[Clinical specimens for PCR detection of syphilis]. / Klinický materiál pro prukaz syfilis metodou polymerázové retezové reakce.

A review is presented of types of clinical specimens used for diagnosis of syphilis by polymerase chain reaction. PCR is a routine method for detection of T. pallidum in swabs of chancres and primary and secondary syphilis mucocutaneous lesions. Whole blood, cerebrospinal fluid, amniotic fluid, aspirate and biopsy specimens, and paraffin-embedded tissues can also be tested by PCR for T. pallidum. However, further research on PCR detection sensitivity at various stages of syphilis is needed before these specimens are used in clinical practice.

[Molecular detection and subtyping of Treponema pallidum subsp. pallidum in clinical specimens]. / Molekulární detekce a typizace Treponema pallidum subsp. pallidum v klinickém materiálu.

An in-house two-step nested PCR amplification targeting the tmpC gene (TP0319, encoding putative membrane lipoprotein) was used for detection of chromosomal DNA of Treponema pallidum subsp. pallidum in clinical specimens. We tested 138 blood serum samples from 111 adult patients with suspected, primary, secondary, early or late latent syphilis. T. p. pallidum DNA was not detected in any of the analyzed specimens. Out of 11 mucocutaneous swabs (7 genital and 4 pharyngeal), 6 collected from 3 patients with primary or secondary syphilis tested positive. One skin swab from a patient with early congenital syphilis was also positive as were his serum and cerebrospinal fluid samples. DNA sequencing of the genes TP0136 and TP0548 from the positive samples revealed two strains with DNA sequences identical to that of T. p. pallidum strain SS14 and two unique previously undescribed T. p. pallidum strains. The advances in molecular typing of T. p. pallidum in clinical specimens will be of relevance to the epidemiology of syphilis and will allow for clinical discrimination between reinfection and syphilitic reactivation.

[Microbiology online--interactive learning and a new discussion forum for microbiology teachers]. / Mikrobiologie on-line--výukové stránky a nové diskusní fórum pro ucitele mikrobiologie.

Rapid development of multiple disciplines of science, increasing numbers of university students and the advent of new learning technologies (e-learning, teleconferences, etc.) have drawn increasing interest in the contents, rationale and forms of learning. New Websites with a discussion forum for microbiology teachers have been available on the Web. In brief, the credo of this project called Forum is: "Teachers can learn from each other". The project is accessible free of charge at www.medmicro.info. Another source intended for free use by both teachers and students is "Learning pages of the Institute of Microbiology, Faculty of Medicine, Masaryk University" with the following sections: encyclopaedia, guidance on practical exercises, video clips and on-line laboratory using web cameras. The learning pages are available in both Czech and English.

[Direct detection of Treponema pallidum in diagnosis of syphilis]. / Prímý prukaz Treponema pallidum v diagnostice syfilis.

Available methods for direct diagnosis of syphilis are summarized with emphasis being on those promising for routine use. Direct detection of the causative agent T. pallidum is limited since the agent is not able to synthesize enzyme cofactors, fatty acids and nucleotides de novo, is completely dependent on its host and thus culture on synthetic media is not feasible. Direct diagnosis of syphilis is based on rabbit infectivity testing (RIT), dark field or fluorescent microscopy and recently also on molecular biological methods used with increasing frequency in routine practice. Suitability and usability of different methods for direct detection of T. pallidum at different stages of syphilis are explained. Except for molecular biological methods, most of detection techniques can only be used at the primary and secondary stages or in early congenital syphilis. Major PCR methods for diagnosis of syphilis are presented. Not all of them are suitable for use in routine practice owing to differences in their sensitivity and design. The polA PCR method appears to be the most promising in this regard.

[Immunoglobulin G avidity in infectious diseases]. / Avidita imunoglobulinu G u infekcních onemocnení.

The paper reviews results reached so far about avidity of immunoglobulins G in viral, parasitic and bacterial infectious diseases. The general introductory part begins with explanation of immunologic basis in maturation of avidity of immunoglobulins G and proceeds up to a survey of methods used for the determination of avidity. It also deals with the modes of evaluation in these methods and comments on their advantages and disadvantages. The part devoted to individual infectious diseases brings the most important information about avidity of immunoglobulins G in specific infections, especially in view of their (even potential) practical medical application, mainly in determining the phase of infection in pregnant women and in patients after transplantation. The highest attention is paid to herpes virus infections, especially the cytomegalous virus and the infections with Epstein-Barr virus, rubella and toxoplasmosis, therefore those infections, which in the given situations clearly demonstrated the contribution of avidity analysis in the solution of interpretation-difficult conditions typical for serology, such as lasting levels of immunoglobulins M and cross-reactivity of antibodies.

Biofilm detection and the clinical significance of Staphylococcus epidermidis isolates.

The ability of Staphylococcus epidermidis to produce biofilm was compared in 147 clinically significant strains repeatedly isolated from blood cultures of patients with bloodstream infection and in 147 strains isolated from skin. The strains were examined for the presence of ica operone, for the ability to form biofilm by Christensen's test-tube method and for the production of slime by Congo Red agar method. The ica operone was found in 92 (62.6 %) blood isolates and in 44 (29.9) isolates from skin. Christensen's test-tube method was positive in 79 (53.7) and 33 (22.4), Congo Red agar method in 64 (43.5) and 31 (21.1) of blood and skin isolates, respectively. All three methods were more frequently positive in clinically significant isolates from blood than in strains isolated from skin. The detection of ica operone and the Christensen's test-tube method showed better correlation with the clinical significance than the Congo Red agar method.

[Contradictory results of passive hemagglutination and immunoenzyme tests in the determination of specific immunoglobulin G in the serodiagnosis of lues]. / Nesouhlasné výsledky pasivní hemaglutinace a imunoenzymatického stanovení specifických imunoglobulinu G v sérodiagnostice lues.

BACKGROUND: With continuing automatization it comes into consideration, which tests should be a part of the syphilis screening. Trying to learn more about the correlation and comparability of passive hemaglutination and imunoenzymatic assay, which are the candidate reactions on the screening test, we analyzed results of the above mentioned reactions in 2319 serum samples sent for diagnostics of syphilis. Special attention was paid to sera with contradictory results in both tests. METHODS AND RESULTS: When results were contradictory the patient's probability of having syphilis and the intensity of the reaction were analysed. Out of 2319 sera samples examined for syphilis 141 (6.1%) specimens belonging to 125 patients were found to provide contradictory results in passive hemagglutination (TPHA) and immunoenzymatic assay for the specific immunoglobulins G (ELISA IgG). In 14 cases (children of syphilitic mothers) only the passively transferred antibodies were found and these samples were excluded from the examined group. Almost four fifths of contradictions (88 out of 111, i.e. 79.3%) were based on the positivity of TPHA with the negativity of ELISA IgG and one fifth only (23 specimens, i.e. 20.3%) concerned the positivity of ELISA IgG with the negativity of TPHA. The relation to syphilis was more obvious in the TPHA-positive patients: in 88 TPHA-positive patients the syphilitics were relatively more common (39 syphilitics, i.e. 44.3%) than in 23 ELISA-positive patients (5 syphilitics, i.e. 21.7%), the difference is statistically significant (p < 0.05). CONCLUSIONS: It is concluded that TPHA seems to be more sensitive, whereas ELISA IgG more specific.

[Detection of Arcanobacterium haemolyticum phospholipase D neutralizing antibodies in patients with acute tonsillitis]. / Prukaz neutralizacních protilátek proti fosfolipáze D Arcanobacterium haemolyticum u nemocných s akutní tonzilitidou.

Neutralization test carried on a non-nutrient blood agar prepared from sheep erythrocytes sensibilized with equi factor of Rhodococcus equi was used for the detection of antibodies against phospholipase D (PLD) of Arcanobacterium haemolyticum. Altogether, 433 sera from 404 patients aged 1 to 25 years with the signs of acute tonsillitis were examined. Antibodies against PLD were found in 28 patients (6.9%). In 116 sera from persons without the signs of tonsillitis the antibodies against PLD were detected in one case (0.9%). The titres reached the values from 4 to 64. Out of 84 paired sera, the significant change of the titre, i.e. at least the four-fold one, was observed five times: three times as the seroconversions, twice as the declines of the titre. The antibodies were found most often in children aged 11 to 15 years, 10 times out of 97 (10.3%). In 253 patients with the tonsillitis, the result of the parallel bacteriologic examinations of throat swabs was at the disposal. A. haemolyticum was successfully isolated in 3 cases, but only owing to the inhibition of staphylococcal hemolysis around its colonies. All three cases were also serologically positive. Consequently, our results show that neutralizing antibodies against PLD of A. haemolyticum can be detected in patients with tonsillitis.

[A diagnostic medium for Arcanobacterium haemolyticum and other bacterial species reacting with hemolytic synergism to the equi-factor of Rhodococcus equi]. / Médium pro diagnostiku Arcanobacterium haemolyticum a dalsích bakteriálních druhu reagujících hemolytickým synergismem s equi-faktorem Rhodococcus equi.

Colonies of Arcanobacterium haemolyticum on common blood agar can be easily overlooked. Therefore a diagnostic medium was developed, on which A. haemolyticum colonies produce a conspicuous zone of complete hemolysis. The medium under question is blood agar prepared from the Columbia Blood Agar Base and 5% washed sheep erythrocytes sensitised with equi factor (EF) of Rhodococcus equi. Optimally, 10 activity units (AU) of EF per 1 ml were used. EF was titrated on a non-nutrient medium consisting of agar Purified (Difco) and 5% washed sheep erythrocytes sensitized with beta-haemolysin (BL) of Staphylococcus aureus, 10 AU/ml. On the same medium, staphylococcal BL was titrated on the basis of its direct haemolytic effect. Despite the very distinct haemolytic reaction of the control strains evident on the diagnostic medium with EF, A. haemolyticum failed to grow from 2.597 throat swabs examined especially for the particular microbe during the a period of two years. However, A. haemolyticum was isolated on this medium twice from 223 swabs from wounds and skin lesions. The proposed medium makes also the rapid diagnosis of species Corynebacterium ulcerans, Dermatophilus congolensis and Listeria monocytogenes on the basis of haemolytic synergism possible.

Production of slime by staphylococcal isolates from blood cultures.

The aim was to examine the ability of staphylococci isolated from blood cultures to produce slime and to compare the slime production of strains considered clinically significant and of strains considered mere contaminants. The ability to produce slime was examined in 359 staphylococcal isolates from blood cultures by the congo red agar method. The clinical significance of an isolate was estimated according to the frequency of its occurrence in a series of blood cultures. Only strains isolated at least twice from the series of two or more blood cultures were considered significant. The slime production was detected in 18 of 32 strains (56.2%) of Staphylococcus aureus, in 61 of 231 strains (26.4%) of S. epidermidis and in 14 of 101 strains (14.6%) of the remaining seven species. Out of 80 strains considered significant, 33 strains (41.2%) produced slime, out of 132 strains considered contaminants, 24 strains (18.2%) were slime producers. The significance of the remaining isolates was non-evaluable. We conclude that the staphylococcal isolates from blood cultures considered clinically significant produced slime more often than the isolates considered mere contaminants.

[Elastase activity of gram-negative non-fermenting bacteria and two variations of a simple method for their detection]. / Elastázová aktivita gramnegativních nefermentujících bakterií a dve varianty jednoduché metody jejího prukazu.

The ability to produce elastase was examined in 1,168 strains of 11 representative genera of Gram-negative non-fermenting bacteria isolated from weakened patients in the course of eight years. Detection of the elastase activity was performed parallelly using the conventional method and its pulverisation variant. The difference between the results accomplished by the classical method and its pulverisation variant (53.9% versus 60.5% detected elastase-positive strains) was highly significant (p < 0.05). Among the examined strains 500 (42.8%) displayed elastase activity. The highest proportion of the elastase positive strains was found in Pseudomonas aeruginosa (84.5%), P. fluorescens (60.5%), P. stutzeri (56.4%), Burkholderia cepacia (43.8%) and Shewanella putrefaciens (39.7%). Elastase activity detected in Burkholderia cepacia, Pseudomonas alcaligenes, P. mendocina, P. putida, P. stutzeri and in Shewanella putrefaciens is apparently described for the first time.

[Comparison of 2 methods of detecting slime production by coagulase-negative staphylococci]. / Srovnání dvou metod prukazu tvorby slizu u koagulázanegativních stafylokoku.

In the series of 212 coagulase-negative staphylococci strains isolated from blood cultures, in which the slime production was tested by means of two methods--a method using agar with Congo red and a Christensen method--S. epidermidis, S. haemolyticus and S. hominis were identified most frequently (in 69.3%, 18.4% and 6.6%, respectively). The slime production was detected by both methods in 28 strains (13%) of four species, most often in S. epidermidis (in 17%). By comparing the Congo red agar method with the Christensen method the sensitivity and specificity of the former was determined (85% and 99%, respectively). Thus the Congo red agar method for the detection of slime production by staphylococci seems to be a reliable tool.