The hyper-IgE syndrome (HIES) is a rare primary immunodeficiency characterized by recurrent skin abscesses, pneumonia, and highly elevated levels of serum IgE. HIES is now recognized as a multisystem disorder, with nonimmunologic abnormalities of the dentition, bones, and connective tissue. HIES can be transmitted as an autosomal dominant trait with variable expressivity. Nineteen kindreds with multiple cases of HIES were scored for clinical and laboratory findings and were genotyped with polymorphic markers in a candidate region on human chromosome 4. Linkage analysis showed a maximum two-point LOD score of 3.61 at recombination fraction of 0 with marker D4S428. Multipoint analysis and simulation testing confirmed that the proximal 4q region contains a disease locus for HIES.

Contamination of hospital water systems with legionellae is a well-known cause of nosocomial legionellosis. We describe a new real-time LightCycler PCR assay for quantitative determination of legionellae in potable water samples. Primers that amplify both a 386-bp fragment of the 16S rRNA gene from Legionella spp. and a specifically cloned fragment of the phage lambda, added to each sample as an internal inhibitor control, were used. The amplified products were detected by use of a dual-color hybridization probe assay design and quantified with external standards composed of Legionella pneumophila genomic DNA. The PCR assay had a sensitivity of 1 fg of Legionella DNA (i.e., less than one Legionella organism) per assay and detected 44 Legionella species and serogroups. Seventy-seven water samples from three hospitals were investigated by PCR and culture. The rates of detection of legionellae were 98.7% (76 of 77) by the PCR assay and 70.1% (54 of 77) by culture; PCR inhibitors were detected in one sample. The amounts of legionellae calculated from the PCR results were associated with the CFU detected by culture (r ‫؍‬ 0.57; P < 0.001), but PCR results were mostly higher than the culture results. Since L. pneumophila is the main cause of legionellosis, we further developed a quantitative L. pneumophila-specific PCR assay targeting the macrophage infectivity potentiator (mip) gene, which codes for an immunophilin of the FK506 binding protein family. All but one of the 16S rRNA gene PCR-positive water samples were also positive in the mip gene PCR, and the results of the two PCR assays were correlated. In conclusion, the newly developed Legionella genus-specific and L. pneumophila species-specific PCR assays proved to be valuable tools for investigation of Legionella contamination in potable water systems.

In a prospective, multicenter study of 342 blood samples from 187 patients with systemic inflammatory response syndrome, sepsis, or neutropenic fever, a new commercial PCR test (SepsiTest; Molzym) was evaluated for rapid diagnosis of bacteremia. The test comprises a universal PCR from the 16S rRNA gene, with subsequent identification of bacteria from positive samples by sequence analysis of amplicons. Compared to blood culture (BC), the diagnostic sensitivity and specificity of the PCR were 87.0 and 85.8%, respectively. Considering the 34 BC-positive patients, 28 were also PCR positive in at least one of the samples, resulting in a patient-related sensitivity of 82.4%. The concordance of PCR and BC for both positive and negative samples was (47 ؉ 247)/342, i.e., 86.0%. In total, 31 patients were PCR/sequencing positive and BC negative, in whom the PCR result was judged as possible or probable to true bacteremia in 25. In conclusion, the PCR approach facilitates the detection of bacteremia in blood samples within a few hours. Despite the indispensability of BC diagnostics, the rapid detection of bacteria by SepsiTest appears to be a valuable tool, allowing earlier pathogen-adapted antimicrobial therapy in critically ill patients.Bloodstream infection is a life-threatening condition with a high mortality rate, especially in intensive care and neutropenic patients (5,19,35,38). Pathogenic bacteria are the most frequent causes of bloodstream infection, although fungi can also be isolated in a minority of patients (7,17,21,32,34). Currently, inoculation of blood cultures (BC) is the standard method for microbiological diagnosis of bloodstream infections. However, the limitations of BC include relatively low sensitivities and a long time-to-result for detection and identification of the pathogen, generally over 2 days, and even longer for fastidious organisms (13,20,27).In contrast, DNA-based procedures may offer faster and more reliable diagnoses (3, 30). PCR amplification of microbial genes, followed by detection of amplified products by gel electrophoresis or real-time PCR monitoring using fluorescent dyes or target-directed fluorescent probes, is a quick process allowing pathogen detection within a few hours (18). Identification of microorganisms can be performed by PCR algorithms, taxon-specific oligonucleotide microarrays, or sequencing amplicons (30).PCR amplification of conserved regions of the bacterial genome, in particular the 16S rRNA gene, combined with sequence analysis is a well-established technique for the identification of bacterial pathogens (18). The main advantages of targeting the 16S rRNA gene are the broad range of pathogens detectable and the independence of this method from the in vitro viability of strains (6). The high sensitivity of detection by PCR of bacterial DNA (15) suggests its use in the diagnosis of bacteremia (16). Initial disadvantages of PCR, notably the incidence of false-positive results from bacterial DNA contaminating PCR reagents (4, 39), have been counteracted by the devel...

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Zinc is a trace element which is essential for immune functions. It directly induces monokine secretion by monocytes; however, effects of zinc on T cells appear contradictory. Apart from enhanced lymphocyte proliferation in peripheral blood mononuclear cells (PBMC), inhibitory properties of high zinc dosages have also been described. In this study, PBMC failed to produce lymphokines like interferon (IFN)-gamma after stimulation with zinc in a serum- and LPS-free cell culture system, whereas monokine secretion [interleukin (IL)-1 beta] occurred. Zinc-uptake studies with the zinc-specific fluorescent probe zinquin revealed that zinc is taken up by PBMC within a few minutes, reaching nearly equal levels in PBMC, isolated monocytes, and T cells. However, if zinc was depleted 1 h after monocyte induction, zinc-free pre-cultured T cells were stimulated to secrete IFN-gamma by zinc-induced monokines. Furthermore, the necessity for a cell-cell interaction between monocytes and T cells for IFN-gamma induction was elucidated. Zinc ions inhibited the proliferation of the IL-1-dependent T cell line D 10N in a dose-dependent manner, suggesting a direct inhibitory effect of zinc. By immunoprecipitation we revealed a specific inhibition of IL-1 receptor-associated protein kinase (IRAK) by zinc ions. Therefore, in contrast to an indirect stimulation of T cells due to zinc-induced monokines, higher concentrations of zinc directly inhibit T cell functions by means of specific inhibition of IRAK and subsequent signaling events such as NF kappa B activation. The divergent effects of zinc on different cell populations, depending on the zinc concentration, could explain contradictory results of zinc stimulation. Furthermore, our data suggest new strategies of specific zinc-mediated immune modulation.

Phenotypic identification of gram-negative bacteria from Cystic Fibrosis (CF) patients carries a high risk of misidentification. Therefore, we compared the results of biochemical identification by API 20NE with 16S rRNA gene sequencing in 88 gram-negative, oxidase-positive rods, other than morphologically and biochemically typical P. aeruginosa, from respiratory secretions of CF patients. The API 20NE allowed correct identification of the bacterial species in 15 out of 88 (17%) isolates investigated. Agreement between the API and the 16S rRNA gene sequencing results was high only in isolates with an API result classified as "excellent identification." Even API results classified as "very good identification" or "good identification" showed a high rate of misidentification (67% and 84%). Fifty-two isolates of morphological and biochemical nontypical Pseudomonas aeruginosa, representing 59% of all isolates investigated, were not identifiable or misidentified in the API 20NE. Therefore, rapid molecular diagnostic techniques like real-time PCR and fluorescence in situ hybridization (FISH) were evaluated in this particular group of bacteria for identification of the clinically most relevant pathogen, P. aeruginosa. The LightCycler PCR assay with a P. aeruginosa-specific probe showed a sensitivity and specificity of 98.1% and 100%, respectively. For FISH analysis, a newly designed P. aeruginosaspecific probe had a sensitivity and specificity of 100%. In conclusion, molecular methods are superior over biochemical tests for identification of gram-negative, oxidase-positive rods in CF patients. In addition, realtime PCR and FISH allowed identification of morphologically nontypical isolates of P. aeruginosa within a few hours.Chronic bacterial colonization of the respiratory tract, leading to exacerbations of pulmonary infection, is the major cause of disease and death in Cystic Fibrosis (CF) patients. The most common pathogen in respiratory secretions of CF patients is Pseudomonas aeruginosa, and Staphylococcus aureus, Haemophilus influenzae, and members of the Burkholderia cepacia complex also play an important role in CF lung disease (13,14,17). Other gram-negative glucose nonfermenters such as Achromobacter xylosoxidans, Ralstonia pickettii, and Stenotrophomonas maltophilia are also occasionally recovered from CF respiratory samples, but their pathogenic significance remains to be fully clarified (4,14,17). Recent studies that applied molecular approaches for the identification of unusual pathogens in CF patients revealed the presence of various rarely or even newly described species belonging to the genera Bordetella, Comamonas, Inquilinus, Pandoraea, Ralstonia,16,22,23). Determination of the clinical relevance of gramnegative bacteria other than P. aeruginosa in CF patients is, however, hampered by the difficult identification of these pathogens by conventional laboratory techniques.Phenotypic identification of bacteria grown from CF patients carries a high risk of misidentification regarding both manual methods and...