Background Slow crack growth can be described in a v (crack velocity) versus K I  (stress intensity factor) diagram. Slow crack growth in ceramics is attributed to corrosion assisted stress at the crack tip or at any pre-existing defect in the ceramic. The combined effect of high stresses at the crack tip and the presence of water or body fluid molecules (reducing surface energy at the crack tip) induces crack propagation, which eventually may result in fatigue. The presence of a threshold in the stress intensity factor, below which no crack propagation occurs, has been the subject of important research in the last years. The higher this threshold, the higher the reliability of the ceramic, and consequently the longer its lifetime.
Background
Slow crack growth can be described in a v (crack velocity) versus K I  (stress intensity factor) diagram. Slow crack growth in ceramics is attributed to corrosion assisted stress at the crack tip or at any pre-existing defect in the ceramic. The combined effect of high stresses at the crack tip and the presence of water or body fluid molecules (reducing surface energy at the crack tip) induces crack propagation, which eventually may result in fatigue. The presence of a threshold in the stress intensity factor, below which no crack propagation occurs, has been the subject of important research in the last years. The higher this threshold, the higher the reliability of the ceramic, and consequently the longer its lifetime.Methods We utilize the Irwin K-field displacement relation to deduce crack tip stress intensity factors from the near crack tip profile. Cracks are initiated by indentation impressions. The threshold stress intensity factor is determined as the time limit of the tip stress intensity when the residual stresses have (nearly) disappeared.
Methods
We utilize the Irwin K-field displacement relation to deduce crack tip stress intensity factors from the near crack tip profile. Cracks are initiated by indentation impressions. The threshold stress intensity factor is determined as the time limit of the tip stress intensity when the residual stresses have (nearly) disappeared.Results We determined the threshold stress intensity factors for most of the all ceramic materials presently important for dental restorations in Europe. Of special significance is the finding that alumina ceramic has a threshold limit nearly identical with that of zirconia.
Results
We determined the threshold stress intensity factors for most of the all ceramic materials presently important for dental restorations in Europe. Of special significance is the finding that alumina ceramic has a threshold limit nearly identical with that of zirconia.Conclusion The intention of the present paper is to stress the point that the threshold stress intensity factor represents a more intrinsic property for a given ceramic material than the widely used toughness (bend strength or fracture toughness), which refers only to  fast  crack growth. Considering two ceramics with identical threshold limits, although with different critical stress intensity limits, means that both ceramics have identical starting points for  slow  crack growth. Fast catastrophic crack growth leading to spontaneous fatigue, however, is different. This growth starts later in those ceramic materials that have larger critical stress intensity factors.
Conclusion
The intention of the present paper is to stress the point that the threshold stress intensity factor represents a more intrinsic property for a given ceramic material than the widely used toughness (bend strength or fracture toughness), which refers only to  fast  crack growth. Considering two ceramics with identical threshold limits, although with different critical stress intensity limits, means that both ceramics have identical starting points for  slow  crack growth. Fast catastrophic crack growth leading to spontaneous fatigue, however, is different. This growth starts later in those ceramic materials that have larger critical stress intensity factors.fastslow

Background Slow crack growth is most suitably described in a  v  (crack velocity) versus  K I  (stress intensity factor) diagram. Slow crack growth in ceramics is attributed to corrosion assisted stress at crack tips or at any defect pre-existing in the ceramic [ 1 ]. The combined presence of body fluid molecules (mainly water), which reduce the surface energy at the crack tip, and the presence of high stresses are the reasons for subcritical crack growth (SCCG) in ceramics. The presence of stress intensities above a critical value (K I  > K Ic ) initiates fast catastrophic crack growth, followed by the deterioration of a dental or a body restoration machined from ceramics. The presence of stress intensities above a threshold value (K I  > K I0 ) initiates SCCG in ceramics, followed by a slow, however continuous, erosion of the strength of a restoration which also may result in final fatigue. In an early stage of ceramic research it was believed that this lower limit for SCCG is very close to zero. In the mean time, however, one has learned that for most ceramic materials the lower limit for SCCG is significantly larger than zero. Indeed, it may even be just below  K Ic . The threshold limit  K I0  corresponds to a crack equilibrium at null crack velocity. Therefore, it allows a safety range of clinical use. The higher the value of  K I0 , the higher the reliability, and hence the lifetime of a restoration. Bio-components should be designed to work in a region of the  v-K I - diagram where the upper border line of that region corresponds to the threshold limit. In the present paper we preferentially focus on those ceramics that are important in dental research. Note, however, that alumina and zirconia have meaning in both fields of application (dentistry and medicine). We use soda lime glass as a well characterized standard and silicon nitride as important in the general field of ceramics. There are several methods available and in the literature extensively described how the threshold limit can be measured. The feasibility of these measurement procedures is mostly demonstrated with the help of soda lime glass as a brittle solid model. In principle, the proper test for existence of a threshold lies in the observation of reversibility of crack growth. The threshold can be regarded as a Griffith quiescent point, where forward and backward fluctuations just balance, i.e., the mean velocity of the crack tip becomes zero. The forward and backward fluctuations take place over discrete energy barriers definable as  G  =  W  = 2 γ , where G is the energy release rate,  W  is the Dupré work of adhesion, and  γ  is the surface energy. If  G  < W  the crack should retract and heal; otherwise it should repropagate [ 2 ]. On the basis of this assumption, the authors in [ 2 ] (see also [ 3 ]) calculate equations prescribing the  v G  characteristics (crack velocity versus mechanical energy release rate; equivalent to  v  -  K I  crack velocity versus stress intensity factor) at specified chemical concentrations and temperatures, which can describe observed  v-G  dependencies. One common experimental method to determine the threshold limit is to measure slow crack growth rate down to velocities as low as 10 -14  m/s. Then one can extrapolate from the vertical branch of the function to the zero velocity limit on the stress intensity factor axis K I , with the intersection K I  equal to K I0  [ 4 - 7 ]. Another method to determine the aforesaid threshold limit is the "interrupted static fatigue test" (ISF-test) [ 8 ]. For a bending experiment, the applied stress is chosen such that a significant fraction of samples fails in a "hold period". Samples that do not fail during this static phase are then fractured by the usual four point bending technique. The threshold is calculated either from the applied stress intensity factor at which 50% of samples fail during the stress hold, or by using the factor applied to the weakest specimen during the stress hold as calculated for various hold times. Once the value of the stress intensity factor becomes independent of hold time, it is equivalent to the threshold [ 9 ]. Another method uses a side grooved specimen with a crack propagating along its length, and under a bending condition similar to four point bending. The crack velocity can be obtained from the rate of load relaxation at constant displacement and the initial crack length. Having established the  v  -  K  diagram, the threshold is determined as described above. For further details refer to [ 10 ]. Other methods may be characterized by the phrase "decay of residual stress" [ 11 ]. Here, the threshold limit can be calculated from the residual stress factor attributed to this decay of residual stress. The current method of measurement used, however, is based on indentation cracking, analogous to other studies also utilizing flaw initiation for starting the test [ 11 - 13 ]. After this start, however, the subsequent procedure is different. A follow up of the decay of residual stress intensities near the crack tip is done over a period of about one year, determining K tip  via the COD for different times after indentation [ 14 ].
Background
Slow crack growth is most suitably described in a  v  (crack velocity) versus  K I  (stress intensity factor) diagram. Slow crack growth in ceramics is attributed to corrosion assisted stress at crack tips or at any defect pre-existing in the ceramic [ 1 ]. The combined presence of body fluid molecules (mainly water), which reduce the surface energy at the crack tip, and the presence of high stresses are the reasons for subcritical crack growth (SCCG) in ceramics.vKIThe presence of stress intensities above a critical value (K I  > K Ic ) initiates fast catastrophic crack growth, followed by the deterioration of a dental or a body restoration machined from ceramics. The presence of stress intensities above a threshold value (K I  > K I0 ) initiates SCCG in ceramics, followed by a slow, however continuous, erosion of the strength of a restoration which also may result in final fatigue. In an early stage of ceramic research it was believed that this lower limit for SCCG is very close to zero. In the mean time, however, one has learned that for most ceramic materials the lower limit for SCCG is significantly larger than zero. Indeed, it may even be just below  K Ic .KIcThe threshold limit  K I0  corresponds to a crack equilibrium at null crack velocity. Therefore, it allows a safety range of clinical use. The higher the value of  K I0 , the higher the reliability, and hence the lifetime of a restoration. Bio-components should be designed to work in a region of the  v-K I - diagram where the upper border line of that region corresponds to the threshold limit.KI0KI0v-KI-In the present paper we preferentially focus on those ceramics that are important in dental research. Note, however, that alumina and zirconia have meaning in both fields of application (dentistry and medicine). We use soda lime glass as a well characterized standard and silicon nitride as important in the general field of ceramics.There are several methods available and in the literature extensively described how the threshold limit can be measured. The feasibility of these measurement procedures is mostly demonstrated with the help of soda lime glass as a brittle solid model.In principle, the proper test for existence of a threshold lies in the observation of reversibility of crack growth. The threshold can be regarded as a Griffith quiescent point, where forward and backward fluctuations just balance, i.e., the mean velocity of the crack tip becomes zero. The forward and backward fluctuations take place over discrete energy barriers definable as  G  =  W  = 2 γ , where G is the energy release rate,  W  is the Dupré work of adhesion, and  γ  is the surface energy. If  G  < W  the crack should retract and heal; otherwise it should repropagate [ 2 ]. On the basis of this assumption, the authors in [ 2 ] (see also [ 3 ]) calculate equations prescribing the  v G  characteristics (crack velocity versus mechanical energy release rate; equivalent to  v  -  K I  crack velocity versus stress intensity factor) at specified chemical concentrations and temperatures, which can describe observed  v-G  dependencies.GWγWγGWv GvKIv-GOne common experimental method to determine the threshold limit is to measure slow crack growth rate down to velocities as low as 10 -14  m/s. Then one can extrapolate from the vertical branch of the function to the zero velocity limit on the stress intensity factor axis K I , with the intersection K I  equal to K I0  [ 4 - 7 ].Another method to determine the aforesaid threshold limit is the "interrupted static fatigue test" (ISF-test) [ 8 ]. For a bending experiment, the applied stress is chosen such that a significant fraction of samples fails in a "hold period". Samples that do not fail during this static phase are then fractured by the usual four point bending technique. The threshold is calculated either from the applied stress intensity factor at which 50% of samples fail during the stress hold, or by using the factor applied to the weakest specimen during the stress hold as calculated for various hold times. Once the value of the stress intensity factor becomes independent of hold time, it is equivalent to the threshold [ 9 ].Another method uses a side grooved specimen with a crack propagating along its length, and under a bending condition similar to four point bending. The crack velocity can be obtained from the rate of load relaxation at constant displacement and the initial crack length. Having established the  v  -  K  diagram, the threshold is determined as described above. For further details refer to [ 10 ].vKOther methods may be characterized by the phrase "decay of residual stress" [ 11 ]. Here, the threshold limit can be calculated from the residual stress factor attributed to this decay of residual stress.The current method of measurement used, however, is based on indentation cracking, analogous to other studies also utilizing flaw initiation for starting the test [ 11 - 13 ]. After this start, however, the subsequent procedure is different. A follow up of the decay of residual stress intensities near the crack tip is done over a period of about one year, determining K tip  via the COD for different times after indentation [ 14 ].Methods Using a micro-hardness testing machine, a Vickers indentation is made on the carefully polished surface of a sample of the ceramic to be investigated. Radial cracks emanate from each of the four indentated corner sources. To determine the stress intensity present at the crack tip due to the indentation, the near crack tip profile is determined using a scanning microscope (ESEM: "Environmental Scanning Electron Microscope"). A specific feature of this technique is that it is carried out at a moderate vacuum (p ≈ 10 -1  mbar). Hence, there is no longer need to sputter the samples with a gold or carbon layer. Our initial attempts to measure the crack opening displacement (COD) showed that sputtering resulted in blurring the crack banks or even partly hiding the crack. Thus, we abandoned those attempts and started again when the ESEM was available. Before the availability of the ESEM it was nearly impossible to precisely measure crack profiles at submicrometer resolution which, however, is mandatory. Images of the crack profiles (Fig.  1 ) were digitally stored and analyzed by imaging software (Paint Shop Pro, V. 6, Jasc Software, Eden Prairie, Maine, USA). Figure 1 Example of a Vickers indentation. Only one of four corners is shown (length of diagonal 115  μ m). With the crack tip as a starting point ( x  = 0) the crack width  2*u(x)  is measured at the distance  x  (COD after  Irwin  [15]; ceramic material for this example: Empress 1). The residual tensions cause crack growth over a long time interval until, at the end of the crack,  K tip  is equal to  K I0 . Crack tip shielding by secondary effects (micro structural elements which toughen material as the crack extends) may slightly distort results (measured  K I0  then lower than true  K I0 ). Insert: idealized COD. The measured profiles can be attributed to the crack opening displacement (COD) near the crack tip [ 14 ]. The near crack tip profiles for stress-free crack surfaces are usually represented by the Irwin K-field displacement relation [ 15 ], with 2u being the total COD,  x  the distance from the crack tip, and the plane strain Young's modulus  E'  =  E /(1- v 2 );( v  = Poisson's constant) being. We assume that there is no crack shielding. Then, in equilibrium, the currently acting crack tip stress intensity factor  K tip  is balanced by the toughness of the material  K Ic  (mode I loading [ 15 ]): and by re-arrangement: If data are taken sufficiently close to the crack tip ( x  ≤ 20  μ m), a linear relationship is experimentally observed between  u(x) 2  and  x .  K tip  can then be calculated from a regression analysis as the slope of a straight line, provided  E'  is known (see below). The residual stresses close to the crack tip initiated by the indentation impression gradually decay over time  t , and one anticipates that they slowly fade away eventually approaching zero. Hence  K tip =  K tip (t)  and it is plausible to assume  K tip ( t →∞) ≈  K I0 . Therefore, in the present work, because of slow crack growth, we take the threshold value of the stress intensity factor as the time limit of the slowly decreasing  K tip  value. Provided that a suitable high resolution scanning microscope is at hand, there is no need of sputtering the samples, and the presently utilized method is very simple. A potential shortcoming, however, is that this method may need many months or even years until the residual stresses are relaxed and the threshold value is reached. The authors concede that they have chosen to consider a somewhat ideal situation since the assumption  K tip ( t →∞) =  K I0  assumes ideal behavior. In real ceramics, especially polycrystalline and composite materials, the crack tip may be shielded from residual load by micro structural elements, which toughen the material in the region just before the crack tip [ 2 ]. This behavior is reminiscent to R-curve behavior. We carried out ESEM analyses of crack profiles after 1 hour and then after up to 420 days, at 5 dates distributed over the whole time interval (Fig.  3 ). After indentation and between two measurements the samples were stored at normal lab environmental conditions (21°C, 65 % humidity). We determined the threshold stress intensity of the following ceramics (Soda lime glass and veneering ceramics as reference): Al 2 O 3 , coarse grained, load of indention 9,9 kg, Young's modulus 350 GPa (Frialit-Degussit, Mannheim/Ludwigshafen, Germany), Cerec Mark II, 4 kg, 69 GPa, HiCeram, 6,9 kg, 107 GPa, VMK 95, 4 kg, 91 GPa (all three Vita, Bad Säckingen, Germany), Cercon Base, 7,9 kg, 210 GPa, CergoGold, 4 kg, 70 GPa (both Degudent-Dentsply, Hanau, Germany), Dicor, 2 kg, 74 GPa (Corning Glass Works, Corning, USA), Empress 1, 5,9 kg, 67 GPa, Empress 2, 5,9 kg, 96 GPa (both Ivoclar, Schaan, Liechtenstein), Lava, 8 kg, 210 GPa (3M-Espe, Seefeld, Germany), Soda lime glass, 2 kg, 73 GPa (Saint Gobain, Aachen, Germany), Si 3 N 4 , 6 kg, 289 GPa and hipped 5%Y 2 O 3 -Zirkon, 8 kg, 210 GPa. The constitution of the soda lime glass was SiO 2  72.65, Al 2 O 3  0.28, MgO 3.98, CaO 8.84, Na 2 O 13.79, K 2 O 0.19, other 0.27.
Methods
Using a micro-hardness testing machine, a Vickers indentation is made on the carefully polished surface of a sample of the ceramic to be investigated. Radial cracks emanate from each of the four indentated corner sources.To determine the stress intensity present at the crack tip due to the indentation, the near crack tip profile is determined using a scanning microscope (ESEM: "Environmental Scanning Electron Microscope"). A specific feature of this technique is that it is carried out at a moderate vacuum (p ≈ 10 -1  mbar). Hence, there is no longer need to sputter the samples with a gold or carbon layer. Our initial attempts to measure the crack opening displacement (COD) showed that sputtering resulted in blurring the crack banks or even partly hiding the crack. Thus, we abandoned those attempts and started again when the ESEM was available. Before the availability of the ESEM it was nearly impossible to precisely measure crack profiles at submicrometer resolution which, however, is mandatory.Images of the crack profiles (Fig.  1 ) were digitally stored and analyzed by imaging software (Paint Shop Pro, V. 6, Jasc Software, Eden Prairie, Maine, USA).The measured profiles can be attributed to the crack opening displacement (COD) near the crack tip [ 14 ]. The near crack tip profiles for stress-free crack surfaces are usually represented by the Irwin K-field displacement relation [ 15 ], with 2u being the total COD,  x  the distance from the crack tip, and the plane strain Young's modulus  E'  =  E /(1- v 2 );( v  = Poisson's constant) being.xE'EvvWe assume that there is no crack shielding. Then, in equilibrium, the currently acting crack tip stress intensity factor  K tip  is balanced by the toughness of the material  K Ic  (mode I loading [ 15 ]):KtipKIcand by re-arrangement:If data are taken sufficiently close to the crack tip ( x  ≤ 20  μ m), a linear relationship is experimentally observed between  u(x) 2  and  x .  K tip  can then be calculated from a regression analysis as the slope of a straight line, provided  E'  is known (see below).xμu(x)xKtipE'The residual stresses close to the crack tip initiated by the indentation impression gradually decay over time  t , and one anticipates that they slowly fade away eventually approaching zero. Hence  K tip =  K tip (t)  and it is plausible to assume  K tip ( t →∞) ≈  K I0 . Therefore, in the present work, because of slow crack growth, we take the threshold value of the stress intensity factor as the time limit of the slowly decreasing  K tip  value. Provided that a suitable high resolution scanning microscope is at hand, there is no need of sputtering the samples, and the presently utilized method is very simple. A potential shortcoming, however, is that this method may need many months or even years until the residual stresses are relaxed and the threshold value is reached.tKtipKtip(t)KtiptKI0KtipThe authors concede that they have chosen to consider a somewhat ideal situation since the assumption  K tip ( t →∞) =  K I0  assumes ideal behavior. In real ceramics, especially polycrystalline and composite materials, the crack tip may be shielded from residual load by micro structural elements, which toughen the material in the region just before the crack tip [ 2 ]. This behavior is reminiscent to R-curve behavior.KtiptKI0We carried out ESEM analyses of crack profiles after 1 hour and then after up to 420 days, at 5 dates distributed over the whole time interval (Fig.  3 ). After indentation and between two measurements the samples were stored at normal lab environmental conditions (21°C, 65 % humidity).We determined the threshold stress intensity of the following ceramics (Soda lime glass and veneering ceramics as reference): Al 2 O 3 , coarse grained, load of indention 9,9 kg, Young's modulus 350 GPa (Frialit-Degussit, Mannheim/Ludwigshafen, Germany), Cerec Mark II, 4 kg, 69 GPa, HiCeram, 6,9 kg, 107 GPa, VMK 95, 4 kg, 91 GPa (all three Vita, Bad Säckingen, Germany), Cercon Base, 7,9 kg, 210 GPa, CergoGold, 4 kg, 70 GPa (both Degudent-Dentsply, Hanau, Germany), Dicor, 2 kg, 74 GPa (Corning Glass Works, Corning, USA), Empress 1, 5,9 kg, 67 GPa, Empress 2, 5,9 kg, 96 GPa (both Ivoclar, Schaan, Liechtenstein), Lava, 8 kg, 210 GPa (3M-Espe, Seefeld, Germany), Soda lime glass, 2 kg, 73 GPa (Saint Gobain, Aachen, Germany), Si 3 N 4 , 6 kg, 289 GPa and hipped 5%Y 2 O 3 -Zirkon, 8 kg, 210 GPa.The constitution of the soda lime glass was SiO 2  72.65, Al 2 O 3  0.28, MgO 3.98, CaO 8.84, Na 2 O 13.79, K 2 O 0.19, other 0.27.Results As examples, Fig.  1  shows a crack starting at the corner of a Vickers indentation (right hand) and Fig.  2  shows a plot representing data for "Cerec Mark II" two days after indentation, as a function of distance from crack tip  x  (2  μ m < x  < 23  μ m), analyzed with the help of Eq. 1'. A linear relationship is observed, from which the value of  K tip  ( t  =  48 h ) =  0,90 MPa√m  was easily and precisely deduced. Fig.  3  shows all  K tip  values determined in an analogous manner for nine examples out of the thirteen investigated ceramics. The gradual decrease of  K tip (t)  due to decaying stress intensities at the crack tip becomes apparent. The manner in which  K tip (t)  decreases suggests an exponential relationship, as the decrease appears to be linear on a logarithmic scale. The truncation of the measurements after about 10 4  hours (for reasons of feasibility) appears somewhat arbitrarily, and it cannot be excluded that a further decay, although very small, may have been missed. Note that due to the apparent exponential relationship, the overestimation of the threshold value  K I0  due to the truncation after 10 4  hours becomes smaller and smaller with time. We plan to do further measurements after another interval of 10 4  hours (417 days). Considering the mathematical aspect, 10 5  hours (11+ years) would make more sense; but such a long interval is obviously not practicable. As already mentioned, this time constraint is a decided disadvantage of our current method to determine the threshold value. Figure 2 Regression analysis representing data for "Cerec Mark II" two days after indentation, analyzed with the help of Eq. 1 ( u(x) 2  as a function of distance from crack tip  x  (2  μ m < x  < 23  μ m)). A linear relationship is observed. Being aware of the these limitations, and having in mind the neglected possible crack tip shielding as discussed above, we identify  K I0 =  K tip ( t →∞). Fig.  4  displays all  K I0  values in comparison with their  K Ic  counterparts. Figure 3 K tip ( t ) values of nine out of the thirteen ceramics investigated. The gradual decrease of  K tip ( t ) with time due to decaying stress intensity at the crack tip becomes apparent. Figure 4 K I0  threshold values (hatched columns) in comparison with their counterpart critical stress intensities,  K Ic  (unfilled columns). Refer also to [22]. In the available literature, values for reference: Al 2 O 3  (K I0  = 2.5 ± 0.2 MPa√m); ZrO 2  (K I0  = 3.1 ± 0.2 MPa√m, both values after [4]); Soda lime glass (K I0  = 0,42 MPa√m, after [11]).
Results
As examples, Fig.  1  shows a crack starting at the corner of a Vickers indentation (right hand) and Fig.  2  shows a plot representing data for "Cerec Mark II" two days after indentation, as a function of distance from crack tip  x  (2  μ m < x  < 23  μ m), analyzed with the help of Eq. 1'. A linear relationship is observed, from which the value of  K tip  ( t  =  48 h ) =  0,90 MPa√m  was easily and precisely deduced. Fig.  3  shows all  K tip  values determined in an analogous manner for nine examples out of the thirteen investigated ceramics. The gradual decrease of  K tip (t)  due to decaying stress intensities at the crack tip becomes apparent. The manner in which  K tip (t)  decreases suggests an exponential relationship, as the decrease appears to be linear on a logarithmic scale. The truncation of the measurements after about 10 4  hours (for reasons of feasibility) appears somewhat arbitrarily, and it cannot be excluded that a further decay, although very small, may have been missed. Note that due to the apparent exponential relationship, the overestimation of the threshold value  K I0  due to the truncation after 10 4  hours becomes smaller and smaller with time. We plan to do further measurements after another interval of 10 4  hours (417 days). Considering the mathematical aspect, 10 5  hours (11+ years) would make more sense; but such a long interval is obviously not practicable. As already mentioned, this time constraint is a decided disadvantage of our current method to determine the threshold value.xμxμKtipt48 h0,90 MPa√mKtipKtip(t)Ktip(t)KI0Being aware of the these limitations, and having in mind the neglected possible crack tip shielding as discussed above, we identify  K I0 =  K tip ( t →∞). Fig.  4  displays all  K I0  values in comparison with their  K Ic  counterparts.KI0KtiptKI0KIcDiscussion K Ic  is the lower limit for (fast) catastrophic crack growth. Stress intensities exceeding this limit cause fast crack growth at supersonic velocity, and eventually result in destruction of ceramic components. This kind of destruction, however, is not the most common or important, since it can be avoided by strictly limiting the stress intensities existing throughout a component by a suitable shape of construction. K I0  is the upper limit of stress intensities for absence of crack growth and the lower limit for (slow) subcritical crack growth (SCCG). Limiting stress intensities such that they stay always below  K I0  means infinite life time for a component, since SCCG becomes irrelevant. Hence, the most favorable characteristic stress intensity values are obvious:  K Ic  as high as possible and  K I0  as close as possible to  K Ic . Such a selection minimizes the extension of the interval in which subcritical crack growth can take place, and it maximizes resistance to catastrophic crack growth due to overloading. Fig.  5  gives a ranking of all ceramics currently tested, based on threshold values related to the corresponding critical values  K I0 /K Ic . Favorable ceramics within their class of toughness are situated at the right hand side of the chart. Note, however, that a perfect ceramic material dependent on the focused area of application has not only a favorable (threshold/critical) stress strength relationship but also a high  K Ic  value. Figure 5 Ranking of all ceramics as imposed by their ratio "threshold value to critical value" ( K I0 / K Ic ). Dicor: see [23]. At first glance zirconia may seem to be a ceramic material superior to alumina, since it has a critical stress intensity factor (Fig.  4 : 9.4 ± 1.5 MPa·√m) which is about three times larger than this of alumina. Values in the literature for zirconia are up to about 8 MPa·√m [ 16 ], compared with 5.4 MPa·√m and [ 5 ]: 5.0 ± 0.2 MPa·√m [ 17 ] for alumina. Naturally, this is a significant advantage when operations near the critical stress of a material are involved. However, in practical applications, stresses having an intermediate level are more common, thus initiating SCCG instead of catastrophic crack growth. Then, if the threshold stress intensities of two ceramics are equal, they are both subject to SCCG at the same rate. Apparently. zirconia vs alumina is an example for such a situation (Fig.  4 ): meaning that both ceramics have equal potential for SCCG. The different behavior of these ceramics is solely rendered to stress bearing capabilities near catastrophic crack growth. At such stresses near  K Ic  zirconia, of course, has properties superior to alumina. It becomes apparent that at moderate stresses alumina and zirconia may be equally suitable choices, and other criteria may become important for favoring the one or the other material. Such reasons may be the ease of shaping, questions of color, ease of veneering, esthetic considerations, availability, and other circumstances. There is one other aspect to be considered when comparing zirconia and alumina. The exponents  n  of SCCG of both ceramics are high (in principle meaning slow SCCG), and the answer to the question of which of the materials has the larger exponent depends on whether static or cyclic behavior is addressed:  n static  = 39 vs 104 and  n cyclic  = 28 vs 16 for Al 2 O 3  and Y-PSZ, respectively [ 16 ]. These parameters show that lifetimes are shortened and crack growth rates are significantly accelerated by cyclic loading compared to static loading. Zirconia is known to be sensitive to humidity, which is a particular important issue when prosthetic and orthopedic applications are considered. It is known that yttria stabilized zirconia ceramics can be destabilized during the process of steam sterilization. This is due to hydrothermal transformation, resulting in surface roughening of the zirconia ceramic femoral heads. These femoral heads may also undergo slow degradation during long term implantation in the human body. This low temperature degradation does not become significant before several years, but it does raise the question of the use of zirconia for load bearing systems [ 4 ]. In conclusion, it can be stated that SCCG of Y-TZP is activated by the influence of water [ 18 , 19 ], however, there is some controversy about this effect [ 20 ]. An analogous statement holds for MgO-partially stabilized zirconia (PSZ) [ 21 ]. Note that concerning the sensitivity to humidity, there is a notable difference between ceramics for dental or for orthopedic applications. Ceramics for dental applications are often veneered by a different ceramic, which means that there is a protective shield against humidity attacking from outside of the ceramic tooth (but not from inside or from the marginal region). Fig.  6  displays an example of a zirconia ceramic material developed for dental applications and which was formerly used. The sensitivity to humidity becomes apparent. Figure 6 Example (linear Weibull plot) for a zirconia based ceramic material developed for dental applications. Samples handled at 60 % relative humidity (lab environmental conditions; diamonds) vs samples stored in aqua dest for 10 days (triangles). The sensitivity to humidity is obvious. The bending strength due to water storage decreases from  σ 63%  = 1,346 MPa to 1,003 MPa (about 25 %). There are some other examples of ceramics for which a large difference in the critical stress intensities is observed whereas the threshold values are very similar. For these ceramics an analogous argument holds, as given above for alumina vs zirconia. From Fig.  4 , for Empress 1 or Empress 2 (e.g.) the following values are measured: K Ic  = 1.17 ± 0.08 MPa·√m or K Ic  = 2.48 ± 0.22 MPa·√m, respectively; and K I0  = 0.83 ± 0.16 MPa·√m or K I0  = 0.94 ± 0.12 MPa·√m, respectively. Again, the critical stress intensity values are largely different, the threshold values, however, are nearly identical. Compare also "Al 2 O 3 " with "Lava" and "Cercon".
Discussion
K Ic  is the lower limit for (fast) catastrophic crack growth. Stress intensities exceeding this limit cause fast crack growth at supersonic velocity, and eventually result in destruction of ceramic components. This kind of destruction, however, is not the most common or important, since it can be avoided by strictly limiting the stress intensities existing throughout a component by a suitable shape of construction.KIcK I0  is the upper limit of stress intensities for absence of crack growth and the lower limit for (slow) subcritical crack growth (SCCG). Limiting stress intensities such that they stay always below  K I0  means infinite life time for a component, since SCCG becomes irrelevant. Hence, the most favorable characteristic stress intensity values are obvious:  K Ic  as high as possible and  K I0  as close as possible to  K Ic . Such a selection minimizes the extension of the interval in which subcritical crack growth can take place, and it maximizes resistance to catastrophic crack growth due to overloading. Fig.  5  gives a ranking of all ceramics currently tested, based on threshold values related to the corresponding critical values  K I0 /K Ic . Favorable ceramics within their class of toughness are situated at the right hand side of the chart. Note, however, that a perfect ceramic material dependent on the focused area of application has not only a favorable (threshold/critical) stress strength relationship but also a high  K Ic  value.KI0KI0KIcKI0KIcKI0/KIcKIcAt first glance zirconia may seem to be a ceramic material superior to alumina, since it has a critical stress intensity factor (Fig.  4 : 9.4 ± 1.5 MPa·√m) which is about three times larger than this of alumina. Values in the literature for zirconia are up to about 8 MPa·√m [ 16 ], compared with 5.4 MPa·√m and [ 5 ]: 5.0 ± 0.2 MPa·√m [ 17 ] for alumina. Naturally, this is a significant advantage when operations near the critical stress of a material are involved. However, in practical applications, stresses having an intermediate level are more common, thus initiating SCCG instead of catastrophic crack growth. Then, if the threshold stress intensities of two ceramics are equal, they are both subject to SCCG at the same rate. Apparently. zirconia vs alumina is an example for such a situation (Fig.  4 ): meaning that both ceramics have equal potential for SCCG. The different behavior of these ceramics is solely rendered to stress bearing capabilities near catastrophic crack growth. At such stresses near  K Ic  zirconia, of course, has properties superior to alumina.KIcIt becomes apparent that at moderate stresses alumina and zirconia may be equally suitable choices, and other criteria may become important for favoring the one or the other material. Such reasons may be the ease of shaping, questions of color, ease of veneering, esthetic considerations, availability, and other circumstances.There is one other aspect to be considered when comparing zirconia and alumina. The exponents  n  of SCCG of both ceramics are high (in principle meaning slow SCCG), and the answer to the question of which of the materials has the larger exponent depends on whether static or cyclic behavior is addressed:  n static  = 39 vs 104 and  n cyclic  = 28 vs 16 for Al 2 O 3  and Y-PSZ, respectively [ 16 ]. These parameters show that lifetimes are shortened and crack growth rates are significantly accelerated by cyclic loading compared to static loading.nnnZirconia is known to be sensitive to humidity, which is a particular important issue when prosthetic and orthopedic applications are considered. It is known that yttria stabilized zirconia ceramics can be destabilized during the process of steam sterilization. This is due to hydrothermal transformation, resulting in surface roughening of the zirconia ceramic femoral heads. These femoral heads may also undergo slow degradation during long term implantation in the human body. This low temperature degradation does not become significant before several years, but it does raise the question of the use of zirconia for load bearing systems [ 4 ]. In conclusion, it can be stated that SCCG of Y-TZP is activated by the influence of water [ 18 , 19 ], however, there is some controversy about this effect [ 20 ]. An analogous statement holds for MgO-partially stabilized zirconia (PSZ) [ 21 ].Note that concerning the sensitivity to humidity, there is a notable difference between ceramics for dental or for orthopedic applications. Ceramics for dental applications are often veneered by a different ceramic, which means that there is a protective shield against humidity attacking from outside of the ceramic tooth (but not from inside or from the marginal region).Fig.  6  displays an example of a zirconia ceramic material developed for dental applications and which was formerly used. The sensitivity to humidity becomes apparent.There are some other examples of ceramics for which a large difference in the critical stress intensities is observed whereas the threshold values are very similar. For these ceramics an analogous argument holds, as given above for alumina vs zirconia. From Fig.  4 , for Empress 1 or Empress 2 (e.g.) the following values are measured: K Ic  = 1.17 ± 0.08 MPa·√m or K Ic  = 2.48 ± 0.22 MPa·√m, respectively; and K I0  = 0.83 ± 0.16 MPa·√m or K I0  = 0.94 ± 0.12 MPa·√m, respectively. Again, the critical stress intensity values are largely different, the threshold values, however, are nearly identical. Compare also "Al 2 O 3 " with "Lava" and "Cercon".Authors' contributions RM conceived in the study, designed the study and drafted the manuscript. POW and FJ carried out the experimental work. All authors read and approved the final manuscript. All authors contributed equally to this work.
Authors' contributions
RM conceived in the study, designed the study and drafted the manuscript. POW and FJ carried out the experimental work. All authors read and approved the final manuscript. All authors contributed equally to this work.


Background Salmonella  spp. with reduced susceptibility to fluoroquinolones have higher than usual MICs to these agents but are still considered "susceptible" by NCCLS criteria. Delayed treatment response to fluoroquinolones has been noted, especially in cases of enteric fever due to such strains. We reviewed the ciprofloxacin susceptibility and clinical outcome of our recent enteric fever cases.
Background
Salmonella  spp. with reduced susceptibility to fluoroquinolones have higher than usual MICs to these agents but are still considered "susceptible" by NCCLS criteria. Delayed treatment response to fluoroquinolones has been noted, especially in cases of enteric fever due to such strains. We reviewed the ciprofloxacin susceptibility and clinical outcome of our recent enteric fever cases.SalmonellaMethods Salmonella enterica  Serotype Typhi ( S . Typhi) and Serotype Paratyphi ( S . Paratyphi) blood culture isolates (1998–2002) were tested against nalidixic acid by disk diffusion (DD) and agar dilution (AD) and to ciprofloxacin by AD using NCCLS methods and interpretive criteria. Reduced fluoroquinolone susceptibility was defined as a ciprofloxacin MIC of 0.125–1.0 mg/L. The clinical records of patients treated with ciprofloxacin for isolates with reduced fluoroquinolone susceptibility were reviewed.
Methods
Salmonella enterica  Serotype Typhi ( S . Typhi) and Serotype Paratyphi ( S . Paratyphi) blood culture isolates (1998–2002) were tested against nalidixic acid by disk diffusion (DD) and agar dilution (AD) and to ciprofloxacin by AD using NCCLS methods and interpretive criteria. Reduced fluoroquinolone susceptibility was defined as a ciprofloxacin MIC of 0.125–1.0 mg/L. The clinical records of patients treated with ciprofloxacin for isolates with reduced fluoroquinolone susceptibility were reviewed.Salmonella entericaSSResults Seven of 21 (33%)  S . Typhi and  S . Paratyphi isolates had reduced susceptibility to fluoroquinolones (MIC range 0.125–0.5 mg/L). All 7 were nalidixic acid resistant by DD (no zone) and by AD (MIC 128- >512 mg/L). The other 14 isolates were nalidixic acid susceptible and fully susceptible to ciprofloxacin (MIC range 0.015–0.03 mg/L). Five of the 7 cases were treated initially with oral ciprofloxacin. One patient remained febrile on IV ciprofloxacin until cefotaxime was added, with fever recurrence when cefotaxime was discontinued. Two continued on oral or IV ciprofloxacin alone but had prolonged fevers of 9–10 days duration, one was switched to IV beta-lactam therapy after remaining febrile for 3 days on oral/IV ciprofloxacin and one was treated successfully with oral ciprofloxacin. Four of the 5 required hospitalization.
Results
Seven of 21 (33%)  S . Typhi and  S . Paratyphi isolates had reduced susceptibility to fluoroquinolones (MIC range 0.125–0.5 mg/L). All 7 were nalidixic acid resistant by DD (no zone) and by AD (MIC 128- >512 mg/L). The other 14 isolates were nalidixic acid susceptible and fully susceptible to ciprofloxacin (MIC range 0.015–0.03 mg/L).SSFive of the 7 cases were treated initially with oral ciprofloxacin. One patient remained febrile on IV ciprofloxacin until cefotaxime was added, with fever recurrence when cefotaxime was discontinued. Two continued on oral or IV ciprofloxacin alone but had prolonged fevers of 9–10 days duration, one was switched to IV beta-lactam therapy after remaining febrile for 3 days on oral/IV ciprofloxacin and one was treated successfully with oral ciprofloxacin. Four of the 5 required hospitalization.Conclusions Our cases provide further evidence that reduced fluoroquinolone susceptibility of  S . Typhi and  S . Paratyphi is clinically significant. Laboratories should test extra-intestinal  Salmonella  spp. for reduced fluoroquinolone susceptibility.
Conclusions
Our cases provide further evidence that reduced fluoroquinolone susceptibility of  S . Typhi and  S . Paratyphi is clinically significant. Laboratories should test extra-intestinal  Salmonella  spp. for reduced fluoroquinolone susceptibility.SSSalmonella

Background Salmonella enterica  Serotype Typhi ( S . Typhi) and Serotype Paratyphi ( S . Paratyphi) with reduced susceptibility to fluoroquinolones are common in India and Southeast Asia [ 1 ]. Such isolates have elevated minimum inhibitory concentrations (MICs) to ciprofloxacin and other fluoroquinolones, although they are still considered "susceptible" using current National Committee for Clinical Laboratory Standards (NCCLS) interpretive criteria [ 2 ]. Reduced susceptibility to fluoroquinolones most often arises from point mutations in the quinolone resistance determining region (QRDR) of the  gyrA  gene which encodes the A subunit of DNA gyrase. These mutations lead to resistance to nalidixic acid, a quinolone, and resistance to this agent can thus be used as an indicator of reduced fluoroquinolone susceptibility [ 3 ]. Although the mechanisms of resistance have been defined, there is still little information on the clinical significance of reduced susceptibility to fluoroquinolones in  Salmonella . Limited published data suggest that treatment of these infections with a fluoroquinolone may result in a delay in clinical response and possibly treatment failure [ 2 ]. As a result of this clinical data, NCCLS currently recommends testing of extra-intestinal  Salmonella  isolates for nalidixic acid resistance as a marker for reduced fluoroquinolone susceptibility [ 4 ]. Others have suggested a re-evaluation of the current fluoroquinolone MIC breakpoints for  Salmonella  [ 2 ] while some recommend differentiating between "low level resistance" (MICs of 0.125 – 1.0 mg/L) and "high level resistance" (MICs >1.0 mg/L) [ 5 ]. A poor response to ciprofloxacin therapy in a patient with typhoid fever at our hospital prompted us to review retrospectively the ciprofloxacin MICs and clinical outcomes of other enteric fever cases that were treated with a fluoroquinolone.
Background
Salmonella enterica  Serotype Typhi ( S . Typhi) and Serotype Paratyphi ( S . Paratyphi) with reduced susceptibility to fluoroquinolones are common in India and Southeast Asia [ 1 ]. Such isolates have elevated minimum inhibitory concentrations (MICs) to ciprofloxacin and other fluoroquinolones, although they are still considered "susceptible" using current National Committee for Clinical Laboratory Standards (NCCLS) interpretive criteria [ 2 ].Salmonella entericaSSReduced susceptibility to fluoroquinolones most often arises from point mutations in the quinolone resistance determining region (QRDR) of the  gyrA  gene which encodes the A subunit of DNA gyrase. These mutations lead to resistance to nalidixic acid, a quinolone, and resistance to this agent can thus be used as an indicator of reduced fluoroquinolone susceptibility [ 3 ].gyrAAlthough the mechanisms of resistance have been defined, there is still little information on the clinical significance of reduced susceptibility to fluoroquinolones in  Salmonella . Limited published data suggest that treatment of these infections with a fluoroquinolone may result in a delay in clinical response and possibly treatment failure [ 2 ].SalmonellaAs a result of this clinical data, NCCLS currently recommends testing of extra-intestinal  Salmonella  isolates for nalidixic acid resistance as a marker for reduced fluoroquinolone susceptibility [ 4 ]. Others have suggested a re-evaluation of the current fluoroquinolone MIC breakpoints for  Salmonella  [ 2 ] while some recommend differentiating between "low level resistance" (MICs of 0.125 – 1.0 mg/L) and "high level resistance" (MICs >1.0 mg/L) [ 5 ].SalmonellaSalmonellaA poor response to ciprofloxacin therapy in a patient with typhoid fever at our hospital prompted us to review retrospectively the ciprofloxacin MICs and clinical outcomes of other enteric fever cases that were treated with a fluoroquinolone.Methods We tested 21  S . Typhi and  S . Paratyphi blood culture isolates recovered from patients at The Ottawa Hospital and The Children's Hospital of Eastern Ontario between 1998–2002. Susceptibility testing was performed using NCCLS methods and interpretive criteria [ 4 , 6 , 7 ]. Disk Diffusion testing was performed using nalidixic acid (30 μg), ampicillin (10 μg), trimethoprim-sulfamethoxazole (1.25 μg/23.75 μg), and chloramphenicol (30 μg) disks (Oxoid, U.K.) on Mueller-Hinton agar plates (PML Microbiologicals, Mississauga, ON). MICs to nalidixic acid (Sigma-Aldrich, St-Louis, MO) and ciprofloxacin (Bayer Inc.) were determined by agar dilution using Mueller-Hinton agar (Becton Dickinson and Company, Sparks, MD). Reduced susceptibility to fluoroquinolones was defined as a ciprofloxacin MIC of 0.125–1.0 mg/L [ 3 ]. We sequenced the QRDR region of the  gyrA  gene for all isolates with reduced susceptibility to fluoroquinolones to detect nucleotide mutations. PCR amplification and DNA sequencing was performed using primers [ 8 ] and conditions previously described [ 9 ]. Sequencing was performed by the Ottawa Genome Centre DNA Sequencing Institute using the Big Dye Terminator v 3.1 method. Sequences were compared to the  gyrA  sequence of the fully susceptible  S.  Typhimurium NCTC 74 (accession number X78977, EMBL GenBank database) [ 8 ] and to 3 fully susceptible  S . Typhi isolates. Pulsed field gel electrophoresis (PFGE) was performed on isolates with reduced susceptibility to fluoroquinolone using  XbaI . Medical records of patients who were treated with ciprofloxacin for infections due to isolates with reduced fluoroquinolone susceptibility were reviewed to determine clinical outcomes.
Methods
We tested 21  S . Typhi and  S . Paratyphi blood culture isolates recovered from patients at The Ottawa Hospital and The Children's Hospital of Eastern Ontario between 1998–2002. Susceptibility testing was performed using NCCLS methods and interpretive criteria [ 4 , 6 , 7 ]. Disk Diffusion testing was performed using nalidixic acid (30 μg), ampicillin (10 μg), trimethoprim-sulfamethoxazole (1.25 μg/23.75 μg), and chloramphenicol (30 μg) disks (Oxoid, U.K.) on Mueller-Hinton agar plates (PML Microbiologicals, Mississauga, ON). MICs to nalidixic acid (Sigma-Aldrich, St-Louis, MO) and ciprofloxacin (Bayer Inc.) were determined by agar dilution using Mueller-Hinton agar (Becton Dickinson and Company, Sparks, MD). Reduced susceptibility to fluoroquinolones was defined as a ciprofloxacin MIC of 0.125–1.0 mg/L [ 3 ].SSWe sequenced the QRDR region of the  gyrA  gene for all isolates with reduced susceptibility to fluoroquinolones to detect nucleotide mutations. PCR amplification and DNA sequencing was performed using primers [ 8 ] and conditions previously described [ 9 ]. Sequencing was performed by the Ottawa Genome Centre DNA Sequencing Institute using the Big Dye Terminator v 3.1 method. Sequences were compared to the  gyrA  sequence of the fully susceptible  S.  Typhimurium NCTC 74 (accession number X78977, EMBL GenBank database) [ 8 ] and to 3 fully susceptible  S . Typhi isolates.gyrAgyrAS.SPulsed field gel electrophoresis (PFGE) was performed on isolates with reduced susceptibility to fluoroquinolone using  XbaI . Medical records of patients who were treated with ciprofloxacin for infections due to isolates with reduced fluoroquinolone susceptibility were reviewed to determine clinical outcomes.XbaIResults Laboratory Seven of 21 isolates or 33% (4 of 12  S . Typhi and 3 of 9  S.  Paratyphi) were nalidixic acid resistant by disk diffusion, all with no zones of inhibition. These isolates were all nalidixic acid resistant with an MIC range of 128->512 mg/L, and demonstrated reduced susceptibility to fluoroquinolones (ciprofloxacin MIC range 0.125–0.5 mg/L). The 14 nalidixic acid susceptible isolates by disk diffusion (zone size range 20–25 mm) were also susceptible by agar dilution (MIC range 2–4 mg/L), and none had reduced susceptibility to fluoroquinolones (ciprofloxacin MIC range 0.015–0.03 mg/L). Multidrug resistance was identified in 5 of 21 isolates (24%) with resistance to ampicillin, trimethoprim-sulfamethoxazole, and chloramphenicol. All 7 isolates with reduced susceptibility to fluoroquinolones had nucleotide point mutations in the  gyrA  gene that resulted in amino acid substitutions: 5 had a Ser83 to Phe change, one had a Ser83 to Tyr change, and one had an Asp87 to Asn change. PFGE of the 7 resistant isolates showed 3 of 4  S . Typhi to have identical patterns. These 3 strains were also multidrug resistant to ampicillin, trimethoprim-sulfamethoxazole and chloramphenicol. The fourth strain, which was susceptible to ampicillin, trimethoprim-sulfamethoxazole and chloramphenicol, was a closely related strain (2 band difference). Two of 3  S.  Paratyphi had identical PFGE patterns, with the third being possibly related (4 band difference) [ 10 ]. All 3 isolates of  S . Paratyphi were susceptible to the other antibiotics tested. Clinical (Table 1- see  Additional file 1 ) Infections with the 7 isolates with reduced susceptibility to fluoroquinolones were all acquired in the Indian subcontinent. Five of the 7 patients with infections due to these isolates were treated initially with ciprofloxacin. The clinical and microbiological findings for these 5 are summarized in Table 1 (see  Additional file 1 ).  S.  Typhi isolates from cases 1, 3 and 4 were also resistant to ampicillin, trimethoprim-sulfamethoxazole and chloramphenicol. The  Salmonella  isolates from the other 2 cases were susceptible to these antibiotics. Of note, 3 of 4 patients with  S . Typhi had fever durations well in excess of the expected 95% upper confidence limit of 3.9 days found in a pooled analysis of multiple studies. These studies involved patients with predominantly nalidixic acid susceptible  S . Typhi who were treated with fluoroquinolones [ 1 ]. The patient infected with  S . Paratyphi with reduced susceptibility to fluoroquinolone was judged to be failing ciprofloxacin, since there was no clinical improvement after 3 days of treatment. Although fever of this duration is common, patients with enteric fever given fluoroquinolones may show some clinical improvement by this time. Three days with no amelioration of symptoms has been used as indicator of a poor response to therapy in some studies [ 11 , 12 ]. Thus, this patient possibly may have had a poor response to therapy due to the elevated ciprofloxacin MIC. None of the 5 had a recurrence of bacteremia after antibiotics were discontinued, and none died.
Results
Laboratory Seven of 21 isolates or 33% (4 of 12  S . Typhi and 3 of 9  S.  Paratyphi) were nalidixic acid resistant by disk diffusion, all with no zones of inhibition. These isolates were all nalidixic acid resistant with an MIC range of 128->512 mg/L, and demonstrated reduced susceptibility to fluoroquinolones (ciprofloxacin MIC range 0.125–0.5 mg/L). The 14 nalidixic acid susceptible isolates by disk diffusion (zone size range 20–25 mm) were also susceptible by agar dilution (MIC range 2–4 mg/L), and none had reduced susceptibility to fluoroquinolones (ciprofloxacin MIC range 0.015–0.03 mg/L). Multidrug resistance was identified in 5 of 21 isolates (24%) with resistance to ampicillin, trimethoprim-sulfamethoxazole, and chloramphenicol. All 7 isolates with reduced susceptibility to fluoroquinolones had nucleotide point mutations in the  gyrA  gene that resulted in amino acid substitutions: 5 had a Ser83 to Phe change, one had a Ser83 to Tyr change, and one had an Asp87 to Asn change. PFGE of the 7 resistant isolates showed 3 of 4  S . Typhi to have identical patterns. These 3 strains were also multidrug resistant to ampicillin, trimethoprim-sulfamethoxazole and chloramphenicol. The fourth strain, which was susceptible to ampicillin, trimethoprim-sulfamethoxazole and chloramphenicol, was a closely related strain (2 band difference). Two of 3  S.  Paratyphi had identical PFGE patterns, with the third being possibly related (4 band difference) [ 10 ]. All 3 isolates of  S . Paratyphi were susceptible to the other antibiotics tested.
Laboratory
Seven of 21 isolates or 33% (4 of 12  S . Typhi and 3 of 9  S.  Paratyphi) were nalidixic acid resistant by disk diffusion, all with no zones of inhibition. These isolates were all nalidixic acid resistant with an MIC range of 128->512 mg/L, and demonstrated reduced susceptibility to fluoroquinolones (ciprofloxacin MIC range 0.125–0.5 mg/L). The 14 nalidixic acid susceptible isolates by disk diffusion (zone size range 20–25 mm) were also susceptible by agar dilution (MIC range 2–4 mg/L), and none had reduced susceptibility to fluoroquinolones (ciprofloxacin MIC range 0.015–0.03 mg/L). Multidrug resistance was identified in 5 of 21 isolates (24%) with resistance to ampicillin, trimethoprim-sulfamethoxazole, and chloramphenicol.SS.All 7 isolates with reduced susceptibility to fluoroquinolones had nucleotide point mutations in the  gyrA  gene that resulted in amino acid substitutions: 5 had a Ser83 to Phe change, one had a Ser83 to Tyr change, and one had an Asp87 to Asn change.gyrAPFGE of the 7 resistant isolates showed 3 of 4  S . Typhi to have identical patterns. These 3 strains were also multidrug resistant to ampicillin, trimethoprim-sulfamethoxazole and chloramphenicol. The fourth strain, which was susceptible to ampicillin, trimethoprim-sulfamethoxazole and chloramphenicol, was a closely related strain (2 band difference). Two of 3  S.  Paratyphi had identical PFGE patterns, with the third being possibly related (4 band difference) [ 10 ]. All 3 isolates of  S . Paratyphi were susceptible to the other antibiotics tested.SS.SClinical (Table 1- see  Additional file 1 ) Infections with the 7 isolates with reduced susceptibility to fluoroquinolones were all acquired in the Indian subcontinent. Five of the 7 patients with infections due to these isolates were treated initially with ciprofloxacin. The clinical and microbiological findings for these 5 are summarized in Table 1 (see  Additional file 1 ).  S.  Typhi isolates from cases 1, 3 and 4 were also resistant to ampicillin, trimethoprim-sulfamethoxazole and chloramphenicol. The  Salmonella  isolates from the other 2 cases were susceptible to these antibiotics. Of note, 3 of 4 patients with  S . Typhi had fever durations well in excess of the expected 95% upper confidence limit of 3.9 days found in a pooled analysis of multiple studies. These studies involved patients with predominantly nalidixic acid susceptible  S . Typhi who were treated with fluoroquinolones [ 1 ]. The patient infected with  S . Paratyphi with reduced susceptibility to fluoroquinolone was judged to be failing ciprofloxacin, since there was no clinical improvement after 3 days of treatment. Although fever of this duration is common, patients with enteric fever given fluoroquinolones may show some clinical improvement by this time. Three days with no amelioration of symptoms has been used as indicator of a poor response to therapy in some studies [ 11 , 12 ]. Thus, this patient possibly may have had a poor response to therapy due to the elevated ciprofloxacin MIC. None of the 5 had a recurrence of bacteremia after antibiotics were discontinued, and none died.
Clinical (Table 1- see  Additional file 1 )
Infections with the 7 isolates with reduced susceptibility to fluoroquinolones were all acquired in the Indian subcontinent. Five of the 7 patients with infections due to these isolates were treated initially with ciprofloxacin. The clinical and microbiological findings for these 5 are summarized in Table 1 (see  Additional file 1 ).  S.  Typhi isolates from cases 1, 3 and 4 were also resistant to ampicillin, trimethoprim-sulfamethoxazole and chloramphenicol. The  Salmonella  isolates from the other 2 cases were susceptible to these antibiotics. Of note, 3 of 4 patients with  S . Typhi had fever durations well in excess of the expected 95% upper confidence limit of 3.9 days found in a pooled analysis of multiple studies. These studies involved patients with predominantly nalidixic acid susceptible  S . Typhi who were treated with fluoroquinolones [ 1 ].S.SalmonellaSSThe patient infected with  S . Paratyphi with reduced susceptibility to fluoroquinolone was judged to be failing ciprofloxacin, since there was no clinical improvement after 3 days of treatment. Although fever of this duration is common, patients with enteric fever given fluoroquinolones may show some clinical improvement by this time. Three days with no amelioration of symptoms has been used as indicator of a poor response to therapy in some studies [ 11 , 12 ]. Thus, this patient possibly may have had a poor response to therapy due to the elevated ciprofloxacin MIC.SNone of the 5 had a recurrence of bacteremia after antibiotics were discontinued, and none died.Discussion Fluoroquinolones have been considered the treatment of choice for enteric fever. As noted, a mean fever clearance time of only 3.9 days (for predominantly nalidixic acid-susceptible strains) has been reported, which is shorter than for other agents [ 1 ]. Calculated clinical and microbiologic failure rates of 2.1% and 0.4%, respectively, are also low in comparison to other antibiotics [ 1 ]. Relapse rates (1.2%) and fecal carriage rates (1.5%) have also been lower than for the traditional drugs, trimethoprim-sulfamethoxazole and chloramphenicol. However, reduced susceptibility to fluoroquinolones jeopardises the usefulness of these agents. It is now seen frequently in  S . Typhi and  S . Paratyphi isolates acquired in the Indian subcontinent and Southeast Asia [ 1 ], where common clones appear to be circulating. In fact, the common PFGE profile of 3 of our  S . Typhi isolates with reduced susceptibility to fluoroquinolones appears to be the same as that described for  S . Typhi strains from India, Pakistan, Bangladesh, and Tajikistan [ 13 ]. The  gyrA  point mutations in our isolates have also been previously described in isolates from these countries [ 1 , 3 ]. Several lines of evidence support the clinical significance of elevated fluoroquinolone MICs in  Salmonella  spp., including case reports, animal experiments, and pharmacodynamic modelling [ 2 ]. In a report of patients treated with short courses of oral ofloxacin [ 11 ], the median time to fever clearance was greater for patients infected with nalidixic acid resistant  S . Typhi than for those with nalidixic acid susceptible strains. In addition, one third of nalidixic acid resistant infections required re-treatment vs. 0.8% of infections due to susceptible strains. The authors concluded that short courses (less than 5 days) of oral fluoroquinolone therapy should not be used for treating nalidixic acid resistant isolates. Our small series adds to the evidence that there is a delayed clinical response to fluoroquinolone therapy in this setting, and demonstrates that fever may be prolonged even with long courses of fluoroquinolone therapy, and even when given parenterally in some cases. Until results from randomized controlled trials of enteric fever due to strains with reduced susceptibility to fluoroquinolones are available, the best treatment regimen is uncertain. Current options include higher dose and longer duration of fluoroquinolones, 3rd generation cephalosporins and azithromycin, either alone or in combination [ 1 ]. If susceptible, agents such as ampicillin, trimethoprim-sulfamethoxazole, and chloramphenicol may also be considered, but rates of resistance to these agents are generally too high to recommend as first-line empiric therapies [ 1 ]. In our series, resistance to all these agents was high among the isolates with reduced fluoroquinolone susceptibility (3 of 7 or 43%). When fluoroquinolones are given, it is important to bear in mind that two pharmacokinetic/pharmacodynamic ratios are predictive of successful treatment with these agents [ 2 ]. These are the ratio of peak serum antimicrobial level to the MIC, and the ratio of the 24-hour area under the serum concentration-versus-time curve to the MIC (AUC/MIC). Higher dosing will maximize these ratios, and thus when oral ciprofloxacin is given for suspected enteric fever, use of 750 mg rather than 500 mg bid seems logical. It is important that microbiology laboratories have procedures to detect  Salmonella  strains that have reduced susceptibility to fluoroquinolones. The current NCCLS guidelines (2004) recommend that all extra-intestinal isolates of  Salmonella  be tested for resistance to nalidixic acid in order to detect reduced fluoroquinolone susceptibility [ 4 ]. In addition, it is recommended that physicians be informed that isolates that test fluoroquinolone "susceptible" but nalidixic acid resistant may not be eradicated with fluoroquinolone therapy. There have also been suggestions to change the NCCLS breakpoints to reflect the risk of treatment failure of  Salmonella  spp. with reduced susceptibility to fluoroquinolones [ 1 ]. This is clearly a difficult decision, since fluoroquinolones have previously been the most active antibiotic class for treatment of enteric fever. Some patients with these isolates may do poorly on short courses and lower doses of fluoroquinolones but potentially could respond adequately to higher doses and longer durations of treatment. Rather than reclassifying all isolates with reduced susceptibility to fluoroquinolones as being resistant to ciprofloxacin, others have recommended differentiating between low-level (MIC of 0.125 – 1.0 mg/L) and high-level (MIC >1.0 mg/L) ciprofloxacin resistance based on the MIC range [ 5 ]. It is not completely clear at present which of these is the optimal approach for laboratory detection of  Salmonella  spp. with reduced fluoroquinolone susceptibility. Not all laboratories routinely perform MICs and many rely on commercially-available automated susceptibility methods that cannot currently detect reduced fluoroquinolone susceptibility. The current NCCLS recommendations to test for nalidixic resistance are easy to implement for routine testing but may have reduced specificity [ 3 ]. Laboratories need to decide which approach is best for their workflow and available resources.
Discussion
Fluoroquinolones have been considered the treatment of choice for enteric fever. As noted, a mean fever clearance time of only 3.9 days (for predominantly nalidixic acid-susceptible strains) has been reported, which is shorter than for other agents [ 1 ]. Calculated clinical and microbiologic failure rates of 2.1% and 0.4%, respectively, are also low in comparison to other antibiotics [ 1 ]. Relapse rates (1.2%) and fecal carriage rates (1.5%) have also been lower than for the traditional drugs, trimethoprim-sulfamethoxazole and chloramphenicol.However, reduced susceptibility to fluoroquinolones jeopardises the usefulness of these agents. It is now seen frequently in  S . Typhi and  S . Paratyphi isolates acquired in the Indian subcontinent and Southeast Asia [ 1 ], where common clones appear to be circulating. In fact, the common PFGE profile of 3 of our  S . Typhi isolates with reduced susceptibility to fluoroquinolones appears to be the same as that described for  S . Typhi strains from India, Pakistan, Bangladesh, and Tajikistan [ 13 ]. The  gyrA  point mutations in our isolates have also been previously described in isolates from these countries [ 1 , 3 ].SSSSgyrASeveral lines of evidence support the clinical significance of elevated fluoroquinolone MICs in  Salmonella  spp., including case reports, animal experiments, and pharmacodynamic modelling [ 2 ]. In a report of patients treated with short courses of oral ofloxacin [ 11 ], the median time to fever clearance was greater for patients infected with nalidixic acid resistant  S . Typhi than for those with nalidixic acid susceptible strains. In addition, one third of nalidixic acid resistant infections required re-treatment vs. 0.8% of infections due to susceptible strains. The authors concluded that short courses (less than 5 days) of oral fluoroquinolone therapy should not be used for treating nalidixic acid resistant isolates. Our small series adds to the evidence that there is a delayed clinical response to fluoroquinolone therapy in this setting, and demonstrates that fever may be prolonged even with long courses of fluoroquinolone therapy, and even when given parenterally in some cases.SalmonellaSUntil results from randomized controlled trials of enteric fever due to strains with reduced susceptibility to fluoroquinolones are available, the best treatment regimen is uncertain. Current options include higher dose and longer duration of fluoroquinolones, 3rd generation cephalosporins and azithromycin, either alone or in combination [ 1 ]. If susceptible, agents such as ampicillin, trimethoprim-sulfamethoxazole, and chloramphenicol may also be considered, but rates of resistance to these agents are generally too high to recommend as first-line empiric therapies [ 1 ]. In our series, resistance to all these agents was high among the isolates with reduced fluoroquinolone susceptibility (3 of 7 or 43%).When fluoroquinolones are given, it is important to bear in mind that two pharmacokinetic/pharmacodynamic ratios are predictive of successful treatment with these agents [ 2 ]. These are the ratio of peak serum antimicrobial level to the MIC, and the ratio of the 24-hour area under the serum concentration-versus-time curve to the MIC (AUC/MIC). Higher dosing will maximize these ratios, and thus when oral ciprofloxacin is given for suspected enteric fever, use of 750 mg rather than 500 mg bid seems logical.It is important that microbiology laboratories have procedures to detect  Salmonella  strains that have reduced susceptibility to fluoroquinolones. The current NCCLS guidelines (2004) recommend that all extra-intestinal isolates of  Salmonella  be tested for resistance to nalidixic acid in order to detect reduced fluoroquinolone susceptibility [ 4 ]. In addition, it is recommended that physicians be informed that isolates that test fluoroquinolone "susceptible" but nalidixic acid resistant may not be eradicated with fluoroquinolone therapy.SalmonellaSalmonellaThere have also been suggestions to change the NCCLS breakpoints to reflect the risk of treatment failure of  Salmonella  spp. with reduced susceptibility to fluoroquinolones [ 1 ]. This is clearly a difficult decision, since fluoroquinolones have previously been the most active antibiotic class for treatment of enteric fever. Some patients with these isolates may do poorly on short courses and lower doses of fluoroquinolones but potentially could respond adequately to higher doses and longer durations of treatment. Rather than reclassifying all isolates with reduced susceptibility to fluoroquinolones as being resistant to ciprofloxacin, others have recommended differentiating between low-level (MIC of 0.125 – 1.0 mg/L) and high-level (MIC >1.0 mg/L) ciprofloxacin resistance based on the MIC range [ 5 ].SalmonellaIt is not completely clear at present which of these is the optimal approach for laboratory detection of  Salmonella  spp. with reduced fluoroquinolone susceptibility. Not all laboratories routinely perform MICs and many rely on commercially-available automated susceptibility methods that cannot currently detect reduced fluoroquinolone susceptibility. The current NCCLS recommendations to test for nalidixic resistance are easy to implement for routine testing but may have reduced specificity [ 3 ]. Laboratories need to decide which approach is best for their workflow and available resources.SalmonellaConclusions Given the accumulating evidence, including our own clinical experience, enteric fever due to  Salmonella  spp. with reduced susceptibility to fluoroquinolones is clinically significant. It is important that laboratories test  S.  Typhi and  S . Paratyphi, as well as other extra-intestinal Salmonella isolates, for reduced susceptibility to fluoroquinolones.
Conclusions
Given the accumulating evidence, including our own clinical experience, enteric fever due to  Salmonella  spp. with reduced susceptibility to fluoroquinolones is clinically significant. It is important that laboratories test  S.  Typhi and  S . Paratyphi, as well as other extra-intestinal Salmonella isolates, for reduced susceptibility to fluoroquinolones.SalmonellaS.SCompeting interests None declared.
Competing interests
None declared.Authors' contributions RS: participated in the design of study and authored first draft; MD: conceived study, coordinated susceptibility testing, and revised manuscript; AM: conceived study and reviewed clinical data; KR: participated in study design and coordinated pulsed field gel analysis; PJ: reviewed clinical data and helped coordinate susceptibilty testing; CB: carried out molecular genetic studies and participated in sequence alignment; BT: conceived study and supervised study, revised manuscript All authors read and approved of the final manuscript.
Authors' contributions
RS: participated in the design of study and authored first draft; MD: conceived study, coordinated susceptibility testing, and revised manuscript; AM: conceived study and reviewed clinical data; KR: participated in study design and coordinated pulsed field gel analysis; PJ: reviewed clinical data and helped coordinate susceptibilty testing; CB: carried out molecular genetic studies and participated in sequence alignment; BT: conceived study and supervised study, revised manuscriptAll authors read and approved of the final manuscript.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here:Supplementary Material Additional File 1 Clinical and microbiological information for patients treated with ciprofloxacin for  S.  Typhi or  S.  Paratyphi isolates with reduced fluoroquinolone susceptibility provides information regarding clinical course for patients with isolates with reduced fluoroquinolone susceptibility as well as antibiotic susceptibility and gene mutation results for these isolates Click here for file
Supplementary Material
Additional File 1
Clinical and microbiological information for patients treated with ciprofloxacin for  S.  Typhi or  S.  Paratyphi isolates with reduced fluoroquinolone susceptibility provides information regarding clinical course for patients with isolates with reduced fluoroquinolone susceptibility as well as antibiotic susceptibility and gene mutation results for these isolatesS.S.Click here for file


Based on sheer numbers, microbes should rule the world. Most don't cause disease, but those that do have the advantage of multiplying and mutating at a much faster rate than any multicellular organism can. So how does a slowly reproducing, trillion-celled organism like a human protect itself? By having the right weapon for the job—and that requires an incredibly diverse arsenal. A new study by a team of researchers from Yale University School of Medicine, Duke University Medical Center, and Mount Sinai School of Medicine demonstrates how the creation of that arsenal depends on a complex series of interactions between key genetic elements and proteins during the formation of the white blood cells called lymphocytes.Two heavy hitters of the immune system—B and T cells—each produce unique protein receptors that specifically recognize and mediate the killing of the variety of potential foreign invaders, or antigens, such as bacteria, viruses, and parasites. (B cells make immunoglobulin, or antibodies, and T cells make T-cell receptors.) But these lymphocytes are unlike other cells: instead of making proteins from genes they inherited, they custom-make their genes by recombining fragments of their genes into new configurations. This genetic reshuffling process, called V(D)J recombination, yields the diversity of molecules necessary to combat the billions of different antigens they might encounter. The V, D, and J refer to different clusters of DNA sequences that follow specific rules of recombination.While the products of recombination vary, the method does not. The fragments are spliced and then reassembled in a highly regulated process directed and controlled by a stretch of DNA (called a recombination signal sequence, or RSS) next to the gene fragment. The recombination process, the researchers show, relies on complex interactions among different parts of the signal sequences and the proteins that regulate them at key steps along the recombination pathway.Each RSS is made up of three components: the nonamer, which controls the ability of proteins to bind to the gene fragments and initiate recombination; the heptamer, which directs the splicing of the gene fragment; and the spacer, which regulates how the gene fragments are recombined. Mutations in the DNA sequence of each of the three RSS components show that all play a critical role in the ability of the gene fragments to recombine appropriately.While it has been established that spacers, as their name suggests, ensure that the space between the nonamer and heptamer is correct, the researchers show that spacers also regulate recombination activity by providing protein-binding sites along the DNA sequences that affect recombination. While the nonamer is the most important determinant of recombination, changes in the spacer, these researchers demonstrate, produced dramatic changes in the ability of the gene fragments to recombine.Past studies have shown that recombination depends on the presence and sequence of specific nucleotides, but the quality of that recombination, the researchers say, can't be understood simply by analyzing those nucleotides in isolation. Generally speaking, highly conserved sequences have functional importance. But it would be a mistake, they suggest, to think that just because a nucleotide sequence isn't highly conserved, it's not biologically important. Using a computer model to predict how different protein-gene interactions affect recombination, the researchers demonstrate that a fuller understanding of the process depends on observing how all these elements—including those that aren't highly conserved—interact throughout the recombination process.


Background Almost one-third of all penile metastases are detected at the same time as a primary tumor, whereas the remaining two-thirds are detected a mean of 18 months after the discovery of the primary tumor. Cutaneous metastasis of transitional cell carcinoma (TCC) is extremely rare and generally accepted as the late manifestation of a systemic spread.
Background
Almost one-third of all penile metastases are detected at the same time as a primary tumor, whereas the remaining two-thirds are detected a mean of 18 months after the discovery of the primary tumor. Cutaneous metastasis of transitional cell carcinoma (TCC) is extremely rare and generally accepted as the late manifestation of a systemic spread.Case presentation We report the first case of simultaneous penile and lung metastases from a primary TCC of the renal pelvis in a 76-year-old man, that occurred 8 years after a left nephroureterectomy.
Case presentation
We report the first case of simultaneous penile and lung metastases from a primary TCC of the renal pelvis in a 76-year-old man, that occurred 8 years after a left nephroureterectomy.Conclusions This case report underscores the importance of physical examinations of the skin of patients who undergo surgical procedures for TCC from bladder as well as from the upper urinary tract, including those seemingly without metastatic disease, because of the possibility of skin and penile metastatic spread.
Conclusions
This case report underscores the importance of physical examinations of the skin of patients who undergo surgical procedures for TCC from bladder as well as from the upper urinary tract, including those seemingly without metastatic disease, because of the possibility of skin and penile metastatic spread.

Background Approximately 300 cases of penile metastases are reported worldwide [ 1 ]. Seventy percent of these metastases have their origin in the genitourinary tract, and the primary tumor is most frequently located in the bladder [ 2 ]. We present the first case of penile metastasis from primary transitional cell carcinoma (TCC) of the renal pelvis, and discuss the striking feature of discovering lung metastasis almost simultaneously.
Background
Approximately 300 cases of penile metastases are reported worldwide [ 1 ]. Seventy percent of these metastases have their origin in the genitourinary tract, and the primary tumor is most frequently located in the bladder [ 2 ]. We present the first case of penile metastasis from primary transitional cell carcinoma (TCC) of the renal pelvis, and discuss the striking feature of discovering lung metastasis almost simultaneously.Case presentation A 76-year-old man with a painful, ulcerative swelling of the glans that had appeared less than 2 months previously was brought to our attention. Physical examination revealed a 3.5-cm fungating lesion involving the upper half of the glans [Fig.  1 ]. The lesion was malodorous with varying amounts of seropurulent discharge, partially ulcerated, erythematous, and slightly itchy. The superficial inguinal lymph nodes were not palpable. No similar lesions were evident on the head, neck, trunk, or arms. The patient had been treated in another hospital 8 years previously for TCC of the left renal pelvis by nephroureterectomy (pT2 Nx M0 G2). Subsequent postoperative follow-ups (every 6 months for the first 3 years after surgery and then once every year) consisting of a clinical examination, total body CT scan, and regular endoscopic examination showed no evidence of recurrence and the patient did well until he noticed the painful penile nodule. Pathological examination of incisional biopsies confirmed the lesion to be urothelial carcinoma [Fig.  2 ]. A subsequent CT workup revealed a 4-cm lesion localized in the upper-right pulmonary lobe and enlarged pelvic lymph nodes suggestive of multiple metastases. The patient began combination chemotherapy (gemcitabine at 1250 mg/m2 on days 1, 8, and 15, and then every 28 days for six courses) and external beam radiotherapy to the mass, which promptly relieved the penile pain. At an 8-month follow-up the patient was still alive with no remarkable changes to the pulmonary and lymph node metastases, or the penile swelling.
Case presentation
A 76-year-old man with a painful, ulcerative swelling of the glans that had appeared less than 2 months previously was brought to our attention. Physical examination revealed a 3.5-cm fungating lesion involving the upper half of the glans [Fig.  1 ]. The lesion was malodorous with varying amounts of seropurulent discharge, partially ulcerated, erythematous, and slightly itchy. The superficial inguinal lymph nodes were not palpable. No similar lesions were evident on the head, neck, trunk, or arms. The patient had been treated in another hospital 8 years previously for TCC of the left renal pelvis by nephroureterectomy (pT2 Nx M0 G2). Subsequent postoperative follow-ups (every 6 months for the first 3 years after surgery and then once every year) consisting of a clinical examination, total body CT scan, and regular endoscopic examination showed no evidence of recurrence and the patient did well until he noticed the painful penile nodule. Pathological examination of incisional biopsies confirmed the lesion to be urothelial carcinoma [Fig.  2 ]. A subsequent CT workup revealed a 4-cm lesion localized in the upper-right pulmonary lobe and enlarged pelvic lymph nodes suggestive of multiple metastases.The patient began combination chemotherapy (gemcitabine at 1250 mg/m2 on days 1, 8, and 15, and then every 28 days for six courses) and external beam radiotherapy to the mass, which promptly relieved the penile pain. At an 8-month follow-up the patient was still alive with no remarkable changes to the pulmonary and lymph node metastases, or the penile swelling.Conclusion Cutaneous metastases from primary TCC of the urinary system are extremely rare and are generally accepted as late manifestations of systemic spread [ 1 ]. Spector et al. (1987) suggested that skin metastases in TCC were the result of increased longevity in successfully treated patients [ 3 ]. Various mechanisms by which lesions may metastasize to the penis have been reported. Recently Bordeau and Lynch and Berger et al. reported three cases of penile metastases from TCC of the bladder in which they assumed that metastatic spread from primary bladder cancer to the penis occurred mainly via the retrograde venous route [ 2 , 4 ]. There are also a few reports of TCC seeding outside the urinary tract after iatrogenic procedures (i.e., partial cystectomy, suprapubic cystostomy, pyelotomy, and laparoscopy) as the cause of cutaneous metastasis [ 1 , 5 , 6 ]. Between 5% and 20% of patients with superficial bladder cancer have vascular or lymphatic spread [ 5 ], and the reported rate of skin metastasis from TCC of the bladder is between 0.2% and 2% [ 7 ]. The rate of skin metastasis from TCC of the renal pelvis is currently unknown, and to the best of our knowledge ours is the first reported case of penile metastasis from TCC of the upper urinary tract. Almost one-third of all penile metastases are generally detected at the same time as a primary tumor, whereas the remaining two-thirds are detected a mean of 18 months after the discovery of the primary tumor [ 8 ]. Because the disease-free survival of this patient was 8 years following the diagnosis of the primary tumor, this case report represents a description of the biology of an unusual tumor. Moreover, the penile metastasis in this case was not the late manifestation of systemic spread, as is usually reported in the literature [ 1 ]; in fact, the patient was accurately evaluated with a standard follow-up scheme. Therefore, it is plausible that the lung metastasis appears almost simultaneously with the penile metastasis. The optimal treatment of penile metastasis requires a multidisciplinary approach that is correlated with the disease extent. The average survival in patients with penile metastasis is 3.9 months from diagnosis and, with extensive surgery and chemotherapy, a survival of 9.2 months has been reported [ 4 ]. This patient was not eligible for platinum-based regimens due to impaired renal function (prior nephrectomy) and advanced age. As a result, he was treated with gemcitabine as a single agent. This treatment has demonstrated objective response rates of 25–29% with minimal toxicity as compared to standard regimens [ 9 - 11 ]. In conclusion, this case report suggests that particular attention should be paid to physical examination of the skin of patients who undergo surgical procedures for TCC from bladder as well as from the upper urinary tract, including those seemingly without metastatic disease, because of the possibility of skin and penile metastatic spread.
Conclusion
Cutaneous metastases from primary TCC of the urinary system are extremely rare and are generally accepted as late manifestations of systemic spread [ 1 ]. Spector et al. (1987) suggested that skin metastases in TCC were the result of increased longevity in successfully treated patients [ 3 ]. Various mechanisms by which lesions may metastasize to the penis have been reported. Recently Bordeau and Lynch and Berger et al. reported three cases of penile metastases from TCC of the bladder in which they assumed that metastatic spread from primary bladder cancer to the penis occurred mainly via the retrograde venous route [ 2 , 4 ]. There are also a few reports of TCC seeding outside the urinary tract after iatrogenic procedures (i.e., partial cystectomy, suprapubic cystostomy, pyelotomy, and laparoscopy) as the cause of cutaneous metastasis [ 1 , 5 , 6 ]. Between 5% and 20% of patients with superficial bladder cancer have vascular or lymphatic spread [ 5 ], and the reported rate of skin metastasis from TCC of the bladder is between 0.2% and 2% [ 7 ]. The rate of skin metastasis from TCC of the renal pelvis is currently unknown, and to the best of our knowledge ours is the first reported case of penile metastasis from TCC of the upper urinary tract.Almost one-third of all penile metastases are generally detected at the same time as a primary tumor, whereas the remaining two-thirds are detected a mean of 18 months after the discovery of the primary tumor [ 8 ]. Because the disease-free survival of this patient was 8 years following the diagnosis of the primary tumor, this case report represents a description of the biology of an unusual tumor. Moreover, the penile metastasis in this case was not the late manifestation of systemic spread, as is usually reported in the literature [ 1 ]; in fact, the patient was accurately evaluated with a standard follow-up scheme. Therefore, it is plausible that the lung metastasis appears almost simultaneously with the penile metastasis.The optimal treatment of penile metastasis requires a multidisciplinary approach that is correlated with the disease extent. The average survival in patients with penile metastasis is 3.9 months from diagnosis and, with extensive surgery and chemotherapy, a survival of 9.2 months has been reported [ 4 ].This patient was not eligible for platinum-based regimens due to impaired renal function (prior nephrectomy) and advanced age. As a result, he was treated with gemcitabine as a single agent. This treatment has demonstrated objective response rates of 25–29% with minimal toxicity as compared to standard regimens [ 9 - 11 ].In conclusion, this case report suggests that particular attention should be paid to physical examination of the skin of patients who undergo surgical procedures for TCC from bladder as well as from the upper urinary tract, including those seemingly without metastatic disease, because of the possibility of skin and penile metastatic spread.Competing interests The author(s) declare that they have no competing interests.
Competing interests
The author(s) declare that they have no competing interests.Authors' contributions GP drafted the manuscript and coordinated the co-authors. IP, MS, PC participated in the sequences alignment. GM carried out the histological features of the lesion. FF participated in the sequences alignment and coordinated the co-authors. All authors read and approved the final manuscript.
Authors' contributions
GP drafted the manuscript and coordinated the co-authors. IP, MS, PC participated in the sequences alignment. GM carried out the histological features of the lesion. FF participated in the sequences alignment and coordinated the co-authors. All authors read and approved the final manuscript.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here:


A developing organism captured on time-lapse video is a wonder to behold. If you're watching a chick embryo, by day 3, you'll see millions of cells engaged in a frenzy of activity, as rapidly dividing cells migrate to new positions, acquire the characteristics of specialized cells, and craft well-defined tissues, organs, and limbs in just under two weeks. In addition to the cells destined for specialization is another important group, stem cells, whose progeny have two very different fates. They can either “self renew”—that is, make identical copies of themselves—or generate intermediate progenitor cells that give rise to mature, differentiated cells.Both differentiation and self renewal are guided by an elaborately regulated genetic program, which transforms embryonic stem cells into the many different cell types that make up the body. Adult stem cells share the hallmark trait of self renewal, but are relatively rare: in bone marrow, the source of hematopoietic, or blood-forming, only an estimated one in 10,000–15,000 cells is an adult hematopoietic stem cell (HSC).Studies that have compared the gene expression profiles of different types of stem cells to identify genetic signatures of “stemness” have found only a limited number of signature genes. And the molecular mechanisms that regulate this so-called potency and the self renewal process have remained obscure. Now, focusing on HSCs, Margaret Goodell and colleagues have undertaken a systematic evaluation of HSC renewal. The study identifies molecular signatures associated with discrete stages of the HSC self renewal cycle and proposes a molecular model of the process.HSC renewal passes through three stages: quiescence, activation and proliferation, and a return to the dormant state. HSCs give rise to both red blood cells, which carry oxygen and carbon dioxide, and white blood cells, which fight infection. Certain stressors—including blood-cell-inhibiting chemotherapy and bone marrow transplants—trigger HSC activation, which induces rapid proliferation, generating both progenitors to deal with the threat and new stem cells that return to quiescence. Once activated by a trigger, dormant HSCs engage a regulatory program that rapidly churns out billions of cells, then puts the brakes on cell division, prompting the return to a nondividing, quiescent state.To understand the genetic programs underlying this process, Goodell and colleagues induced proliferation in HSCs (with the chemotherapeutic drug, 5-fluorouracil, or 5FU), then allowed the cells to return to quiescence, so they could characterize the changes in gene expression that occurred during each stage. They compared these time-specific patterns to the gene expression profiles of naturally proliferating fetal mouse HSCs (which undergo massive proliferation) and quiescent adult mouse HSCs (which hardly divide at all) to find genes associated with the two different states.Genes were grouped into proliferating or quiescent groups based on when they were expressed after 5FU treatment, and these groupings were refined based on comparisons to previously published HSC gene expression data. Functional analysis of these genes found a bias toward genes involved in cell division processes in the proliferation stage and toward cell division inhibitors in the quiescent stage, supporting the logic of the groupings. To understand the activation process at a global level, the authors employed some novel analysis strategies, including the “Gene Ontology” (GO) system for classifying genes.With these results, Goodell and colleagues constructed a model of the HSC self renewal cycle: quiescent HSCs maintain a “state of readiness,” molecularly speaking, that allows a quick response to environmental triggers. A stressor (like the chemotherapy mentioned above) triggers a “prepare to proliferate” state—a kind of pregnant pause—and then the proliferation machinery kicks in, going through an early and late phase before quiescence returns. By shedding light on the molecular mechanisms of stem cell renewal, this study will aid efforts to develop stem-cell-based clinical therapies, which depend on replicating the HSC self renewal cycle to replenish diseased or damaged tissue, and will ultimately guide efforts to grow stem cell colonies outside the body, a long-standing goal that would have many clinical applications. The authors suggest their findings may also be relevant to studies of cancer stem cells, tumor cells with self renewal properties.