Extrahepatic Detection Of Hepatitis C Virus Biology Essay

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Hepatitis C virus infection is a worldwide disease with several long-term complications including cirrhosis, end-stage liver disease, and hepatocellular carcinoma. It is known to be responsible for both hepatic and extrahepatic manifestations suggesting that chronic HCV infection should be considered as a systemic disease. They appear to be associated primarily with autoimmune or lymphoproliferative states.

Aim of the work: The aim of this work was to try to detect the HCV-RNA in extrahepatic site "in gingival tissue" in patient with chronic HCV infection.

Patients and Methods: This study was conducted on thirty patients with chronic HCV infection and ten healthy subjects as controls. They included 26 males and 14 females. Patients were classified according to the Child-Pugh score. All patients and controls were subjected to thorough history and complete physical examination .Routine laboratory investigations were done for all subjects including: complete blood picture, fasting blood glucose, Liver function tests , renal function tests and serum lipids profile .Diagnosis of hepatitis C viral infection was done by the positive results of serum samples for HCV antibodies by ELISA and detection of HCV-RNA by the polymerase chain reaction .All patients were subjected to the examination with abdominal ultrasonography All patients were subjected to liver biopsy with conventional staining with Haematoxylin and Eosin. Histopathological diagnosis included grading and staging of chronic hepatitis according to the histological activity index. All patients were examined for serum sample, liver and gingival biopsies and HCV-RNA was detected by the PCR assay.

Results: The study included two groups; patients and controls. Patients were subdivided according to the level of HCV viremia into three subgroups; subgroup (A, B and C) with low, moderate and high viral loads respectively. The serum alanine transaminase (ALT) levels were elevated significantly in 66.6% of patients with positive correlation with the level of viremia (P value <0.05). All subjects underwent gingival biopsies revealing that 40% of patients were positive for HCV RNA with a statistically significant correlation between the presence of the HCV- RNA in the gingival tissues and different grades of viremia (P>0.01) .

Conclusion: The detection of HCV in gingival tissue may assume an extrahepatic reservoirs of HCV with important implications for transmission, disease progression and effective treatment

INTRODUCTION

Hepatitis C virus (HCV) infection is a worldwide disease that is characterized by a preferential chronic evolution with mild to severe liver disease. Several long-term complications may develop in patients with chronic HCV infection including cirrhosis, end-stage liver disease, and hepatocellular carcinoma (HCC).(1)

HCV infection is known to be responsible for

both hepatic and extrahepatic manifestations (EHMs) suggesting that chronic HCV infection should be considered as a systemic disease. The association of some EHMs with HCV is very close, while for others, is strongly suspected and slightly indicated by anecdotal data in some cases. They appear to be associated primarily with autoimmune or lymphoproliferative states.(2)

Chronic HCV infection can directly or indirectly affect a number of organs other than the

liver. A minority of patients with chronic HCV infection develops significant extrahepatic manifestations of the disease such as HCV related lymphoproliferative disorders whose prototype is mixed cryoglobulinaemia which represent the most closely related extrahepatic manifestations of HCV infection.(3) Other HCV associated disorders include nephropathies, thyropathies, sicca syndrome, idiopathic pulmonary fibrosis, porphyria cutanea tarda, lichen planus, diabetes, chronic polyarthritis, cardiopathy and atherosclerosis. These wide series of extrahepatic manifestations, based on available data, have a link with the virus and some of these extrahepatic diseases is only suggested and needs further confirmation. A pathogenetic link between HCV infection and some extrahepatic manifestations was confirmed by their responsiveness to antiviral therapy.(4) HCV-RNA is often present in saliva of HCV infected patients with plasma viral load being the only known predictable factor. Further studies on salivary HCV-RNA are needed.(5)

The aim of this work was to try to detect the HCV-RNA in extrahepatic site "in gingival tissue" in patients with chronic HCV infection.

METHODS

This study was conducted on thirty patients with chronic HCV infection and ten healthy subjects as controls. They were selected from the inpatients and outpatients' clinics of the Internal Medicine Department of Menoufiya University hospital as well as the Main Alexandria University Hospital. They included 26 males and 14 females .Their ages ranged between 26 and 51 years. Patients were classified according to the Child-Pugh score.

ISSN 1110-0834Patients with renal failure, alcoholics, viral hepatitis other than HCV infection, schistosomiasis as well as those with any chronic illness (other than chronic liver disease) were excluded from the study. As well, patients other than Child-Pough A classification of liver cirrhosis were excluded. All patients and controls were enrolled during the same period and informed consent was taken to participate in the study.

All patients and controls were subjected to thorough history taking with special emphasis on symptoms of chronic liver disease such as abdominal pain, enlarged abdomen, lower limb edema, fatigue, loss of weight and jaundice. Complete physical examination was stressing on signs of liver cell disease such as jaundice, pallor, ascites, hepatomegaly, splenomegaly and lower limb edema as well as signs of hepatic encephalopathy. Routine laboratory investigations were done for all subjects including: complete blood picture, fasting blood glucose, renal function tests (blood urea and serum creatinine) and serum lipids profile (serum cholesterol and serum triglycerides). Liver function tests included serum alanine transaminase (ALT), aspartate transaminase (AST), serum albumin, serum bilirubin and prothrombin time and concentration. Diagnosis of hepatitis C viral infection was done by the positive results of serum samples for HCV antibodies by ELISA and detection of HCV-RNA by the polymerase chain reaction (PCR). All patients were subjected to examination with abdominal ultrasonography laying stress on the stigmata of chronic liver disease and their complications. All patients were subjected to liver biopsy using the

tru cut needle and the liver tissues specimens, after preparation, were subjected to conventional staining with Haematoxylin and Eosin. Histopathological diagnosis included grading (the degree of the necroinflammatory changes) and staging (the degree of the liver fibrosis) of chronic hepatitis according to the histological activity index (HAI).

All patients were examined for serum sample, liver and gingival biopsies and HCV-RNA was detected by the PCR assays.(6) Blood was collected from the patients by vacuum venipuncture, using a dry 10 mL tube. The serum was separated, centrifuged, aliquoted and stored at -20°C until the time of assay.

The tissues samples (liver and gingiva) have been fixed in neutral buffered formalin immediately after removal at 4 â-¦C for 10-12 hrs and was then embedded in paraffin according to a routine standard protocol for pathological specimen. Paraffin blocks were stored at room temperature for 2 and 3 weeks.(7)

RNA extraction was performed.(8) Briefly, sections of 4µm x 7.4µm, 2µm x 7.4µm , and 1µm x 7.4µm of stored tissues were deparaffinized in xylene at 65 â-¦C, followed by washes in ethanol. Controls consisted of an empty paraffin blocks and formalin fixed paraffin-embedded gingival tissue of a patient who was tested negative for anti HCV in serum.

All paraffin blocks were cut at the same microtome with single use disposable blades. Tissue was digested overnight in a lytic buffer containing 500 µg/mL proteinase K, 1% sodium dodecyl sulfate , 20 mmol/L Tris-HCl (pH 8.0), and 5 mmol/L ethylenediaminetetraacetic acid (pH 8.0).

RNA was extracted by phenol/chloroform in the presence of 300 mmol/L sodium acetate (pH 5.2) , followed by precipitation with isopropanol and yeast tRNA (10 mg/mL). The resulting pellet was washed in 70% ethanol, dried and resolved in 25µL diethyl pyrocarbonate-treated water. One microlitre RNAse inhibitor was added resulting in a RNA extraction volume of 26 µL. Prior to cDNA synthesis and nucleic acid amplification, serial dilution of 5µL extracted RNA was performed by adding diethyl pyrocarbonate-treated water. Dilution steps were 1:1, 1:10, 1:100, 1:1000, 1:10,000, 1:50,000 and 1:100,000 for all three samples of extracted RNA per patient. After RNA extraction and serial dilution, qualitative HCV RNA testing of liver and gingival biopsy specimens was performed using the Cobas Amplicor HCV version 2.0 (Roche Diagnostics Inc., Mannheim, Germany) assay.

Statistical analysis:

The collected data were tabulated & analyzed using SPSS Statistical package version (10) on IBM compatible computer. Qualitative data was expressed as number and percentage (No & %) and analyzed by applying Chi-square test (X2). Quantitative data was expressed as mean and standard deviation (X±SD) and analyzed by applying student t-test for comparison of two groups of normally distributed variables & f-test (ANOVA) for more than two groups where post-hoc test was used to detect the significance for each two groups. All these tests were used as tests of significance at 5% level. P value < 0.05 was considered statistically significant & P value > 0.05 was considered statistically non significant.

RESULTS

This study was conducted on 40 subjects, classified into 2 groups as follows:

Group I: included 30 patients with chronic hepatitis C infection of Child A cirrhosis. Patients were further subdivided according to the level of HCV viremia into three subgroups: subgroup (A): 8 patients with low viral load (600 - 2.5Ã-105 IU/ml), subgroup (B): 14 patients with moderate viral load (2.5Ã-105 - 2.5Ã-106 IU/ml) and subgroup (C): 8 patients with high viral load (more than 2.5Ã-106 IU/ml).

Group II: included 10 healthy subjects as controls. They had been confirmed negative for HCV infection by PCR testing.

Age and sex distribution:

There was no statistically significant difference between the two groups as regards age and sex.

Clinical Findings:

The most frequent symptoms in the studied group of patients were right hypochondrial pain, dyspepsia, abdominal distension, constipation and easy fatigability while the most frequent signs were abdominal distension, hepatomegaly and splenomegaly. Non had ascites, jaundice or signs of hepatocellular failure.

Imaging study:

On abdominal ultrasonography, enlarged liver was detected in 12 patients (40%). The sonographic evidence of cirrhosis was found in 20 patients (66.6%). Spleen was enlarged in 2 patients (6.6%). Ascites was absent in all patients .No focal lesions had been detected in the studied groups of patients.

Hematological studies:

The mean hemoglobin value, white blood cell count and platelet count were significantly lower in the studied group of patients compared to that of controls.

Liver function tests:

The serum ALT (IU/ml) levels were elevated in 20 patients (66.6%) with a high statistically significant difference compared to the control group (P value <0.001). They were distributed as 4 (50%) in subgroup A, 8 (57.1%) for subgroup B and 8 (100%) for subgroup C. (P value <0.05). The serum AST (IU/ml) levels were elevated in 6 patients of group I (20%), distributed as 2 and 4 patients in subgroups B and C respectively. There was a statistical significant difference between patients and controls. (P value <0.05).

There were no significant differences between both groups as regards mean serum albumin and mean serum bilirubin. The mean prothrombin activity was significantly lower in group I patients than that of the control group. (P value <0.05).

The mean fasting blood glucose level in group I was statistically significantly lower than that of the control group. (P value <0.05).

The mean serum creatinine level in group I was statistically significantly higher than that of the control group. Meanwhile, no statistically significant difference could be detected on comparing the mean blood urea levels in the two studied groups.

No statistically significant difference could be detected on comparing the mean cholesterol and triglycerides levels in the two studied groups.

All subjects included in this study underwent gingival biopsies .The results revealed that 12 patients (40%) were positive for HCV RNA, and 18 patients (60%) was negative. All controls (100%) were negative. Also liver biopsies was done to group I patients and all (100%) was positive for HCV RNA. (Table I)

The positive gingival biopsies for HCV RNA were distributed as 0/8(0%) in subgroup A (low viremia), 6/14(42.9%) in subgroup B (moderate viremia), and 6/8(75%) in subgroup C (high viremia) with a statistically significant correlation between the presence of the HCV- RNA in the gingival tissues and different grades of viremia (P>0.01) (Table II). HCV- RNA was present in the liver biopsies of patients regardless level of serum PCR. (Table III)

There was a significant correlation between the serum level of HCV RNA in the studied group of patients and blood hemoglobin level in the subgroups A, B and C. On the other hand, there was no significant relation between the serum level of HCV RNA and platelets count in the three subgroups. There was a significant relation between serum level of HCV RNA and blood leucocytic count in the three subgroups.

Regarding the relation between the serum level of HCV RNA and serum transaminases levels. ALT elevation was detected in 50%, 57% and 100% of patients with low, moderate and high viremia respectively. As well, AST elevation was detected in 0%, 14.35 and 50% for low, moderate and high viremia respectively. No significant relation was detected between serum level of HCV RNA and serum albumen and serum total bilirubin levels. On the other hand, there was a high significant relation between the serum level of HCV RNA and prothrombin concentration (Table IV).

There was a high significant relation between the serum level of HCV RNA and fasting blood sugar in the three subgroups.

There was no significant relation between the serum level of HCV RNA and serum creatinine level in the three subgroups. On the other hand, there was a high significant relation between serum level of HCV RNA and serum level of urea in the three subgroups.

There was no significant relation between the serum level of HCV RNA and serum cholesterol and triglycerides levels in the three subgroups.

Table I: Prevalence of HCV RNA positive gingival biopsy.

Gingival PCR

Total

+ ve

- ve

No

%

No

%

No

%

Case

12

40.0

18

60.0

30

75.0

Control

0

00.0

10

100.0

10

25.0

Total

12

30.0

28

70.0

40

100.0

+ve = positive -ve = negative No. = Number

Table II: Relationship between serum PCR level and presence of gingival HCV

Gingival

PCR

Serum PCR

X 2

P value

Low

Moderate

High

No

%

No

%

No

%

+ Ve

0

0.00

6

42.90

6

75.00

16.53

<0.01

- Ve

8

100.0

8

57.10

2

25.00

+ve = positive -ve = negative NO. = Number

Table III: Relationship between serum PCR level and presence of liver HCV:

Liver

PCR

Serum PCR

Low

Moderate

High

No

%

No

%

No

%

+ Ve

4

50.00

4

28.60

2

25.00

- Ve

0

0.00

0

0

0

0.00

Not done

4

50.00

10

71.40

6

75.00

+ve = positive -ve = negative NO. = Number

Table IV: Relationship between serum PCR level and liver profile

Liver

profile

Serum PCR

Test of

significance

P value

Low

Moderate

High

No

%

No

%

No

%

ALT

Normal

Elevated

4

4

50.0

50.0

6

8

42.9

57.1

0

8

0.0

100.0

AST

Normal

Elevated

8

0

100.0

0.0

12

2

85.7

14.3

4

4

50.0

50.0

Albumin

X ± SD

3.8 ± 0.20

3.7 ± 0.27

3.6 ± 0.27

F test: 0.49

>0.05

Bilirubin

X ± SD

1.7 ± 0.44

1.8 ± 0.39

2.1 ± 0.39

F test: 1.23

>0.05

PC

X ± SD

80.7 ± 7.30

78.0 ± 3.20

71.7 ± 3.20

F test: 10.5

<0.01*

ALT = Alanine Transaminase, AST = Aspartate Transaminase

PC = Prothrombin Concentration

DISCUSSION

Chronic hepatitis C is the most common cause of chronic liver disease and cirrhosis, and the most common indication for liver transplantation in developed countries. Researchers have long sought to determine whether HCV replicates outside the liver, because finding HCV RNA in extrahepatic reservoirs has important implications for transmission, disease progression and effective treatment. Up to 40-74% of patients infected with HCV might develop at least one extrahepatic manifestation during the course of their disease. However, contradictory data related to extrahepatic HCV infection and replication have been reported and few in situ detection of HCV in extrahepatic tissues are controversial. Many reports support the results of our studies. So far, there have been few reports on the status of extrahepatic HCV infection and replication in hepatitis C patients and these studies need to be extended. Whether HCV can bring about cell injury of infected extrahepatic tissues is not well understood because of the lack of extrahepatic histopathological observation.

In the context of the search for an extrahepatic reservoir of HCV replication, the strand-specific detection of the negative-strand HCV RNA has contributed to clarify which may be the major sites of HCV tropism. Recently, real-time PCR has been described for viral load monitoring in serum and in liver samples(9,10) and it was shown in a previous study that this technology could be used as a very reliable and highly sensitive method to quantify the viral load of HCV in serum.(11)

The aim of this work was to try to detect the HCV-RNA in the extrahepatic site "in gingival tissue" in patient with chronic HCV infection. This study was carried on 30 patients with chronic HCV infection and 10 healthy persons of matched age and sex as a control group.

In the present study, there was a statistically significant difference between patients and controls regarding the presence of HCV RNA outside the liver as extracted from the gingival tissues with a positive overall prevalence of 40% in the infected patients. Lower prevalence was reported in the previous studies.(12,13) Meanwhile, no significant relationship was detected between the serum PCR and its presence in the liver tissues, as it was detected in all cases regardless level of serum PCR and also if gingival HCV is positive or negative. These results are in agreement with other data suggesting that the levels of extrahepatic HCV infection and replication were relatively low as compared with those of livers; and there were differences in the status of HCV replication among different extrahepatic tissues.(14) In the present study, there was a significant relation between serum levels of HCV RNA and their detection in the gingival biopsies and this coincides with other data where HCV-RNA was detected in subjects with the highest serum viral load.(15)

The detection of HCV in gingival tissue may assume the oral rout of HCV transmission. This finding is in agreement with Tetsuro Suzuki et al(16) which quantitatively determined HCV RNA in oral fluids from dental patients, including some patients with oral diseases, and demonstrated frequent detection of HCV in the saliva and gingival crevicular fluid (GCF). Juan José Arrieta et al, (2001) had demonstrated that HCV infects and replicates in epithelial cells from salivary glands of patients with Sjögren's syndrome or chronic sialadenitis.(17)

The infected extrahepatic tissues might act as a reservoir for HCV and play a role in both HCV persistence and reactivation of infection. HCV as an etiological agent replicating and expressing viral proteins in extrahepatic tissues contributes to EHM associated with chronic HCV infection.

An important feature of HCV is that the virus avoids immune elimination. A consequence is chronic infection, an accumulation of circulating immunocomplexes and autoimmune phenomena. HCV shows a particular lymphotropism other than hepatic tropism, that is responsible for many EHM.(18) Mixed Cryoglobulinemia (MC) is the most known and studied syndrome associated with HCV infection. It is a systemic vasculitis that may involve the skin, kidney and nervous system. A frequent reported association is that between HCV infection and non-Hodgkin lymphoma. The cryoglobulinemia may be the intermediary disorder, in fact, some persistent forms of cyoglobulinemia can switch over to a more aggressive haematologic disorder.(19)

Whatever the sources or mechanisms are, the findings obtained provide important implications for medical personnel regarding HCV transmission in health care settings as well as for HCV epidemiology, as the origin of the viral infection remains unclear in up to 40% of cases.(16) Although HCV is a hepatotropic virus, convincing evidence of HCV lymphotropism has been demonstrated in tissue culture.(16) Is likely that several possible sources discussed above are involved in HCV penetration into the saliva and GCF. Generally, periodontal inflammation increases the excretion of BMC-rich GCF. There is also a possibility that HCV exists within mucosal epithelial cells. HCV has been identified in the mucosal tissue as well as salivary glands of anti-HCV-positive patients with oral lichen planus using various techniques including in situ hybridization, strand-specific RT-PCR and immunohistochemistry.(20)

Peripheral blood mononuclear cells (PBMC) have been shown to harbor low-titer of HCV replication and the viral HCV replication in these cells is further confirmed by the analysis of the viral quasispecies. Different subsets of circulating leukocytes have been analyzed and HCV replication seems to be confined to polymorphonuclear leukocytes, monocytes, macrophages and B (but not T) lymphocytes. (21-26)

It has been argued that the PBMC reservoir may be involved in the graft reinfection by HCV after liver transplantation. Moreover, the HCV replication in cells involved in the immune response may have dramatic consequences as far as pathogenesis and oncogenesis are concerned. It has been shown that HCV may interact in vitro with the apoptotic cascade leading to resistance to the apoptotic stimulus.(27,28) If this interaction occurs in lymphocytes and other cells of the immune system, this may lead to loss of immune competence. Defects in the control of the immune system homeostasis by apoptosis are relevant for the pathogenesis of both autoimmune and lymphoproliferative disorders which are frequently associated with HCV infection.(29,30) HCV does not seem, however, to replicate in B-cell non-Hodgkin lymphoma tissue.(31,32)

The presence of HCV RNA in PBMC has

been questioned by other investigators.(33,34) The disparate results reported in the literature may

be due to the difficulties in detecting minute amounts of negative-strand HCV RNA in such cells as suggested by the fact that negative-strand

HCV RNA becomes more easily detectable in PBMC of immunosuppressed patients who are characterized by higher HCV replication levels as in case of confection with HIV-1(33) or after liver transplantation.(35)

Other organs seem to support low-level HCV replication such as bone marrow, lymph nodes, spleen, pancreas, thyroid and adrenal glands.(33) On the contrary, no negative-strand HCV RNA has been found in kidney, lung, muscle, spinal cord, skin affected by vasculitis secondary to mixed cryoglobulinemia or lichen ruber planus.(36) Extrahepatic manifestations are frequent in HCV infection but any correlation between HCV replication at extrahepatic sites and disease expression is premature at present. Taken together, the above results seem to confirm that the liver is the major reservoir of HCV replication. This is in agreement with indirect kinetics data from the liver transplantation model(37) and extrahepatic sites contribute little to the total viral load.(38) However, the lowlevel replication of HCV in these compartments may significantly affect the HCV disease expression even though the evidence in favor of this is at present exclusively experimental.

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