28 June 2012
Fatal anthrax infection in a heroin user from southern Germany, June 2012
Blood cultures from a heroin user who died in June 2012, a few hours after hospital admission, due to acute septic disease, revealed the presence of Bacillus anthracis. This report describes the extended diagnosis by MALDI-TOF and real-time PCR and rapid confirmation of the anthrax infection through reference laboratories. Physicians and diagnostic laboratories were informed and alerted efficiently through the reporting channels of German public health institutions, which is essential for the prevention of further cases.
In early June 2012, a case of anthrax infection was identified in an injecting drug user in Germany. Anthrax wasn’t suspected initially and the patient died on the day of hospital admission. Two days later anthrax was confirmed and the relevant authorities were informed. This report underlines the importance of considering anthrax as a possible diagnosis in injecting heroin users presenting with fever or sepsis at emergency rooms and of the rapid management of such cases.
Clinical case description
In early June 2012 an injecting drug user in their 50s presented at the emergency department of a hospital in the south of Germany, with a two-day history of worsening swelling and reddening at an injection site, nausea and dyspnoea. The patient had been on oral substitution therapy for two years. Moreover, a history of chronic hepatitis C infection with liver cirrhosis was reported. In the next hours after admission to hospital, the patient developed respiratory failure and was transferred to the intensive care unit (ICU) where they were ventilated mechanically. An elevated white blood cell count (15.9 cells/nL), anaemia (haemoglobin 10.4 g/dL), thrombocytopenia (38 cells/nL), elevated procalcitonin (1.05 ng/mL) and hypokalaemia (2.5 mmol/L) were observed. Elevated liver enzymes, lowered coagulation parameters and extremely high levels of D-dimers (>36,364 ng/mL) were pointing to multi-organ failure. Blood and urine cultures were sent to the Institute of Medical Microbiology and Hygiene, University of Regensburg. The patient’s condition worsened and they died on the day of admission due to a septic shock with multi-organ failure and massive disseminated bleeding. At the time, there was no clinical suspicion of anthrax.
Blood cultures (Becton Dickinson, Heidelberg, Germany) turned positive after 53 minutes of incubation. Gram-stained microscopy showed non-branching Gram-positive bacilli growing in chains. Subcultures presented typical growth of aerobic spore-forming bacilli without haemolysis. Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) identification revealed Bacillus cereus (Bruker Daltonics, Bremen, Germany). The patient’s history led to reanalysis with the Bruker ’SR Database’ that contains so-called security-relevant organisms, which correctly identified B. anthracis.
An initial set of molecular diagnostic tests was performed for confirmation at the Institute of Medical Microbiology and Hygiene, University of Regensburg. Briefly, a loopful of cells was suspended in 500 µL of detergent buffer. The buffer consisted of TE (pH 7.5) containing 0.5% Triton X-100 and 0.25% Tween 20. The suspension was heated at 95°C for 30 minutes with occasional shaking, sonicated for 1 minute, and heated again at 95°C for 30 minutes. After 10 minutes centrifugation at 11,000 × g, the supernatant was passed through a 0.2 µm sterile filtration membrane. Extracting genomic DNA from a boiled bacterial culture proved to be reproducible, easy-to-perform and rapid before . The DNA was directly used as template for a series of real-time PCR assays performed with the Light Cycler PCR system (Roche Diagnostics, Mannheim, Germany). The real-time assays included an in-house protocol for pan-bacterial 16S rDNA amplification and the LightMix kit B. anthracis (Cat. No: 40-0252-16, TIB Molbiol, Berlin, Germany). The kit is designed to detect the pagA gene as marker for the plasmid pXO1 and a B. anthracis-specific segment of the bacterial rpoB gene for species identification, using hybridisation probes. Early crossing points (around cycle 18) and the specific melting points of the respective target genes pagA and rpoB were observed in the melting curve analysis, indicating the presence of B. anthracis carrying at least the virulence plasmid pXO1 (Figure).
Figure. Real-time PCR amplification plots (A) and melting curve analysis (B), anthrax infection, Germany, June 2012
Box 1. Timeline of events, fatal case of anthrax infection, Germany, June 2012
To substantiate the initial test results, an aliquot of the DNA preparation was sent to the Bundeswehr Institute of Microbiology in Munich. The initial PCR results were confirmed and extended using PCR assays for the capC gene (marker for the second virulence plasmid pXO2) and an additional chromosomal marker highly specific for B. anthracis (dhp61) . First results of molecular genotyping of the strain showed close relationship to strains from a large anthrax outbreak among IDUs in Scotland .
The District Health Office was informed about the suspected case of human B. anthracis infection immediately after obtaining the PCR results. Their experts got involved in the management of the case in close contact with the diagnostic institutions, the police authorities and the Task Force Infectiology of the Bavarian Health and Food Safety Authority (LGL). Health officials considered contaminated heroin or cutting agents mixed with the heroin as possible source of the infection. Further investigations by the German police authorities were initiated immediately.
The competent public health authorities at national level were informed immediately about the confirmation of B. anthracis. The information on the occurrence of the case was distributed to the public health authorities in all 16 German federal states, at international level through the Early Warning and Response System (EWRS) of the European Commission and via ProMED-mail and according to the International Health Regulations (IHR). In Bavaria, the medical associations were informed. Substance abuse counselling agencies were contacted nationally and at European level through the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) in order to spread the information among drug users. Additional information and materials were published by the public health institutes on their websites.
Identification of the second case
Two weeks after the first case was admitted to hospital, a second case of anthrax was identified in an IDU from the same region as the first case. The patient is stable under antibiotic therapy after surgical debridement . The raised level of awareness created with the first case lead to a much faster workflow in the laboratory analysis in the second case. B. anthracis was confirmed three hours after blood cultures turned positive.
Injectional anthrax has first been reported 1988 as fourth route of infection besides cutaneous, gastrointestinal and inhalational anthrax infections . The first anthrax case related to injecting drug use was described 2000 from Norway . There were no subsequent reports of injectional anthrax until 10 December 2009 when anthrax was identified in blood cultures from two injecting drug users from Glasgow, Scotland . In the following months an increasing number of cases were identified . By the end of the outbreak in December 2010, there were 47 confirmed cases of injectional anthrax (including 13 deaths), 35 probable cases (including one death) and 37 possible cases in Scotland and five cases including four deaths in England . There were two confirmed cases in Germany related to this outbreak, including one fatal case . The favoured outbreak hypothesis assumed that heroin had been in contact with goat skin contaminated with anthrax spores during transportation to Scotland . Risk factors for infection were longer injection history, receiving opioid substitution therapy, and alcohol consumption . All cases of injectional anthrax reported so far including the case presented here were not associated with the typical black escharseen in patients with cutaneous anthrax .
Because B. anthracis is seen very rarely in Germany and other developed countries, laboratory staff and clinicians should raise their attention when Gram-positive bacilli growing in chains are detected in clinical specimens (Box 2).
Box 2. Recommendations and lessons learnt from the fatal case of anthrax infection, Germany, June 2012
B. anthracis cannot be reliably distinguished from B. cereus by growth characteristics, bacterial cell morphology or biochemical methods. The applicability of MALDI-TOF-MS for the identification of B. anthracis was demonstrated elsewhere . Because of safety regulations, B. anthracis and other potential bioterroristic agents are not included in the manufacturer’s (Bruker Daltonics) database. As in our case, the isolate is classified as B. cereus with the standard databases. Using a special database, containing the missing spectra, B. anthracis is identified correctly. The manufacturer discourages the standard use of the B. anthracis spectra due to misidentification of members of the B. cereus group. Consequently, the result ’B. cereus’ in combination with a patient’s history of injecting drug use should lead to further diagnostic steps. To differentiate between B. anthracis and non-anthracis Bacillus species harbouring anthrax-specific virulence plasmids, PCR targeting a chromosomal marker should be performed in addition to PCR assays covering the virulence plasmids pXO1 and pXO2. Non-pathogenic B. anthracis strains not containing plasmids can be identified using this combination as well [2, 14].
Health professionals and diagnostic laboratories should consider anthrax as a possible diagnosis in injecting heroin users presenting with fever or sepsis at the emergency room. The observed re-emergence of drug-related anthrax in Germany supports the hypothesis that heroin may provide a continuing entry route of B. anthracis into western Europe.
We would like to thank the clinical colleagues at the collaborating Hospital Barmherzige Brüder, Regensburg, for providing further details of the case, H. Körber from the GA Regensburg for coordinating anamnestic investigations and reporting activities on district/local level, Silvia Förster, for her excellent technical assistance in performing the various PCR assays, as well as Jürgen Wenzel for his helpful comments on the manuscript. Moreover, we would like to thank Olfert Landt from TIB Molbiol, Berlin, for his continuous support and for providing specialized primer and hybridization probe oligonucleotides at very short notice for supplementary testing of the bacterial strain.
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