Limitations of the germ theory.
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Bacteriological Techniques↗
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Can the API (RAPID) Coryne system be used for identification of rapidly growing mycobacteria?
Fifty-six strains of rapidly growing mycobacteria (RGM) and 14 strains of aerobic actinomycetes as quality controls (QC) were tested in the API (RAPID) Coryne system version 2. Both groups yielded codes with low identification scores, considerable overlaps, and similar diagnoses. No species-specific codes were observed. Thus, the system would not be useful for the identification of RGM.
Actinomycetales↗
[Clinical importance of Mycobacterium kansasii and evaluation of the need to identify nontuberculous mycobacteria].
BACKGROUND: The aim of this study was to evaluate the clinical importance of a single isolation of Mycobacterium kansasii in order to determine whether a single isolation of nontuberculous mycobacteria is sufficient to diagnose mycobacteriosis or whether multiple isolations are required, in which case, it would be better to wait until a second isolation is produced before undertaking identification of all nontuberculous mycobacteria. PATIENTS AND METHODS: Between 1992-1998, we studied the characteristics of 77 isolates of M. kansasii because it is the nontuberculous mycobacterium most frequently associated with disease. Our hospital has 500 beds but processed the samples from the Principe de Viana Outpatient Center and those from the district hospitals of Tudela and Estella for mycobacteria. During the study period, a mean of 3,900 specimens were processed per year. The isolates came from 22 episodes in 21 patients (19 males and 2 females). The specimens were cultured using standard techniques and confirmation was performed using gene probes (Gen-Probe, San Diego, California). RESULTS: Of the 22 episodes, positive cultures were obtained on multiple occasions in 15 (68.1%) and a single isolation was obtained in 7. All isolations came from respiratory samples. Isolations were obtained from blood on only 2 occasions (both in HIV-positive patients). Direct bacilloscopy was positive in 60% of cases with multiple isolations and negative in all cases of single isolation. Multiple isolations were found in 14 patients, 7 of whom were HIV-positive and with advanced immunodepression and 7 were HIV-negative. Of these, two were alcoholics and the remaining patients had chronic pulmonary diseases. All treated patients showed initial improvement after specific therapy and negative bacilloscopies and cultures. None of the patients with a single isolation, including two HIV-positive patients, showed clinical repercussions. CONCLUSION: In our experience, a single isolation of M. kansasii was without clinical importance, even in two HIV-positive patients. Given that when the criteria of the American Thoracic Society for the diagnosis of mycobacteriosis are followed, in general, multiple mycobacteria should be isolated, a strategy of not undertaking the identification of all nontuberculous mycobacteria until a second isolation is produced can be established, unless there is serious clinical indication to suggest otherwise.
Adult↗
[Multiplex PCR in clinical microbiology].
The introduction of molecular biology methods in clinical microbiology laboratories brings important insights to obtain sensitive and specific diagnoses as fast as possible. These methods are not intended for replacement but for complement of the already applied microbiologic methods. The integrated analyses of all of them is bringing to the most feasible and efficient results. Within the molecular techniques applied, polymerase chain reaction (PCR) has acquired a great diagnostic value, permitting the identification of etiologic agents and the fast and sensitive detection of their virulence and resistance genotypes. Since some years ago, the development of the so called multiplex PCRs has gained deep interest. Those are reactions that get the simultaneous single tube amplification of different target sequences, allowing the simultaneous detection and identification of various genes of interest. In the present article, the most relevant applications of multiplex PCR for clinical microbiology are summarized.
Bacterial Infections↗
[The Pneumococcal Observatory for the Central Region, 1 April 1999 to 31 March 2000].
Seven hundred and ninety six strains of pneumococcus were collected in the Centre region, from 15 laboratories, between 1st April 1999 and 31st of March 2000. Data were processed, using 4th dimension software, and concerned age, file number, consultation/hospitalisation, sample type, susceptibility to oxacillin (5 micrograms), results of the E-test for benzylpenicillin, amoxicillin, cefotaxime and results of the routine disc diffusion test. Strains with reduced susceptibility to benzylpenicillin (PRSP) were collected by the co-ordinating centre to perform MICs by the reference agar dilution test and serotyping. Out of 796 strains, 450 strains (56.7%) were categorised as PRSP and 400 of them were studied by the co-ordinating centre. Forty two percent of the samples originated from lungs, followed by 19.5% from blood samples, 15% from ear pus (85.7% PRSP) and 2.5% from CSF. Thirty nine percent of the patients were female. 36.6% were children under sixteen (70.1% PRSP) and 62.4% were adults (49.2% PRSP). Out of 400 PRSP 106 (26.5%) were characterised as resistant and 294 (73.5%) as intermediate to benzylpenicillin. Compared to the agar dilution test, 90% of the PRSP studied by E-test had a MIC value for benzylpenicillin within +/- 1 dilution. Thirty six strains of PRSP were resistant to amoxicillin (9% of the PRSP) and 10 (2.5% of the PRSP) to cefotaxime. Serotyping was done on 375 strains. The serotypes encountered were the following: 23 (26.9%), 14 (22.1%), 19 (19.5%), 6 (12.8%), 9 (9.9%) and 15 (5.1%).
Adolescent↗
A PCR assay used to study aerosol transmission of Actinobacillus pleuropneumoniae from samples of live pigs under experimental conditions.
The study describes a polymerase chain reaction (PCR) assay for the detection of Actinobacillus pleuropneumoniae. The test is based on the amplification of the omlA gene coding for an outer membrane protein of A. pleuropneumoniae. To test the specificity of the reaction, 19 other bacterial species related to A. pleuropneumoniae or isolated from pigs were assayed. They were all found negative in the PCR assay. The detection threshold of the test was 10(2) A. pleuropneumoniae CFU/assay. The test was then applied to the detection of A. pleuropneumoniae from tonsillar biopsies and tracheobronchial lavage fluids of pigs without a culture step. The detection of A. pleuropneumoniae in these samples was performed by PCR, by conventional culture and by bacteriology with immunomagnetic beads. The number of samples that were found positive by PCR was almost three times higher than the number of samples from which A. pleuropneumoniae was isolated by both bacteriological techniques. The detection of A. pleuropneumoniae in these samples allowed us to demonstrate its aerosol transmission to pigs under experimental conditions. The trial involved 18 specific pathogen free pigs. Six pigs, infected with A. pleuropneumoniae, were located in a unit A, together with four non-infected animals (contact pigs). Eight non-infected pigs (reporter pigs) were located in a unit B, adjacent to A. We detected A. pleuropneumoniae in samples from infected animals but also from 'contact' (unit A) and 'reporter' (unit B) pigs. The results of this study show that the simple preparation of the samples followed by the PCR assay may be a useful tool for epidemiological studies.
Actinobacillus Infections↗
Use of polymerase chain reaction to detect Brucella abortus biovar 1 in infected goats.
The polymerase chain reaction (PCR) was used to diagnose goat brucellosis and compare its sensitivity against some of the most commonly used serological and bacteriological techniques. Twenty two female and one male out of 300 clinically healthy, mixed-breed goats were randomly chosen from a ranch located at Marín, Nuevo León, Mexico. Milk and blood samples were taken from each animal and used to obtain both microbiological cultures and DNA of the pathogen, and sera was tested against Rose Bengal antigen (RBT). Results showed that 86% of the blood samples were positive on the PCR test, while 60% were positive on the serological test. The pathogen was isolated from only one blood culture. Sixty four percent of the milk samples were positive on PCR tests, but failed to yield bacteria in culture. Biochemical and PCR specific assay demonstrated that Brucella abortus biovar 1 was associated with the infection. This study demonstrates the higher sensitivity of PCR over RBT and blood culture and its potential towards a rapid identification of Brucella strains.
Agglutination Tests↗
Practical approach to the identification of clinically relevant Enterococcus species.
Enterococci have become important nosocomial pathogens, with Enterococcus faecalis and then Enterococcus faecium predominating. Because of the emergence of glycopeptide (vancomycin and teicoplanin) resistance in enterococci, laboratories have been required to screen for resistant strains and to identify them to the species level. This has resulted in the need for accurate identification of species less commonly associated with clinical infections, such as Enterococcus casseliflavus and Enterococcus gallinarum, which are inherently resistant to the glycopeptides. Studies evaluating commonly used commercial identification systems, have found error rates for enterococcal species identification of 2-21% for E. faecalis, 5-9% for E. faecium, and 14-79% for other species. Reporting errors may have adverse effects on the management of clinical infections, as well as in the control of multidrug-resistant strain outbreaks. The purpose of this document is to present a simplified approach to the identification of Enterococcus species that uses a combination of rapid, readily available, and inexpensive tests.
Anti-Bacterial Agents↗
Infective endocarditis--a 25 year overview of diagnosis and therapy.
Diagnosis and management of infective endocarditis have significantly changed in the past 25 years. Improved bacteriologic techniques have allowed detection of cases of infective endocarditis caused by unusual organisms. Bactericidal therapy has become available for patients with gram-negative endocarditis and antimicrobial therapy has improved. Echocardiography has become an important diagnostic and management aid, and cardiac valve replacement has dramatically improved the outlook for many patients.
Anti-Bacterial Agents↗
Development of a sensitive DNA microarray suitable for rapid detection of Campylobacter spp.
Campylobacter is the most common cause of human acute bacterial gastroenteritis worldwide, widely distributed and isolated from human clinical samples as well as from many other different sources. To comply with the demands of consumers for food safety, there is a need for development of a rapid, sensitive and specific detection method for Campylobacter. In this study, we present the development of a novel sensitive DNA-microarray based detection method, evaluated on Campylobacter and non-Campylobacter reference strains, to detect Campylobacter directly from the faecal cloacal swabs. The DNA-microarray method consists of two steps: first, both universal bacterial sequences and specific Campylobacter sequences (size range: 149-307 bp) are amplified and fluorescently labeled using multiplex-PCR, targeting the 16S rRNA, the 16S-23S rRNA intergenic region and specific Campylobacter genes. Secondly, the Cy5 labeled PCR-amplicons are hybridised to immobilised capture probes on the microarray. The method allows detection of three to thirty genome equivalents (6-60 fg DNA) of Campylobacter within 3 h, with a hands on time of only 15 min. Using the DNA-microarrays, two closely related Campylobacter species, Campylobacter jejuni and Campylobacter coli could be detected and differentiated directly from chicken faeces. The DNA-microarray method has a high potential for automation and incorporation into a dedicated mass screening microsystem.
Animals↗
Listeria: growth, phenotypic differentiation and molecular microbiology.
The identification of Listeria species is based on a limited number of biochemical markers, among which absence or presence of hemolysis and arylamidase are used to differentiate between L. monocytogenes and L. innocua. The CAMP (Christie, Atkins, Munch-Petersen) test must be interpreted with caution. Chromogenic media are based on both the specific chromogenic detection of phosphatidylinositol phospholipase C and the xylose fermentation and give specific and direct identification of L. monocytogenes and L. ivanovii. Isolates of L. monocytogenes with atypical properties require tools of molecular biology for final identification. Serotyping, although not allowing speciation, serves a useful purpose for confirming the genus diagnosis Listeria. Polymerase chain reaction is particularly useful when prior administration of antimicrobial agents compromises culture. For clinical specimens the importance of trying to isolate the pathogen as a prerequisite for an epidemiological work-up and finally for prevention of further cases cannot be overstressed.
Animals↗
Development of an evanescent-field fibre optic sensor for Escherichia coli O157:H7.
An intensity-modulated fibre optic sensor was developed for Escherichia coli O157:H7. The interaction between the whole natural bacteria and the guided lightwave was carried out by means of evanescent-field coupling. A correlation between optical response and the current number of bacteria was achieved. The device sensitivity had been calibrated for initial number of bacteria (N(0)) from 10-800. The sensor sensitivity was 0.016 (+/-0.001) dB/h/N(0). The sensing mechanism starts together with the log phase leading the present sensor response to be five to ten times faster than conventional bacteriological techniques.
Biosensing Techniques↗
A method for measuring the cleaning effect of flushing disinfectors.
A method is presented with which the mechanical cleaning effect of flushing disinfectors can be estimated independently of the thermal disinfecting effect of the hot flushing water. This makes it possible to specify the demands to be placed on the disinfecting effect of flushing with water of 85 degrees C or more. Bacillus stearothermophilus spores suspended in faeces were used as indicators because of their non-sensitivity to the hot-water temperature. Their elimination by flushing could thus be attributed to the mechanical effect of the water and not to the disinfecting effect of the temperature. A simple bacteriological technique was used, and the elimination factor (EF) was calculated as the ratio of the number of micro-organisms in the contamination before and after flushing. By using flushing water below 50 degrees C for 130 s the EF on a bedpan was about 10(4)-10(6). The effect of flushing with water of the same temperature for only half that time was somewhat weaker and when the temperature was raised to 85 degrees C after half the flushing time the effect was somewhat stronger. It can be presumed that the conventional disinfecting phase with hot (85 degrees C) water for about 45 s in the commonly used flushing units could be substantially shortened and the costs of their use thereby reduced.
Disinfection↗
THE MEASUREMENT OF BACTERIAL RESPIRATION ON PIG SKIN WITH MICRO-RESPIROMETERS.
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Animals↗
ASSOCIATION OF BACTERIAL L-PHASE ORGANISMS IN CHRONIC INFECTIONS.
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Allergy and Immunology↗
Bacterial sex timely.
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Bacteriological Techniques↗
Influence of infection at the time of root filling on the outcome of endodontic treatment of teeth with apical periodontitis.
This study investigated the role of infection on the prognosis of endodontic therapy by following-up teeth that had had their canals cleaned and obturated during a single appointment. The root canals of 55 single-rooted teeth with apical periodontitis were thoroughly instrumented and irrigated with sodium hypochlorite solution. Using advanced anaerobic bacteriological techniques, post-instrumentation samples were taken and the teeth were then root-filled during the same appointment. All teeth were initially infected; after instrumentation low numbers of bacteria were detected in 22 of 55 root canals. Periapical healing was followed-up for 5 years. Complete periapical healing occurred in 94% of cases that yielded a negative culture. Where the samples were positive prior to root filling, the success rate of treatment was just 68%--a statistically significant difference. Further investigation of three failures revealed the presence of Actinomyces species in each case; no other specific bacteria were implicated in failure cases. These findings emphasize the importance of completely eliminating bacteria from the root canal system before obturation. This objective cannot be reliably achieved in a one-visit treatment because it is not possible to eradicate all infection from the root canal without the support of an inter-appointment antimicrobial dressing.
Bacteria↗
Identification of atypical Aeromonas salmonicida: inter-laboratory evaluation and harmonization of methods.
The atypical isolates of Aeromonas salmonicida are becoming increasingly important as the frequency of isolation of bacteria belonging to this group continues to rise. The primary object of this study was to compare and evaluate the results obtained in various laboratories concerning the biochemical identification of atypical Aer. salmonicida before and after standardization of media and methods. Five laboratories examined 25 isolates of Aer. salmonicida from diverse fish species and geographical locations including the reference strains of Aer. salmonicida subsp. salmonicida (NCMB 1102) and Aer. salmonicida subsp. achromogenes (NCMB 1110). Without standardization of the methods, 100% agreement was obtained only for two tests: motility and ornithine decarboxylase. The main reason for the discrepancies found was the variation of the incubation time prior to reading the biochemical reactions. After standardization, improvement was obtained with the identification; however, disagreement was still observed between the different laboratories. These findings demonstrate the difficulties involved in a proper identification of atypical Aer. salmonicida and also that data presented in the literature on various strains of Aer. salmonicida are not readily comparable. This paper seems to be the first on standardization of microbiological tests for identification of fish pathogens and the results obtained show the need for standardization of methods both within and between laboratories.
Aeromonas↗