Arghya Majee

Alois Würger

Collective thermo-electrophoresis of charged colloids

Keywords: numbers: 66.10.C, 82.70.-y, 47.57.J-

Thermally driven colloidal transport is to a large extent due to the thermoelectric or Seebeck effect of the charged solution. We show that, contrary to the generally adopted single-particle picture, the transport coefficient depends on the colloidal concentration. For solutions that are dilute in the hydrodynamic sense, collective effects may significantly affect the thermophoretic mobility. Our results provide an explanation for recent experimental observations on polyelectrolytes and charged particles, and suggest that for charged colloids collective behavior is the rule rather than the exception.

I. INTRODUCTION

Transport in macromolecular or colloidal dispersions is mainly driven by interface forces [START_REF] Derjaguin | Surface Forces[END_REF][START_REF] Anderson | [END_REF][3][4]. Because of the rather short-ranged flow pattern induced in the surrounding fluid, these forces do not result in hydrodynamic interactions, in contrast to diffusion and sedimentation. As a consequence, nearby beads hardly see each other, and their transport velocity is independent of concentration [5]; for the same reason, free-solution electrophoresis of polyelectrolytes does not depend on the molecular weight [START_REF]Capillary Electrophoresis[END_REF][START_REF] Viovy | [END_REF]. Similar results have been obtained for thermal diffusion of high polymers [8][9][10][11].

Recent experiments on thermophoresis in charged colloids, however, dress a rather different picture and indicate that the single-particle description fails in several instances: Contrary to expectation, the transport velocity due to a temperature gradient,

u = -D T ∇T, (1)
was found to depend on the volume fraction of particle dispersions and on the chain length N of macromolecular solutions: (i) Data on sodium polystyrene sulfonate (NaPSS) [12] and single-stranded DNA [13] at constant polymer content but variable N, reveal that the mobility D T becomes smaller for larger molecules; e.g., in the range from 50 to 48000 base pairs, that of DNA decreases by a factor of 5. These findings are obtained at low concentration where the molecular mean distance is much larger than the gyration radius. (ii) Regarding particle suspensions, experiments on 70-nanometer silica beads [14] and 26-nm latex spheres [15] in a weak electrolyte, show that at a volume fraction of 2%, D T is significantly reduced with respect to the zero-dilution value.

In the present work we show that these experimental findings arise from an interaction mechanism that has been overlooked so far, i.e., the collective thermoelectric response of the composite system. By treating the salt ions and the dispersed colloid on an equal footing, we find that both the thermoelectric field and the mobility D T vary with the colloidal concentration. Depending on the electrolyte strength and the valency of the macroions, collective effects may occur at low dilution, that is, for particle dispersion with negligible pair potential and polymer solutions where neighbor chains do not overlap.

Thermally driven motion of charged colloids is very sensitive to the solvent composition. From previous work it emerges that two rather different mechanisms contribute to the velocity [15,16],

u = -µ T ∇T + µE. (2)
The first term arises from the local particle-solvent interactions in a non-uniform temperature. As first pointed out by Ruckenstein [17], the temperature gradient deforms the electric double layer and induces a pressure gradient opposite to ∇T . The resulting thermoosmotic surface flow toward higher T drives the particle to the cold side; the overall picture is similar to electroosmotic effects in an electric field [18]. The coefficient µ T ∝ εζ 2 /ηT depends on the ζ-potential, and the solvent permittivity ε and viscosity η; different prefactors occur in the limits of small and large particles [17][18][19][20][21][22][23][24][25]. This form agrees rather well with the observed salinity dependence [26], yet fails in view of the strong variation with T reported for various systems [12,13], thus suggesting the existence of an additional, so far poorly understood contribution to µ T .

The present work deals with the second term in (2), which accounts for the Seebeck effect of the charged solution or, in other words, for electrophoresis in the thermoelectric field E with the mobility µ = εζ/η. Due to their temperature dependent solvation forces, ions migrate along or opposite to the thermal gradient. As a consequence, surface charges develop at the cold and warm boundaries of the vessel and give rise to a macroscopic electric field E = -ψ∇T /T ; see Fig. 1. The thermopotential parameter ψ is related to the Seebeck coefficient S = -ψ/T ; for electrolytes S attains values of several 100µV/K, which is by one to two orders of magnitude larger than in common metals [START_REF] Rowe | Thermoelectrics Handbook-Macro to Nano[END_REF].

II. THERMOPHORETIC MOBILITY

We consider a dispersion of negatively charged particles or macromolecules of valency -Z and concentration n, in a monovalent electrolyte solution of ionic strength n 0 with a constant temperature gradient ∇T . According to the general formulation of non-linear thermodynamics the currents of colloid and salt ions are linear functions of generalized forces [START_REF] De Groot | Non-equlibrium Thermodynamics[END_REF]; the latter can be expressed through thermal and concentration gradients. The current of colloidal macroions is given by

J = -D∇n + nu, (3)
where the first term on the right-hand side accounts for normal diffusion and the second one for transport with the drift velocity [START_REF] Anderson | [END_REF]. The densities of small ions account for the counterions released by the colloidal particles and the added salt. the salinity. The mobile ion currents

J ± = -D ± ∇n ± + 2n ± α ± ∇T T ∓ n ± eE k B T (4)
comprise normal diffusion with coefficients D ± , thermal diffusion with the reduced Soret parameters α ± , and electrophoresis with the Hückel mobility for monovalent ions. In ( 3) and ( 4) we have added an electric field term; it is important to note that E is not an external field but arises from the kinetics of the mobile charges and is proportional to the applied temperature gradient. A similar phenomenon occurs in a non-uniform electrolyte, where the electric field is proportional to the salinity gradient and to the difference of the ionic diffusion coefficients D ± [START_REF] Anderson | [END_REF][START_REF] Prieve | [END_REF].

The numbers α ± describe the drift of positive and negative salt ions in a temperature gradient. The values for the most common ions have been determined by Agar from thermopotential measurements of electrolyte solutions [31]; our notation and Agar's "heat of transport" Q * ± are related through α ± = Q * ± /2k B T . Typical values range from α ≈ 0 for Li + to α ≈ 3 for OH -; those of the most common ions are given in Table I. Eqs. ( 3) and ( 4) provide the currents as functions of the generalized thermodynamic forces, that is, of the concentration and temperature gradients [START_REF] De Groot | Non-equlibrium Thermodynamics[END_REF]. We are interested in the steady state characterized by

J ± = 0 = J. (5)
For later use we give of a resulting relation for the electric field. Inserting the drift velocity (2) and superposing the three equations ( 5) such that the concentration gradients result in the gradient of the charge density, ∇ρ = e∇(n + -n --Zn), and collecting terms proportional to E and ∇T , one has

E = e 2n + α + -2n -α --ZnT µ T /D εκ 2 ∇T T + ∇ρ εκ 2 , (6)
with the shorthand notation κ 2 = e 2 (n

+ + n -+ ZnT µ T /D)/εk B T .
In order to determine the four unknowns ∇n ± , ∇n, E, the three equations ( 5) need to be completed by a fourth condition; it is provided by Gauss law

divE = ρ/ε (7)
which relates E and the charge density ρ = e(n + -n --Zn), and thus closes the above set of equations.

B. Small-gradient approximation

The above Eqs. ( 5) and ( 7) are non-linear in the concentrations and thus cannot be solved as they stand. The salt and colloid concentrations vary very little through the sample; the relative changes δn/n and δn ± /n ± between the hot and cold boundaries are proportional to the reduced temperature difference δT /T . Since in experiment, the ratio δT /T is much smaller than unity, we may safely replace the concentrations n and n ± in the coefficients of ( 6) with constants n and n± ; the latter are defined as the colloidal and salt concentrations at ∇T = 0.

Formally, this small-gradient approximations corresponds to neglecting terms that are quadratic in the small quantities ∇n ± , ∇n, E, and ∇T . This approximation has been used, more or less explicitly, in previous works on the thermoelectric effect [30,31] and in recent applications in colloidal thermophoresis [15,16,32]. Moreover, various works on the osmotic flow driven by externally imposed gradients of charged solutes resorts to the same approximation, albeit with the salinity change ∇n 0 instead of the temperature gradient [START_REF] Anderson | [END_REF][START_REF] Prieve | [END_REF]33].

C. Bulk thermoelectric field

The above relations ( 5)-( 7) describe both the bulk properties of a macroscopic sample and boundary effects such as the surface charges that develop at the hot and cold boundaries; see Fig. 1. The thickness of the surface layer is given by the Debye length and thus in the range between one and hundred nanometers. This is much smaller than the sample size. Thus we discard surface effects and discuss the bulk behavior only; a full evaluation including surface effects is given in the Appendix.

In a macroscopic sample the net charge density vanishes because of the huge electrostatic energy. With ρ bulk = 0, Gauss' law imposes [START_REF] Viovy | [END_REF] imposes a constant electric field; its explicit expression is readily obtained from ( 6)

E = -ψ ∇T T , (8)
with the shorthand notation for the coefficient of ∇T/T

ψ = -e 2n + α + -2n -α --Z nT µ T /D εκ 2
and κ2 = e 2 (n + + n-+ Z nT µ T /D)/εk B T . Note that we have used the small-gradient approximation and replaced the colloidal and ion concentrations with their mean values.

Although it is not always mentioned explicitly, the argument of zero bulk charge density has been used in previous works on the Seebeck effect of electrolytes [15,16,[30][31][32] and, more generally, for colloidal transport in non-equilibrium situations involving thermal or chemical gradients [START_REF] Anderson | [END_REF][START_REF] Prieve | [END_REF]33].

D. Zero-dilution limit

We briefly discuss the case of a very dilute suspension where the colloidal charges are negligible for the electrostatic properties. Putting n → 0 the electric field (8) we have

ψ 0 = -(α + -α -) k B T /e and E 0 = (α + -α -) k B ∇T e .
This expression has been used previously in [15,16,30,32]. Note that the parameter κ -1 reduces to the usual exprerssion of the Debye screening length.

Inserting the thermoelectric field E in the drift velocity (2) and comparing with (2) defines the thermophoretic mobility

D 0 T = µ T + εζψ 0 ηT . (9)
Not surprisingly it is independent of the colloidal concentration. The parameter ψ 0 and the macroscopic thermopotential U = ψ 0 δT /T between the hot and cold vessel boundaries, are given by the steady state of the electrolyte solution. With the numbers of Table I, one finds the values ψ 0 = -15 mV and +70 mV for NaCl and NaOH solutions, respectively. Thus one expects D 0 T to change its sign upon replacing one salt by the other [16]. This is confirmed by a very recent study on sodium dodecylsulfate (SDS) micelles, where the electrolyte composition NaCl 1-x OH x was varied at constant ionic strength [32]; increasing the relative hydroxide content x from 0 to 1 resulted in a linear variation of the Soret coefficient S T and a change of sign at x ≈ 1 2 [32].

E. Collective effects on the electric field E

Now we derive the main result of this paper, that is, the dependence of E and D T on the colloidal concentration and, in the case of polyelectrolytes, on its molecular weight. As two important parameters we define the ratio of the colloidal charge density and the salinity,

φ = Z n n 0 , (10)
and the ratio of colloidal electrophoretic mobility µ and diffusion coefficient D,

ξ = k B T e |µ| D . (11)
In the following we assume a negative surface potential. For typical colloidal suspensions, the charge ratio is smaller than unity, φ ∼ 0.1, whereas the parameter ξ may exceed 10 2 .

Rewriting the coefficient ψ in (8) in terms of the dimensionless quantities φ and ξ, we have

ψ = - 2(1 + φ)α + -2α --φT µ T /D 2 + φ + φξ k B T e . (12)
Eq. ( 12) shows how the thermoelectric field arises from the competition of the Soret motion of the mobile ions and the colloidal solute. In the low-dilution limit φ → 0 the first term in the numerator reduces to (α + -α -) which corresponds to the response of the electrolyte solution discussed in previous work [15,16,32]. The φ-dependent term in the numerator becomes relevant where φ ∼ D/T µ T and, in particular, may change the sign of ψ and thus of the thermoelectric field. With typical values T µ T ∼ 10 -9 m/s 2 one has D/T µ T = 10 -3 for micron-size particles (and polyelectrolytes of a gyration radius.of 1 µm), and D/T µ T = 10 -1 for 10nanometer beads. This means that, at typical colloidal densities, the thermoelectric field is essentially determined by the macroions. The denominator in (12) results in an overall decrease when augmenting the colloidal concentration.

F. Collective effects on the mobility DT

Now we determine the steady-state thermophoretic mobility. Plugging the value of the electric field E given in (8) into the drift velocity (2) and comparing with (1), we get

D T = D 0 T 1 + φ 2+φ ξ . ( 13
)
where D 0 T is defined by Eq. ( 9) albeit with a modified parameter

ψ 0 = - (1 + φ)α + -α - 1 + φ/2 k B T e . (14)
The mobility and its dependence on the ratio φ constitute the main result of this paper. According to (9), the sign of D T is determined by the competition of the bare mobility µ T and the Seebeck term proportional to ζψ 0 . Since φ < 1 in most cases, the numerator of ( 13) is rather similar to the dilute case discussed above (9). A much more striking variation arises from the denominator of (13). For typical values of the charge ratio φ ∼ 0.1, collective effects set in where 1 2 φξ ∼ 1, in other words where ξ is of the order of 20. For high polymers (N = 10 3 ...10 6 ) and colloidal particles in the range from ten nanometers to a micron, the parameter ξ takes values between 10 and 10 3 . This simple estimate suggests collective effects to occur in many systems. A detailed comparison with experiment is given in the following section.

In the limit of zero dilution φ → 0 one readily recovers the expression (9). The opposite case of a saltfree system leads to

D T = D 0 T 1 + ξ , (φ → ∞)
with ψ 0 determined by the counterions only. In view of the large values of ξ mentioned above, one expects a strong reduction of the mobility in the salt free case.

III. COMPARISON WITH EXPERIMENT

We discuss Eq. ( 13) in view of recent experiments on colloidal suspensions. At relevant values of the charge ratio (φ ∼ 0.1) the numerator hardly differs from that of the dilute case. Thus in the following we focus on the reduction of D T due to the denominator.

A. Polyelectrolytes

We start with experimental findings on polyelectrolytes at constant volume fraction but variable molecular weight. In their study of 2 g/l of NaPSS in a 100 mM/l NaCl solution, Iacopini et al. found a significant variation with the chain length [12]: Fig. 2a shows the data measured at 30 • C for molecules of 74, 160, and 360 repeat units, with an overall decrease of the mobility by 40 percent. The same factor has been found in the temperature range from 15 to 35 • The solid line represents collective effects arising from the denominator of Eq. ( 13). It has been calculated with the double-layer term in the small-bead limit, assuming the monomer to be small as compared to the Debye length (R < λ) [22][23][24],

µ T = - dε dT ζ 2 3η ,
and with the Hückel-limit electrophoretic mobility µ =

ξ = e|ζ| k B T R ℓ B . ( 15
)
The theoretical curve of Fig. 2a is calculated with the parameters ζ = -27 mV, nN = 10 mM/l, and φ = 0.1. Its variation arises only from the gyration radius R = ℓN 1-ν K N ν ; we have used the usual exponent ν = 3 5 , the size of a monomer ℓ = 0.4 nm, and the number of monomers per segment N K = 10. The dashed line indicates the mobility in the short-chain limit. The theoretical expression (13) provides a good description of the reduction of D T with increasing chain length.

As a second example, DNA in 1 mM/l Tris buffer shows a similar behavior; its mobility decreases by a factor of 5 over the range from N =50 to 48500 base pairs per molecule [13]. The overall DNA content was kept constant, N n = 50µM/l, with a charge ratio φ = 0.05. Eqs. ( 13) and ( 15) provide a good fit to these data, albeit with a somewhat too small exponent ν ≈ 0.4. In view of this discrepancy one should keep in mind the rather complex electrostatic properties of polyelectrolytes.

The reduction observed for both NaPSS and DNA cannot be explained by hydrodynamic effects. Interchain interactions are of little significance because of the low dilution. Indeed, the effective volume fraction of the polymer coils hardly attains a few percent, nR 3 ∼ 10 -2 ; thus [12]. The solid line is calculated from Eq. ( 13) with the parameters as given in the main text. The dependence on N arises from the gyration radius R. (b) Volume fraction dependence of DT of a dispersion of 70-nm silica beads in a solution of 30µM/l sulphorhodamine B. The data are from Ghofraniha et al. [14]; the fit curves are obtained from ( 13) and ( 16), with different values of the reduced virial coefficient B/V , where V = 4 3 πR 3 is the particle volume. nearby chains do not overlap and leave both the viscosity and the diffusion coefficient unchanged. Regarding hydrodynamic interactions of beads of the same molecule, it is known that they enhance the electrophoretic mobility in (2) and ( 9) with increasing chain length. Yet this effect occurs for short polyelectrolytes and saturates for chains longer than the size of the screening cloud [35]; for the examples studied here, it would enhance D T in the range N < 40. We conclude that hydrodynamic effects may ruled out as an explanation for the reduction shown in Fig. 2a. Finally we discuss electrostatic single-particle effects. The electrophoretic mobility in saltfree solution has been found to decrease slightly at higher concentration, because of the increase of the overall ionic strength and the shorter screening length [36,37]. In the present case, however, the weight fraction of the polyelectrolyte is constant, and so is the overall charge density. Thus the electrostatic properties of the solution are the same for different chain lengths.

B. Colloidal particles

Now we discuss the concentration dependent mobility D T that has been reported for dispersions of solid particles in weak electrolytes. Ghofraniha et al. studied silica particles (R = 35 nm) in a 30 µM/l solution of the negatively charged dye sulpho-rhodamine B [14]. The data shown in Fig. 2b reveal a significant decrease with the colloidal volume fraction; at 3% D T is reduced to less than half of the zero-dilution value. The negative sign of the measured D T indicates that the thermoelectric contribution εψ 0 ζ/ηT to (9) overtakes the Ruckenstein term [16] µ T = εζ 2 3ηT .

The negative surface potential ζ implies that the thermopotential parameter of the sulpho-rhodamine solution is positive, ψ 0 > 0.

The curves in Fig. 2b are calculated from ( 11) and ( 13) with ψ 0 = 10 mV, which is comparable to common salts and weaker than the values of NaOH and tetraethylammonium [15,32]. The rather small D T suggests that the particles are weakly charged; we use Z = 30 and ζ = -10 mV. The dashed line gives the mobility D 0 T in the zero-dilution limit, whereas the solid lines are given by (13).

In addition to the explicit concentration dependence in terms of the parameter φ, one has to take into account that, even at moderate colloidal volume fraction, the Einstein coefficient D is not constant. Indeed, cooperative diffusion of charged particles arises from the electrostatic pair potential Φ(r) and, to a lesser extent, from hydrodynamic interactions [START_REF] Russel | Colloidal Dispersions[END_REF]. To linear order in the concentration, the virial expansion for the Einstein coefficient reads as

D = D 0 (1 + 2nB), (16)
with the parameter

B = 1 2 dV 1 -e -Φ/kBT .
For hard spheres the virial coefficient is given by the particle volume, B = 4V with V = 4 3 πR 3 The electrostatic pair potential results in an effective interaction volume V = 4 3 π(R + χ 2 λ) 3 , where λ is the Debye length and χ a numerical factor [26,32,34]; for small and highly charged particles in a weak electrolyte, the repulsive forces may enhance the virial coefficient by one or two orders of magnitude. On the other hand, hydrodynamic interactions contribute a negative term B/V ∼ -6.5 and reduce the Einstein coeffient accordingly [START_REF] Russel | Colloidal Dispersions[END_REF]. Our discussion of the data of Ref. [14] is restricted to volume fractions up to 3%; at higher concentration the measured D saturates and the linear approximation ceases to be valid. In units of the particle volume V , the measured virial coefficient reads B/V = 20 [14]; the best fit of the mobility data is obtained with B/V = 14. This value is much larger than that of hard spheres and thus indicates the importance of electrostatic repulsion. The concentration of mobile charge carriers n 0 = 30µM/l leads to a screening length of about 50 nm. With χ ∼ 2 in the above expression for the effective volume, one finds a virial coefficient close to the measured value. As an illustration of the effect of collective diffusion on D T , we plot Eq. ( 13) for these three values: Though the variation of D T with B is not neglegible, it is significantly weaker than that of the thermoelectric effect.

As a second experiment we mention data by Putnam and Cahill on latex beads of radius R = 13 nm in an electrolyte solution of 2mM/l ionic strength [15]; varying the volume fractions from 0.7 to 2.2 wt%, these authors observed a reduction of D T by about 10 percent. With a valency of Z ∼ 50 one finds that, at the highest particle concentration n = 4µM/l, the charge ratio φ does not exceed 10 percent.

Finally we address the concentration dependence observed by Guarino and Piazza for the Soret coefficient S T = D T /D of SDS micelles [26]. Its decrease with the SDS content, is well described by collective diffusion according to (16). In a very recent measurement, Vigolo et al. vary the electrolyte composition NaCl 1-x OH x and thus the diffusion parameter of the anion in ( 14), α -= (1 -x)α Cl + xα OH [32]. The observed linear dependence of S T on x confirms the crucial role of the thermopotential. Unfortunately there are no mobility data for micelles; thus at present it is not possible to determine whether their D T is subject to collective effects similar to those of polyelectrolytes and solid beads.

IV. SUMMARY AND CONCLUSION

In summary, charged colloids in a non-uniform temperature show collective transport behavior mediated by the Seebeck effect of both colloidal and salt ions. For large particles and macromolecules, cooperative effects set in at rather low concentration, where hydrodynamic interactions are absent and where the charge ratio φ is much smaller than unity. The criterion for the onset of collective behavior, φξ ∼ 1 in (13), involves the ratio of the electrophoretic mobility and the Einstein coefficient; by contrast, the criterion for cooperative diffusion, Bn ∼ 1, depends on the pair potential of the solute particles. The discussed examples suggest that the collective thermoelectric effect is generic for colloids at ordinary concentrations. This issue could be relevant for microfluidic applications of thermophoresis.

We conclude with a remark on the thermoelectric field given in Eq. ( 12). Both its magnitude and its sign can be tuned by chosing the appropriate electrolyte and adjusting the charge ratio. With a thermal gradient of less than one Kelvin per micron, E may attain values of 100 V/m. Thus the thermoelectric effect could be used for applying electric fields in microfluidic devices. Local laser heating would permit to realize almost any desired spatiotemporal electric-field pattern.

Helpful and stimulating discussions with D.G. Cahill, R. Piazza, D. Braun, and N. Ghofraniha are gratefully acknowledged.

V. APPENDIX

The thermoelectric field (8) has been derived by using the charge neutrality of the bulk of a macroscopic sample. Here we give a derivation based on the steady state, Gauss' law, and the electrostatic boundary conditions. Resorting to the small-gradient approximation, we replace the coefficients in [START_REF]Capillary Electrophoresis[END_REF] by their mean values and thus have

E = -ψ ∇T T + ∇ρ ε κ2 .
From Gauss's law [START_REF] Viovy | [END_REF] one has ∇ρ/ε = ∇ 2 E and thus obtains a differential equations for the thermoelectric field E with a constant inhomogeneity -(ψ/T )∇T ,

E - ∇ 2 E κ2 = -ψ ∇T T .
The solution E = E inh + E h consists of two contributions. The inhomogeneous term E inh = -(ψ/T )∇T accounts for the macroscopic Seebeck effect. The remaining one E h is related to surface charges at the cold and hot boundaries of the sample. The homogeneous equation ∇ 2 E h = κ2 E h is solved by the exponential function,

E h = A + e κz + A -e -κz ,
where z is the coordinate in the direction of the temperature gradient. Its range is -1 2 L ≤ z ≤ 1 2 L with the sample size L.

The electrostatic boundary conditions require that the electric field vanishes at z = ± 1 2 L. Putting E = 0 and solving for the coefficients of E h , one readily finds A ± = -1 2 E inh / cosh(κL/2) and the thermoelectric field

E = - ψ T ∇T 1 - cosh(κz) cosh(κL/2)
.

Both E and the corresponding charge density ρ are illustrated in the right panel of Fig. 1. The field vanishes at the boundaries and reaches its constant bulk value (8) within a few screening lengths κ-1 . The parameter κ-1 takes values in the range between 1 and 100 nanometers and thus is much smaller than the size of sample L. Even in microfluidic devices, κL is in general larger than 10 3 . In real systems a more complex picture may emerge from the surface roughness of the boundaries, the solute size, and surface charges of other origin. Note that such additional effects do not affect the bulk electric field (8) and thus are irrelevant for the results of this paper.

2 3

 2 εζ/η. Inserting the diffusion coefficient D = k B T /6πηR and the Bjerrum length ℓ B = e 2 /4πεk B T in (11), we have

FIG. 2 :

 2 FIG. 2: Comparison with measured data. (a) Variation of DT with the chain length N of a polyelectrolyte at fixed volume fraction. The data on 2 g/l NaPSS in a 100 mM/l NaCl solution at 30 • C are taken from Iacopini et al. [12]. The solid line is calculated from Eq. (13) with the parameters as given in the main text. The dependence on N arises from the gyration radius R. (b) Volume fraction dependence of DT of a dispersion of 70-nm silica beads in a solution of 30µM/l sulphorhodamine B. The data are from Ghofraniha et al.[14]; the fit curves are obtained from (13) and (16), with different values of the reduced virial coefficient B/V , where V =4 3 πR 3 is the particle volume.

TABLE I :

 I Reduced Soret coefficient α ± of several salt ions at room temperature. The values of the heat of transport Q * This schematic view exaggerates the surface layers, which are much thinner in real systems. The right panel shows the spatial variation of the net charge density ρ, the thermoelectric field

		cold	warm
			charge density ρ
			thermoelectric field E
			thermopotential U
		FIG. 1: Thermoelectric effect in a colloidal suspension of
		charged particles in salt solution. In the example presented,
		the Soret parameters are such that negative and positive ions
		accumulate at the cold and warm boundaries, respectively.
		In the left panel, vertical dashed lines indicate the thick-
		ness of the surface layers of about one Debye length λ.
		±
	are taken from Ref. [31]. The parameters α± are calculated from α± = Q * ± /2kBT .
	Ion	H + Li + K + Na + OH -Cl -
	Q * i (kJ/Mol) 13.3 0.53 2.59 3.46 17.2 0.53
	αi	2.7 0.1 0.5 0.7 3.4 0.1

E, and the thermopotential U ; dashed lines indicate the zero of the ordinate. Note the non-zero surface charges at the cold and hot boundaries. The present paper discusses the bulk behavior only, where ρ = 0 and where E is constant.

A. The steady state


Jérémy Lecoeur

Feng Wang

Min Li

Rui Chen

Malcolm J Li

Benoit M Avison

Dawant

Min Li

Chen

Rui Li

Malcom J Avison B C , Benoit

M Dawant

Sub millimeter coregistration of functional maps accross imaging sessions

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction -fMRI is ideally suited for longitudinal studies of structural and functional brain plasticity. It is now feasible to collect functional brain maps with spatial resolutions of ~100 m in ultra-high field systems. While these data sets are intrinsically coregistered for studies collected within a session from immobilized animals, the accuracy of coregistration across imaging sessions (or within, in the presence of head motion) is limited by the accuracy with which structural and functional images can be coregistered The goal of this study is to develop an accurate and reliable registration process for longitudinal acquisitions of functional MRI scans providing partial brain coverage at very high resolution in order to quantify functional and structural changes across time Methods -Structural and functional images were collected from five squirrel monkeys across multiple sessions separated by weeks to months. In each session, three types of image volumes were acquired: a) BOLD and/or CBV-weighted functional echo planar images (EPI) runs, centered on the somato-sensory cortex, comprising 4 contiguous slices oriented obliquely with respect to the scanner frame of reference (in-plane resolution 0.27 x 0.27 mm, 2 mm slice thickness); b) a high resolution anatomical slab of 4 slices covering the same space, having the same orientation as the functional slab (in-plane resolution 68.5 x 68.5 m, slab thickness 2 mm); c) an anatomical image covering the whole head (resolution: 0.5x0.5x0.5 mm). All images were acquired with the same 3 cm surface coil on anesthetized monkeys under an IACUC approved protocol. Focal single activations were generated in area 3b of primary somatosensory cortex by delivering seven 30 sec duration blocks of vibrotactile stimulation, alternated with 30 sec rest periods, to digits 1 and 3. We assume that geometric distortion is minimal for the anatomical slabs. The various transformations required to register these images are thus all rigid body transformations. The easiest approach would be to simply register slabs acquired during different sessions to each other with a standard intensity-based algorithm. However incomplete overlap of slabs across sessions led to unsatisfactory performance with intensity-based techniques. The solution we developed to address these issues involved four main steps: (1) register the anatomical slab to the full head volume acquired during the same session; (2) register full head volumes across sessions;

(3) apply distortion correction to the functional maps; (4) apply the overall transformation computed to the distortion corrected functional maps.

Using a combination of mutual information [1] intensity-based rigid-body registration [2] with information retrieved from the scanner (Euler angles used to acquire the scan and center of the volume) as a starting point, we were able to accurately register the anatomical slab to the full head scan. Since signal attenuation is severe with the surface coils, and because the coil is not placed at the exact same position relative to the brain from acquisition to acquisition, regions of the brain show variable signal attenuation across acquisitions and direct intensity based registration between full head scans was unreliable. We therefore generated gradient images, and used mutual information on these gradient images of the full head scans to register them together between the two timestamps Since the EPI data sample the same volume as the anatomical slab, the overall transform from anatomical slab 1 to anatomical slab 2 was used on functional maps computed from the average of the three EPI runs without, and then with distortion correction

Results -Fig. 1a-c show the two original anatomical slabs before registration and the result of registration. The green lines represent landmarks from slab 1 (sulci, vessels, etc.) projected on slab 2 after registration. Anatomic coregistration of the slab across two sessions in five monkeys was accurate to 30 m (s.d. 15 m). Fig. 1d-g show the same slice of both slabs with the BOLD functional maps overlaid before registration and following registration of raw and distortion-corrected functional maps. Fig. 1h-k present the equivalent results for the CBV-base dfunctional maps. As shown in Fig. 1 f, j, this rigid body coregistration of raw functional maps, while accurate to ~1 mm across sessions, was nonetheless significant, and presumably reflects the effects of residual distortion in the EPI images. Correction of these residual distortions led to excellent (< 100 m) coregistration of the activation centers maps obtained by BOLD and CBV mapping.

Discussion/Conclusion -To the best of our knowledge, this is the first registration method proposed for the longitudinal registration of ultrahigh resolution functional maps with sub-millimeter coregistration accuracy. Furthermore, these results suggest that high resolution fMRI mapping has the capability to identify functional reorganization in cortex with sub-millimeter accuracy in longitudinal studies. 187-198 (1997).

  -[1] Studholme, C., Hill, D. and Hawkes, D., "An overlap invariant entropy measure of 3D medical image alignment," Pattern Recognition 32, 71-86 (1999). [2] Maes, F., Collignon, A., Vandermeulen, D., Marchal, G. and Suetens, P., "Multimodality image registration by maximization of mutual information," IEEE Transactions on Medical Imaging 16(2),

Figure 1 -

 1 Figure 1 -Results of registration. From left to right: Session 1, session 2 and result of registration of session 1 images to session 2. Top panel: anatomical slabs. Middle panel: BOLD maps. Bottom panel: CBV maps. Colors lines on right column images are features from session 1 projected on session 2 images after registration.


Apostolos P Athanasiadis

Menelaos Zafrakas

Christos Arnaoutoglou

Aikaterini Karavida

Panayiota Papasozomenou

Basil C Tarlatzis

Keywords: Prenatal Diagnosis, Ectopic Kidney, Intrathoracic Kidney, Congenital Diaphragmatic , Ultrasonography Prenatal Diagnosis, Ectopic Kidney, Intrathoracic Kidney, Congenital Diaphragmatic Hernia, Obstetrics, Ultrasonography Prenatal Diagnosis, Ectopic Kidney, Intrathoracic Kidney, Renal Ectopy, Congenital Diaphragmatic Hernia, Obstetrics, Ultrasonography

HAL is

INTRODUCTION

Renal ectopia is a rare congenital malformation, caused by malpositioning of the kidney during embryogenesis. Most cases are located in the pelvic and lumbar regions, due to disruption of the normal migration of the kidneys before the 8 th embryonic week. Thoracic kidney is extremely rare, located in a higher position, possibly due to a different pathogenetic mechanism. Its prevalence is estimated to be less than 0.01% [1,2]. Rare cases, of acquired thoracic renal ectopia following severe trauma, have been also reported in adults [3]. Congenital thoracic kidney is generally considered to be a sporadic malformation and it is not associated with increased risk of recurrence. Males tend to be affected more often than females (63% vs. 37%, respectively) and thoracic kidneys are found more often in the left hemithorax than in the right (62% vs. 38%, respectively) [1,4]. Extremely rare cases of postnatally diagnosed bilateral congenital thoracic kidneys have been also reported [5]. Thoracic kidneys are classified into four groups: thoracic renal ectopia with 1) closed diaphragm, 2) eventration (i.e. relaxation) of the diaphragm, 3) diaphragmatic hernia, which is subdivided into congenital diaphragmatic defects and acquired hernia, such as Bochdalek hernia and 4) traumatic rupture of the diaphragm [6,7]. We report a rare case of thoracic kidney diagnosed prenatally on ultrasound at 22 weeks, with delayed ultrasound manifestation of associated congenital diaphragmatic hernia (CDH) in the 27 th week of pregnancy. A 25-year-old white woman, G1, was referred for routine anomaly scan at 22 weeks gestation. Ultrasound equipment (Voluson 730 Expert Diamond, GE Healthcare, Kretztechnik, Zipf, Austria) with RAB 4-8 abdominal convex probe (4-8MHz) was used. Previous obstetrical, medical and family history was unremarkable. The woman was a non-smoker, she did not consume any alcohol during pregnancy and she had never been exposed to drugs or toxins. The dating scan in the first trimester was compatible with dates and the nuchal translucency was normal.

On ultrasound examination at 22 weeks, the left kidney was absent from the ipsilateral renal fossa, while the left hemithorax contained a mass with mixed echogenicity, causing a mediastinal shift to the opposite side (Figure 1). The right kidney was normally located, but its pelvis was dilated to 16 mm. No further malformations were identified at this point. Based on these findings, the initial diagnosis was isolated ectopic thoracic kidney; this was confirmed by TUI, colour flow Doppler, demonstrating the abnormally high origin of left renal artery and 3Dsurface reconstruction scan. Amniocentesis revealed a normal male karyotype (46, XY), and fetal echocardiography was also normal. Fetal MRI using 1.5T equipment (Philips Intera Enterprise MRI System, Philips Medical Systems, Best, The Netherland) confirmed diagnosis of isolated intrathoracic kidney; notably there were no signs of diaphragmatic hernia (Figure 2). On follow-up ultrasound at 27 weeks, the appearance of bowel in the thoracic cavity suggested the presence of associated CDH (Figure 3). Parents were counselled by a paediatric surgeon about neonatal surgical management and decided to continue pregnancy. Prenatal management included serial ultrasound scans in order to monitor fetal growth and assess the fetal heart. Elective caesarean section was performed at 34 weeks, due to symptoms of labour (uterine contractions and cervical dilation), in order to facilitate planning of neonatal surgery. Steroids were administered 24hr prior the delivery and an ex utero intrapartum treatment (EXIT) procedure were used according to our institutional guidelines. A male infant weighing 2,650g was delivered with Apgar scores 7 and 8 at 1 and 5 minutes, respectively. The neonate was initially transferred to the Neonatal Intensive Care Unit (NICU) due to respiratory distress and surgery was performed within two hours from delivery. The herniated viscera, i.e. the left kidney, part of the spleen and part of the small intestine, were carefully re-positioned into the abdominal cavity and the diaphragmatic defect was closed with primary repair. The infant remained postoperatively in the NICU for almost three months, due to respiratory distress and sepsis, and it was discharged in good condition on day 93, weighing 3,680 g. Today, two years after neonatal surgery, the child is in good general health, with normal physical and mental development and without any long term complications. Recent, scintigraphy with Tc-99m MAG-3 and Tc-99m DMSA (Gyroscan T5-NT, Philips Medical Systems, Eindhoven, The Netherlands) showed that both kidneys are adequately functioning.

DISCUSION

Thoracic renal ectopia is a very rare congenital malformation. In a study including 15,919 autopsies in children, there was only one case of intrathoracic kidney among 22 cases of renal ectopia [START_REF] Campbell | Renal ectopy[END_REF]. Prenatal diagnosis of ectopic thoracic kidney has been only rarely reported. An extensive search of the contemporary literature revealed only two such cases [2,[START_REF] Masturzo | Prenatal diagnosis of an ectopic intrathoracic kidney in right-sided congenital diaphragmatic hernia using color Doppler ultrasonography[END_REF] (MEDLINE; 1970-October 2009; English language; search terms: "Ectopic Kidney", "Intrathoracic Kidney", "Thoracic Kidney" and "Prenatal Diagnosis"); both cases were diagnosed during the third trimester of pregnancy, whereas diagnosis in the present case was set at 22 weeks gestation. It is notable that in the present case diaphragmatic hernia, in contrast to previous cases reported, was not present at the time of prenatal diagnosis. Though a rare anomaly, thoracic kidney should be considered in the differential diagnosis of intrathoracic masses, particularly it should be differentiated from pulmonary sequestration [START_REF] Masturzo | Prenatal diagnosis of an ectopic intrathoracic kidney in right-sided congenital diaphragmatic hernia using color Doppler ultrasonography[END_REF].

Underlying diaphragmatic hernia should be always suspected in cases of thoracic renal ectopia, since this condition may manifest later, either in the third trimester, as in the present case, or postnatally, as previously described [1,[START_REF] Keles | Late-presenting congenital diaphragmatic hernia associated with ectopic thoracic kidney[END_REF]. It is well established that fetal MRI provides useful information in cases of CDH about the herniated viscera [START_REF] Hubbard | Prenatal MRI evaluation of congenital diaphragmatic hernia[END_REF], as it might demonstrate more accurately hypointense organs.

Its main limitation is in cases of a right-sided defect, in which MRI might not be helpful in distinguishing liver from renal parenchyma, as both organs are hypointense. However, it should be noted that in two of the three reported cases [2], including the present case, fetal MRI was carried out and neither revealed the presence of diaphragmatic hernia.

Prenatal management of ectopic kidneys includes amniocentesis for fetal karyotyping, since such malformations might be associated with fetal chromosomal anomalies [START_REF] Navarro | Unusual cause of lung and renal disease in a baby with trisomy 21[END_REF][START_REF] Chu | Intrathoracic kidney in a case of trisomy 18[END_REF]. Furthermore, ectopic kidneys might be associated with other malformations, including rib, cardiovascular, craniofacial, CNS and gastrointestinal anomalies [2,[START_REF] Sumner | Intrathoracic kidney--diagnosis by ultrasound[END_REF][START_REF] Pelizzo | Gastric volvulus associated with congenital diaphragmatic hernia, wandering spleen, and intrathoracic left kidney: CT findings[END_REF], and thus fetal echocardiography and detailed anomaly scan are mandatory. It should be noted however, that there is no consistent pattern of anomalies associated with renal ectopia [16].

Unlike other types of renal ectopia, intrathoracic kidneys usually function normally and very often no medical or surgical treatment is required [5]. Most patients are asymptomatic, and this condition may be discovered incidentally, even in adults [17,18]. Due to the limited number of prenatally diagnosed cases of ectopic intrathoracic kidney with associated CDH and despite the fact that post-surgical outcome was favorable in all three reported cases [2,[START_REF] Masturzo | Prenatal diagnosis of an ectopic intrathoracic kidney in right-sided congenital diaphragmatic hernia using color Doppler ultrasonography[END_REF], including the one reported here, no firm conclusions can be drawn about elective surgical treatment in the neonatal period.

SHORT TITLE / RUNNING HEAD

Fetal thoracic kidney with diaphragmatic hernia.

INTRODUCTION

Renal ectopia is a rare congenital malformation, caused by malpositioning of the kidney during embryogenesis. Most cases are located in the pelvic and lumbar regions, due to disruption of the normal migration of the kidneys before the 8 th embryonic week. Thoracic kidney is extremely rare, located in a higher position, possibly due to a different pathogenetic mechanism. Its prevalence is estimated to be less than 0.01% [1,2]. Rare cases, of acquired thoracic renal ectopia following severe trauma, have been also reported in adults [3]. Congenital thoracic kidney is generally considered to be a sporadic malformation and it is not associated with increased risk of recurrence. Males tend to be affected more often than females (63% vs. 37%, respectively) and thoracic kidneys are found more often in the left hemithorax than in the right (62% vs. 38%, respectively) [1,4]. Extremely rare cases of postnatally diagnosed bilateral congenital thoracic kidneys have been also reported [5]. Thoracic kidneys are classified into four groups: thoracic renal ectopia with 1) closed diaphragm, 2) eventration (i.e. relaxation) of the diaphragm, 3) diaphragmatic hernia, which is subdivided into congenital diaphragmatic defects and acquired hernia, such as Bochdalek hernia and 4) traumatic rupture of the diaphragm [6,7]. We report a rare case of thoracic kidney diagnosed prenatally on ultrasound at 22 weeks, with delayed ultrasound manifestation of associated congenital diaphragmatic hernia (CDH) in the 27 th week of pregnancy. A 25-year-old white woman, G1, was referred for routine anomaly scan at 22 weeks gestation. Ultrasound equipment (Voluson 730 Expert Diamond, GE Healthcare, Kretztechnik, Zipf, Austria) with RAB 4-8 abdominal convex probe (4-8MHz) was used. Previous obstetrical, medical and family history was unremarkable. The woman was a non-smoker, she did not consume any alcohol during pregnancy and she had never been exposed to drugs or toxins. The dating scan in the first trimester was compatible with dates and the nuchal translucency was normal.

On ultrasound examination at 22 weeks, the left kidney was absent from the ipsilateral renal fossa, while the left hemithorax contained a mass with mixed echogenicity, causing a mediastinal shift to the opposite side (Figure 1). The right kidney was normally located, but its pelvis was dilated to 16 mm. No further malformations were identified at this point. Based on these findings, the initial diagnosis was isolated ectopic thoracic kidney; this was confirmed by TUI, colour flow Doppler, demonstrating the abnormally high origin of left renal artery and 3Dsurface reconstruction scan. Amniocentesis revealed a normal male karyotype (46, XY), and fetal echocardiography was also normal. Fetal MRI using 1.5T equipment (Philips Intera Enterprise MRI System, Philips Medical Systems, Best, The Netherland) confirmed diagnosis of isolated intrathoracic kidney; notably there were no signs of diaphragmatic hernia (Figure 2). On follow-up ultrasound at 27 weeks, the appearance of bowel in the thoracic cavity suggested the presence of associated CDH (Figure 3). Parents were counselled by a paediatric surgeon about neonatal surgical management and decided to continue pregnancy. Prenatal management included serial ultrasound scans in order to monitor fetal growth and assess the fetal heart. Elective caesarean section was performed at 34 weeks, due to symptoms of labour (uterine contractions and cervical dilation), in order to facilitate planning of neonatal surgery. Steroids were administered 24hr prior the delivery and an ex utero intrapartum treatment (EXIT) procedure were used according to our institutional guidelines. A male infant weighing 2,650g was delivered with Apgar scores 7 and 8 at 1 and 5 minutes, respectively. The neonate was initially transferred to the Neonatal Intensive Care Unit (NICU) due to respiratory distress and surgery was performed within two hours from delivery. The herniated viscera, i.e. the left kidney, part of the spleen and part of the small intestine, were carefully re-positioned into the abdominal cavity and the diaphragmatic defect was closed with primary repair. The infant remained postoperatively in the NICU for almost three months, due to respiratory distress and sepsis, and it was discharged in good condition on day 93, weighing 3,680 g. Today, two years after neonatal surgery, the child is in good general health, with normal physical and mental development and without any long term complications. Recent, scintigraphy with Tc-99m MAG-3 and Tc-99m DMSA (Gyroscan T5-NT, Philips Medical Systems, Eindhoven, The Netherlands) showed that both kidneys are adequately functioning.

DISCUSION

Thoracic renal ectopia is a very rare congenital malformation. In a study including 15,919 autopsies in children, there was only one case of intrathoracic kidney among 22 cases of renal ectopia [START_REF] Masturzo | Prenatal diagnosis of an ectopic intrathoracic kidney in right-sided congenital diaphragmatic hernia using color Doppler ultrasonography[END_REF][START_REF] Campbell | Renal ectopy[END_REF]. Prenatal diagnosis of ectopic thoracic kidney has been only rarely reported. An extensive search of the contemporary literature revealed only two such cases [2,[START_REF] Masturzo | Prenatal diagnosis of an ectopic intrathoracic kidney in right-sided congenital diaphragmatic hernia using color Doppler ultrasonography[END_REF] (MEDLINE; 1970-October 2009; English language; search terms: "Ectopic Kidney", "Intrathoracic Kidney", "Thoracic Kidney" and "Prenatal Diagnosis"); both cases were diagnosed during the third trimester of pregnancy, whereas diagnosis in the present case was set at 22 weeks gestation. It is notable that in the present case diaphragmatic hernia, in contrast to previous cases reported, was not present at the time of prenatal diagnosis. Though a rare anomaly, thoracic kidney should be considered in the differential diagnosis of intrathoracic masses, particularly it should be differentiated from pulmonary sequestration [START_REF] Masturzo | Prenatal diagnosis of an ectopic intrathoracic kidney in right-sided congenital diaphragmatic hernia using color Doppler ultrasonography[END_REF].

Underlying diaphragmatic hernia should be always suspected in cases of thoracic renal ectopia, since this condition may manifest later, either in the third trimester, as in the present case, or postnatally, as previously described [1,[START_REF] Keles | Late-presenting congenital diaphragmatic hernia associated with ectopic thoracic kidney[END_REF]. It is well established that fetal MRI provides useful information in cases of CDH about the herniated viscera [START_REF] Hubbard | Prenatal MRI evaluation of congenital diaphragmatic hernia[END_REF], as it might demonstrate more accurately hypointense organs.

Its main limitation is in cases of a right-sided defect, in which MRI might not be helpful in distinguishing liver from renal parenchyma, as both organs are hypointense. However, it should be noted that in all three reported cases of prenatally diagnosed thoracic kidney [1,[START_REF] Masturzo | Prenatal diagnosis of an ectopic intrathoracic kidney in right-sided congenital diaphragmatic hernia using color Doppler ultrasonography[END_REF], including the present case, fetal MRI did not reveal the presence of diaphragmatic hernia. However, it should be noted that in two of the three reported cases [2], including the present case, fetal MRI was carried out and neither revealed the presence of diaphragmatic hernia.

Prenatal management of ectopic kidneys includes amniocentesis for fetal karyotyping, since such malformations might be associated with fetal chromosomal anomalies [START_REF] Navarro | Unusual cause of lung and renal disease in a baby with trisomy 21[END_REF][START_REF] Chu | Intrathoracic kidney in a case of trisomy 18[END_REF]. Furthermore, ectopic kidneys might be associated with other malformations, including rib, cardiovascular, craniofacial, CNS and gastrointestinal anomalies [2,[START_REF] Sumner | Intrathoracic kidney--diagnosis by ultrasound[END_REF][START_REF] Pelizzo | Gastric volvulus associated with congenital diaphragmatic hernia, wandering spleen, and intrathoracic left kidney: CT findings[END_REF], and thus fetal echocardiography and detailed anomaly scan are mandatory. It should be noted however, that there is no consistent pattern of anomalies associated with renal ectopia [16].

Unlike other types of renal ectopia, intrathoracic kidneys usually function normally and very often no medical or surgical treatment is required [5]. Most patients are asymptomatic, and this condition may be discovered incidentally, even in adults [17,18]. Due to the limited number of prenatally diagnosed cases of ectopic intrathoracic kidney with associated CDH and despite the fact that post-surgical outcome was favorable in all three reported cases [2,[START_REF] Masturzo | Prenatal diagnosis of an ectopic intrathoracic kidney in right-sided congenital diaphragmatic hernia using color Doppler ultrasonography[END_REF], including the one reported here, no firm conclusions can be drawn about elective surgical treatment in the neonatal period.

In conclusion, this is a prenatally diagnosed thoracic kidney in the second trimester of pregnancy, with delayed ultrasound manifestation of an associated congenital diaphragmatic hernia.

8 . 9 . 15 .

 8915 Masturzo B, Kalache KD, Cockell A, et al. Prenatal diagnosis of an ectopic intrathoracic kidney in right-sided congenital diaphragmatic hernia using color Doppler ultrasonography. Ultrasound Obstet Gynecol 2001;18:173. Campbell MF. Renal ectopy. J Urol 1930;24:187-189. 10. Keles S, Artac H, Elmaci M, et al. Late-presenting congenital diaphragmatic hernia associated with ectopic thoracic kidney. Eur J Pediatr 2006;165:571. 11. Hubbard AM, Crombleholme TM, Adzick NS, et al. Prenatal MRI evaluation of congenital diaphragmatic hernia. Am J Perinatol 1999;16:407. 12. Navarro A, Jimenez J, Rios T, et al. Unusual cause of lung and renal disease in a baby with trisomy 21. Pediatr Pulmonol 2005;40:173. 13. Chu IW, Yeh SJ, Lin YC. Intrathoracic kidney in a case of trisomy 18. Turk J Pediatr 2008;50:176. 14. Sumner TE, Volberg FM, Smolen PM. Intrathoracic kidney--diagnosis by ultrasound. Pediatr Radiol 1982;12:78. Pelizzo G, Lembo MA, Franchella A, et al. Gastric volvulus associated with congenital diaphragmatic hernia, wandering spleen, and intrathoracic left kidney: CT findings. Abdom Imaging 2001;26:306.

10 16.

 10 Hampton LJ, Borden TA. Ureteropelvic junction obstruction in a thoracic kidney treated by dismembered pyeloplasty. Urology 2002;60:164. 17. Singh P, Vijjan V, Gupta M, et al. Percutaneous nephrolithotomy of a staghorn stone in thoracic ectopic kidney. Int J Urol 2007;14:558. 18. Sundaram V, Vidhyashree SA, Pratap B, et al. A male patient with right-sided thoracic kidney, diabetes mellitus, hearing loss and renal dysfunction. Int Urol Nephrol 2007;39:959-62.

Figure 1 :

 1 Figure 1: Transverse and sagittal sections of the fetal thorax on prenatal ultrasound

Figure 2 :

 2 Figure 2: Sagittal section of the fetal thorax with Magnetic Resonance Imaging

Figure 3 :

 3 Figure 3: Transverse section of the fetal thorax on prenatal ultrasound at 27 weeks,

1 TITLE

 1 Prenatal diagnosis of thoracic kidney in the 2nd trimester with delayed manifestation of associated diaphragmatic hernia.

10 16.

 10 Hampton LJ, Borden TA. Ureteropelvic junction obstruction in a thoracic kidney treated by dismembered pyeloplasty. Urology 2002;60:164. 17. Singh P, Vijjan V, Gupta M, et al. Percutaneous nephrolithotomy of a staghorn stone in thoracic ectopic kidney. Int J Urol 2007;14:558. 18. Sundaram V, Vidhyashree SA, Pratap B, et al. A male patient with right-sided thoracic kidney, diabetes mellitus, hearing loss and renal dysfunction. Int Urol Nephrol 2007;39:959-62.

Figure 1 :

 1 FIGURE LEGENDS

Figure 2 :

 2 Figure 2: Sagittal section of the fetal thorax with Magnetic Resonance Imaging

Figure 3 :

 3 Figure 3: Transverse section of the fetal thorax on prenatal ultrasound at 27 weeks,

Figure 1 Figure 2 Figure 3

 123 Figure 1 Transverse and sagittal sections of the fetal thorax on prenatal ultrasound at 22 weeks, demonstrating the ectopic kidney (K) in the left hemithorax, compressed left lung (L), shift of the heart (H) to the right, diaphragm (D) and absence of viscera in the thorax. 251x141mm (300 x 300 DPI)


Tatiana ; Dupinay

Iserm

Karin ; Restorp

John Wiley

Sons

Helene Norder

Keywords: molecular epidemiology, human migation, genotyping, HBV, Africa

The prevalence of hepatitis B virus (HBV) markers was investigated in 566 inhabitants aged 15 to 55 41 years from a sugar cane region, Sirama, and from a village, Mataipako, in Northern Madagascar. 42 Serological markers of past or present infection were significantly higher in Sirama, 74 % versus 55 %. 43

There was no difference in the prevalence of chronic HBsAg carriers, 8.7 versus 8.5 % between the 44 two regions. Sequencing the S gene in 45 strains revealed a predominance of genotype E, in 53 %, 45 followed by subgenotype A1 in 22 %, and genotype D in 18 %. Phylogenetic analyses of the genotype 46 E strains showed homology with West African strains. All A1 isolates were similar to Malawi strains. 47

Most genotype D strains were subgenotype D7 and related to strains from Somalia and Tunisia. One 48 genotype D strain formed a branch between Pacific D4 and African D7 strains at neighbour-joining 49 analysis. The precore stop mutant was found in 33 % of the genotype D strains, 17 % of E but not in 50 any A1 strain. The high prevalence and low variability of genotype E strains in only two villages, 51 indicates a rather recent introduction of this genotype into Madagascar from West Africa, possibly 52 through migration or slave trade. The wider spread and genetic relationship of genotype D with East 53 African and Austronesian strains indicate an earlier introduction of this genotype. Molecular 54 epidemiology of HBV may thus be used to complement linguistic and genetic studies on past human 55 migrations in Africa.

Hepatitis B virus (HBV) is a human pathogen causing both acute and chronic hepatitis. HBV is a 58 major public health concern and approximately two billion people worldwide have serological markers 59 of past or present infection. More than 350 million people are chronically infected according to the 60 World Health Organisation (WHO). In addition, WHO estimations indicate that approximately one 61 third of all cases of cirrhosis and half of all cases of hepatocellular carcinoma can be attributed to 62 chronic HBV infection worldwide. The prevalence of chronic infection varies throughout the world 63 with South East Asia, China, Amazon Basin and Africa carrying the highest burden of disease. 64

Parenteral and sexual transmissions are globally the most common modes of transmission. In endemic 65 countries the predominant routes of transmission are perinatal transmission from mother to child 66 during birth in Asia, and horizontal transmission between children in Africa. A global hepatitis B 67 immunization program has been recommended by WHO since 1991. 68 69 Madagascar is a country with an ethnically diverse population divided into 22 tribes and a growth rate 70 of 2,8 % per year [START_REF] Hewitt | Beta-globin haplotype analysis 409 suggests that a major source of Malagasy ancestry is derived from Bantu-speaking Negroids. 410[END_REF][START_REF] Mafrezi | Evolution and structure of the population of Madagascar[END_REF]. Madagascar is classified as an 71 area of high endemicity for HBV, and about 5% of the urban population and 30 % of the rural 72 population are chronic HBsAg carriers [START_REF] Boisier | Hepatitis B virus infection in general 371 population in Madagascar: evidence for different epidemiological patterns in urban and in rural 372 areas[END_REF]Migliani et al., 2000a;Migliani et al., 73 2000b;Morvan et al., 1994). Hepatitis B vaccination is the most effective strategy for prevention of 74 hepatitis B. However, in Madagascar laboratory facilities are insufficient and the use of hepatitis B 75 vaccine is limited (Migliani et al., 2000b). 76 77 HBV is a small, double-stranded DNA virus and the prototype of the hepadnavirus family (Ganem & 78 Varmus, 1987). HBV strains are classified into eight genotypes, designated A-H (Arauz-Ruiz et al., 79 2002;[START_REF] Naumann | Identification of a 479 new hepatitis B virus (HBV) genotype from Brazil that expresses HBV surface antigen subtype 480 adw4[END_REF]Norder et al., 1993;[START_REF] Okamoto | Point mutation in the S gene 495 of hepatitis B virus for a d/y or w/r subtypic change in two blood donors carrying a surface 496 antigen of compound subtype adyr or adwr[END_REF][START_REF] Stuyver | A new 540 genotype of hepatitis B virus: complete genome and phylogenetic relatedness[END_REF]. Each 80 genotype has distinct geographical distribution. Genotype A is common in North-Western Europe, 81

North America, and Africa. Genotypes B and C are predominant in East-and South East Asia, Oceania 82 and among aboriginal populations in Australia. Genotype D is found worldwide and is predominant in 83 the Mediterranean, Middle East, and India, whereas genotype E has so far been confined to 84 populations in western parts of sub-Saharan Africa apart from one report from India (Norder et al., 85 2004;[START_REF] Singh | First report of 536 genotype e of hepatitis B virus in an Indian population[END_REF]. Genotype G has been identified in samples from several countries, and is As a part of a schistosomiasis morbidity study, sera were collected from 563 inhabitants aged 15 to 55 126 years living in a rural area in Northern Madagascar [START_REF] Leutscher | Coexistence of urogenital schistosomiasis and sexually 436 transmitted infection in women and men living in an area where Schistosoma haematobium is 437 endemic[END_REF][START_REF] Ramarakoto | Ultrasonographical findings in the 513 urogenital organs in women and men infected with Schistosoma haematobium in northern 514 Madagascar[END_REF]. 127

Participants were from the Mataipako village and from five villages (Ambodikatakata, Ambodimanga, 128 Ankatoko, Antsaboahitra, and Tanambao) in the neighboring Sirama sugercane plantation area close to 129 the major town Ambilobe. Two-hundred-fifty-one samples (43 %) were from Mataipako and 322 from 130 the Sirama villages (Table I). Diagnostics Systems, New Brunswick, NJ, USA, and DiaSorin, Saluggia, Italy). Samples were also 141 tested for anti-HBc (Wellcozyme, Abbott Diagnostics, Maidenhead, England, and Anti-HBc PLUS 142 BioRad, Hercules, CA, US). HBeAg and anti-HBe were determined with HBeAg-Ab Plus BioRad 143 Monolisa, and anti-HBs was detected with the EIA Anti-HBs BioRad (BioRad LaboratoriesAB,144 Hercules, CA, US). 145 146 Extraction of HBV DNA, PCR amplification and sequencing of the S gene, the pre-core and the core 147 promoter regions. 148 HBV DNA was extracted from 50 µl serum by the standard phenol-chloroform method. Briefly, 50 µl 149 serum were incubated for 2 hours at 42°C with 250 mg/ml of proteinase K (Sigma, St Louis, MO, US), 150 1% sodium dodecylsulphate, 2,5 mM disodium EDTA (Sigma, St Louis, MO,US), 25 mM sodium 151 acetate and 0.25 µg/ml tRNA. The DNA was subsequently extracted with phenol and with a 1:1 152 mixture of phenol:chloroform. Thereafter the DNA was precipitated in absolute ethanol with 0.15 M of 153 potassium acetate and dissolved in 40 µl of milli-Q water. 154 155 PCR amplification of the S-region was performed by using primers hep75 and 73b in the first PCR 156 and hep3/hep33 and hep4/hep34 for nesting [START_REF] Norder | Molecular basis of hepatitis B virus serotype variations 485 within the four major subtypes[END_REF]. The pre-core region was amplified 157 with primer promC and hep68 [START_REF] Sendi | T1764G1766 core promoter 531 double mutants are restricted to Hepatitis B virus strains with an A1757 and are common in 532 genotype D[END_REF], and nested with primers hep67 and hep68 (Arauz-158 Ruiz et al., 1997). For amplification of the core promoter region, primers hep64 and hep70 were used 159 in the first round of PCR and hep64 and hep54 in the nested PCR [START_REF] Sendi | T1764G1766 core promoter 531 double mutants are restricted to Hepatitis B virus strains with an A1757 and are common in 532 genotype D[END_REF]. 160 161 Purified products were used as templates in the sequencing reaction using the dideoxy-nucleotide 162 chain-termination method with an ABI PRISM BigDye terminator cycle-sequencing reaction kit 163 (version 3; Applied Biosystems, Foster City, CA, US). The primers used in the PCR were used as 164 sequencing primers. All amplification products were sequenced bi-directionally. 165

An ABI PRISM 3100 genetic analyser (Applied Biosystems) was used for electrophoresis and data 166 collection. 167 168

Phylogenetic analysis 169

Sequences obtained were edited manually using the SeqMan program in the LASERGENE package 170 (DNASTAR,Inc,Madison, WI). The sequences were thereafter aligned with the corresponding region 171 in sequences retrieved from GenBank. 172 173 Phylogenetic analysis was carried out with the PHYLIP program package version 3.53 (Felstenstein, 174 1993). Evolutionary distances were estimated with the DNADIST program using the F81 model. 175 Phylogenetic trees were constructed using UPGMA and neighbour-joining (N-J) algorithm in the 176 PHYLIP package. Genotypes and subgenotypes were determined by phylogenetic analysis of the 177 amplified fragments of the S gene with sequence from previously genotyped and subgenotyped strains 178 [START_REF] Norder | Genetic diversity of hepatitis B virus strains derived 483 worldwide: genotypes, subgenotypes, and HBsAg subtypes[END_REF]. The deduced amino acid sequence of the S gene region was used to determine 179 the subtype, which was assessed from the substitutions at codons 122, 127, and 160 (Norder et al., 180 1992;[START_REF] Okamoto | Point mutation in the S gene 495 of hepatitis B virus for a d/y or w/r subtypic change in two blood donors carrying a surface 496 antigen of compound subtype adyr or adwr[END_REF]. 181 182

Results

183

Seroprevalence of hepatitis B markers 184

There was an overall high prevalence of HBV markers (66 %). It was significantly higher in the 185 Sirama villages, 74 % versus Mataipako, 55 %, (p<0.0001; Fisher´s exact test; Table I), whereas there 186 was no major difference between males and females (69% versus 65%, p=0.34). In all, 370 inhabitants 187 had HBV markers, 322 (57 %) were negative for HBsAg but had anti-HBc and/or anti-HBs as markers 188 of past infection, while 48 (8.5 %) individuals were positive for HBsAg (Table I). Anti-HBs as only 189 serological marker was found in nine (1.6 %) inhabitants (Table I). There was a significantly higher 190 rate of HBV markers among males in the Sirama villages compared to Mataipako village, 70% versus 191 1c). The 226 D2 and D7 strains formed separate clades also in the N-J analysis, although in this analysis the D1 and 227 D3 strains were split into two and three clades, respectively (fig. 1c). The D4 and D7 strains formed 228 the first split in the N-j tree, although this was not the case in the UPGMA tree. Six D1 and two D2 229 strains classified based on complete genomes, could not be classified into a subgenotype by UPGMA 230 but belonged to their respective subgenotypes in the N-J analysis (figs 1b; 1c). On the other hand there 231 were five D1 strains based on complete genomes that were classified as D1 in the UPGMA tree but 232 could not be classified into a subgenotype by N-J (figs 1b; 1c). Two genotype D strains from 233

Madagascar were not classifiable with any of the analyses. One, 1500-02, from a female from 234 Mataipako encoding ayw4, was found between genotype D and E strains in the N-J tree (fig 1c). The 235 other strain, 600-02, from a male from Ambodimanga specified ayw2. This strain was similar to a 236 strain from aboriginal populations in Australia in the UPGMA tree and formed a separate branch 237 between the Asian D4 and the African D7 strains in the N-J tree (figs 1b; 1c). 238 239The 24 genotype E isolates were found intermixed with strains from West Africa in the phylogenetic 240 tree (Fig. 1d). Fourteen of the Madagascar strains were found in two clades. In the major clade, 11 of 241 the Madagascar strains were intermixed with strains from Cameroon, Nigeria, Ivory Coast, and Mali 242 (Fig. 1d). Eight of these strains were from Mataipako, and four from two villages in the Sirama region, 243 Tanambao and Ankatoko. In the second cluster 4 isolates from Tanambao were intermixed with strains 244 from Congo (Fig 1d). 245 246Pre-core and core promoter region 247

The pre-core region was sequenced in 36 strains. A premature stop codon related to the G1896A 248 mutation was observed in six sequences (16.6%). This pre-core mutant was found in three of seven 249 investigated genotype D7 strains and in three of 18 genotype E strains. It was not found in any of nine 250 genotype A strains investigated. All strains with the pre-core stop mutant were from patients with anti-251 HBe. Another pre-core mutation, G1896T, converting Trp 1896 to Leu 1896 , was found in two genotype D 252 strains and in one genotype E strain. This variant was found in two patients with anti-HBe and in one 253 patient lacking HBe markers. 254

255

The core promoter region was amplified and sequenced in 16 strains, three were genotype A, another 256 three were genotype D, and 10 were genotype E strains. All genotype D and E strains expressed A 1757 , 257 and the genotype A strains expressed G 1757 . One genotype A strain had C1766T and T1768A 258 The prevalence of hepatitis B markers in the rural populations from the Northern region of Madagascar 263 was high, although not as high as the prevalence described in other rural population in the centre of 264 Madagascar; where up to 18 % were HBsAg positive [START_REF] Boisier | Hepatitis B virus infection in general 371 population in Madagascar: evidence for different epidemiological patterns in urban and in rural 372 areas[END_REF]Migliani et al., 2000a;265 Morvan et al., 1994). Only 1.6 % of the inhabitants from this study had anti-HBs only which strongly 266 suggest that implementation of control programs for hepatitis B in these areas is warranted. The 267 present findings on the epidemiological features of persons infected with hepatitis B such as age, sex, 268 and origin showed that the prevalence of infection appears to be higher among males from rural 269 villages. 270 271 In this study, there was a high frequency of HBeAg positive HBsAg carriers (42 %), mainly among 272 those infected with genotypes A1 and E. This prevalence of HBeAg was higher than that from other 273 parts of East and Central Africa as Zimbabwe, Cameroon, Ethiopia, Benin and the Central African most predominant in the central highlands, while the coastal populations often are of African origin 294 [START_REF] Chow | Genetic characterization of 385 four Austronesian-speaking populations[END_REF][START_REF] Regueiro | Austronesian 519 genetic signature in East African Madagascar and Polynesia[END_REF][START_REF] Hurles | The dual origin of the Malagasy in Island 412 Southeast Asia and East Africa: evidence from maternal and paternal lineages[END_REF]. 295 296The origin of the population may also be reflected by the prevalent HBV genotypes. The subgenotype 297 A1 strains were similar and mainly found in two villages. This subgenotype dominates in all East 298

African countries including South Africa, Malawi, Tanzania, Uganda, and Somalia (Kramvis & Kew, 299 2007). The isolates from Madagascar were similar to Malawi isolates, suggesting that this subgenotype 300 was introduced into Madagascar from East Africa. Geographically Malawi is close to the North 301 Western part of Madagascar and historically, in the nineteenth century, the trade route from Malawi to 302 the western coast of Madagascar became exploited increasingly for slave trading. 303

304

The genotype E strains in this study showed high sequence similarity to West African strains, where 305 this genotype is confined. Previous studies have shown a low genetic diversity of genotype E strains 306 isolated from a large geographical area in West Africa [START_REF] Kramvis | Full genome analysis of 422 hepatitis B virus genotype E strains from South-Western Africa and Madagascar reveals low 423 genetic variability[END_REF][START_REF] Quintero | Molecular epidemiology of hepatitis B virus in Afro-511 Venezuelan populations[END_REF]. 307

Several studies have proposed that this low genetic divergence together with few findings of genotype 308 E in the Americas indicates a recent introduction of genotype E into the human population (Mulders et 309 al., 2004;[START_REF] Odemuyiwa | 492 Phylogenetic analysis of new hepatitis B virus isolates from Nigeria supports endemicity of 493 genotype E in West Africa[END_REF][START_REF] Suzuki | Distribution of hepatitis B virus (HBV) genotypes 548 among HBV carriers in the Cote d'Ivoire: complete genome sequence and phylogenetic 549 relatedness of HBV genotype E[END_REF]. The genotype E strains in this study were 310 mainly from inhabitants in two villages, which may indicate a recent introduction, and that sufficient 311 time has not yet passed for its subsequent spread into the rest of the country. The inhabitants in the 312 two villages were working in the rice fields, and they may be slave descendant with origin from 313 mainland Africa. West African origin of some Malagasy populations is supported by genetic studies on 314 Y-chromosomal polymorphism and mitochondrial sequence diversities and on β-globulin haplotypes 315 [START_REF] Hurles | The dual origin of the Malagasy in Island 412 Southeast Asia and East Africa: evidence from maternal and paternal lineages[END_REF][START_REF] Hewitt | Beta-globin haplotype analysis 409 suggests that a major source of Malagasy ancestry is derived from Bantu-speaking Negroids. 410[END_REF]. In addition, hepatitis C virus genotype 2 strains, common in 316 West Africa, have been isolated from Malagasy populations, further supporting previous human 317 migrations from West Africa to Madagascar [START_REF] Markov | Phylogeography and 450 molecular epidemiology of hepatitis C virus genotype 2 in Africa[END_REF]. 318 319 Interestingly, 24 % of the strains in this study were genotype D, and found in all five villages with 320 HBsAg positive individuals. The majority were subgenotype D7, which are also found in Somalia and 321 Tunisia. This is consistent with the subdivision of the previous classified D4 strains into two 322 subgenotypes, D4 represented by strains from the Australia and Western Pacific, and D7 with North 323 African strains [START_REF] Meldal | A novel hepatitis B virus subgenotype, 454 D7, in Tunisian blood donors[END_REF][START_REF] Schaefer | Under Construction: Classification of Hepatitis B Virus 529 Genotypes and Subgenotypes[END_REF]. The D4 and D7 strains formed the first split 324 in the genotype D phylogenetic tree, when N-J algorithm was used, as they do when complete 325 genomes are analysed [START_REF] Meldal | A novel hepatitis B virus subgenotype, 454 D7, in Tunisian blood donors[END_REF][START_REF] Norder | Genetic diversity of hepatitis B virus strains derived 483 worldwide: genotypes, subgenotypes, and HBsAg subtypes[END_REF][START_REF] Tallo | D2: major subgenotype of hepatitis B virus in Russia and the Baltic region[END_REF]. N-J analyses of 326 genotype D S -genes thus seems better to mirror the tree obtained from complete genomes than 327 UPGMA. The split of genotype D strains into two main branches, one with D4 and D7 and one with 328 the other D subgenotypes, with D5 as first spit, might support that genotype D has followed two waves 329 of two human migrations out of Africa. The D4 subgenotype being linked to the early settlement of 330 Papua-Australia-Melanesia followed by a later wave of settlement reaching India supported by India, 331 being populated before Europe, and D5 being the first split in this branch. 332 333 There were in this study five different D7 strains, all showing close relationship with strains from 334 Somalia. It has been proposed that there has been a migration wave into Madagascar from central East 335 Africa based on human genetic and linguistic studies [START_REF] Dahl | Bantu substratum in Malagasy[END_REF][START_REF] Hewitt | Beta-globin haplotype analysis 409 suggests that a major source of Malagasy ancestry is derived from Bantu-speaking Negroids. 410[END_REF]Hurles et al., 336 2005). Interestingly, there was one genotype D strain similar showing a distant genetic relationship to 337 a strain from Australia, and formed a separate branch between the African D7 and the Asian D4 338 strains, suggesting also Asian import of genotype D into Madagascar. This is in agreement with 339 genetic studies showing a South East Asian and Pacific origin of some Malagasy populations (Hurles 340 et al., 2005;[START_REF] Migot | 462 HLA class II haplotype studies bring molecular evidence for population affinity between 463 Madagascans and Javanese[END_REF][START_REF] Soodyall | Polynesian' mtDNA in the Malagasy[END_REF]. The dissemination of genotype D in the 341 majority of the villages and its higher genetic variability compared to genotype A and

  123 , Karin Restorp 4 , Peter Leutscher 5,6 , Dominique Rousset 7 , Isabelle 13 Chemin 1 , Rene Migliani 5 , Lars Magnius 4 , Heléne Norder 4* .

  the Sirama region, and was similar to strains from East Europe (Figs 1b;

  core promoter region, and one genotype E strain had a triple mutation, A1762C, 259 G1764T and C1766G. All other strains had wild type promoter sequence. 260 261 Discussion 262

  have contributed to Madagascar's gene pool, those of Asian origin are 293

Figure 1

 1 Figure 1 Dendrograms based on phylogenetic analysis of 681 nucleotides of the S gene in a) UPGMA tree with 10 genotype A strains from Madagascar; b) UPGMA tree with 11 genotype D strain from Madagascar; c) Neighbor-Joining tree with 11 genotype D strains from Madagascar and d) UPGMA tree with 24 genotype E strains from Madagascar together with sequences of the corresponding region obtained from GenBank. Sequences from GenBank are indicated with accession numbers. The designation and origin of each strain are indicated at the nodes of the branches. The strains from Madagascar are indicated by arrows.

Table I .

 I Age, sex and hepatitis B serological markers of investigated inhabitants in villages with mainly farmers (Sirama) and non-farmers (Mataipako) in Northern Madagascar. Figures in parenthesis are number of individuals with anti-HBs as the only marker.

			Males			Females			TOTAL
				HBsAg				HBsAg				HBsAg
				neg				neg				neg
				anti-				anti-				anti-
				HBC	%			HBc	%			HBc	%
				and/or	with			and/or	with			and/or	with
				anti-	total			anti-	total			anti-	total
			HBsAg	HBs	mark		HBsAg	HBs	mark		HBsAg	HBs	mar
	Age	N	pos	pos	ers	N	pos	pos	ers	N	pos	pos	kers
	Sirama
	15-19	27 3	19	81% 37	1	22 (1)	62% 64	4	41 (1)	70%
	20-24 25-29 30-34 35-39 40-44	28 3 35 5 20 2 20 2 17 1	F o r F o r F o r F o r F o r F o r 86% 20 22 (1) 77% 26 21 14 80% 32 11 (1) 65% 21 13 82% 15 F o r	0 3 3 1 1	13 (1) 16 16 11 12	65% 48 73% 61 59% 52 57% 41 87% 32	3 8 5 3 2	34 (1) 38 (1) 30 22 (1) 25	71% 62% 67% 54% 78%
	E, indicate an earlier introduction of genotype D into the Malagasy populations than the other two identified genotypes. P e e r P e e r P e e r P e e r P e e r P e e r 18 2 15 (1) 94% 6 1 5 100 % 24 3 20 (1) 165 18 115 (3) 81% 157 10 95 (2) 67% 322 28 P 210 (5) 74% 96% e 10.9% 70% 6.4% 60% 8.7% 65% Matai-342 343 344 45-Sub-total pako e r
	345 346 347 348 349 15-19 20-24 25-29 30-34 35-39 40-44 45-	It is well known that the prevalence of the different HBV genotypes and subgenotypes vary in geographical areas and correlate strongly with ethnicity. Studying the genetic variability of the HBV strains in endemic regions may thus be helpful when studying migration patterns of populations of different origins. This study showed that molecular epidemiology of HBV in the North-western part of R e v i e R e v i e R e v i e R e v i e R e v i e R e v i e 21 1 4 24% 18 1 8 (1) 50% 39 2 12 (1) 36% 35 6 16 (1) 63% 26 2 15 65% 61 8 31 (1) 64% 19 1 11 63% 17 2 7 53% 36 3 18 R 58% 14 1 6 50% 15 1 10 73% 29 2 16 e 62% 22 1 12 59% 14 1 6 50% 36 2 18 55% 16 2 8 (1) 62% 7 1 1 29% 23 3 9 (1) 52% 9 0 4 (1) 44% 8 0 4 50% 17 0 8 (1) 47% v i e
	350 351 Sub-total	Madagascar confirms linguistic and human genetic studies on human migrations in Africa. 8.8% 45% 7.6% 8.3% w w w w w w 136 12 61 (3) 54% 105 8 51 (1) 56% 241 20 w	46% 112 (4) 55%
	TOTAL
	15-19	48 4	23	56% 55	2	30 (2)	58% 103 6	53 (2)	57%
	20-24	63 9	37 (1) 73% 46	2	28 (1)	65% 109 11	65 (2)	70%
	25-29	54 6	33 (1) 72% 43	5	23	65% 97	11	56 (1)	69%
	30-34	34 3	20	68% 47	4	26	64% 81	7	46	65%
	35-39	42 3	23 (1) 62% 35	2	17	54% 77	5	40 (1)	58%
	40-44	33 3	21 (1) 73% 22	2	13	68% 55	5	34 (1)	71%
	45-	27 2	19 (2) 78% 14	1	9	71% 41	3	28 (2)	76%
	TOTAL 301 30	176 (6) 68% 262 18	146 (3) 62% 563 48	322 (9) 66%
			10%				6.7%				8.5%
													18 19

Table II .

 II HBeAg and anti-HBe in sera from 48 HBsAg positive inhabitants in the Sirama region and Mataipako

		HBsAg postive	HBeAg positive	HBeAg negative	HBeAg negative
				Anti-HBe	Anti-HBe positive	Anti-HBe
				negative			negative
		Male	Female	Male	Female	Male	Female	Male	Female
	Ambodikatakata	3	1	2	0	1	1	0	0
	Ambodimanga*	6	2	4	0	2	2	0	0
	Ankatoko Tanambao Sirama subtotal Mataipako	2 7 18 12	1 6 10 8 F o r 1 3 10 5	0 4 4 1	1 3 7 7	1 2 6 6	0 1 1 0	0 0 0 1
	Total	30	18	15 P	5	14	12	1	1
	Total both sexes		48	20 e e r	26		2
					R
					e
						v i e
						w
									20

Table III .

 III The infecting HBV genotypes in strains from 45 HBsAg positive inhabitants in five villages in

	Northern Madagascar
				Genotype
				Male/ Female
		HBsAg +	A/ayw1	D/ayw2	D/ayw3	D/ayw4	D/ayw2	E/ayw4	Total
			A1	D7	D2
	Ambodikatakata	3/1	2/0	1/1	0/0	0/0	0/0	0/0	3/1
			F o r
				P
				e
				e r
					R
					e
						v i e
							w

Journal of Medical Virology

John Wiley & SonsJournal of Medical Virology

Acknowledgements 352

This work was supported by a bilateral program financed by Swedish Institute scholarship (grant no:

A2 0.01 3178-87 (AY738813) 2713-87 (AY738814) 57-94n (U91810) B1-89 (M74498) tar. (AY738812) pfdw294 (M57663) s3-1 (AY161142) pat567 (AF418676) 744-92 e17 (U87731) e20 (U87728) 78-adw2 (AF297621) e30 (U87733) e14 (U87727) 8903-86 (AY738810) 5409-89 (AY738811) Ir (U87743) 8423-87 (AY738809) 3138-87 (AY738808) 83

AF143304) 7720-G8 (AF143298) AF132443 1889-07 a317 (Z72478) 10552-85 (AY738819) 18720-70 (AY738818) HD1402 (AF143302) HD1406 (AF143303) sd4767 (AF209403) st21-a (AY168427) Len (AY738817) Wil (AY738816) sn1288 (AF065113) sn4222 (AF065115) wp2380 (AF061526) jv1802 (AF209397) mh5274 (AF209399) p08 (U87726) 925-92 (AY738815) HBVKL (AY034878) 8017 (U87744) S50225 (S50225) as9419 (AF209392) hbv991 (X51970) 1385 (U87739)) ui004 (AF209405) 8-22 (AY152726) A23 (U87735) cp595 (AF208866) 41-adw2 (AF297619) sn2882 (AF065114) pal3660 (AF208873) pal4783 (AF208875) s9-1 (AY161161) 7782 (U87746) 1007-93 s37-z (X69458) 84-adw2 (AF297625) 1449 (U87745) h510 (U87740) 1046-85 (AY738807) PLC-PRF5 (X04820) 705.seq 682.seq 654-02.seq 697-02-680.seq 1031-02 1025-02 HBV-Mala30 (AB076685) HBV-Mala9 (AB076686) HBV-Mala4 (AB076688) HBV-Mala25 (AB076684) 723.seq 650.seq e06 (U87736) HBV-Mala8 (AB076689) 987.seq HBV-Mala3 (AB076687) 1149 (U87852)


Peter ; Neville

Nevill

Jeff Turner
email: jkturner76@hotmail.com

Andrew Godkin

Jerry Kingham

Chin Lye Ch

Clinical characteristics of hepatitis E in a "non-endemic" population. 1 2

Keywords: Acute hepatitis, Epidemiology, Indigenous, Jaundice, Liver function tests

show a group mortality rate of 4.2%, similar to that reported in endemic countries. 42

HEV results in a severe and occasionally fatal hepatitis. Testing for hepatitis E is now 43 recommended in any patient presenting with acute hepatitis of unknown aetiology.

Hepatitis E (HEV) is a ribonucleic acid (RNA) virus with predominant faecal oral 29 spread. Traditionally in Western Europe it is associated with travel to endemic 30 countries, but an increasing number of locally acquired cases have been reported 31 throughout England. Patients presenting with acute non -travel associated HEV 32 infection in south Wales over a 25 month period were monitored, in an attempt to 33 understand the clinical picture and epidemiology in our patient population. 24 patients 34 were identified with non -travel associated HEV infection and studied prospectively. 35

Patient demographics, symptoms and serial laboratory results were recorded. There 36 was a male:female ratio of 3:1, with a median patient age of 65.5 years old. Patients 37 developed a significant icteric hepatitis (median peak bilirubin -139 µmol/l, median 38 peak AST -1973 IU/l and ALT -2021 IU/l), with liver function remaining abnormal 39 for approximately 7 weeks. All patients in whom HEV RNA was isolated were 40 infected with genotype 3. 46% of patients presented during winter months. The data the development of more specific serological assays that this earlier epidemic was 85 recognised as being due to ET-NANBH [START_REF] Worm | Hepatitis E: an overview[END_REF]. This virus was named 86

formally hepatitis E after isolation of cDNA sequences in 1990 [Reyes et al., 1990] 87 and was placed in the family Caliciviridae, but later placed into the family 88 Hepeviridae (genus Hepevirus) [START_REF] Lu | Phylogenetic analysis of global hepatitis E virus 361 sequences: genetic diversity, subtypes and zoonosis[END_REF]. 89

It is a positive sense, single stranded, non -enveloped RNA virus that usually 90 results in a self-limiting hepatitis, similar to infection with hepatitis A virus. However 91 chronic carriage has been reported in infected patients with the human 92 immunodeficiency virus type 1 and organ-transplant recipients [START_REF] Dalton | Persistent carriage of 338 hepatitis E virus in patients with HIV infection[END_REF]93 Kamar et al., 2008]. Hepatitis E virus is transmitted by the faecal-oral route as 94 demonstrated by volunteer studies, and there is increasing evidence of zoonotic 95 transmission [Balayan et al., 1983;[START_REF] Dalton | Autochthonous hepatitis E in southwest 328 England[END_REF][START_REF] Ijaz | Non-travel-associated hepatitis E in England and Wales: 346 demographic, clinical, and molecular epidemiological characteristics[END_REF]. 96

Hepatitis E is clearly recognised as a cause of viral hepatitis in travellers to 97 endemic countries. There has been increasing awareness and reporting of cases of 98 HEV infection in England acquired sporadically, however information regarding 99 infection in Wales remains limited [START_REF] Dalton | Autochthonous hepatitis E in southwest 328 England[END_REF] RNA was detected. RT-PCR was performed using a technique described previously 121 [START_REF] Meng | A novel virus in swine is closely related to the 365 human hepatitis E virus[END_REF]. Following PCR product purification, strands were sequenced 122 using PCR internal primers, generating nucleoside sequences for analysis [Ijaz et al., 123 2005]. 124

125

RESULTS 126

Twenty-four patients (18 male and 6 female), with a median age of 65.5 years 127 (range: 47 -81 years) were studied. Jaundice and reduced appetite were the 128 presenting symptoms reported most frequently (Table I clinically and 48% developed a coagulopathy. The median prothrombin time 171 prolongation was 3.2 seconds (range: 0 -25 seconds). The median time period from 172 reported symptom onset until normalisation of the bilirubin was 50 days, AST/ALT 173 was 49 days and ALP was 47 days. 69% of patients had a persisting 174 hyperbilirubinaemia at 4 weeks following the reported onset of symptoms. RNA 175 extraction was not attempted in one patient. RNA was detected in 52% of the 176 remaining cases, all of whom were genotype 3. Three patients were Hepatitis A or 177 The majority of published data regarding indigenous HEV infection in the UK 182 are derived from England, where the question has been raised whether cases of HEV 183 were secondary to local outbreaks or more widespread infection [Dalton et al., 2007 184 and2008]. Results from the present study support the suggestion that HEV may be 185 widespread throughout the UK and that previous lack of serological testing for this 186 virus accounts for its under diagnosis. Increasing awareness has resulted in a 187 progressive increase in the number of cases diagnosed over recent years [Lewis et al., 188 2008]. This study demonstrates a male preponderance of infection with HEV and 189 median age at presentation of 65.5 years old, which conforms with previous data 190 published from England and Europe [START_REF] Dalton | Autochthonous hepatitis E in southwest 328 England[END_REF][START_REF] Renou | A national survey of acute hepatitis E in France[END_REF]. Lewis et 191 al [2008] reported similar results for England and Wales from 2005, however regional 192 data were not included for comparison. The UK seroprevalence data are limited. HEV 193 IgG seroprevalence rates of 3.9 -8.8% have been reported in Inner City London, with 194 a more recent study demonstrating rates of 16% in blood donors from Southwest 195 England [START_REF] Bernal | A community prevalence study of antibodies to 324 hepatitis A and E in inner-city London[END_REF][START_REF] Dalton | Autochthonous hepatitis E in Southwest England: natural 333 history, complications and seasonal variation, and hepatitis E virus IgG 334 seroprevalence in blood donors, the elderly and patients with chronic liver disease[END_REF]. The seroprevalence rates in other 196 developed countries have varied between 21.3% of blood donors in the USA and 197 Europe and 17% of outpatients reviewed with an acute hepatitis in Madrid [Quiroga et 198 al., 1996;[START_REF] Thomas | Seroreactivity to hepatitis E virus in areas where the disease is not endemic[END_REF]. 199

Patients present commonly with symptoms of jaundice or reduced appetite 200 (Table I), however these are not specific to hepatitis E and occur commonly with 201 other forms of acute hepatitis. They also develop marked hepatic damage, with 202 transaminases elevated an average of fifty fold. The time period from the reported 203 that hepatitis E may be transmitted by undercooked pork and boar meat, which is now 232 available more widely [START_REF] Dalton | Autochthonous hepatitis E in southwest 328 England[END_REF]]. This is one potential mechanism of HEV 233 acquisition leading to the increase in cases diagnosed during the summer months. 234 Mortality rates of 0.5 -4.0% have been reported in endemic countries, but 235 remain poorly studied in non -endemic regions [START_REF] Aggarwal | Hepatitis E: an overview and recent advances in 310 clinical and laboratory research[END_REF]. 236

The group mortality rate in this study was 4.2%, which is identical to that reported 237 previously in a national retrospective study in France [START_REF] Renou | A national survey of acute hepatitis E in France[END_REF]. Higher 238 mortality rates have been reported in other parts of the United Kingdom [Dalton et al., 239 2008]. 240

In conclusion, hepatitis E usually results in severe but self -limiting acute 241 hepatitis with approximately seven weeks from reported onset of symptoms until 242

  HEV) was named initially 'enterically transmitted non-A non-B 82 hepatitis' (ET-NANBH). One of the first documented epidemics of this type of viral 83 hepatitis started in India in 1955. It was not until 1980 during a further epidemic and 84

  results requested between December 2004 -2006, with a 108 complete clinical history and examination performed on all patients by a hepatologist. 109 Acute HEV infection was diagnosed in patients by the following criteria: 110 (1) no history of foreign travel within the preceding 3 months, (2) clinical and 111 biochemical evidence of an acute hepatitis, (3) reactive serology for both anti -HEV 112 IgM and anti -HEV IgG by enzyme-linked immunosorbent assay (Genelabs 113 Technologies, Redwood City, USA) ± an increasing titre of anti -HEV IgG and/or 114 detection of serum HEV RNA by reverse transcriptase-polymerase chain reaction 115 (RT-PCR). 116 Serial laboratory results were recorded and other causes of viral hepatitis 117 excluded (hepatitis A, cytomegalovirus and Epstein -Barr virus IgM, hepatitis B 118 surface antigen, hepatitis C PCR and adenovirus titres). HEV serology was carried out 119 at the Viral Reference Laboratory, Colindale, London and genotyping attempted when 120

  normalisation of liver function. Mortality rates are similar in endemic and non -243 endemic countries. It is an important cause of infection acquired locally in south 244 Wales as in other parts of the UK. The misconception that hepatitis E only affects 245 travellers to endemic areas has resulted historically in its under testing and diagnosis. 246 The more recent increasing awareness of indigenous infection has led to an increasing 247 number of cases diagnosed. Reported seasonal variation within the UK varies and 248 requires further evaluation. Testing for hepatitis E is recommended in any patient 249 presenting with acute hepatitis, abnormal liver function of unknown aetiology and 250 patients with chronic liver disease who decompensate for no identifiable reason. 251 252 Acknowledgements: We would like to thank Dr T Zimbwa (Singleton Hospital) and 253 A Gill and S Puritz (Medical students -University Hospital of Wales) for their help 254 with data collection, as well as support from the microbiology services in our 255 hospitals, with particular thanks to Dr McCracken & Dr J Salmon.

Journal of Medical Virology

Competing interests: The authors have no financial or other conflicts of interest to