N G Almarza

Eva G Noya

Csic Noya

CONFERENCE on the Statistical Mechanics of Liquids

Keywords: patchy models, simulation, phase equilbria PACS numbers:

Phase transitions of a lattice-model for patchy particles

with tetrahedral symmetry

Simple anisotropic models are representative of interactions in a large variety of systems, such a water [1][2][3], proteins [4][5][6] or anisotropic colloids, and both theoretical and simulation studies of those models have contributed to improve our understanding of the behaviour of such systems. For example, for proteins it has been recently shown that the metastable fluidfluid phase separation experimentally measured for several proteins can be quantitatively reproduced by simple anisotropic models [7].

During the last few years there has been a renewed interest in these simple models, which is partly motivated by recent advances in colloidal science that allowed the synthesis of colloids with anisotropic shapes or interactions [8,9]. Indeed it is now possible to design colloids with a quite accurate control on the location of the patches on their surface [10].

Theoretical and simulation studies on simple anisotropic models revealed that they can exhibit a complex phase behaviour [11][12][13][14][15][16][17].

The use of lattice models has proven to be very fruitful [18][19][20][21][22][23][24][25][26][27][28] in the analysis of the phase diagram of simple models with complex phase behaviour, with the advantage that simulations can be performed for larger system sizes with higher accuracy. For example, it has been shown that it is possible to design associating lattice models that reproduce some of the behaviour of water, including the density and diffusion anomalies [22,27,28]. Another interesting example is provided by a lattice model with two repulsive ranges, which predicts the existence of a body-centred-cubic (BCC) solid that exhibits reentrant melting, i.e. the BCC solid can melt either upon compression and upon expansion in a region of the phase diagram [24]. These two examples illustrate that simple lattice models are able to capture the complex behaviour of simple models.

In this work, we will compute the phase diagram of a lattice model that mimics the behaviour of patchy particles consisting on a spherical repulsive core with four interaction sites or patches in its surface with tetrahedral symmetry. The behaviour of tetrahedral particles has already been studied in the past using off-lattice models with the main objective of finding the appropriate conditions at which a solid with diamond structure could be obtained [29][30][31][32][33]. The interest in obtaining a colloidal crystal with diamond structure arises from its predicted optical band gap that makes it an ideal material for many applications in photonics [34][35][36]. The studies performed so far have shown that the phase diagram of tetrahedral particles exhibits multiple stable solid phases, that can include the diamond structure, a BCC solid, which is obtained by interpenetrating two diamond sublattices, and, at high pressures, a face-centred-cubic (FCC) structure. Both in the diamond and BCC solids the number of bonds is maximized, i.e. the four patches on a particle point directly at four nearest neighbours. As a consequence both solid structures have a similar potential energy. It has also been found that the stability of the diamond solid is very sensitive to the range of the potential [31][32][33], the stability of the diamond decreasing with respect to the BCC solid when the range of the potential increases and even disappearing from the phase diagram for long ranged models [33]. The stability of the diamond crystal for short ranged models has been attributed to the higher vibrational entropy of the diamond crystal as compared to that of the BCC solid [32,33].

We will check how much of the complex behaviour of the tetrahedral models in the continuum can be captured by a lattice model. The use of a lattice model will allow us to explore in detail the phase diagram as a function of the parameters of the model. The results obtained for the lattice model will be compared to those obtained by Romano et al. [31,32] and Noya et al. [33] using two different off-lattice models. This paper is organized as follows: In the following section, we introduce our lattice model. In section III we describe the procedures to compute the phase diagram of the system, paying attention to the limits of both low and high temperature, and high density.

In section IV the different phase diagrams are presented, and their topology is analyzed as a function of the parameters of the model. Finally in section V we discuss the analogies and differences between the results for lattice and off-lattice models and present the main conclusions of the work.

II. THE MODEL

The model proposed in this work will be denominated as the lattice patchy model (LPM) in reference to the off-lattice models that we try to mimic. It combines features of the socalled lattice hard sphere (LHS) models [20] with the orientationally dependent interactions of the three-dimensional associating lattice gas (ALG) model proposed by Girardi et al. [22,28] The model is defined on a simple cubic lattice with side length L = 8n, where n is a positive integer. The sites on the lattice can be either occupied by one particle or empty. A hard-core interaction between particles excludes the simultaneous occupation of two sites i and j located at distances shorter than r ij < σ = 2 √ 3d 0 , where d 0 is the distance between two nearest neighbours (NN) sites on the reference cubic lattice.

The total potential energy of the system can be calculated as:

U = i<j [V LHS (r ij ) + V p (r ij , S i , S j )] (1)
where

V LHS (r ij )/k B T = ∞ if r ij < σ
V p (r ij , S i , S j ) =    ǫ 0 -(ǫ + ǫ 0 )τ ij i τ ji j ; for r ij = σ 0 ; for r ij = σ ; ( 2
)
where τ ij i is equal to one if there is a patch of particle i pointing to particle j and zero otherwise. In this work, we took ǫ > 0, i.e. the interaction between two particles, each one with a patch pointing to the other particle, is always attractive. The parameter ǫ 0 (|ǫ 0 | < ǫ) accounts for the interaction between two particles when one or both of them do not have a patch pointing to the other particle.

All throughout this paper reduced units are used. Energies will be given in units of ǫ and distances are given in units of d 0 .

A. Justification of the model

The phase diagram of the ALG model on a BCC lattice has already been computed [28].

It has been found that it stabilizes a low density and a high density solid which are the lattice analogues of the diamond and BCC solids found for the off-lattice models that we try to mimic [31][32][33]. However, at high temperatures, transitions between the low density and high density ordered structures and between the fluid and high density solid become continuous, something that does not happen for the off-lattice tetrahedral models [28]. This can be avoided by including a hard-core in the interactions using the LHS model. The Here and throughout the paper the densities are expressed as packing fractions η = ρ/ρ 0 , where ρ = N/V is the density (V = L 3 is the volume of the system, i.e., the number of sites in the underlying lattice) and ρ 0 is the density at close packing.

For the model potential described above, we expect that at high temperature the system will behave as the corresponding LHS model, whereas at low temperature the patch interactions will influence the phase behaviour. The known phase behaviour of the ALG and LHS models makes advisable, therefore, to consider in addition to the anisotropic interactions of the ALG model some excluded volume effects in order to attain a lattice model with phase behaviour resembling that of the off-lattice tetrahedral patchy models. Moreover the choice of σ * = 2 √ 3, was found to be required (instead of σ * = √ 3) to assure that the density of the BCC phase stays well above that of the low density solid at any temperature. If either the ALG model [27] or σ * = √ 3 version of our model are used, then one finds that the transition between the low density and the high density solids becomes continuous at some temperature range.

III. COMPUTATION OF THE PHASE DIAGRAM

In this section we present the procedures used in the computation of the phase diagram, and the corresponding results. The phase diagram was computed using several simulation techniques that include Grand Canonical Monte Carlo, Grand Canonical Wang-Landau, and Gibbs-Duhem-like integration (GDI). The essential features of the methodology can be found elsewhere [24,25,37,38] and thus only a brief description is given here.

Given the relative complexity of the phase diagram, we first pay attention to the transitions occurring in the limits of close packing, and of high and low temperature, and the order-disorder transitions occurring at high density. Later we will focus on the global computation of equilibria between the fluid, low density solid, and high density solid phases at intermediate temperatures.

A. The system at close packing

At close packing conditions, the model considered in this work becomes equivalent to the ALG model at full occupancy. In the limit of T → ∞ the interactions are dominated by the repulsive core and the orientation of the patches is random (between the two possible discrete orientations), whereas at T = 0 the system is orientationally ordered, with each particle forming bonds (patch-patch interaction) with four of its eight neighbour particles located at a distance σ. The average potential energy per particle in these two limit cases is given by:

U/N =    -2ǫ + 2ǫ 0 ; η = 1, T = 0 -ǫ + 3ǫ 0 ; η = 1, T = ∞. (3)
Between these two limits the system exhibits an order-disorder transition at a finite temperature T * c = k B T c /ǫ that depends on the value of ǫ * 0 = ǫ 0 /ǫ. The order-disorder transition at close packing can be computed by realizing that it is possible to map the system at full occupancy to the antiferromagnetic Ising model (AFIM) on the BCC lattice. The procedure is similar to that reported for a two dimensional model of self-assembled rigid rods [39]. The energy of the system at full occupancy can be calculated as a sum of the energies of N four-particle plaquettes, with N being in this context equivalent to the maximum number of particles (N = N max ).

In Fig. 1, we show how the four-particle plaquettes can be defined. The plaquette depicted in the figure is associated to the site labelled as 0. All the plaquettes required to give account of the potential energy of the system can be built by translation of the origin of the plaquette (label 0) to each site of the BCC lattice. According to the definition of the orientation variables S i , a particle on the plaquette has a patch oriented to one of its NNs. If S i = 1 the patch on site i points to the site i + 1 (anticlockwise), whereas if S i = -1 the patch points to the site i -1 (clockwise).

It is then straightforward to compute the energy of the plaquette as a function of the values of S i for the current model at η = 1, and the corresponding values for the AFIM on the BCC lattice with NN interactions, and coupling constant J [40]. The results for representative configurations are gathered in table I.

Plaquette ++++ + + +-+ + --+ -+- LPM 4ǫ 0 3ǫ 0 -ǫ 3ǫ 0 -ǫ 2ǫ 0 -2ǫ AFIM 4J 0 0 -4J
The values of the potential energy in both models for a given plaquette configuration are related by: Therefore the Helmholtz energies of the two systems are related by:

u AFIM plaq = 4J ǫ + ǫ 0 u LPM plaq + 4J ǫ -3ǫ 0 ǫ + ǫ 0 . (4)
A AF IM (N, T I ) = 4N J ǫ -3ǫ 0 ǫ + ǫ 0 + A LP M (N, T ); (5)
with:

T = ǫ + ǫ 0 4J T I (6)
Using the standard reduced temperatures for the AFIM and LPM as

T * (AF IM ) c = k B T (AF IM ) c
/J, and T * c = k B T c /ǫ, respectively, the critical temperatures of both models are related by:

T * c = 1 + ǫ * 0 4 T * (AF IM ) c . (7)
Precise values of T * (AF IM ) c

on the BCC lattice can be found in the literature [40].

B. The limit of high temperature

At high temperature, k B T >> ǫ, the phase behaviour of the system is basically controlled by the hard-core interactions. The LHS model has already been studied in Ref. 20. However, in order to check our programs and to get a more accurate value of the equilibrium conditions we have carried out a series of Grand-Canonical Wang-Landau simulations [25,37] of the LHS model with σ * = 2 √ 3.

We have considered the following system sizes: L = 12, 16, 20, 24, 32, 36, and 40. For

(L) = [< η 2 > -< η > 2 ]
1/2 . The results are presented in table II.

The finite-size results for βµ t (L), η m (L), and δη(L) for systems of L ≥ 24 are well represented by linear functions of the inverse of the volume;

y t (L) = y t + aL -3 . (8)
Using Eq. ( 8) we get the following estimates for the transition at the thermodynamic limit: These results agree with those reported by Panagiotopoulos [20], which expressed in our units are βµ t = 2.86(2), η f = 0.545(2); η s = 0.942 (2). Note that the chemical potential definition used in this work differs from that given in Ref. [20]. In our case in the limit of very low density: βµ ≃ ln(N/L 3 ) = ln(ρd 3 0 ). In the limit of high temperature: β = 0, the canonical partition function of the LPM can be related to the canonical partition function of the LHS model as:

βµ t = 2.
Q LP M (β = 0, N, V ) = 2 N Q LHS (N, V ). ( 9
)
where N ≤ V /32. The factor 2 N appears due to the two possible orientations that a particle can exhibit in the patchy model. Therefore the chemical potential of the two models at the fluid-solid transition are related by:

βµ LP M t = βµ LHS t -ln 2 ≃ 2.1608 ± 0.0001. ( 10
)
C. Order-disorder transition of the high density solid

Given the features of the model, and the limits at close-packing and high temperature, one expects that the BCC phase will undergo a transition between an orientationally ordered BCC solid (o-BCC phase) and a disordered BCC solid (d-BCC phase) . This transition is expected to be continuous, and like that at η = 1, to belong to the three-dimensional Ising universality class. In order to compute the critical line, η c (T ) or µ c (T ), using the standard simulation procedures [41,[START_REF] Landau | A Guide to Monte Carlo Simulations in Statistical Physics[END_REF] it is very useful to find the appropriate order parameters. Firstly we can consider an order parameter to check whether the particles in the system exhibit a true BCC structure. This can be achieved by considering that the close packing BCC structure has a degeneracy g = 32. Accordingly, it is possible to define 32 sub-lattices so that each one contains L 3 /32 sites and represents one of the 32 possible degenerate configurations of the system.

The lattice points are defined by three integers (k x , k y , k z ) with 0 ≤ k α < L. Given a reference site k 0 = (k 0x , k 0y , k 0z ), the points belonging to the same sublattice are given by:

(k x , k y , k x ) =    k 0 + (4l, 4m, 4n) ; Sites A : t = +1 k 0 + (4l + 2, 4m + 2, 4n + 2) ; Sites B : t = -1 (11)
where l, m, and n are integers. The reference points k 0 are chosen to be those that fulfill

0 ≤ k x ≤ 1, 0 ≤ k y ≤ 3, 0 ≤ k z ≤ 3.
The points on the sublattice are classified in two types according to the parameter t.

For a given configuration of the system, we define a BCC order parameter, q, as:

q = N max SL N , (12)
where N max SL is the number of particles in the most populated sublattice and N is the total number of particles in the system. BCC phases are easily identified using q. For instance at the LHS limit the parameter q of the BCC phase at the transition is q ≃ 0.9987. This high value of q indicates that in the BCC solid most of the occupied positions are on the same sublattice, so that it is feasible to locate the order-disorder transition of the BCC lattice by considering a second order parameter, m, that takes into account exclusively the orientation of the particles staying at sites of the predominant sublattice. The parameter m is defined as:

m = i t i S i N max SL ( 13
)
where the sum is carried out only over the sites of the predominant sublattice, and S i = 0 for empty sites on the sublattice. In order to compute the critical line corresponding to the orientationally order-disorder transition of the BCC structure, we performed for different values of βµ a series of Grand Canonical Monte Carlo simulations around the expected critical temperature T c (βµ), for several system sizes. The pseudo-critical temperatures for each system size T c (L, βµ) can be estimated by following the behaviour of the fourth order Binder cumulant of the order parameter [START_REF] Landau | A Guide to Monte Carlo Simulations in Statistical Physics[END_REF]: g 4 =< m 4 > / < m 2 > 2 , as T c (L, βµ) is defined as the temperature at which g 4 reaches the universal value expected for the three dimensional Ising universality class for cubic boxes with periodic boundary conditions: g c 4 ≃ 1.604 [43]. The results for different system sizes at a given value of βµ can then be used to estimate the critical properties at the thermodynamic limit: T c (βµ) and η c (βµ); by fitting the simulation results using [41,[START_REF] Landau | A Guide to Monte Carlo Simulations in Statistical Physics[END_REF]:

T c (L, βµ) = T c (βµ) + aL -(1+ω)/ν . ( 14
)
η c (L, βµ) = η c (βµ) + aL -1/ν . ( 15
)
The values of the critical exponents, ν and ω, were taken from Ref. [43]. In table III some points of the order disorder transition are given for different values of the non-patch interaction ǫ 0 . The critical lines given in Table III can be fitted to equations of the form: IV: Fits of the critical lines for the order-disorder transition of the BCC solid. In the last column we include estimates computed from Eq. ( 7) in conjunction with data for the Ising model in a BCC lattice taken from Ref. [40]. The second column, labelled as T * c (η = 1), refers to the value of T * c in the limit of η → 1. This coincides with the T 0 parameter, but its value is given separately to indicate the statistical error in this limit. The parameters of the fits to Eq. 16 obtained for different values of ǫ * 0 are given in Table IV. The value of the critical temperature at close packing (extrapolated using Eq. ( 16) for η = 1) can be used to check the consistency of the data by comparing with the result obtained from the mapping to the AFIM in a BCC lattice, for which T * (AF IM ) c ≃ 6.3542 [40],

T c (η) = T 0 + a 1 (1 -η) + a 2 (1 -η) 2 . ( 16
)
ǫ * 0 T * c (η = 1) T 0 a 1 a 2 T * c (η =
using Eq. ( 7). Note that on a BCC lattice the order-disorder transition is coincident for the ferromagnetic Ising model and the AFIM. As shown in Table IV the agreement between the results from both routes is excellent.

D. The limit of low temperature

The most stable phase at some given thermodynamic conditions corresponds to that that minimizes the Grand Potential Φ:

Φ = U -µN -T S, (17)
with S being the entropy. At T = 0 the Grand Potential reduces to [28]:

Φ = U -µN, (18)
which can be divided by N max at both sides to obtain:

φ = U/N max -µη (19)
The plausible phases at zero temperature are a diluted gas, an ordered diamond phase, and the ordered BCC phase. It is straightforward to compute the Grand Potential for each one of these phases at zero temperature: φ g (µ) = 0, φ D (µ) = -ǫ -µ/2, and φ BCC = -2ǫ + 2ǫ 0 -µ. Phase transitions can then be simply calculated by equating the Grand Potential of these phases, from which the chemical potential at coexistence can be obtained.

In Figure 2, the stability regions for the three considered phases at very low temperatures are shown as a function of the parameter ǫ 0 /ǫ that controls the non-patch interactions in the model. The diamond phase becomes the thermodynamically stable phase at intermediate values of the chemical potential (or pressure) for ǫ 0 > 0.

E. Phase transitions at intermediate temperatures

The discontinuous transitions between the gas (fluid), the diamond and the BCC solid at finite temperatures have been calculated by a GDI. The variable of integration was chosen by numerical convenience in each region of the phase diagram. At low temperatures, we apply the differential equations given by:

dµ = µ T - ∆U T ∆N dT, (20)
and

dT = µ T - ∆U T ∆N -1 dµ, (21)
whereas in the integrations from the high temperature limit we used: where ∆ indicates the difference of the value of a given property in the two phases at equilibrium.

d(βµ) = ∆U ∆N dβ. (22
The integration of the above differential equations was carried out using the fourth-order Runge-Kutta method. We have checked the consistency of the results by performing GDI from both the low and high temperature limits. The calculations shown in the figures were performed using systems of size L = 40 (N max = 2000). The system size dependence was checked by conducting calculations also for L = 24, and L = 32. No significant differences were found between the results for different system sizes.

In order to avoid the possible effects that the continuous order-disorder transition in the BCC solid might have on the gas-BCC coexistence curve, small values of the integration step δ(β) were considered in the GD integration from β = 0. For all the cases considered, a good coalescence of different curves at triple points was obtained, which is a signature of the accuracy of the GDI results.

The GDI results were also checked by comparing the coexistence points with those obtained from Grand Canonical Wang Landau simulations at some selected temperatures [24,25].

IV. PHASE DIAGRAMS

Using the techniques described in previous sections we have computed the phase diagram of the model for several values of ǫ * 0 . The topology of the phase diagrams can be classified in four types. We will describe them in order of growing complexity.

A. Attractive non-patch interaction -1 < ǫ * 0 < 0

In Figure 3 the T -η projection of the phase diagram for ǫ * 0 = 0.00 is shown. This diagram is representative of the model in the range -1 < ǫ * 0 < 0, i.e., for attractive nonpatch interactions. At all temperatures there is a phase transition between a translationally disordered phase (labelled as fluid), and a high density phase with translational order. In the high density phase, particles are basically ordered in a BCC structure (most of the particles occupy sites of the predominant BCC sublattice). At densities below η = 1, some defects are present in the system, mostly vacant sites on the BCC sublattice, but also some are oriented in such a way that the patch-patch orientation is favored. The bond framework defined by the patch-patch interaction can be interpreted in terms of two diamond structures [28]. The orientational transition between the low and high temperature BCC phase is found to be continuous, defining a critical line starting at η = 1 and ending when it meets the BCC branch of the fluid-BCC transition.

A low density phase with diamond structure is not stable in any region of the phase diagram for attractive non-patch interaction. The absence of an energy penalty associated to the interpenetration of the two diamond sublattices to obtain a BCC structure favours the stability of BCC over the diamond structure due to the higher density of the former.

In the particular case of ǫ * 0 = 0 the diamond and BCC solids are degenerate at µ/ǫ = -2 and zero temperature (see Fig. 2). However at any finite temperature the diamond is never stable, so that at finite temperature the phase diagram for ǫ * 0 = 0 is representative of the behaviour for ǫ * 0 < 0.

B. Slightly repulsive non-bonding interaction 0 ≤ ǫ * 0 < 0.71

As it can be seen in Figure 2 for ǫ 0 ≥ 0 the diamond phase becomes stable in a certain range of chemical potentials at T = 0. At finite temperatures this translates into a certain range of temperatures at which the diamond phase is thermodynamically stable. As one increases further the temperature, the low entropy of this phase makes it unstable with respect to the fluid-BCC equilibrium. In Figure 4 we show a typical phase diagram of this region. The stability of the diamond phase ends at a triple point in which fluid, diamond and ordered BCC phases coexist. As for the case ǫ 0 < 0 the BCC phase exhibits an orientational order-disorder transition, which is shifted to higher temperatures, with a dependence for fixed packing fraction that can be approximately described by T c (η) ∝ (1 + ǫ * 0 ). An interesting result is that a small non-patch repulsion (i.e., a small positive value of As the value of ǫ 0 increases from ǫ * 0 = 0.71, some qualitative changes happen in the phase diagram. In addition to the aforementioned increase of the stability region of the diamond phase, and the displacement of the order-disorder transition of the high density solid to higher temperatures, the density of the fluid branch of the high temperature fluid-BCC equilibrium increases with ǫ 0 . As a direct consequence this fluid density can reach values similar to or greater than the density range of the stability of the diamond phase (η ≃ 0.5) when approaching the temperature region where diamond phase is thermodynamically stable. As a result, one can find for ǫ * 0 ≥ 0.71 a stable fluid denser than the diamond phase. This is illustrated in the phase diagram represented in Figure 5. It can be seen that there is a certain temperature range where the diamond phase can melt either by increasing or reducing the chemical potential (or the pressure). In other words, for the present model, the low density solid phase can melt under isothermal compression. This is usually known as reentrant melting and it occurs in real associating liquids (like water) [44]. For the off-lattice tetrahedral models that we try to mimic, reentrant melting have also been observed when the range of the interactions is below a given value [32]. Reentrant melting have also been observed for other lattice models [24,25]. behaviour is basically controlled by the attractive patch-patch interactions, whereas at the high temperature limit the hard core interactions are responsible for the phase transitions.

At intermediate temperatures, and for high values of ǫ 0 , the interaction energy between pair of particles separated by a distance 2 √ 3d 0 are still relevant, but the role of entropy makes that this interaction becomes on average repulsive. Under this view one can guess that the system could find thermodynamic stability by looking for configurations with distances between neighbour particles slightly above 2 √ 3d 0 . The search of the structure of such possible phases, besides being a difficult task, is out of the scope of the present work.

V. CONCLUSIONS

The phase diagram of a lattice patchy model that combines a repulsive core plus four attractive sites in a tetrahedral arrangement has been calculated by computer simulation.

This model was chosen to mimic the behaviour of off-lattice models of tetrahedral particles.

The use of a lattice model has allowed us to explore a wide range of parameters of the model. Keeping the patch-patch interactions attractive (ǫ = 1), non-patch interactions varied from attractive (ǫ 0 = -1.0) to highly repulsive (ǫ 0 = 1.0). It was found that for attractive non-patch interactions only a high density solid is obtained with BCC structure that is orientationally ordered at low temperatures and orientationally disordered at high temperatures. When the repulsion is increased, reentrant behaviour is found in the fluid-low density solid coexistence line. A low density solid with diamond structure only becomes stable for repulsive non-patch interactions. Interestingly a quite small repulsion is enough to stabilize the diamond solid on a quite large region of the phase diagram. This suggests that adding an isotropic repulsion beyond the hard core of the colloidal particles could be used in experiments to stabilize the diamond structure.

When comparing the lattice patchy model with the models of tetrahedral particles in the continuum that have been studied by Romano et al. [31,32] and by Noya et al. [33],

both would correspond to non-interacting or slightly attractive non-patch interactions if one looks at the model potential. Both consist of a repulsive core plus four attractive sites and neither of them include non-patch interactions that disfavour the formation of a BCC structure. However, at finite temperatures, it has been found that the diamond structure can be stabilized over the BCC solid for short ranged models due to the higher vibrational entropy of the diamond structure. The interactions between the two diamond sublattices in the BCC reduce its vibrational entropy and this effect is more pronounced for short ranged models, for which the distance between the two sublattices decreases. The simple lattice model described here neglects the vibrational entropy. However, the lower vibrational entropy of the BCC solid can be mimicked by introducing a repulsion energy between the two sublattices, which in our model implies a positive value of ǫ 0 . Obviously this is an oversimplification because entropic effects become larger with temperature, something that can not be picked up with an energy penalty term. Following this recipe, long range off-lattice models are mapped by the lattice model with attractive non-patch interactions whereas short range models in the continuous can be related to positive values of ǫ 0 , the value of ǫ 0 increasing with decreasing the range of the potential. It can be seen that with this mapping the phase diagram of the lattice model bears some resemblance with the low temperature region of off-lattice models. For attractive non-patch interaction the diamond is absent in the phase diagram, similarly to long range patchy models in the continuum [33].

For low repulsive non-patch interactions the diamond is stabilized, mimicking the behaviour of patchy models when the range is decreased [31,33]. Finally, the reentrant melting of the diamond structure that it is observed for very short ranged models in the continuum also appears for lattice models with medium repulsive non-patch interactions [32].

Obviously the underlying lattice imposes constraints and there are other features of the phase diagram that cannot be reproduced, which in this case correspond mainly to the high temperature and high density limits and are of less interest. As an example, the lattice model considered in this work cannot form the FCC structure found at high pressures [32,33] due to the constraints imposed by the lattice.

In summary, it has been shown that lattice models are able to reproduce much of the behaviour of continuous patchy particles. The advantage of lattice models is that due to its simplicity it is possible to explore the model parameter space more quickly and simulations can be performed for larger system sizes. [43] H. W. J. Blöte, E. Luijten, and J. R. Heringa, J. Phys. A: Math. Gen. 28, 6289 (1995).

[44] O. Mishima, J. Chem. Phys. 132, 234502 (1993).

  and zero otherwise, with k B being the Boltzmann constant and T the temperature. V p represents the patch interaction, which is similar to that proposed in the ALG model[22,28]. In brief, each particle has four bonding arms (or four patches) distributed in a tetrahedral arrangement. The patch interaction is considered to occur only between particles separated by a distance r ij = σ. For σ = 2 √ 3d 0 , there are eight sites in the lattice at a distance σ from a central occupied site that are located at positions [±2, ±2, ±2]d 0 with respect to the central site. Two possible configurations, S i = ±1, are possible for the bonding arms, in which the four patches are pointing at directions {(111)(1 11 )( 11 1)( 11 1)} or {( 111 ), (11 1)(1 11)( 111)}. This patch interaction can be expressed mathematically as:

3 .

 3 LHS model on a simple cubic lattice forms a BCC structure at close packing when the exclusion parameter σ is a multiple of √ 3d 0 . The LHS models with σ * = σ/d 0 = √ 3 and σ * = 2 √ 3 are known to exhibit a discontinuous solid-fluid transition (order-disorder)[20]. The BCC structure formed can contain a number of defects if their densities are less than the corresponding close packing densities, which are ρ 0 d 3 0 = 1/4 for σ * = √ 3, and ρ 0 d 3 0 = 1/32 for σ * = 2 √ The density of solid phase at the transition increases considerably when increasing the value of σ, being η s ≃ 0.52 for σ * = √ 3, and η s ≃ 0.94 for σ * = 2 √ 3 (see Ref. 20).

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1 FIG. 1 :

 11 FIG.1: Construction of four site plaquettes of the system. The coordinates of the labelled points in terms of the underlying lattice are r 0 = (0, 0, 0), r 1 = (2, 2, 2), r 2 = (0, 0, 4) and r 3 = (-2, 2, 2).On the right hand side, a projection of the plaquette (as seen from the front) is shown. Thick lines on the left panel (lines on the right panel) connect pairs of interacting sites.

  each system size estimates of the order-disorder transition have been obtained by computing the value of βµ(L) that maximizes the density fluctuations, µ being the chemical potential, and β ≡ (k B T ) -1 . Let us designate as βµ t (L) such value. At those conditions we computed the average packing fraction η m (L) and the average fluctuation of the same quantity δη

  8539(1), η m = 0.74326(1), δη = 0.19873(1). The packing fractions of each phase in coexistence can also be obtained: η f = η m -δη = 0.5445(1), η s = η m + δη = 0.9420(1).

  Values of |m| close to one indicate that the particles in the BCC lattice are orientationally ordered (o-BCC), whereas values close to zero indicate that the BCC lattice is orientationally disordered (d-BCC).

Page 12 of 22 URLFIG. 2 :

 222 FIG. 2: Stable phases as function of µ and ǫ 0 , at zero temperature. o-BCC means ordered BCC solid.

  particles. Paying attention to the orientational degrees of freedom, the BCC solid undergoes a phase transition at intermediate temperatures. At low temperature the particles

ǫ * 0 )

 0 is enough to get a considerable temperature range of stability of the diamond phase. For instance, the triple point corresponding to the largest temperature with diamond stability appears at T * T P ≃ 0.293 for ǫ * 0 = 0.01, whereas for ǫ * 0 = 0.001, T * T P ≃ 0.225.

DFIG. 3 :FIG. 4 :

 34 FIG. 3: Phase diagram for ǫ * 0 = 0. o-BCC means ordered BCC solid and d-BCC disordered BCC solid.

FIG. 5 :

 5 FIG. 5: Phase diagram for ǫ * 0 = 0.80. Labelling of regions as in previous figures, D means diamond phase, and F fluid phase.

22 Page 22 of 22 URL

 2222 : http://mc.manuscriptcentral.com/tandf/tmph Molecular Physics

TABLE I :

 I

Potential energy of four-site plaquettes in the LPM, and antiferromagnetic Ising model (AFIM) for representative plaquette configurations. The plus and minus signs correspond to orientations S i = +1 and S i = -1, respectively, for each of the four sites on a plaquette.

TABLE II :

 II Estimates of the fluid-solid transition for the lattice hard sphere model with σ * = 2

				√	3
	for different system sizes
	L N max βµ t (L)	η m (L)	δη(L)
	40 2000 2.849560(15) 0.743096(3) 0.198738(4)
	36 1458 2.847931(11) 0.743025(3) 0.198740(2)
	32 1024 2.845391(11) 0.742931(2) 0.198746(3)
	28 784 2.841230(18) 0.742765(3) 0.198758(3)
	24 432 2.833723(20) 0.742455(4) 0.198788(3)
	20 250 2.819105(27) 0.741881(5) 0.198812(4)
	16 128 2.786989(42) 0.740899(7) 0.198612(5)
	12 54	2.704225(55) 0.739716(6) 0.196946(7)

TABLE III :

 III Estimates of the order-disorder transition for the BCC lattice

		ǫ * 0 = 0
	βµ	0.5	1.0	1.5	2.0	2.5	3.0
	T * c	1.4541(7) 1.5048(6) 1.5370(4) 1.5567(5) 1.5687(4) 1.5766(4)
	η c	0.9260(2) 0.9548(1) 0.9723(1) 0.9830(1) 0.9896(1) 0.9937(1)
		ǫ * 0 = 0.2
	βµ	1.5	2.0	2.5	3.0	3.5
	T * c	1.7638(8) 1.8192(7) 1.8523(5) 1.8736(8) 1.8860(5)
	η c	0.9356(2) 0.9610(1) 0.9762(1) 0.9854(1) 0.9911(1)
		ǫ * 0 = 0.8
	βµ	3.0	3.5	4.0	4.5	5.0
	T * c	2.6504(4) 2.7367(11) 2.7852(10) 2.8141(9) 2.8320(7)
	η c	0.9374(1) 0.9637(1) 0.9782(2) 0.9867(1) 0.9919(1)

TABLE

URL: http://mc.manuscriptcentral.com/tandf/tmph Molecular Physics

Acknowledgments

The authors gratefully acknowledge the support from the Dirección General de Investi-

Científica y Técnica under Grants No. MAT2007-65711-C04-04 and FIS2010-15502, from the Dirección General de Universidades e Investigación de la Comunidad de Madrid under Grant No. S2009/ESP-1691 and Program MODELICO-CM and from CSIC under Grant No. 200980I099.


S Perkhofer

C Lass-Flörl

M Hell

G Russ

R Krause

M Hönigl

C Geltner

J Auberger

G Gastl

M Mitterbauer

B Willinger

P Knöbl

G Resch

R Waldner

A Makrai

G Hartmann

M Girschikofsky

R Greil

The Nationwide Austrian Aspergillus Registry: a prospective data collection on epidemiology; therapy and outcome of invasive mould infections in immunocompromised and/or immunosuppressed patients

Keywords:

The Nationwide Austrian Registry: a prospective data collection on epidemiology; therapy and outcome of invasive mould infections in immunocompromised and/or immunosuppressed patients

A c c e p t e d M a n u s c r i p t 3 ABSTRACT A prospective, observational, multicentre study was performed to assess the incidence, diagnosis, epidemiology and outcome of invasive mould infections (IMIs) reported to the Nationwide Austrian Aspergillus Registry. In total, 186 cases were recorded, corresponding to an annual incidence of 42 cases/1000 patients at risk or 2.36 cases/100 000 inhabitants. Patients with acute myelogenous leukaemia (34%) and lung transplant recipients (17%) are currently at highest risk for IMI, followed by a mixed population with impaired immunity (14%). In total, 34%, 30% and 36% were proven, probable and possible cases of IMI. Predominant pathogens were Aspergillus spp. (67%), followed by the zygomycetes (28%). Voriconazole was the most frequently administered agent (38%), followed by caspofungin (20%) and posaconazole (19%). Eighty patients (43%) received antifungal prophylaxis for 7 days, 30% of whom (24 patients) suffered from a breakthrough infection. The overall crude 12-week mortality was 34%. Multivariate analysis showed that outcome and survival did not correlate with the status of fungal disease, breakthrough infection, fungal species or age (P > 0.05). Aspergillosis remains the most commonly identified IMI among immunocompromised and/or immunosuppressed patients, but other moulds constitute a significant problem. Survival from IMIs appears to have improved and the main challenge is to overcome breakthrough fungal infections.

A c c e p t e d M a n u s c r i p t

Introduction

Immunocompromised and immunosuppressed patients have a high risk of acquiring invasive fungal infections (IFIs) owing to cytopenia, B-and T-cell deficiencies specific to neoplastic diseases, as well as receipt of therapies to prevent and treat organ rejection in solid organ transplant recipients and for suppression of graft-versus-host disease in allogeneic stem cell or bone marrow recipients [START_REF] Neofytos | Epidemiology and outcome of invasive fungal infection in adult hematopoietic stem cell transplant recipients: analysis of Multicenter Prospective Antifungal Therapy (PATH) Alliance registry[END_REF][START_REF] Singh | Aspergillus infections in transplant recipients[END_REF][START_REF] Singh | Trends in the epidemiology of opportunistic fungal infections: predisposing factors and the impact of antimicrobial use practices[END_REF][4][START_REF] Malani | Changing epidemiology of rare mould infections: implications for therapy[END_REF]. During the past two decades, the epidemiology of IFIs has changed: fluconazole prophylaxis has been successfully used to prevent Candida albicans infection [START_REF] Marr | Itraconazole versus fluconazole for prevention of fungal infections in patients receiving allogeneic stem cell transplants[END_REF], and mould infections have become more common [START_REF] Marr | Epidemiology and outcome of mould infections in hematopoietic stem cell transplant recipients[END_REF]. This may be due to the fact that treatment of neoplastic diseases has changed substantially, including more aggressive therapies in patients with haematopoietic malignancies, the extension of the age range treated, e.g. with myelosuppressive or myeloablative therapies in acute leukaemia and myelodysplastic syndromes, and a higher number of subsequent lines of treatment for relapse, e.g. in many types of leukaemias and lymphomas [START_REF] Malani | Changing epidemiology of rare mould infections: implications for therapy[END_REF][START_REF] Marr | Epidemiology and outcome of mould infections in hematopoietic stem cell transplant recipients[END_REF][START_REF] Upton | Emergence of opportunistic mould infections in the hematopoietic stem cell transplant patient[END_REF][START_REF] Patterson | Advances and challenges in management of invasive mycoses[END_REF]. These medical developments also apply to patients suffering from solid tumours. The trends and facts apparently lead to a prolongation of life in many of these neoplastic conditions, but simultaneously the time and severity of immunosuppression may also increase, thereby predisposing to the acquisition of IFI. In addition, changes in transplantation practices, including the sources used for stem cells, conditioning regimens and strategies to diagnose and treat IFI, have likely impacted the epidemiology and outcomes of infections [START_REF] Upton | Emergence of opportunistic mould infections in the hematopoietic stem cell transplant patient[END_REF][START_REF] Patterson | Advances and challenges in management of invasive mycoses[END_REF]. Most recent data are limited to single-centre studies usually with a high patient volume and experience as well as a focus on allogeneic transplantation [10,[START_REF] Pappas | Amphotericin B lipid complex in the treatment of invasive fungal infections: results of the Collaborative Exchange of Antifungal Research (CLEAR), an industry supported patient registry: introduction[END_REF]. Few data are available on the incidence, prophylaxis and A c c e p t e d M a n u s c r i p t 5 treatment of IFI in countries with a homogeneous health system and registries covering many centres with a different medical focus and specialisation.

The Nationwide Austrian Aspergillus Registry (NAAR) is such a prospective, multicentre, observational study group that collects data and monitors trends in the epidemiology, diagnosis, treatment and outcomes of invasive mould infections (IMIs) in Austria. This long-term initiative was undertaken with the overarching goal of shaping improvement of care for patients with IMIs and to assess the epidemiology and outcomes of IMIs in Austria.

Material and methods

Case identification and data collection

The NAAR includes 12 major medical centres in Austria that have been chosen for participation based on the number of patients treated for IMI per year and the diversity of patients. Participating centres include care providers such as general oncology and/or haemato-oncology departments as well as stem cell and solid organ transplantation units. The registry collects data from routine clinical practice to address endpoints in four broad research areas of IMI, namely epidemiology, diagnosis, treatment and outcome. This study was performed by reviewing data from centres that reported at least four adults (age 19 years) with an IMI during a 1-year period (2007)(2008). The project is strictly observational, focusing on data collection.

Patient data are kept anonymous and are entered prospectively into electronic case Mycoses Study Group (EORTC/MSG) Consensus Group [START_REF] De Pauw | Revised definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group[END_REF]. Variables collected included demographic characteristics, underlying diseases, relevant and specific characteristics in case of transplantation, as well as type of IMI, treatment and outcome. Underlying diseases and characteristics included data on the type and state of haematological malignancy or other underlying disease as well as use of corticosteroids (within 30 days before the diagnosis) and other immunosuppressive agents. Furthermore, receipt of prior antifungal therapy (within 30 days before the diagnosis of infection), the fungal genus and species, diagnostic modalities, site of infection, history of antifungal therapy and therapeutic procedures were required to be reported. IMIs were defined in accordance with published guidelines [START_REF] De Pauw | Revised definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group[END_REF]. The therapeutic protocols used by the centres were similar and reflected current Infectious Diseases Society of America (IDSA) and/or European Conference on Infections in Leukemia (ECIL) guidelines [START_REF] Walsh | Treatment of aspergillosis: clinical practice guidelines of the Infectious Diseases Society of America[END_REF][START_REF] Maertens | Primary antifungal prophylaxis in leukaemia patients[END_REF]. The outcome of IMI therapy was recorded (i.e. 3-4 weeks after diagnosis) as complete or partial response, stable condition or worsening condition as assessed by investigators and as described elsewhere [START_REF] Pappas | Amphotericin B lipid complex in the treatment of invasive fungal infections: results of the Collaborative Exchange of Antifungal Research (CLEAR), an industry supported patient registry: introduction[END_REF][START_REF] Post | Invasive fungal infections in allogeneic and autologous stem cell transplant recipients: a single-center study of 166 transplanted patients[END_REF]. Breakthrough infection was defined as occurrence of proven fungal infection while on antifungal prophylaxis for at least 7 days. Survival was evaluated at 12 weeks. Each participating centre also contributed data on all newly months for patients whose records had been completed.

Statistical analyses

Comparison between categorical variables was performed by Fisher's exact test or  2 test. Regression analysis with analysis of variance (ANOVA) was used to determine trends in incidence. A P-value of <0.05 was considered statistically significant.

Results

A total of 217 patients were prospectively enrolled and records for 186 patients were completed by January 2008. Baseline demographic characteristics and patient categories are described in Table 1. The vast majority of infected patients suffered from haematological malignancies (68%), with a smaller number of patients who had undergone allogeneic stem cell (20%) or lung transplantation (17%). The third most important group consisted of mixed individuals with and without known risk factors Infecting fungal pathogens are shown in Table 2, with Aspergillus fumigatus being the most frequently isolated species (59%). Culture and microscopy was the most common diagnostic tool in identifying IMI in this series (64%), followed by computed tomography (CT) (58%). The diagnosis was proven in 63 cases (34%) and was considered probable in 56 cases (30%) (Table 3). The galactomannan assay [in serum and/or bronchoalveolar lavage fluid (BAL) specimens] was applied in 26% of cases, polymerase chain reaction (PCR) in 16% and the -D-glucan assay in 3%.

Azoles, echinocandins and polyenes were used as single agents either sequentially or in combination. Voriconazole was the most commonly used antifungal agent (38%), followed by caspofungin (20%) and posaconazole (19%). A minority of patients received itraconazole (1%) or amphotericin B deoxycholate (4%). Lipid formulations of amphotericin B plus posaconazole in combination were most frequently administered in the case of zygomycetes. Combination treatment, defined as more than one antifungal agent used sequentially or/and concomitantly, was administered in 67 patients (36%). Treatment with voriconazole plus caspofungin was the most frequently used combination regimen (47 patients; 70%). Eighty patients received antifungal prophylaxis for 7 days (range 7-56 days, mean 21 days), of whom 30% (24 patients) had a breakthrough fungal infection. An adjunct procedure (e.g. surgical intervention) was performed in 6 patients (3%).

Overall, 93 patients (50%) improved, 54 (29%) were stable and 39 (21%) deteriorated; the 12-week assessment estimated a mortality rate of 34% (see Table 3). In total, 186 cases of IMI were recorded, which corresponds to an annual incidence of 42 cases/1000 patients at risk or to 2.36 cases/100 000 inhabitants.

Multivariate analysis showed that outcome and survival did not correlate with the status of fungal disease, breakthrough infection, fungal species or age (P > 0.05).

Combination treatment, applied as first line in 19% of cases, was not superior compared with monotherapy. Survival and outcome were superior with voriconazole compared with the other drugs (P < 0.01) (see Fig. 2). In this analysis, invasive aspergillosis was the most commonly encountered IMI, presenting predominantly as pneumonia in patients with haematological malignancies and as tracheobronchitis in organ transplant recipients. The second most important fungi were the zygomycetes, followed by a small number of uncommon moulds. Occurrence of infections due to zygomycetes was not focused on a single centre, but rather was reported from several institutions. This trend, first described in patients who received transplants after 1995, probably reflects the inclusion of higher risk patients in modern tumour therapies, the increasing number of subsequent lines of antineoplastic treatment applied, and the better tolerability of treatment by young olds and older olds with an increasing number of patients at risk, as well as by the increased use of higher rates of chemoimmunotherapies. In addition, higher risk patients are undergoing stem cell transplantation with incompletely matches donors leading to a more immunosuppressed patient population in this group. Moreover, extensive use of prophylactic treatment with agents that have activity against Aspergillus spp. [START_REF] Upton | Emergence of opportunistic mould infections in the hematopoietic stem cell transplant patient[END_REF][START_REF] Maertens | Primary antifungal prophylaxis in leukaemia patients[END_REF][START_REF] Almyroudis | Zygomycosis in solid organ transplant recipients in a tertiary transplant center and review of the literature[END_REF][START_REF] Kontoyiannis | Zygomycosis in a tertiary-care cancer center in the era of Aspergillus-active antifungal therapy: a case-control observational study of 27 recent cases[END_REF] may have favoured a Within the setting of organ transplantation recipients, this epidemiological trend was strongest, displaying the highest incidence (8%) with rare mould infections. Overall, incidences of IMI are comparable with other studies, showing patients with acute myeloid leukaemia being at highest risk for IMI [START_REF] Chamilos | Invasive fungal infections in patients with hematologic malignancies in a tertiary care cancer center: an autopsy study over a 15-year period (1989-2003)[END_REF][START_REF] Pagano | The epidemiology of fungal infections in patients with hematologic malignancies: the SEIFEM-2004 study[END_REF]. The third most important group at risk consists of mixed individuals with various types of underlying immunodeficiency. Patients with prolonged ICU stay and use of steroids, neutrophil deficiencies or dysfunctions, and chronic granulomatous diseases are an emerging group for IMI and are definitively on the rise. In this patient population, most of the group had no underlying haematological malignancies and suffered from a primary mould infection.

Discussion

Culture and microscopy was the most common diagnostic tool in identifying IMIs in this series (64%). In several Austrian centres, we attempt to perform CT-guided percutaneous lung biopsies to confirm the diagnosis [START_REF] Lass-Flörl | The value of computed tomography-guided percutaneous lung biopsy for diagnosis of invasive fungal infection in immunocompromised patients[END_REF]. This intervention appears to be mandatory in our patient population, as we record a high rate of infections due to Aspergillus terreus and zygomycetes [START_REF] Lass-Flörl | Epidemiology and outcome of infections due to Aspergillus terreus: 10-year single centre experience[END_REF]. Aspergillus terreus is amphotericin Bresistant yet this drug is mandatory for zygomycoses. A similar diagnostic approach is widely used in other Austrian institutions. The diagnosis was proven in 63 cases (34%) and probable in 56 cases (30%). However, there is a need to improve diagnostic approaches, as only a limited number of other diagnostic tests were used to establish early diagnosis. The galactomannan assay (in serum and/or BAL specimens) was applied in 26% of cases, PCR in 16% and the -D-glucan assay in A c c e p t e d M a n u s c r i p t prophylaxis, this approach might be helpful for timely and sensitive diagnosis.

The outcome of current treatment regimens (3-4 weeks after diagnosis) was satisfactory compared with other studies [START_REF] Aisner | Treatment of invasive aspergillosis: relation of early diagnosis and treatment to response[END_REF][START_REF] Bodey | Fungal infections in cancer patients: an international autopsy study[END_REF][START_REF] Herbrecht | Voriconazole versus amphotericin B for primary therapy of invasive aspergillosis[END_REF]. Overall, 50% of patients improved, 29% were stable and 21% deteriorated. Of note, conventional amphotericin B was minimally used in this study. As this reflects practice observed in 12 different medical centres, it appears that conventional amphotericin B has largely been replaced by lipid formulations of amphotericin B and the new third-generation azoles.

Combination therapy, either sequential or concomitant, was frequently observed in 36% of patients. This reflects the real-life situation and possibly reflects the high rate of breakthrough infections observed. However, combination treatment was not superior for outcome and/or survival compared with single drug use. Voriconazole appears to be associated with improved outcome and survival (P < 0.01); a similar trend for outcome was for posaconazole.

Here we report a 12-week mortality rate of 34% among patients with IMI, which is markedly improved compared with rates from prior reports of outcomes during the 1990s [START_REF] Anaissie | Opportunistic mycoses in the immunocompromised host: experience at a cancer center and review[END_REF][START_REF] Denning | Epidemiology and pathogenesis of systemic fungal infections in the immunocompromised host[END_REF][START_REF] Pfaller | Epidemiological typing methods for mycosis[END_REF]. These data on aspergillosis are consistent with recently reported data [START_REF] Neofytos | Epidemiology and outcome of invasive fungal infection in adult hematopoietic stem cell transplant recipients: analysis of Multicenter Prospective Antifungal Therapy (PATH) Alliance registry[END_REF][START_REF] Horn | Presentation of the PATH Alliance registry for prospective data collection and analysis of the epidemiology, therapy, and outcomes of invasive fungal infections[END_REF][START_REF] Auberger | Significant alterations in the epidemiology and treatment outcome of invasive fungal infections in patients with hematological malignancies[END_REF]. Improved outcomes likely reflect improved diagnostic and therapeutic modalities compared with older patient series. However, in these studies mortality was highest in patients with zygomycoses [START_REF] Neofytos | Epidemiology and outcome of invasive fungal infection in adult hematopoietic stem cell transplant recipients: analysis of Multicenter Prospective Antifungal Therapy (PATH) Alliance registry[END_REF]. This is in contrast to the present report where IMI-related deaths occurred to the same extent for infections due to Aspergillus and zygomycetes.

A c c e p t e d M a n u s c r i p t 13 Outcome and survival did not correlate with the underlying fungal disease status, yet it was significantly better in patients receiving voriconazole. The reason might lay in the fact that most of the infections were due to A. fumigatus and that this azole has excellent anti-Aspergillus activity [START_REF] Herbrecht | Voriconazole versus amphotericin B for primary therapy of invasive aspergillosis[END_REF]. Moreover, voriconazole was primarily used as first-line treatment and other drugs as rescue.

However, in this registry, with all the limitations of its nature, it has been shown that breakthrough IMIs occurred frequently in patients receiving antifungal prophylaxis.

Aspergillus and zygomycetes were the main organisms, however the reason for this high rate (30%) of breakthrough infection is not clear yet and several causes might be responsible. First, the distinction between baseline infections (those present on or immediately before starting treatment) and breakthrough infections (those with an onset while on treatment) may be difficult to ascertain [START_REF] Martino | Empirical antifungal therapy in patients with neutropenia and persistent or recurrent fever of unknown origin[END_REF]. Second, serum drug concentrations may be insufficient to prevent infections [START_REF] Müller | HPLC analysis of the antifungal agent posaconazole in patients with haematological diseases[END_REF] as patients do not follow the strict guidelines for oral drug uptake. Third, diagnosis has improved to such an extent and allows for more sensitive and timely diagnosis and more tailored therapy [START_REF] Lass-Flörl | The changing face of epidemiology of invasive fungal diseases in Europe[END_REF]. In an earlier study, it was shown that >50% of infections were identified at autopsy [START_REF] Groll | Trends in the postmortem epidemiology of invasive fungal infections at a university hospital[END_REF]. So far, breakthrough aspergillosis in patients receiving empirical or prophylactic antifungal treatment using an agent with anti-Aspergillus activity is well known. For example, breakthrough aspergillosis was observed in 1.0-18% of patients receiving conventional amphotericin B, lipid-based amphotericin B, voriconazole or itraconazole and caspofungin [START_REF] Martino | Empirical antifungal therapy in patients with neutropenia and persistent or recurrent fever of unknown origin[END_REF][START_REF] Trifilio | Breakthrough zygomycosis after voriconazole administration among patients with hematologic malignancies who receive hematopoietic stem-cell transplants or intensive chemotherapy[END_REF][START_REF] Lafaurie | High rate of breakthrough invasive aspergillosis among patients receiving caspofungin for persistent fever and neutropenia[END_REF][START_REF] Walsh | Voriconazole compared with liposomal amphotericin B for empirical antifungal therapy in patients with neutropenia and persistent fever[END_REF][START_REF] Vehreschild | A double-blind trial on prophylactic voriconazole (VRC) or placebo during induction chemotherapy for acute myelogenous leukaemia (AML)[END_REF][START_REF] Cornely | Posaconazole vs. fluconazole or itraconazole prophylaxis in patients with neutropenia[END_REF][39]. Recently, a rate of breakthrough invasive aspergillosis among patients receiving caspofungin for persistent fever and neutropenia was recorded as 13% [START_REF] Lafaurie | High rate of breakthrough invasive aspergillosis among patients receiving caspofungin for persistent fever and neutropenia[END_REF]. However, statements are somewhat limited on this topic as we have no access to the overall data on how Other registries or studies have also gathered information on IMIs but have been hindered by a of observations limited in terms of patient type, therapies used or geographic areas [START_REF] Marr | Epidemiology and outcome of mould infections in hematopoietic stem cell transplant recipients[END_REF]10,[START_REF] Pappas | Amphotericin B lipid complex in the treatment of invasive fungal infections: results of the Collaborative Exchange of Antifungal Research (CLEAR), an industry supported patient registry: introduction[END_REF]. In contrast, the NAAR is a registry that allows the collection and analysis of high-quality data obtained from targeted centres with substantial clinical experience and is facilitated by the use of electronic data capture and reporting software. The wide distribution of investigating centres across Austria, the variety of patient categories and the enrolment of more than 150 patients with IMI per year make this registry a significant tool for the study of common, rare and emerging IMIs. This registry has some limitations. Because this is a strictly observational project, the generated results might have limited clinical significance. It is likely that patients with possible infections are underrepresented in the database because of the challenges in ascertainment. Despite its limitations, the NAAR is a network of medical institutions collecting significant information regarding IMIs in Austria. As enrolment continues, additional data will be analysed and published, which will provide valuable information regarding the epidemiology, therapy and outcomes of IMIs.

In conclusion, this study shows that aspergillosis remains the most commonly identified IMI among immunocompromised and/or immunosuppressed patients, but

  The registry has been approved by the Ethics Committee of Innsbruck Medical University, one of several lead ethics committees in Austria. All patients with a diagnosis of proven, probable or possible IMI were eligible for enrolment according to the definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases

  during the survey period to gain an insight into the current incidence of IMI in the centres deemed representative of Austria. After completion of an individual patient record, the record was locked and was available for inclusion in the clinical reports. For a complete patient record, the following requirements were defined in advance: (i) completion of follow-up for 12 weeks or until death or discharge; (ii) no outstanding data queries by the central coordinator(s) (C.L.-F. and J.A.); and (iii) a patient record sign-off by the site investigator. Participating sites also had the option to enter follow-up data at 12

  A c c e p t e d M a n u s c r i p t 8 (14%) (see Fig. 1). Underlying factors for this pooled group are prolonged Intensive Care Unit (ICU) stay associated with a high number of antibiotic drugs and/or need for ventilation, parenteral nutrition or central line access, neutrophil deficiencies or dysfunctions, and chronic granulomatous diseases. Overall, 89 patients (48%) received treatment targeting T-cells and 56 patients (30%) were treated with corticosteroids. Most of the culture-positive IMIs (proven and probable) were caused by Aspergillus spp. (60 patients; 67%), followed by zygomycetes (25 patients; 28%).

  and itraconazole were the drugs (preferably orally) administered, and zygomycetes and A. fumigatus were the predominant pathogens.

  This prospective, observational, multicentre study was performed to evaluate the contemporary incidences, epidemiological characteristics, clinical presentations and outcomes of IMIs in 186 adults during the period 2007-2008. Restriction to proven and probable infections recorded an annual incidence of 19 cases/1000 patients at risk and 1.08 cases/100 000 inhabitants for Austria. Patients with haematological malignancies and organ transplant recipients were at highest risk. The third most important group consisted of a mixed population with various underlying risk factors, including mainly ICU patients. Important findings also included an improvement in IMI-associated mortality, although there was a considerable rate of breakthrough fungal infections.

  a network of medical institutions gathering and sharing information to increase understanding of the incidence, epidemiology, therapy and outcomes of IMIs in Austria.The ability to capture and display information on demographics, epidemiology, treatment and outcomes of patients from multiple clinical centres greatly enhances the timely dissemination of accurate and detailed clinical information and current approaches to therapy.

  zygomycoses. So far, zygomycoses were observed mainly in tertiary care centres, showing obvious differences in patients at risk or in clinical approaches.

A

  c c e p t e d M a n u s c r i p t 14 many patients received successful prophylaxis. None the less, our Austrian-wide rate of breakthrough infection needs to be investigated in more detail. So far, breakthrough infections while on an azole (e.g. posaconazole) did not exclude beneficial treatment with another azole (e.g. voriconazole).

A

  c c e p t e d M a n u s c r i p t 15 other moulds constitute a significant problem. Survival from IMIs appears to have improved compared with previous reported data. Overall, the main challenge is to overcome breakthrough fungal infections. A mixed patient population with various underlying host defects comprise an emerging and important group for IMIs. Funding C-LF has received research funding from Gilead, AESCA Pharma and Pfizer. SP has received research funding from Pfizer. Competing interests C-LF has served as a consultant or advisor to Gilead and Pfizer and has been on the speakers' bureau for Pfizer, Gilead and Merck Sharp & Dohme. All other authors declare no competing interests.Ethical approvalResearch Ethics Committee of Innsbruck Medical University, Innsbruck, Austria (Judgement's reference no. UN2891).

  Ullmann AJ, Lipton JH, Vesole DH, Chandrasekar P, Langston A, Tarantolo SR, et al. Posaconazole or fluconazole for prophylaxis in severe graft-versus-host disease. N Engl J Med 2007;25:335-47.

A c c e p t e d M a n u s c r i p t 22 Fig. 1 .

 221 Fig. 1. Overview of underlying diseases in 186 patients with invasive mould

Fig. 2 .

 2 Fig. 2. Distribution of antifungal agents used in 186 patients with invasive fungal

  ., standard error; HEPA, high-efficiency particulate air.a Data are number (%) of patients, unless otherwise indicated.b Underlying diseases and immunological risk factors were not mutually exclusively (i.e. patients could have more than one factor).

A c c e p t e d M a n u s c r i p t 1 3 Clinical

 13 Table chain reaction; BAL, bronchoalveolar lavage fluid.a Administration of an antifungal drug for at least 1 week before clinical signs and symptoms of fungal infection.

A c c e p t e d M a n u s c r i p t

Aspergillus fumigatus 59

Aspergillus flavus 4

Aspergillus terreus 3

Aspergillus niger 1

Zygomycetes 28

Absidia spp. 10

Mucor spp. 9

Rhizopus spp. 9

Other moulds 4

A c c e p t e d M a n u s c r i p t


C Van Nieuwkoop
email: c.van_nieuwkoop@lumc.nl

P L Den Exter

H W Elzevier

J Den Hartigh

J T Van Dissel

Intravesical gentamicin for recurrent urinary tract infection in patients with intermittent bladder catheterisation

Keywords:

A c c e p t e d M a n u s c r i p t ABSTRACT Clean intermittent catheterisation (CIC) of the bladder is used to imitate normal bladder emptying in patients with bladder dysfunction. CIC is associated with urinary tract infection (UTI) that may be difficult to treat in the case of antimicrobial resistance. The aim of this study was to establish the effect and safety of intravesical gentamicin treatment in such settings. In 2009, intravesical gentamicin treatment was started in selected patients. Here we describe our experience with two patients treated until March 2010. Two patients using CIC suffering recurrent UTI with multiresistant Escherichia coli were treated with daily administration of 80 mg intravesical gentamicin. On treatment they appeared asymptomatic. During 8-and 9month follow-up they were free of UTI, urine cultures were negative and there were no side effects. A systematic review was conducted through searches of PubMed and other databases. Clinical trials that met the eligibility criteria and displayed the efficacy or safety of intravesical aminoglycoside treatment in patients using CIC were studied. Study selection was performed by two independent reviewers. Eight studies were included for review. Owing to study heterogeneity, a meta-analysis could not be performed. Of four controlled studies using neomycin or kanamycin, two demonstrated a significant reduction in bacteriuria, whilst two other trials did not. One case series on neomycin/polymyxin showed that the majority of patients still developed bacteriuria. Three case series using gentamicin all pointed towards a significant reduction in bacteriuria and UTIs. There were no clinically relevant side effects reported but follow-up in all studies was limited. Although data are limited, intravesical treatment with gentamicin might be a reasonable treatment option in selected patients practicing CIC who suffer recurrent UTIs with highly resistant microorganisms.

A c c e p t e d M a n u s c r i p t

Introduction

Clean intermittent catheterisation (CIC) of the bladder was introduced by Lapides in 1971 to manage children with neurogenic bladder dysfunction [START_REF] Lapides | Clean, intermittent selfcatheterization in the treatment of urinary tract disease[END_REF]. To date, this technique has become an important tool for the management of patients with bladder dysfunction. CIC will ideally be performed every 4-6 h, with the aim of mimicking normal emptying of the bladder and thus preventing overflow incontinence and dilatation of the upper urinary tract with potential loss of renal function. For patients requiring long-term or chronic catheterisation, CIC is considered the preferable method of urinary catheterisation since it is associated with less complications, in particular urinary tract infections (UTIs), compared with chronic indwelling bladder catheterisation [START_REF] Shekelle | Systematic review of risk factors for urinary tract infection in adults with spinal cord dysfunction[END_REF]. However, even with CIC, colonisation of the bladder frequently develops as has been shown by the presence of significant bacteriuria in 61% of urine samples from patients practicing CIC [START_REF] Bakke | Physical predictors of infection in patients treated with clean intermittent catheterization: a prospective 7-year study[END_REF]. Asymptomatic bacteriuria itself does not require antimicrobial treatment, but once symptoms occur it is considered to represent UTI, which is the major complication of CIC [START_REF] Tenke | European and Asian guidelines on management and prevention of catheterassociated urinary tract infections[END_REF][START_REF] Wyndaele | Complications of intermittent catheterization: their prevention and treatment[END_REF]. Not surprisingly, recurrent UTI frequently occurs, requiring antimicrobial treatment that, in addition to the use of prophylactic antibiotics to prevent new episodes, supports the emergence of resistant organisms and further limits antimicrobial treatment options. In this respect, administration of intravesical gentamicin following CIC has the advantage of treating the site of infection with high local antibiotic concentrations [START_REF] Wan | Intravesical instillation of gentamicin sulfate: in vitro, rat, canine, and human studies[END_REF].

Here we report two patients practicing CIC suffering recurrent UTI due to multiresistant Escherichia coli. When other treatments had failed, these patients were eventually successfully treated with intravesical instillation of gentamicin. In addition, a systematic review was performed to evaluate the effectiveness and potential and 'intermittent catheterization'. For these three subjects, all relevant keyword variations were used, not only keyword variations in the controlled vocabularies of the various databases but also the free-text word variations of these concepts. In general, the search consisted of the combination of the following terms: aminoglycosides, gentamicin, neomycin, kanamycin, butirosin sulfate, sisomicin, hygromycin, amikacin, dibekacin, nebramycin, metrizamide, framycetin, paromomycin, ribostamycin, puromycin, spectinomycin, streptomycin, dihydrostreptomycin sulfate, streptothricins, streptozocin, netilmicin, tobramycin, AND intravesical administration, instillation, irrigation AND intermittent catheterization or A c c e p t e d M a n u s c r i p t urinary catheterization. This search strategy was optimised for all consulted databases, taking into account the differences in the various controlled vocabularies as well as differences in database-specific technical variations (e.g. the use of quotation marks).

Methods

Search strategy

Study selection

Two reviewers (PLdE and CvN) independently determined study eligibility on the basis of the published abstracts; if eligible or indeterminate, the full article was retrieved for further review. Studies were excluded if they were reviews, editorials, guidelines, non-human studies or written in non-English language. All retrieved articles were discussed by the two reviewers and based on their consensus final selection for inclusion in the systematic review was made.

Data extraction

The following data were extracted from each study: A c c e p t e d M a n u s c r i p t

Statistical and microbiological methods

Although the aim was to perform a meta-analysis, the interventions evaluated in the selected studies were too heterogeneous, thus this was not feasible.

Interobserver agreement between the two reviewers with regard to study selection was assessed using the Cohen  test, in which a  value of 0.41-0.60 corresponds to fair agreement, 0.61-0.80 to good agreement, 0.81-0.92 to very good agreement and 0.93-1.00 to excellent agreement [START_REF] Byrt | How good is that agreement?[END_REF].

Urine cultures were performed using standard microbiological methods, and resistance to antimicrobials was defined using European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria [START_REF]Clinical breakpoints[END_REF].

Results

In 2009, intravesical treatment with gentamicin was started in selected patients practicing CIC who developed recurrent UTIs with a gentamicin-susceptible uropathogen but who otherwise had very limited treatment options. Here we describe our experience with two patients treated until March 2010.

Case 1

An otherwise healthy 68-year-old male had a history of neurogenic bladder of unknown aetiology requiring CIC for almost 10 years. Urodynamic evaluation demonstrated a neurogenic acontractile detrusor function as defined by the International Continence Society [START_REF] Haylen | An International Urogynecological Association (IUGA)/International Continence A c c e p t e d M a n u s c r i p t Society (ICS) joint report on the terminology for female pelvic floor dysfunction[END_REF]. Over the years he frequently suffered from UTIs, A c c e p t e d M a n u s c r i p t with predominant complains of dysuria and lower abdominal pain. Urinary cultures almost constantly grew E. coli. He was usually treated with oral courses of nitrofurantoin, ciprofloxacin and trimethoprim/sulfamethoxazole (SXT). However, in the past few years the frequency of UTI increased to once every month.

To evaluate the therapeutic options for these recurrent UTIs, he was referred to the infectious diseases outpatient department of Leiden University Medical Center (Leiden, The Netherlands). At that time, urine and semen cultures simultaneously grew a multiresistant E. coli [extended-spectrum -lactamase (ESBL)-negative, resistant to ampicillin, cefuroxime, ciprofloxacin, nitrofurantoin, SXT and tetracycline, and sensitive to ceftriaxone, gentamicin and fosfomycin] and Enterococcus faecalis.

Computed tomography (CT) and ultrasonography of the urinary tract were normal. By exclusion, chronic bacterial prostatitis was diagnosed. Because he clinically responded well to self-treatment with SXT (960 mg twice a day), it was decided to continue this for 6 weeks as the urine culture turned negative; repeated semen culture still grew E. faecalis in the absence of bacteriuria. However, 2 weeks after a new episode of UTI developed with the same E. coli strain. For the next few months he was treated with prostatic massage combined with ceftriaxone, but this strategy appeared to be unsuccessful both clinically and microbiologically.

In an attempt to suppress the recurrent UTIs, it was decided to start treatment with intravesical gentamicin after sterilisation of the urine with fosfomycin (one dose of 3 g). Informed consent was obtained. A solution of 80 mg gentamicin in 20 mL of normal saline was instilled once daily after catheterisation. The solution was left overnight in the bladder until the next catheterisation. Thus, high-dose gentamicin A c c e p t e d M a n u s c r i p t was locally in the bladder for ca. 8 h per day. This treatment was well tolerated.

During 9 months of follow-up he has been free of UTI while he continued the daily intravesical gentamicin treatment. Urine cultures performed every 3 months and one semen culture performed after 3 months were all sterile. Repetitive urinalysis ruled out leukocyturia. Serum levels of gentamicin remained undetectable (<0.3 mg/L) and renal function was stable. The urine gentamicin trough level measured just before the next catheterisation was 0.6 mg/L. Cystoscopic evaluation after 9 months of gentamicin bladder instillation revealed a normal uroepithelium bladder.

Case 2

A 70-year-old female visited the outpatient clinic because of recurrent UTIs. Her medical history revealed type 2 diabetes mellitus, diabetic neuropathy and osteoarthritis leading to wheelchair-limited mobility. She had suffered from recurrent UTIs for more than 20 years, occasionally complicated by pyelonephritis. She was allergic to nitrofurantoin.

Ultrasonography revealed a post-void residual volume of 500 mL. Urodynamic evaluation demonstrated a neurogenic acontractile detrusor function that was ascribed to diabetic neuropathy [START_REF] Haylen | An International Urogynecological Association (IUGA)/International Continence A c c e p t e d M a n u s c r i p t Society (ICS) joint report on the terminology for female pelvic floor dysfunction[END_REF]. She started to perform CIC four to six times daily.

During the next year she had frequent episodes of symptomatic UTI. Finally, she required antibiotic UTI treatment (fosfomycin) every month to suppress symptoms of dysuria and lower abdominal pain. Repetitive urine cultures revealed that her bladder was invaded with a multiresistant E. coli (ESBL-positive, resistant to -lactams, ciprofloxacin and SXT, and sensitive to nitrofurantoin, fosfomycin and gentamicin).

Because treatment options were limited, twice-daily gentamicin (80 mg in 20 mL of A c c e p t e d M a n u s c r i p t normal saline) intravesically was then started after sterilising the urine with one dose of fosfomycin (3 g) and obtainment of informed consent. Intravesical gentamicin was left in the bladder until the next catheterisation; thus, high-dose gentamicin was locally in the bladder twice daily for ca. 4-6 h. During 8 months of follow-up she was free of UTI while she continued the intravesical gentamicin treatment. Urine cultures performed every 2 months were all sterile. Repetitive urinalysis ruled out leukocyturia. Serum levels of gentamicin were undetectable and renal function was stable. The urine gentamicin level measured in a morning urine sample after the last gentamicin had been instilled the evening before was >100 mg/L. On one occasion she temporally stopped instilling the gentamicin solution owing to motor disabilities. Within 2 weeks she developed a symptomatic UTI that was successfully empirically treated with fosfomycin by her primary care physicians; a urine culture was not done.

After 8 months of treatment she fractured her forearm and thus was not able to perform CIC. A chronic indwelling bladder catheter was placed and the intravesical gentamicin treatment was stopped. Soon after the bladder became colonised with another non-resistant E. coli strain (ESBL-negative and sensitive to -lactams, ciprofloxacin, SXT, nitrofurantoin, fosfomycin and gentamicin).

Study selection

The search strategy revealed 117 references. Of these, ten articles were selected for further review by one reviewer and nine by the other reviewer. Interobserver agreement between the reviewers was excellent, with a  value of 0.94 (P < 0.001).

After reviewing and discussing the ten articles, two articles were excluded: one article appeared to be a review that reported no original data [START_REF] Harper | An appraisal of 12 solutions used for bladder irrigation or instillation[END_REF] and the other article did A c c e p t e d M a n u s c r i p t not primarily assess the efficacy or safety of aminoglycoside bladder instillations [START_REF] Mulcahy | Management of neurogenic bladder in infancy and childhood[END_REF].

Details of the literature search and study selection are outlined in Fig. 1.

Study characteristics

Characteristics of the included studies are presented in Table 1. Overall, the level of evidence of all studies was limited. Five studies included hospitalised adult patients with newly diagnosed neurogenic bladder dysfunction requiring intermittent catheterisation in order to evaluate the prophylactic value of antibiotic bladder instillations in preventing bacteriuria [START_REF] Haldorson | Nonvalue of neomycin instillation after intermittent urinary catheterization[END_REF][START_REF] Rhame | Urinary tract infections occurring in recent spinal cord injury patients on intermittent catheterization[END_REF][START_REF] Pearman | The value of kanamycin-colistin bladder instillations in reducing bacteriuria during intermittent catheterisation of patients with acute spinal cord injury[END_REF][START_REF] Anderson | Prophylaxis of bacteriuria during intermittent catheterization of the acute neurogenic bladder[END_REF][START_REF] Pearman | Comparison of the efficacy of 'Trisdine' and kanamycin-colistin bladder instillations in reducing bacteriuria during intermittent catheterisation of patients with acute spinal cord trauma[END_REF]. The primary outcome assessed in these studies was the presence of significant bacteriuria. Either neomycin or kanamycin were the aminoglycosides of interest.

Two studies included children with neurogenic bladder and gentamicin bladder instillations [START_REF] Wan | Intravesical instillation of gentamicin sulfate: in vitro, rat, canine, and human studies[END_REF][START_REF] Defoor | Safety of gentamicin bladder irrigations in complex urological cases[END_REF]. Both were case series that indicated a reduction of bacteriuria and UTI with intravesical gentamicin.

Finally, one study described case reports involving outpatient adult females with recurrent UTI [START_REF] Mcguire | Treatment of intractable bacterial cystitis with intermittent catheterization and antimicrobial instillation: case report[END_REF]. All four patients were diagnosed with haemorrhagic cystitis and had a residual volume on urodynamic evaluation. CIC was started with gentamicin bladder instillations. There were no UTIs reported on treatment.

Efficacy of intravesical aminoglycoside

Because of the heterogeneous nature of the included studies and the lack of a comparative arm in most studies, performing a meta-analysis or data pooling was A c c e p t e d M a n u s c r i p t inappropriate. Therefore, the main outcomes of the individual trials are summarised in Table 1.

In two trials with a control group, including a randomised controlled trial (RCT), aminoglycoside instillation of the bladder significantly reduced the incidence of bacteriuria [START_REF] Pearman | The value of kanamycin-colistin bladder instillations in reducing bacteriuria during intermittent catheterisation of patients with acute spinal cord injury[END_REF][START_REF] Anderson | Prophylaxis of bacteriuria during intermittent catheterization of the acute neurogenic bladder[END_REF]. Another RCT and a retrospective case-control study could not demonstrate any difference in outcome [START_REF] Haldorson | Nonvalue of neomycin instillation after intermittent urinary catheterization[END_REF][START_REF] Pearman | Comparison of the efficacy of 'Trisdine' and kanamycin-colistin bladder instillations in reducing bacteriuria during intermittent catheterisation of patients with acute spinal cord trauma[END_REF].

In a prospective case series, all 10 children had sterile urine after 1 week of treatment, whereas E. coli was yielded in three of the urine cultures at baseline [START_REF] Wan | Intravesical instillation of gentamicin sulfate: in vitro, rat, canine, and human studies[END_REF].

Breakthrough UTI occurred in 26% of the children examined by Defoor et al. [START_REF] Defoor | Safety of gentamicin bladder irrigations in complex urological cases[END_REF]. All those were on low-dose gentamicin (14.4 mg once daily), suggesting that a higher dosage of gentamicin is required to prevent UTI [START_REF] Defoor | Safety of gentamicin bladder irrigations in complex urological cases[END_REF].

Safety of intravesical aminoglycoside

Reports upon safety were scarce and poor. None of trials that investigated neomycin instillation presented details on drug toxicity or adverse effects. Pearman [START_REF] Pearman | The value of kanamycin-colistin bladder instillations in reducing bacteriuria during intermittent catheterisation of patients with acute spinal cord injury[END_REF] stated that haematuria or other clinical evidence of chemical cystitis did not occur in any of the patients who received kanamycin/colistin instillations. None of the children investigated had detectable gentamicin serum levels [START_REF] Wan | Intravesical instillation of gentamicin sulfate: in vitro, rat, canine, and human studies[END_REF][START_REF] Defoor | Safety of gentamicin bladder irrigations in complex urological cases[END_REF]. Small increases in serum creatinine levels were observed in 3 of 80 children evaluated by Defoor et al. [START_REF] Defoor | Safety of gentamicin bladder irrigations in complex urological cases[END_REF], but this was ascribed to underlying renal disease; no side effects, allergic reactions or clinical toxicities were documented.

A c c e p t e d M a n u s c r i p t

Discussion

In this study, we demonstrated that intravesical gentamicin treatment was successful and safe in two patients practicing CIC who suffered from recurrent UTI. In one case it even suppressed chronic bacterial prostatitis. In addition, the systematic review summarises the available literature on the effectiveness and safety of intravesical aminoglycoside administration in patients practicing CIC.

The concept of intravesical drug delivery lies in the uroepithelium, a transitional epithelium lining the inner surface of the bladder. This layer is known to exhibit a tough barrier function that allows the instillation of potentially toxic drugs to achieve a localised pharmacological effect whilst avoiding systemic effects [START_REF] Tyagi | Recent advances in intravesical drug/gene delivery[END_REF]. Diffusion of aminoglycosides across the uroepithelium is even more limited because of their polar cationic nature. Thus, by administrating aminoglycosides intravesically, high antimicrobial concentrations can be achieved with minimal concern of adverse effects such as nephrotoxicity or ototoxicity. Furthermore, it may thus prevent the development of antimicrobial resistance. Because in our patients the bladder was infected with a multiresistant E. coli, we opted to treat them intravesically with relatively high dosages of gentamicin to prevent further emergence of resistance.

Compared with previous studies, the dosage (80 mg) used was considerably higher [START_REF] Wan | Intravesical instillation of gentamicin sulfate: in vitro, rat, canine, and human studies[END_REF][START_REF] Mcguire | Treatment of intractable bacterial cystitis with intermittent catheterization and antimicrobial instillation: case report[END_REF][START_REF] Defoor | Safety of gentamicin bladder irrigations in complex urological cases[END_REF]. This may have been overtreatment, but based on the literature review we can conclude that the optimal dosage remains unclear. However, even with the high dose used, no systemic absorption of gentamicin was observed as serum levels were repeatedly undetectable. This supports the hypothesis that systemic side effects are not to be expected.

A c c e p t e d M a n u s c r i p t

Neomycin was the first aminoglycoside to be administered directly into the bladder, initially to prevent bacteriuria in hospitalised patients with indwelling devices by means of continuous or intermittent irrigation [START_REF] Harper | An appraisal of 12 solutions used for bladder irrigation or instillation[END_REF]. However, this has not been advocated since Warren et al. [START_REF] Warren | Antibiotic irrigation and catheter-associated urinary-tract infections[END_REF] could not show any benefit in patients with indwelling catheters.

The review has some limitations that are predominantly related to the quantity, quality, design and heterogeneity of the original literature on this topic. In general, the few included studies had methodological flaws, the studies were heterogeneous and the sample sizes were small. Consequently, the evidence summarised in this review is not sufficient to draw definite conclusions on the effectiveness and safety of aminoglycoside instillations. As a tool to prevent bacteriuria in hospitalised patients, some benefit has been suggested but the results are not consistent [START_REF] Haldorson | Nonvalue of neomycin instillation after intermittent urinary catheterization[END_REF][START_REF] Pearman | The value of kanamycin-colistin bladder instillations in reducing bacteriuria during intermittent catheterisation of patients with acute spinal cord injury[END_REF][START_REF] Anderson | Prophylaxis of bacteriuria during intermittent catheterization of the acute neurogenic bladder[END_REF]. Furthermore, most studies included in this review did not differentiate between symptomatic and asymptomatic bacteriuria. The clinical relevance of intravesical aminoglycoside treatment therefore remains unclear in most of the studies. An exception might be the intravesical installation of gentamicin, as the three studies all point towards a significant reduction of bacteriuria and UTI in selected patients with recurrent UTI [START_REF] Wan | Intravesical instillation of gentamicin sulfate: in vitro, rat, canine, and human studies[END_REF][START_REF] Mcguire | Treatment of intractable bacterial cystitis with intermittent catheterization and antimicrobial instillation: case report[END_REF][START_REF] Defoor | Safety of gentamicin bladder irrigations in complex urological cases[END_REF]. Combined with our own experience in two patients, we therefore consider intravesical gentamicin to be a reasonable treatment option in selected patients. This may contradict a recently published international guideline on the management of catheter-associated UTI [START_REF] Hooton | Diagnosis, prevention, and treatment of catheter-associated urinary tract infection in adults: 2009 International Clinical Practice Guidelines from the Infectious Diseases Society of America[END_REF] that recommends not using catheter irrigation with antimicrobials. However, this recommendation is predominantly based on studies in patients with a chronic indwelling urinary catheter in which intravesical installation of antimicrobials is more like a rinsing of the drainage bag and catheter A c c e p t e d M a n u s c r i p t rather than active treatment of bladder infection. In contrast, in our experience highdose gentamicin remained in the bladder for over 8 h per day when left following intermittent catheterisation; 80 mg gentamicin in 20 mL of normal saline (=4000 mg/L) was left in the bladder and, assuming a volume of 500 mL will be catheterised at the next catheterisation, the urine gentamicin level will still be 160 mg/L as was confirmed by Case 2. Therefore, we consider the potential development of antimicrobial resistance to be unlikely.

Regarding the safety of intravesical gentamicin, in 90 children reported in the literature there was no systemic absorption [START_REF] Wan | Intravesical instillation of gentamicin sulfate: in vitro, rat, canine, and human studies[END_REF][START_REF] Defoor | Safety of gentamicin bladder irrigations in complex urological cases[END_REF]. Previously, negligible absorption of neomycin during post-operative irrigation through indwelling devices was reported [START_REF] Chamberlain | The absorption of antibiotics from the bladder[END_REF]. However, caution is still warranted as two reports have noted neomycin ototoxicity following bladder irrigation through indwelling catheters [START_REF] De Jong | Neomycin toxicity in bladder irrigation[END_REF][START_REF] Gerharz | Neomycin-induced perception deafness following bladder irrigation in patients with end-stage renal disease[END_REF].

Nevertheless, it should be emphasised that serum neomycin levels were not monitored whilst all these patients had end-stage renal disease. Moreover, there are no data on long-term follow-up of patients treated with intravesical aminoglycosides and one study suggested that it may disrupt the uroepithelium [START_REF] Elliott | Bladder irrigation or irritation?[END_REF]. The duration of intravesical gentamicin treatment should therefore be determined based on an individual risk-benefit assessment.

In summary, we conclude that intravesical treatment with gentamicin appears to be safe and effective in treating and suppressing UTI in patients practicing CIC. In selected patients with very limited treatment options for UTI, such intravesical gentamicin treatment might therefore be considered as a reasonable alternative to improve patients' quality of life. Potential side effects should be closely monitored as b A solution of chlorhexidine gluconate 0.01% added with ethylene diamine tetra-acetic acid disodium salt and Tris buffer.

A

  c c e p t e d M a n u s c r i p t complications of this unusual regimen to suppress recurrent UTI in patients undergoing intermittent catheterisation.

A

  comprehensive literature search was performed to identify all clinical trials reporting intravesical UTI treatment with aminoglycosides in patients using intermittent catheterisation and assessing the efficacy or safety of this intervention. The search was performed on 3 March 2010. In co-operation with a trained librarian, the following databases were searched: PubMed (1949 to February 2010); EMBASE (OVID version, 1980 to March 2010); Web of Science (1945 to March 2010); Cochrane Library (1990 to March 2010); CINAHL (EBSCOhost version, 1982 to January 2010); Academic Search Premier (EBSCOhost version, 1865 to March 2010); and ScienceDirect (1823 to March 2010). The search strategy consisted of the AND combination of three main subjects, 'aminoglycosides', 'intravesical administration'

  (i) year of publication; (ii) study design; (iii) patient population; (iv) details of intervention; (v) number of participants; (vi) detection of aminoglycoside serum levels; and (vii) clinical and microbiological outcomes. The two reviewers independently extracted the data and discussed the final outcome. The quality of each individual study was determined using the level of evidence classification of the Oxford Centre for Evidence-Based Medicine (http://www.cebm.net).

Fig. 1 . 1 1

 111 Fig. 1. Flow diagram of articles searched, excluded, reviewed and included in the

Acknowledgments

The authors would like to thank Jan W. Schoones, medical librarian of Leiden University Medical Center, for assistance in performing the literature search.

Funding

None.

A c c e p t e d M a n u s c r i p t long-term data are lacking. Although the overall evidence remains limited, these data suggest that further studies on intravesical gentamicin treatment should be performed in patients using CIC who suffer UTIs with highly resistant microorganisms.

Competing interests

None declared.

Ethical approval

Not required. Edited Figure 1