The ability of the Biodex Stability System to distinguish level of function in subjects with a second-degree ankle sprain

OBJECTIVE: To assess the capacity of the Biodex Stability System using a one-leg stance protocol to differentiate between injured and non-injured limbs and between level of disabilities. DESIGN: Cross-sectional study. SETTING: Military and civilian clinic. SUBJECTS: Thirty-four individuals with a second-degree lateral ankle sprain and 36 healthy subjects. METHODS AND MEASURES: Subjects were tested on the Stability System 30 days after injury using a one-leg stance protocol in the dynamic limit-of-stability mode. All subjects also filled out a disability questionnaire (Lower Extremity Functional Scale). The groups were compared based on the overall dynamic limit-of-stability score and its relationship with the score on the disability questionnaire was also examined. RESULTS: The overall dynamic limit-of-stability scores (subjects with a lateral ankle sprain: 13.0% (5.5)-26.0% (9.2); healthy subjects: 16.9% (7.9)-27.9% (9.6)) clustered in the lower end of the theoretical range of 0-100%. Statistically significant differences in the overall dynamic limit-of-stability scores were found between the injured and non-injured limbs but group differences were small and clinically not relevant. No significant relationships were found between the overall dynamic limit-of-stability scores and the Lower Extremity Functional Scale scores (58.2 (11.8)) of the subjects with a lateral ankle sprain. CONCLUSIONS: The one-leg stance protocol carried out in the dynamic limit-of-stability mode is very challenging and offers a very limited capacity to differentiate between injured and non-injured limbs. The main outcome of the Stability System does not appear to be a good indicator of the functional capacity of people with a lateral ankle sprain.     

Sensitive HPLC-fluorescence method for irinotecan and four major metabolites in human plasma and saliva: application to pharmacokinetic studies

BACKGROUND: We developed gradient HPLC methods for quantification of the antimitotic drug irinotecan (CPT-11) and its four metabolites, SN-38, SN-38 G, 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]-carbonyloxycamptothecin (APC), and 7-ethyl-10-[4amino-1-piperidino]-carbonyloxycamptothecin (NPC), as the sum of the lactone and carboxylate forms, in human plasma and saliva. Camptothecin was used as internal standard. METHODS: The sample pretreatment involved protein precipitation with methanol-acetonitrile (50:50 by volume) followed by acidification with hydrochloric acid to convert the lactone ring-opened form into its lactone form, quantitatively. HPLC separation was performed on a Xterra RP18 column. The excitation wavelength was 370 nm, and the emission wavelength was set at 470 nm for the first 24 min and then at 534 nm for the next 4 min. The stabilities of irinotecan and its four metabolites in plasma, saliva, and acidic extracts were also investigated under various conditions. RESULTS: Assays were linear in the tested range of 0.5-1000 micro g/L. For the five analytes, limits of quantification were 0.5 micro g/L in both matrices. The interassay imprecision (as relative standard deviation) was 3.2-14% in plasma and 2.6-5.6% in saliva. Assay recoveries ranged from 92.8% to 111.2% for plasma and 100.1% to 104.1% for saliva. Mean extraction recovery from plasma or saliva was 90%. CONCLUSION: The developed assay can be used to determine pharmacokinetic parameters for CPT-11, SN-38, SN-38 G, APC, and NPC in plasma and saliva from patients with metastatic colorectal cancer.     

Temporary and definitive external fixation of war injuries: use of a French dedicated fixator

Purpose

External fixation is the recommended stabilization method for both open and closed fractures of long bones in forward surgical hospitals. Specific combat surgical tactics are best performed using dedicated external fixators. The Percy Fx© (Biomet) fixator was developed for this reason by the French Army Medical Service, and has been used in various theatres of operations for more than ten years.

Methods

The tactics of Percy Fx© (Biomet) fixator use were analysed in two different situations: for the treatment of French soldiers wounded on several battlefields and then evacuated to France and for the management of local nationals in forward medical treatment facilities in Afghanistan and Chad.

Results

Overall 48 externals fixators were implanted on 37 French casualties; 28 frames were temporary and converted to definitive rigid frames or internal fixation after medical evacuation. The 77 Afghan patients totalled 85 external fixators, including 13 temporary frames applied in Forward Surgical Teams (FSTs) prior to their arrival at the Kabul combat support hospital. All of the 47 Chadian patients were treated in a FST with primary definitive frames because of delayed surgical management and absence of higher level of care in Chad.

Conclusion

Temporary frames were mostly used for French soldiers to facilitate strategic air medical evacuation following trauma damage control orthopaedic principles. Definitive rigid frames permitted achieving treatment of all types of war extremity injuries, even in poor conditions.     

Detection of specific chromosomal aberrations in urine using BCA-1 (oligo-CGH-array) enhances diagnostic sensitivity and predicts the aggressiveness of non-muscle-invasive bladder transitional cell carcinoma

Introduction

Bladder carcinoma (B-TCC) is the fifth most prevalent carcinoma in the United States (US) or Europe. In addition, B-TCC is the most expensive carcinoma per patient between diagnosis and death, because of its 50–80 % recurrence rate. B-TCC is an optimal carcinoma for which to detect DNA alterations in urine, which is easily obtainable. Chromosomal aberrations in tumors have been closely related to the carcinogenesis process.

Material and Methods

We developed a highly specific and sensitive oligo-CGH-array for the diagnosis and follow-up of B-TCC, based on the detection of chromosomal aberrations in urine samples. One hundred and sixty-four urine samples were analyzed. The qualitative results, including chromosomal aberrations, were obtained. Quantitative results are expressed as a percentage of chromosomal alterations on the autosomes.

Results

From the urine samples, we were able to differentiate B-TCC from non-malignant conditions with an accuracy of 100 % for patients without history of B-TCC. For follow-up of B-TCC in clinical practice, at least a deletion (8p; 9p; 9q) or a cut-off of >2 % of chromosomal imbalance was considered as a positive test. According to our criteria, 100 % of high-grade tumors were diagnosed, and the sensitivity to predict positive cystoscopy was 95 % (specificity 73 %). A cut-off >9 % was a strong signature of high-grade TCC (odds ratio 53 CI 95 % 7–417; p = 0.0002).

Conclusion

We developed a sensitive clinical tool for the detection of B-TCC using DNA extracted from patient urine. This tool is also able to identify low-grade B-TCC and identify high-risk patients harboring a high-grade disease.     

Transfer of learning and patient outcome in simulated crisis resource management: a systematic review

Purpose

Simulation-based learning is increasingly used by healthcare professionals as a safe method to learn and practice non-technical skills, such as communication and leadership, required for effective crisis resource management (CRM). This systematic review was conducted to gain a better understanding of the impact of simulation-based CRM teaching on transfer of learning to the workplace and subsequent changes in patient outcomes.

Source

Studies on CRM, crisis management, crew resource management, teamwork, and simulation published up to September 2012 were searched in MEDLINE^®, EMBASE?, CINAHL, Cochrane Central Register of Controlled Trials, and ERIC. All studies that used simulation-based CRM teaching with outcomes measured at Kirkpatrick Level 3 (transfer of learning to the workplace) or 4 (patient outcome) were included. Studies measuring only learners’ reactions or simple learning (Kirkpatrick Level 1 or 2, respectively) were excluded. Two authors independently reviewed all identified titles and abstracts for eligibility.

Principal findings

Nine articles were identified as meeting the inclusion criteria. Four studies measured transfer of simulation-based CRM learning into the clinical setting (Kirkpatrick Level 3). In three of these studies, simulation-enhanced CRM training was found significantly more effective than no intervention or didactic teaching. Five studies measured patient outcomes (Kirkpatrick Level 4). Only one of these studies found that simulation-based CRM training made a clearly significant impact on patient mortality.

Conclusions

Based on a small number of studies, this systematic review found that CRM skills learned at the simulation centre are transferred to clinical settings, and the acquired CRM skills may translate to improved patient outcomes, including a decrease in mortality.     

Unilateral posterior cervical spinal cord infarction due to spontaneous vertebral artery dissection

Context

Ischemia of the cervical spinal cord is a rare complication of spontaneous vertebral artery dissection (VAD) and usually involves the ventral portion. We describe a less evocative clinical presentation and images of unilateral posterior spinal cord infarction due to spontaneous VAD in order to facilitate early diagnosis.

Findings

A previously fit 30-year-old man presented with persistent headaches and proximal motor deficit of the right arm. He was diagnosed with spontaneous dissection of both vertebral arteries, with occlusion of the right one, and the right carotid artery. Neurological examination also revealed a right C2–C3 tactile sensory loss, with unilateral proprioceptive deficit below. Brain images revealed small bilateral cerebellar infarcts which could not be responsible for the clinical symptoms. Magnetic resonance imaging of the spinal cord showed a right posterior cervical spinal cord infarction. The patient achieved nearly complete recovery after several weeks of anticoagulation and rehabilitation.

Conclusion and clinical relevance

Infarction of the caudal portion of the cervical spinal cord, especially unilateral, caused by spontaneous VAD, has rarely been described and is certainly under-diagnosed due to less suggestive symptoms, like unilateral and mainly sensory deficit. Nevertheless, early diagnosis of this condition is important to guide patient management and rehabilitation.     

Estimating Endogenous Dopamine Levels at D2 and D3 Receptors in Humans using the Agonist Radiotracer [11C]-(+)-PHNO

Using positron emission tomography (PET) and an acute dopamine depletion challenge it is possible to estimate endogenous dopamine levels occupying dopamine D_2/3 receptors (D_2/3R) in humans in vivo. Our group has developed [¹¹C]-(+)-PHNO, the first agonist radiotracer with preferential in vivo affinity for D₃R. Thus, the use of [¹¹C]-(+)-PHNO offers the novel possibility of (i) estimating in vivo endogenous dopamine levels at D_2/3R using an agonist radiotracer, and (ii) estimating endogenous dopamine levels at D₃R in extrastriatal regions such as the substantia nigra, hypothalamus, and ventral pallidum. Ten healthy participants underwent a [¹¹C]-(+)-PHNO PET scan under baseline conditions and another under acute endogenous dopamine depletion achieved via oral administration of alpha-methyl-para-tyrosine (64 mg/kg). [¹¹C]-(+)-PHNO binding was sensitive to acute dopamine depletion, allowing in vivo estimates of endogenous dopamine in D₂R-rich regions (caudate and putamen), mixed D_2/3R-rich regions (ventral striatum and globus pallidus), and extrastriatal D₃R-rich regions (hypothalamus and ventral pallidum). Dopamine depletion decreased self-reported vigor, which was correlated with the reduction in dopamine levels in the globus pallidus. [¹¹C]-(+)-PHNO is a suitable radiotracer for use in estimating endogenous dopamine levels at D₂R and D₃R in neuropsychiatric populations.     

Soft spots and their structural signature in a metallic glass

In a 3D model mimicking realistic Cu₆₄Zr₃₆ metallic glass, we uncovered a direct link between the quasi-localized low-frequency vibrational modes and the local atomic packing structure. We also demonstrate that quasi-localized soft modes correlate strongly with fertile sites for shear transformations: geometrically unfavored motifs constitute the most flexible local environments that encourage soft modes and high propensity for shear transformations, whereas local configurations preferred in this alloy, i.e., the full icosahedra (around Cu) and Z16 Kasper polyhedra (around Zr), contribute the least.Metallic glasses (MGs) have an inherently inhomogeneous internal structure, with a wide spectrum of atomic-packing heterogeneities (1–4). As a result, an a priori identification of structural defects that carry atomic rearrangements (strains) under imposed stimuli such as temperature and externally applied stresses has always been a major challenge (3–6). In several quasi-2D or 3D models of amorphous solids (such as jammed packings of soft spheres interacting via repulsive potentials or colloidal particles), low-frequency vibrational normal modes have been characterized, and it has recently been demonstrated that some of these modes are quasi-localized (7–14). A population of “soft spots” has been identified among them in terms of their low-energy barriers for local rearrangements (13, 14), correlating also with properties in supercooled liquids such as dynamic heterogeneity (15–17). However, it is not certain where the soft spots are in realistic MGs (18), in terms of an explicit correlation with local atomic packing and topological arrangements (18–20). In particular, there is a pressing need to determine whether it is possible to identify shear transformation zones, i.e., the local defects that carry inelastic deformation (21, 22). Accomplishing this would permit the characterization of MG microstructure in a way that directly ties atomic configuration with mechanical response beyond the elastic regime. We will show here that there is indeed a correlation between soft modes and atoms that undergo shear transformations, and both have their structural signature in specific atomic packing environments defined in terms of coordination polyhedra (3).Fig. 1 displays the vibrational density of states (V-DOS), D(ω), calculated from the eigen-frequencies obtained by normal mode analysis of the Cu₆₄Zr₃₆MG prepared with a cooling rate of 10⁹ K/s (Methods). The main peak stays around 14 meV and becomes only slightly narrower (or wider) when the cooling rate used to prepare the MG is slower (or faster), as seen in Fig. S1; the glasses cooled at slower rates exhibit fewer low-frequency (or low-energy) vibrational modes. The blue portion in Fig. 1 indicates the 1% lowest-frequency normal modes, which will be summed over in our calculations of the participation fraction, P_i, in soft modes (Methods). Those low-frequency vibrational modes are confirmed to be quasi-localized, similar to previous work on 2D models (15), as they involve a compact group of atoms on the basis of the amplitude distribution of their corresponding eigenvectors (also see the contour maps in Fig. 4).Open in a separate windowFig. 1.V-DOS of the inherent structure for Cu₆₄Zr₃₆ MG produced with the cooling rate of 10⁹ K/s. The blue portion indicates the 1% lowest frequency normal modes that were summed over to calculate the participation fraction (in soft modes) of atoms.Open in a separate windowFig. 4.Contoured maps showing the spatial distribution of participation fraction P_i (see sidebar) for Cu and Zr atoms in the Cu₆₄Zr₃₆ metallic glass with the cooling rate of 10⁹ K/s. The four sampled representative thin slabs (A–D) each has a thickness of 2.5 Å. White spots superimposed in the maps mark the locations of atoms that have experienced clear shear transformations (Methods) under AQS to a strain of 5%.We first demonstrate that certain types of coordination polyhedra, specifically those geometrically unfavored motifs (GUMs), contribute preferentially to the quasi-localized soft modes identified above, whereas the geometrically preferable clusters at this alloy composition represent the short-range order that participate the least. To establish the connection between the low-frequency modes and atomic packing structure, we analyze the latter first from the perspective of Cu-centered coordination polyhedra (23), in terms of the P_i of Cu atoms that are in the center of different types of polyhedra. In Fig. 2A, from left to right, each solid bar represents a bin that contains 10% of all of the Cu atoms, in ascending order from the lowest to the highest P_i. In addition, the 1% Cu atoms with the lowest P_i and the top 1% with the highest P_i are displayed on either end, each with a separate bar. The Cu atoms in full icosahedra (with Voronoi index <0, 0, 12, 0>) dominate the lowest P_i, which is consistent with the notion that full icosahedra are the short-range order most energetically and geometrically comfortable and hence least likely to participate in soft spots at this MG composition (23). Specifically, ∼98% of the Cu atoms with the 1% lowest participation fraction are enclosed in <0, 0, 12, 0>, which is much greater than the average value that ∼40% of Cu atoms center full icosahedra in this MG sample (23). In stark contrast, the local configurations on the other end of the coordination polyhedra spectrum, i.e., the GUMs (see examples below) that deviate considerably from the coordination number (CN) = 12 full icosahedra and their close cousins (Fig. 2), are not found at all among the atoms with the lowest 1% participation fraction. For the 1% of Cu atoms with the highest participation fraction, GUMs account for as high as 63%, whereas the share of full icosahedra is as low as only 1.1%. This observation clearly indicates that atoms involved with soft spots in low-frequency normal modes (i.e., soft modes) are those with the most unfavorable local coordination polyhedra.Open in a separate windowFig. 2.Atoms at the center of different types of (A) Cu-centered and (B) Zr-centered coordination polyhedra contribute differently to low-frequency normal modes. Each solid bar contains 10% of all of the Cu (or Zr) atoms; from left to right, the bins are ordered from the lowest to the highest participation fraction. Two additional bars describe the makeup of atoms contributing to the lowest 1% participation fraction and the highest 1% participation fraction, respectively. The latter is seen to be dominated by Cu (or Zr) atoms in GUMs.We also examined the dependence on local environments for Zr atoms. A plot analogous to Fig. 2A, this time for Zr-centered coordination polyhedra, is shown in Fig. 2B. From left to right, each solid bar represents a bin that contains 10% of all of the Zr atoms, in ascending order from the lowest to the highest P_i. In addition, the 1% Zr atoms with the lowest P_i and the top 1% with the highest P_i are displayed on either end, each with a separate bar. The most favorable Zr-centered Kasper polyhedra in this MG are of the Z16 type (<0, 0, 12, 4>) (23). Interestingly, for the Zr atoms with the 1% lowest participation fraction, ∼75% of them are enclosed in <0, 0, 12, 4>, which is much greater than the sample average of ∼17% in this MG (23). In contrast, GUMs that deviate considerably from the CN = 16 Kasper polyhedra and their close cousins (Fig. 2) only constitute ∼5%. Conversely, for the 1% of Zr atoms with the highest participation fraction, GUMs account for as high as 76%, whereas the share of Z16 clusters is as low as 1.6%.We now illustrate the GUMs, i.e., the typical types of coordination polyhedra that are strongly correlated with the soft modes. Fig. 3 A and B illustrates the local environments of the top five Cu atoms and Zr atoms, respectively, i.e., those with the highest participation fractions. For these five Cu-centered GUMs, the coordination polyhedra have Voronoi indices of <0, 0, 12, 2>, <0, 4, 4, 4>, <0, 6, 0, 6>, <0, 4, 4, 3>, and <0, 3, 6, 2>. For the five Zr GUMs, they are <1, 3, 4, 4>, <1, 2, 6, 5>, <0, 2, 9, 4>, <0, 3, 7, 4>, and <0, 4, 5, 6>. Clearly, they are among the polyhedra that deviate most significantly from the geometrically preferable Frank-Kasper polyhedra <0, 0, 12, 0> (for Cu) and <0, 0, 12, 4> (for Zr). Specifically, they are non-Kasper polyhedra and contain an increased density of extrinsic (e.g., fourfold) disclinations (3) at the favored CN, or clusters (including Kasper polyhedra) with unfavorable (too large or too small) CNs. In fact, those Zr-centered GUMs even contain sevenfold bonds, e.g., <1, 3, 4, 4> is actually <1, 3, 4, 4, 1> (except for these Zr-centered GUMs, the fifth digit is zero in the Voronoi indices for all the other coordination polyhedra in this work). From the perspective of either constituent element, Cu or Zr, these are the most geometrically unfavored clusters at the given alloy composition and atomic size ratio. According to ref. 24, transverse vibrational modes associated with defective (more disordered) soft structures could also be an origin of the boson peak [the excess rise in the D(ω) at low-frequency vibrational modes].Open in a separate windowFig. 3.Configurations of five different (A) Cu-centered and (B) Zr-centered polyhedra, in which the center atoms are the top five atoms with the highest participation fractions for each constituent species. These are representatives of GUMs in this MG. Orange spheres are for Cu atoms and silver ones for Zr atoms.The next task at hand is to correlate the relaxation events with vibrational modes. In a 2D sheared model glass, Manning et al. (14) recently associated low-frequency vibrational modes with soft spots where particle rearrangements are initiated. Here we use a similar analysis on our 3D realistic Cu₆₄Zr₃₆ glass. The contoured maps of participation fraction P_i for all of the (Cu and Zr) atoms inside four representative slabs, each with a thickness of 2.5 Å (roughly the average atomic spacing), are plotted in Fig. 4 A–D. We notice that the P_i distributions are heterogeneous: atoms that participated the most in soft modes tend to aggregate together, with a typical correlation length of ∼1 nm. For a direct comparison, the local atomic rearrangements in sheared Cu₆₄Zr₃₆ MG [under athermal quasi-static shear (AQS) to a global shear strain γ = 5%, well before global yielding/flow of the entire sample at γ ∼ 12%] are superimposed in Fig. 4 A–D, where white spheres represent the (Cu or Zr) atoms that have experienced the most obvious shear transformations (indicated by their large and simultaneous jumps of Dmin2 that are clearly above other atoms; Methods, Fig. S2, and SI Text). The distribution of these atoms is also inhomogeneous and, interestingly, almost always overlaps with the regions with high P_i. This observation is consistent with the correlation between quasi-localized low-frequency modes and low energy barriers (13). Fig. 5 displays the locations of all such Cu and Zr atoms in the simulation model, which are about 2% of the total number of atoms in the box. Two features are highly noteworthy. First, they cluster into patches (only 6 atoms are exceptions, being isolated in a group of <3 atoms), each comprising 10–40 atoms (Cu in orange and Zr in gray color). Second, the atoms in each cluster record a simultaneous jump in Dmin2. Taken together, the spatial and temporal correlations clearly indicate that these are the clusters of atoms that each has been through a well-defined shear transformation. The several representative cases in Fig. 4 (and Fig. S3) give a visual illustration of the correlation that, under imposed deformation, the most obvious shear transformations have a strong tendency to arise from the collection of atoms involved in soft modes. Each group (cluster) of the activated atoms in Fig. 5 centers a shear transformation zone.Open in a separate windowFig. 5.Cluster of atoms that have undergone obvious shear transformations (Methods) (24) in Cu₆₄Zr₃₆ MG sheared to γ = 5%. Atoms in each cluster are activated at the same time, as indicated by their simultaneous jump in ΔDmin2 at the same shear strain γ. Two such shear transformation zones are circled, with the Inset displaying the overlapping ΔDmin2 jumps of the atoms involved in each cluster.Note here that not all of the regions with the highest participation fraction P_i would undergo shear transformation for a particular loading condition, as seen in Fig. 4 and Fig. S3. One should keep in mind that such a local structure–property correlation in an amorphous system is expected to be statistical (better perceived in Fig. 6), rather than deterministic with a one-to-one correspondence (12, 14). The soft spots are only candidates for potential shear transformation zones. The ones actually activated are not necessarily the softest, and would be determined by the loading direction and local stress field interacting with the anisotropy of the soft spots. The statistical correlation is obvious for the entire range of imposed γ, from 2% to 10%. The contour maps similar to those in Fig. 4 for γ = 10% (before global yielding) are shown in Fig. S3. As another way to see this correlation, we present in Fig. 6 a plot correlating the average participation fraction with Dmin2 (with respect to the undeformed configuration) for γ from 2% to 10%. Each data point is an average for 5% of all of the atoms inside a bin (each bin contains atoms grouped in ascending participation fraction). Obviously, the atoms with higher participation in soft mode contribute more to the nonaffine deformation and therefore shear transformations. This trend persists throughout the entire range of strains we studied and is therefore statistically valid for all the atoms in the metallic glass.Open in a separate windowFig. 6.Correlation between the average Dmin2 (with reference to undeformed configuration) with participation fraction P_i for all of the (Cu and Zr) atoms in the Cu₆₄Zr₃₆ MG deformed to different γ levels (2–10%). Each data point is the average for 5% of all of the atoms, sorted in the order of increasing P_i.In conclusion, we identified soft spots in an MG. They are soft in the sense that the atoms (Cu and Zr in our case) in those local environments participate preferentially in soft vibrational modes and at the same time they have the highest propensity to undergo shear transformations. These two aspects are found to be strongly correlated: shear transformations in an MG preferentially occur at localized soft modes. In the language of the potential energy landscape, we established a correlation between the curvature at the bottom of the basin (stiffness) with the barrier for transitions between basins (energy barrier against reconfiguration). Importantly, we showed that both have a common signature in the local atomic packing environments: the GUMs are the local configurations most prone to instability. The GUMs, as the most disordered atomic arrangements, hence tend to constitute or center the “liquid-like regions” often hypothesized in the literature (4, 5, 25). They tend to be soft and fertile for shear transformations. Such a correlation, albeit statistical (not all soft modes or GUMs would be activated to undergo shear transformations for a given stress state/magnitude and loading duration), is very useful and important as a step forward in establishing a concrete structure–property relationship for MGs, i.e., a direct connection between short-range order and vibrational soft modes, as well as stress-induced atomic rearrangements. The spatial distribution of nanometer-scale patches observed in Fig. 4 and Fig. S3 (a 3D view from outside the MD box is in Fig. S4), in terms of property (soft spots) and corresponding structure (GUMs), may also help explain the origin of the heterogeneity in local elastic modulus and local viscoelasticity recently mapped out in experiments (26–28).     

Multiplex PCR for Detection of Seven Virulence Factors and K1/K2 Capsular Serotypes of Klebsiella pneumoniae

A single multiplex PCR assay targeting seven virulence factors and the wzi gene specific for the K1 and K2 capsular serotypes of Klebsiella pneumoniae was developed and tested on 65 clinical isolates, which included 45 isolates responsible for community-acquired severe human infections. The assay is useful for the surveillance of emerging highly virulent strains.