Thomas Holzmann

Rapid detection and molecular differentiation of toxigenic Corynebacterium diphtheriae and Corynebacterium ulcerans strains by LightCycler PCR.

ABSTRACT The systemic symptoms of diphtheria are caused by the tox-encoded diphtheria toxin (DT) which is produced by toxigenic Corynebacterium spp. Besides the classical agent C. diphtheriae, the zoonotic pathogen C. ulcerans has increasingly been reported as an emerging pathogen for diphtheria. The reliable detection of toxigenic Corynebacterium spp. is of substantial importance for both diphtheria surveillance in the public health sector and the clinical workup of a patient with diphtherialike symptoms. Since the respective tox genes of C. diphtheriae and C. ulcerans differ from each other in both DNA and amino acid sequence, both tox genes should be covered by novel real-time PCR methods. We describe the development and validation of a LightCycler PCR assay which reliably recognizes tox genes from both C. diphtheriae and C. ulcerans and differentiates the respective target genes by fluorescence resonance energy transfer (FRET) hybridization probe melting curve analysis.

Cerebellar cysticercosis caused by larval Taenia crassiceps tapeworm in immunocompetent woman, Germany.

Human cysticercosis caused by Taenia crassiceps tapeworm larvae involves the muscles and subcutis mostly in immunocompromised patients and the eye in immunocompetent persons. We report a successfully treated cerebellar infection in an immunocompetent woman. We developed serologic tests, and the parasite was identified by histologic examination and 12s rDNA PCR and sequencing.

Injectional anthrax in heroin users, Europe, 2000-2012.

To the Editor: Anthrax is a global zoonotic disease, but human infections are rare in countries of Western Europe. During 2009–2010, a total of 119 (47 laboratory-confirmed) drug-abuse–related cases of anthrax were reported in the United Kingdom and Germany (1). In these patients, the disease had an unusual manifestation. In contrast to acquiring the disease through typical routes of infection, leading to cutaneous, inhalation, or gastrointestinal anthrax, these patients became infected by injecting heroin (1–3). The term injectional anthrax was then coined to describe this new mode of infection. Patients with injectional anthrax show severe symptoms, and death rates are high. Of the 47 patients with confirmed cases of injectional anthrax acquired during the 2009–2010 outbreak, 19 died from the disease (1). However, this outbreak was not the first report of death caused by B. anthracis in an injectional drug (heroin) user; the disease was described in 1 person who died in Norway in 2000 (3). Attempts to directly connect the United Kingdom cases to batches of anthrax-contaminated heroin were unsuccessful. No viable B. anthracis or DNA could be retrieved from the investigation’s drug samples. However, PCR- or culture-positive samples were obtained from some patients and used for genotyping (2). Later, researchers from Arizona and the United Kingdom worked together to use a high-resolution molecular approach to genotype the 2009–2010 outbreak strains. This information was used to obtain insight into the epidemiology and likely geographic origin of the European outbreak strains. All patients were infected by a single B. anthracis strain type (2,4) that belonged to the large Trans-Eurasian clade of B. anthracis. The whole genome of a representative strain of this type, Ba4599, had previously been sequenced (2). Strains related to strains associated with those isolated from European drug users, which belong to the A.Br. 008/011 canSNP cluster but are still genetically distinct, have so far only been identified from Turkey (2). However, more isolates from other relevant regions need to be investigated to confirm the likely geographic source. In June 2012, after a 20-month gap, 2 new cases of injectional anthrax in heroin consumers were reported in Bavaria (5,6). Additional cases have been reported since then from Germany, Denmark, the United Kingdom, and France, leading to 26 deaths as of August 2013 (7). Molecular phylogenetic methods were used to determine the genetic relatedness of these strains with Ba4599 (8,9). Genotyping results using canonical single nucleotide polymorphisms (SNPs) (Figure) (2,4) placed all of these strains along branch A.Br.008 within the Trans-Eurasian group of B. anthracis (10). Further hierarchical fine-scale typing of the isolates by interrogating SNPs that were discovered from the heroin-associated strain Ba4599 (2) indicated that all isolates are identical at these SNPs (Figure) (7). The initial strain isolated in Norway in 2000 also falls into this group (7). In addition, analysis by multiple locus variable number tandem repeats suggested that all investigated strains are closely related, differing at just 2 markers (7). Thus, we conclude that all injectional anthrax isolates likely came from the same source. Figure Diagram of single nucleotide polymorphism (SNP) assays used for bioforensic genotyping of heroin-associated Bacillus anthracis strains. Shown are the results of PCR-based SNP assays performed to elucidate the phylogenetic position of strains. Indicated ... This reemergence of drug-related anthrax in Europe strengthens the view that heroin may provide a continuing route of entry for B. anthracis into Western Europe (2). Ideally, this unfortunate deadly incident could offer an opportunity to sensitize heroin users to the risks for severe infection and to educate public health officials to be vigilant for this rare disease. This study also shows the power of molecular genotyping approaches for trace-back analysis of infectious disease agents.

Contact-free inactivation of Trichophyton rubrum and Microsporum canis by cold atmospheric plasma treatment

AIM Cold atmospheric plasma (CAP) has already proven efficient at disinfection of microorganisms including biofilms. The objective of the present study is to assess the efficacy of CAP against the dermatophytes Trichophyton rubrum and Microsporum canis in vitro. MATERIALS & METHODS T. rubrum and M. canis were exposed to CAP for different treatment times and time intervals in vitro. Treatment with ciclopirox olamine or UVC radiation (0.120 J/cm(2)) served as controls. CAP was generated by the surface microdischarge technology. Fungal colony growth was measured upon CAP treatment. RESULTS Repeated daily CAP treatments of 10 min demonstrated an inhibition of growth during the treatment period of 9 days. Single CAP treatment sessions for 5, 8 and 10 min, as well as treatments for 5 or 8 min daily, resulted in less fungal growth inhibition. UVC radiation treatment failed, but not ciclopirox olamine. CONCLUSION CAP shows promising potential for future application in the treatment of dermatophyte infections.

Injection Anthrax—a New Outbreak in Heroin Users

BACKGROUND Injection anthrax is a rare disease that affects heroin users and is caused by Bacillus anthracis. In 2012, there were four cases in Germany, one of which was fatal, as well as a small number of cases in other European countries, including Denmark, France, and the United Kingdom. Three cases among drug users occurred in Germany in 2009/2010, in the setting of a larger outbreak centered on Scotland, where there were 119 cases. CASE PRESENTATION AND CLINICAL COURSE: We present three cases of injection anthrax, two of which were treated in Regensburg and one in Berlin. One patient died of multi-organ-system failure on the day of admission to the hospital. The others were treated with antibiotics, one of them also with surgical wound debridement. The laboratory diagnosis of injection anthrax is based on the demonstration of the pathogen, generally by culture and/or by polymerase chain reaction, in material removed directly from the patients wound. The diagnosis is additionally supported by the detection of specific antibodies. CONCLUSION Injection anthrax may be viewed either as an independent disease entity or as a special type of cutaneous anthrax with massive edema, necrotizing fasciitis in many cases, and about 30% mortality. It has appeared in recent years among heroin users in various European countries. In patients with suggestive clinical presentation and a history of heroin use, anthrax infection must be suspected early, so that the appropriate diagnostic tests can be performed without delay. Timely treatment can be life-saving. It is therefore important that physicians--and the individuals at risk--should be well-informed about this disease.

Draft Genome Sequence of Bacillus anthracis UR-1, Isolated from a German Heroin User

We report the draft genome sequence of Bacillus anthracis UR-1, isolated from a fatal case of injectional anthrax in a German heroin user. Analysis of the genome sequence of strain UR-1 may aid in describing phylogenetic relationships between virulent heroin-associated isolates of B. anthracis isolated in the United Kingdom, Germany, and other European countries.

UPLC-MS/MS Method for Therapeutic Drug Monitoring of Ten Antibiotics used in Intensive Care Units

A large variation in the levels of different ß-lactams and other antibiotics used in critically ill patients has been documented. The aim of this study is to establish and validate a fast, ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous analysis of ten antibiotics (Meropenem, Cefepime, Ceftazidime, Piperacillin, Benzylpenicillin, Ampicillin, Flucloxacillin, Linezolid, and Sulfamethoxazole/Trimethoprim) in human plasma according to European Medicines Agency (EMA) guidelines. Protein precipitation with ice-cold methanol containing 9 isotopically labeled internal standards was used for sample clean up. Antibiotics were detected, following a 4-minute gradient separation, in multiple reactions monitoring (MRM) using API 4000 instrument equipped with electrospray source operating in positive ion mode. The lower limit of quantification was 0.1 mg/L for Meropenem, Ceftazidime, Piperacillin, Ampicillin, Flucloxacillin, and Sulfamethoxazole; 0.05 mg/L for Cefepime, Benzylpenicillin, and Trimethoprim; and 0.02 mg/L for Linezolid. The method proved to be precise and accurate and applicable for therapeutic drug monitoring and other pharmacokinetic studies.

Screening of ESBL-producing Enterobacteriacae concomitant with low degree of transmission in intensive care and bone marrow transplant units

Abstract Background: Extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E) are spreading worldwide in both hospital and community settings. In this study, the molecular epidemiology and the transmission modalities of ESBL-E in intensive care- and bone marrow transplant were investigated. Methods: All patients included in this study were screened for presence of ESBL-E on admission and weekly. Relevant β-lactamase genes were identified by PCR and sequencing. Results: A total of 669 patients were included in this study. On admission, ESBL-producing Escherichia coli were detected in 49 (7.3%) patients and ESBL-producing Klebsiella pneumoniae in one patient. The most common ESBL types among E. coli isolates were CTX-M-15 (38.8%) and CTX-M-1 (38.8%). Furthermore, 12 of 49 (24.5%) ESBL-producing E. coli could be assigned to the epidemic clone ST131. A single patient acquired ESBL-producing E. coli during the hospital stay but cross-transmission could not be demonstrated. Among 1095 environmental samples none revealed ESBL. Conclusions: Our results suggest that early detection of ESBL-producing Enterobacteriaceae and consequent implementation of basic hygiene measures and contact isolation may reduce the transmission rate during the hospital stay.

Injektionsmilzbrand - neu aufgetretene Fälle bei Heroinabhängigen

BACKGROUND Injection anthrax is a rare disease that affects heroin users and is caused by Bacillus anthracis. In 2012, there were four cases in Germany, one of which was fatal, as well as a small number of cases in other European countries, including Denmark, France, and the United Kingdom. Three cases among drug users occurred in Germany in 2009/2010, in the setting of a larger outbreak centered on Scotland, where there were 119 cases. CASE PRESENTATION AND CLINICAL COURSE: We present three cases of injection anthrax, two of which were treated in Regensburg and one in Berlin. One patient died of multi-organ-system failure on the day of admission to the hospital. The others were treated with antibiotics, one of them also with surgical wound debridement. The laboratory diagnosis of injection anthrax is based on the demonstration of the pathogen, generally by culture and/or by polymerase chain reaction, in material removed directly from the patients wound. The diagnosis is additionally supported by the detection of specific antibodies. CONCLUSION Injection anthrax may be viewed either as an independent disease entity or as a special type of cutaneous anthrax with massive edema, necrotizing fasciitis in many cases, and about 30% mortality. It has appeared in recent years among heroin users in various European countries. In patients with suggestive clinical presentation and a history of heroin use, anthrax infection must be suspected early, so that the appropriate diagnostic tests can be performed without delay. Timely treatment can be life-saving. It is therefore important that physicians--and the individuals at risk--should be well-informed about this disease.

Diphtheria Outbreak in Amerindian Communities, Wonken, Venezuela, 2016–2017

In February 2017, a diphtheria outbreak occurred among Amerindians of the Pemón ethnic group in Wonken, Venezuela. A field investigation revealed ≈10 cases; clinical presentation did not include cutaneous or neurologic signs or symptoms. To prevent future outbreaks in Venezuela, Amerindian communities need better access to vaccination and healthcare.