Farming exposure in childhood, exposure to markers of infections and the development of atopy in rural subjects.

BACKGROUND: Within the context of the hygiene hypothesis, we aimed to study the potential association between farming-related risk factors and Toxoplasma gondii (T. gondii) as well as Helicobacter pylori (H. pylori) seropositivity. METHODS: The study included questionnaire data and serum samples of 321 young adults living in a rural environment. Serum samples were analysed for specific IgE to a common panel of aeroallergens (SX1) as well as IgG against T. gondii and H. pylori. RESULTS: Regular contact with animal stables before the age of 3 years (odds ratio (OR) (95% confidence interval): 2.0 [1.0; 4.0]) and unpasteurized milk consumption at age 6 years (1.8 [1.0; 3.3]) were the strongest risk factors for T. gondii infection. None of the farming-related factors were significantly associated with H. pylori infection. Current consumption of raw farm milk was not significantly associated with H. pylori infection (2.1 [0.8; 5.3]). Regular contact with animal houses before the age of 7 years was the strongest predictor for atopy (0.49 [0.26-0.96]). The reduction in risk could not be further decreased by any other factor under consideration. After adjustment for animal house contact, the OR for atopy was decreased by raw milk consumption and H. pylori infection in an additive manner. CONCLUSION: Exposure to farming environments in childhood might predict T. gondii seropositivity in rural subjects. Nevertheless, the strongest predictor for atopy in rural subjects seems to be regular contact with farm animals. Whether T. gondii infection is an intermediate factor in the association between farm contact and atopy needs to be confirmed in larger studies.

Mixed lung infection by Legionella pneumophila and Legionella gormanii detected by fluorescent in situ hybridization.

A mixed infection by Legionella pneumophila and a nonpneumophila Legionella species was detected in a lung biopsy specimen obtained from a patient with atypical pneumonia by fluorescent in situ hybridization (FISH). This result was confirmed by polymerase chain reaction (PCR). Sequencing of PCR products confirmed mixed infection by L. pneumophila and L. gormanii. Culture for Legionella spp. was negative and serology showed a rise only in IgG anti- Legionella pneumophila titer. To our knowledge, this is the first report of a mixed infection by L. pneumophila and a non-pneumophila Legionella species detected by FISH. Because FISH is a rapid and culture independent method that detects specific microorganisms in biopsy specimens it is recommended, in particular, for the detection of fastidious bacteria.

Evaluation of BACTEC MGIT 960 and BACTEC 460TB systems for recovery of mycobacteria from clinical specimens of a university hospital with low incidence of tuberculosis.

Clinical samples obtained over a period of 8 months (n = 2,624) were processed in parallel with the BACTEC 460TB system, with the MGIT 960 system, and in Löwenstein-Jensen (LJ) medium, resulting in the recovery of 127 mycobacteria. Recovery rates in combinations of the BACTEC 460TB or MGIT 960 system with LJ were, respectively, 94.7 and 94.7% for Mycobacterium tuberculosis complex (n = 57) and 91.4 and 70.0% for nontuberculous mycobacteria (n = 70). Contamination rates, elevated in the MGIT 960 system, were associated with patients (cystic fibrosis) and type of material but not with transport time. Detection time was reduced in the MGIT 960 system.

Conservative management of biliary obstruction due to Fasciola hepatica.

We report a case of temporary biliary obstruction due to fascioliasis. This case report shows that in Central Europe, fascioliasis is one of the differential diagnoses of abdominal pain, especially if it is associated with eosinophilia. Successful medical treatment is possible even with obstruction of the bile duct.

Culture independent and rapid identification of bacterial pathogens in necrotising fasciitis and streptococcal toxic shock syndrome by fluorescence in situ hybridisation.

Fluorescence in situ hybridisation (FISH) targeted to ribosomal RNA is well established for studies in environmental microbiology. Initial applications of this technique in the field of medical microbiology showed that FISH is also a suitable means for the rapid, reliable and cultivation-independent identification of bacterial pathogens. In particular, for infectious diseases that follow a fulminant live-threatening course, such as sepsis or necrotising fasciitis (NF), a fast and reliable detection technique is of great importance. This study describes the development of an rRNA-targeted oligonucleotide set covering more than 95% of the pathogens associated with NF. These probes were tested with a broad collection of target and non-target organisms and found to be highly specific. Subsequently, the FISH approach was applied for the direct detection of bacterial pathogens in clinical samples. Two cases of NF and one case of streptococcal toxic shock syndrome (STSS) were analysed. FISH correctly identified almost all pathogens present in the samples examined within 2-3 h. However, Proteus mirabilis, which was identified in one sample by conventional methods was detected as a rod-shaped bacteria but could not be identified by FISH, since no specific probe was available for this particular organism. In contrast, identification of pathogens in these samples by conventional laboratory methods took 48-72 h. Furthermore, in one patient with pre-sampling antimicrobial therapy bacteria could not be grown from any of the samples. FISH unequivocally revealed the presence of Streptococcus pyogenes in affected tissue samples from this patient. In an experimental setting we demonstrated that FISH readily identifies S. pyogenes cells rendered non-cultivable by antibiotic treatment.

Cost reduction after introduction of a multidisciplinary infectious disease service at a German university hospital.

BACKGROUND: In 1997 an infectious disease service (IDS) similar to those in the US was established at a university hospital in Munich, Germany. PATIENTS AND METHODS: We assessed the economic impact of the new policy by performing a cost comparison analysis. Inpatients with pneumonia, skin infections/cellulitis, urinary tract infections (UTI) and bacteremia/sepsis were assigned to two groups: patients from a 6-month period after the establishment of the IDS (post-IDS group) were compared with similar patients before the implementation of the ID-service (pre-IDS group). Costs of microbiological investigation (MB), antibiotic treatment (AB), clinical imaging (CI), total costs and length of antibiotic therapy were analyzed. RESULTS: Patients with UTIs in the post-IDS group had 39% fewer MBs (p<0.05) than patients in the pre-IDS group, resulting in a 33% decrease in average MB costs (p<0.05). In the total group, in which subgroups with pneumonia, skin infection and UTI were summarized, the post-IDS group had 37% fewer MBs (p<0.05) resulting in MB cost reductions of 34% (p<0.05). There were no significant differences in expenditures for AB and CI and in the average length of antibiotic therapy. CONCLUSION: This study shows that continuous consultation by an IDS does not increase diagnostic and treatment costs, but results in significant cost reductions.

Pulmonary toxoplasmosis in bone marrow transplant recipients: report of two cases and review.

Toxoplasma gondii may cause disseminated disease in bone marrow transplant (BMT) recipients. Pulmonary toxoplasmosis in BMT patients is rarely described. Mortality rates of >90% have been previously reported. Since pulmonary toxoplasmosis is extremely difficult to diagnose, it is very often detected only at autopsy. Two cases of pulmonary toxoplasmosis in BMT recipients that were diagnosed by visualization of T. gondii tachyzoites in bronchoalveolar lavage fluid and by a new semi-nested PCR method amplifying 18S rRNA from bronchoalveolar lavage fluid are presented, and the literature on pulmonary toxoplasmosis in BMT patients is reviewed.

Invasion and intracellular development of the human granulocytic ehrlichiosis agent in tick cell culture.

Human granulocytotropic ehrlichias are tick-borne bacterial pathogens that cause an acute, life-threatening illness, human granulocytic ehrlichiosis (HGE). Ehrlichias within neutrophil granulocytes that invade tick bite sites are likely ingested by the vector, to be transmitted to another mammalian host during the tick's next blood meal. Thus, the cycle of replication and development in the vector is prerequisite to mammalian infection, and yet these events have not been described. We report tick cell culture isolation of two strains of the HGE agent directly from an infected horse and a dog and have also established a human isolate from HL60 culture in tick cells, proving that the blood stages of the HGE agent are infectious for tick cells, as are those replicating in the human cell line HL60. This required changes to the culture system, including a new tick cell line. In tick cell layers, the HGE agent induced foci of infection that caused necrotic plaques and eventual destruction of the culture. Using the human isolate and electron microscopy, we monitored adhesion, internalization, and replication in vector tick cells. Both electron-lucent and -dense forms adhered to and entered cells by a mechanism reminiscent of phagocytosis. Ehrlichial cell division was initiated soon after, resulting in endosomes filled with numerous ehrlichias. During early development, pale ehrlichias with a tight cell wall dominated, but by day 2, individual bacteria condensed into dark forms with a rippled membrane. These may become compacted into clumps where individual organisms are barely discernible. Whether these are part of an ehrlichia life cycle or are degenerating is unknown.

First-glance diagnosis of Strongyloides stercoralis autoinfection by stool microscopy.

We report a case of autoinfection due to Strongyloides stercoralis in a 27-year-old Ethiopian AIDS patient living in Germany for nearly 3 years. This case was diagnosed on the basis of a single-view field in microscopy of a freshly obtained formalin-fixed stool specimen showing both rhabditiform and filariform larvae. The diagnosis of autoinfection by microscopy is discussed in detail.

Anti-recombinant V antigen serum promotes uptake of Yersinia enterocolitica serotype 08 by macrophages.

Phagocytosis resistance even in the presence of opsonizing antibodies is a key feature of pathogenic Yersinia spp. Nevertheless, antibodies against the secreted V antigen and the outer membrane protein YadA are known to mediate protection against Y. enterocolitica serotype 08 in a mouse model with intravenous infection. To investigate the impact of anti-V antigen serum on the interaction of Y. enterocolitica and phagocytic cells, gentamicin kill assays and immunofluorescence staining were performed. In contrast to anti-YadA, the presence of V antigen-specific antibodies resulted in an increased uptake of yersiniae by macrophages. The inhibition of phagocytosis by cytochalasin D suppressed the anti-V antigen-mediated uptake. The uptake-promoting effect of anti-V antigen was more distinct for macrophages than for polymorphonuclear leukocytes. The findings of the passive immunization experiments using an orogastric infection model were in agreement with those of cell-culture experiments. In the first 3 days of infection both antisera exhibit no protective effect on the multiplication of the bacteria in the Peyer's patches. Only mice passively immunized with anti-V antigen survived lethal oral infections with Y. enterocolitica serotype 08. Taken together, the results support the assumption that V antigen might be part of the translocation apparatus and that anti-V antigen inhibits the Yop translocation. In addition, antisera against in-frame-deleted recombinant V antigen were generated. Protection experiments using these antisera suggested that the type-specific region (amino acids 225-232) of the V antigen might not be a protective epitope.

PCR for detection and identification of Abiotrophia spp.

Members of the genus Abiotrophia, formerly known as nutritionally variant streptococci, are important pathogens causing septicemia and endocarditis. Cultivation and biochemical differentiation of Abiotrophia spp. are often difficult. Based on 16S rRNA sequences, two PCR assays for detection and identification of Abiotrophia spp. were developed. The first PCR assay was positive for all Abiotrophia spp. Subsequently performed restriction fragment length polymorphism analysis allowed the verification of the PCR amplicons and the differentiation of the three species. The second PCR assay was positive only for A. elegans, the most fastidious species of Abiotrophia. Both PCR assays were shown to be specific and sensitive and should facilitate the identification of Abiotrophia spp.

In vitro and in vivo expression studies of yopE from Yersinia enterocolitica using the gfp reporter gene.

The Yersinia outer protein YopE belongs to the translocated effector proteins of pathogenic yersiniae. We constructed various truncated yopE genes fused to gfp (encoding the green fluorescent protein) to study yopE gene expression and YopE-GFP translocation of Y. enterocolitica in cell culture and mouse infection models. The hybrid gene fusions were co-expressed in Y. enterocolitica (i) on a low-copy plasmid in the presence of the virulence plasmid pYV08 (in trans configuration) and (ii) after co-integration by homologous recombination of a yopE-gfp-carrying suicide plasmid into pYV08 (co-integrate configuration). After 30min of infection of HEp-2 cell monolayers, extracellularly located yersiniae began to emit green fluorescence after excitation. In contrast, internalized bacteria were weakly fluorescent. Translocation of YopE-GFP into HEp-2 cells by attached yersiniae was visualized by optical sectioning of fluorescent HEp-2 cells using confocal laser scanning microscopy and was confirmed by immunoprecipitation of cytosolic YopE-GFP from selectively solubilized HEp-2 cells. The co-translocation of other Yops was not significantly impaired by YopE-GFP as shown by YopH/YopE-mediated suppression of the oxidative burst of infected neutrophils. The time course of yopE-gfp expression (in trans as well as in the co-integrate configuration) in the HEp-2 cell infection model as well as after in vitro induction was studied using a highly sensitive CCD camera and a flow cytometer. Similar results were obtained with a YopE-LUC (firefly luciferase) protein fusion as reporter. After intraperitoneal, intravenous and orogastrical infection of Balb/c mice with the recombinant yersiniae strains, green fluorescing bacteria could be visualized microscopically in the peritoneum, the spleen, the liver and in the Peyer's patches. However, only weakly fluorescent yersiniae were observed in the intestinal lumen. These results were quantified by flow cytometric measurements. The application of gfp as a reporter gene turned out to be promising for the study of protein translocation by protein type III secretion systems and differential virulence gene expression in vivo.

Contribution of the Mn-cofactored superoxide dismutase (SodA) to the virulence of Yersinia enterocolitica serotype O8.

Enteric pathogens harbor a set of enzymes (e.g., superoxide dismutases [SOD]) for detoxification of endogenous and exogenous reactive oxygen species which are encountered during infection. To analyze the role of the Mn-cofactored SOD (SodA) in the pathogenicity of yersiniae, we cloned the sodA gene of Yersinia enterocolitica serotype O8 by complementation of an Escherichia coli sodA sodB mutant and subsequently constructed an isogenic mutant by allelic exchange. Sequence analysis revealed an open reading frame that enabled the deduction of a sequence of 207 amino acids with 85% identity to SodA of E. coli. In a mouse infection model, the sodA null mutant was strongly attenuated in comparison to its parental strain. After intravenous infection, the survival and multiplication of the mutant in the spleen and liver were markedly reduced. In contrast, inactivation of sodA had only minor effects on survival and multiplication in the gut and Peyer's patches, as could be demonstrated in the orogastric infection model. The reduction in virulence was accompanied by a low but significant increase of susceptibility of the soda mutant to bacterial killing by polymorphonuclear leukocytes (PMN) and an alteration of the intracellular chemiluminescence response of PMN. These results suggest that the resistance of Y. enterocolitica to exogenous oxygen radicals produced by phagocytes involves the Mn-cofactored SOD. The important role of sodA for the pathogenicity of Y. enterocolitica could also be due to detoxification of endogenous, metabolically produced oxygen radicals which are encountered by extracellular enteric pathogens during the invasion of the host.

Passive immunity to infection with Yersinia spp. mediated by anti-recombinant V antigen is dependent on polymorphism of V antigen.

The V antigen is a 37-kDa secreted polypeptide encoded on the 70-kb virulence plasmid of pathogenic Yersinia spp. Besides having regulatory functions, it is known to be a virulence factor and a protective antigen. DNA sequencing of the most common serotypes of human pathogenic Yersinia enterocolitica and Y. pseudotuberculosis revealed that two evolutionary distinct types of V antigen exist in Yersinia spp. One type is represented by Y. enterocolitica serotype 08 strains WA, WA-314, and NCTC 10938 (designated LcrV-YenO8); the other type comprises Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica serotypes O3, O9, and O5,27 (LcrV-Yps). A hypervariable region between amino acids 225 and 232 represents the main difference between the two types. By raising monospecific antisera against both types of V antigen (anti-rVO8 and anti-rVO3), we were able to demonstrate that, in general, passive immunization of mice against a challenge with yersiniae was possible with both anti-Y. enterocolitica V antigen sera. However, anti-V antigen serum was protective only if the immunizing V antigen was the same type as the V antigen produced by the infective strain. The failure of the American V antigen type represented by Y. enterocolitica serotype O8 to protect against Yersinia spp. carrying the other V antigen type (LcrV-Yps) could be an explanation for the presence of plague foci in American countries.

Deletion of amino acids 29 to 81 in adhesion protein YadA of Yersinia enterocolitica serotype O:8 results in selective abrogation of adherence to neutrophils.

In order to analyze the multiple functions of the yersinia adhesin YadA in more detail, we constructed an N-terminally truncated YadA protein (deletion of amino acids [aa] 29 to 81) of Yersinia enterocolitica serotype 0:8. The region aa 29 to 81 of YadA is located between the signal sequence and the amino-terminal hydrophobic domain (aa 80 to 101), which is involved in surface polymerization and collagen binding. The deletion of aa 29 to 81 (resulting in YadADelta29-81) had no effect on the well-known features of YadA such as autoagglutination, serum resistance, HEp-2 cell adherence, binding of collagen, and binding of the complement-inhibiting factor H. In contrast to this, mutant WA(pYVO8-A-Delta29-81), producing the truncated YadADelta29-81 had lost the ability to adhere to polymorphonuclear leukocytes and to induce an oxidative burst. This functional deficiency was comparable to that of a yadA-null mutant (K. Ruckdeschel, A. Roggenkamp, S. Schubert, and J. Heesemann, Infect. Immun. 64:724-733, 1996). Moreover, mutant WA(pYVO8-ADelta29-81) turned out to be attenuated in virulence comparably to the yadA-null mutant, as demonstrated with orogastrically and intravenously infected mice. In summary, this study shows that specific functions of YadA (i) can be impaired by designed mutations and (ii) are important in distinct stages of the infection process.