Low prevalence of human metapneumovirus and human bocavirus in adult immunocompromised high risk patients suspected to suffer from Pneumocystis pneumonia.

BACKGROUND: Novel respiratory viruses were discovered in the last years predominantly in children. Until now information on newly identified respiratory viruses in immunosuppressed adult patients is limited. Here we investigated immunocompromised adults with suspected Pneumocystis jirovecii pneumonia (PCP) for new respiratory viruses. METHODS: Bronchoalveolar lavage (BAL) samples of 128 patients with atypical pneumonia (HIV-infected n=50, hematological malignancy n=51, immunosuppressive treatment n=27) were prospectively evaluated for P. jirovecii and retrospectively for new respiratory viruses (HMPV, HBoV, HCoV-NL63/SARS/HKU1). RESULTS: P. jirovecii was detected in 26/128, bacteria in 10, fungi in four, Influenza A in two patients. Novel respiratory viruses were found in only two/128 patients with hematological malignancy, of those one patient with HBoV-infection and one with HMPV-infection, respectively. No pathogens were detected in 82/128 patients. The one patient with detection of hMPV and clinical diagnosis of atypical pneumonia died of pulmonary failure. CONCLUSION: Human bocavirus and human metapneumovirus are rarely involved in atypical pneumonia in immunocompromised adult patients with suspected PCP, but may contribute to severe respiratory failure.

Analysis of the beta-tubulin gene from Vittaforma corneae suggests benzimidazole resistance.

We amplified, cloned, and sequenced the beta-tubulin gene of Vittaforma corneae, a microsporidium causing human infections. The beta-tubulin gene sequence has a substitution at Glu(198) (with glutamine), which is one of six amino acids reported to be associated with benzimidazole sensitivity. Benzimidazoles were assayed for antimicrosporidial activity and showed poor parasite inhibition.

[Illness and death of Wolfgang Amadeus Mozart (1756-1791). A contribution to the Mozart Year 2006]. / Krankheit und Tod Wolfgang Amadeus Mozarts (1756-1791) : Ein Beitrag zum Mozart-Jahr 2006.

In the Mozart year 2006 also medicine has to deal with the music genius W.A. Mozart. It has been intensely discussed for a long time whether Mozart was poisoned, whether he deceased from a certain illness, or by self-medication and/or medical procedures. Consequently, his death caused wild speculations and adventurous assertions. Many authors assume that Mozart was a chronically ill man all his life. However, most of Mozart's described illnesses were harmless, and his unbelievable amount of work proves his extraordinary efficiency. The exact cause of death remains speculative. The story of a tall stranger dressed in somber gray who assigned Mozart to write a Requiem Mass and the circumstances of Mozart's funeral have additionally contributed to the generation of legends. However, if one gathers all known facts, there is no evidence that Mozart was murdered, and the exact cause of his death remains unclear.

An ultrastructural and molecular study of Tubulinosema kingi Kramer (Microsporidia: Tubulinosematidae) from Drosophila melanogaster (Diptera: Drosophilidae) and its parasitoid Asobara tabida (Hymenoptera: Braconidae).

Tubulinosema kingi is a pathogen of Drosophila spp. that was originally described 40 years ago. Although Drosophila melanogaster is widely used as a model organism for biological research, only limited data about microsporidia infecting Drosophila have been published so far and very little is known about the ultrastructure of T. kingi. In this study, we present the results of ultrastructural and molecular examinations of T. kingi. The whole life cycle took place in direct contact with the host cell cytoplasm and all examined life cycle stages contained a diplokaryon. Very few membrane elements were present in early merogonial stages, but their number and order of arrangement increased as the life cycle proceeded. The cell membrane of meronts had a surface coat of tubular elements that encircled the cell. Later, numerous electron-dense strands without any ornamentation accumulated on the plasma membrane, indicating that cells had entered sporogony. The cell membrane of sporonts was covered by electron-dense material. The polar filament in the spores was slightly anisofilar with the last three or four coils being smaller in diameter. The polar filament has 10 to 14 coils which were arranged predominantly in a single row, but in many spores, one winding of the coiled polar filament was located inside the outer coils. In some spores, the polar filament was irregularly arranged in two or even three rows. Molecular analysis showed that all Tubulinosema spp. are closely related and form a clade of their own that is distinct from the Nosema/Vairimorpha clade. All these ultrastructural and molecular features are in concordance with the family Tubulinosematidae and the genus Tubulinosema which reinforces the recent reclassification of this microsporidium.

Transfer of the members of the genus Brachiola (microsporidia) to the genus Anncaliia based on ultrastructural and molecular data.

Two microsporidian genera, AnncaliiaIssi, Krylova, & Nicolaeva 1993 and BrachiolaCali et al. 1998, possess a Nosema-type life cycle and unique cell surface ornamentations, which include precocious electron-dense coating of the plasmalemma and a variety of secretory structures deposited on the parasite surface and scattered in the host cell cytoplasm. Comparative analysis of ultrastructure of Anncaliia meligethi (the type species of the genus Anncaliia) and of B. vesicularum and B. algerae (the best-studied members of the genus Brachiola) clearly demonstrated that these microsporidia share many distinctive morphological features. The comparison of small subunit ribosomal DNA sequences showed high sequence identity of A. meligethi and B. algerae. Phylogenetic analyses indicated that the rDNA sequences of A. meligethi clustered with those of B. algerae suggesting a close relatedness of these microsporidia. The combination of molecular and morphological data provided clear evidence that these microsporidia belong to the same genus and therefore, warranted emendation of the genus Anncaliia and establishments of the following new combinations: Anncaliia vesicularum nov. comb., Anncaliia algerae nov. comb., Anncaliia connori nov. comb., and Anncaliia gambiae nov. comb. The generic name Brachiola is submerged according to the rule of priority.

Detection of polyomavirus BK and JC in children with kidney diseases and renal transplant recipients.

BACKGROUND: Reactivation of polyomavirus is a known reason for severe renal dysfunction in adult renal transplant recipients. Testing for polyomavirus DNA in plasma has been described as a sensitive and specific method to discover viral nephropathy in adult patients. We were now interested in polyomavirus status in a pediatric patient setting. METHODS: Plasma and urine samples were obtained from 80 children including 38 children after renal transplantation (group 1), 7 children with different kidney diseases receiving immunosuppressive treatment (group 2) and 35 children with different kidney diseases not receiving immunosuppressive treatment (group 3). A nested polymerase chain reaction method was used for amplification of polyomavirus DNA fragments. Differentiation between JC and BK virus was done by digestion with restriction endonucleases. RESULTS: Polyomavirus DNA was detected in the urine sample of 19 of 38 (50%) renal transplant recipients (group 1), of 1 of 7 (14%) patients from group 2 and in none of the 35 patients of group 3. Plasma samples from 3 (8%) of group 1 patients and from 1 child each of group 2 (14%) and group 3 (3%) were tested positive for polyomavirus DNA. CONCLUSION: Urinary polyomavirus excretion seems to be more frequent in pediatric patients with kidney diseases receiving immunosuppressive treatment and after renal transplantation than in children with various kidney diseases without immunosuppressive treatment.

In vitro cultivation of an insect Microsporidian Tubulinosema ratisbonensis in mammalian cells.

Tubulinosema ratisbonensis is a microsporidian pathogen of Drosophila melanogaster belonging to the family Tubulinosematidae. The microsporidia in this family mainly cause infections in invertebrate hosts, but two members of this family, Brachiola vesicularum and Brachiola algerae, have been found to cause infections in humans as well. Moreover, B. algerae can be transmitted to immunodeficient mice and grows in mammalian cell cultures. Thus, the examination of the opportunistic properties of other members of the family Tubulinosematidae is important. Spores of T. ratisbonensis, isolated from infected fruit flies, were used to inoculate mammalian and insect cell cultures. Parasite growth was only seen in human lung fibroblasts. No growth was seen in Vero cells or insect cell cultures. Comparison of growth kinetics at 31 degrees C and 37 degrees C showed that there were fewer and smaller parasitic foci in cultures incubated at 37 degrees C. Transmission electron microscopy revealed the typical ultrastructure of T. ratisbonensis, and scanning electron microscopy showed oval or slightly pyriform spores, with some spores having extruded their polar tubes. The PCR-amplified sequences of rDNA fragments from infected cell cultures were 100% identical to the original T. ratisbonensis rRNA sequence. As T. ratisbonensis is able to proliferate in mammalian cell cultures, it may have the opportunistic properties of other members of the family Tubulinosematidae.

How do microsporidia invade cells?

Microsporidia are obligate intracellular eukaryotic parasites that utilize a unique mechanism to infect host cells. One of the main characteristics of all microsporidia is that they produce spores containing an extrusion apparatus that consists of a coiled polar tube ending in an anchoring disc at the apical part of the spore. With appropriate conditions inside a suitable host, the polar tube is discharged through the thin anterior end of the spore, thereby penetrating a new host cell for inoculating the infective sporoplasm into the new host cell. This method of invading new host cells is one of the most sophisticated infection mechanisms in biology and ensures that the microsporidia enter the host cell unrecognized and protected from the host defence reactions. Recent studies have shown that microsporidia gain access to host cells by phagocytosis as well. However, after phagocytosis, the special infection mechanism of the microsporidia is used to escape from the maturing phagosomes and to infect the cytoplasm of the cells. Gaining access to cells by endocytosis, and escaping destruction in the phago-/endo-/lysosome by egressing quickly from the phagocytic vacuole to multiply outside the lysosome, is a common phenomenon in biology that has been evolved several times during evolution. How this is put into execution by the microsporidia is an inimitable principle by which an obligate intracellular organism has managed this problem. The extrusion apparatus of the microsporidia has obviously ensured the success of this phylum during evolution, resulting in a group of obligate intracellular organisms, capable of infecting almost any type of host and cell.

Blockade of TNF does not alter oxygen burst and phagocytosis of human neutrophils in patients with rheumatoid arthritis.

Clinical trials evaluating tumor necrosis factor alpha (TNF-alpha) binding agents in patients with rheumatoid arthritis (RA) have demonstrated significant efficacy in reducing symptoms of disease and slowing radiographic progression. However, infectious complications are the most severe and common adverse effects of anti-TNF therapy. The functional capacities of neutrophils (PMNs) as the first line of defense in bacterial and fungal infections are enhanced by soluble TNF as a potent neutrophil primer. The aim of this study was to assess the influence of in vivo TNF blockade on oxygen burst (OB) and phagocytosis of human neutrophils. PMNs were derived from 20 patients with RA on anti-TNF-alpha therapy and 13 patients using conventional DMARDs. By flow cytometry we measured OB upon stimulation with Escherichia coli and N-formyl-1-methionyl-1-leucyl-phenylalanine (FMLP) with and without priming with granulocyte-colony stimulating factor (G-CSF) and/or TNF-alpha using dihydrorhodamine (DHR) 123. Phagocytosis of fluorescein isothiocyanate (FITC)-labeled E. coli was also assessed by flow cytometry. Thirty-three healthy volunteers served as controls. Upon stimulation with E. coli and FMLP, there was no significant difference in OB between the two patient groups and healthy controls. Priming was effective in all groups. Phagocytosis of E. coli by PMNs was equally effective in controls and patients independent from the treatment regimen. These data show that OB, phagocytosis and responsiveness to priming with TNF and G-CSF of PMNs are not impaired in patients with RA treated with anti-TNF agents in comparison with patients on conventional DMARDs or healthy controls. Thus, the infectious complications observed in patients with TNF blockade cannot be explained by functional impairment of PMNs; however, the neutralization of TNF as a potent primer of neutrophil response may increase the susceptibility for infections in these patients.

Morphological and molecular investigations of Tubulinosema ratisbonensis gen. nov., sp. nov. (Microsporidia: Tubulinosematidae fam. nov.), a parasite infecting a laboratory colony of Drosophila melanogaster (Diptera: Drosophilidae).

A new species of microsporidia from Drosophila melanogaster was investigated by light and electron microscopy and by ribosomal RNA (rRNA) sequencing. This microsporidium and the previously described Nosema kingi and Nosema acridophagus have been transferred to the new genus Tubulinosema gen. nov. with the following characters: nuclei are in diplokaryotic arrangement during the life cycle. All stages are in direct contact with the host cell cytoplasm, slightly anisofilar polar tube with the last coils being smaller in diameter arranged in one or two rows on both sides of the diplokaryon and small tubuli on the surface of late meronts. Spores are oval or slightly pyriform. Thick endospore wall, thinner over anchoring disc. This new genus and the genus Brachiola have been placed in a new family Tubulinosematidae fam. nov. Phylogenetic analysis of small subunit rRNA sequences by different methods placed Tubulinosema spp. in one clade with the genus Brachiola forming its sister clade, which is distant from the clade containing the true Nosema spp. including Nosema bombycis.

Uptake of Encephalitozoon spp. and Vittaforma corneae (Microsporidia) by different cells.

Microsporidia are obligate intracellular parasites infecting a broad range of vertebrates and invertebrates. Various microsporidian species induce different clinical pictures in humans. The reason for this is not clear. It has been speculated that the different microsporidian species are transmitted by various routes, thus causing infections in different organs. Another possibility is that the diverse microsporidia have different tropisms to organ-specific cells, thus causing various diseases. In this study, we investigated the uptake of microsporidian spores by different cells with an immunofluorescence staining technique to investigate whether there is a difference between microsporidian species as well as between different cells. Using this technique, we were able to distinguish between intra- and extracellular microsporidian spores. All examined cell lines were able to internalize microsporidian spores, but the extent of internalization differed significantly between the cells. Although the results showed some patterns that correlate with the distribution of the parasites in humans, the different clinical pictures cannot be sufficiently explained by this phenomenon, so it seems more likely that the various clinical manifestations caused by the different microsporidian species are a consequence of different infection routes rather than of different affinities of the microsporidian species to different cells.

Quantitation of microsporidia in cultured cells by flow cytometry.

BACKGROUND: Microsporidia are obligate intracellular protozoan parasites that emerged as major opportunistic pathogens in humans since the onset of the AIDS pandemic. In the present study, we investigated whether FCM is a useful method for the quantitation of intracellular microsporidian spores in cultured cells. METHODS: Microsporidia (Encephalitozoon cuniculi) were grown in cell cultures and various cell-lines were coincubated with microsporidian spores at different multiplicities of infection, as well as for different periods of time. After permeabilization of the cells, intracellular spores were stained with a polyclonal anti-E. cuniculi serum and a FITC-labeled secondary antibody. Stained cells were analyzed on a flow cytometer and results were compared with those of fluorescence microscopy. RESULTS: Noninfected cells showed a lower fluorescence, while the relative fluorescence observed for infected cells was significantly higher. The cell population with the more intense fluorescence, representing cells with internalized microsporidian spores, increased with the multiplicity of infection as well as over time. Results of FCM and fluorescence microscopy were in excellent agreement for all experiments. CONCLUSIONS: We have developed a flow cytometric assay to detect and quantify cells with intracellular microsporidian spores. This method is easy to use, highly reproducible, and should be useful for future research.

Current treatment strategies for patients with Hodgkin's lymphoma and HIV infection.

Hodgkin's lymphoma is the most common non-AIDS-defining tumor diagnosed in HIV-infected patients. Although the introduction of highly active antiretroviral therapy (HAART) led to a decreased incidence of several malignancies among HIV-infected patients, the incidence of HIV-associated Hodgkin's lymphoma (HIV-HL) has been persistent in recent years. Its unusually aggressive tumor behavior includes a higher frequency of unfavorable histologic subtypes, high stage and extranodal involvement by the time of presentation and poor therapeutic outcome, in comparison with Hodgkin's lymphoma outside the HIV setting. Treatment of HIV-HL is challenging considering the underlying immunodeficiency caused by HIV itself and may increase the risk of opportunistic infections by inducing further immunosuppression. To address this delicate vulnerability of the HIV-infected host, tailored regimens, which are less aggressive than standard regimens for HIV-negative hosts, have been applied to achieve tumor control. The introduction of HAART has opened a new perspective in the treatment of HIV-associated malignancies. The improved control of HIV infection and the subsequently improved survival rates of HIV-infected patients has changed the goal from tumor control to cure and new treatment approaches with more potent regimens need to be evaluated to improve survival and quality of life in HIV-HL.

[Infectious diseases-Part II: New agents, resistances, and treatment strategies]. / Infektiologie 2004-Teil II. Neue Substanzen, Resistenzen und Therapien.

A number of new antimicrobial agents have been licensed in the last years, most prominently a number of new antiviral and antimycotic agents. The development of new antibacterial agents has slowed down, new agents are mainly targeted at a small but rapidly growing number of patients infected with bacteria resistant to the current antibiotics. The rates of resistance against antibiotics are rapidly rising, especially for gram-positive cocci and, in addition, for gram-negative nosocomial agents. Rising rates of resistances are further present in chronic viral infections, e. g., HIV infection. Instruments to monitor and minimize the rates of resistances are necessary. Important changes are to be found in a number of infectious complications. For patients with sepsis a new therapeutic principle, drocretogrin, has been found to reduce mortality. Additionally, low-dose corticosteroids in selected patients, close control of blood sugar and other interventions have shown to be effective. Corticosteroids have proven to be effective as well in the reduction of complications in adults with bacterial meningitis, most pronounced in pneumococcal meningitis. With new drugs licensed for the treatment of malaria and changes in the epidemiology, the guidelines for therapy and prevention have been reformulated.

[Infectious diseases-part I: epidemiology]. / Infektiologie 2003-Teil IEpidemiologie.

The outstanding issue regarding recent trends in the epidemiology of infectious diseases is the epidemic occurrence of severe acute respiratory syndrome (SARS) in 2003. SARS is caused by a novel coronavirus, presumably originating from wild cats. This new agent was rapidly identified and characterized, the outbreak was terminated in early summer 2003 after implementation of strict infectious control measures. Especially the high rate of complications and nosocomial infections has caused severe public health problems in China and Canada. The number of persons infected with HIV is still rising rapidly globally. New regions with rapid spread include the countries of the former Soviet Union in East Europe and Central Asia. The introduction of modern antiretroviral therapy in countries with high prevalence is making slow progress, with no visible impact on transmission dynamics so far. The WHO has implemented a number of programs to eradicate or eliminate targeted infectious diseases. Polio eradication and elimination of neonatal tetanus are making slow progress, obstacles to the control of these diseases are ongoing armed conflicts in regions with high prevalence and under financing of programs. Lack of funding is especially obvious regarding programs for the control and therapy of malarial infections. The numbers of patients newly identified with new variant Creutzfeldt-Jakob disease are not rising, but rather constant or even declining, making a large epidemic of vCJD unlikely.