A real-time PCR assay for the differentiation of Candida species.

AIMS: We established a real-time PCR assay for the detection and strain identification of Candida species and demonstrated the ability to differentiate between Candida albicans the most common species, and also Candida parapsilosis, Candida glabrata, Candida tropicalis and Candida dubliniensis by LightCycler PCR and melting curve analysis. METHODS AND RESULTS: The DNA isolation from cultures and serum was established using the QIAmp Tissue Kit. The sensitivity of the assay was ≥ 2 genome equivalents/assay. It was possible to differentiate all investigated Candida species by melting curve analysis, and no cross-reaction to human DNA or Aspergillus species could be observed. CONCLUSIONS: The established real-time PCR assay is a useful tool for the rapid identification of Candida species and a base technology for more complex PCR assays. SIGNIFICANCE AND IMPACT OF THE STUDY: We carried out initial steps in validation of a PCR assay for the detection and differentiation of medically relevant Candida species. The PCR was improved by generating PCR standards, additional generation of melting curves for species identification and the possibility to investigate different specimens simultaneously.

[Primary vitrectomy with intraocular antibiotic application in postoperative endophthalmitis]. / Primäre vitrektomie mit intraokularer antibiotikaapplikation bei akuter postoperativer endophthalmitis.

PURPOSE: Pars plana vitrectomy with intravitreal antibiotic application is an established procedure for treating postoperative endophthalmitis. The presented study analyzes our own results with this treatment as well as the role of adjuvant systemic steroid treatment. METHOD: We analyzed the data of 34 consecutive patients with postoperative endophthalmitis from January 2000 to March 2006. Thirty-two patients underwent vitrectomy and intravitreal application of antibiotics, and two patients received intravitreal antibiotics only. All patients received intravitreal dexamethasone and systemic antibiotics, and 12 patients received the systemic treatment with prednisolone. The effect of vitrectomy with respect to final visual acuity and the rate of postoperative complications were analyzed. The vitreous was microbiologically examined. Postoperative follow-up time ranged from 2 weeks to 24 months. RESULTS: Endophthalmitis followed cataract surgery with intraocular lens implantation in 30 patients (89%) and followed pars plana vitrectomy in four patients (11%). Positive cultures were obtained in 19 (56%) patients. Visual acuity improved in 31 (91%) patients. At their final examinations, 27 (79%) patients had gained visual acuity of 0.05 or better. CONCLUSION: Immediate vitrectomy in combination with intraocular antibiotics and steroid administration resulted in preservation of ambulatory vision in most of the patients. Systemic postoperative therapy with steroids seems to be associated with better final visual acuity.

Biocompatibility of bacterial contaminated prosthetic meshes and porcine dermal collagen used to repair abdominal wall defects.

BACKGROUND AND AIMS: A contaminated or infected surgical site is considered a contraindication for the use of the nonabsorbable alloplastic materials employed to repair abdominal wall defects. Therefore, the biocompatibility of new prosthetic materials was investigated. MATERIALS AND METHODS: Meshes measuring 1.5x1.5 cm made of conventional and titanium-coated polypropylene, polyglycol, or porcine dermal collagen were implanted under the abdominal wall of 96 rats (eight groups of 12 animals each) employing the inlay technique. Implantation of all four materials was performed both under semisterile conditions and bacterial contamination of the mesh. The meshes were explanted after 28 days. RESULTS: All the materials implanted under semisterile conditions were incorporated into the abdominal wall with only few intraabdominal adhesions (mean adhesion scores: 1.0, 1.2, 1.0, 0.8 points, respectively, not significant). With the porcine dermal collagen, proliferation rate and the proportion of inflammatory cells were statistically lower (p<0.01). In the bacterial contamination group, all meshes were associated with a suppurating infection and strong adhesions between the bowel and mesh, which were most prominent in the case of dermal collagen (mean adhesion scores: 1.6, 1.7, 1.7, and 1.9 points, respectively, not significant). In this group, two animals died of peritonitis. In comparison with the other materials, the proliferation rate was significantly elevated (p=0.03). No significant differences were seen between the other materials employed. CONCLUSION: Irrespective of the material employed, implantation of alloplastic meshes in an abdominal wall contaminated with bacteria, is associated with suppurating infections, in particular in the case of the membrane-like porcine dermal collagen. Nonabsorbable alloplastic meshes and dermal skin grafts should therefore not be used to repair infected abdominal wall defects.

[Appropriate microbiological diagnostics]. / Sinnvolle Mikrobiologische Diagnostik.

In the industrialized world the threat of infectious diseases is mainly due to nosocomial infections and multi-resistant agents. In this context, microbiological evaluations have not only a benefit for the individual patient, but also allow to evaluate the local epidemiologic situation. However, quality and benefits are often compromised by incorrect specimen collection. This review attempts to summarize diagnostic procedures, collection and transport of appropriate specimens and relevant causative agents for prominent clinical manifestations of infectious diseases.

[Neurosyphilis and neuroborreliosis. Retrospective evaluation of 22 cases]. / Neurosyphilis oder Neuroborreliose. Retrospektive Auswertung von 22 Kasuistiken.

We present laboratory data from 22 patients suspected of having neurosyphilis. In two cases the suspicion could not be confirmed, and in 20 cases neurosyphilis was detected. The sera from 17 patients were also assayed for Borrelia-specific antibodies. Suspicious immunoglobulin G antibody indices were detected in nine cases and a suspicious immunoglobulin M antibody index in one. In six of these, stored CSF/serum pairs were available to specify the antibodies by immunoblotting. This allowed for the identification of one patient apparently infected by both Borrelia spp. and Treponema pallidum. In all cases of newly suspected neurosyphilis, we recommend considering neuroborreliosis at the same time.

Multicenter evaluation of strategies for serodiagnosis of primary infection with Toxoplasma gondii.

The diagnostic performance of single-serum assays for toxoplasma-specific immunoglobulin (Ig)M. IgA. IgG, and IgE antibodies and of different combinations of such antibody assays in 20 European reference centers was assessed. A panel of 276 sera, of which 73 came from patients who seroconverted within 3 months (acute infection), 49 from patients who had seroconverted 3-12 months earlier (convalescence), and 154 from subjects who had two IgG-positive samples obtained 12 months apart (past infection), was tested with 20 toxoplasma-antibody assays and 195 combinations. In general, every assay with high diagnostic sensitivity showed low diagnostic specificity, i.e. no assay performed alone could reliably distinguish acute from past infection. Furthermore, no single assay (or combination) could separate convalescence from the other stages of toxoplasma infection. However, excellent diagnostic performances were reached by sequential use of highly sensitive IgM assays and methods examining IgG avidity or stage specificity. IgA or IgM assays were less suitable for confirmation of toxoplasma-IgM positivity. This study documents the strength of test combinations in assessing the stage of toxoplasma infection.

Prevalence of Chlamydia specific antibodies among obstetric and gynecological outpatients in Gondar, north-west Ethiopia.

A total of 533 obstetric and gynecological outpatients who visited the dedicated clinics in Gondar, Ethiopia, between April and August 1995 were investigated for Chlamydia-specific antibodies. Relevant epidemiological and clinical data were collected using a pretested questionnaire, clinical examinations and laboratory tests. Earlier, using an antigen specific ELISA technique, we had reported that only 33 (6%) of these women were positive for C. trachomatis in cervical swabs. In this study, we evaluated the prevalence of C. trachomatis-specific antibodies in serum samples of the same patients collected at the time the cervical swabs were taken. Employing a genus-specific ELISA for the detection of chlamydial LPS antibodies, 90% of the sera were found to be positive for IgG, while 49% of the sera contained IgA and 28% IgM antibodies. For 436 of the positive sera, species specificity of the antibodies was determined by means of a micro-immunofluorescence assay. Antibodies against C. trachomatics were present in 75% and antibodies against C. pneumoniae in 69% of these sera. Forty-nine percent of the patients carried antibodies against both species. The antibody based assay estimated that 13.9% of the patients had acute genital chlamydial infection (antigen specific IgM and the simultaneous presence of C. trachomatis specific IgG, IgM and IgA). C. trachomatis antigen detection in cervical swabs apparently underestimated the real frequency of acute infections. Seroprevalence of C. pneumoniae antibodies in the Ethiopian patients was similar to that in industrialized countries.

Serological evidence of Chlamydia pneumoniae lipopolysaccharide antibodies in atherosclerosis of various vascular regions.

BACKGROUND: The role of Chlamydia pneumoniae in the pathogenesis of atherosclerosis has so far mainly been investigated in patients suffering from coronary heart disease; the other vascular regions have virtually been ignored. The aim of this study was to carry out a statistical survey of serological markers of a C. pneumoniae infection in patients with different patterns of atherosclerosis manifestation. PATIENTS AND METHODS: 340 patients were examined for the atherosclerotic alteration of peripheral arteries of the lower limbs, carotid arteries and coronary arteries by ultrasound scan and/or angiography. Immunoglobulin(Ig)G and IgA-rELISA were used to measure chlamydial lipopolysaccharide antibodies. Species determination was performed using the IgG micro-immunofluorescence test. RESULTS: 24.0% of atherosclerotic cases (A) and 52.3% of controls (C) were negative for C. pneumoniae lipopolysaccharide antibodies (p = 0.00002). By contrast, 45.1% of atherosclerotic cases and 16.9% of controls were positive for both IgG and IgA (p = 0.00002). The mean antibody titers of the atherosclerosis group were higher than in the control group (IgG positive xAIgG = 344, xCIgG = 272; IgG and IgA positive xAIgG = 576, xCIgG = 486 and xAIgA = 120, xCIgA = 91). Concerning atherosclerosis manifestation in various vascular regions, no significant differences were found between IgG and IgA antibody titers and prevalence. CONCLUSIONS: The results show that a persistent C, pneumoniae infection with evidence of lipopolysaccharide immunoglobulin G and A is equally associated with the atherosclerotic alteration of coronary arteries, carotid arteries and peripheral arterial occlusive disease, irrespective of the severity of atherosclerosis and with no predisposition to any particular vascular region.

A second European collaborative study on polymerase chain reaction for Toxoplasma gondii, involving 15 teams.

In order to investigate the accuracy and practicability of the polymerase chain reaction (PCR) in the antenatal diagnosis of congenital toxoplasmosis, a collaborative study involving 15 European laboratories was performed under the auspices of the Biomed 2 Programme of the European Community. Each team received 12 aliquots (four negative, eight positive) of 'artificial samples' made of amniotic fluid spiked with tachyzoites of the RH strain of Toxoplasma gondii. Each team performed its own PCR protocol (all were different). Nine of the 15 laboratories were able to detect a single parasite, but two of the 15 found all samples negative. Four of the 15 laboratories found one or more control samples to be falsely positive. This study highlights the lack of homogeneity between PCR protocols and performance and underlines the need for an external quality assurance scheme which could provide 'reference' samples that could be used by any laboratory wanting to establish and maintain an accurate diagnostic test based on PCR.

[Acquired toxoplasmosis with cerebral involvement and subsequent hearing loss]. / Erworbene Toxoplasmose mit zerebraler Beteiligung und nachfolgender Hörstörung.

In immunocompetent patients the postnatally acquired toxoplasmosis is usually a mild or asymptomatic disease. CNS manifestations are rare, e.g. in patients with HIV infection or in patients with other types of immunosuppression. We report on a 9-year old healthy boy, who was hospitalized after one week with subfebrile temperatures and headache with clinical signs of encephalitis and unilateral deafness. We diagnosed a toxoplasmosis and treated the patient with pyrimethamine and sulfadiazine. While most of the signs and symptoms disappeared rapidly the deafness persisted.

Bacterial cytolysin perturbs round window membrane permeability barrier in vivo: possible cause of sensorineural hearing loss in acute otitis media.

The passage of radioiodinated streptolysin-O (SLO) and albumin through the round window membrane (RWM) was studied in vivo. When applied to the middle ear, SLO became quantitatively entrapped in this compartment and no passage to the cochlea occurred. However, flux of radioiodinated albumin through the toxin-damaged RWM was observed. We propose that the passage of noxious macromolecules, such as proteases, from a purulent middle-ear effusion may be facilitated by pore-forming toxins, resulting in cochlear damage and sensorineural hearing loss.

[Pars plana vitrectomy in Borrelia burgdorferi endophthalmitis]. / Pars-plana-Vitrektomie bei Borrelia burgdorferi-Endophthalmitis.

BACKGROUND: Ocular manifestations of Lyme borreliose present with unusual forms of conjunctivitis, keratitis, optic nerve disease, uveitis, vitritis and rarely endophthalmitis. CASE REPORT: A 57-year-old man working as logger in Sax-ony-Anhalt suffering from an endophthalmitis on his left eye was referred to us. The vision of his left eye was intact light perception and hand motions. The slit-lamp examination revealed severe inflammation of the anterior chamber with hypopyon, posterior synechiae, and opacity of the posterior lens capsule. Funduscopy showed no red reflex, no retinal details. In the local hospital serum analysis was performed and showed in Western-Blot IgM- and IgG-antibodies against Borrelia burgdorferi. Despite of intravenous application of ceftriaxon for 14 days panuveitis persisted, and endophthalmitis developed when antibiotic therapy was finished. RESULTS: During pars plana vitrectomy a sharply delineated cystic lesion containing yellowish fluid was revealed, and creamy yellow fluid was aspirated. Microscopically in hematoxylineosin stained slides of the aspirate structures consistent with Borrelia burgdorferi were found. Postoperatively vision increased to 1/15. Despite of a second intravenous ceftriaxon treatment for 14 days we observed a retinal vasculitis in the follow up of 6 months. CONCLUSIONS: Despite intravenous ceftriaxon-therapy borrelia burgdorferi must have survived in the vitreous body. Further investigations are required with respect to the use of other antibiotics or immunosuppressives.

Breakdown of the round window membrane permeability barrier evoked by streptolysin O: possible etiologic role in development of sensorineural hearing loss in acute otitis media.

Sensorineural hearing loss is a common sequela of acute and chronic otitis media, and the round window membrane (RWM) is currently being considered as a major route for noxious agents to pass from the middle ear cavity to the cochlea. Streptococcus pneumoniae, a major causative agent of otitis media, and Streptococcus pyogenes A produce molecularly related toxins, pneumolysin and streptolysin O (SLO), that form large pores in target membranes. In this study, we analyzed the effects of SLO on the permeability of the RWM. Resected RWMs from a total of 104 guinea pigs were embedded between two chambers of an in vitro system. One chamber was designated as the tympanal (cis) compartment, and the other was designated as the inner ear (trans) compartment. The permeability of normal and SLO-damaged RWMs towards Na+, [14C]mannitol, and proteins was investigated. SLO evoked permeability defects dose dependently in the RWM with fluxes of both Na+ and [14C]mannitol being demonstrable over a time span of up to 8 h. Serum proteins and radioiodinated SLO were also shown to pass through the damage RWM. Scanning electron microscopy revealed the morphological correlates to these results. We propose that damage to the RWM by potent pore-forming cytolysins leads to leakage of ions from the perilymph. Ionic disequilibrium and passage of noxious macromolecules to the cochlea could contribute to disturbances of the inner ear function.

[Congenital syphilis]. / Beitrag zur Problematik der Lues connata.

Four infants with Lues connata, three with the early stage of the disease (patients 1-3), are reported. Diagnosis was made after exclusion of other diseases. Initially an infectious disease was expected, since anemia, leucocytosis, thrombocytopenia, hepatomegaly and/or splenomegaly and a bad condition were found. In two patients bone structure was abnormal. Elevated serum concentrations of liver enzymes (ALAT, ASAT) were the indication for liver biopsy in one patient, in whom an accompanying hepatitis was diagnosed. Treatment was performed with penicillin, no JARISCH-HERXHEIMER reaction was observed. The Lues tests were negative during pregnancy but a displacental transfer of pathogenic agents could be assumed. Patient 4 was diagnosed at 9 months of age. Infection of the mother probably occurred in the last 6 weeks of pregnancy. It can not be decided if the baby has a connatal or acquired Lues. The titer decrease of the CMT-test after the end of the penicillin therapy is a marker for a successful treatment. If treatment was started at 2 years of age a total clinical recovery can be expected. The case reports demonstrate that negative Lues test during pregnancy do not exclude Lues connata in newborns. The Lues diagnosis should be considered if an infectious disease in a newborn can not be diagnosed. A general Lues serodiagnostic test is recommended in all newborns before they leave the obstetrics department.

Modulating the microbial colonization of the gastrointestinal tract by oral administration of defined Escherichia coli strains. I. Influencing the biotope by means of metabolic drift mutants of Escherichia coli.

Through the selection of spontaneous metabolic drift mutants (selection marker RifR) of an Escherichia coli strain (O6:H2 SmR) of known settling capacity in conventional mice, it was attempted to obtain clones with positively optimized in vitro characteristics, which may exert a promotive influence upon the in vivo colonizing behaviour. Using 512 drift mutant strains (E. coli O6:H2 SmR RifR) we were able to establish positive optimizations, at an over-aleatory rate, for each of the in vitro characters tested (haemagglutination capacity of rabbit, guinea pig, and fowl erythrocytes; overgrowing power of mouse-adapted wild-type E. coli strains; formation of biomass with exclusive utilization of dextrose, lactose, fructose, adonitol, salicin, rhamnose, mannose; multiplying power; quantitative motility; and capacity to synthesize mucus). A higher settling rate (larger number of animals in which the test strain shared greater than or equal to 50% of the Enterobacteriaceae population than that obtained with the initial strain) could be established for one out of the 62 clones tested in vivo (a rise from 20% to 55%). The higher settling rate was associated with combinations of various functional parameters and not with an improvement of any of the individual functions. Despite the increase in settling rate relative to the number of experimental animals used in these studies, it was not generally possible to obtain a more than 3 days' dominance of the test strain within the lac+ Enterobacteriaceae. This is considered to be due primarily to the incipient synthesis of secretory IgA.

Modulating the microbial colonization of the gastrointestinal tract by oral administration of defined Escherichia coli strains. II. Influencing the biotope by means of geographically unrelated Escherichia coli strains.

An effective increase in the rate of colonization can be accomplished, in analogy with the pathogenetic mechanism of what is known as travelers' disease, through the administration of geographically unrelated strains. As compared to Escherichia coli strains isolated in the GDR, metabolic drift-optimized and Ethiopian strains showed an increase from 20 to 50%. It was not, however, possible to maintain a greater than or equal to 50% share in the population of Enterobacteriaceae over a period of more than up to three days. Immunologic mechanisms are considered to be responsible for this.

Modulating the microbial colonization of the gastrointestinal tract by oral administration of defined Escherichia coli strains. III. Lipopolysaccharide-specific IgA in the intestine after oral administration of Escherichia coli.

The assumption that local immunologic mechanisms are responsible for findings on the specific microbial colonization of the gastrointestinal tract of mice, is confirmed by results described in the present communication. Quantitative examination of intestinal lavage fluids after oral administration of germs by ELISA showed that (i) there is a critical dose above which a significant increase of IgA synthesis is observable; (ii) low antibody levels could be detected already in the untreated control group. It may be assumed that--regardless of the biological characteristics of the administered strain, namely, substrate and receptor competition--the stimulability of local immunity provides against permanent monocolonization with displacement of the autochthonous Enterobacteriaceae in adult and immunocompetent hosts.

[CSF diagnosis in syphilis]. / Liquordiagnostik bei Syphilis.

Treponemal antibodies in CSF are often to be found by FTA-ABS and TPHA, also in cases without syphilis cerebrospinalis. The checking of the CSF barrier allows--together with the TPHA-index--the diagnostic of the syphilis cerebrospinalis. Antibodies against treponema pallidum have been proved in 15 patients at different stages of syphilis until 30 years after treatment, but the titres were low. Only in one case we have found an active syphilis cerebrospinalis by the help of the TPHA- and IgG-index.