Ultrasound assessment of testicular volume - An interobserver variability study.

BACKGROUND: Ultrasonography measurement of the testicles and subsequent calculation of the testicular volume is recommended as a part of a standard scrotal ultrasound examination. The interobserver variability of testicular volume measurement has implications for surgical recommendations. Therefore, this study aimed to investigate the interobserver variability in the measurement of testicular volume. METHODS: Interobserver variability was established by comparing testicular measurements performed by two observers on the same patient during the same clinical appointment. The observers were blinded to each other's measurements. Testicular volume was calculated using the Lambert formula: length x width x height x 0.71. A total of three observers, A, B and C, participated in the study. The observers had between 4 to 20 years' experience with scrotal ultrasound examinations. RESULTS: In total, 24 patients' were included (48 testicles). The patient´s mean age was 43 years (range 19-75 years). The overall mean right testicular volume was 19.8 ml (range 7.3-31.6 ml), and the left was 20.1 ml (range 7.1-36.1 ml). The interclass correlation coefficient (ICC) between observer A and B was excellent (ICC= 0.98, CI:0.92-0.99), between observer A and C, was excellent (ICC=0.91, CI: 0.77-0.97) and between B and C good (ICC=0.82, CI:0.51-0.93). CONCLUSION: Variability in estimating testicular volume is low, with interobserver agreement ranging from good to excellent. Ultrasound provides a highly reproducible tool to determine testicular volume.

Spermatocytic tumors in 2 patients aged 50 and 77 years: 2 case reports and brief review of the literature.

Testicular cancer is predominantly diagnosed in young men aged 15-35 years. However, there are some rare tumors such as spermatocytic tumors that are seen more often in the older male population. Spermatocytic tumors have previously been known as spermatocytic seminomas in the scientific literature. We report the cases of 2 patients aged 50 and 77 years both diagnosed with spermatocytic tumors. In this paper we will discuss the ultrasound and histopathology features of these tumors and review the literature of spermatocytic tumor cases.

Diffusion-Weighted MRI as a Quantitative Imaging Biomarker in Colon Tumors.

PURPOSE: To assess the use of quantitative diffusion-weighted MRI (DW-MRI) as a diagnostic imaging biomarker in differentiating between benign colon adenoma, early, and advanced cancer of the colon, as well as predicting lymph node involvement, and finally comparing mucinous-producing colon cancer with adenomas and non-mucinous colon cancer. METHOD: Patients with a confirmed tumor on colonoscopy were eligible for inclusion in this study. Using a 3.0 Tesla MRI machine, the main tumor mean apparent diffusion coefficient (mADC) was obtained. Surgically resected tumor specimens served as an endpoint, except in mucinous colon cancers, which were classified based on T2 images. RESULTS: A total of 152 patients were included in the study population. The mean age was 71 years. A statistically significant mADC mean difference of -282 × 10-6 mm2/s [-419--144 95% CI, p < 0.001] was found between colon adenomas and early colon cancer, with an AUC of 0.80 [0.68-0.93 95% CI] and an optimal cut off value of 1018 × 10-6 mm2/s. Only a small statistically significant difference (p = 0.039) in mADC was found between benign tumors and mucinous colon cancer. We found no statistical difference in mADC mean values between early and advanced colon cancer, and between colon cancer with and without lymph node involvement. CONCLUSION: Quantitative DW-MRI is potentially useful for determining whether a colonic tumor is benign or malignant. Mucinous colon cancer shows less diffusion restriction when compared to non-mucinous colon cancer, a potential pitfall.

Interobserver Reliability and the Sigmoid Takeoff-An Interobserver Study.

Background: Colorectal cancer is the second most common cancer worldwide. The sigmoid takeoff is the landmark where the colon sigmoid curves toward the sacrum viewed from sagittal magnetic resonance imaging (MRI). The purpose of this study was to assess interobserver variability in the assessment of the anal verge and anorectal junction in patients diagnosed with rectal cancer on magnetic resonance imaging (MRI). Materials and Methods: The rectal MRI examinations were performed using a 1.5- or 3.0-tesla unit using an anterior coil and a standard scan protocol. Two senior radiologists assessed MRI scans from patients under investigation for rectal cancer. The two observers assessed the anal verge and takeoff in cm independently. Difference in agreement between the observers were evaluated using intraclass correlation (ICC) and graphically by Bland-Altman plots. Results: The study population (n = 122) included 68 (55.7%) female and 54 (44.3%) male subjects. The overall median age was 69.5 years (range 39-95 years). There was perfect agreement between the two observers when defining rectal tumor above or below the takeoff landmark. The reliability of measuring the distance from the anal verge to the sigmoid takeoff was 0.712. Conclusion: Overall, the study found a moderate reliability in assessing the location of the sigmoid takeoff, with a low difference in the distance measuring, as well as a good consensus concerning the determination of tumors in relation to the sigmoid takeoff. Routine implementation of this information within the report seems reasonable.

MRI interobserver reliability in rectal tumor angulation.

BACKGROUND: When rectal tumors are examined using magnetic resonance imaging (MRI) the perpendicular angulation of the axial T2-weighted image to the tumor axis is essential for a correct measure of the shortest distance between tumor and mesorectal facia. PURPOSE: The purpose of this study was to determine the interobserver variability in rectal tumor angulation between a radiologist and a radiographer. MATERIAL AND METHODS: Two observers performed the angulation independently. All MRI examinations were performed using an MRI 1.5 Tesla unit. A Bland-Altman plot was used to assess the interobserver variance and Intraclass correlation coefficient (ICC) statistic was used to assess the interobserver reliability. RESULTS: MRI was performed in 55 patients with rectal cancer during a one-year period (25 (45.5%) women and 30 (54.5%) men). The median age was 71 years (range 46-87 years). The rectal tumor mean length was 3.9 cm. The interobserver reliability was good (ICC = 0.83, 95% confidence interval 0.72-0.90). CONCLUSION: Radiographers receiving training will be able to perform MRI rectal tumor angulation.

Ultrasound and contrast enhanced CT imaging of a colon mesentery leiomyosarcoma.

Primary leiomyosarcoma of the colon mesentery is an extremely rare neoplasm, and only a small number of cases have been reported. We describe a case of leiomyosarcoma originating in the colonic mesentery, in a 68-year-old woman. Ultrasound showed a heterogeneous mass with varying vascularization in the left fossa. Central areas of the mass were hypoechoic, without detectable vascularization. Contrast enhanced computed tomography (CECT) of chest and abdomen showed a contrast enhanced tumour, with central non-enhanced areas. The tumour was radically resected and histopathology showed primary leiomyosarcoma. Two years after primary surgery, follow-up CECT revealed a local recurrence, which was re-resected. Subsequent follow-up CECT since have shown no sign of recurrence.

Plant history and soil history jointly influence the selection environment for plant species in a long-term grassland biodiversity experiment.

Long-term biodiversity experiments have shown increasing strengths of biodiversity effects on plant productivity over time. However, little is known about rapid evolutionary processes in response to plant community diversity, which could contribute to explaining the strengthening positive relationship. To address this issue, we performed a transplant experiment with offspring of seeds collected from four grass species in a 14-year-old biodiversity experiment (Jena Experiment). We used two- and six-species communities and removed the vegetation of the study plots to exclude plant-plant interactions. In a reciprocal design, we transplanted five "home" phytometers (same origin and actual environment), five "away-same" phytometers (same species richness of origin and actual environment, but different plant composition), and five "away-different" phytometers (different species richness of origin and actual environment) of the same species in the study plots. In the establishment year, plants transplanted in home soil produced more shoots than plants in away soil indicating that plant populations at low and high diversity developed differently over time depending on their associated soil community and/or conditions. In the second year, offspring of individuals selected at high diversity generally had a higher performance (biomass production and fitness) than offspring of individuals selected at low diversity, regardless of the transplant environment. This suggests that plants at low and high diversity showed rapid evolutionary responses measurable in their phenotype. Our findings provide first empirical evidence that loss of productivity at low diversity is not only caused by changes in abiotic and biotic conditions but also that plants respond to this by a change in their micro-evolution. Thus, we conclude that eco-evolutionary feedbacks of plants at low and high diversity are critical to fully understand why the positive influence of diversity on plant productivity is strengthening through time.

Long-term ultrasound follow-up in patients with small gallbladder polyps.

INTRODUCTION: Gallbladder polyps often have a benign appearance by ultrasonography. Even so, the current guideline recommends follow-up in gallbladder polyps lesser-than 6 mm. The aim of this study was to investigate long-term follow-up growth of polyps in patients with a polyp size lesser-than 6 mm in a ten-year cohort. METHODS: Abdominal ultrasonography reports from 2007 to 2009 were reviewed, including reports on patients diagnosed with a gallbladder polyp (polyp size lesser-than 6 mm) during the 2007-2009 period. The patients were invited to a final follow-up ultrasonography of the gallbladder conducted during October 2019 to February 2020. A total of 154 patients were included (100 women and 54 men). RESULTS: In 53 patients (34.4%), the polyp was not visible at the ultrasonography follow-up. Gallbladder polyps were confirmed in 101 (65.6%) patients. A total of 49 patients had a single polyp (31.8%) and 52 (33.8%) patients had multiple polyps. The median size of the gallbladder polyp was 4 mm (range: 2.0-5.9 mm) at baseline compared with 4 mm (range: 1.7-15.0 mm) at the follow-up. A total of 15 patients experienced polyp growth of 2 mm or more. None developed gallbladder cancer. CONCLUSIONS: Our study showed that gallbladder polyps lesser-than 6 mm has a low probability of increasing in size. None of the patients with small polyps developed gallbladder cancer. FUNDING: none. TRIAL REGISTRATION: not relevant.

Determinants of Deadwood-Inhabiting Fungal Communities in Temperate Forests: Molecular Evidence From a Large Scale Deadwood Decomposition Experiment.

Despite the important role of wood-inhabiting fungi (WIF) in deadwood decomposition, our knowledge of the factors shaping the dynamics of their species richness and community composition is scarce. This is due to limitations regarding the resolution of classical methods used for characterizing WIF communities and to a lack of well-replicated long-term experiments with sufficient numbers of tree species. Here, we used a large scale experiment with logs of 11 tree species at an early stage of decomposition, distributed across three regions of Germany, to identify the factors shaping WIF community composition and Operational Taxonomic Unit (OTU) richness using next generation sequencing. We found that tree species identity was the most significant factor, corresponding to (P < 0.001) and explaining 10% (representing 48% of the explainable variance) of the overall WIF community composition. The next important group of variables were wood-physicochemical properties, of which wood pH was the only factor that consistently corresponded to WIF community composition. For overall WIF richness patterns, we found that approximately 20% of the total variance was explained by wood N content, location, tree species identity and wood density. It is noteworthy that the importance of determinants of WIF community composition and richness appeared to depend greatly on tree species group (broadleaved vs. coniferous) and it differed between the fungal phyla Ascomycota and Basidiomycota.

Predicting HCAHPS scores from hospitals' social media pages: A sentiment analysis.

BACKGROUND: Social media is an important communication channel that can help hospitals and consumers obtain feedback about quality of care. However, despite the potential value of insight from consumers who post comments about hospital care on social media, there has been little empirical research on the relationship between patients' anecdotal feedback and formal measures of patient experience. PURPOSE: The aim of the study was to test the association between informal feedback posted in the Reviews section of hospitals' Facebook pages and scores on two global items from the Hospital Consumer Assessment of Healthcare Providers and Systems (HCAHPS) survey, Overall Hospital Rating and Willingness to Recommend the Hospital. METHODOLOGY/APPROACH: We retrieved star ratings and anecdotal comments posted in Reviews sections of 131 hospitals' Facebook pages. Using a machine learning algorithm, we analyzed 57,985 comments to measure consumers' sentiment about the hospitals. We used regression analysis to determine whether consumers' quantitative and qualitative postings would predict global measures from the HCAHPS survey. RESULTS: Both number of stars and the number of positive comments posted on hospitals' Facebook Reviews sections were associated with higher overall ratings and willingness to recommend the hospital. The findings suggest that patients' informal comments help predict a hospital's formal measures of patient experience. CONCLUSION: Consistent with crowd wisdom, ordinary consumers may have valid insights that can help others to assess patient experience at a hospital. Given that some people will judge hospital quality based on opinions voiced in social media, further research should continue to explore associations between anecdotal commentary and a variety of quality indicators. PRACTICE IMPLICATIONS: Administrators can tap into the wealth of commentary on social media as the forum continues to expand its influence in health care. Comments on social media may also serve as an early snapshot of patient-reported experiences, alerting administrators to problems that may appear in subsequent HCAHPS survey results.

Francisella tularensis and other bacteria in hares and ticks in North Rhine-Westphalia (Germany).

Tularemia is a zoonotic disease caused by the bacterium Francisella tularensis. The disease can be transmitted to humans through contact with infected animals such as the European brown hare (Lepus europaeus) and ticks as vectors. The aim of this study was to isolate F. tularensis from ticks and hares in North Rhine-Westphalia using cysteine heart agar to determine their genetic relatedness and to identify other bacteria that grow on this medium. 848 European brown hares and 1556 questing ticks (all Ixodes ricinus) from forests were tested using cultivation and MALDI-TOF mass spectrometry or partial 16S rRNA gene sequencing. The majority of F. tularensis isolates from hares (n=24; 96%) and genomic F. tularensis DNA recovered from ticks belonged to the basal genetic clade IV and subclade B.18. These isolates were sensitive to erythromycin and were assigned to biovar I. Only a single strain isolated from a hare was assigned to basal clade I (B.12/B.35). All isolates were sensitive to tetracycline, doxycycline, streptomycin, gentamicin, chloramphenicol, and ciprofloxacin. Only 4 tick pools were positive for F. tularensis and cultivation was not successful in any of the pools. Most of the other isolated bacteria belonged to the order Bacillales with 36 Staphylococcus isolates, 9 Bacillus isolates and 8 Paenibacillus isolates. Prominent members of Enterobacterales were represented by different genera like Pantoea, Erwinia, Raoultella etc. Several of the bacterial species were soil or plant-associated, but some of the bacterial species were found in I. ricinus for the first time. Our results showed that F. tularensis was detected only in few ticks of an endemic area, but ticks were also infected by several other bacteria with zoonotic potential. Therefore, a wider spectrum of pathogens should be considered if a patient was bitten by a tick.

Antibiotic susceptibility in vitro of Francisella tularensis subsp. holarctica isolates from Germany.

Background: Tularaemia is a zoonotic disease caused by the bacterium Francisella tularensis. In Germany, the disease is still rare (e.g. 34 human cases reported in 2015). There is a lack of data about the susceptibility of F. tularensis strains to antibiotics, because many cases are diagnosed using serological assays only. Objectives: The antibiotic susceptibility in vitro of F. tularensis subsp. holarctica strains isolated in Germany was assessed to determine whether the currently recommended empirical therapy is still adequate. Methods: A total of 128 F. tularensis strains were investigated that were collected between 2005 and 2014 in Germany from wild animals, ticks and humans. All isolates were genotyped using real-time PCR assays targeting canonical SNPs, and antibiotic susceptibility was tested using MIC test strips on agar plates. MIC values were interpreted using CLSI breakpoints. Results: The strains were susceptible to antibiotics commonly recommended for tularaemia therapy, i.e. aminoglycosides (MIC90 values: gentamicin 1 mg/L; streptomycin 4.0 mg/L), tetracyclines (MIC90 values: tetracycline 0.5 mg/L; doxycycline 1.5 mg/L) and quinolones (MIC90 value: ciprofloxacin 0.064 mg/L). Chloramphenicol (MIC90 value: 3.0 mg/L) may be of value in treatment of tularaemia meningitis. Ninety-four isolates were susceptible to erythromycin, which defines biovar I (genotypes B.4 and B.6); 34 were resistant (biovar II; genotype B.12). Conclusions: The F. tularensis isolates investigated in this study showed the typical antibiotic susceptibility pattern that was previously observed in other countries. Therefore, recommendations for empirical antibiotic therapy of tularaemia can remain unchanged. However, antibiotic susceptibility testing of clinical isolates should be performed whenever possible.

A human macrophage-hepatocyte co-culture model for comparative studies of infection and replication of Francisella tularensis LVS strain and subspecies holarctica and mediasiatica.

BACKGROUND: Francisella tularensis, a gram-negative bacterium replicates intracellularly within macrophages and efficiently evades the innate immune response. It is able to infect and replicate within Kupffer cells, specialized tissue macrophages of the liver, and to modulate the immune response upon infection to its own advantage. Studies on Francisella tularensis liver infection were mostly performed in animal models and difficult to extrapolate to the human situation, since human infections and clinical observations are rare. RESULTS: Using a human co-culture model of macrophages and hepatocytes we investigated the course of infection of three Francisella tularensis strains (subspecies holarctica--wildtype and live vaccine strain, and mediasiatica--wildtype) and analyzed the immune response triggered upon infection. We observed that hepatocytes support the intracellular replication of Franciscella species in macrophages accompanied by a specific immune response inducing TNFα, IL-1ß, IL-6 and fractalkine (CX3CL1) secretion and the induction of apoptosis. CONCLUSIONS: We could demonstrate that this human macrophage/hepatocyte co-culture model reflects strain-specific virulence of Francisella tularensis. We developed a suitable tool for more detailed in vitro studies on the immune response upon liver cell infection by F. tularensis.

Isolation and propagation of the animal rotaviruses in MA-104 cells--30 years of practical experience.

A total of 136 rotavirus positive samples from diarrhoeic animals of different species were submitted for isolation and cultural propagation of rotavirus on MA-104 cells. The samples were collected from animals with diarrhoea, between 1980 and 2010, originating from herds or farms located in several parts of Germany. Rotaviruses of species A were isolated from 102 faecal samples in cultures of MA-104 cells under the following conditions: pre-treatment of virus with trypsin, incorporation of trypsin into culture medium, use of roller cultures, and centrifugation of the samples on the cells. The cell culture adapted viruses produced a cytopathic effect, accompanied by the release of cells from the glass surface of the cultivation vessels. After 10 passages the virus isolates yielded titres between 10(5.5) and 10(7.5)ml(-1) TCID50. Isolation and serial propagation of the virus in MA-104 cells was confirmed by immunofluorescence assay, transmission electron microscopy, and polyacrylamide-gel electrophoresis of viral dsRNA. Eight (5.9%) of the electrophoretic profiles were characteristic of species B or D rotaviruses, which were not replicated in MA-104 cells.

Detection of rotavirus species A, B and C in domestic mammalian animals with diarrhoea and genotyping of bovine species A rotavirus strains.

Rotaviruses (RVs) are a major cause of neonatal diarrhoea in humans and animals worldwide. In this study, 425 faecal samples were collected between 1999 and 2013 from diarrhoeic livestock and companion animals at different locations in Germany and tested for RVs. A previously published real-time RT-PCR assay was optimized for detection of a larger variety of RV species A (RVA) strains, and real-time RT-PCR assays for detection of RV species B (RVB) and C (RVC) were newly developed. The detection limits of the assays were 1.54×10(2), 3.95×10(2) and 3.60×10(3) genome copies for RVA, RVB and RVC, respectively. RVA was identified in 85.2% of bovine samples, 51.2% of porcine samples, 50.0% of feline samples, 43.2% of equine samples and 39.7% of canine samples. RVB was found in 3.0% of bovine samples, 2.7% of equine samples and 1.6% of porcine samples. RVC was detected in 31.0% of porcine samples, 21.7% of feline samples, 9.0% of canine samples and 6.0% of bovine samples. For genotyping, 101 RVA-positive bovine samples were further analysed by semi-nested RT-PCR. Genotype combination G6P[5] was most frequently detected (67.3% of samples), followed by G6P[11] (13.9%), G10P[5] (4.0%), G8P[11] (3.0%), G6P[1] (1.0%), and G10P[11] (1.0%). Mixed RVA infections were detected in 5.9% of samples; no or incomplete typing was possible in 4.0% of the samples. This first overview on RV species and RVA genotypes in diarrhoeic livestock and companion animals from Germany indicates a broad circulation of a large variety of RVs.

Hare-to-human transmission of Francisella tularensis subsp. holarctica, Germany.

In November 2012, a group of 7 persons who participated in a hare hunt in North Rhine-Westphalia, Germany, acquired tularemia. Two F. tularensis subsp. holarctica isolates were cultivated from human and hare biopsy material. Both isolates belonged to the FTN002-00 genetic subclade (derived for single nucleotide polymorphisms B.10 and B.18), thus indicating likely hare-to-human transmission.

Performance of seven serological assays for diagnosing tularemia.

BACKGROUND: Tularemia is a rare zoonotic disease caused by the Gram-negative bacterium Francisella tularensis. Serology is frequently the preferred diagnostic approach, because the pathogen is highly infectious and difficult to cultivate. The aim of this retrospective study was to determine the diagnostic accuracy of tularemia specific tests. METHODS: The Serazym®Anti-Francisella tularensis ELISA, Serion ELISA classic Francisella tularensis IgG/IgM, an in-house ELISA, the VIRapid® Tularemia immunochromatographic test, an in-house antigen microarray, and a Western Blot (WB) assay were evaluated. The diagnosis tularemia was established using a standard micro-agglutination assay. In total, 135 sera from a series of 110 consecutive tularemia patients were tested. RESULTS: The diagnostic sensitivity and diagnostic specificity of the tests were VIRapid (97.0% and 84.0%), Serion IgG (96.3% and 96.8%), Serion IgM (94.8% and 96.8%), Serazym (97.0% and 91.5%), in-house ELISA (95.6% and 76.6%), WB (93.3% and 83.0%), microarray (91.1% and 97.9%). CONCLUSIONS: The diagnostic value of the commercial assays was proven, because the diagnostic accuracy was >90%. The diagnostic sensitivity of the in-house ELISA and the WB were acceptable, but the diagnostic accuracy was <90%. Interestingly, the antigen microarray test was very specific and had a very good positive predictive value.

Serological investigation of wild boars (Sus scrofa) and red foxes (Vulpes vulpes) as indicator animals for circulation of Francisella tularensis in Germany.

Tularemia outbreaks in humans have recently been reported in many European countries, but data on the occurrence in the animal population are scarce. In North America, seroconversion of omnivores and carnivores was used as indicator for the presence of tularemia, for the European fauna, however, data are barely available. Therefore, the suitability of wild boars (Sus scrofa) and red foxes (Vulpes vulpes) as indicators for the circulation of F. tularensis in Germany was evaluated. Serum samples from 566 wild boars and 457 red foxes were collected between 1995 and 2012 in three federal states in Central Germany (Hesse, Saxony-Anhalt, and Thuringia). The overall rate of seropositive animals was 1.1% in wild boars and 7.4% in red foxes. In conclusion, serological examination of red foxes is recommended, because they can be reliably used as indicator animals for the presence of F. tularensis in the environment.

Comparison of bacterial culture and polymerase chain reaction (PCR) for the detection of F. tularensis subsp. holarctica in wild animals.

Detection of the zoonotic pathogen Francisella tularensis subsp. holarctica (EF tularensis) in wild animals with culture techniques as well as polymerase chain reaction were compared and discussed on the basis of the investigation of 60 animals. The samples originated from 55 European brown hares (Lepus europaeus), two red foxes (Vulpes vulpes) and one each from a wild rabbit (Oryctolagus cuniculus), a European beaver (Castor fiber), and a lemur (Lemur catta). When comparing the growth of 28 F. tularensis isolates on the cysteine blood agar and the modified Martin-Lewis-agar used in this study, cultivation was successful for 26 isolates on both media, but for two isolates only on the cysteine blood agar. Out of 43 carcasses 19 tested positive in bacteriological culture and PCR. Two culture positive samples of tonsils originating from foxes could not be confirmed by PCR, although PCR was positive in 22 samples that missed growth of F. tularensis. Comparative studies on cultural detection of E. tularensis were performed on samples of 16 hares from lung, spleen, liver and gut and in one case with a peritoneal swab. In at least one of these localizations cultivation of the pathogen was successful. Detection rate was reduced to 94% (15 of 16 hares) considering only the results of the cultures of the lungs and spleens. For a sensitive and rapid detection of F. tularensis subsp. holarctica, the PCR is a suitable method thereby avoiding hazardous multiplying of the pathogen. However, cultivation of F. tularensis is often a prerequisite for further studies on antibiotic resistance patterns of the pathogen, molecular epidemiological and pathological analyses of tularaemia.

German Francisella tularensis isolates from European brown hares (Lepus europaeus) reveal genetic and phenotypic diversity.

BACKGROUND: Tularemia is a zoonotic disease caused by Francisella tularensis that has been found in many different vertebrates. In Germany most human infections are caused by contact with infected European brown hares (Lepus europaeus). The aim of this study was to elucidate the epidemiology of tularemia in hares using phenotypic and genotypic characteristics of F. tularensis. RESULTS: Cultivation of F. tularensis subsp. holarctica bacteria from organ material was successful in 31 of 52 hares that had a positive PCR result targeting the Ft-M19 locus. 17 isolates were sensitive to erythromycin and 14 were resistant. Analysis of VNTR loci (Ft-M3, Ft-M6 and Ft-M24), INDELs (Ftind33, Ftind38, Ftind49, RD23) and SNPs (B.17, B.18, B.19, and B.20) was shown to be useful to investigate the genetic relatedness of Francisella strains in this set of strains. The 14 erythromycin resistant isolates were assigned to clade B.I, and 16 erythromycin sensitive isolates to clade B.IV and one isolate was found to belong to clade B.II. MALDI-TOF mass spectrometry (MS) was useful to discriminate strains to the subspecies level. CONCLUSIONS: F. tularensis seems to be a re-emerging pathogen in Germany. The pathogen can easily be identified using PCR assays. Isolates can also be identified within one hour using MALDI-TOF MS in laboratories where specific PCR assays are not established. Further analysis of strains requires genotyping tools. The results from this study indicate a geographical segregation of the phylogenetic clade B.I and B.IV, where B.I strains localize primarily within eastern Germany and B.IV strains within western Germany. This phylogeographical pattern coincides with the distribution of biovar I (erythromycin sensitive) and biovar II (erythromycin resistance) strains. When time and costs are limiting parameters small numbers of isolates can be analysed using PCR assays combined with DNA sequencing with a focus on genetic loci that are most likely discriminatory among strains found in a specific area. In perspective, whole genome data will have to be investigated especially when terrorist attack strains need to be tracked to their genetic and geographical sources.