Seroprevalence of campylobacteriosis and relevant post-infectious sequelae.

Post-infectious sequelea such as Guillain Barré syndrome (GBS), reactive arthritis (RA), and inflammatory bowel disease (IBD) may arise as a consequence of acute Campylobacter-enteritis (AE). However, reliable seroprevalence data of Campylobacter-associated sequelae has not been established. The objectives of this study were, first, to identify the most specific and sensitive test antigen in an optimized ELISA assay for diagnosing a previous Campylobacter-infection and, second, to compare the prevalence of anti-Campylobacter antibodies in cohorts of healthy blood donors (BD), AE, GBS, RA, and IBD patients with antibodies against known GBS, RA and IBD triggering pathogens. Optimized ELISAs of single and combined Campylobacter-proteins OMP18 and P39 as antigens were prepared and sera from AE, GBS, RA and IBD patients and BD were tested for Campylobcter-specific IgA and IgG antibodies. The results were compared with MIKROGEN™-recomLine Campylobacter IgA/IgG and whole cell lysate-immunoblot. Antibodies specific for Helicobacter pylori, Mycoplasma pneumoniae, Yersinia enterocolitica, and Borrelia afzelii were tested with commercial immunoblots. ROC plot analysis revealed AUC maxima in the combination of OMP18 and P39 for IgA and in the P39-antigen for IgG. As a result, 34-49 % GBS cases, 44-62 % RA cases and 23-40 % IBD cases were associated with Campylobacter-infection. These data show that Campylobcater-seropositivity in these patient groups is significantly higher than other triggering pathogens suggesting that it plays an important role in development of GBS and RA, and supports the hypothesis that recurrent acute campylobacteriosis triggers IBD.

Chemotaxis in Campylobacter jejuni.

Chemotaxis is the common way of flagellated bacteria to direct their locomotion to sites of most favourable living conditions, that are sites with the highest concentrations of energy sources and the lowest amounts of bacteriotoxic substances. The general prerequisites for chemotaxis are chemoreceptors, a chemosensory signal-transduction system and the flagellar apparatus. Epsilonproteobacteria like Campylobacter sp. show specific variations of the common chemotaxis components. CheV, a CheW-like linking-protein with an additional response regulator (RR) domain, was identified as commonly used coupling scaffold protein of Campylobacter jejuni. It attaches the histidine autokinase (CheAY), which also has an additional RR-domain, to the chemoreceptors signalling domains. These additional RR-domains seem to play an important role in the regulation of the CheAY-phosphorylation state and thereby in sensory adaptation. The Campylobacter-chemoreceptors are arranged into the three groups A, B, and C. Group A contains membrane-anchored receptors sensing periplasmic signals, group B consists only of one receptor with two cytoplasmic ligand-proteins representing a bipartite energy taxis system that senses pyruvate and fumarate, and group C receptors are cytoplasmic signalling domains with mostly unknown cytoplasmic ligand-binding proteins as sensory constituents. Recent findings demonstrating different alleles of the TLP7 chemoreceptor, specific for formic acid, led to an amendment of this grouping.

[Campylobacteriosis of man : livestock as reservoir for Campylobacter species]. / Die Campylobacteriose des Menschen : Nutztiere als Reservoir für Campylobacter-Arten.

Over the last few years, infections with Campylobacter have significantly increased in Europe and Germany and these bacteria have even surpassed Salmonella as the most prevalent bacteria, causing gastroenteritis. Especially contamination during the handling and consumption of meat products seems to be the most important risk factor which plays a prominent role for transmission to man. In addition, contact with pets and other animals, drinking raw or improperly pasteurized milk, and the tenacity of Campylobacter in different environments, especially water, have also to be considered for an adequate risk assessment. Besides gastroenteritis, arthralgia, and Guillain-Barré syndrome are important clinical complications of Campylobacter infections in man. At the same time, it is mostly unclear why the course of infection in man and in reservoir animals differs significantly, especially as only a few classical bacterial virulence factors have been identified so far. For these reasons, the development of efficient prevention strategies is of utmost importance in order to control campylobacteriosis.

Improved clinical laboratory identification of human pathogenic yeasts by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

The key to therapeutic success with yeast infections is an early onset of antifungal treatment with an appropriate drug regimen. To do this, yeast species identification is necessary, but conventional biochemical and morphological approaches are time-consuming. The recent arrival of biophysical methods, such as matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), in routine diagnostic laboratories holds the promise of significantly speeding up this process. In this study, two commercially available MALDI-TOF MS species identification systems were evaluated for application in clinical diagnostics, using a geographically diverse collection of 1192 clinical yeast and yeast-like isolates. The results were compared with those of the classical differentiation scheme based on microscopic and biochemical characteristics. For 95.1% of the isolates, all three procedures consistently gave the correct species identification, but the rate of misclassification was greatly reduced in both MALDI-TOF MS systems. Furthermore, several closely related species (e.g. Candida orthopsilosis/metapsilosis/parapsilosis or Candida glabrata/bracarensis) could be resolved by both MALDI-TOF MS systems, but not by the biochemical approach. A significant advantage of MALDI-TOF MS over biochemistry in the recognition of isolates novel to the system was observed. Although both MALDI-TOF MS systems employed different approaches in the database structure and showed different susceptibilities to errors in database entries, these were negligible in terms of clinical usefulness. The time-saving benefit of MALDI-TOF MS over biochemical identification will substantially improve fungal diagnostics and patient treatment.

Is the Campylobacter jejuni secretory protein Cj0069 a suitable antigen for serodiagnostics?

Campylobacter spp. is the most common bacterial pathogen of gastroenteritis worldwide. Poultry is the main reservoir and consequently the main origin of infections for humans. As a consequence of a primary Campylobacter infection which typically manifests as diarrhea, there is an increased risk to suffer from post-infectious complications such as reactive arthritis, neuropathia, myositis or a Guillain-Barré Syndrome. Usually the verification of acute campylobacteriosis is made by stool culture. In contrast, post-infectious complications can be diagnosed by serological assays. Since most of them are based on whole cell lysates, an insufficient specificity results from cross-reactions between related species. Therefore, the use of recombinant antigens becomes more and more favorable. Campylobacter is able to secrete a number of proteins, which are amongst others necessary for cell invasion and therefore play a crucial role for virulence. One of these, Cj0069, has a similar specificity and sensitivity in the detection of anti-Campylobacter jejuni IgG compared to the well-established antigens OMP18 and P39. This makes it a suitable antigen for diagnosing C. jejuni post-infectious complications.

Comparison of different protocols for DNA preparation and PCR for the detection of fungal pathogens in vitro.

Although a large number of different PCR protocols for the detection of fungal DNA from clinical samples have been described, a generally recognised standardisation has not yet been developed. In a first step, we compared six different methods to isolate DNA under in vitro conditions from Aspergillus fumigatus, Candida albicans and Saccharomyces cerevisiae with respect to efficiency and expenditure of time. To this end, methods were tested that are based on both mechanical and enzymatic/thermic lysis. Thereby, enzymatic/thermic lysis were shown to be superior to mechanical lysis, although these methods of DNA isolation were more time consuming. The subsequent comparison of three different PCR protocols showed real-time PCR to be the most sensitive method.

Expression and localization of type III secretion-related proteins of Chlamydia pneumoniae.

The entire developmental cycle of the obligate intracellular bacteria Chlamydia pneumoniae takes place within the inclusion body. As many gram negative bacteria, Chlamydia possess a type III-secretion system (TTSS), which allows them to target effector molecules into the host cell. The expression and localization of several proteins constituting the TTSS apparatus and of proteins supposed to be secreted by the TTSS have been investigated. For the TTSS-constituting proteins, we selected representatives such as YscN (ATPase), LcrE (putative "lid" of the TTSS) and LcrH1 (postulated to be a chaperone). Furthermore, we focused on the putative effector proteins IncA, IncB, IncC, Cpn0809 and Cpn1020. Expression of these proteins was detected by reverse transcriptase-PCR followed by immunoblot analysis using antisera that were generated against the corresponding recombinant proteins. Thereby, expression could be detected on the RNA and/or protein level. Intracellular localization of proteins under investigation was determined by immunofluorescence assays using the respective antisera. YscN was shown to be distributed equally throughout the inclusion body, whereas LcrE gave a more prominent staining of the inclusion membrane. IncA was detected mainly on the membrane of the inclusion body, whereas IncB and IncC were shown to be located within the inclusion. Immunofluorescence assays with antisera raised against Cpn0809 and Cpn1020 showed completely different labeling. Signals corresponding to Cpn0809 and Cpn1020 were distributed within the host cell rather than inside the inclusions. Taken together, the different localization patterns of the effector proteins indicate differences in function and interplay with the host cell.

Toxoplasma gondii inhibits the in vitro induced apoptosis of HL-60 cells.

The integrity of the host cell may represent an important prerequisite for the intracellular survival and development of obligate intracellular pathogens. In the present study, we investigated the influence of infections with the protozoan parasite Toxoplasma gondii on the rate of apoptosis in the human leukemia cell line HL-60. After infection with T. gondii tachyzoites of the strain NTE and in uninfected controls, less than 2% of the host cells showed typical signs of apoptosis, i.e. condensation of chromatin after staining with Hoechst 33258 or internucleosomal DNA fragmentation after agarose gel electrophoresis of genomic DNA. After treatment with 0.1 to 0.5 microg/ml actinomycin D for up to 16 hours, HL-60 cells considerably underwent apoptosis. However, this actinomycin D-induced apoptosis was clearly reduced after concomitant infection with T. gondii as shown by staining with Hoechst 33258 and by DNA fragmentation assay. Inhibition of apoptosis by the intracellular pathogen T. gondii might be recognized as an evasion mechanism that enables intracellular survival and establishes long-lasting persistence.

Characterization of human endogenous retrovirus type K virus-like particles generated from recombinant baculoviruses.

The family of human endogenous retrovirus type K (HERV-K) comprises members with long open reading frames (ORF) for retroviral proteins. The existence of a biologically active provirus with replicative capacities has not yet been demonstrated. To confirm the assumption that HERV-K codes for the previously observed retrovirus-like particles (human teratocarcinoma-derived virus, HTDV) in human teratocarcinoma cells, we have constructed recombinant full-length HERV-K cDNA-based baculoviruses with gag, pro, pol, and env ORFs. Two viral constructs were used for infections of insect cells, one bearing 67 bp of the 5' untranslated region upstream of the 5' splice donor (SD) site and of the retroviral genes, the second omitting the SD sequence. For both recombinant viruses, indirect immunofluorescence and laser scan analyses revealed expression of HERV-K Gag protein. Electron microscopy studies demonstrated efficient production of virus-like particles (VLPs) at the cytoplasmic cell membranes. These VLPs are morphologically identical with the HTDV phenotype. In immunoelectron microscopy of ultrathin frozen sections, anti-HERV-K Gag antibodies specifically reacted with HERV-K VLPs. In Western blots, in addition to the 76-kDa precursor protein, the putative major core protein with an apparent molecular mass of 32 kDa exhibited predominant immunoreactivity with anti-Gag antiserum. In contrast, neither HERV-K Env nor cORF proteins could be detected due to inefficient mRNA splicing. Purified particles from insect cell culture supernatants tested in an ultrasensitive reverse transcriptase assay revealed weak polymerase activity. The data demonstrate that HERV-K codes for retroviral particles of the HTDV phenotype.

Reactivation of chronic toxoplasmosis: is there a link to strain-specific differences in the parasite?

The protozoan parasite Toxoplasma gondii comprises three clonal lineages that are associated with the clinical outcome in infected individuals. Whereas group C strains are mainly found in animals, group A and B strains are associated with human disease (Howe and Sibley, 1995). An increased level of transcripts of the tachyzoite-specifically expressed gene SAG1 could be identified in group A T. gondii strains compared to group B strains. Since SAG1-mediated host-cell invasion seems to be important for parasite replication, the observed higher replication rate in group A T. gondii strains might explain the association with clinically overt symptoms at the acute stage in patients who are infected with this group of parasite strains. The presence of external stress factors, such as interferon-gamma (IFN-gamma)-mediated nitric oxide (NO) formation has been identified to stabilize the cyst stage, most likely by activation of promoter(s) which drive the expression of genes encoding bradyzoite-specific antigens. Reactivation of chronic toxoplasmosis thus might occur in the absence of external stress factors, as has been observed in AIDS patients with decreases levels of IFN-gamma. Since group B T. gondii strains might form more cysts in infected individuals due to an increased potential to convert into bradyzoites, reactivation with resulting toxoplasmic encephalitis could be a more common event in those AIDS patients who were infected with persistent cysts of this group of parasite strains.

Specific suppression of false-positive signals in the product-enhanced reverse transcriptase assay.

The recently developed ultrasensitive reverse transcriptase (RT) test involving a PCR step can detect minute amounts of RT in single retroviral particles and is 10(6) times more sensitive than conventional RT assays. We have found that different DNA-dependent DNA polymerases like DNA Polymerase I from Escherichia coli and eukaryotic enzymes like DNA polymerase alpha and gamma exhibit RT-like activities in this assay, whereas DNA polymerase beta and Klenow enzyme show only minor activities. To discriminate false-positive signals caused by DNA-dependent DNA polymerases in the RT-PCR assay, we have included increasing amounts of activated DNA in the RT reactions. This modification of the assay leads to complete inhibition of aberrant but not authentic RT activities. This improvement specifies the RT-PCR assay as the most sensitive tool for the detection of even very rare replication-competent retroviruses and of related enzymatic activities indigenous, for example, for products of endogenous retrovirus-like RT genes in cell extracts.

HERV-K: the biologically most active human endogenous retrovirus family.

The human genome contains a wide variety of endogenous retrovirus-like sequences. The human endogenous retrovirus type K (HERV-K) family comprises 30-50 members per haploid genome in humans and is highly conserved in Old World monkeys and apes. Some proviruses are displaying open reading frames (ORF) with coding capacity for viral particles. HERV-K sequences most likely code for the previously described human teratocarcinoma-derived virus (HTDV) and correlated expression of HERV-K Gag has been demonstrated by immunoelectron microscopy studies. Protease, but not yet reverse transcriptase (RT), enzymatic activity was demonstrated for recombinant HERV-K proteins. However, an ultrasensitive RT assay revealed specific polymerase activity associated with the HTDV particles. HERV-K transcription is specifically regulated by viral long terminal repeats and RNA is expressed at low steady-state levels in a variety of human tissues and tumours. In teratocarcinoma cell lines, HERV-K is highly expressed in a complex pattern showing full-length as well as subgenomic envelope (env) and two alternatively spliced small transcripts. The doubly spliced 1.8-kb mRNA codes for cORF protein which resembles Rev of HIV-1 and is located in the nucleolus. In addition, the cORF sequence acts as a leader and is essential for effective expression of glycosylated HERV-K Env protein. Although HERV-K sequences code for all necessary retroviral proteins, infectious particles could not yet be demonstrated. The putative implication of HERV sequences in pathophysiological processes, for example, testicular malignancies, remains to be elucidated.