[Laboratory survey on the incidence of Pneumocystis jirovecii - obviously a peculiar fungus, but also a rare pathogen?]. / Laborerhebung zur Häufigkeit von Pneumocystis jirovecii - Zwar ein besonderer Pilz, aber auch ein seltener Erreger?

Pneumocystsis jirovecii is a peculiar fungus for a variety of reasons. This opportunistic pathogen multiplies in humans only under certain conditions; a defect in the T-cell defense system creates a predisposition to this infection. In 2010 a data survey (IFT as well as PCR) from a few laboratories in Germany revealed 412 positive individuals. Even if only a few patients test positive for the colonization stage of this pathogen, the sheer number of individuals testing positive for other stages of infection indicate that the incidence of pneumocystosis in immunocompromised patients in Germany is underestimated.

[A 25-year-old patient with corneal infiltration]. / 25-jähriger Patient mit Hornhautinfiltrat.

Filamentous fungal keratitis represents a serious infection of the eye. When corneal infiltrates appear, particularly in those who wear contact lenses, mycological assessment should already be performed initially so that filamentous fungal keratitis can be recognized early and treated. Keratitis caused by Fusarium responds well in most cases to topical therapy with ketoconazole or other antimycotic agents so that surgical intervention is only necessary in advanced or treatment-refractory cases.

[Epidemiological aspects of gastrointestinal infections]. / Epidemiologische Aspekte gastrointestinaler Infektionen.

BACKGROUND AND OBJECTIVES: The aim of this study was to investigate seasonal patterns and age-associated trends of the main bacterial, viral, and parasitic enteric pathogens in Southwest Germany. PATIENTS AND METHODS: From January 2002 through December 2008 a total of 99,057 patients were tested for Norovirus, Rotavirus, bacterial pathogens, Cryptosporidium parvum (C. parvum), and Giardia lamblia (G. lamblia). RESULTS: All these pathogens were detected throughout the whole year. But there were distinctive seasonal patterns of activity of the following pathogens being detected: norovirus was detected mainly from September through April. The highest rotovirus activity was observed from December through June. But bacterial pathogens und C. parvum were found mainly from June to November. The percentage of positive results during the months with the highest activity was 10 - 49% for norovirus, 25% - 41% for rotavirus, 14 - 18% for bacterial infection and 3 - 4 % for C. parvum. G. lamblia and adenovirus were found throughout the year in 7 - 15% and 3 - 10% of samples, respectively. Moreover, the detection rate of different pathogens depended on patient age. In infants younger than one year, rotavirus, norovirus and adenovirus were most frequently isolated pathogenes. Stool samples from kindergarden- and school-age children were positive largely for bacterial pathogens such as Salmonella and Campylobacter particularly in late summer or early autum. In patients older than 60 years, norovirus, rotavirus, and toxin producing Clostridium difficile strains were the most common pathogens. CONCLUSIONS: In view of the age and season related frequency of detection of enteric pathogens, a step-by-step diagnosis of gastrointestinal tract infections is recommended. Considering that most pathogens are detected sporadically over the whole year, the analysis of negative samples should be appropriately expanded. The knowledge of seasonal occurrence can also be applied to improve the application of hygienic measures.

Antimicrobial resistance of Neisseria gonorrhoeae isolates from the Stuttgart and Heidelberg areas of southern Germany.

The aim of the present study was to determine prospectively the antimicrobial susceptibility of Neisseria gonorrhoeae strains collected in southern Germany (Heidelberg and Stuttgart areas). Sixty-five N. gonorrhoeae strains, isolated between July 2004 and June 2005 from patients with uncomplicated gonorrhoea, were tested. Minimum inhibitory concentrations of penicillin, tetracycline, ciprofloxacin, azithromycin, spectinomycin, ceftriaxone, and cefixime were determined by the E test. All isolates were fully susceptible to ceftriaxone, cefixime, and spectinomycin. However, 21.5% (14/65), 29.2% (19/65), and 47.7% (31/65) of isolates were resistant to penicillin (>2.0 mg/l), tetracycline (>2.0 mg/l), and ciprofloxacin (>1.0 mg/l), respectively. Critical MICs of azithromycin (>1.0 mg/l, as defined by the Neisseria Reference Laboratory at the Centers for Disease Control) were found for five (7.7%) N. gonorrhoeae isolates. These data indicate a high prevalence of N. gonorrhoeae strains resistant to the antimicrobial agents currently used to treat gonococcal infections in the Heidelberg and Stuttgart areas. Even though the findings may not be representative of the general population in Germany, they nevertheless illustrate the need to establish an antimicrobial resistance surveillance system in order to control gonorrhoea effectively.

Sensitivity of a new commercial enzyme-linked immunospot assay (T SPOT-TB) for diagnosis of tuberculosis in clinical practice.

Diagnosis of active and latent tuberculosis (TB) remains a challenge; however, over the last few years, a new approach based on detecting Mycobacterium tuberculosis-specific T cells has shown much promise. In particular, there is substantial published evidence showing that the detection of ESAT-6- and CFP-10-specific T cells using the ex vivo enzyme-linked immunospot technique is a marked improvement over the existing tuberculin skin test. This technique, which detects gamma interferon-producing T cells, is now available as the commercial assay T SPOT-TB (Oxford Immunotec, Oxford, UK). In the present study, the usefulness of the T SPOT-TB test for diagnosis of TB in "real-world" clinical practice was investigated. Ninety patients of a southern German referral centre for TB with confirmed or suspected TB were randomly selected for this study. The results of the T SPOT-TB test were compared with the results of conventional diagnostic tools. The T SPOT-TB test detected 70 of 72 patients with pulmonary or extrapulmonary TB, indicating a sensitivity of 97.2% (95% confidence interval, 90.3-99.7). For 45 of these patients, tuberculin skin test (TST) results were also available. Only 40 (89%) of these 45 patients were positive in the TST compared to all 45 (100%) in the T SPOT-TB test (p=0.056). Among 12 of 90 patients for whom active TB disease was ruled out, the T SPOT-TB test was negative for 11 (92%), allowing the rapid exclusion of TB in patients suspected to have active TB disease. The T SPOT-TB test is a sensitive assay for detection of TB and represents a useful addition to the diagnostic algorithm available for detecting TB in low-incidence settings.

[Real-time PCR assay for rapid detection of clarithromycin-resistant helicobacter pylori in gastric biopsy specimens]. / Schneller Nachweis von clarithromycinresistenten Helicobacter pylori in der Magenbiopsie mittels Real-Time-PCR.

The main cause for failure of Helicobacter pylori eradication therapy is resistance to clarithromycin which is due to point mutations. The use of real-time PCR allows the detection of these mutations directly on biopsy specimens within a few hours. In our routine laboratory, we compared LightCycler PCR to conventional detection and susceptibility testing of H. pylori by culture. PCR showed a positive result for H. pylori in 74 specimens. PCR was confirmed by culture in 69 specimens. In five specimens which were positive by PCR but negative on culture the (13)C urea breath test confirmed the PCR results. Sensitivity and specificity of our LightCycler assay for the detection of H. pylori in biopsy specimens were both 100 %. In 26 out of 68 specimens conventional susceptibility testing yielded resistance to clarithromycin. Corresponding point mutations were found in 24 of these specimens. Compared to culture, PCR gave a false-resistant, respectively, a false sensitive result in one specimen each. In another specimen, culture yielded a resistant strain whereas PCR detected both a resistant mutant and the wild-type strain. From two other specimens clarithromycin-sensitive strains were cultured but both a wild-type strain and a mutant were detected by PCR. Sensitivity and specificity of LightCycler PCR for resistance to clarithromycin were 96.2 % and 97.6 %, respectively. This assay had an accuracy comparable to culture and could be performed within 3 hours, allowing it to be used before the administration of H. pylori eradication therapy.

Diagnosis of extrapulmonary tuberculosis by Gen-Probe amplified Mycobacterium tuberculosis direct test.

A total of 294 specimens collected from nonrespiratory sites of 268 patients were tested for direct detection of Mycobacterium tuberculosis complex by the Gen-Probe Amplified Mycobacterium tuberculosis Direct Test (AMTD). The specimens included ascitic, pleural, pericardial, and synovial fluids, abscess aspirates, and tissue and lymph node biopsy samples, as well as gastric aspirates and cerebrospinal fluid samples. All samples were processed by the N-acetyl-L-cysteine-sodium hydroxide decontamination procedure prior to testing. Twenty samples showed acid-fast bacilli on auramine staining, and 48 samples were positive by AMTD, 9 of which were negative for M. tuberculosis complex by culture. After reviewing the patients clinical charts to resolve discrepancies, the test result of one cerebrospinal fluid sample was considered to be false positive by AMTD. The overall sensitivity, specificity, positive predictive value, and negative predictive value were 83.9, 99.6, 97.9, and 96.3%, respectively. No significant differences were found when AMTD results obtained with specimens of nonrespiratory origin were compared with assay results obtained with samples of respiratory origin (P > 0.05). In conclusion, our results demonstrate that AMTD performs equally well with all types of specimens.

Murine model of Bartonella henselae infection in the immunocompetent host.

Bartonella henselae is an emerging pathogen causing cat scratch disease, bacillary angiomatosis, and peliosis hepatis. Progress in understanding the pathogenesis of and the immune response to these infections has been limited by the lack of an animal model. Following intraperitoneal infection of C57BL/6 mice with B. henselae, organs were cleared of cultivatable bacteria within 6 days. In contrast, B. henselae DNA could be detected in liver tissue for at least 3 months. Liver tissue showed granulomatous inflammation reaching its highest degree of intensity during the fourth week of infection and resolving within 12 weeks postinfection. This mouse model is applicable to the study of the pathogenesis of B. henselae and the immune response to this pathogen in the immunocompetent host.

Bartonella henselae-specific cell-mediated immune responses display a predominantly Th1 phenotype in experimentally infected C57BL/6 mice.

Immune responses of the immunocompetent host to Bartonella henselae infection were investigated in the murine infection model using C57BL/6 mice. Following intraperitoneal infection with human-derived B. henselae strain Berlin-1, viable bacteria could be recovered from livers and spleens during the first week postinfection, while Bartonella DNA remained detectable by PCR in the liver for up to 12 weeks after infection. Granulomatous lesions developed in livers of infected mice, reached maximal density at 12 weeks after infection, and persisted for up to 20 weeks, indicating that B. henselae induced a chronic granulomatous hepatitis in the immunocompetent murine host. T-cell-mediated immune responses were analyzed in vitro by means of spleen cell proliferation and cytokine release assays as well as analysis of immunoglobulin G (IgG) isotypes. Spleen cells from infected mice proliferated specifically upon stimulation with heat-killed Bartonella antigen. Proliferative responses were mainly mediated by CD4+ T cells, increased during the course of infection, peaked at 8 weeks postinfection, and decreased thereafter. Gamma interferon, but not interleukin-4, was produced in vitro by spleen cells from infected animals upon stimulation with Bartonella antigens. Bartonella-specific IgG was detectable in serum of infected mice by 2 weeks, and the antibody concentration peaked at 12 weeks postinfection. IgG2b was the prominent isotype among the Bartonella-specific serum IgG antibodies. These data indicate that B. henselae induces cell-mediated immune responses with a Th1 phenotype in immunocompetent C57BL/6 mice.

Efficacy of different methods for detection of low Cryptosporidium parvum oocyst numbers or antigen concentrations in stool specimens.

The detection of Cryptosporidium parvum oocysts in stool specimens by acid-fast (AF) stains or immunofluorescence assays (IFA) requires the presence of large numbers of oocysts. To determine whether new commercially available enzyme immunoassays (EIAs) are more sensitive alternatives, three EIAs, a direct IFA, and the modified cold Kinyoun AF stain were compared, particularly with respect to detection of low oocyst numbers or antigen concentrations. Thirty-one negative and 31 calf stool-enriched human stool specimens were tested. One EIA method detected only nine positive specimens, demonstrating a sensitivity significantly less (p < 0.0001) than that of the IFA, the AF stain, and the other two EIAs. No differences could be found with respect to specificity. In addition, serial dilutions of 28 patients' stool samples containing cryptosporidian oocysts were prepared and examined using two EIAs, IFA, and the AF stain. One EIA yielded significantly inferior results (p < 0.0001), whereas the other one and the two microscopic methods did not differ significantly in either part of the study. The results indicate that the new EIAs do not exhibit higher sensitivities for detection of Cryptosporidium parvum than the two routinely used microscopic methods. Thus, for most laboratories, the IFA or AF stain may still represent the preferred method for the diagnosis of cryptosporidiosis.

Characterization of Bartonella henselae isolated from bacillary angiomatosis lesions in a human immunodeficiency virus-infected patient in Germany.

Infections with Bartonella (Rochalimaea) henselae can result in a variety of clinical entities, including bacillary angiomatosis in immunocompromised hosts. The fastidious nature of this bacterium has so far prevented the culture of many clinical isolates. We report the recovery of the first European B. henselae isolate associated with bacillary angiomatosis. The isolate was cultured in a frozen skin biopsy specimen from a human immunodeficiency virus (HIV)-infected patient and was characterized by means of biochemical, bacteriologic, immunologic, and molecular biological methods including pulsed-field gel electrophoresis. This strain was compared with two B. henselae strains isolated in the United States to determine the relationship between the isolates. We found that it was phenotypically and genotypically indiscernible from B. henselae Houston-1, a blood culture isolate from an HIV-infected patient in Houston. These data suggest that one B. henselae clone is associated with human infections in Europe and the United States.

A new acid-fast trichrome stain for simultaneous detection of Cryptosporidium parvum and microsporidial species in stool specimens.

The detection in stool specimens of Cryptosporidium parvum and microsporidia, the most frequent parasitic pathogens causing diarrhea in AIDS patients, until now has depended on two different staining methods. However, since double infections occur and minimization of laboratory costs is mandatory, development of a method for simultaneous detection of these parasites appeared desirable. We report on a new, inexpensive, and easy-to-perform staining procedure to demonstrate both acid-fast oocysts of C. parvum and other coccidia, as well as microsporidial spores. This acid-fast trichrome stain yields results comparable to those obtained by the Kinyoun and modified trichrome methods and considerably reduces the time necessary for microscopic examination.