Roland Grunow

Tularaemia in Europe: An epidemiological overview

Tularaemia exists endemically in most European countries. In some areas, such as Finland and Sweden, outbreaks comprising hundreds of cases are recorded at least once a decade. In other areas, outbreaks of such a magnitude occur only occasionally, except in times of war. Between outbreaks, the natural reservoir of the causative agent, Francisella tularensis, is unknown. The organism replicates intracellularly in protozoans. An association of tularaemia to natural water may be of significance in locating the reservoir. Epidemiological work has to date been slow, but is now facilitated by the development of new molecular methods. Due to a variation in numbers of short sequence-tandem repeats in the bacterial genome, individual strains of F. tularensis can today be distinguished.

Detection of Francisella tularensis in Biological Specimens Using a Capture Enzyme-Linked Immunosorbent Assay, an Immunochromatographic Handheld Assay, and a PCR

ABSTRACT The early detection of Francisella tularensis, the causative agent of tularemia, is important for adequate treatment by antibiotics and the outcome of the disease. Here we describe a new capture enzyme-linked immunosorbent assay (cELISA) based on monoclonal antibodies specific for lipopolysaccharide (LPS) of Francisella tularensis subsp. holarctica and Francisella tularensis subsp. tularensis. No cross-reactivity with Francisella tularensis subsp. novicida,Francisella philomiragia, and a panel of other possibly related bacteria, including Brucella spp.,Yersinia spp., Escherichia coli, andBurkholderia spp., was observed. The detection limit of the assay was 103 to 104 bacteria/ml. This sensitivity was achieved by solubilization of the LPS prior to the cELISA. In addition, a novel immunochromatographic membrane-based handheld assay (HHA) and a PCR, targeting sequences of the 17-kDa protein (TUL4) gene of F. tularensis, were used in this study. Compared to the cELISA, the sensitivity of the HHA was about 100 times lower and that of the PCR was about 10 times higher. All three techniques were successfully applied to detect F. tularensis in tissue samples of European brown hares (Lepus europaeus). Whereas all infected samples were recognized by the cELISA, those with relatively low bacterial load were partially or not detected by PCR and HHA, probably due to inhibitors or lack of sensitivity. In conclusion, the HHA can be used as a very fast and simple approach to perform field diagnosis to obtain a first hint of an infection with F. tularensis, especially in emergent situations. In any suspect case, the diagnosis should be confirmed by more sensitive techniques, such as the cELISA and PCR.

The Genome of a Bacillus Isolate Causing Anthrax in Chimpanzees Combines Chromosomal Properties of B. cereus with B. anthracis Virulence Plasmids

Anthrax is a fatal disease caused by strains of Bacillus anthracis. Members of this monophyletic species are non motile and are all characterized by the presence of four prophages and a nonsense mutation in the plcR regulator gene. Here we report the complete genome sequence of a Bacillus strain isolated from a chimpanzee that had died with clinical symptoms of anthrax. Unlike classic B. anthracis, this strain was motile and lacked the four prohages and the nonsense mutation. Four replicons were identified, a chromosome and three plasmids. Comparative genome analysis revealed that the chromosome resembles those of non-B. anthracis members of the Bacillus cereus group, whereas two plasmids were identical to the anthrax virulence plasmids pXO1 and pXO2. The function of the newly discovered third plasmid with a length of 14 kbp is unknown. A detailed comparison of genomic loci encoding key features confirmed a higher similarity to B. thuringiensis serovar konkukian strain 97-27 and B. cereus E33L than to B. anthracis strains. For the first time we describe the sequence of an anthrax causing bacterium possessing both anthrax plasmids that apparently does not belong to the monophyletic group of all so far known B. anthracis strains and that differs in important diagnostic features. The data suggest that this bacterium has evolved from a B. cereus strain independently from the classic B. anthracis strains and established a B. anthracis lifestyle. Therefore we suggest to designate this isolate as “B. cereus variety (var.) anthracis”.

Comparison of enzyme-linked immunosorbent assay, Western blotting, microagglutination, indirect immunofluorescence assay, and flow cytometry for serological diagnosis of tularemia.

ABSTRACT The serodiagnostic efficiencies of five different approaches to detecting antibodies (immunoglobulins G, A, and M) developed in clinically proven infections with Francisella tularensis have been assessed. Fifty serum samples from patients suffering from tularemia during an outbreak in Sweden were compared with samples from 50 healthy blood donors (controls) by using an enzyme-linked immunosorbent assay (ELISA), microagglutination (MA), Western blotting (WB), an indirect immunofluorescence assay (IIFA), and flow cytometry (FC). ELISA, WB, and FC were based on the use of preparations of lipopolysaccharides (LPS) of the live vaccine strain of Francisella tularensis subsp. holarctica (ATCC 29684) as a capture antigen. Whole methanol-fixed bacteria were used for IIFA and MA. Optimized protocols yielded a diagnostic sensitivity and specificity of 100% for WB, MA, and FC, 98% for ELISA, and 93% for IIFA. A total of 6,632 serum samples from individuals between the ages of 18 and 79 years, representatively recruited from all regions of Germany, were screened to estimate and confirm the positive predictive value (PVpos) of the ELISA. Serum samples from 15 (0.226%) individuals tested positive for F. tularensis-specific antibodies by ELISA and confirmatory WB. The resulting prevalence-dependent PVpos of 10.2% and specificity of 98.1% were consistent with our findings for tularemia patients and controls. We conclude that the combined usage of a screening ELISA and a confirmatory WB based on LPS as a common antigen, as well as the MA, is a suitable serodiagnostic tool, while the quality of the IIFA is hampered by subjective variations of the results. FC is a promising new approach that might be improved further in terms of multiplex analyses or high-throughput applications.

Shedding and Transmission of Novel Influenza Virus A/H1N1 Infection in Households—Germany, 2009

Essential epidemiologic and virologic parameters must be measured to provide evidence for policy/public health recommendations and mathematical modeling concerning novel influenza A/H1N1 virus (NIV) infections. Therefore, from April through August of 2009, the authors collected nasopharyngeal specimens and information on antiviral medication and symptoms from households with NIV infection on a daily basis in Germany. Specimens were analyzed quantitatively by using reverse transcriptase-polymerase chain reaction. In 36 households with 83 household contacts, 15 household contacts became laboratory-confirmed secondary cases of NIV. Among 47 contacts without antiviral prophylaxis, 12 became cases (secondary attack rate of 26%), and 1 (8%) of these was asymptomatic. The mean and median serial interval were 2.6 and 3 days, respectively (range: 1-3 days). On average, the authors detected viral RNA copies for 6.6 illness days (treated in time = 5.7 days, not treated in time = 7.1 days; P = 0.06), but they estimated that most patients cease to excrete viable virus by the fifth illness day. Shedding profiles were consistent with the number and severity of symptoms. Compared with other nasopharyngeal specimen types, nasal wash was the most sensitive. These results support the notion that epidemiologic and virologic characteristics of NIV are in many aspects similar to those of seasonal influenza.

Field detection of Francisella tularensis.

A field investigation was undertaken following an outbreak of water-borne tularemia in Northern Norway. Francisella tularensis bacterial cellular components were analysed by rapid immunochromatography (RI)-testing, enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). Water from 1 reservoir, fed from a rapid stream, tested negative. From another reservoir, 2 of a chain of 3 wells tested negative. The third well, at the end of the chain, contained lemming (Lemmus lemmus) carcasses and gave ample proof of F. tularensis contamination. We concluded that the origin of the outbreak was dead, infective lemming carcasses in the water sources. For the various sampling materials, the RI-test proved itself particularly handy and versatile, compared with the ELISA and the PCR.A field investigation was undertaken following an outbreak of water-borne tularemia in Northern Norway. Francisella tularensis bacterial cellular components were analysed by rapid immunochromatography (RI)-testing, enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). Water from 1 reservoir, fed from a rapid stream, tested negative. From another reservoir, 2 of a chain of 3 wells tested negative. The third well, at the end of the chain, contained lemming (Lemmus lemmus) carcasses and gave ample proof of F. tularensis contamination. We concluded that the origin of the outbreak was dead, infective lemming carcasses in the water sources. For the various sampling materials, the RI-test proved itself particularly handy and versatile, compared with the ELISA and the PCR.

Evaluation of clinical, laboratory, and therapeutic features of 145 tularemia cases: the role of quinolones in oropharyngeal tularemia†

Tularemia outbreaks have occurred in various regions of Turkey in recent years. In this study, clinical (145 patients) and laboratory (97 patients) features of patients with oropharyngeal tularemia were evaluated during the tularemia outbreak in the district of Gölcük in Kocaeli, Turkey. We analyzed the risk factors for therapeutic failure and prolonged recovery time, and compared the efficacy of three antibiotic groups, namely aminoglycoside, tetracycline and quinolone. The most common physical sign and laboratory findings in patients were lymphadenopathy (LAP) and increased erythrocyte sedimentation rate, respectively. Treatment failure was observed in 55 of the 145 (38%) patients during one‐year follow‐up and the most successful results were obtained in the quinolone group. It was determined that antimicrobial therapy initiated 14 days after onset of symptoms was a statistically significiant risk factor, reducing the success rate (p=0.0001, OR=13.10, 95% CI=5.69–30.15) and prolonging the recovery period (p=0.001, OR=3.23, 95% CI=1.63–6.40) in oropharyngeal tularemia cases. These results suggest that antimicrobial treatment should be started early, and quinolones such as moxifloxacin and ciprofloxacin seem to be new alternatives in the treatment of oropharyngeal tularemia.

Characterization of Yersinia Using MALDI-TOF Mass Spectrometry and Chemometrics

Yersinia are Gram-negative, rod-shaped facultative anaerobes, and some of them, Yersinia enterocolitica, Yersinia pseudotuberculosis, and Yersinia pestis, are pathogenic in humans. Rapid and accurate identification of Yersinia strains is essential for appropriate therapeutic management and timely intervention for infection control. In the past decade matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) in combination with computer-aided pattern recognition has evolved as a rapid, objective, and reliable technique for microbial identification. In this comprehensive study a total of 146 strains of all currently known Yersinia species complemented by 35 strains of other relevant genera of the Enterobacteriaceae family were investigated by MALDI-TOF MS and chemometrics. Bacterial sample preparation included microbial inactivation according to a recently developed mass spectrometry compatible inactivation protocol. The mass spectral profiles were evaluated by supervised feature selection methods to identify family-, genus-, and species-specific biomarker proteins and--for classification purposes--by pattern recognition techniques. Unsupervised hierarchical cluster analysis revealed a high degree of correlation between bacterial taxonomy and subproteome-based MALDI-TOF MS classification. Furthermore, classification analysis by supervised artificial neural networks allowed identification of strains of Y. pestis with an accuracy of 100%. In-depth analysis of proteomic data demonstrated the existence of Yersinia-specific biomarkers at m/z 4350 and 6046. In addition, we could also identify species-specific biomarkers of Y. enterocolitica at m/z 7262, 9238, and 9608. For Y. pseudotuberculosis a combination of biomarkers at m/z 6474, 7274, and 9268 turned out to be specific, while a peak combination at m/z 3065, 6637, and 9659 was characteristic for strains of Y. pestis. Bioinformatic approaches and tandem mass spectrometry were employed to reveal the molecular identity of biomarker ions. In this way, the Y. pestis-specific biomarker at m/z 3065 could be identified as a fragment of the plasmid-encoded plasminogen activator, one of the major virulence factors in plague infections.

First indication for a functional CRISPR/Cas system in Francisella tularensis

Francisella tularensis is a zoonotic agent and the subspecies novicida is proposed to be a water-associated bacterium. The intracellular pathogen F. tularensis causes tularemia in humans and is known for its potential to be used as a biological threat. We analyzed the genome sequence of F. tularensis subsp. novicida U112 in silico for the presence of a putative functional CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system. CRISPR/Cas systems are known to encode an RNA-guided adaptive immunity-like system to protect bacteria against invading genetic elements like bacteriophages and plasmids. In this work, we present a first indication that F. tularensis subsp. novicida encodes a functional CRISPR/Cas defence system. Additionally, we identified various spacer DNAs homologous to a putative phage present within the genome of F. tularensis subsp. novicida-like strain 3523. CRISPR/Cas is also present in F. tularensis subsp. tularensis, holarctica, and mediasiatica, but these systems seem to be non-functional.

Seroprevalence study of Francisella tularensis among hunters in Germany

In 2005 and 2006, Francisella tularensis unexpectedly reemerged in western Germany, when several semi-free-living marmosets (Callithrix jacchus) in a research facility died from tularemia and a group of hare hunters became infected. It is believed that hunters may have an elevated risk to be exposed to zoonotic pathogens, including F. tularensis. A previous cross-sectional study of the German population (n=6883) revealed a prevalence of 0.2%. Here, we investigated 286 sera from individuals mainly hunting in districts with emerging tularemia cases (group 1) and 84 sera from a region currently not conspicuous for tularemia (group 2). Methods included standard enzyme-linked immunosorbent assay (ELISA), Western blot analysis and indirect immunofluorescence assay. We found five out of the 286 hunters (1.7%; 95% CI 0.6-4.0%) in group 1 positive with standard ELISA and Western blot, but none in the Berlin area (group 2; 95% CI 0-0.04%). Group 1 showed an elevated risk for hunters to be seropositive for F. tularensis compared with the cross-sectional study (OR=7.7; P<0.001). This indicates a higher prevalence for tularemia in hunters of a suspected endemic region of Germany.