Review Articles

An Evaluation of HIV Pathogenicity and Treatment Using Glycobiology

The Vegetative State: A Review of Etiology and Prognostic Factors

An Evaluation of HIV Pathogenicity and Treatment Using Glycobiology

Aleksandra Leligdowicz, B.Sc. *
To whom correspondence should be addressed: 418 Ave des Pins Ouest, Apt 34, Montreal, Quebec H2W 1S2, Canada. E-mail address: alelig@po-box.mcgill.ca.

INTRODUCTION
Over 50 million people are infected with the Human Immunodeficiency Virus (HIV) worldwide (1). Caring for the millions of people living with HIV and AIDS is essential. Over 40 therapies are approved by the Food and Drug Administration and as a result, the death rate has fallen dramatically across Europe and the United States. However, AIDS is increasingly a disease of the poor and medicines are where the problem is not, while the problem is where the medicines are not (2). Terminating the HIV epidemic is of utmost significance especially to developing countries, which account for over 95% of new HIV infections (1). Therefore, controlling the virus is important not only to the state of world health, but also to global development.

Glycobiology, the study of carbohydrates, is an interesting topic to medical research. Critical biological processes, including regulation of the growth and mobility of cells, immune responses, and responses of cells to hormones and growth factors, all depend on carbohydrates. In addition, viruses, including HIV, use cell-surface carbohydrates to get into cells and initiate infections. The aim of this review is to demonstrate how the expanding research in glycobiology can be applied to understanding the structure of HIV, as well as developing potential treatment approaches.

ESSENTIALS OF GLYCOBIOLOGY
One of the post-translational modifications that occurs on proteins after they have begun to be translated in the endoplasmic reticulum (ER) is N-linked addition of carbohydrate chains, resulting in the formation of glycoproteins (figure 1)

Glycoproteins contain one or more carbohydrate residues. The carbohydrate moieties of glycoproteins help to determine the tertiary structure of a protein. In addition, they may serve as biological labels, marking the proteins for different fates. This is possible because the number of possible permutations and combinations of monosaccharide types and glycosidic linkages in an oligosaccharide is astronomical (4). Each oligosaccharide can therefore present a different face that can be recognized by not only specific enzymes but more importantly for HIV infection, by specific receptors.

OVERVIEW OF HIV INFECTION
The human immunodeficiency virus can be classified as types one (HIV-1) and two (HIV-2). HIV-1 is older, more infectious, and accounts for the vast majority of HIV worldwide while HIV-2 is present mainly in Western Africa (5). Since HIV -1 accounts for the majority of the infections worldwide and since it is 3.55-fold more infectious than HIV-2, it will be the virus type of focus of this review.

HIV-1 is the causative agent for the acquired immunodeficiency syndrome, or AIDS. Infection begins when HIV interacts with a host cell surface membrane protein (CD4) via a viral envelope glycoprotein. The glycoprotein is an oligomer of extracellular (gp120) and transmembrane (gp41) glycoproteins (6). The gp120 is responsible for virion binding to CD4 receptors of host cells, whereas gp41 mediates fusion of the HIV virus and the host cell membrane. The fusion of membranes allows the viral particle to enter the cell by receptor-mediated endocytosis. Once the virus is inside the cell, it releases its RNA genome and the enzyme reverse transcriptase (RT). RT reverse transcribes the viral RNA into DNA. The HIV DNA moves into the nucleus where it is inserted into the host cell's genome. New HIV virions are made by the host cell, which replicates and transcribes the HIV DNA, and translates the viral transcript as well. Newly formed virions are released from the host cell, ready to infect the next CD4+ host cell (T helper-cell).


Figure 1. Steps in the formation of N-linked oligosaccharides in the lumen of the rough endoplasmic reticulum ER and the various compartments of the Golgi complex. The attachment of oligosaccharides is via an N-glycosidic bond to the side chain of an asparagine residue on the protein. The asparagine is part of the sequon Asn-X-Thr/Ser. The sequon is recognized by an enzyme that transfers the oligosaccharide from dolichol to Asn in the peptide. The first seven sugars (2 GlcNAc's and 5 Mannoses), are transferred one at a time to the dolichol-PP on the cytosolic side of ER. The oligosaccharide then flips across the ER membrane and the rest of the monosaccharides are added to the glycan on the luminal side of the membrane. After the oligosaccharide is assembled, it is transferred to an asparagnine residue on the nascent peptide. Once the glycan is attached, it undergoes removal steps mediated by glucosidases (glucose cleaving enzymes) that take off the terminal glucose residues. The protein is then shipped to the Golgi complex, where most of the terminal mannose units are removed by mannosidases and a variety of sugars. Various Glycosylation pathway inhibitors are listed above the enzymes associated with the steps in the pathway (3). (click for larger image)

The precursor to gp120 and gp41 is gp160. This glycoprotein is heavily glycosylated in the ER lumen, where disulfide formation also occurs. The gp160 is cleaved intracellularly by a host protease in the late Golgi compartment to yield the noncovalently linked gp120 and gp41 complexes (figure 2 a, b, c), which are then transported to the host plasma membrane. At the surface, the complexes can be used by the emerging virus when it buds out from the cell to equip the virus for infection of new CD4 cells. In addition, the gp120-gp41 complex in the host plasma membrane can bind a CD4 receptor on an uninfected cell, resulting in cell fusion (6).

HIV GLYCOPROTEIN STRUCTURE
THE USE OF MUTATED RECOMBINANT HIV GLYCOPROTEINS TO STUDY THEIR STRUCTURE
The structure of the HIV envelope glycoproteins (gp120 and gp41) is important for developing drugs that can function to eradicate the virus. An understanding of the chemical nature and the processing of the oligosaccharide chains attached to the envelope proteins, and the identification of the sites where post-translational modification occurs on the gp160 precursor glycoprotein can serve as an invaluable tool for terminating the spread of the virus within the host.


Figure 2. Structure of HIV a) Structural view of the viral envelope (0.1 µm in diameter). Embedded in the envelope is a protein that consists of an outer protruding cap (gp120) and a stem (gp41) (10) . b) Close up view of the structure of gp41(11). c) X-ray crystallography image of gp120. Here, two views of the gp120 show the total topography of the glycoprotein. In white and yellow are the glycosylation sites(12). (click for larger image)

Glycosylation of gp160 is extensive. The gp120 portion of gp160 has approximately 24 potential N-linked glycosylation sites (Asn-X-Thr/Ser) (7) and the weight of the glycans make up over 50% of the molecular mass of the gp120 glycoprotein(6, 8) (figure 2). In contrast, the gp41 ectodomain is poorly glycosylated, with only four to five potential sites for N-linked glycosylation (6). No evidence for O-linked glycosylation of the envelope proteins has been observed (9).

To study N-linked oligosaccharide structures on the HIV gp120, Mizouchi et al. used chronically HIV-infected lymphoblastoid (H9) cells and sequenced the glycans after they were released from proteins by hydrazinolosis. The conclusion from these studies is that the number of possible oligosaccharide structures present on gp120 outnumbers the 24 potential glycosylation sites present on the glycoprotein. Therefore, alternative structures occur on some of the glycosylation sites and numerous glycosylation variants of the gp120 are produced even in one cell line (13). The varied glycosylation is beneficial for the virus because it allows the virus to escape the immune system's response to the original invading virus by always appearing to the immune system as a new pathogen.

The importance of glycosylation sites in the infectivity of the virus has been investigated in both gp120 and gp41. This has been achieved by mutating the DNA sequences that code for the portion of glycoprotein important in its interaction with CD4 molecules on host cells. The results indicate that multiple mutations of glycosylation sites are required to alter the function of the gp120/gp41 complex (6). For example, the complete removal of glycan clusters from gp41 accomplished by altering the asparagines of the N-linked glycosylation sequons in positions 621, 630, and 642 to serine residues, abrogated the viral ability to carry out fusion completely (6). Mutated gp41 co-migrated with deglycosylated forms of wild-type gp41 on electrophoretic gels (14), suggesting that the deletion of the glycosylation sites contributes to the lack of glycosylation of the mutated proteins. These studies prove that for a successful HIV infection, the viral envelope proteins must be properly glycosylated so that the folding of the glycoproteins is correct and so that gp120 can interact with a CD4 host membrane receptor and allow the subsequent entry of the virus into the host cell. If gp41 is incompletely glycosylated, membrane fusion between the viral envelope and the host plasma membrane cannot occur (14). This finding may be useful in the construction of drugs that focus on the alteration of the oligosaccharide part of the HIV glycoprotein structure during its processing, decreasing the number of HIV particles that are capable of infecting CD4+ cells.

Additional studies involving mutated forms of the gp41 portion of gp160 have demonstrated that the transport of mutated forms of gp160 from the cis to the medial Golgi is slow and the transport to the trans Golgi is impaired. Therefore, since cleavage occurs in the trans part of the Golgi, cleavage reactions were severely impaired in the mutated gp160 at the gp120-gp41 junction. Gp160 lacking gp41 carbohydrates demonstrated that proteins lacking glycans are arrested in the Golgi following biosynthesis. Therefore, the glycan components of the glycoprotein are important in the intracellular transport and processing of the gp160 (5).

N-linked carbohydrates within the epitope portion of the HIV gp120 are important in the recognition of the HIV CD4 receptor. Support for this comes from a study by Botarelli et al. who immunized T-cells with a recombinant non-glycosylated gp120. When the immunized cells were presented with a wild-type glycosylated form of gp120, the cells' T-cell receptor did not recognize the glycosylated gp120 and did not develop a rapid immune response to it. To investigate the reason for the slow recognition of glycosylated glycoprotein, the epitope portion of the gp120 bound to the T-cell was mapped. It was found that the epitope region of gp120 contained two asparagines residues which are sites for N-linked glycosylation (8). Therefore, the carbohydrates on the wild-type gp120 prevented epitope recognition by the immunized cells that had a T-cell receptor that was specific for the glycosylated form of gp120 used in the immunization.

THE USE OF OLIGOSACCHARIDE PROCESSING INHIBITORS TO STUDY HIV GLYCOPROTEIN STRUCTURE
Previous studies have relied on utilizing glycobiology tools to better understand the mechanism of HIV infectivity and the source of its immunogenicity. For example, N-linked glycosylation processing inhibitors can be used for studying the importance of protein glycosylation in HIV pathogenesis, replication, target-cell infectivity, and syncytium formation (15). The effect of oligosaccharide processing inhibitors can be determined by electrophoretic mobility. Proteins with the lowest molecular weight travel the furthest on the electrophoretic gel. In studies where gp120 is processed in the presence of glucosidase inhibitors (castanospermine or 1-deoxynojirimycin), there is a decrease in gp120 mobility. However, when mannosidase inhibitors (1-deoxymannojirimycin or swainsonine) are present, glycoprotein mobility is increased (15). These results suggest that the final glycan products found on gp120 are smaller that the glycan products seen when glucosidase inhibitors are added and bigger than high mannose chains that are seen when mannosidase inhibitors are introduced into the media.

Inhibitors of oligosaccharide processing can also be used to study the steps in the processing of envelope glycoproteins in cells. For example, treating chronically HIV-infected cells with tunicamycin (an inhibitor that completely abrogates the addition of glycans to N-linked glcosylation sites of the protein) severely inhibits the glycosylation of envelope proteins. Treatment with deoxynojirimycin, an inhibitor of glucosidase I in the rough ER, inhibits proteolytic cleavage of gp160 (9, 15). Inhibitors of mannosidase I and mannosidase II (deoxymannojirimycin and swainsonine respectively) allow for the processing of the gp160 to gp120 and gp41. However, the virions made by cells treated with mannosidase inhibitors are significantly less infectious than virions synthesized by untreated cells. This suggests that proteolytic cleavage of gp160 takes place in infected cells when the glycoprotein has mannose-rich oligosaccharide structures. However, for the release of infections virions, the trimming of glucose residues and the primary trimming of mannose are both necessary (15). Therefore, oligosaccharide processing inhibitors can be used to decrease the infectivity of new HIV virions since the viral particles would contain improperly glycosylated glycoproteins.

HIV THERAPY BASED ON CURRENT KNOWLEDGE OF GLYCOBIOLOGY
HIV is a very complicated pathogen due to its ability to mutate at a fast rate. It introduces difficulty for the immune system, which continually sees the virus as foreign, posing challenges to treatment. Research in the field of HIV has contributed to a wealth of therapeutic options. Current treatments can be classified into the following classes: (i) nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) (i.e. zidovudine (AZT), (ii) non-nucleoside reverse transcriptase inhibitors (NNRTIs) (i.e. nevirapine); and (iii) protease inhibitors (PIs) (i.e. saquinavir). Other steps in the HIV replicative cycle that are potential targets for chemotherapeutic intervention include viral adsorption, viral entry, virus-cell fusion, proviral DNA integration, and viral mRNA transcription (16). Additionally, the expanding field of glycobiology proposes additional treatment options.

HIV DRUG THERAPY BASED ON INHIBITORS OF OLIGOSACCHARIDE PROCESSING
Application of carbohydrate analogues such as tunicamycin (inhibitor of the synthesis of the dolichol-linked oligosaccharide precursor), castanospermine and 1-deoxynojirimycin (DMN, glucosidase inhibitor), as well as 1-deoxymannojirimycin (mannosidase I inhibitor), has been shown to attenuate the HIV-1 infectivity invitro. These inhibitors oligosaccharide processing are ineffective in preventing the formation of a cytoplasmic continuity (syncytia) between two cells when gp120/gp41 complexes are targeted to the infected cell's plasma membrane and bind to CD4 receptors of neighboring CD4+ cells (15). However, N-glycosylation is critical in HIV pathogenesis at the level of viral binding and fusion to uninfected CD4+ cells.


Figure 3. Formulas of N-linked oligomannose molecules including Man(6) and Man(9). Isomers of Man(7) and Man(8) can be deduced by adding mannose termini to the Man(6) structure (18). (click for larger image)

The gp120/gp41 HIV envelope complex must be folded properly for it to be pathogenic. Proper folding is mediated by oligosaccharides on the gp120/gp41 complex. Therefore, glycosylation inhibitors can be applied to alter the fusogenicity of the gp120/gp41 complex since improperly folded proteins cannot induce fusion. For example, DNM inhibits the cellular a-glucosidase I-II activity, blocking the trimming of the glycan precursor at the level of the ER (i.e. the cleavage of three glucose residues from Glc(3)Man(9)GlcNAc(2) precursor glycan, see figure 3). In the presence of DNM, glycoproteins with abnormally glycosylated oligomannosidic moieties are produced. Treatment of HIV-infected lymphocyte cultures with DNM inhibited the spread of the virus (16). This was possible because although the gp120/pg41 complex synthesized in the presence of DNM could bind CD4 receptors on human lymphocytes, it was not able to induce membrane fusion. N-butyl DNM, an alkyl substituted variant of DNM, has shown antiviral effects at doses with no reported cell cytotoxicity (3). Phase I clinical trials on n-butyl DNM in twenty-nine patients, however, showed no significant trends in CD4+ cell counts, and Grade II elevations in liver function, leucopenia, and neutropenia were observed in some patients, calling the study to a halt before the dose-escalading tolerance trial could be completed (17).

HIV DRUG THERAPY BASED ON OLIGOSACCHARIDE STRUCTURE
If the glycan structure composition of the HIV glycoprotein gp120 can be determined, the interaction between gp120 and CD4+ cell can become a potentially useful target in HIV therapy. To study the gp120 epitopes involved in interaction with the CD4 receptor, three N-linked glycosylation sites on the gp120 in the vicinity of the epitope recognized by the host T-cell were removed (7). Mutant gp120 with one, two, and three N-linked glycans missing were analyzed. The conclusions made were that two of the three glycosylation sites were heterogeneous in the structural composition of glycans. However, the site located next to the T-cell epitope had a large, high mannose structure with more than 11 mannose units and it covered a large part of the surface of gp120 (7). The finding that high mannose glycan structure present in the epitope of the gp120 is recognized by the T-cell has allowed for the creation of cyanovirin-N (CV-N), a drug that inactivates diverse strains of HIV at the level of cell fusion by virtue of its interaction with specific N-linked oligosaccharides on gp120.

CV-N is a 11kDa monomeric protein derived from cyanobacteria. CV-N binds to glycosylated gp120 but not to unglycosylated gp120. When the ligand which binds to CV-N was studied by examining the binding of gp120 to a CV-N affinity column, only Man-8 and Man-9 glycoforms (figure 3) were preferentially retained on the affinity column. Studies have found that adding free Man-8 and Man-9 oligosaccharides partially inhibited binding of CV-N to gp120 (2). When a mixture of carbohydrates that structurally represent N-linked carbohydrates found on the gp120 was added, it was found that CV-N specifically recognizes with nanomolar affinity Man(9)GlcNAc(2) and Man(8)GlcNAc(2) (18). Viruses sensitive to CV-N exhibit an abundant exposure of high mannose oligosaccharides on their surfaces (figure 3) (19). All of the above findings suggest that the part of gp120 which is recognized by CV-N is the high mannose glycan portion of the glycosylated gp120.

CV-N is unlike other lectins that have been reported to have anti-HIV activity mediated through interactions with gp120. Classical carbohydrate-interacting lectins, such as concanavalin A and wheat-germ agglutinin, associate with gp120 in a monosaccharide-specific manner and are inhibited by the presence of exogenous monosaccharides. However, CV-N/gp120 interaction is not inhibited by high concentrations (i.e. 10,000-fold excess) of monosaccharides (19). This observation suggests that CV-N/gp120 interactions are defined by a more complex oligosaccharide-specific binding. In addition, the carbohydrate structures recognized by CV-N are rare in normal human tissue. Therefore, CV-N is a potential oligosaccharide-specific therapeutic agent for the treatment of HIV as well as other pathogens with high mannose ligands on their outer surface.

Besides CV-N, other drugs have also been designed based on the principle that high mannose chains are essential for HIV infection. For example, Pradmicin A, an antifungal antibiotic isolated from Actnomdura hibisca, and its derivative, BMY-28864, have the ability to inhibit HIV infection in vitro (20). Similar to the CV-N studies, the inhibitory effects of these drugs were suppressed by the addition of high mannose type oligosaccharides. Therefore, targeting oligosaccharide chains of the envelope glycoprotein is a possible way to block HIV infection.

THERAPY BASED ON OLIGOSACCHARIDE STRUCTURE TO CONSTRUCT A POTENT HIV VACCINE
An effective way of combating HIV is via vaccination. To construct a successful vaccine, the HIV gp120 antigen needs to be recognized by the helper T-cell. In order to generate vaccines that will induce a strong immune response, the HIV gp120 antigen needs to be made more foreign. In one vaccination study, gp120 was made more immunogenic by neuraminidase treatment, which removed terminal salicylic acids from the carbohydrate side-chains of the glycoprotein, producing an asialoglycoprotein (21). Neuraminidase treatment exposed terminal galactose residues so that they could be recognized by galactose receptors on antigen presenting cells. Evidence for the use of galactose receptors became apparent when galactose is added to cultures of antigen presenting cells. Extrinsic galactose, when added, competes with asialo-glycoproteins for binding to galactose receptors. Thus, the antigenicity of the HIV glycoprotein gp120 can be enhanced by exposing galactose residues on the gp120 since these glycoproteins can be internalized by antigen presenting cells containing galactose receptor. Uptake of the proteins with terminal galactose units results in a quicker immune response because asialoglycoproteins are more quickly recognized as foreign (4).

When constructing an HIV vaccine, it is therefore worthy to explore glycobiology to produce a vaccine with a more immunogenic antigen. If a more antigenic form of gp120 could be administered to a patient, the patient may be able to mount a stronger immune response against the HIV.

INHIBITION OF HIV GP120 BINDING TO CD4 MOLECULES BY NATURAL GLYCOSAMINOGLYCANS
It is a fact that HIV is spread via body fluids. Past studies of HIV inhibitors have suggested that the presence of inhibitory macromolecules in the body fluids that do not transmit the virus could be responsible for the inability for these fluids to function as a vehicle for HIV transmission. Other body fluids that serve as a route of HIV transmission, such as breast milk, have been found to contain compounds that could limit the rate of postnatal vertical transmission for HIV in breast-fed infants of HIV-infected mothers. The proposed reason for the lack of viral transmission by some body fluids is the presence of glycosaminoglycans (22).

Previous studies have proven that non-antibody glycoconjugates from human milk can inhibit the binding of many pathogenic microorganisms. According to Newburg et al, glycosaminoglycans found in human milk represent a novel class of naturally occurring molecules that are capable of inhibiting viral binding to host CD4 receptors (22).

Glycosaminoglycans are heteropolysaccharides; they are linear polymers made of repeating disaccharide units. One of the monosaccharides is always N-acetylglucosamine or N-acetylgalactosamine, and the other is a uronic acid such as glucuronic acid. One or more of the hydroxy groups of the amino sugars can be sulfonated (4), producing glycosaminoglycans such as dermatan sulfate, heparan sulfate, or chondroitin sulfate.

Sulfonated molecules, including sulfonated glycosaminoglycans (GAG), have been shown to inhibit the binding and replication of HIV. The classes of GAG found in human milk include heparin, heparan sulfate, chondroitin sulfate, and dermatan sulfate .

To determine the types of glycosaminoglycans that are most important in the inhibition of HIV replication, Newburg et al analyzed milk from thirty women. To test for the inhibitory nature of each glycosaminoglycan from human milk, isolated GAGs, CD4 and gp120 molecules were mixed and allowed to interact. Glycosaminoglycan cleaving enzymes (lyases) were then added to cleave the linear glycosaminoglycan polymeric backbone and the reaction mixtures were tested for the loss of inhibitory action. The greatest loss in the inhibition of the gp120 and CD4 interaction was seen when chondroitinase AC was added (22). This suggests that the specific glycosaminoglycan component of human milk responsible for the inhibition of HIV binding and replication is chondroitin sulfate.

Further research on sulfated glycosaminoglycans could provide valuable treatment methodologies to terminate the HIV epidemic. Based on the studies of sulfated glycosaminoglycans, synthetic sulfonated polymers that share the gross features of GAGs could be synthesized such that they contain anti-HIV activity.

CONCLUSION
New antiretroviral therapies prolong the lifespan of people infected with HIV. Correspondingly, the number of AIDS-related deaths has declined. Despite the availability of drug therapies, HIV is still a challenge. Glycobiology is a field with foreseeable therapeutic alternatives for HIV. Glycobiology-related treatments such as inhibitors of oligosaccharide processing, treatments based on blocking the reactive sites of gp120, vaccinations, and natural methods using glycosaminoglycans to combat the deadly virus, further add to the list of potential antiretroviral treatments. Combining these treatments with current antiretroviral methods could result in synergistic anti-HIV effects. However, further testing of the glycobiology-related therapies is needed to determine the non-specific effects on other cellular processes and before the therapies can be made available for the HIV-affected population.

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Aleksandra M. Leligdowicz is a second year medical student at the McGill University Faculty of Medicine. In 2000, she completed a BSc degree in molecular biology at the University of Manitoba and Deakin University, Australia. In October 2004, she will begin research in Immunology and Epidimiology of HIV-2 at the Institute of Molecular Medicine at Oxford University as a Rhodes Scholar.

The Vegetative State: A Review of Etiology and Prognostic Factors

Rachel Davison*
* To whom correspondence should be addressed: Framlington Place University of Newcastle upon Tyne Medical School Newcastle upon Tyne England NE1 7RU
E-mail: R.E.Davison@ncl.ac.uk

INTRODUCTION

While the advent of cardiopulmonary resuscitation during the 1960s was a breakthrough for medical science, some survivors remained in a limbo state of 'waking unconsciousness'. It was Jennett and Plum (1), in 1972, who termed this condition the persistent vegetative state (PVS) and described the vegetative state (VS) as:

The absence of any adaptive response to the external environment, the absence of any evidence of a functioning mind which is either receiving or projecting information in a patient who has long periods of wakefulness

In short, the VS is a clinical condition of unawareness of self and environment but with retained wakefulness. Efforts to predict the outcome of patients in a vegetative state began around this time due to the concern that large numbers of patients surviving in a VS would be costly and use resources that could be more effectively spent elsewhere (2).

The incidence of the VS is unknown, partly because of the rarity of the condition and partly because of the lack of accepted universal diagnostic criteria. Estimates range from 0.4-1.1/100,000 people throughout the world (3,4). Moreover, until 10 years ago, the VS was not a codable diagnosis in either the International Classification of Diseases or most health agencies. Studies at the time suggested that the prevalence in the United States alone was around 10,000-25,000 adults and 4000-10,000 children (3,4).

The VS causes distress to family and friends and creates difficulties for the doctors involved. To the non-medically trained, a patient in the VS may appear to be alive and functioning; for example they may seem to smile or turn to sound despite the fact that by definition the patient is not aware. This may cause conflict between the family of the patient and the doctors who are trying to explain what the VS is and the likely outcome. This may be further complicated by the fact that prediction of recovery is an inexact science. Doctors cannot give a definite answer and the family may prefer to simply watch and wait while they feel there is still hope. To provide the care that the patient needs, the doctor must have the ability to predict the best possible outcome and also to recognize when it would be more humane to withdraw medical interventions and let nature take its course. The ethical dilemmas regarding quality of life and best intentions are many and complex, and as such, are beyond the scope of this review. This paper reviews the research investigating the vegetative state (VS), in terms of its etiology and prognostic factors that may be indicative of the outcome for patients in the VS. Furthermore, this review identifies the methods by which prediction of prognosis may be possible.

METHODS

A literature search was conducted in Medline (from 1993) and EMBASE (from 1980) to identify suitable papers. The databases limited the choice of date selection; the selected dates were chosen to identify only the most up to date work. The keywords used were PVS, VS, (persistent) vegetative state, (permanent) vegetative state, children and (persistent, permanent) vegetative state, coma or life support care, combined with prognosis or prediction. The aim in the use of the keywords was to detect all relevant articles in the given time period. In all, about one hundred articles were identified but not all of the papers were applicable to the aims of this review. The reference lists of relevant articles were scrutinised to detect any additional studies that had not been already identified. The articles most applicable to the subject were selected and the information within them collated, with careful attention being paid to the methods of the systematic reviews and critical analysis of the original studies that warranted inclusion.

CONSCIOUSNESS AND THE VEGETATIVE STATE

There are two components to consciousness (3):

1. WAKEFULNESS
Clinical work indicates that the midline structures in the upper pons, midbrain and thalamus (the reticular activating system or RAS) are necessary for wakefulness. They are activated by arousal and influence the cerebral cortex directly. Wakefulness is essentially not being asleep; therefore it comprises acts that one would not do while asleep such as opening one's eyes and looking around.

2. AWARENESS

The cerebral cortex and its projections to the major subcortical nuclei are considered to be the root of awareness with its content being the mass of information that it processes from the external environment. Awareness comprises behaviours that indicate that a person comprehends the outside environment (e.g.: communication and understanding).

As one of the main links between the two component areas, the thalamus is crucial to the preservation of consciousness (3,5). Unconsciousness therefore implies global (or total) unawareness. Awareness requires wakefulness, but wakefulness can be present without awareness. In the comatose state both awareness and wakefulness are lacking, while in the VS wakefulness is preserved and awareness is not (3).

If the VS lasts for a month, it is termed continuing or persistent VS (3,6). The consensus is that the terminology changes to that of permanent VS when the condition is deemed to be irreversible, no recovery seems possible and further treatment is considered futile. This decision is usually taken once a year has elapsed in traumatic aetiologies and after three months in non-traumatic cases (4-6). However, as with all clinical judgements, it is based on probabilities. While persistent VS is a diagnosis, permanent VS is a prognosis. In practise, the terms are commonly used interchangeably and the acronym PVS is used for both conditions (3,5-7). Therefore, the acronym VS will be used in this review, and distinguished where necessary if specific reference is being made to a persistent or permanent state. Recovery from the VS is classified by the Glasgow Outcome Scale (GOS, see Table 1), with a good recovery indicated by a GOS score of 4 or 5.

DIAGNOSING THE VS

Before a diagnosis of the VS can be made, an established cause must be found and all reversible factors that may be contributing (e.g. metabolic disturbances, sedatives, anaesthetics or neuromuscular blocking drugs) eliminated (5).

The Multi-Society Task Force (MSTF) on persistent VS defined the following diagnostic criteria, which are widely acknowledged by the medical community (3):

1. No evidence of awareness of self or environment and an inability to interact with others.
2. No evidence of sustained, reproducible, purposeful or voluntary behavioural responses to visual, auditory, tactile or noxious stimuli.
3. No evidence of language comprehension or expression.
4. Intermittent wakefulness manifested by the presence of sleep-wake cycles.
5. Sufficiently preserved hypothalamic and brainstem autonomic functions to permit survival with medical and nursing care.
6. Bowel and bladder incontinence.
7. Variably preserved cranial nerve reflexes (pupillary, oculocephalic, corneal, vestibulo-ocular and gag) and spinal reflexes.

Patients in the VS are usually not immobile. There may be apparent semi-coordinated movements such as scratching, moving hands towards noxious stimuli (such as during mouth care) and reflex grasping. There is flexor withdrawal after a delay; inflicting a painful stimulus such as pressing the supraorbital ridge causes a stereotyped flexing of limbs as during assessment of Glasgow Coma Scale status (GCS, see Table 2). Movements are slow, dystonic and obviously abnormal. Neck movements may provoke reflex postural alterations. There may be chewing and grinding of teeth, and food and liquid placed into mouth may be swallowed. Patients in the VS may retain the response of turning their head or moving their eyes to sound, but eye fixation and tracking is not demonstrated. They may smile or appear to shed tears, and may grunt or groan (vocalise) but never speak (verbalise) (3,5-7). Although this may be disturbing to family and carers, it should be remembered that by definition patients in the VS have no awareness, and therefore it is the opinion of experts on the subject that they cannot feel any pain (4-7).

DIAGNOSTIC PROBLEMS

Determining cognitive awareness in another person can only ever be an educated guess as there are no tests that can confirm the presence or absence of inner awareness (3,6-8). Repeated assessment is therefore essential, especially if there is some doubt over whether the behavioural patterns necessary for diagnosis of the VS are present.

The diagnosis of the VS is difficult to make in infants younger than three months because they have a limited capacity to show higher cognitive functions; the differentiation between voluntary and involuntary responses may also be unreliable until this age. The concept of the VS cannot be applied to preterm infants because of developmental immaturity and the lack of consistently recognisable sleep-wake cycles. The exception is infants born with severe developmental malformations such as anencephaly and hydranencephaly where there is minimal or no cerebral cortex and therefore no awareness. These infants are categorised as being in a VS congenitally (3,8).

PATHOPHYSIOLOGY AND ETIOLOGY OF THE VS

The VS is largely characterised by a functioning brainstem with no input from the cerebral hemispheres due to either disconnection or damage of two main types (3):

1. Acute, e.g. head injury, hypoxic-ischaemic damage following cardiopulmonary arrest, metabolic disturbances.
2. Chronic, e.g. degenerative processes such as Alzheimer's disease, congenital defects such as anencephaly.
The VS can be caused by a vast array of conditions; with any insult to the body that causes damage to the cerebral cortex being a potential cause of the VS. Three main patterns of brain pathology are seen at autopsy (3,9):
1.Diffuse Axonal Injury (DAI), which is extensive subcortical axonal injury that virtually isolates the cortex from other parts of the brain. It is most commonly due to the shearing forces in trauma or sometimes to hypoxic-ischaemic insults and is the most common pathological feature seen.
2.Extensive laminar necrosis is due to acute global cerebral ischaemia or hypoxia. Multifocal or diffuse necrosis is seen with almost invariable involvement of the hippocampus, hypothalamus and brainstem.
3.Relatively selective thalamic necrosis is an uncommon observation that may follow acute global ischaemia. Specific anatomical boundaries are not well described.

Mixtures of all three lesions are commonly seen with additional focal lesions depending on the precipitant of the insult. Acutely inflicted hypoxic-ischaemic insults and shearing forces are therefore shown to have a devastating impact on the brain, as these are the most frequently seen pathologies at autopsy. There have also been reports of rare isolated lesions of the brainstem or hypothalamus alone causing the VS but these are not well studied (3).

The diagnosis of permanent VS is made by identification of cause, fulfilling diagnostic criteria and lasting for at least a set amount of time (three months for non-traumatic aetiologies, one year for traumatic) (6). Therefore, there are two aspects to the prediction of whether the VS will be permanent or whether recovery is possible - the etiology of brain insult and the duration of the VS to date.

1. ETIOLOGY OF BRAIN INSULT

Outcome of coma is directly related to its cause (2), which can be separated into two aetiologies: traumatic (e.g. road traffic accidents, falls) and non-traumatic (e.g. cardiac or pulmonary arrest, anoxic- ischaemic, metabolic).

Traumatic
Traumatic etiology of brain injury is the better of the two categories. The MSTF collated data from several similar studies of traumatic brain injury giving information on outcome for 434 patients (4). Recovery of consciousness varied with time, with 88% of those who recovered (46% of all patients studied) doing so within the first six months, and reaching 99.98% at one year. After this, recovery was rare. Only seven of the 434 (0.02%) recovered after this time, between one and three years after the injury. Five of them remained severely disabled, one was moderately disabled and the status of the seventh was undeterminable. Five of the seven were under 30 years, suggesting that age is another important confounding factor in the prediction of prognosis. Accordingly, those under 40 had a greater chance of recovering within three months without severe disability (4). Children in traumatic coma generally have a better prognosis than adults in a similar condition, although recovery of function is comparable (4,10). Data from several similar studies showed that 62% of children had regained consciousness at one year following injury, compared to 52% of adults (4,8).

Traumatic brain injury is also associated with a poor chance of a good functional recovery as described by the GOS (see Table 1). Using the collated data (4), from the 434 patients in a VS, of the 52% who had recovered consciousness by one year, 28% had severe disability, 17% moderate disability and only 7% had made a good recovery. Of those who made a good recovery over half showed signs of improvement within the first three months and all within six months. Those who recovered consciousness but remained disabled all began to show signs of improvement three to six months after the brain injury. This indicates that a later recovery was almost always associated with severe disability (4).

Non-traumatic
Non-traumatic etiology carries a much poorer prognosis than its traumatic counterpart. Collecting data on the outcome of 169 patients in a systematic analysis (4) showed 85% or more died within the first month or remained in a VS. Of the remaining 15% who recovered consciousness (11% of which were within the first three months, and the other 4% by six months), only one patient (0.6%) made a good recovery. One year after the injury, 32% remained in persistent VS and 53% had died. A larger study of 500 patients by Levy et al. (11) found that at one year, 73% were dead or in a VS and of the remaining 27%, 11% were severely disabled, 4% were moderately disabled and 12% (2.4% of the total) had made a good recovery. They agreed that most improvement occurred within the first month and that the longer the VS persisted, the smaller the chance of recovery.

There is very little evidence to suggest that there is a consistent relationship between age and prognosis in non-traumatic coma, mainly due to a lack of data (4,11). Shewmon (10) suggests that in children (under 16 years), the outcome is either full neurological recovery or remaining in a VS with very few outcomes in-between, with ratios ranging from 50:50 to 70:30 in favour of intact functioning. The commonest cause of non-traumatic coma in children is near drowning, and Shewmon (10) postulates that childrens' brains are more protected against anoxic-ischaemic damage due to their body temperature falling faster because of their smaller size. No evidence was found to confirm this, but it appears that children are less susceptible to anoxic or ischaemic injury and have a greater potential for neurological recovery than adults. As a result of this, the observation period in children is often allowed to be longer than the standard three months that adults are given before their VS is declared permanent (8).

Specific etiology

The underlying cause of the coma has been shown to relate to outcome in many studies (2,4,11). Metabolic and diffuse disorders carried a better prognosis than hypoxic-ischaemic causes (2,4,11). Cerebrovascular disease such as subarachnoid haemorrhage or stroke and other disorders causing structural brain damage carried the worst prognosis of all (2,4,11). The incidence of a VS was also observed to be higher following anoxic-ischaemic injury; for 20% of the patients studied this was the best outcome that they ever achieved (GOS 2) (2,11). Drug overdose also carried a favourable prognosis due to the reversibility of their effects, despite a bleak outlook at initial assessment that was considered to be due to depressive effects on the brainstem (2).

2. DEPTH AND DURATION OF COMA

The longer the patient remains in a coma, the poorer their chance of recovery and the greater the chance that they will enter a VS (2,11). A week is often used as a cut-off point; by that time the chance of a moderate or good recovery is only 6-7% and almost half of those who are still unconscious will be in a VS (2,11). By convention, one month after the brain injury the patient is in a persistent VS (6) and the chance of recovery is small. However, studies by Andrews (12) and Dubroja and colleagues (13) have shown that recovery is possible beyond this time. In one case, recovery began three years after the initial insult (13). Whilst this shows that higher functioning can be regained beyond predictions, it should be noted that these studies only looked at small groups and that no one in them made a complete recovery. There have been other documented cases of very late recovery (4) but in all of them the level of function was far from independent. Such outcomes may be undesirable to some, but to others this may be an acceptable quality of life.

PROBLEMS WITH PROGNOSIS ACCURACY

Predicting prognosis in the VS is an inexact science and there are faults that are common to all the studies considered. The rarity of the condition itself (often resulting in studies with small sample sizes) coupled with the inability to conduct true prospective studies has led to many of the published papers lacking sufficient power to demonstrate their value. There are inevitable confounding factors in the studies, such as patients dying of non-neurological disease during the studies and the fact that in many studies no distinction is made between the VS and death, or the VS is combined with severe disability as a non-acceptable outcome. The studies are also limited by ethical considerations, as ideally patients would be kept alive indefinitely. Doctors are bound to act in their patient's best interests if the patient cannot express their own views. It can be argued, that keeping a patient in the VS simply for means of a scientific study is not in the patient's best interests and therefore is unethical. Short follow-up times are also commonplace. In addition, the studies face the problem of self-fulfilling prophecies [i.e. if a patient is predicted to have a poor outcome, it has been shown that therapies are often less aggressive and the next of kin are more likely to ask for withdrawal of medical support (2)].

Improvement on previous studies is difficult. Despite recognised problems with their methodology, it is not easy to correct them. We cannot alter the small sample sizes or the lack of prospective studies because of the rarity of the condition itself and because the ethical problems remain the same. This will most likely continue to be a problem unless a large scale, multicentre trial is organised with the cooperation of a large number of major neurological centres around the world.

Age

As has already been implied, the age of the patient may hold prognostic significance. Shewmon (10) states that children (under 16 years) have a better chance of recovering consciousness, although their functional recovery is often equivalent to that of adults. He asserts that the mortality rate from severe head injury declines with increasing age in childhood, reaching a trough at 14-15 years and then rising with age throughout adulthood. However, others have shown that outcomes worsen with rising age, even in childhood, which is possibly related to age specific differences in types of trauma (e.g. falls are more common in young children and road traffic accidents in older children) (4). Shewmon (10) also suggests that children may continue to recover long after adults have reached a plateau, possibly due to their retained potential for further growth and development. They may also be more likely to be offered long-term life support than adults because of the fact that people see them as more 'worthwhile' of the use of resources (4). Some may believe that death is preferable to survival with a severe disability, but Shewmon (10) insists that children and their families adapt better to physical and mental disabilities than most adults do. However, many of these views were difficult to substantiate as children and neonates tend to be excluded from studies on the basis that an accurate and consistent diagnosis of a VS is difficult to make, especially in the youngest children due to a limited capacity to show higher cognitive function (3,14). This is a complex issue with a small amount of applicable research literature available, making it difficult to conclude if any advantage is offered by a younger age.

At the other extreme of life, superficially it would appear that there is a relationship between increasing age and mortality in the VS (15), but after adjustment for the severity of the illness and co-morbidity it no longer appears to exist (11,15). Although the age of the patient does not seem to directly contribute to the prediction of outcome from the VS, it may act as a substitute for other important and otherwise unmeasurable cofactors that do (15) such as pre-existing co-morbidity (e.g. cardiovascular disease) and reduced physiological reserve. Therefore, age should still be taken into account in prediction, but not as the deciding factor in where rationing of resources may be an issue and could lead to claims of ageism.

RECOVERY FROM THE VS

There are two dimensions of recovery from the VS (4):

1. Recovery of consciousness
Verified by consistent evidence of self and environment, interaction with others and voluntary behavioural responses.
2. Recovery of function
Characterised by communication, ability to learn and perform adaptive tasks, mobility, self-care and participation in recreational or vocational activities.

Recovery of consciousness may occur without recovery of function, but the converse is never true (4).

The VS may be a transient stage in the recovery from coma or it may persist until death (6). The average duration of survival is 2-5 years and mortality for adults has been quoted at 82% at three years and 95% at five years (3,4). Studies have shown that the duration of survival is similar between children and adults (5-7 years), but in infants under one year it is much shorter, with estimates at a maximum of four years (4,8). There have been cases reported of patients being alive in the VS many years after this (48 years is the longest known case) (7), but the probability of prolonged survival in the VS (i.e. longer than fifteen years) has been estimated at less than 1 in 15,000 to 75,000 (4). The cause of death in the VS is most commonly infection (usually of the respiratory or urinary tract) or generalised systemic failure. Underlying comorbidity (such as ischaemic heart disease) and other unknown causes also claim a small proportion of patients but exact figure are not recorded (4).

OTHER ANCILLIARY TESTS

Other tests alone can neither diagnose nor predict if the VS will be permanent. However, when used in conjunction with the clinical examination, they can provide useful supportive information (3).

Imaging Studies (Neuroimaging)

There are no established patterns seen on neuroimaging that have been proven to predict outcome (3,9) and Bates (2) feels that their value in prediction is no better than that of the basic clinical signs. Neuroimaging methods often document lesions so severe and diffuse that awareness is highly improbable, given our current understanding of the anatomy and physiology of the brain. Several studies have documented that patients with serial abnormal scans do not recover consciousness and have progressive brain atrophy (3,7).

Magnetic resonance imaging is proven to be more sensitive than computerized tomography (CT) for detection of traumatic and ischaemic cerebral lesions (9). Kampfl et al. (9) found that although multiple lesions were commonly seen in the VS, additional injuries to specific parts of the brain were of particular importance to its persistence. Patients with lesions of the corpus callosum and dorsolateral upper brainstem had a 214-fold and 7-fold higher probability, respectively, for non-recovery (i.e. VS becoming permanent). However, the study sample size was too small to definitively prove these findings (9,16).

Cerebral Metabolic and Blood Flow Studies

Functional assessment of brain activity has been investigated as an aid to prediction. There is evidence that cerebral blood flow (CBF) is decreased in patients in established VS, with estimates at 10-50% of normal (3,9,17). However, it is accepted that measurement of CBF in the acute phase is of no prognostic significance (3) and it does not reliably predict if recovery is possible.

Cerebral metabolic activity has also been implicated in the prediction of outcome. Using positron emission tomography, a collection of studies (3,9) demonstrated a decreased cerebral metabolic rate of only 40-60% of normal in 20% of adults in permanent VS (3). Unfortunately, the limited power of these studies (due to small sample sizes) means that as yet, there is insufficient evidence to incorporate cerebral metabolic rates or CBF studies into routine practise (3,7,9).

Electrophysiology: EEGs and Evoked Potentials

The transition from coma to the vegetative state is not characterised by obvious EEG changes; it is a clinical diagnosis (2). Most patients in persistent VS show diffuse generalised polymorphic delta or theta activity, which is un-reactive to sensory stimuli. In other patients, alpha activity is the most obvious EEG feature, or some low background activity is all that can be detected (2,3). Epileptiform and seizure activity is rare in VS.

A similar pattern is seen in children although the EEG activity may be of a lower voltage and more discontinuous (3). Before three months, the EEG pattern is termed 'neonatal' and is very different to the EEG seen after this time ('mature' EEG) and throughout the rest of adulthood. This transition is termed encephalisation and is when the child's brain changes from mainly reflex subcortical functioning to cortically mediated cognition. Once this change has taken place, the same prognostic patterns apply to children as to adults, but before this, little information can be gained from EEG due to a lack of research on the topic (7,10).

Recovery from the VS may be seen on EEG recordings as decreasing delta and theta activity and reappearance of a reactive alpha rhythm. However, this pattern has also been seen in patients who clinically remain in the VS, suggesting that it is not always predictive of recovery (3).

Sedative, anticonvulsant or anaesthetic drugs may cause depression of brain activity and lead to misdiagnosis on EEG (11). This again emphasises the need to eliminate all reversible causes of coma and to repeatedly assess the patient. However, the technical problems of performing such a measure in a busy intensive care ward with numerous potential sources of electrical interference can cause much of its practical value to be lost (2).

The most sensitive and reliable form of evoked potentials (EPs) in both adults and children are somatosensory evoked potentials (SSEPs) (3). Many studies (2,3,14,18,19) have produced consistent results as to their value but their clinical use is not yet widely adopted due to the belief that it is difficult to obtain accurate and reliable results in the intensive care setting (2,18). The advantage SSEPs have over EEGs is that sedating drugs minimally affect them (18). Many of the studies into SSEPs are not fully blinded, therefore any predictions of death or disability have been criticised as to being, to some extent, a self-fulfilling prophecy. Sleigh et al. (18) carried out a fully blinded study to counteract these claims and to show that SSEPs could be recorded in an ICU where dedicated neurophysiological personnel are not present. They found that bilaterally normal SSEPs were associated with a good outcome (i.e. recovery from the VS) but if they were of reduced amplitude, slowed conduction time or absent altogether, the prognosis was poor. Indeed, if SSEPs were absent bilaterally this carried the worse prognosis, being highly predictive of failure to regain consciousness (i.e. death or permanent VS). However, they were less predictive in traumatic aetiologies due to the structural damage that can ensue during the brain insult. Chen et al. (19) agreed with these findings, giving the poor prognostic factors of low amplitude or absent SSEPs positive predictive values of 100% and 89% respectively. However, as with their EEG findings, whereas negative factors were highly specific, positive ones were not, meaning that although absent or delayed SSEPs accurately predicted a poor outcome, normal SSEPs did not automatically predict a good recovery.

It has been suggested by Yingling et al. (20) that some brainstem EPs can be of predictive value, in particular P300 evoked responses. They suggest that the presence of P300 could indicate the integrity of brain systems that mediate cognitive functions, even in the absence of consciousness or overt behavioural responses. Unfortunately, the work that they have published to show its value was on a very small number of patients and therefore does not have adequate power to alter clinical practise at this time. Its diagnostic value is also limited by the fact that brainstem auditory EPs have been shown to be preserved when SSEPs are absent meaning that the predicted outcome does not alter from performing SSEPs alone - the best outcome possible is survival in the VS or death (3). Therefore, the presence of P300 cannot necessarily be correlated with outcome.

RESPONSES TO STIMULI

Motor or eye movements and facial responses such as grimacing in response to various stimuli are commonly seen stereotyped patterns. They are reflexive responses mediated at deep subcortical levels rather than as learned voluntary acts. Therefore, they do not indicate any degree of awareness and the family of the VS patient should be informed of the possibility of their occurrence, to prevent the creation of false hopes (3,7).

GENETIC FACTORS

A small number of studies have suggested that some people may have a genetic predisposition to poor outcome from traumatic injury. The 4 allele of apolipoprotein E (apoE) has been shown to be associated with increased mortality. Sorbi et al. (21) stated that deposition of amyloid -protein (A ) in the brain occurs in one third of individuals who die shortly after a severe brain injury. They found that the apoE- 4 allele occurred in a higher frequency in those who did not recover consciousness within a month (entered a persistent VS). For those who did recover, the frequency of the allele was comparable to that of the control population, suggesting a genetic susceptibility to the fatal effect of a head injury. However, their follow up was only for this month, so it is unclear as to whether the frequency of the allele was any higher in those who entered permanent VS. Also, as their study only included 16 patients, their findings have very little statistical power and are therefore inconclusive for the time being.

CONCLUSIONS

Predicting the outcome from the VS is a difficult challenge that, as yet, does not appear to be resolved. Factors mainly influencing prognosis remain etiology and duration. Traumatic brain insults have a better prognosis, nearly half of the patients studied recovered consciousness within six months, compared to only 15% of non-traumatic etiology (4). Patients were also more likely to make a good recovery from traumatic aetiologies in comparison to non-traumatic (4,11). Recovery after a year was rare in both groups (2,4,11).

From the work considered, there is a possibility that age may come into the equation. This is largely inconclusive in the younger age groups, but in the older patients, age may act as a substitute for other unmeasurable factors such as comorbidity, and thus should be taken into account when determining prognosis (4). The evidence is less conclusive in children in comparison to adults, but it appears that a similar pattern is seen in terms of functional recovery. There is evidence to suggest that children may have a better chance of recovering consciousness than adults due to the developmental potential of their brains, especially in non-traumatic etiology (4,10). False predictions of poor outcome should be avoided because this may lead to withdrawal of support in patients with the potential to recover.

Attempts have been made to treat the VS by various techniques, e.g. dopaminergic agonists, direct electrical stimulation of the brain, anticholinergics, GABA agonists, catecholaminergic anatgonists, and serotonergic agonists. However, none of these have been proven to be effective, despite seemingly encouraging preliminary studies (4,7).

Therefore, the evidence would suggest that routine clinical practise in assessment of VS patients should remain repeated clinical examinations to attempt to detect any changes in their awareness. Although criticised for the possible technical difficulties in recording, EEGs and SSEPs should be performed fairly regularly during this period. A CT scan on admission is necessary, as are the standard blood tests, to detect any potentially reversible causes of the coma.

Once a state of permanent VS is reached, little improvement can be expected. Discussion needs to take place with the family to determine the level of treatment that will be given for the duration of their lives; this is mostly preventative (e.g. avoiding contractures, pressure sores, etc). The decision to withdraw artificial nutrition and hydration is not an easy one and patients usually die within ten to fourteen days of acute dehydration and electrolyte imbalance (4,7). In the UK, it remains obligatory to seek a legal ruling to do so unless there is a clear advance directive (5). However, decisions should not be taken before a year has elapsed as the chances of recovery are small but they are a realistic possibility, although the best outcome available is usually severe disability (5,12,13).

Improvement on previous studies is difficult, if not impossible. More work is needed on the subject of prognosis in the VS to allow definitive guidelines to be agreed upon but the same problems of small sample sizes and a low incidence of the condition persists. This will most likely continue to be a problem until a large scale, multicentre trial is organised with the cooperation of major neurological centres throughout the world. This may help to alleviate some of the methodological issues but will be a massive project requiring funding which may not be possible as yet.

The VS is a complex condition that ultimately can only benefit from continued large scale studies into potential treatments and methods of accurately predicting outcome.

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Rachel Elizabeth Davison is in her final year of a medical degree (MB BS) at the University of Newcastle upon Tyne in the United Kingdom.