Significance of certain blood and saliva indicators in the diagnosis of fibrosis and the prediction of its course in patients with chronic diffuse liver diseases

The dissertation is devoted to the search for and evaluation of the informativeness of non-invasive laboratory indicators applicable to the diagnosis of liver fibrosis and to the prediction of its progression to cirrhosis in patients with chronic diffuse liver diseases. The work focuses on comparing the content of iron, gamma-glutamyl transpeptidase and ornithine decarboxylase in the blood and saliva of patients at the stages of fibrosis F2-3 and cirrhosis, as well as on the comparative detection of HCV RNA in whole, isolated and concentrated saliva at different levels of blood viral load. Using methods of mathematical modelling, quantitative criteria have been developed for the combined change of serum and salivary indicators, which make it possible to diagnose fibrosis and to predict its further course. It has been shown that the investigation of saliva is not inferior in informativeness to the investigation of blood serum and can be used as a non-invasive tool for dynamic monitoring of the course of hepatic fibrogenesis, as well as for non-invasive diagnosis of HCV infection and for monitoring of the level of viremia.

Relevance of the problem

Liver cirrhosis is an important medical and social problem of the present day. The disease is the most frequent cause of death among patients with non-neoplastic diseases of the gastrointestinal tract [Khazanov A. I. et al., 1998]. According to the World Health Organization, in 2002 liver cirrhosis caused 786 thousand deaths worldwide (on average, 12.6 per 100 thousand population). Within the structure of mortality from digestive system diseases, liver cirrhosis accounted for 40%, and within the overall mortality structure in the countries of the European Region it accounted for 1.8%, ranking eighth among causes of death [The worlds health report, 2004]. Liver cirrhosis also contributes substantially to in-hospital mortality, exceeding its average figure by a factor of 60. Seventy percent of those who die from liver cirrhosis are of working age (16-65 years) [Pavlov A. I. et al., 2005; Bobrov A. N. et al., 2006]. Chronic hepatitis and liver cirrhosis occupy the 2nd-4th places among causes of hospitalization and loss of working capacity in the population aged 20-60 years [Levitan B. N. et al., 2002].

Among the causes of chronic diffuse liver diseases (CDLD) and of the fibrosis and subsequently liver cirrhosis (LC) that develop in their outcome, researchers place alcohol first (35.5-40.9% of cases) and hepatitis C virus second (19.1-25.1% of cases) [Khazanov A. I. et al., 2001; Khazanov A. I., 2005; Galimova S. F. et al., 2001; Sherlock S., Dooley J., 2002; Ignatova T. M., 2002]. Within the mortality structure from liver cirrhosis, alcoholic cirrhosis accounts for 42.8-63.4% of cases and HCV cirrhosis for 8.6-11.8% [Plyusnin S. V. et al., 2002; Pavlov A. I. et al., 2005; Bobrov A. N. et al., 2006].

According to T. Poynard and Y. Ratzin (2002), 10% of the world population can be classed as alcohol abusers. Liver cirrhosis develops in 10-20% of alcohol abusers [Zeitz G., 2001; Sherlock S., Dooley J., 2002].

At present more than 200 million people in the world are infected with the hepatitis C virus (HCV). It is anticipated that by 2015-2020 this number will double. Chronic hepatitis C is characterized by a continuously progressive course, with the formation of liver cirrhosis in 12.5% of patients over 20 years and in 20-30% of patients over 30 years [Armstrong G. L. et al., 2000; Seeff L. B., 1999]. The rates of progression of liver fibrosis in HCV infection follow an exponential course; that is, with an increase in the duration of infection the rate of disease progression rises [Burnevich E. Z., 2005]. At the present moment the proportion of patients at the stage of liver cirrhosis among all those infected with HCV amounts to 10-25%, and by 2015-2020 a significant increase is expected in the frequency of detection of chronic hepatitis C (CHC) at the stage of cirrhosis, and the frequency of HCV-associated hepatocellular carcinoma and "hepatic" death will also rise substantially [Seeff L. B., 1999; Benvegnu L., 2004]. In the USA and in European countries, hepatitis C accounts for 20% of all cases of acute hepatitis, 70% of chronic hepatitis, 65% of hepatocellular carcinoma, 40% of liver cirrhosis and 30% of liver transplantations [Nikitin I. G., 2002].

Early diagnosis of liver fibrosis and cirrhosis is extremely important, since researchers have shown that timely initiated etiopathogenic therapy in chronic diffuse liver diseases leads to regression of fibrosis and halts the development of liver cirrhosis [Kershenobich D., Concepcion Gutierrez-Ruiz M., 2005; Otare H., 2000; Tutsumi M., Takase S., 2000; Malekzadeh R. et al., 2004; Dienstag J. L. et al., 1999; Kweon Y. O. et al., 2001; Farci P. et al., 2004; Sato A., Suzuki H., 1998; Lau D. T.-Y. et al., 1998; Metwally M. A. et al., 2003; Shiratori Y. et al., 2000; Poynard T. et al., 1999; Pol S. et al., 2004; Poynard T., 2002]. In addition, the possibility is being discussed and the efficacy of antifibrotic therapy proper is being studied [Granstein R. D., 1990; Baroni G. S. et al., 1996; Nelson D. R. et al., 2000; Kobayashi T., 2000; Hernandez E. et al., 2002; Stickel F. et al., 2002; Rombouts K. et al., 2003; Losach R. G., 2003; Nie L. et al., 2004].

Needle biopsy, which currently has the status of the "gold standard" for the diagnosis of pathological processes in the liver, may, due to so-called "sampling errors", give a false impression of the nature of the changes in the organ. Moreover, there is always a risk of severe complications associated with this invasive diagnostic procedure [Galimova S. F. et al., 2001; Pinzani M., 2002; Severov M. V. et al., 2003]. In this connection the development of alternative, non-invasive methods for the assessment of hepatic fibrogenesis is extremely urgent. Traditional instrumental methods of investigation (ultrasonography, radioisotope study, computed tomography) are of ancillary value in the diagnosis of pre-cirrhotic stages of the disease, owing to the absence of clear criteria for the assessment of fibrosis at early stages of its development. To address the task of earlier non-invasive diagnosis of liver fibrosis, in recent years a sonoelastograph apparatus has been created in Japan [Sanada M. et al., 2000], in France the "Fibroscan" (Echosens company) [Sandrin L., 2003; Saito H., 2004], and Italian researchers have proposed an automated system for the quantitative assessment of liver fibrosis using the computer program "FibroQuant" [Caballero T. et al., 2001]. However, at present, for various reasons, including economic ones, these techniques have not become widespread and remain far from application in broad clinical practice.

The studies by Bayer Diagnostics (2000) and T. Poynard (2000) were based on the measurement in the systemic circulation of serum markers of fibrogenesis — components of the extracellular matrix [Galimova S. F. et al., 2001; Pinzani M., 2002]. Despite the demonstrativeness of the results, the broad introduction of these techniques has been hindered by economic reasons and by the insufficient specificity of the developed indicators for liver tissue. At present the search continues for new biochemical serum markers of fibrosis based on the assessment, in the course of fibrogenesis, of changes in the main functions of the liver, the state of hepatocyte membranes and the content of growth factors [Bychkova V. I. et al., 2003; Makarov V. K., 2003; Tsaregorodtseva T. M. et al., 2004; Pavlov Ch. S. et al., 2006; Yagoda A. V. et al., 2006; Neuman M. et al., 1999]. The principal requirement for the tests being developed remains their accessibility for application in broad clinical practice.

In the literature there are indications that virological markers of hepatitis C have been determined not only in blood but also in saliva [Chernetsova O. V. et al., 2003; Vorobyev O. V., 2000; Roy K. M. et al., 1998; Taliani G. et al., 1997; Kage M. et al., 1997]. The results of these studies are ambiguous, and furthermore the tasks set in those works did not include assessment of the viral load of blood at different stages of the pathological process in the liver (fibrosis, cirrhosis).

No works on the determination of biochemical indicators of liver fibrosis and cirrhosis in saliva were found in the literature available to us.

Meanwhile, the investigation of saliva has a number of advantages over routine methods of laboratory diagnosis using venous or capillary blood. These are, above all, the simplicity and convenience of material collection, the non-invasive and painless nature of the procedure, and the absence of a risk of infection [Denisov A. B., 2003]. In work with specific contingents of those examined (for example, persons who inject drugs intravenously), saliva is a considerably more preferable material for investigation than blood, owing to the psychological characteristics of the personality and the state of peripheral veins of such patients [Ongarbaev A. B. et al., 2004].

A distinction should be made between saliva and oral fluid. Saliva proper is the secretion obtained directly from the ducts of the salivary glands. The product of secretion of the salivary and mucous glands, together with desquamated epithelial cells, leukocytes, microorganisms and also gingival fluid, forms the oral fluid [Denisov A. B., 2003]. It is also termed whole saliva, as opposed to isolated saliva obtained from the excretory duct of any single salivary gland. The composition of the oral fluid is determined by the activity and permeability of the hematosalivary barrier [Komarova L. G., Alekseyeva O. P., 1994]. We have not found in the literature any works on the detection of HCV RNA using additional techniques (isolated sampling from the excretory ducts of salivary glands). Among the scientific developments of the D. I. Ivanovsky Research Institute of Virology of the Russian Academy of Medical Sciences we were able to find a description of a study on the concentration of saliva for the purpose of obtaining and purifying a recombinant polypeptide — the internal core protein of hepatitis C virus (amino acids 1-150) expressed in E. coli [Petrakova N. V. et al., 1997], which allowed us to rely on this technique in concentrating saliva in order to increase the sensitivity of the method of HCV RNA detection in it.

These data determined the relevance and the aim of the work, which is devoted to the study of possible biochemical serum and salivary indicators of liver fibrosis and of its progression to cirrhosis, as well as to the determination of the viral load of blood and the relationship between the level of viremia and the possibility of detecting HCV RNA in saliva in HCV fibrosis and HCV cirrhosis of the liver.

Aim: To study the possibility of diagnosing and predicting the course of fibrosis in patients with chronic diffuse liver diseases on the basis of changes in certain biochemical serum and salivary indicators and the detection of HCV RNA in saliva.

Tasks:

1. To carry out a comparative assessment of clinical manifestations of chronic diffuse liver diseases with different stages of fibrosis — F2-3 and F4 (cirrhosis).

2. To show the dependence of quantitative changes of iron, gamma-glutamyl transpeptidase and ornithine decarboxylase in blood and saliva on the presence of a fibrotic process in patients with chronic diffuse liver diseases.

3. To study the possibility of diagnosing fibrosis and predicting its course on the basis of combined changes in the content in blood and saliva of iron, gamma-glutamyl transpeptidase and ornithine decarboxylase in patients with chronic diffuse liver diseases at the stage of fibrosis F2-3 and at the stage of cirrhosis, using methods of mathematical modelling.

4. To evaluate the informativeness of saliva investigation relative to that of blood investigation in the diagnosis of fibrosis and the prediction of its course in patients with chronic diffuse liver diseases at the stage of fibrosis F2-3 and at the stage of cirrhosis on the basis of combined changes in iron, gamma-glutamyl transpeptidase and ornithine decarboxylase.

5. To carry out a comparative assessment of the results of determination of HCV RNA in saliva in comparison with the level of viral load of blood in patients with chronic hepatitis C at the stage of liver fibrosis F2-3 and at the stage of cirrhosis.

6. To compare the possibilities of detecting HCV RNA in whole, isolated and concentrated saliva in patients with chronic hepatitis C.

Scientific novelty:

The study established for the first time that the outcome of a chronic diffuse liver disease (fibrosis F2-3 or F4 — cirrhosis) can be predicted from the combined changes in the content of iron, gamma-glutamyl transpeptidase and ornithine decarboxylase in blood or saliva. For the first time, quantitative criteria have been developed for the combined change of serum or salivary levels of iron, gamma-glutamyl transpeptidase and ornithine decarboxylase, which make it possible, with a probability of 90-95%, to diagnose fibrosis and to predict its further course in a patient with a chronic diffuse liver disease.

For the first time, the high informativeness of saliva investigation in predicting the development of liver fibrosis F2-3 and cirrhosis has been demonstrated using quantitative biochemical criteria, which is not inferior to the informativeness of serum investigation.

For the first time, a dependence has been shown of the frequency of HCV RNA detection in saliva on the level of viral load of blood in patients with chronic hepatitis C at the stage of liver fibrosis F2-3 and at the stage of cirrhosis.

For the first time, it has been established that chronic hepatitis C at the stage of liver fibrosis F2-3 and at the stage of cirrhosis is characterized by high viremia, which is accompanied by a level of viral load in saliva sufficient for detection by modern methods of investigation.

For the first time, the priority informativeness of whole saliva (oral fluid) for the detection of HCV RNA in comparison with isolated saliva from the excretory duct of the parotid gland has been demonstrated.

Practical significance of the work:

The developed biochemical serum and salivary criteria of liver fibrosis and cirrhosis can be used in non-invasive investigations aimed at identifying, among patients with chronic diffuse liver diseases, those with fibrosis who need early etiopathogenic or pathogenetic therapy, as well as for non-invasive dynamic monitoring of the course of the process of hepatic fibrogenesis in such patients.

In patients with chronic hepatitis C at the stage of liver fibrosis F2-3 and at the stage of cirrhosis, the results of HCV RNA detection in saliva can be used for non-invasive diagnosis of this pathology and for non-invasive monitoring of the level of viremia.

This will make it possible to avoid possible complications of invasive diagnostic techniques and to increase the effectiveness of therapy through its early initiation, improving the prognosis for patients.

Main propositions for defense:

1. The determination of iron, gamma-glutamyl transpeptidase and ornithine decarboxylase in the blood or saliva of a person examined for chronic diffuse liver disease, and a comprehensive analysis of their levels using the quantitative criteria developed by us, makes it possible, with a probability of > 95%, to diagnose the development of liver fibrosis.

2. In the course of dynamic observation of a patient previously diagnosed with liver fibrosis, the determination of iron, gamma-glutamyl transpeptidase and ornithine decarboxylase in blood or saliva and a comprehensive analysis of their levels using the quantitative criteria developed by us makes it possible, with a probability of 90-95%, to predict the transition of the process to cirrhosis.

3. The method of saliva investigation for the non-invasive diagnosis and prediction of the course of liver fibrosis is not inferior in informativeness to the investigation of blood.