P Amat

W Puech

S Druon

J P Pedeboy

Lossless 3D steganography based on MST and connectivity modification

Keywords:

a b s t r a c t Data hiding has become increasingly important for many applications, like confidential transmission, video surveillance, military and medical applications. In this paper we present a new approach of 3D object data hiding without changing the position of vertices in the 3D space. The main idea of the proposed method is to find and to synchronize particular areas of the 3D objects used to embed the message. The embedding is carried out by changing the connectivity of edges in the selected areas composed of quadruples. The proposed approach of data hiding in 3D objects is based on minimum spanning tree (MST). This method is lossless in the sense that the positions of the vertices are remaining the same before and after embedding. Moreover the method is blind and does not depend of the order of the data in the files. This approach is very interesting when the 3D objects have been digitalized with high precision.

Introduction

With the evolution of computer networks, transmissions involving 3D downloadable objects are becoming commonplace. To be universally readable, these 3D objects must meet international standards even if we want to bring new features to these products. It is, therefore, important to follow standard formats 1 to manipulate these 3D objects. In our work, these new features relate not only to the protection of 3D objects during their transmission and archiving but also the enrichment of these objects with their meta-data without increasing the size of objects and synchronizing the metadata in a hierarchical manner. The representation mostly used for 3D objects is the surface mesh. This representation, consisting of vertices, edges and faces, is widely supported by all the graphics rendering cards. Many data hiding methods are based on this representation. Data hiding methods can be divided into two categories, spectral or spatial, depending on the domain used to embed the hidden data. Irrespective of the embedding domain, existing watermarking and steganography methods for 3D objects are usually based on the modification of vertices in the 3D mesh for embedding the data. For many applications, such as medical or industrial manufacturing, the 3D objects are acquired with great precision and accuracy and any change in the position of the vertices may well prove catastrophic. In this paper we propose a data hiding method wherein the embedding is not based on the modification of the position of vertices of the 3D objects. Few of the previous works propose to hide data in 3D objects without changing the position of vertices in the 3D space. The new approach presented in this paper proposes a method to hide data in 3D objects based on the minimum spanning tree (MST). This method is lossless in the sense that the positions of the vertices are unchanged. The method is blind and does not depend on the ordering of the data in the files. Moreover the embedding capacity is significant and can be practically as high as 0.128 bit/vertex. The method enables the embedding, in a 3D object, of meta-data like the author's name, the date of creation of the object, a 2D or 3D logo, color of the object or the texture of the object itself. It should be noted that the method resists geometric transformations such as rotation, translation and uniform scaling. The proposed method, based on a better selection of quadruples, is an improvement of a previous method presented in [START_REF] Amat | Lossless data hiding method based on MST and topology changes of 3D triangular mesh[END_REF]. We compare our new proposed method with the previous one by showing the improvement in the embedding capacity.

The rest of this paper is divided into the following sections. In Section 2, we present a brief literature survey on different data hiding methods applied to 3D objects. Section 3 explains the method and in Section 4, we present and discuss the results of our method when applied to real 3D objects. Conclusion as well as the perspective are given Section 5.

Previous work

Depending on the type of application, watermarking methods can be classified according to certain mutually antagonistic constraints. These constraints include perceptual transparency, robustness and embedding capacity. This list is not exhaustive and in many applications, factors like security or time complexity should also be taken into account. Based on the embedding domain, data hiding can be carried out in either the spatial or in the transform domain. Another criterion can be the need (non-blind) or otherwise (blind) of the original 3D object. If only a few original parameters are needed for extracting the message, then the method can be termed as semi-blind. Totally blind methods may require the use of a secret key to retrieve the hidden message. In this case, the secret key is not only independent of the hidden content but also of the 3D object itself. A watermarking algorithm must be known to everyone, even if it uses a secret key, in order to respect the principle of Kerckhoffs [START_REF] Kerckhoffs | La cryptographie militaire[END_REF].

In this paper, we propose to classify the 3D data hiding methods into two categories. The first of these categories is based on the scheduling of the object primitives or on the connectivity and topology of the 3D objects to be marked. In this first category, the positions of the vertices constituting the original 3D object are unchanged. The second category is based on the spatial displacement of the vertices of the 3D object or on the geometric deformations of other primitives constituting 3D objects, whether in the spatial or in the transform domain.

Methods based on primitives, connectivity or topology

This first category presents data hiding and steganography methods based on the scheduling of the object primitives or on the modification of connectivity or topological features of the 3D objects to be watermarked. In general, these changes are carried out in the spatial domain.

The embedding methods of this category are based on the organization of data with respect to the file comprising the 3D object. Ichikawa et al. [START_REF] Ichikawa | Redundancy in 3D polygon models and its application to digital signature[END_REF] propose two watermarking methods which modify the organization of data to embed a hidden message. The first method modifies the order of triangles in a list of triangles, while the second method modifies the order of triplets of vertices forming a triangle.

The triangle strip peeling symbol sequence (TSPS) watermarking algorithm of Ohbuchi et al. is based on a topological change [START_REF] Ohbuchi | Watermarking three-dimensional polygonal models[END_REF]. To embed information in the triangle mesh, this algorithm proposes to detach a band of triangles of the original mesh. The first step of this algorithm is to choose, in the original mesh, a starting edge e of the initial triangle. From this edge of departure and the embedded message a strip of triangles is generated. The choice of the next triangle to be added to the band is based on the value of the bit to be embedded and the triangle scan order (clockwise or anticlockwise). This allows us to index all the edges to 1 or a 0 depending on their scan order in the triangle. In this manner the rest of the edges, and therefore the triangles generating the band, are chosen as a function of the message to be embedded. The next step of this algorithm is to detach this band of triangles by duplicating all the vertices of the band except those of the e. The embedded mesh is represented with a hole in the surface covered by the band of triangles containing the message. The message extraction is carried out by first searching the edge e and then scanning the associated band of triangles. The disadvantage of this method is that the characteristics of the object change with the introduction of the holes. Another disadvantage is that the file size increases with the addition of new vertices. Moreover, one can notice that this embedding is localized in the sense that the message embedded is not distributed across the mesh of the object, and it is easy to spot the duplicated vertices in the file and thus find the added message.

Mao et al. have developed a method to introduce smaller triangles into those initial triangles of the original 3D object in which the information is embedded [START_REF] Mao | Watermarking 3D Geometric Models Through Triangle Subdivision[END_REF]. With this approach, each edge of an initial triangle is divided into two edges by the vertex of a small triangle inserted. The ratio between the lengths of the two new edges is used to achieve the embedding of a bit. To initiate the process, two sides of a triangle are selected through a pseudo-random number generator. After embedding all the three edges of a triangle, the adjacent triangle of the latest used edge, if it exists and if it has not already been embedded, is chosen for the continuation of the embedding process. The maximal embedding capacity of this method is 2 bits per triangle (0.667 bit/vertex). This method can have a strong embedding capacity since the division step of a triangle can be repeated several times. For extraction, the two edges of the initial triangle are found by using the key and the triangles are scanned as has been done in the embedding step. The major disadvantage of this method is that new vertices are added leading to an increase in the file size. In addition, the watermarked areas are visible because an embedded triangle generates four smaller in the triangles. Lastly, the adjacency requirement of triangles2 is no longer respected along the watermarked area.

One can notice that the position of the vertices constituting the original 3D object remains unchanged in these methods. However, some methods propose to add additional vertices, and most of these methods are very fragile and do not resist the manipulation, like the reversal of two lines, of the file containing the original data. Our method presented in Section 3 belongs to this category but is robust to data file manipulation, spreads over the message in the whole object and does not add vertices.

Methods based on geometric modifications of the 3D object

This second family of watermarking is based on the change in the geometry of the 3D object. This may be in the form of change in the position of the vertices, or alteration of the normals or faces of the triangles. Unlike the first family of watermarking, which applies only in the spatial domain, this second category may be applied in both the spatial and transform domains.

In the spatial domain, Ohbuchi et al. propose different watermarking approaches, such as tetrahedral volume ratio (TVR) and triangle similarity quadruple (TSQ) [START_REF] Ohbuchi | Watermarking three-dimensional polygonal models[END_REF]. The TVR uses the ratio of the volumes of two tetrahedrons. In this method, a reference tetrahedron is needed to compare the volume of two tetrahedra. The tetrahedron used is constituted by an edge and its two incident triangles. The scanning path of the connectivity graph is based on a spanning tree of vertices. The edges of the constructed tree are used to generate and reference tetrahedra and their order of scan. The vertex flood algorithm of Benedens [START_REF] Benedens | Geometry-based watermarking of 3D models[END_REF] is based on the change in the allowable distance of the vertices at the center of mass of the object. In another method, Benedens (triangle flood) uses the connectivity and geometry information to generate a path of the mesh of the object [START_REF] Benedens | Two high capacity methods for embedding public watermarks into 3d polygonal models[END_REF]. In this method the message embedding is done by modifying the position of vertices to alter the height of the triangles. The method proposed by Bors uses local geometric configuration of vertices for selecting embedding areas [START_REF] Bors | Watermarking mesh-based representations of 3-D objects using local moments[END_REF]. Each area is represented by a vertex and all its adjacent triangles. Bors calculates an ellipsoidal neighborhood for this vertex. To encode a 0, the vertex is moved out of its neighborhood and to encode a 1 it is internally displaced. Cayre and Macq propose a technique whereby the embedding is carried out by projecting the apex of a triangle on its base and watching whether it belongs to the left or right half of the base [START_REF] Cayre | Data hiding on 3D triangle meshes[END_REF]. The selection of the embedding areas is made by a unique path in the mesh with a secret key. The method of Wagner [START_REF] Wagner | Robust watermarking of polygonal meshes[END_REF] seeks to embed the hidden data along the length of local Laplacian vectors. To that end, Wagner alters the local curvature of the object. The use of NURBS (non-uniform rational B-spline) was also explored for the watermarking of 3D objects. Benedens has proposed a method of 3D object watermarking using NURBS and changing weights and vectors of nodes in a manner that preserves the overall geometry [START_REF] Benedens | Affine invariant watermarks for 3D polygonal and NURBS based models[END_REF]. The method of Lee et al. proposes to embed the watermark no more in the 3D object, directly, but in the virtual images derived from it [START_REF] Lee | Watermarking for 3D NURBS Graphic Data[END_REF]. In this article, for affecting a watermarking, three virtual images are constructed by uniformly sampling the 3D object and recording the coordinates of vertices thus obtained. The method of Agarwal and Prabhakaran is a blind method, robust and resistant to cropping [START_REF] Agarwal | Robust blind watermarking mechanism for point sampled geometry[END_REF]. This approach is based on a method of clustering. The algorithm divides the vertices into clusters by the application of the closest neighbor heuristic. Within each cluster the vertices are organized in the form of a graph. In a cluster, vertices with at least to incident edges are called cluster heads. Vertices other than the cluster heads, i.e. the leaflets, are utilized for embedding. The distance between the cluster head and the leaflets, to be watermarked, is partitioned into intervals. The embedding is carried out by modifying this interval by decreasing the euclidean distance between the cluster head and the leaflet.

In the transform domain, Ohbuchi et al. propose a method that employs a Laplacian matrix of the 3D mesh in order to pass from the spatial domain to the frequency domain [START_REF] Ohbuchi | A frequency-domain approach to watermarking 3D shapes[END_REF]. In the wavelet domain, the method of Kanai et al. uses a wavelet decomposition mesh and embed the information bit by changing the LSBs of certain wavelet coefficients [START_REF] Kanai | Digital watermarking for 3D polygons using multiresolution wavelet decomposition[END_REF]. Yin et al. too propose a wavelet domain algorithm [START_REF] Yin | Robust mesh watermarking based on multiresolution processing[END_REF]. The algorithm is based on a multiresolution decomposition of the 3D object mesh developed by Guskov et al. [START_REF] Guskov | Multiresolution signal processing for meshes[END_REF]. The method proposed by Praun et al. uses a Kernel space to embed information in the low frequency surface components [START_REF] Praun | Robust mesh watermarking[END_REF]. Wang et al. offer a hierarchical watermarking method. Thanks to the wavelet decomposition, a watermark robust to geometric attacks and a high capacity watermark are embedded into different resolution levels of the mesh by modifying the values of the wavelet coefficients [START_REF] Wang | Hierarchical blind watermarking of 3D triangular meshes[END_REF]. Rondao-Alface et al. use and improve the robustness of very good methods of watermarking based on histograms and perform the robustness against cropping [START_REF] Rondao-Alface | Blind and robust watermarking of 3D models: how to withstand the cropping attack?[END_REF]. Bennour and Dugelay have proposed two embedding methods, one blind and the other non-blind, with the aim of embedding a 3D object such that the mark is always present, in one form or another, in the resulting 2D images [START_REF] Bennour | Watermarking of 3D objects based on 2D apparent contours[END_REF]. Recently, two surveys have been developed in [START_REF] Rondao-Alface | From 3D mesh data hiding to 3D shape blind and robust watermarking: a survey[END_REF][START_REF] Wang | Three-dimensional meshes watermarking: review and attack-centric investigation[END_REF].

The proposed method

In this section we describe a new method of embedding data in 3D objects. Our method is based on a 3D model represented by cloud of vertices and a list of edges corresponding to a triangular mesh of the surface. The main idea of the method is to find and synchronize specific areas that can be used to embed the message. The embedding of data is based on the modification of the vertex connectivity in the selected areas. These modifications have implications for changing the structure of triangles in these areas. This approach of data hiding has the advantage of not changing the initial position of the vertices of the 3D model. This invariant position of the vertices enables us to make the embedding robust to affine transformations of such types like rotation, translation or change of scale. The method is based on the concept of minimum spanning tree (MST).

Our approach consists of three steps. The first step is to construct a MST covering the vertices of the initial 3D model. The second step necessitates a scanning of the constructed MST to select and synchronize areas for embedding the data. The starting vertex of this step is selected on the basis of a secret key. From a principal component analysis (PCA) of the 3D object, the secret key allows us, in the coordinate system of the object, to locate the starting vertex of the MST. We employ the PCA in an attempt to obtain the three principal directions of the 3D object. To determinate them, the moments are calculated and we obtain coefficients of variance and covariance. This set of coefficients is represented by a 3 Â 3 covariance matrix and an analysis of this matrix gives three eigenvalues and three associated eigenvectors. These vectors denote directions providing maximal variance of the 3D object. Associated with the centroid of the 3D object, these directions form a frame of reference adapted to select the first vertex. As a result of the PCA, a reorganization of the file does not disturb the location of the starting vertex which is dependent, therefore, only on the secret key. The embedding areas must meet a number of requirements due to the connectivity of MST and the geometry of the areas. The third step involves embedding of the message itself. This is done by modifying the connectivity of triangles in the selected areas. Fig. 1 illustrates the overview of the method.

Section 3.1 is about the definition of a MST and its construction. We show, in Section 3.2, how to select embedding areas in order to synchronize them with the message. The data embedding process is described in Section 3.3. Section 3.4 depicts that the selected embedding areas are subject to geometrical constraints. Finally, Section 3.5 describes how to extract the message embedded in a watermarked 3D file.

Minimum spanning tree

Definition

Let G be a graph consisting of a set of vertices and the connecting edges. An edge of G can be represented by (a,b), where a and b are the corresponding vertices. Let A be a undirected connected graph without any cycle which implies that there is no path linked to any vertex of A that would revert back to the same vertex. This graph has thus two characteristics:

If an edge removed, it is no longer connected. If an edge is added, it forms a cycle.

For graph G, there is at least one tree spanning A. It is a partial graph which has the same vertices as G and that the overall relationship is included in the relationships in G.

The cost of graph G is the sum of all the weights of the edges of G. If the sum of weights of the edges of a tree A spanning G is the lowest among all the possible spanning trees of G then A is called the minimum spanning tree (MST) of G. In our method, the weights used to calculate the MST are the Euclidean distances between the vertices of the 3D object. The peculiarity of MST is that it is unique, although there is an ambiguity in the methods of construction when a vertex has two edges of the same weight. The method chooses the edge it encounters first in its walk over the vertices. If the scheduling of vertices in the file is changed then modifications in the MST may also be likely. To correct this ambiguity a calculation of distance between the two vertices of the two identical edges and the center of mass of the 3D object is utilized. In this way MST is always unique regardless of the reorganization of vertices in the file of the 3D object. It is this peculiarity of MST that we refer to Section 3.2 to synchronize the message with the 3D model. We also rely this feature to embed the data as presented in Section 3.3.

Construction of the MST

There are many algorithms for MST construction, the most popular being the Prim's algorithm [START_REF] Prim | Shortest connection networks and some generalisations[END_REF] and Kruskal's algorithm [START_REF]On the shortest spanning tree of a graph and the traveling salesman problem[END_REF].

Prim's algorithm gradually constructs a subgraph A of G by choosing the edge of minimum weight locally at each step, hoping it to be a part of the global solution [START_REF] Prim | Shortest connection networks and some generalisations[END_REF]. Initially, A is empty and a vertex s of G is chosen as a starting vertex. A is thus composed of only s at the start. Let E be the set of vertices connected to s in A and let F is the set of remaining vertices. Initially E is empty, and F contains all the vertices of G. At each step, an edge is added to A provided that this edge has minimum weight among all the edges having a vertex x in F and the other in E. This must not form a cycle. Thus vertex x comes out of the F and is included in E. The algorithm is stopped when F is rendered vacant. Fig. 2b presents a result of Prim's algorithm when applied to the example given in Fig. 2a.

Kruskal's algorithm gradually constructs a subgraph A of G

by first sorting all the edges depending on their weight and then selecting those which do not form cycle in A [START_REF]On the shortest spanning tree of a graph and the traveling salesman problem[END_REF]. For this, the vertices are chosen one at a time and sorted in ascending order according to the weight of the set of those edges that link a vertex s to others. For each sorted edge (s, s 0 ) check whether the list of neighbors of s in A is different from those of s 0 in A. If no vertex neighbor is common, then the edge (s, s 0 ) is added to A.

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Fig. 1. Overview of the method of MST-based embedding.

Selection and synchronization of embedding areas

Synchronization in the embedding process is achieved by identifying the specific connectivities present in the MST. Once MST is built, two phases are required to synchronize the message to be embedded. The first phase is to define the manner in which MST is to be scanned. Indeed, the tree is unique, but the way of scanning depends on the vertex to start with. In our approach this vertex is selected by the use of a secret key that brings us security of the message to be embedded into the 3D model. One can say that the number of ways to read the MST is equal to the number of nodes in the 3D model. Thus the greater the number of nodes, higher is the system security. 3 Once the scanning pattern of MST is defined, specific areas are identified for the perspective embedding of data. Such areas correspond to edges of the MST belonging to particular areas of the 3D mesh as presented in Fig. 3. These areas correspond to a specific edge of the MST which have two triangles with specific configurations. There are two configurations allowing to select the areas used for the embedding step. The first configuration is represented by two triangles with a common edge corresponding to the edge of MST. The second configuration is represented by two triangles with a common edge and the two vertices which do not belong to this common edge correspond to two nodes of the current edge of MST. These quadruples, composed of two triangles and synchronized with two nodes of the MST, therefore, represent the areas where the message bits are to be embedded. Fig. 3 illustrates the selection and synchronization of three quadruples which can be used to embed data. Fig. 3a presents the construction of the MST starting from the node A. Fig. 3b illustrates the corresponding triangles we can find in the file of the 3D object. From the MST and these triangles, we can detect and synchronize three quadruples illustrated in Fig. 3c. For example, the first quadruple is based on the edge number 2 and the two triangles using this edge such common edge.

The embedding method presented in Section 3.3 directly utilizes these detected and synchronized areas. However, as we will explain in Section 3.4, some part of the quadruples cannot be used for embedding. In fact, some areas do not meet certain constraints imposed by the data hiding method and others cannot be used for reasons of not being imperceptible. The MST, due to its construction, will allow us to resist the affine transformations such as rotation, translation or scale variation. This is made possible through our embedding technique which does not alter the position of the vertices in the 3D object. The number of selected areas depends not only on the number of vertices in the 3D object and threshold values for the various constraints but also on the overall mesh of the object. We show, in Section 4, how many areas are selected, at an average, based on all these parameters.

Data hiding step

This section details the embedding step. In Section 3.2 we presented how to locate embedding areas in the MST. These areas are based on edges of the MST and need triangles with common edge. These areas are therefore quadruples. To add one bit of the message in a selected quadruple we look at the edge of the two included triangles in the quadruple.

An example of areas used for embedding data is presented Fig. 4. Embedding of a 0-bit is realized by constructing the common edge of the two triangles on the edge of the original MST as presented in Fig. 4b. In contrast the embedding of a 1-bit, the common edge of the two triangles is constructed so as not to be superimposed over the initial edge of the MST as presented in Fig. 4c. In Fig. 5, from the selected quadruples illustrated in Fig. 3, we show the embedding of the message ''100''. Note that the change takes place in the mesh connectivity and not in that of the MST. This method of embedding permits not to change the position of the vertices of the 3D model. This is an important point for the synchronization and allows us to rebuild the same MST during the extraction of the message. However, this method of embedding imposes certain constraints on the quadruples to be chosen. Modification in the mesh connectivity can lead not only to errors in it but also to visual defects in the surface. We explain in Section 3.4 why these constraints are to be respected and why some quadruples cannot be used to embed the data.

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Constraints on the selection of the embedding areas

In this section we present the constraints applied to quadruples resulting from the synchronization step. In fact, some of these quadruples cannot be used for the data embedding. These constraints are concerned with coplanarity, convexity and overlapping.

Coplanarity constraint

The first constraint is the coplanarity of the quadruples. The underlying fact is that any change in the connectivity of the two triangles forming one quadruple also changes the angle formed between these two triangles. Consequently, the data hiding on a non-coplanar quadruple affects the surface and therefore the visual aspect of the 3D model. In an ideal situation, the message should be embedded only in quadruples which are strictly coplanar. However, the number of quadruples complying strictly to this coplanar criterion is very limited. To increase the embedding capacity, we introduce in our approach a threshold of tolerance, S c , on the coplanarity of the chosen quadruples. This threshold allows us to reach a compromise between the embedding capacity and the quality of the 3D model. The higher the threshold, higher will be the embedding capacity at the expense of visual rendering. Conversely, lower threshold will lead to lower embedding capacity and hence smaller visual degradation of the 3D object. Through the threshold S c , adjustment is thus possible between the embedding capacity and the quality of the visual rendering of the 3D model.

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Let the quadruple Q 1234 is formed by two triangles T 123 and T 234 . First of all, the normal N 1 to plane P 123 and the normal N 2 to plane P 234 , as well as angle A 1 between the N 1 and N 2 is calculated. In the second step, the common edge of the two triangles in the quadruple is modified to simulate an inverse embedding. The new angle A 2 between the normals of the triangles is calculated. Fig. 6 illustrates the calculation of the angles A 1 and A 2 . The quadruple will be retained only if both the angles are smaller than the already fixed threshold S c :

Q 1234 is retained if and only if maxðA 1 ,A 2 Þ oS c : ð1Þ
3.4.2

. Convexity constraint

The second constraint imposes that the quadruples used for the data hiding are convex. For this constraint, we assume that the selected quadruples already obey the coplanarity constraint. Fig. 4 illustrates a convex quadruple where a 0-bit and a 1-bit are embedded as shown in Fig. 4b andc, respectively. In this case, despite the change in triangle connectivity, the quadruple responds to the same characteristics and covers the same geometric space for the two embedding cases. Fig. 7a illustrates a case of non-convex quadruple formed by the planes P 123 and P 234 . We note that the modification of the triangle connectivity when embedding a bit modifies the surface covered by the previous quadruple. This modification introduces an error on the surface of the quadruple. This error is represented by the surface P 134 in Fig. 7b. It is to prevent this error on the surface that constraint of convexity was introduced.

The measurement of this constraint is a combination of the two following procedures: Vectors between vertices V i et V (i mod 4) + 1 of the quadruples are calculated for i 2 f1, . . . ,4g. Angles a i between two successive vectors are then calculated.

Q 1234 is selected only if a i o180 3 for i 2 f1, . . . ,4g: ð2Þ
This allows to eliminate all quadruples having obtuse angles or three collinear vertices.

The second measure prevents us from having quadruples too close to a triangular form when three out of four vertices are aligned. In that case the data hiding would make reveal too disproportional triangles. A threshold of tolerance S t is chosen so that the proportion of both triangles of one quadruple respects a certain value. To this end we calculate the length of edges V 23 and V 14 and the distance D between the centers of these two edges. A quadruple is thus retained if and only if:

Dr S t  V 14 and D rS t  V 23 : ð3Þ
For this constraint, compared to the previous method presented in [START_REF] Amat | Lossless data hiding method based on MST and topology changes of 3D triangular mesh[END_REF], we have a better formalization of this second measure. Thus, with this new constraint we can keep more quadruples than with the previous method.

Overlapping constraint

The third constraint concerns the overlapping of the quadruples. To avoid the recovery of quadruples when a quadruple is selected for embedding then the four nodes of the MST are marked and will no longer be used to search another quadruple. Finally we retain only the quadruples satisfying the presented constraints. Stability is also a strong constraint for data hiding. In fact, the connectivity change of triangles for the data hiding creates new triangles and therefore new quadruples. These new quadruples may well be susceptible to the constraints imposed and thus create new candidates for embedding which can interfere with the data hiding process. To avoid this problem, when all the neighbors of a node are covered even if no quadruple is selected for embedding, then the node is marked as no longer utilizable to search another quadruple. In the previous method presented in [START_REF] Amat | Lossless data hiding method based on MST and topology changes of 3D triangular mesh[END_REF] we did not mark the nodes, consequently in order to extract the correct hidden message we eliminated too many quadruples. The embedding method presented in Section 3.3 is then applied only to the selected quadruples. The constraints restrict the maximum number of quadruples we can watermark to the theoretical limit of N/4, where N is the number of vertices of the 3D object. The maximum theoretical embedding capacity equals then to 0.25 bit/vertex.

Message extraction

To extract the hidden message, the first step is to rebuild the MST from the vertices of the model. We thus run a PCA on the vertices to determine the starting vertex of the MST using the secret key. Since our embedding method does not change the position of the vertices, the rebuilt MST is the same as was for the embedding step. The second step is to locate the embedding areas, using the same constraints as had been for embedding. Once these two steps are performed, then extraction of the message from these areas is easy. For this we compare the position of the common edge of the two triangles forming a quadruple with the current edge of the MST. If the common edge forms a part of the MST then the extracted bit is a 0, otherwise it is a 1 as shown in Fig. 4. Note that the reconstruction of MST requires the use of a secret key to protect hidden information. Fig. 8 illustrates the process of extraction of the message. The MST of the object is unique, but the quadruples selected for embedding depend among them on the manner of scanning the MST. If N is the number of vertices in the 3D object, the complexity of this method is N. To increase the complexity of the method, the key can be used to scramble the order of bits in the message. The complexity will then be N Â 2 n , with n r N being the number of quadruples used for the embedding of the hidden data.

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Experimental results

In this section we apply and analyze our method to 12 3D objects having a number of vertices and different characteristics. Table 1 shows the characteristics of the 3D objects used.

Fig. 9 represents the mesh triangulation of the 3D object called Julius composed of 43039 vertices and 85145 triangles. In this section, to illustrate the results of quadruple search, we rely on this model. Fig. 10a-d illustrate respectively the results of the quadruples search with our method for a threshold S c =11, S c =51, S c =101 and S c =301. With S c =11, 777 quadruples are selected for the data embedding, while 2921, 4261 and 5517 quadruples are selected with respectively S c =51, S c =101 and S c =301.

In order to illustrate the results we obtained, we compare the original 3D object illustrated Fig. 9 with the watermarked 3D objects illustrated Fig. 11a-d. We notice for these images of the watermarked 3D objects that when the threshold of coplanarity S c is high the quadruples that had been reversed to achieve the embedding can become visible. The maximal embedding capacity is observed for the object Julius, with S c =301 the embedding capacity equals to 0.128 bit/vertex. On the other hand when one lowers the threshold of coplanarity these quadruples are less visible. For the given 3D object with the coplanarity threshold of S c =11 one can note that it is not visually possible to see the difference with the original 3D object.

To illustrate the result of embedding conducted on a mesh, we present in Fig. 12 magnified a portion of the mesh. Fig. 12a shows a mesh before embedding, whereas Fig. 12b shows the same mesh wherein the set of the common edges of the selected quadruples for embedding have been reversed. If one considers that the selected quadruples used to embed bits of the message are convex and coplanar, then the change in connectivity made at the watermarking of these quadruples will not change the surface of the 3D model. The criteria to be taken into account in the analysis of results are the embedding capacity, analysis of the error on the surface generated by the embedding of hidden message and the resistance of our method to noise due to transformation.

The first criterion regarding the embedding capacity, calculated in bit per vertex, allows us to evaluate the quality of our method. As illustrated in Fig. 13, the embedding capacities change for the 3D models presented in Table 1 for various values of the coplanarity threshold, S c . The x-axis represents the type of the 3D object, from the smallest to the largest, and the y-axis represents the embedding capacity in bit/vertex. For various resolutions of the same object we note that the embedding capacity increases with the object size. If the coplanarity threshold S c is increased, the difference in the embedding capacities in the objects no longer hold. Fig. 14 also illustrates this criterion wherein the x-axis represents the threshold of coplanarity S c used for the quadruples search and the y-axis represents the mean embedding capacity calculated for the set of 3D objects given in Table 1. The second analysis criterion consists of assessing the error made on the surface depending on the threshold of coplanarity S c and therefore the number of embedded bits. As stated previously, increasing the threshold can increase the embedding capacity but this is done at the expense of the visual quality of the surface. To evaluate the error in the watermarked surface relative to the surface of the original object, a calculation is performed to quantify the errors made at the embedding of a bit in a quadruple. The standard method for the calculation of the error is the Hausdorff's geometric distance which uses the calculation of the Euclidean distance between two vertices on a mesh. There are many methods for calculating the error between two surfaces. Alliez et al. utilize volumetric information to achieve the calculation of the error between two 3D objects [START_REF] Alliez | Mesh approximation using a volume-based metric[END_REF]. Kim et al. calculate the distance based on the curves [START_REF] Kim | Discrete differential error metric for surface simplification[END_REF]. Rondao, De Craene and Macq use a metric based on the estimate of the energy distortion [START_REF] Rondao-Alface | Shape quality measurement for 3D watermarking schemes, in: security, steganography, and watermarking of multimedia contents VIII[END_REF]. Lavoue ´et al. measure the distortion between two 3D objects using the concept of structural similarity [START_REF] Lavoue | Perceptually driven 3D distance metrics with application to watermarking[END_REF]. To measure the error with Hausdorff's geometric distance, we used in this work the algorithm proposed by the software Metro [START_REF] Cignoni | METRO: measuring error on simplified surfaces[END_REF].

ARTICLE IN PRESS

ARTICLE IN PRESS

Fig. 15 shows the surface error values as a function of the threshold of coplanarity S c . This error calculation has been applied to 3D models. Depending on the threshold S c , Fig. 15 illustrates errors obtained with the method of Hausdorff. In all the cases we note that the error increases with the coplanarity threshold. We can also remark that the error are not the same as a function of the watermarked 3D object. Indeed, the error is larger for three particular objects which are lady shoe, male shoe and Chinese dragon. For the two shoes, these distortions are due to the original surface of these objects composed of triangles very disorganized compared to the other 3D objects presented in Table 1. The object Chinese dragon is the more complex object we have tested because a lot of high curvatures are near flat areas which have been watermarked.

The third criterion allows us to evaluate the robustness of the proposed method to noise. Fig. 16 represents the robustness of our method against the noise added to the vertices in the 3D object. The x-axis represents the magnitude of added noise and y-axis the percentage of correct quadruples detected. One can observe that with a low amplitude noise, 100% of the quadruples are selected. However, with an increase in the amplitude of the noise, the number of correct quadruples detected declines fairly rapidly throughout except for one object which seem to be more robust to noise. This object called bunny 1 is the smallest object we have tested and its embedding capacity is very small. All the other objects with a larger embedding capacity have been faster disturbed by an adding of noise. We can notice that object the more complex, Chinese dragon, is the less robust to noise of all the objects we have tested.

In order to analyze the reliability of the proposed method, even if we have a large complexity for the extraction due to the use of a secret key, we have done a receiver operating characteristic (ROC) analysis. The ROC curves presented in Fig. 17 illustrate the sensibility and the specificity of our method by calculating the false positives and false negatives during the extraction step. We have watermarked the object Julius with S c =151, in this case 4816 bits are hidden. For several values of the noise, we changed the threshold value S c between 11 and 301 to extract the message. We note that when you change the value of S c , then false positives and false negatives appear. This is due to the fact that when we change the value of S c , the constraints, described Section 3.4, particularly the overlapping constraint, do not select the same quadruples. This analysis shows that the proposed method is very sensitive to noise.

We have compared our method with previous methods presented in Section 2. We have embedded the same message of 5517 bits in the object Julius with the method of Mao et al. [START_REF] Mao | Watermarking 3D Geometric Models Through Triangle Subdivision[END_REF]. With the method of Mao et al., the watermarked object is no more robust than with our method and the distortion measured with the Hausdorff's geometric distance is larger than with our method. Moreover, the size of the watermarked 3D object has increased. We have also compared our method with the method presented by Cayre and Macq [START_REF] Cayre | Data hiding on 3D triangle meshes[END_REF]. For the same message, the method of Cayre and Macq is more robust to noise, but the distortion is a hundred times larger than with our method.

ARTICLE IN PRESS

Conclusion and perspective

In this paper we presented a new 3D steganography method. The peculiarity of the method lies in the fact that there is no change in the position of the vertices in the 3D object. In this sense the approach is lossless. For specific applications, like industrial, medical, military or cultural heritage applications, the fact to not move the vertices is very important. The proposed method enables the embedding, in a 3D object, of meta-data like the author's name, the date of creation of the object, a 2D or 3D logo, color of the object or the texture of the object itself. For example, if a texture is embedded in a 3D object, when someone receives the watermarked 3D object, if he does not have the algorithm and the corresponding key he can visualize the 3D object with a standard software but only if he has algorithm and the key he will be able to see the 3D object with texture. This method, based on MST scanning, synchronizes the embedding areas with the 3D model. Contrary to the previous methods belonging to this category, our method spreads the message all over the 3D object and does not add any new vertices. Moreover, our approach is robust to data file manipulation. The originality of the proposed method is to synchronize the hidden message with quadruples selected from the MST. This method is robust to attacks like rotation, translation and scaling but very sensitive to noise. The method is blind and the embedding capacity as a function of the threshold of coplanarity S c allows us to hide large message. Indeed, the embedding capacity, on the average, is close to 0.06 bit/vertex and can reach 0.128 bit/vertex for particular objects. In perspective we are planning to develop a new method robust to noise based on the presented one. A new idea may be to change positions of vertices during the MST scanning to select quadruples. But in that eventually, the method will not be lossless and the application domains cannot be the same.

ARTICLE IN PRESS

Fig. 2 .

 2 Fig. 2. (a) Cloud of initial vertices (43,039 vertices) and (b) calculation of the MST with Prim's algorithm.

Fig. 3 .

 3 Fig. 3. Example of areas forming quadruples selected to embed data: (a) construction of the MST starting to A, (b) the corresponding triangles in this area and (c) selection and synchronization of three quadruples.

Fig. 4 .

 4 Fig. 4. (a) A selected quadruple based on an edge of the MST, (b) embedding of a 0-bit and (c) embedding of a 1-bit.

Fig. 5 .

 5 Fig. 5. Data hiding of the message ''100''.

Fig. 6 .

 6 Fig. 6. Calculation of the angles A 1 and A 2 between two triangles forming a quadruple.

Fig. 7 .

 7 Fig. 7. (a) Triangles T 123 and T 234 before embedding and (b) triangles T 124 and T 134 after embedding.

Fig. 8 .

 8 Fig. 8. Overview of the extraction method.

Fig. 9 .

 9 Fig. 9. Triangular mesh (43,039 vertices and 85,145 triangles).

Fig. 10 .

 10 Fig. 10. Quadruple selection for the (43,039 vertices and 85,145 triangles): (a) with a threshold S c =11, 777 quadruples are selected, (b) with a threshold S c =51, 2921 quadruples are selected, (c) with a threshold S c =101, 4261 quadruples are selected and (d) with a threshold S c =301, 5517 quadruples are selected.

Fig. 11 .

 11 Fig. 11. Watermarked 3D Object, Julius (43,039 vertices and 85,145 triangles: (a) coplanarity threshold S c =11 (777 bits embedded), (b) coplanarity threshold S c =51 (2921 bits embedded), (c) coplanarity threshold S c =101 (4261 bits embedded) and (d) coplanarity threshold S c =301 (5517 bits embedded).

Fig. 12 .

 12 Fig. 12. (a) Zoom in a area of the mesh before embedding, (b) zoom in the same area of the mesh after a reversal of all the common edges of the triangles of the selected quadruples.

Fig. 13 .

 13 Fig. 13. Embedding capacity of the proposed method as a function of the 3D objects and the threshold S c .

Fig. 14 .

 14 Fig. 14. Comparison of the average of the embedding capacity as a function of the threshold S c between a previous method [3] and the proposed method.

Fig. 16 .

 16 Fig. 16. Effect of noise on the detection of the quadruples.

Fig. 17

 17 Fig. 17.

  Fig. 17.

Fig. 15 .

 15 Fig. 15. Error as a function of S c calculated by Hausdorff's Method.

Table 1

 1 Characteristics of the 3D objects used.

	3D Object	bunny	dragon	happy	male shoe
	Number of vertices	453/1887/8146	5203/22,982	7102/32,316	12,606
	Number of triangles	948/3851/16,301	11,102/47,794	15,536/67,240	25,208
	3D Object	Chinese dragon	lady shoe	Julius	horse
	Number of vertices	20,184	31,088	43,039	48,485
	Number of triangles	40,364	62,168	85,145	96,966

Each edge of a mesh must have exactly two sides adjacent to an edge inside the mesh and one if it is a border of the mesh. P. Amat et al. / Signal Processing: Image Communication 25 (2010) 400-412

P. Amat et al. / Signal Processing: Image Communication 25 (2010) 400-412

The key can also serve as a seed for a PRNG to randomly scatter the message in the 3D object.

Author's personal copy

We can point out that the embedding capacity increases with the coplanarity threshold S c . In Fig. 14 we also compare the embedding capacities of our proposed method with a previous one [START_REF] Amat | Lossless data hiding method based on MST and topology changes of 3D triangular mesh[END_REF]. With this new method, we can observe that we doubled the embedding capacity irrespective of the coplanarity threshold.


Anne-Laure Foucher

Thierry Chanier

Christine Rodrigues

John Fynn

Aurélie Bayle

Ciara R Wigham

Chanier, T A.-L Foucher

What learning scenarios for virtual worlds and CLIL?

Keywords: CLIL, virtual worlds, language learning, collaborative tasks, intercultural

come

There is a strong case to be made for using a CLIL approach in the learning of foreign languages: learners are exposed to authentic tasks and authentic samples of language whilst their language needs and interests in their specific fields of study are catered for in a very real way. This would result in learners being given optimum conditions for foreign language learning i.e. where language use is meaningful and also calls upon cognitive operations with real issues at stake [START_REF] Coyle | CLIL: Content and Language Integrated Learning[END_REF].

In addition, the use of virtual worlds for foreign language learning is today well researched. Sufficiently so that, in spite of the technical problems that may arise during virtual learning sessions and the cognitive overhead for both learners and teachers entering into these virtual worlds, the following advantages have been recognized: -reduced apprehension in expressing oneself in the FL [START_REF] Schwienhorst | Evaluating tandem language learning in the MOO: discourse repair strategies in a bilingual internet project[END_REF] ; -richness of the target language input thanks to the physical presence of avatars providing verbal, para-verbal and non-verbal data [START_REF] Schwienhorst | Evaluating tandem language learning in the MOO: discourse repair strategies in a bilingual internet project[END_REF] ; -the possibility of organizing collaborative group work allowing learner engagement and thus facilitating situated learning based on experience [START_REF] Dalgarno | What are the Learning Affordances of 3-D virtual Environments[END_REF].

Connecting a CLIL approach to virtual worlds would therefore seem fertile in its principle. We would like to show the relevance of this connection whilst insisting, however, on the need for a rigorous learning scenario design which genuinely takes into account the potential advantages of virtual worlds whilst avoiding the pitfalls of language and content integration that has been insufficiently thought out [START_REF] Coonan | Some issues in implementing CLIL[END_REF]. To do this we will draw on experimentation undertaken in the European project Archi21 where collaborative tasks in the foreign language, set up with a CLIL approach, have been successfully completed by students of Architecture and Design in Second Life.


Dr Emmet Edward Mcgrath
email: e.mcgrath@sheffield.ac.uk

PhD; Paul B Anderson
email: paul.anderson@sth.nhs.uk

M A Frcp

Keywords:

The investigation of pleural effusion has been greatly assisted by advancements in pleural fluid analysis. In the case of tuberculous pleural effusion, diagnosis traditionally requires the demonstration of acid fast bacilli in the pleural space using microbiological or histological techniques. In recent years there has been progress in pleural fluid analysis in suspected tuberculous effusions with particular interest in adenosine deaminase and interferon-γ. These individual tests are quite sensitive and specific, however data is sparse on the benefits multiple parameter testing may have when analysed in combination. We reviewed the literature to investigate the evidence for multiple parameters testing, both biochemical and clinical, in the evaluation of tuberculous effusion.

Diagnostic tests for tuberculous pleural effusion

Dear Editor, Thank you for giving us the opportunity to submit a revised manuscript. I very much appreciated the reviewers' comments and have made every effort to address them. I have highlighted the changes to the manuscript in red font and will specifically address each comment below chronologically. As ever, we would be only too delighted to make any further changes the reviewers' recommend.

Introduction

Tuberculosis continues to be an important cause of morbidity and mortality in developing countries. Areas of high epidemiologic burden (annual incidence >25/100,000) include South Africa 480/100,000 and Swaziland 1,155/100,000 which contrast with the low burden areas of the United States and Monaco where annual incidence is 4.6 and 2/100,000 respectively [START_REF] Porcel | Tuberculous pleural effusion[END_REF][START_REF]Global tuberculosis control: surveillance, planning, financing[END_REF]. With pleural fluid analysis, acid fast bacilli are detected in <5% of cases, whereas mycobacterial culture of fluid has a sensitivity of <60% with wait times up to 2 months for culture results [START_REF] Bueno | Cytologic and bacteriologic analysis of fluid and pleural biopsy specimens with Cope's needle. Study of 414 patients[END_REF][START_REF] Seibert | Tuberculous pleural effusion. Twenty-year experience[END_REF].

Other methods traditionally used for TB lung infection also have disappointing diagnostic ability. The culture of spontaneous or induced sputum has a sensitivity of <30% and 52% respectively and approximately one third of patients with tuberculous pleural effusion have a negative tuberculin skin test [START_REF] Chakrabarti | Pleural tuberculosis[END_REF].

Histological analysis and mycobacterial culture of closed pleural biopsied tissue have traditionally been the gold standard investigation with sensitivities of 71-80% and 39-79% respectively [START_REF] Villegas | Evaluation of polymerase chain reaction, adenosine deaminase, and interferon-gamma in pleural fluid for the differential diagnosis of pleural tuberculosis[END_REF]. Combining both together has a detection rate of 91.3% for TB [START_REF] Valdes | Tuberculous pleurisy: a study of 254 patients[END_REF]. The introduction of thoracoscopy has had a very important impact on diagnosis which was particularly highlighted by Diacon et al who performed a direct comparative study of the diagnostic tools used in tuberculous pleurisy and found that sensitivity of histology, culture and combined histology/culture was 66, 48 and 79%, respectively for closed needle biopsy and 100, 76 and 100%, respectively for thoracoscopy. Both were 100% specific. [START_REF] Diacon | Diagnostic tools in tuberculous pleurisy: a direct comparative study[END_REF]. Apart from achieving superior histological samples, the technique of thoracoscopically biopsying large areas of abnormal pleura also increases the likelihood of positive diagnosis especially when macroscopic abnormalities such as "tapioca pleura" are observed. This macroscopic appearance combined with the microscopic appearance of granulomas often allows the commencement of therapy while culture results are awaited. Thoracoscopy, unlike other forms of pleural biopsy, also facilitates the removal of pleural fluid thereby relieving troublesome dyspnoea.

In more recent times, additional pleural fluid biomarkers have been discovered including adenosine deaminase (ADA) which reportedly has a sensitivity of 88% and a specificity of 85.7% and interferon-γ (IFN-γ) which has a sensitivity of 85.7% and a specificity of 97.1%. These studies were reported from an area where prevalence was 59 per 100,000 population [START_REF] Villegas | Evaluation of polymerase chain reaction, adenosine deaminase, and interferon-gamma in pleural fluid for the differential diagnosis of pleural tuberculosis[END_REF][START_REF]Global Tuberculosis Database[END_REF]. A more recent meta-analysis of 63 studies found that ADA has a sensitivity and specificity of 92% and 90% respectively and a metaanalysis of 22 studies found that IFN-γ has a sensitivity and specificity of 89% and 97% respectively [START_REF] Liang | Diagnostic accuracy of adenosine deaminase in tuberculous pleurisy: a meta-analysis[END_REF][START_REF] Jiang | Diagnostic value of interferongamma in tuberculous pleurisy: a metaanalysis[END_REF]. Acceptance of these tests for the diagnosis of pleural TB has not been universal and remains contentious in some countries with diagnostic performance very variable between populations [START_REF] Hooper | Interferon-gamma release assays for the diagnosis of TB pleural effusions: hype or real hope?[END_REF].

Used individually, the different cheap and easy to perform pleural fluid tests have a wide range of sensitivity and specificity. We hypothesised that these tests used in combination might improve the ability to diagnose this condition.

Methods

In this review, we searched for publications describing pleural fluid test combinations that may help with the diagnosis of tuberculous pleural effusion. Publications from a MEDLINE search and references from relevant articles were reviewed on the clinical investigation of tuberculous pleural effusion. All articles that adhered to the criteria of multiple tests or parameter combinations in the diagnosis of tuberculous effusions were included in this review. Papers were separately selected by author 1 (EMG) and author 2 (PA) with common papers included in the review. Papers found by only one author were assessed by both authors together for their suitability. Keywords searched were tuberculous effusion, pleural fluid, diagnosis, adenosine deaminase, interferonγ, pleuritis and empyema

Results

As summarised in Table 1, twelve studies describing the use of combination fluid/clinical parameter analysis were found (6, 13-23). Here we describe and appraise these studies in chronological order. San Jose et al analysed the use of serum and pleural ADA and lysozyme in tuberculous pleurisy in 271 patients in Spain, of which 53 had tuberculosis. They found that pleural ADA had a sensitivity of 100% and a specificity of 93% for the diagnosis of TB and that combining the use of pleural ADA and total protein in the form of a ratio increased the specificity to 95% at the expense of a reduction in sensitivity to 93%. Using a ratio involving pleural fluid ADA and lysosyme also increased the specificity to 95% with a sensitivity of 80%. A ratio of pleural fluid lysosyme to total protein had a sensitivity of 60% and a specificity of 42% [START_REF] José | Diagnostic value of adenosine deaminase and lysozyme in tuberculous pleurisy[END_REF].

While these combinations improved specificity, it was at the expense of a reduction in sensitivity. This was a retrospective study involving a small number of tuberculosis cases. The authors used robust statistics but failed to describe parameters such as age and sex. There is also little information given regarding the diagnostic methods used in the tuberculous effusion group. The cases appear to be a mix of microbological/histological and clinical diagnosis, the latter having not been proven.

The ADA cut-off was different to the other studies described at 43U/l.

Muranishi et al investigated the simultaneous measurement of ADA activity and tuberculostearic acid (TSA) in pleural effusions for the diagnosis of tuberculous pleuritis. Pleural effusions from 18 patients with tuberculous pleuritis, 16 patients with suspected tuberculous pleuritis, 14 patients with malignant effusions, 6 patients with parapneumonic effusions, 5 with cardiac failure related effusions, 3 with collagen disease related effusions and 5 miscellaneous effusions were studied. They found that when they analysed pleural fluid ADA and TSA in combination, sensitivity increased from 56% (ADA alone) to 83%. Specificity remained at 76% [START_REF] Muranishi | Simultaneous measurements of adenosine deaminase activity and tuberculostearic acid in pleural effusions for the diagnosis of tuberculous pleuritis[END_REF]. The authors felt that the simultaneous measurement of both these parameters was useful in the diagnosis of pleural effusions. There were a number of weaknesses with this study. It was a very small study and the sensitivity and specificity were relatively low when compared to the more recent data. It was retrospective and included cases where definitive proof of TB was absent relying solely on high clinical suspicion. Therefore, some of the suspected TB cases will have been due to other aetiologies. They used an ADA cut-off level of 50 U/l which differs from many of the other studies and did not describe the study population adequately (age/sex).

In 1993 in Brazil, De Olivereia et al studied the usefulness of pleural fluid ADA in combination with pleural fluid lymphocyte proportion in 276 patients of which 54 had TB. They found that using these tests in combination resulted in a sensitivity of 90.7% and a specificity of 97.7%. They concluded that the use of these tests in combination was a highly efficient diagnostic strategy of low cost that merits wider use [START_REF] Oliveira | Pleural fluid adenosine deaminase and lymphocyte proportion: clinical usefulness in the diagnosis of tuberculosis[END_REF].While this study benefitted from its prospective nature, there were a number of important weaknesses. Only 48% of the TB cases included in the study were actually proven either by microbiology or histology. Cardiac failure and very small effusions were excluded from this study which has implications on the interpretation of the results.

Only 61% of the neoplastic effusions had positive cytological or histological evidence of malignancy. The balance was made up of presumed malignancy. The ADA cut-off level was different to many other previous studies at 40U/l. Finally, the statistical analysis was not well explained.

Villena et al measured the concentration of pleural ADA and the ratio of pleural lysozyme (PL) to serum lysozyme (SL) in consecutive patients (49 tuberculous and 179 nontuberculous) [START_REF] Villena | Rapid automated determination of adenosine deaminase and lysozyme for differentiating tuberculous and nontuberculous pleural effusions[END_REF]. Using sensitivity and specificity curves, they established cut-off values at 33 U/L (units per litre) for ADA and 1.7 for the PL/SL ratio. The sensitivity of ADA activity for tuberculous effusion was 90% with a specificity of 85%. Combining ADA with the PL/SL ratio enhanced specificity to 99% [START_REF] Villena | Rapid automated determination of adenosine deaminase and lysozyme for differentiating tuberculous and nontuberculous pleural effusions[END_REF].

However, they concluded that these markers should only suggest a diagnosis and point the clinician in the direction of pleural biopsy and pleural fluid cultures, and that values for ADA and lysozyme ratios are not, alone or in combination, sensitive or specific enough to replace pleural biopsy or culture of pleural fluid for the diagnosis of tuberculous empyema. This work had a number of good qualities. It was prospective. All patients followed the exact same diagnostic algorithm with difficult cases discussed in a blinded manner by a multidisciplinary panel. However, on the negative side, this study included cases where definitive histology or microbiology was not forthcoming and clinical suspicion alone was used. The non-tuberculous group was approximately 3.5 times the size of the tuberculous group. The findings were only relevant when diagnosing TB cases from a mix that includes parapneumonic, neoplastic and transudative effusions only. The ADA cut-off level of 33 U/l was again different to other studies. The analysis of pleural fluid lysozyme is also a contentious issue with discrepancies in results noted in previous work, possibly attributed to the method of analysis (turbimetric v lysoplate). In this work the sensitivity of lysozyme was reported as much higher than previous reports. Again, in this study, there was inadequate description of the study population. Statistical analysis was robust and explained.

Burgess et al analysed the differential cell counts and ADA on 303 of 472 consecutive pleural fluid samples after hemothorax, transudate and unsuitable samples were excluded. They found that when ADA levels were combined with the lymphocyte/neutrophil ratio results, the accuracy of identifying TB increased compared with isolated ADA analysis [START_REF] Burgess | Combined use of pleural adenosine deaminase with lymphocyte/neutrophil ratio. Increased specificity for the diagnosis of tuberculous pleuritis[END_REF]. When a positive ADA result was combined with a lymphocyte neutrophil ratio of 0.75 or greater, the sensitivity, specificity, positive predictive value, negative predictive value, and accuracy for the identification of TB were calculated at 88%, 95%, 95%, 88%, and 92%, respectively [START_REF] Burgess | Combined use of pleural adenosine deaminase with lymphocyte/neutrophil ratio. Increased specificity for the diagnosis of tuberculous pleuritis[END_REF]. The authors concluded that pleural fluid which met these criteria was very suggestive of TB. The good qualities of this work included the fact that it was a prospective study with a higher number of TB cases that were evenly distributed between men and women. The ADA detection method followed the well established Giusti method (used by all but one of the listed studies) although the cut-off used was 50 U/l [START_REF] Giusti | Adenosine deaminase[END_REF]. The main problems with the work include the inclusion of suspected rather than definitive (microbiology or histology) cases. Transudates were excluded from the study affecting its sensitivity and specificity when compared with other studies. Conclusions can only be drawn on exudative effusions. The study population was 68% mixed race with a low number of Caucasian patients (16%), therefore the findings may not be as relevant in the Caucasian population. The statistics used were not well described.

Villegas et al evaluated the individual use of ADA, IFN-γ levels and polymerase chain reaction (PCR) in the diagnosis of tuberculous effusion in 140 cases of pleural effusion. They found that these tests had a sensitivity of between 73%-88% when used alone, but that sensitivity and specificity increased when all three were used in combination [START_REF] Villegas | Evaluation of polymerase chain reaction, adenosine deaminase, and interferon-gamma in pleural fluid for the differential diagnosis of pleural tuberculosis[END_REF]. Incidentally, they also found that a lower age and fever was associated with TB related effusion. Pleural biopsy was also performed and had a sensitivity of 64.3% and a specificity of 98%. The authors concluded that their findings supported the use of new and more efficient diagnostic strategies in the management of pleural TB. This was a prospective study with a good male/female balance and a good mix of TB and non TB cases. The ADA was analysed in a standardised fashion (Giusti), however like the previous studies described, the cut-off level was not standardised with a cut-off level of 45.3 U/l. Only 42 patients had confirmed TB, the rest were included due to high clinical suspicion. There was a significant frequency of false-negative results of PCR in the study (26%). This is most likely attributable to the inefficient recovery of genomic DNA and the extraction of DNA from frozen samples. Nonetheless, this will have had a significant effect on the results of this study. Statistical analysis was robust and well explained.

Porcel et al retrospectively reviewed 106 patients with tuberculous effusions and 286 patients with malignant effusions over a 9 year period. They used two scoring systems, using clinical and laboratory variables where the main difference was the inclusion (model 2) or exclusion (model 1) of ADA [START_REF] Porcel | Differentiating tuberculous from malignant pleural effusions: a scoring model[END_REF]. They found that using the scoring system from model 1 which was calculated by attributing scores to the results of measured variables including an adenosine deaminase ≥ 40 U/L (5 points), age <35 years (2), temperature ≥ 37.8 degrees C (2), and pleural fluid red blood cell count < 5 x 10 9 /L (1).

Model 2 involved a scoring system based on model 1 (excluding ADA) plus the following: no history of malignancy (3), pleural protein ≥ 50 g/L (1), and pleural fluid to serum lactate dehydrogenase ratio ≥ 2.2 (1). Total scores ≥ 5 in model 1 and ≥ 6 in model 2 resulted in a sensitivity and specificity of 95% and 94% and 97% and 91% respectively in the discrimination of tuberculous from malignant effusions [START_REF] Porcel | Differentiating tuberculous from malignant pleural effusions: a scoring model[END_REF]. This work while involving larger numbers, was retrospective and again included cases of high clinical suspicion rather than exclusively involving confirmed cases. The other important weakness with this work was that their diagnostic tree method was not validated by the authors.

Ghanei et al investigated the combination of pleural fluid ADA, LDH and lymphocyte to neutrophil ratio. In the combination of three methods, with a positive result by either three of the methods considered to be indicative of a positive diagnosis of pleural TB, ADA activity plus LDH levels plus lymphocyte to neutrophil ratio in the pleural fluid yielded a sensitivity of 100%. This combination requiring all three methods to be positive for diagnosis to be made was 100% specific for pleural TB.

They also found that using a combination of pleural fluid ADA and LDH yielded a sensitivity of 91.4% and a specificity of 100% [START_REF] Ghanei | Simple method for rapid diagnosis of tuberculosis pleuritis: a statistical approach[END_REF]. This was a very small study where only 17 patients had a tuberculous effusion. They concluded that using these parameter combinations could lead to a diagnosis of pleural TB with an acceptable degree of confidence. While this was a prospective study with a good male/female balance and well described robust statistical method, the main criticism with this work is the small number of confirmed TB cases which were compared to a non TB group that was approximately 3.5 times its size [START_REF] Burgess | Combined use of pleural adenosine deaminase with lymphocyte/neutrophil ratio. Increased specificity for the diagnosis of tuberculous pleuritis[END_REF]. The ADA detection method was as per Giusti but again the cut-off level was different to other studies at 47 U/l.

Neves et al prospectively analysed 215 patients with pleural effusions from 1997-2001, of which 104 were tuberculous, and found that the analysis of ADA, total serum leukocytes, percentile of lymphocytes, protein and duration of disease combined, yielded a sensitivity and specificity of > 95%. [START_REF] Neves | Predictive model for the diagnosis of tuberculous pleural effusion[END_REF]. This was a retrospective study which excluded a large proportion of cases due to inadequate specimen collection or inappropriate handling of ADA specimens. ADA detection was as per Giusti with a cut-off of 39 U/l. Of the TB cases included in the study, less than 10% of pleural fluid cultures and 58% of biopsy cultures were positive for TB. Acid fast staining was negative in all cases. Like Porcel's work from 2003, the main weakness of this study was that the authors did not validate their predictive model. Statistics were well described and thee was a good balance between TB and non TB cases in the study.

Daniil et al investigated the measurement of ADA, IFN-γ, C-reactive protein (CRP), carcinoembryonic antigen, interleukin-6, tumour necrosis factor-α and vascular endothelial growth factor concentration in pleural fluid and found that the combination of ADA and CRP increased the percentage of correctly classified individuals as having tuberculous rather than malignant or parapneumonic effusions in 92% of cases [START_REF] Daniil | Discrimination of exudative pleural effusions based on multiple biological parameters[END_REF]. The study involved 72 patients of which only 12 were tuberculous. The authors recommended further and larger studies in this area. While this was a prospective study using robust statistical method, it was extremely small. ADA detection was again as per Giusti with a cut-off of 42.2 U/l. The study involved twice as many males as females. The non TB group was approximately 4.5 times the size of the TB group and the non TB malignant group was highlighted by the unusual absence of lymphomatous effusions which will have had an impact on the sensitivity and specificity.

In 2008 Porcel et al, having already published in this area in 2003, described a decision tree for differentiating tuberculous from malignant effusions. They retrospectively compared clinical and pleural features of 238 adults with pleural effusion who satisfied diagnostic criteria for tuberculosis or malignancy [START_REF] Porcel | A decision tree for differentiating tuberculous from malignant pleural effusions[END_REF]. They found that the combination of age > 35 years, pleural fluid ADA > 38 U/L, temperature ≥ 37.8 o C, and a pleural fluid LDH > 320 U/L yielded a sensitivity of 92.2% and a specificity of 98.3% allowing them to conclude that the use of this decision tree could help in the differential diagnosis of malignant and tuberculous effusions but not of other forms of effusions [START_REF] Porcel | A decision tree for differentiating tuberculous from malignant pleural effusions[END_REF]. This was a good sized retrospective study which only involved confirmed TB cases and not suspected ones. Importantly, it included a derivation cohort followed by a validation cohort. The limitations of this work are few but involve the important fact that this model is only useful in differentiating TB from malignancy and not from other forms of effusions. The ADA detection system was not like those of the previous studies described and the cut-off used with the detection system was 38 U/l. While it is limited by its retrospective nature, the study involved only objective clinical characteristics and laboratory parameters strengthening the data. The author's model is only relevant in areas of high prevalence and could lead to very different findings if used in the USA for example, where prevalence is 3.4 /100,000.

Lastly, Kupeli et al have recently examined concurrent measurement of ADA and dipeptidylpeptidase (DPP) IV (T cell associated enzyme) activity in the diagnosis of tuberculous pleural effusion [START_REF] Kupeli | Concurrent measurement of adenosine deaminase and dipeptidyl peptidase IV activity in the diagnosis of tuberculous pleural effusion[END_REF]. They found that using both of these markers together improved the sensitivity, specificity and diagnostic accuracy (77%, 94% and 91% respectively), when compared to analysing these markers separately [START_REF] Kupeli | Concurrent measurement of adenosine deaminase and dipeptidyl peptidase IV activity in the diagnosis of tuberculous pleural effusion[END_REF]. This study involved 87 patients of which 18 had TB. Apart from the small number of patients involved, Kupeli et al included known transudates in their study thus increasing the diagnostic yield of the test. They concluded that in an area of high prevalence, ADA levels in combination with that of DPP can be useful in the diagnosis of tuberculous pleural effusion with high specificity and accuracy. The strengths of this study include its prospective nature, robust statistics and its focus on definite TB cases only. Like many of the previous studies it is limited by its extremely small number of TB cases [START_REF] Porcel | Differentiating tuberculous from malignant pleural effusions: a scoring model[END_REF]. The ADA analysis method was also different to other studies as was the ADA cut-off level of 40 U/l. While this work was performed in an area of high prevalence, the age of the TB group was young with an age range of 17 -30 yrs and the male to female ratio was 3:1. Another limitation of the work is that parameter testing of each case occurred depending on the clinical scenario rather that blindly testing all cases similarly.

Marker combinations

Conclusion

The combination of tests and biomarkers to diagnose tuberculous pleural effusion in endemic areas where resources are limited or in cases where traditional investigation is negative but suspicion is high certainly has merit. Even in cases where ADA is unavailable, the use of clinical and demographic information in combination with a cell differential count, in high incidence settings, can yield a diagnosis of TB with a high predictive value [START_REF] Trajman | Novel tests for diagnosing tuberculous pleural effusion: what works and what does not?[END_REF]. This concept has been further advanced with a number of groups recommending the use of pleural fluid and clinical parameter combinations in the diagnosis of tuberculous pleural effusions which we described in this review.

We believe there are a number of advantages to using such scoring systems. They are also useful in those highly suspicious few patients that do not yield a definitive diagnosis after closed pleural biopsy or fluid analysis and culture.

While the advantages described above might appear obvious it is also important to highlight a number of disadvantages with these tests. Firstly, the specificity of ADA is relatively low when compared to, for example, thoracoscopy and therefore it is important to remember that ADA in lymphocyte rich effusions has been described in rheumatoid arthritis, mesothelioma, psittacosis, brucellosis, histoplasmosis, coccidiomycosis, bronchoalveolar carcinoma, chlamydia and mycoplasma pneumonia, Mediterranean fever and in the majority of patients with empyema [START_REF] Light | Tuberculous pleural effusions[END_REF][START_REF] Ferrer | Pleural tuberculosis[END_REF][START_REF] Sharma | Adenosine deaminase in diagnosis of tuberculosis pleural effusion[END_REF][START_REF] Galborini | Adenosine deaminase in the diagnosis of tuberculous pleural effusion: is it really an ideal test; a word of caution[END_REF].

The benefits described in the studies in figure 1 have in the majority of cases been performed in areas where TB prevalence is high and data is therefore lacking from countries with lower prevalence. As ADA makes up part of the combination in all the studies reviewed, it is important to highlight that that ADA is less sensitive in countries of low TB prevalence and may lead to an under-diagnosis of pleural TB (however TB may be excluded particularly if ADA is negative and suspicion is moderate or low) which can lead to further organ spread in 65% of patients [START_REF] Roper | Primary serofibrinous pleural effusion in military personnel[END_REF]. It is also possible that false positive cases will be commenced on anti-tuberculous therapy resulting in significant drug related adverse effects. Importantly the Infectious Diseases Society of America (IDSA) recommends the demonstration of M.

tuberculosis in the sputum, pleural fluid or pleural biopsy specimen as their gold standard in diagnosing the condition and clearly do not recommend the replacement or substitution of this standard by pleural fluid and clinical combination tests [START_REF] Blumberg | AmericanThoracic Society/Centers for Disease Control and Prevention/Infectious Diseases Society of America: treatment oftuberculosis[END_REF].

Additionally, one of the most important disadvantages of using these combinations alone in the diagnosis of pleural TB is the fact that these combinations bypass the culture of fluid or tissue and therefore drug susceptibility testing is not performed in an era when drug resistant TB is increasing. In the USA, Baumann et al described resistance to a first line treatment drug in 9.9% of pleural TB patients from 1993-2003 (32). This can lead to treatment failure and clinical deterioration. Certainly the use of combination tests may allow the commencement of four drug therapy while pleural fluid culture is awaited but if this is non-diagnostic, pleural biopsy will still be required especially in those groups who have a higher rate of drug resistance [START_REF] Baumann | Pleural tuberculosis in the United States: incidence and drug resistance[END_REF].

Also, the process of testing for these markers does not offer any immediate therapeutic benefit unlike thoracoscopy which is not only diagnostic but allows the removal of large pleural fluid collections giving immediate relief to the patient. Other advantages highlighted earlier included microscopic and macroscopic examination of tissue leading to early drug testing prior to culture results and drug susceptibility testing on biopsied tissue.

While the studies listed here appear to demonstrate a benefit of combination (particularly those involving ADA) over single parameter testing, the authors feel that there is currently insufficient evidence available to conclude whether the use of While we await these studies, we believe that combination tests may be used in current practice in areas of high prevalence to steer the physician towards a diagnosis allowing therapy to start and continue or be adjusted once definitive proof has been achieved with culture or histology. They may also be useful in endemic areas with poor resources, such as the Sub Saharan region where diagnosis is often made clinically or empirically as no other resources are available [START_REF] Reuter | Tuberculous pericarditis and HIV infection in Africa[END_REF].

Clearly, the use of a combination of parameters to diagnose tuberculous effusions has great potential for both physicians and patients involved. The benefits in the acute setting, in patients with negative initial results despite high clinical suspicion, in frail patients unable to undergo pleural biopsy combined with their cheap cost are considerable in regions where TB is endemic. Their use in areas where the incidence of TB is low is less valid. As a result, further clinical assessment of these combinations through larger studies and randomised trials may validate their general and routine use in medical practice and in particular identify a robust combination that yields the highest sensitivity and specificity.

  Mycobacterium tuberculosis (TB) related pleural effusion results from infiltration of the pleural space by Mycobacterium tuberculosis antigens or bacilli. Traditional investigation involves microscopic examination for acid fast bacilli (AFB), fluid culture, cytological examination of fluid for inflammatory cells and microbiological and histological examination of biopsied tissue.

  clinical and biochemical parameter combinations should routinely be used to diagnose tuberculous effusions. Studies have involved low numbers of patients and all have recommended the use of different parameters thus lacking consensus. Other problems with the studies include the variable prevalence rates associated with the different countries where the research took place, the different ADA detection methods and cut-off levels used, the variable study design (retrospective versus prospective), the regular inclusion of unproven TB cases in the studies as well as the lack of gold are required to validate their use and studies in areas of low TB prevalence are also warranted to investigate the suitable use of these combination tests in these areas. In particular the study by Porcel in 2008 would benefit from validation in an area of low prevalence as it was one of the stronger studies performed by a world recognised research group with a good demographic distribution, robust statistics and large study size. Studies which intend to include ADA might benefit from using the more specific ADA2 isoenzyme which may increase the sensitivity and specificity further. Furthermore, while at best these combinations may strongly suggest a diagnosis in endemic areas warranting commencement of drug therapy, their use as a replacement to the demonstration of M. tuberculosis in culture or histology will require randomised control trials comparing these parameters with the gold standard of thoracoscopic biopsy for microbiology and histology. If they performed well in these trials, their regular use may be validated however the problem still arises regarding susceptibility testing in areas of high drug resistance.

Table 1 . Studies examining the role of combination tests in the diagnosis of tuberculous pleural effusion.

 1

		Result	Author	Year	Compared	Study	Patient	Prev.
					with biopsy	design	no.	per
					or culture			100/000
	ADA and fluid lysozyme	↑ specificity to 95%	San Jose	1992	No	Pro	271	39
		P < 0.05
	ADA and fluid	↑ sensitivity to 83%	Muranishi 1992	No	Pro	67	58
	tuberculostearic acid	P < 0.05
	ADA and fluid lymphocyte	↑ sensitivity to 90.7%	De	1993	No	Pro	276	108
	proportion	↑ specificity to 97.7%	Oliveira
		P < 0.05
	ADA, ratio of pleural to serum	↑ specificity to 99%	Villena	1996	No	Pro	228	32
	lysozyme	P < 0.05
	ADA, ratio of fluid	↑ sensitivity to 88%	Burgess	1996	No	Pro	303	480
	lymphocytes to neutrophils	↑ specificity to 95%
		P < 0.05
	ADA and IFN-γ, PCR (any 2	↑ sensitivity to 90.5%	Villegas	2000	Yes	Pro	140	50
	markers combined)	P < 0.05
	ADA, fever, age, fluid RBC	↑ sensitivity to 95%	Porcel	2003	No	Retro	392	32
		↑ specificity to 94%
		P < 0.05
	ADA, fluid LDH, ratio of fluid	↑ sensitivity to 100%	Ghanei	2004	No	Pro	88	32
	lymphocytes to neutrophils	(1 of 3 +ve)
		↑ specificity to 100%
		(all 3+ve)
		P < 0.05
	ADA, fluid % lymphocytes,	↑ sensitivity > 95%	Neves	2007	No	Pro	215	59
	serum leukocyte count, fluid	↑ specificity > 95%
	protein, symptom duration	P < 0.05
	ADA and fluid CRP	↑ specificity > 91.7%	Daniil	2007	No	Pro	72	16
		P < 0.05
	ADA, fluid LDH, age,	↑ sensitivity to 92.2%	Porcel	2008	No	Retro	238	32
	temperature	↑ specificity to 98.3%
		P < 0.05
	ADA, fluid	↑ sensitivity to 77%	Kupeli	2009	No	Pro	87	33
	dipeptidylpeptidase IV	↑ specificity to 94%
		P < 0.05

Marker combinations