Assay for hepatitis C virus in peripheral blood ...

Clinica Chimica Acta 283 (1999) 1–14

Assay for hepatitis C virus in peripheral blood mononuclear cells enhances sensitivity of diagnosis and monitoring of HCV-associated hepatitis a, a b Mostafa K. El-Awady *, Somayia M. Ismail , Mohamed El-Sagheer , Yousry A. Sabour a , Khalda S. Amr a , Essam A. Zaki c a

Department of Human Genetics, National Research Center, Dokki, Tahrir St. 12311, Cairo, Egypt b Department of Microbiology, National Research Center, Dokki, Tahrir St. 12311, Cairo, Egypt c Genetic Engineering and Biotechnology Research Institute ( GEBRI), Mubarak City for Scientific Research, P.O. Box 5, Manchyet El Aluma, El Agami, Alexandria, Egypt Received 24 July 1998; received in revised form 14 December 1998; accepted 29 December 1998

Abstract Hepatitis C virus (HCV) is a major etiological factor in chronic hepatitis affecting up to 24% of blood donors in Egypt. Since fluctuating levels of HCV RNA loads, including undetectable values, have been frequently observed in sera of chronic hepatitis patients, this study was designed to assess the sensitivity of PCR amplification for the plus- and minus-RNA strands in peripheral blood mononuclear cells (PBMC) compared to single serum PCR assay. Since the latter test detects viremia in only 79.5% of seropositive cases, the highest sensitivity for HCV diagnosis was achieved (93.20% when applying the combined triple test including PCR amplification of plus-strand in serum, together with plus-strand in PBMC and minus-strand in PBMC.The results of this study indicate that the triple test provides significant information on extrahepatic replication of HCV in a sizable sample of seropositive subjects (429 cases) and improves the assessment of HCV viremia. The cost / effectiveness and speed were upgraded by using capillary / air rapid thermal cycler. The use of the triple assay in HCV diagnosis and post-therapy monitoring is recommended.  1999 Elsevier Science B.V. All rights reserved.

*Corresponding author. Tel.: 1 20-2-336-2609; fax: 1 20-2-360-1036. E-mail address: [email protected] (M.K. El-Awady) 0009-8981 / 99 / $ – see front matter  1999 Elsevier Science B.V. All rights reserved. PII: S0009-8981( 99 )00007-8

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M.K. El-Awady et al. / Clinica Chimica Acta 283 (1999) 1 – 14

1. Introduction Hepatitis C virus (HCV) is a small RNA virus that is the major causative agent of post-transfusional non-A non-B hepatitis [1–3]. The genome consists of a single ORF (open reading frame) encoding a large polyprotein precursor that is cleaved to yield individual structural and non-structural viral proteins [2,4]. HCV is taxonomically related to flaviviruses and pestiviruses. By analogy with these viruses, the HCV genome is presumed to replicate by using the negativestrand as a template [4,5]. HCV infection is a major etiological cause of transfusion-associated hepatitis [6–8]. In Egypt, it represents the highest donor infection rates recorded so far, where 14–24% of blood donors are anti- HCV positive [9–13]. Monitoring the viremia pre- and post-antiviral therapy through the detection of viral RNA by use of qualitative reverse transcription-polymerase chain reaction (RT-PCR) and various quantitative methods has become the most frequently used and sensitive technique. Although these quantitative assays accurately determine the amount of virus in plasma or serum, HCV is also found in bone marrow and peripheral blood mononuclear cells (PBMCs) [14–17], suggesting that the virus may have an extrahepatic localization and pathogenetic role. These cellular reservoirs of HCV are not detected by plasma RNA detection methods [18]. In addition, a major problem associated with assessment of viral loads in serum samples is that fluctuating levels of RNA are commonly observed including periods during which viremia is undetectable [19–23]. Consequently, routine serum or plasma measurements of HCV RNA may not provide an accurate determination of the total HCV load present in peripheral blood. The accurate determination of HCV RNA is important, since high serum levels of HCV RNA appear to correlate with a poor response to interferon therapy [24]. The aim of this work is to assess HCV viremia by use of simultaneous detection of plus and minus RNA strands in PBMC, together with serum RNA. The advantage of the triple assay of HCV RNA in serum and in PBMC (both plus and minus strands) over serum RNA and other possible combined assays in assessment of the status of HCV viremia in 429 infected patients was evaluated.

2. Material and methods

2.1. Patients The subjects were 429 HCV seropositive patients including 347 males and 82 females. Patients were referred to the medical service unit at the National research Center over a period of 1 year. No patients received antiviral therapy throughout the study. Three patients (one healthy carrier and two with chronic


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hepatitis) were referred weekly to assess fluctuation of viral loads and intracellular replication during a 9-week period.

2.2. Methods Anti-HCV antibodies were assayed using IgG third generation commercial kits (Sorin Biomedical, Italy).

2.3. Isolation of PBMC and extraction of RNA Peripheral blood cell samples were isolated according to [25]. Briefly, peripheral blood samples were diluted into 5 vol of a freshly made red blood cell lysis buffer (38.8 mmol / l NH 4 C1, 2.5 mmol / l K 2 HCO 3 , 1 mmol / l EDTA, pH 8.0), incubated at room temperature for 10 min and nucleated cells precipitated and washed in the same buffer to remove adherent viral particles before lysis in 4 mol / l guanidinium isothiocyanate containing 25 mmol / l sodium citrate, 0.5% sarcosyl and 0.1 mol / l b-mercaptoethanol. Cellular RNA was extracted using the single-step method described originally by Chomczynski and Sacchi [26] and modified by Fong et al. [27] and Goergen et al. [28].

2.4. RT-PCR of genomic and antigenomic strands of HCV Reverse transcription-nested PCR was carried out according to Lohr el al. [25] with few modifications. Retrotranscription was performed in 25 ml reaction mixture containing 20 units of reverse transcriptase (Finnzyme) with either 400 ng of total PBMC RNA or 3 ml serum as template, 40 units of RNasin, (Clonetech, USA), a final concentration of 0.2 mmol / l from each dNTP (Promega, USA) and 50 pmol of primer SRl (for plus-strand) or 50 pmol of primer SF1 (for minus strand), incubated at 428C for 60 min, and then denatured at 988C for 10 min. Amplification of the highly conserved 59-UTR sequences was done using two rounds of polymerase chain reaction with two pairs of nested primers. First round amplification was done in 10 ml reaction containing 20 pmol from each of SR1 and SF1, a final concentration of 0.2 mmol / l from each dNTP, 1 ml 10 3 Ficol buffer containing 5 mg BSA, 10 mol / l MgCl 2 (Idaho Technology, USA), 1 unit of Taq DNA polymerase (Primzyme, Germany) and 1 ml of reverse transcription product. Reaction components were aspirated in capillary tubes that were flame sealed and placed in the rapid air cycler (Idaho Technology, USA). The thermal cycling protocol was as follows: initial denaturation was one cycle for 15 s at 948C, followed by 40 cycles of the following denaturation at 948C for zero time, annealing at 558C for zero time and extension at 728C for 15 seconds. The second round of amplification was similar to the first round, except for 10 3 cresol red buffer containing 5 mg

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BSA, 20 mmol / l MgCl 2 (Idaho Technology, USA), 20 pmol from 1TS and SF2 nested primers and 1 ml from the first round PCR. The total 10 ml of amplified products were resolved on 3% agarose gel. Ethidium bromide fluorescence was visualized by UV. Successful amplification of HCV sequences yields a fragment of 183 bp. Primer sequences are as follows; SR1 (339–321): 59 tgc acg gtc tac gag acc t 39; SF1 (82–101): 59 gcc atg gcg tta gta tga gt 39; 1TS (287–267): 59 gcg acc caa cac tac tcg gct 39; SF2 (105–124): 59 gtg cag cct cca gga ccc 39. To control for false detection of negative-strand HCV RNA [29] and known variations in PCR efficiency [30,31], specific control assays and rigorous standardization of the reaction were employed. Specific control assays were included: (i) cDNA synthesis without RNA templates to exclude product contamination, (ii) cDNA synthesis without RTase to exclude Taq polymerase RTase activity, (iii) cDNA synthesis and PCR step done with only the reverse or forward primer to confirm no contamination from mixed primers. These controls were consistently negative. In addition, cDNA synthesis was carried out using only one primer present followed by heat inactivation of RTase activity at 958C for 1 h, in an attempt to diminish false detection of negative-strand prior the addition of the second primer.

3. Results

3.1. Fluctuation of HCV viremia To examine the phenomenon of temporary disappearance of serum HCV RNA, three patients (two with chronic hepatitis and one healthy carrier) were subjected to a weekly assay of HCV RNA in serum as well as the detection of both genomic and antigenomic RNA strands in PBMC. These results (Table 1) demonstrated that the two patients with chronic hepatitis displayed intermittent negativity of serum HCV viremia. Patient 2 (chronic hepatitis) had no detectable viral RNA over the fourth and fifth weeks of the study. RNA was again detectable in week 6, then disappeared during the seventh and eighth weeks. HCV RNA disappeared from serum of patient (1) (chronic hepatitis) only during week 5. It is worth noting that, on one hand, minus RNA strand was mostly absent in PBMC concomitantly with absent viremia in both cases. On the other hand, the temporarily negative serum viremia could not be observed in patient (3) (healthy carrier) with any apparent correlation with PBMC viral status.

3.2. Sensitivity, reproducibility and specificity of the nested RT-PCR To establish the sensitivity, reproducibility and specificity of the nested RT-PCR used in this study, we analyzed three patients with various profiles of


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Table 1 Longitudinal weekly analysis of HCV RNA plus-strand in sera and plus and minus RNA strands in PBMC in two patients with chronic hepatitis (1 and 2) and one healthy carrier (3) Weeks

Patient 1

Patient 2

Patient 3

Serum

PBMC ( 1 )

PBMC (2)

Serum

PBMC ( 1 )

PBMC (2)

Serum

PBNMC ( 1 )

PBMC (2)

1

1

2

2

1

1

1

1

2

1

2

1

1

1

1

1

1

1

2

1

3

1

1

1

1

1

1

1

1

1

4

1

1

1

2

1

1

1

2

2

5

2

1

2

2

1

2

1

1

2

6

1

1

2

1

2

1

1

1

2

7

1

2

1

2

1

2

1

1

1

8

1

1

2

2

1

2

1

2

2

9

1

1

1

1

1

1

1

2

2

viral localization and replication and a healthy blood donor (Figs. 1–4). Patient 1 (Fig. 1), had positive-strand HCV RNA in PBMC and no viremia. The RNA was extracted and the cDNA was synthesized following the protocol described above, retested in triplicate, using primer SR 1 (for plus-strand) and SF1 (for minus-strand) and different annealing temperatures. Plus-strand HCV RNA was only detected using annealing temperatures of 55 and 578C, respectively, while all samples that employed SF1 (for minus-strand) did not show amplification product, suggesting the absence of false-negative strand either by false priming during cDNA synthesis or contamination from mixed primers. Patient 2 (Fig. 2) was identified as having replicating stage of HCV RNA with no viremia. Negative-strand HCV RNA was only amplified using annealing temperatures of

Fig. 1. Sensitivity of the polymerase chain reaction for detection of HCV positive- and negativestrand RNA in PBMC using different annealing temperatures. This patient had positive-strand HCV RNA in PBMC.

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Fig. 2. Sensitivity of the polymerase chain reaction for detection of HCV positive- and negativestrand RNA in PBMC using different annealing temperatures. This patient had negative-strand HCV RNA in PBMC.

53 and 558C, respectively, with the failure of SR1 (for plus-strand) to show amplification product, thus confirming the absence of false priming during cDNA synthesis or contamination from mixed primers. Patient 3 (Fig. 3) had both mature and replicating HCV RNA in PBMC with no viremia. Plus-strand HCV RNA was solely detected using annealing temperatures of 55 and 578C, while negative-strand HCV RNA was visible using annealing temperatures of 53 and 558C, thus confirming high level of reproducibility of the method. In addition, it substantiates the specificity of strand amplification. Fig. 4 represents a healthy blood donor who was anti-HCV-negative and showed no amplification

Fig. 3. Sensitivity of the polymerase chain reaction for detection of HCV positive- and negativestrand RNA in PBMC using different annealing temperatures. This patient had both positive- and negative-strand HCV RNA in PBMC.


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Fig. 4. Sensitivity of the polymerase chain reaction for detection of HCV positive- and negativestrand RNA in PBMC using different annealing temperatures. A healthy blood donor who was anti-HCV negative.

product, confirming the exclusion of HCV RNA contamination during sample preparation, RNA extraction and RT-PCR.

3.3. Sensitivity of HCV RNA assays To evaluate the sensitivity of HCV RNA detection assays in seropositive patients, we compared the results obtained from six different assays: (1) serum PCR of plus-strand (S), PBMC plus-strand (PBMCp), PBMC minus-strand (PBMCm), S 1 PBMCm (double assay 1), S 1 PBMCp (double assay 2) and S 1 PBMCp 1 PBMCm (triple assay). The data presented in Table 2 show the following: using S assay, HCV-RNA was detected in 341 (79.50%) patients while 88 (20.5%) seropositive patients demonstrated negative viremia. When using PBMCp assay the presence of plus-strand HCV RNA was detected only in 295 (68.8%) and was undetectable in 134 patients (31.2%). However, in the case of employing PBMCm assay the minus-strand HCV RNA was detected in PBMC RNA derived from 199 subjects (46.4% whom might be identified as patients with replicative virus, whereas 230 patients (53.60%) lacked minus-strand HCV RNA in their PBMCs. To evaluate the sensitivity of double assay 1 based on detection of S 1 PBMCm; HCV RNA was detected in 347 patients (81%) with an additional six positives (1.4%), whereas 82 patients (19.1%) had undetectable HCV RNA. Employing the double assay 2 S 1 PBMCp, greatly improved the sensitivity over S and double assay 1 in diagnosing HCV infection where 394 (91.8%) patients demonstrated plus-strand in serum and / or in PBMC with additional 53 positives (12.3%). The greatest sensitivity possible in this study in diagnosing HCV infection was


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Table 2 Relative sensitivities of HVC RNA PCR assays in sera and PBMC (plus- and / or minus-stands RNA) in 429 consecutive seropositive individuals a

No. positive with one test S PBMCp PBMCm Additional positives with second test S 1 PBMCp S 1 PBMCm Additional positives with all three tests S 1 PBMCm 1 PBMCp a

P

N

341 (79.5%) 295 (68.8%) 199 (46.4%)

88 (20.5%) 134 (31.2%) 230 (53.6%)

53 (12.3%) 6 (1.4%) 59 (13.7%)

N, negative; P, positive.

achieved when employing the triple assay, S 1 PBMCp 1 PBMCm where 400 (93.2%) out of 429 HCV seropositive patients demonstrated HCV in serum and / or PBMC RNA, with an additional 59 positives (13.7%). The remaining 29 cases (6.8%) represent this fraction of seropositive HCV patients in whom the presence of HCV virions could not be documented even by the triple assay.

4. Discussion HCV is known to circulate in peripheral blood in a variety of forms [32,33]. Consequently, quantitative measurements of plasma or serum do not provide a complete estimate of the circulating peripheral blood HCV burden [18]. The accurate determination of HCV RNA is important, since high serum levels of HCV RNA appear to correlate with a poor response to interferon therapy [24]. In addition, a major problem is associated with assessment of viral loads in serum samples in that fluctuating levels of RNA are commonly observed including periods during which viremia is undetectable [19–23]. Furthermore, disappearance of HCV RNA from patient’s serum may not be a reliable predictor of sustained response [34,35]. This may be due to HCV contained in the peripheral blood cells or to the failure of current commercial plasma or serum assays to detect HCV RNA still present in peripheral blood [18]. The aim of the current study is, therefore, to assess the HCV viremia by use of


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simultaneous detection of plus- and minus-RNA strands in PBMC besides serum RNA. The advantage of the triple assay of HCV RNA in serum and in PBMC (both plus- and minus-strands) over serum RNA and other possible combined assays in assessment of the status of HCV viremia in 429 infected patients was evaluated. The results of our study suggest that the sensitivity for HCV detection was significantly increased by employing the triple assay of HCV RNA in serum and in PBMC (both plus- and minus-strand) in comparison with serum RNA. The S assay derived from 429 HCV seropositive individuals could only detect viremia in 79.5% of cases. When detecting the presence of plus-strand HCV RNA in PBMC, only 68.8% cases were detected. On the other hand, in the case of detecting the minus-strand HCV RNA in PBMC, only 46.4% were documented. The sensitivity of double assay 1 based on detection of S 1 PBMCm; HCV RNA was increased by the detection of an additional six positives (1.4%), whereas the double assay 2 S 1 PBMCp, had greatly improved the sensitivity over S and double assay 1 by the detection of an additional 53 positives (12.3%). However, the greatest sensitivity possible in this study in diagnosing HCV infection was achieved when employing the triple assay, S 1 PBMCp 1 PBMCm, where 400 (93.2%) out of 429 HCV seropositive patients demonstrated HCV in serum and / or PBMC RNA with an additional 59 positives (13.7%). The finding of relatively increased sensitivity for HCV detection in PBMC compared to single serum is consistent with a recent study by Schmidt et al. [18] that indicated PBMC contain a significant proportion of HCV that is not identified by standard plasma or serum RNA detection methods. However, the results of previous studies have been conflicting: some authors have reported inconstant presence of HCV in B and T lymphocytes [36,37], but using specific monoclonal antibody and flow cytometry, they found the presence of HCV core antigen in monocytes but not in lymphocytes [16]. Nevertheless, recently various authors have established the presence of HCV RNA in PBMC [18,38,39]. A major inherent problem in HCV diagnosis is the fluctuating nature of the viral load throughout the course of infection and even at post-antiviral therapy. In this study, we evaluated the sensitivity of PCR assay in serum samples (S) derived from 429 HCV seropositive individuals. The S assay could only detect viremia in 79.5% of cases. The remaining 20.5% of seropositive individuals who demonstrated no detectable RNA may constitute those subjects who had immune programming to HCV antigens following acute infection that did not progress to chronic hepatitis. The presence of confirmed antibodies may reflect ongoing viremia [40], although this may also indicate past infection [41]. Furthermore, this 20.5% fraction constitutes those patients with viremia but only assayed during periods where viral RNA was below detection limits by the sensitive PCR technology. The longitudinal study of serum viral RNA on the three HCV

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patients has confirmed the nature of intermittent negativity of HCV viremia in the two chronic hepatitis patients, but not in the healthy carrier subject. Such temporary disappearance of HCV RNA from serum lasted for a maximum of 2 weeks. However, our analysis does not rule out the possibility of positive viremia over short intervals within the 2-week period. These results are consistent with earlier reports on HCV RNA fluctuation [19–23] and may establish the presence of such phenomenon in HCV subtypes pertinent to the Egyptian population. It seems that the viral RNA fluctuations become a standard phenomenon in documented chronic HCV diseases. This explanation is supported by the early experimental infection of chimpanzees where viral RNA fluctuation became evident upon entering into chronicity [42]. Several laboratories have reported evidence of HCV replication not only in liver but also in PBMC of infected individuals [37,42–44], but not in others [45,46], and it has been suggested that active replication of HCV in PBMC represents a large extrahepatic reservoir for viral replication [47,48]. Our data provide support for this concept by suggesting that an estimated 46.4% of HCV-infected subjects has extrahepatic replication. This finding signifies the importance of detecting minus-strand HCV RNA in PBMC for the understanding of the life cycle of HCV and might pave the way for a reliable marker in monitoring viral responses to antiviral therapy. Recently, Cabot et al. [49] analyzed the quasispecies of HCV in paired liver and serum samples from human patients with chronic hepatitis C and demonstrated that the HCV strain detected in the serum does not necessarily reflect the strain replicating in the liver. Furthermore, Gretch et al. [50] reported the emergence of viral strains, which have reduced hepatotropism after liver transplantation in patients infected with HCV. These findings taken with our results firmly suggest that replication of HCV in PBMC is a large extrahepatic reservoir for viral replication. Negative HCV RNA strands were detected with the nested RT-PCR [25] in the present study. Doubts have been raised as to the validity of negative-strand HCV detection [29]. To circumvent this problem, Gungi et al. [51] developed a strand-specific RT-PCR strategy combined with chemical modification of RNA samples at the 39 end and Lanford et al. [45] developed a highly strand-specific rTth method of RT-PCR. It should be noted that our experiments described above were not carried out using either of the aforementioned methods. However, four lines of evidence strongly suggest that the strand amplification of the positive- and negative-strand HCV RNA employed in this study were specific. First, to control for false detection of negative-strand HCV RNA [29] and known variations in PCR efficiency [30,31], specific control assays and rigorous standardization of the reaction were employed (see Section 2). Second, positive- and negative-strand RNA were amplified using nested PCR [25]. Nested PCR provides the opportunity for only the legitimate product to be


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amplified in the second round of amplification using either 1TS or SF2 internal primers, thus eliminating the opportunity for false priming. Third, when the reaction was carried out using different RNA templates (taken from different patients), no duplications were found, but specific variability consistent with variability of various RNA templates. These results are further supported by the fact that cases solely reporting negative-strand and not positive-strand HCV RNA. Finally, when different annealing temperatures were employed, specific and distinct conduct of primer SR1 (for positive-strand) and SF1 (for negativestrand) was observed indicating the presence of different RNA templates. These various lines of evidence confirm a high level of the specificity of strandamplification. In addition, it substantiates the high level of reproducibility. A healthy blood donor who was anti-HCV negative and showed no amplification product, indicating the exclusion of HCV RNA contamination during sample preparation, RNA extraction and RT-PCR. In conclusion, our data demonstrate that employing the triple assay, PCR amplification of plus-strand in serum 1 plus-strand in PBMC 1 minus-strand in PBMC, can relatively increase the sensitivity. Consequently, this method promotes a practical clinical advantage over current plasma or serum detection systems. In addition, it is recommended for post-therapy monitoring and in screening programs in human populations. The cost effectiveness was successfully enhanced using the capillary tubes in the rapid air cyclers where it was possible to save up to 80% of PCR expenses and to perform the thermal amplification in approximately one-ninth of the time required in the metal block cyclers.

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