Detection of hepatitis C virus in serum and peripheral blood mononuclear cells by two reverse transcription polymerase

Over 75% of patients with acute hepatitis C (HCV) will develop into chronic infection associated with chronic hepatitis, end stage liver disease or hepatocellular carcinoma.^1,2 HCV infection is usually ignored because the level of HCV in serum of some patients is very low and most patients with chronic HCV infection are asymptomatic. Some patients with persistent abnormal liver function may be infected with HCV although HCV RNA and HCV antibodies in serum are negative because their HCV load is under the current detectable limit or sometimes HCV is absent in serum but present in the liver or peripheral blood mononuclear cells (PBMCs) and other tissues.^3-5 Replication of HCV in PBMCs causes relapse and chronic infection.^6-9 Therefore PBMCs play an important role in replication, relapse and chronic infection of HCV. We detected HCV RNA in serum and PBMCs by real time-fluorescent quantitative reverse transcription-polymerase chain reaction (FQ-RT-PCR) and nested reversed transcription-polymerase chain reaction (nested RT-PCR) to evaluate the sensitivity of HCV detection and to emphasize the importance of PBMCs for diagnosis and treatment of HCV infection.

METHODS

Patients
Patients with abnormal liver function were from Outpatient Department and Inpatient Department of Infectious Disease of First Affiliated Hospital of Zhengzhou University from 2004 to 2006. All patients gave informed consents before the tests. Patients with hepatitis B virus/human immunodeficiency virus infection and continuous alcohol or drug abuse were excluded. Patients enrolled in the study were divided into two groups randomly. In group I (180 patients), HCV RNA was detected by FQ-RT-PCR and group II (180 patients) by nested RT-PCR.

Extraction of RNA
Sera of patients were collected and stored at -80˚C. Human PBMCs were isolated with lymphocyte separation medium from peripheral blood by density gradient centrifugation on Percoll (Shanghai Chemical Reagents Co, China). Cells were washed three times with phosphate buffered saline (PBS) pH 7.0, then were diluted in 2 ml of ddH₂O. HCV RNA was extracted from 50 µl of serum and 50 µl of diluted PBMCs. Total RNA was prepared from serum and PBMCs by TRIzol reagent (PG Biotech, China) according to the manufacturer's instructions.

FQ-RT-PCR
HCV RNA concentrations were quantitated by real time RT-PCR assay (Light Cycler II; Roche Diagnostics, Germany) according to the protocol provided by the test kit (PG Biotech, China). The sensitivity limit of the method was about 1000 genome copies/ml.

Nested RT-PCR
Qualitative analysis of HCV RNA was done by nested RT-PCR. The primers from 5'-noncoding region were used for cDNA synthesis and PCR amplification. The primers were as follows: outside sense primer 5'-GGGGCGACACTCCACCATA-3', outside antisense primer 5'-CCTATCAGGCAGTACCACAAGG-3'; inside sense primer 5'-ACACTCCACCATAGATCACTCCC-3', inside antisense primer 5'-CGCGACCCAACAC- TACTCG-3'. HCV RNA was extracted from 50 µl of serum and 50 µl dilution of PBMCs and copied into cDNA by reverse transcription (RT). Two milligram RNA from serum or PBMCs of healthy people, were taken as negative control. The RT reaction condition was at 42˚C for 60 minutes. The first cycle reaction conditions: denaturation at 94˚C for 4 minutes, followed by 30 cycles at 94˚C for 30 seconds, 55˚C for 45 seconds and 72˚C for 1 minute. The second PCR was performed with 2 µl product of the first PCR reaction in the same condition as the first cycle. PCR productions were visualized on agarose gels stained with the fluorescent dye ethidium bromide following electrophoresis.

Statistical analysis
All data were analyzed by SPSS 10.0. Chi-square test was used to compare proportion of the two groups. All tests were performed two sided, P<0.05 was considered statistically significant.

RESULTS

HCV RNA was positive if patient's HCV RNA was positive in serum or in PBMCs. Of 180 patients in group I, 106 (58.9%) were positive for HCV RNA by FQ-RT-PCR; in which 84 patients (46.7%) in serum, 63 patients (35.0%) in PBMCs and 41 patients (22.8%) in serum and PBMC. In group II, 136 of 180 patients (75.5%) had positive HCV RNA by nested RT-PCR, in which 105 patients (58.3%) in serum, 98 patients (54.4%) in PBMCs and 67 patients (37.2%) in serum and PBMC. Total rate of positive HCV RNA detected by nested RT-PCR was higher than FQ-RT-PCR (χ²=11.34, P<0.05). Rate of positive HCV RNA in serum and PBMCs detected by nested RT-PCR were also higher than FQ-RT-PCR (χ²=4.9, P<0.05; χ²=13.76, P<0.05, respectively. Table). Nested RT-PCR is taken as standard method to detect all patients with hepatitis C. By nested RT-PCR, rate of positive HCV RNA in PBMCs, only in serum and both in serum and PBMCs were 22.8%, 27.9%, and 49.3% respectively (Fig. 1). By FQ-RT-PCR, rate of positive HCV RNA in PBMCs, only in serum and both in serum and PBMCs were 16.2%, 31.6% and 30.2% respectively (Fig .2). Of patients with hepatitis C, 22.1% were negative by FQ-RT-PCR but positive by nested RT-PCR (Fig. 2).

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Table. Positive HCV RNA in serum and PBMCs by FQ-RT –PCR and nested RT-PCR

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Fig.1. Constituent ratio of the results of nested RT-PCR

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Fig. 2. Constituent ratio of the results of FQ-RT-PCR

Patients with positive HCV RNA were divided into three groups: groups of patients whose HCV RNA were only positive in serum, PBMCs and in serum and PBMC. Patients with positive HCV RNA in PBMCs were not always with positive HCV RNA in serum and vice versa. If we make the diagnosis of HCV infection by positive serum HCV RNA only, some of the patients were bound to miss detection.

DISCUSSION

Our results showed that some of the patients negative for HCV RNA in serum or PBMCs by FQ-RT-PCR turned out to be positive by nested RT-PCR, especially in PBMC detection. Detection rate of positive HCV RNA was higher in nested RT-PCR than that of FQ-RT-PCR confirming that nested RT-PCR was the more sensitive in detecting HCV RNA and the extent of HCV infection may be grossly underestimated by FQ-RT-PCR. It is strongly recommended to analyse for HCV RNA by nested RT-PCR in serum and especially in PBMCs whenever we encounter patients with suspected nonalcoholic steatohepatitis, cryptogenic cirrhosis or increased aminotransferase of unknown origin.

HCV is one of the major causes of hepatitis related to blood transfusion.^10,11 A more disturbing concern, however, arises with regard to procedures used in blood transfusion. Blood donors with low HCV load in serum or PBMCs are always negative HCV for antibody and normal for aminotransferase, so HCV infection cannot be excluded only by assessing aminotransferase and HCV antibody. We should revaluate the methods of detection for transfused blood. To keep the blood transfusion safe, donors with occult HCV infected should be excluded efficiently.^12-14 Occult HCV infected donors negative for HCV RNA in serum could be discovered positive HCV RNA in PBMCs easily by nested RT-PCR without liver biopsy. Therefore, it is necessary to detected HCV RNA in serum and PBMCs by nested RT-PCR anytime before we make the negative diagnosis of HCV infection.

Relapse following end of treatment of antiviral therapy is common. Patients “cured” after antiviral treatment with persistent viral response may have occult HCV infection in liver or PBMCs. HCV can finish replication within PBMCs.⁷ Thus HCV in PBMCs may be one of the causes of relapse after antiviral therapy. Perhaps nonclearance of HCV RNA in PBMCs is a predictor of persistent response to antiviral therapy and is a reference to formulate the duration of antiviral therapy in chronic hepatitis C.

In conclusion, nested RT-PCR is appropriate for diagnosis of HCV infection, and FQ-RT-PCR is fit for monitoring HCV load. Detection of HCV RNA in serum and PBMC can discover patients infected with HCV maximally. Detection of HCV RNA in PBMCs may be important to assess the virological response to interferon treatment and to predict relapse after antiviral therapy.

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