with almost half of all disease outbreaks still go unidentified, primarily as a result of inadequate monitoring and surveillance. Though significant improvements have been made in refining molecular methods for detecting infectious agents, a majority of these methods are being employed only on clinical samples where pathogen densities are much higher than those found in environmental and food samples. Comparative evaluations of the various protocols in terms of cost, sensitivity, specificity, speed, and reproducibility need to be undertaken so that the true applicability of these methods can determined. In the future, molecular methods, especially gene amplifications and in situ hybridizations, will find increasing applications in the differentiation of viable and non-viable organisms, in predicting antimicrobial resistance, and in the identification and characterization of unculturable microorganisms. Though molecular detection methods will not totally replace conventional methods, they will significantly enhance our ability to detect microbial pathogens rapidly.

Animals↗
PubMed2002

Validity of detection of microbial growth in buildings by trained dogs.

Microbial growth in buildings may evoke respiratory and other symptoms in the occupants and promote decay of construction materials. The decay in wood is usually caused by dry-rot fungus, leading to the decomposition of cellulose and lignin. There are also some mold fungi and bacteria that can use wood as a nutrient. In this study, two trained dogs were used to detect microbial growth present in buildings. The rot fungi Serpula lacrymans, Coniophora puteana and Antrodia sinuosa were used in the training. In addition to decay samples, pieces of healthy birch, pine and imbued wood were used as controls. Another experiment was made using bacteria (Streptomyces sp.). In these experiments, a total of 100 decay, 75 control and 25 bacteria samples were used. The dogs detected 75% of the decay and 60% of the bacteria samples. Some (0-24%) control samples were also expressed as positive. Since the dogs identified also the bacteria samples without any specific training, a new test with some mold strains (Cladosporium, Botrytis, Trichoderma, Penicillium, Aspergillus) was carried out. The dogs found all the decay, mold and bacteria samples but only one sample of healthy wood. The use of dogs to detect mold or decay damage appears to have high specificity and high positive predictive value, but low sensitivity.

Animals↗
PubMed2001

Fluorogenic substrates for the detection of microbial nitroreductases.

AIMS: To synthesize and evaluate fluorogenic substrates for the detection of microbial nitroreductases. These substrates, all based on 7-nitrocoumarin, may be reduced to form fluorescent aminocoumarins. METHODS AND RESULTS: Thirty pathogenic microbial strains, including both bacteria and yeasts, were examined for nitroreductase activity in a whole-cell assay. All strains readily reduced each of the seven substrates to generate fluorescence, suggesting the widespread presence of nitroreductase activity in pathogenic bacteria. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: These novel substrates facilitate the direct detection of nitroreductase activity and have potential as sensitive indicators of microbial growth.

Bacteria↗
PubMed1996

Free-living spirochetes from Cape Cod microbial mats detected by electron microscopy.

Spirochetes from microbial mats and anaerobic mud samples collected in salt marshes were studied by light microscopy, whole mount and thin section transmission electron microscopy. Enriched in cellobiose-rifampin medium, selective for Spirochaeta bajacaliforniensis, seven distinguishable spirochete morphotypes were observed. Their diameters ranged from 0.17 micron to > 0.45 micron. Six of these morphotypes came from southwest Cape Cod, Massachusetts: five from Microcoleus-dominated mat samples collected at Sippewissett salt marsh and one from anoxic mud collected at School Street salt marsh (on the east side of Eel Pond). The seventh morphotype was enriched from anoxic mud sampled from the north central Cape Cod, at the Sandy Neck salt marsh. Five of these morphotypes are similar or identical to previously described spirochetes (Leptospira, Spirochaeta halophila, Spirochaeta bajacaliforniensis, Spirosymplokos deltaeiberi and Treponema), whereas the other two have unique features that suggest they have not been previously described. One of the morphotypes resembles Spirosymplokos deltaeiberi (the largest free-living spirochete described), in its large variable diameter (0.4-3.0 microns), cytoplasmic granules, and spherical (round) bodies with composite structure. This resemblance permits its tentative identification as a Sippewissett strain of Spirosymplokos deltaeiberi. Microbial mats samples collected in sterile Petri dishes and stored dry for more than four years yielded many organisms upon rewetting, including small unidentified spirochetes in at least 4 out of 100 enrichments.

Massachusetts↗
PubMed1983

A rapid and sensitive paper electrophoresis assay for the detection of microbial siderophores elicited in solid-plating culture.

A rapid and sensitive assay for the detection of microbial siderophores (iron-binding compounds) is described. Nine representative fungal and bacterial cultures including Ustilago sphaerogena, Penicillium sp., Fusarium roseum, Rhodotorula pilimanae, Bacillus subtilis W 23, Bacillus subtilis W 168, Bacillus megaterium, Azotobacter vinelandii OP, and Escherichia coli B, were nutritionally stressed for iron by sequential transfers on iron-deficient solid-plating media. In response to Fe-stress conditions, the microorganisms excreted siderophore compounds into the extracellular solid culture medium. The solid agar matrix effectively concentrated and restricted the migration of the siderophore compounds to the region immediately adjacent to colonial growth. Agar-block samples from this region were removed and placed at the origin of an electrophoresis paper strip. The resultant absorbed material from the agar-block sample was subjected to high-voltage paper electrophoresis which separated the siderophore compounds by size and molecular net charge. Phenolic acid ("catechol")-type siderophores were detected by fluorescence under uv light. Hydroxamic acid-type siderophores were visualized by spraying the electrophoretogram with ferric iron solution.

Bacteria↗
PubMedDate not supplied

Optimizing detection of microbial sepsis: a comparison of culture systems using packaged sets with directions for blood collection.

Detection of microbial sepsis is an important aspect of medical practice. To facilitate the recovery of bacteria and fungi we evaluated 10,933 complete blood culture sets integrating the automated ESP system (Difco) with a manual system using the Isolator (Wampole) and Thiol broth bottle (Difco). To improve compliance with the recommended procedure for submitting specimens, we packaged the three components as a unit that included brief instructional material. A three-component (one ESP bottle, one Thiol bottle, and one Isolator tube) package containing specific instructions was used. The aerobic ESP performance was compared with the Isolator, and the anaerobic ESP with the Thiol bottle. A hypothetical assessment involving all three components was also made. The cultures were processed under routine laboratory conditions. Charts were reviewed from specimens with discrepant results to ascertain true positives versus contaminant cultures in the discordant culture sets. There were 896 organisms recovered from 393 patients. Of these, 421 were in the aerobic compared with 475 in the anaerobic portion of the study. ESP enhanced the recovery of Streptococcus pneumoniae (P < .05). The Isolator was superior for the recovery of yeasts (.05 < P < .1). Using the packaged system, the compliance rate for complete three-component culture sets was 95.2%, with a 34.4% resultant increase in positive blood cultures from only a 5.7% increase in sets. Packaged culture media including instructions enhance appropriate collection of complete sets, increase the number of recovered organisms, and improve medical practice.

Aerobiosis↗
PubMed2005

Roadmap to approval: use of an automated sterility test method as a lot release test for Carticel, autologous cultured chondrocytes.

BACKGROUND: In February 2004, FDA approved a supplement to our biologics license for Carticel, autologous cultured chondrocytes, to use the BacT/ALERT microbial detection system as an alternative to the compendial sterility test for lot release. This article provides a roadmap to our approval process. The approval represents more than 4 years of development and validation studies comparing the Steritest compact system to the BacT/ALERT microbial detection system. METHODS: For this study, freshly cultured chondrocytes were prepared from a characterized cell bank. Microbial isolates were prepared from either American Type Culture Collection (ATCC) strains or from in-house contaminants. For each test condition, a suspension of chondrocyte cells and test organisms was inoculated into both aerobic media (SA standard adult culture bottles, FA FAN, tryptic soy broth) and anaerobic media (SN standard adult culture bottles, FN FAN, fluid thioglycollate media) and tested for sterility using the Steritest compact system (Millipore, Bedford, MA, USA) and the BacT/ALERT microbial detection system (bioMerieux, Durham, NC, USA). Negative control bottles were inoculated with chondrocytes and no microorganisms. All bottles were incubated for 14 days and read daily. Bacterial growth was determined by either visual examination of Steritest canisters or detection of a positive by the BacT/ALERT system. A gram stain and streak plate were used to confirm positive bottles and negative bottles after 14 days. RESULTS: The detection of a positive by either the Steritest compact system or the BacT/ALERT system was summarized for each organism in each validation study. Data generated from studies reducing the incubation temperature from 35 degrees C to 32 degrees C improved detection times in the automated method compared with the compendial method. Other improvements included the use of FAN aerobic and anaerobic media to absorb the gentamicin contained in the culture media of prepared chondrocyte samples. Chondrocytes alone did not generate positive results in either the compendial method or the automated method. DISCUSSION: Data from validation studies support the use of the BacT/ALERT microbial detection system as an alternative sterility test for Carticel.

Bacteria↗
PubMed2002

Novel methods for the detection of microbial antibodies in oral fluid.

Compared with blood, oral fluid has several advantages as a sample for antibody detection. It is simple, safe, painless, and cheap to collect. The only drawback is that while the antibody profiles indicate those in blood, they are at lower concentrations. Antibody capture assays are the method of choice for the detection of microbial antibodies in oral fluid, but their relative lack of sensitivity when based on conventional immunoassay techniques has mostly limited their use to epidemiological applications. Immuno-PCR and time-resolved fluorescence offer more sensitive detection systems that could be applied to oral fluid specimens. We review antibody detection in oral fluid and discuss immuno-PCR and time-resolved fluorescence as candidate systems. Both have the potential to broaden the applications of oral fluid testing to clinical diagnostics.

Antibodies↗
PubMedDate not supplied

Detection and identification of previously unrecognized microbial pathogens.

Features of a number of important but poorly explained human clinical syndromes strongly indicate a microbial etiology. In these syndromes, the failure of cultivation-dependent microbial detection methods reveals our ignorance of microbial growth requirements. Sequence-based molecular methods, however, offer alternative approaches for microbial identification directly from host specimens found in the setting of unexplained acute illnesses, chronic inflammatory disease, and from anatomic sites that contain commensal microflora. The rapid expansion of genome sequence databases and advances in biotechnology present opportunities and challenges: identification of consensus sequences from which reliable, specific phylogenetic information can be inferred for all taxonomic groups of pathogens, broad-range pathogen identification on the basis of virulence-associated gene families, and use of host gene expression response profiles as specific signatures of microbial infection.

Animals↗
PubMed2001

SdiA of Salmonella enterica is a LuxR homolog that detects mixed microbial communities.

Proteins of the LuxR family detect the presence of N-acylhomoserine lactones (AHLs) and regulate transcription accordingly. When AHLs are synthesized by the same species that detects them, the system allows a bacterium to measure the population density of its own species, a phenomenon known as quorum sensing. The sdiA genes of Escherichia coli and Salmonella enterica serovar Typhimurium are predicted to encode LuxR homologs. However, these species do not appear to synthesize AHLs or any other molecule detected by SdiA. It has previously been demonstrated that overexpression of sdiA results in the activation of the ftsQAZ locus in E. coli and four other loci in Salmonella serovar Typhimurium. Here we report that transcriptional fusions to these five loci fall into two classes. The first class requires overexpression of sdiA for activation. The second class responds to sdiA expressed from its natural position in the chromosome if the appropriate AHLs are added to the culture. The only member of the second class is a series of Prck-luxCDABE fusions in Salmonella serovar Typhimurium. SdiA responds with highest sensitivity to AHLs that have a keto modification at the third carbon and an acyl chain length of 6 or 8 (half-maximal response between 1 and 5 nM). Growth of Salmonella in proximity to species known to synthesize these AHLs results in sdiA-dependent activation of the Prck-luxCDABE fusions. SdiA appears to be the first AHL receptor discovered that detects signals emanating exclusively from other species.

4-Butyrolactone↗
PubMed1998

Detection of microbial pathogens in shellfish with multiplex PCR.

Multiplex PCR amplification of uidA, cth, invA, ctx, and tl genes was developed enabling simultaneous detection in shellfish of Escherichia coli, an indicator of fecal contamination and microbial pathogens, Salmonella typhimurium, Vibrio vulnificus, V. cholerae, and V. parahaemolyticus, respectively. Each of the five pairs of oligonucleotide primers was found to support PCR amplifications of only its targeted gene. The optimized multiplex PCR reaction utilized a PCR reaction buffer containing 2.5 mM MgCl2 and primer annealing temperature of 55 degrees C. Oyster tissue homogenate seeded with these microbial pathogens was subjected to DNA purification by the Chelextrade mark 100 (BioRad) method. The sensitivity of detection for each of the microbial pathogens was </=10(1)-10(2) cells following a "double" multiplex PCR amplification approach. Amplified target genes in a multiplex PCR reaction were subjected to a colorimetric GeneCombtrade mark (BioRad) DNA-DNA hybridization assay. This assay was rapid and showed sensitivity of detection comparable to the agarose gel electrophoresis method. The colorimetric GeneCombtrade mark assay avoids use of hazardous materials inherent in conventional gel electrophoresis and radioactive-based hybridization methods. Multiplex PCR amplification, followed by colorimetric GeneCombtrade mark DNA-DNA hybridization, has been shown to be an effective, sensitive, and rapid method to detect microbial pathogens in shellfish.

Animals↗
PubMed1994

The value of amniotic fluid interleukin-6 determination in patients with preterm labor and intact membranes in the detection of microbial invasion of the amniotic cavity.

OBJECTIVES: Our purpose was to (1) determine the value of amniotic fluid interleukin-6 determination in the detection of microbial invasion of the amniotic cavity and (2) compare interleukin-6 to other rapid tests in the evaluation of preterm labor. STUDY DESIGN: Amniotic fluid interleukin-6 was determined quantitatively by enzyme-linked immunosorbent assay in 91 amniotic fluid specimens obtained by amniocentesis in 89 patients with preterm labor. Amniotic fluid cultures for aerobes, anaerobes, and mycoplasma species were performed. Receiver-operator characteristic curve analysis, logistic regression analysis, and Cox's proportional-hazards model were used to explore the relationship between several explanatory and outcome variables. Diagnostic index values of interleukin-6, glucose level, Gram stain, leukocyte esterase, and limulus amebocyte lysate assay for prediction of a positive amniotic fluid culture, preterm delivery, clinical infection, and neonatal sepsis were calculated. RESULTS: The prevalence of positive amniotic fluid cultures was 13% (12/89). The median interleukin-6 concentration in patients with positive cultures was 241.8 ng/ml, in contrast to 0.291 ng/ml in patients with negative cultures (p. < 0.005). Sensitivity and specificity of an interleukin-6 level > or = 6.17 ng/ml was 75% and 79%, in contrast to that of glucose, < or = 12 mg/dl (83% and 86%) for a positive amniotic fluid culture and sensitivity (p = 0.26, z test). All patients with an interleukin-6 level > 6.17 ng/ml were delivered preterm, in contrast to 85.2% of patients with a glucose level < or = 12 mg/dl. When all rapid tests and clinical parameters were considered simultaneously in the logistic model, only interleukin-6 maintained a significant relationship to preterm birth (odds ratio 35, p = 0.003). Cox's proportional analysis demonstrated a strong relationship between interleukin-6 and the amniocentesis-to-delivery interval after clinical variables were controlled for (hazard ratio 3.01, p < 0.00001). CONCLUSION: Amniotic fluid interleukin-6 determination may be a useful adjunct to our armamentarium of rapid tests to exclude infection and predict delivery in patients with preterm labor and intact membranes.

Amniocentesis↗
PubMed2003

The use of NASBA for the detection of microbial pathogens in food and environmental samples.

The isothermal amplification method nucleic acid sequence-based amplification (NASBA), which amplifies RNA, has been reported as useful for the detection of microbial pathogens in food and environmental samples. Methods have been published for Campylobacter spp., Listeria monocytogenes and Salmonella enterica ser. Enteritidis in various foods and for Cryptosporidium parvum in water. Both 16S rRNA and various mRNAs have been used as target molecules for detection; the latter may have advantages in allowing specific detection of viable cells. Most of the methods to detect pathogens in foods have employed enrichment in nutrient medium prior to NASBA, as this can ensure sensitivity of detection and encourage the detection of only viable target cells. Although a relatively recent method, NASBA has the potential for adoption as a diagnostic tool for environmental pathogens.

Animals↗
PubMed1986

Comparison of the standard pour plate procedure and the ATP and Limulus amebocyte lysate procedures for the detection of microbial contamination in intravenous fluids.

The intrinsic and extrinsic microbial contamination of large-volume parenterals has been associated with bacteremias in hospitalized patients. When epidemiologic data suggest the association of contaminated intravenous (i.v.) fluids with disease, appropriate laboratory methods must be devised to analyze quickly the suspect fluid. A study was undertaken to compare three laboratory test methods (standard pour plate [SPP] technique, ATP procedure, and Limulus amebocyte lysate [LAL] assay) for detecting the presence of microorganisms in artificially contaminated i.v. fluid. SPP proved the most sensitive of the assay techniques used, but a 24-h period was required for microbial growth. The LAL assay appeared more sensitive in detecting i.v. contamination than the ATP method. These studies suggest that the LAL and ATP methods can be used for the rapid detection of microbial contamination in i.v. fluid. SPP and LAL procedures would have practical laboratory application when the contamination of i.v. fluids is suspected, and they are more sensitive in detecting microbial contamination in these solutions than the ATP method.

Adenosine Triphosphate↗