Bacterial gastroenteritis caused by the putative zoonotic pathogen Campylobacter lanienae: First reported case in Germany.

Foodborne campylobacteriosis is the most common cause of human bacterial enteritis in Germany. Campylobacter jejuni and Campylobacter coli are the main causative agents for enteric disease, but a number of other species are involved, including rare ones. These rare Campylobacter spp. are emerging zoonotic pathogens in humans due to increasing international movement of supplies, livestock and people. Campylobacter lanienae was first isolated from healthy abattoir workers in Switzerland and at first its pathogenic potential for humans was considered to be low. Recently, the first case of Campylobacter lanienae -associated human enteritis was reported in Canada. Here, we describe a case of mild Campylobacter lanienae -associated enteritis with subsequent asymptomatic excretion in a butcher. The isolate is available at the TLV strain collection (no. TP00333/18). This first reported case of human Campylobacter lanienae campylobacteriosis in Germany demonstrates the agent's likely zoonotic pathogenicity.

Innovative and Highly Sensitive Detection of Clostridium perfringens Enterotoxin Based on Receptor Interaction and Monoclonal Antibodies.

Clostridium perfringens enterotoxin (CPE) regularly causes food poisoning and antibiotic-associated diarrhea; therefore, reliable toxin detection is crucial. To this aim, we explored stationary and mobile strategies to detect CPE either exclusively by monoclonal antibodies (mAbs) or, alternatively, by toxin-enrichment via the cellular receptor of CPE, claudin-4, and mAb detection. Among the newly generated mAbs, we identified nine CPE-specific mAbs targeting five distinct epitopes, among them mAbs recognizing CPE bound to claudin-4 or neutralizing CPE activity in vitro. In surface plasmon resonance experiments, all mAbs and claudin-4 revealed excellent affinities towards CPE, ranging from 0.05 to 2.3 nM. Integrated into sandwich enzyme-linked immunosorbent assays (ELISAs), the most sensitive mAb/mAb and claudin-4/mAb combinations achieved similar detection limits of 0.3 pg/mL and 1.0 pg/mL, respectively, specifically detecting recombinant CPE from spiked feces and native CPE from 30 different C. perfringens culture supernatants. The implementation of mAb- and receptor-based ELISAs into a mobile detection platform enabled the fast detection of CPE, which will be helpful in clinical laboratories to diagnose diarrhea of assumed bacterial origin. In conclusion, we successfully employed an endogenous receptor and novel high affinity mAbs for highly sensitive and specific CPE-detection. These tools will be useful for both basic and applied research.

Antibody response using six different serological assays in a completely PCR-tested community after a coronavirus disease 2019 outbreak-the CoNAN study.

OBJECTIVES: Due to a substantial proportion of asymptomatic and mild courses, many severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections remain unreported. Therefore, assessment of seroprevalence may detect the real burden of disease. We aimed to determine and characterize the rate of SARS-CoV-2 infections and the resulting seroprevalence in a defined population. The primary objective of the study was to assess SARS-CoV-2 antibody seroprevalence using six different IgG-detecting immunoassays. Secondary objectives of the study were: (a) to determine potential risk factors for symptomatic versus asymptomatic coronavirus disease 2019 courses, and (b) to investigate the rate of virus RNA-persistence. METHODS: CoNAN is a population-based cohort study performed in the community Neustadt am Rennsteig, Germany, which was quarantined from 22 March to 5 April after six SARS-CoV-2 cases were detected in the village's population. The SARS-CoV-2 outbreak comprised 51 cases and 3 deaths. The CoNAN study was performed from 13 May to 22 May 2020, 6 weeks after a SARS-CoV-2 outbreak. RESULTS: We enrolled a total of 626 participants (71% of the community population) for PCR and antibody testing in the study. All actual SARS-CoV-2 PCR tests were negative. Fifty-two out of 620 (8.4%) participants had antibodies against SARS-CoV-2 in at least two different assays. There were 38 participants with previously PCR-confirmed SARS-CoV-2 infection. Of those, only 19 (50%) displayed anti-SARS-CoV-2 antibodies. We also show that antibody-positive participants with symptoms compatible with a respiratory tract infection had significantly higher antibody levels then asymptomatic participants (EU-assay: median 2.9 versus 7.2 IgG-index, p 0.002; DS-assay: median 45.2 versus 143 AU/mL, p 0.002). Persisting viral replication was not detected. CONCLUSIONS: Our data question the relevance and reliability of IgG antibody testing to detect past SARS-CoV-2 infections 6 weeks after an outbreak. We conclude that assessing immunity for SARS-CoV-2 infection should not rely on antibody tests alone.

[Prevalence of multiresistant gram-negative bacteria in inhabitants of long-term care facilities in 2019 in Thuringia]. / Prävalenz von multiresistenten gramnegativen Erregern bei Bewohnern von stationären Pflegeeinrichtungen 2019 in Thüringen.

BACKGROUND: Multidrug-resistant gram-negative bacteria (MDRGN) pose an emerging threat in German hospitals and in the outpatient sector. However, only few studies have investigated the prevalence of MDRGN in nonhospital settings and the associated risk factors for colonization. OBJECTIVE: In our study we determined the prevalence of MDRGN in inhabitants of long-term care facilities (LTCFs) and associated risk factors for colonization in the region Weimar, Weimarer Land, and Jena. METHODS: Between May and August 2019, deep rectal swabs were taken from 307 inhabitants of 13 facilities and examined microbiologically for the presence of MDRGN. Furthermore, using a standardized questionnaire, the characteristics of the inhabitants were collected and their association with the likelihood for colonization with MDRGN was analyzed. RESULTS: MDRGN were found in 59 swabs, predominantly Escherichia coli (95%). The weighted prevalence of extended spectrum beta-lactamase-producing bacteria was 19.1% and for MDRGN with additional resistances to fluoroquinolones was 12.3%. Resistances to carbapenems or carbapenemases were not found. Multivariable as well as univariable analysis recognized the presence of chronic wounds to be a potential risk factor (OR: 2,66 [95 %-CI: 1,54-4,60]). Additionally, the univariable analysis detected the necessity of a wheelchair and the accommodation in double rooms as risk factors. DISCUSSION: The prevalence of MDRGN found in our study is similar to findings of previous German studies. The result shows the importance of strict compliance with basic hygiene guidelines for all inhabitants of LTCFs for the prevention of transmission of MDRGN.

Analysis of an echovirus 18 outbreak in Thuringia, Germany: insights into the molecular epidemiology and evolution of several enterovirus species B members.

In October and November 2010, six children and one woman were presented with symptoms of aseptic meningitis in Jena, Thuringia, Germany. Enterovirus RNA was detected in the cerebrospinal fluid of all patients by RT-PCR, and preliminary molecular typing revealed echovirus 18 (E-18) as causative agent. Virus isolates were obtained from stool samples of three patients and several contact persons. Again, most isolates were typed as E-18. In addition, coxsackievirus B5 (CV-B5) and echovirus 25 (E-25) were found to co-circulate. As only few complete E-18 sequences are available in GenBank, the entire genomes of these isolates were determined using direct RNA-sequencing technology. We did not find evidence for recombination between E-18, E-25 or CV-B5 during the outbreak. Viral protein 1 gene sequences and the cognate 3D polymerase gene sequences of each isolate and GenBank sequences were analysed in order to define type-specific recombination groups (recogroups).

[Case report: Analysis of the critical time delays from the fever increase to the first antimycotic dose during the diagnosis of candidemia]. / Fall-Bericht: Analyse der kritischen Zeitverzögerungen vom Fieberanstieg bis zur ersten Antimykotika-Gabe bei der Diagnostik einer Candidämie.

In a secondary care hospital in Germany with an in-house microbiology laboratory, an 84-year-old female patient with candidemia was selected for determination of the time periods required for each diagnostic step preceding the application of the first dose of antimycotic treatment. The total time from the beginning of fever to the first dose of fluconazole was 25 hours and 10 minutes. The largest delay occurred between the positive signal of the blood culture bottle during the night and the start of the work-up of the positive blood culture bottle in the morning. Improvements appear to be possible in both clinical and laboratory settings by optimizing pre-analytical, analytical, and post-analytical procedures.

Evaluation of Mueller-Hinton-agar as a simple medium for the germ tube production of Candida albicans and Candida dubliniensis.

Candida albicans is the most frequently isolated yeast species from clinical specimens. A classical rapid presumptive differentiation from non-albicans species is based on its ability to produce germ tubes after incubation in human serum. The only non-albicans Candida species producing germ tubes is Candida dubliniensis. In this study, we evaluated Mueller-Hinton-agar (MH-agar) as a medium for germ tube formation of C. albicans and C. dubliniensis. A total of 859 yeast isolates from stool samples, including 632 strains of C. albicans, 10 C. dubliniensis and 217 other yeast strains from 20 different species, were grown on Sabouraud glucose (2%) agar at 37 degrees C for 24-72 h. Species were identified by standard methods. For the germ tube test (GTT), an inoculum from a single colony was streaked onto a MH-agar plate and covered by a sterile coverslip. After incubation at 37 degrees C for 2 h, the MH plates were examined using a light microscope at x200. The GTT was positive in 578 of 632 C. albicans strains (sensitivity 91.5%), in six of 10 C. dubliniensis strains (sensitivity 60.0%), and in none of the other yeast strains. MH-agar is a suitable medium for the GTT and the presumptive identification of C. albicans. It is safer to use than human serum and is widely available in microbiology laboratories.

[Impact of antifungal prophylaxis on the gastrointestinal yeast colonisation in patients with haematological malignancies]. / Einfluss einer Antimykotika-Prophylaxe auf die gastrointestinale Besiedlung mit Sprosspilzen bei hämatologischen Patienten.

Patients with haematological malignancies are at high risk for developing invasive Candida infections. They are often colonised with Candida spp. in the gastrointestinal (GI) tract. In order to prevent infection, the prophylactic use of antifungal agents has been established. The widespread use of fluconazole may lead to the emergence of resistant Candida isolates. We studied the yeast colonisation of the GI tract in patients with haematological malignancies receiving antifungal prophylaxis (AP) in comparison with healthy controls. The study cohort included 46 neutropenic patients with 52 stool samples under 52 episodes of AP and 110 healthy controls. The patients received amphotericin B orally (n = 8), amphotericin B and fluconazole (n = 7), amphotericin B and itraconazole (n = 5), fluconazole orally (n = 15) and itraconazole orally (n = 17). Yeasts were cultured from the stool samples of 63.5% of the patients and 60% of the controls with a mean yeast load of 1.6 x 10(3) and 0.4 x 10(3) cfu g(-1), respectively (P = 0.045). Patients and controls had a low faecal yeast load of 10(3) to 10(4) cfu g(-1) in 19.3% and 37.3%, respectively (P = 0.021), and yeast overgrowth of >10(5) cfu g(-1) in 28.9% and 10.9%, respectively (P = 0.004). The rate of Candida albicans was 32.6% and 54.1% in the patients and controls, respectively (P = 0.021). The rates of fluconazole-resistant yeast species were higher in the patient group than in the control group: C. glabrata 20.9% vs. 11.7% (P = 0.168), C. krusei 25.6% vs. 4.7% (P = 0.001). Not a single patient under AP suffered from proven or probable invasive candidosis. In conclusion, oral AP in haematological patients resulted in a higher colonisation rate with fluconazole-resistant Candida species but efficiently prevented invasive candidosis.

Effect of nasal antifungal therapy on nasal cell activation markers in chronic rhinosinusitis.

OBJECTIVE: To examine the effect of nasal antifungal treatment on eosinophil cationic protein (ECP) and tryptase levels in samples of nasal lavage fluid from patients with chronic rhinosinusitis and nasal polyps. DESIGN: Prospective double-blind placebo-controlled clinical trial. SETTING: Tertiary surgical center. PATIENTS: Subjects with severe chronic rhinosinusitis and nasal polyps. Of 120 screened patients, 76 were eligible. Six patients withdrew because of minor adverse events, and 10 dropped out for other reasons. In total, 60 patients completed the study according to the study protocol. INTERVENTIONS: Nasal treatment with amphotericin B or saline control for 8 weeks. MAIN OUTCOME MEASURES: Nasal lavages were performed before and after treatment. Fungal elements were assessed by culture and with different polymerase chain reaction assays. Levels of ECP and tryptase were determined by fluorescent enzyme immunoassay. RESULTS: No correlation between cell activation markers and fungus detection was observed before treatment (all P>.20). Nasal amphotericin B treatment had no effect on levels of ECP (P = .17) or tryptase (P = .09) in nasal lavage samples. Moreover, successful fungus eradication, defined as fungus detection before but not after treatment, did not influence nasal ECP or tryptase levels (all P>.40). CONCLUSION: Neither topical amphotericin B therapy nor fungal state before and after treatment had any significant influence on activation markers of nasal inflammatory cells in chronic rhinosinusitis.

Intrinsic in vitro susceptibility of primary clinical isolates of Aspergillus fumigatus, Aspergillus terreus, Aspergillus nidulans, Candida albicans and Candida lusitaniae against amphotericin B.

A total of 60 clinical fungal isolates from patients without prior amphotericin B treatment and three control strains were evaluated for their intrinsic susceptibility to amphotericin B (AmB) using microdilution, Etest and disc diffusion assays, on three media each, Roswell Park Memorial Institute (RPMI) 1640, Antibiotic Medium 3 (AM3) and High Resolution Medium. The fungal strains included isolates of Aspergillus fumigatus (n = 10), Aspergillus terreus (n = 12), Aspergillus nidulans (n = 9), Candida albicans (n = 6) and Candida lusitaniae (n = 23). The A. terreus strains were significantly less susceptible to AmB than the A. fumigatus strains in all nine experimental settings (P-values ranging from 0.009 to <0.00001). The A. nidulans strains were equally susceptible to AmB as the A. fumigatus strains in seven of nine experimental settings and less susceptible in two (microdilution performed on RPMI and AM3, P = 0.01 and 0.007). The C. lusitaniae strains were equally susceptible to AmB as the C. albicans strains in seven of nine experimental settings and more susceptible in two (microdilution and Etest, both performed on AM3, P = 0.01 and 0.0002). Thus, we confirmed that A. terreus is intrinsically less susceptible to AmB than A. fumigatus. In contrast, nine German clinical isolates of Aspergillus nidulans were found equally susceptible to AmB as 10 isolates of A. fumigatus. Furthermore, we found 23 German clinical isolates of C. lusitaniae from patients without prior treatment with AmB equally susceptible to AmB as C. albicans.

In vitro susceptibility of 15 strains of zygomycetes to nine antifungal agents as determined by the NCCLS M38-A microdilution method.

The in vitro susceptibility of 15 strains of six genera of zygomycetes including Rhizopus oryzae (Rhizopus arrhizus), Rhizopus stolonifer, Mucor circinelloides (three), Absidia corymbifera (three), Rhizomucor pusillus (three), Cunninghamella bertholletiae (two), and Syncephalastrum racemosum (two) to nine antifungal agents were determined by the NCCLS M38-A broth microdilution method. Geometric means of the minimal inhibitory concentrations (MIC) were: amphotericin B 0.07 mg l(-1) (range 0.03-0.5 mg l(-1)), nystatin 0.83 mg l(-1) (range 0.25-4 mg l(-1)), itraconazole 0.59 mg l(-1) (range 0.03 to >8 mg l(-1)), voriconazole 6.50 mg l(-1) (range 2 to >8 mg l(-1)), ciclopiroxolamine 1.59 mg l(-1) (range 0.5-4 mg l(-1)), and amorolfine 9.19 mg l(-1) (range 1 to >16 mg l(-1)). All strains were resistant to 5-flucytosine, fluconazole (MIC >64 mg l(-1)) and caspofungin (MIC >16 mg l(-1)).

Fungus culture and PCR in nasal lavage samples of patients with chronic rhinosinusitis.

Chronic rhinosinusitis (CRS) affects approximately 15 % of the adult population in industrialized countries. Fungi have been recognized as important pathogens in CRS in the immunocompromised host. Recently, fungi have been detected in more than 90 % of nasal lavages (NLs) in immunocompetent patients with CRS. Employing NLs of immunocompetent patients with CRS in the present study, the detection rates for fungi by culture techniques were compared with the results of different fungus-specific PCR assays. Standard fungal cultures were performed on NLs from 77 patients with CRS. NLs were also tested for the presence of fungal DNA by a panfungal assay with and without specific probes for Candida spp. and Aspergillus spp./Penicillium spp., and an Aspergillus-specific nested PCR assay. Nineteen of the 77 samples (25 %) grew fungi. Fungus-specific DNA was detected in 34 of 77 NLs (44 %). Twelve samples were positive for both culture and panfungal PCR, whereas seven specimens grew fungi in culture, but were negative in panfungal PCR, and an additional seven samples were positive in panfungal PCR, but negative in culture. The combination of culture and all employed PCR assays detected fungi in 39 patients (50 %). This study demonstrated that PCR and conventional culture techniques could be complementary diagnostic techniques to detect fungi in nasal specimens from CRS patients.

Topical antifungal treatment of chronic rhinosinusitis with nasal polyps: a randomized, double-blind clinical trial.

BACKGROUND: Recently, fungal elements were suspected to be the causative agent of chronic rhinosinusitis, and benefits of topical amphotericin B therapy have been reported. OBJECTIVE: The effects of amphotericin B versus control nasal spray on chronic rhinosinusitis were compared in a double-blind, randomized clinical trial. METHODS: Patients with chronic rhinosinusitis were administered 200 microL per nostril amphotericin B (3 mg/mL) or saline nasal spray 4 times daily over a period of 8 weeks. The response rate, defined as a 50% reduction of pretreatment computed tomography score, was the primary outcome variable. Additional outcome variables included a symptom score, a quality of life score, and an endoscopy score. Before and after treatment, nasal lavages were pretreated with dithiothreitol and examined for fungal elements by PCR and standard culture techniques. RESULTS: Seventy-eight patients were included, and 60 patients finished the study per protocol. In the control group, no positive response (0 of 32) was observed, and 2 of 28 patients responded in the amphotericin B group (P>.2). The symptom scores were distinctly worse after amphotericin B therapy (P <.005). The other parameters investigated did not differ remarkably between the treatment groups. CONCLUSION: Nasal amphotericin B spray in the described dosing and time schedule is ineffective and deteriorates patient symptoms.

First report of a case of meningitis caused by Cryptococcus adeliensis in a patient with acute myeloid leukemia.

Cryptococcus adeliensis is a recently described new fungal species which has been isolated from decaying algae in Terre Adelie, Antarctica. We report the first known case of meningitis caused by C. adeliensis in a patient with acute myeloid leukemia undergoing allogeneic peripheral blood stem cell transplantation.

Cross-reactivity of the PLATELIA CANDIDA antigen detection enzyme immunoassay with fungal antigen extracts.

We studied the specificity of the PLATELIA CANDIDA Ag enzyme immunoassay by using 130 isolates of 63 clinically relevant fungal species. Antigen extracts of seven Candida spp. (Candida albicans, C. dubliniensis, C. famata, C. glabrata, C. guilliermondii, C. lusitaniae, and C. tropicalis) repeatedly yielded positive reactions (>0.5 ng/ml). Geotrichum candidum and Fusarium verticillioides were found to yield borderline-positive reactions (0.25 to 0.50 ng/ml). Antigen preparations from the other 54 fungal species, including yeasts, molds, dermatophytes, and dimorphic fungi, did not cross-react in the assay.

Fungal diseases.

In this chapter, we present concise reviews on the clinical manifestations of the complete set of human fungal infections known today. Emphasis is given to the clinical symptoms. The classification corresponds to the body sites affected, from systemic to superficial. Within the groups, the order of presentation follows the order of prevalence and overall importance of the fungal infections. Short paragraphs on the ecology of the causative fungi and the epidemiology of the corresponding diseases, including the mode of aquisition, the susceptible population, and the geographical distribution, precede each clinical entity.

Diagnosis of fungal diseases.

In this chapter, we focus on diagnostic laboratory methods that are necessary and suitable for providing physicians with a timely and accurate diagnosis of fungal diseases. After discussing some pre-analytical aspects, the complete set of methods, i.e., microscopy, histopathology, culture, antigen detection, DNA detection, and antibody detection, is concisely described. Identification techniques depend on the fungal group involved. Therefore, separate paragraphs are dedicated to the identification of yeasts and filamentous fungi, which include moulds, dermatophytes, and dimorphic fungi.

Performance of an IS6110-based PCR assay and the COBAS AMPLICOR MTB PCR system for detection of Mycobacterium tuberculosis complex DNA in human lymph node samples.

We compared the performance of two PCR assays, an IS6110-based in-house protocol and the COBAS AMPLICOR MTB PCR (COBAS MTB) system, for the detection of Mycobacterium tuberculosis complex in 43 human lymph node samples from 40 patients. For the in-house PCR and the COBAS MTB assays, respectively, sensitivities were 87.5% versus 45.5% (P < 0.05), specificities were 100.0% versus 91.3% (P > 0.05), and inhibition rates were 4.8% versus 19.5% (P < 0.05). For the COBAS MTB system, additional N-acetyl-L-cysteine-NaOH pretreatment of the samples changed neither the inhibition rate nor the sensitivity significantly.

[Invasive aspergillosis: results of an 8-year study]. / Invasive Aspergillosen: Ergebnisse einer 8-Jahres-Studie.

We analysed retrospectively 90 cases of invasive aspergillosis (IA) which occurred at the University Hospital and the Thoraxklinik gGmbH Heidelberg between 1991 and 1998. 71 cases were histologically proven, 19 were probable diseases. There were 49 male and 41 female patients, with a mean age of 51.5 years (range 16 days to 80 years). Underlying diseases were: hematological malignancies in 52% (n = 47; 24 with acute leukemia), solid organ transplantation (n = 11; 9 liver, 1 kidney, 1 heart), solid cancer (n = 10), others (n = 21), and in one case no underlying disease was diagnosed. Only 54 cases (60%) were correctly diagnosed as IA during lifetime of the patients. In 59 cases (65%) only the lung was affected, 25 patients suffered from disseminated IA, in 6 patients only extrapulmonary lesions were present. 11 patients underwent lung surgery, 63 patients received antimycotic drugs (44 amphotericin B, 15 fluconazole, 4 itraconazole), 21 were not treated antimycotically. 68 patients (71%) died, from these 30 (36%) due to IA during remission of the underlying disease. The laboratory methods showed the following sensitivities, respectively: microscopy by calcofluor white staining 17%, culture 69%, Aspergillus-PCR from respiratory tract samples and biopsies 95%, galactomannan antigen detection by latex agglutination 28%, by enzyme immunoassay 59%, Aspergillus antibody detection 23%.

Disseminated Penicillium marneffei infection in an HIV-positive female from Thailand in Germany.

We report the case of a 33 year old Thai female, who was married in Germany for eight years and used to travel to Thailand every year for several weeks. She presented with abdominal and back pain, prolonged fever, generalized lymphadenopathy, and a recent history of oral thrush. She was diagnosed HIV positive with initial CD4 counts of 18/µl and an HI virus load of 59.000 copies/ml. Antiviral therapy was installed with zidovudin, lamivudin, and efavirenz. Abdominal CT scans revealed greatly enlarged abdominal lymphnodes. Fine needle aspirates of cervical and retroperitoneal lymphnodes, sputum samples, blood samples, and a bone marrow biopsy were microscopically positive for Penicillium marneffei and grew P. marneffei. The isolates were sensitive to amphotericin B, flucytosine, itraconazole, and fluconazole. Both universal and specific fungal polymerase chain reaction assays were positive in various samples. Serum Aspergillus galactomannan antigen, which is known to crossreact with P. marneffei, was elevated and subsequently used for monitoring of therapy. With antifungal treatment (intravenous amphotericin B 0.6 mg/kg/d for two weeks, oral itraconazole 400 mg/d for 10 weeks and 200 mg/d as maintenance therapy), the fever declined in 6 days, the size of the enlarged lymphnodes gradually decreased in the CT scans, and the initial abdominal and back pain vanished.