INTRODUCTION
Hepatitis C virus (HCV) is an RNA virus responsible for chronic infection in about 200 million individuals worldwide1. It is estimated that 2.7 million Americans have chronic HCV infection (2) Patients are often unaware that they have contracted the virus until the appearance of long-term consequences of the infection, primarily cirrhosis and hepatocellular carcinoma3,4.
Recent findings suggested that Hepatitis C virus infection has been associated with both organ-specific (thyroiditis, diabetes) and systemic autoimmune diseases5 the high prevalence of HCV infection has led to increasing interest in the many extrahepatic manifestations of this disease. There have been conflicting reports regarding the association between HCV infection and neuropsychiatric and cognitive abnormalities in HCV infected dividuals6.
Chronic hepatitis C virus (HCV) infection may be associated with psychiatric and cognitive abnormalities7,8 and there is evidence that HCV is neurotropic and may cause Neuropsychological (NP) impairment even in the absence of advanced liver disease9.
HCV serostatus was a significant predictor of NP performance both globally and in the areas of learning, abstraction, and motor skills, with trends in speeded information processing and delayed recall.9
We therefore considered whether HCV infection has an early effect on the central nervous system, resulting in neuropsychological and psychiatric abnormalities before appearance of disease's full blown picture.
Inclusion Criteria:
Twenty six individuals with serologically proven positive HCV who were discovered accidentally during blood donation or routine investigations in El-Minia University Hospital.
Exclusion Criteria:
1- Patients with either clinical or laboratory signs of hepatic decompensation or significant cirrhosis, to exclude hepatic encephalopathy. We also excluded other hepatic diseases as malignancy or autoimmune liver disease.
2- Treatment with interferon Alfa (as it is associated with a constellation of symptoms, including depression, memory impairment and cognitive slowing.)
3- General medical conditions or Neurological disorders with expected neuropsychological impairment
4- Psychiatric disorders and major drug usage or Intra Venous Drug Usage (IVDU).
Controls:
Fifty age and sex matched healthy control subjects were recruited from volunteer hospital staff. They were proven to be HCV negative using ELISA test.
Methods:
All patients underwent:
I) Clinical assessment and Neurological examination.
II) Laboratory tests including: *Routine investigations:
1. Liver function tests (serum bilirubin, ALT, AST, albumin and total proteins), fasting and post prandial blood glucose and renal function tests: urea and creatinine.
2. Prothrombin time and concentration and complete blood count.
3. ELISA For HIV 1 & 2, HBVsAg, HBVcAb, and HCVAb.
Special investigations:
a) Antinuclear antibodies (ANA), antismooth muscle antibodies (ASMA) and antimitochondrial antibodies (AMA) by the indirect immunofluorescence technique.
b) Identification and enumeration of CD4 andCD8 T-lymphocyte subsets by Flowcytometry EPICS-XL: Total and differential white blood cell counts and T-cell subset distributions were analyzed on fresh EDETA blood samples. Counts were obtained with a Sysmex NE 1500 fully automated. Test procedure10: For each sample, 4 tubes were prepared, labeled 1st, 2nd, 3rd and 4th. The 1st tube is the control negative for CD4, the 2nd tube is the test for CD4, the 3rd tube is the control negative for CD8 and the 4th tube is the test for CD8. To each control tube we added 10 ul of Dako FITC-conjugated monoclonal antibody of the same isotype as negative control then we added 10 ul of the corresponding DaKo-conjugated monoclonal mouse antihuman CD4 in the 2nd and CD8 in the 4th tubes. To each tube we added 100 ul of patient's blood. Vortexed gently and incubated for 30 minutes in the dark. Then we added and mixed 2 ml of freshly prepared diluting lysing reagent (1:10 D.W) in each tube. The tubes were centrifuged at 3000 rpm for 5 minutes. Then the supernatant was removed leaving pellets at the bottom containing the cells to be analyzed. The samples were ready for flow cytometric analysis after adding 0.5 ml of PBS.
C) Quantitative Real Time PCR for the detection of HCV RNA and viral load using (Applied Biosystems 7500 Fast Real-Time PCR System, CA, USA).
Isolation (Extraction) of RNA: serum samples from patients was used to extract HCV viral RNA using the fully automated QIAcube instrument and the specific kits; Qiagen columns (QIAamp Viral RNA Kits; Qiagen Inc), according to the manufacturer’s protocol; HCV one step quantitative RT-PCR using TaqMan® probe- based technology11: HCV RNA detection using polymerase chain reaction (PCR)on Real-Time PCR instrument is based on the use of kit contains reagents and enzymes for the reverse transcription and specific amplification of a 240 pb region of the HCV genome in fluorescence detector (FAM)(Kits were supplied from Applied Biosytems). The amplified product is detected via monitoring the fluorescence intensities during the PCR run (i.e. in real-time)12.
The reaction mix was performed in a final volume of 25 µl containing 8.5 µl of the extracted RNA and 16.5 µl of the Master Mix containing: 2xRT-PCR buffer, HCV forward primer, HCV reverse primer and HCV probe, and 25xRT-PCR Enzyme Mix in addition to the forward and reverse primers of the Internal positive control and its probe (which is a system supplied with the kit allowing both to control the RNA isolation procedure and to check for possible PCR inhibition).13 The TaqMan PCR cycling conditions were 10 minutes at 45°C (RT Incubation), 10 minutes at 95°C (Taq activation), 45 cycles of denaturation at 95°C for 15 seconds, annealing and extension at 60° C for 45 seconds. With these primers and conditions, we were able to detect as little as one viral template. The final quantification of the DNA was performed by the software provided by the manufacturer and was defined as IU/ml.14
III) Abdominal ultrasound and Liver Biopsy:
To exclude hepatic cirrhosis and hepatic focal lesions.
IV) Electroencephalography (EEG):
Using 16 channel digital Nihon Kohden machine.
V) Neuropsychological assessment:
1- Montreal Cognitive Assessment (MoCA):
A cognitive screening tool with proven validity to assist in the detection of mild cognitive impairment (MCI)15. The final version of the MoCA used in this study is a 30-point test, including short-term memory recall, executive function tests, sustained attention task, serial subtraction task, digits forward and backward, language tasks, and orientation to time and place.16
2- Mini Mental State (MMS) examination:
MMS is used to assess: Orientation to time and place, Instantaneous recall, Short term memory, Serial subtraction or reverse spelling, Constructional capacities (copying a design) and Use of language17.
3- The Hamilton Anxiety Scale (HAS):
It is a 14-item test measuring the severity of anxiety symptoms. For the 14 items, the values on the scale range from zero to four; according to the severity of anxiety. The total anxiety score ranges from 0 to 56.18 Persons with generalized anxiety disorder and panic disorder tend to have a total anxiety score of above 20.
4- Montgomery-Åsberg Depression Rating Scale (MADRS):
MADRS is designed to measure the degree of severity of depressive symptoms. It is a 10-item checklist. Since there is a comparative lack of emphasis on somatic symptoms, the scale is useful for the assessment of depression in people with physical illness19. The following mean scores correlates with global severity measures: very severe, 44; severe, 31; moderate, 25; mild, 15; and recovered, 7.
5- Impact of Illness Scale (IIS):
A brief scale, which measures the degree that an illness is perceived to restrict psychosocial functioning. The measure is shown to have high internal consistency in a variety of samples. The IIS is a reliable and valid measure of psychosocial impact of illness that may be applicable in a wide range of sociocultural setting20.
Statistical Analysis:
The advanced statistical package for social science [SPSS] for MS windows (version 11.0) was utilized to furnish the statistical analysis for the study.
Simple descriptive statistical tests (Mean and Standard deviation) are used to describe the numerical values of the sample. To test the 2- tailed significance of differences in means, Student t-test for independent samples for 2 groups and one-way analysis of variance (ANOVA) test for comparison between more than 2 groups were used and spearmen correlation test. A probability of (p) ≤ 0.05 is accepted as significant.
RESULTS
This study included 26 patients who were diagnosed accidenlly to have HCV ;18 males and 8 females, their ages ranged from 20 to 59 years (mean=35.85±11.39)
Both general medical and neurological examinations showed no abnormalities in all patients.
Laboratory findings are summarized in Table (1) all within normal ranges. Both CD4 and CD8 were significantly lower than controls (P<0.001) Table (2).
Abdominal ultrasound excluded cirrhosis and focal lesions. Liver biopsies had been performed and all individuals had mild inflammation only, in absence of cirrhosis or significant fibrosis.
EEG was normal in 18 patients, and showed epileptiform changes in 8 patients; one of them had right focal changes, five showed left focal changes and two with generalized epileptiform changes. None of our patients had triphasic waves. Viral load was higher among patients with epileptiform EEG changes, however this was statistically insignificant. And no significant difference in cognitive functions between patients with normal or epileptiform EEG changes.
The scores of MoCA ranged between 14 and 30 (mean=22.50±4.27) indicating mild cognitive impairment (MCI) according to Naseraddin.16 Scores were significantly lower than controls (p<0.006). Only 5 patients had normal scores (above 25.2) those patients showed no significant differences in lab variables in comparison to patients with MCI.
Attention, naming, memory, fluency, abstraction, orientation (dominant hemisphere functions) were the most affected domains. Visuospatial function and concentration were lower among patients but this difference was statistically insignificant (Table 3); while Table (4) shows comparison between males and females, showing nearly significant higher scores of MoCA and MMS in males (p= 0.07 and 0.06 respectively).
MMS scores ranged between 10 and 30 (mean=22.11±4.78), they were highly significantly lower than controls and were significantly correlated to scores of MoCA (r=0.679 & p=0.0001)
MADRS scores ranged between zero and 32 (mean=13.42±11.62); the mean was significantly higher than controls (p<0.0001). MADRS was normal in 8 patients, another 3 had recovered depression, 7 mild, 4 moderate and 4 had severe depression, while none of our patients had very severe depression. Degree of depression was not correlated with any of the laboratory variables though the liver enzymes were higher among depressed patients. MADRS scores were significantly correlated with HAS (r=0.561 & p=0.003) and with scores of IIS (r=0.589 &p=0.002)
HAS scores ranged between zero and 29 (mean=5.46±7.97), only 3 patients had scores above 20 which signify anxiety (p=0.15). While 14 were completely normal and the rest of patients had insufficient scores to diagnose anxiety.
IIS scores ranged between zero and 19 (mean=3.40±5.67) Scores were not correlated with lab variables.
Correlations between neuropsychological tests and laboratory variables were tested, there was significant correlation between MADRS, HAS and AST levels (r=0.395 and 0.421 respectively) (Table 5).