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Introduction: Large epidemiological studies require a test system which is rapid, reliable and can be employed for screening maximum number of pathogens at a time. A multiplex-PCR assay has been developed for simultaneous detection and identification of three most common hepatitis virus infections i.e. hepatitis B, C and E in patients with various liver diseases. Methods: A total of 54 serum samples were collected from patients with different liver diseases attending the medical out patient department and wards of Lok Nayak Hospital and associated MAM College, New Delhi during February 2007 and March 2008. All these subjects were also evaluated for serological markers of HBV, HCV and HEV. All samples were tested first by uniplex PCR and followed by multiplex RT-PCR for identification of both DNA and RNA viral genomes using a mixture of three pairs of specific primers for hepatitis B, C and E viruses, respectively. A representative samples positive by uniplex/multiplex RT-PCR were also confirmed by direct nucleotide sequencing. Results: The specificity of multiplex PCR was 100% with high sensitivity 89%, 87%, 74% for HBV, HCV and HEV, respectively. The sensitivity and specificity of RT-multiplex PCR demonstrated a good correlation with that of uniplex PCR. Conclusion: The study suggests that multiplex RT-PCR can serve as a simple and reliable assay for rapid, sensitive and cost-effective method for simultaneous detection of super-infections with HEV particularly in Asian countries as a cause of decompensation of chronic liver disease.
Keywords: Multiplex PCR; Hepatitis B virus; Hepatitis C virus; Hepatitis E virus, liver diseases
Nucleic acid amplification technologies have helped in reliable detection of various infectious agents particularly viruses which are otherwise difficult to detect by standard methods. Amplification of up to several million fold of low-copy-number DNA or RNA genome allows one to make an early and rapid diagnosis of bacterial or viral infections [1-3]. The diagnosis of viral hepatitis poses a unique problem, since causative agents belong to both DNA (hepatitis B) and RNA (hepatitis A, C, D, E, and G) viruses. But it is important to establish the exact etiological agent for better prognostication and management, especially with the advent of anti-viral therapies. The clinically most important viruses that need frequent detection are HBV, HCV and HEV, which may occur as single or multiple infections. Hepatitis B virus (HBV) causes post transfusion acute viral hepatitis as well as chronic hepatitis, and viral DNA has been detected in a small but appreciable percentage of chronic HBsAg-negative hepatitis cases. Hepatitis C virus (HCV), on the other hand is an important cause of chronic hepatitis. Hepatitis E virus (HEV) has been established as the sole cause of endemic hepatitis in Afro-Asian countries and the most important cause for fulminant hepatitis particularly in pregnant women from developing countries [4,5]. Hepatitis E is endemic in many developing countries and occurs both in sporadic forms and in epidemics. The case-fatality rate usually ranges between 0.2% and 4% in the general population but can be as high as 8% among pregnant women [5-7]. Several large water-borne outbreaks have been reported, especially from Africa, the Indian subcontinent, and Southeast Asia [6,8-10]. Developed countries where predominantly sporadic cases have been reported [e.g., the United States, the United Kingdom, or the Netherlands] are not considered to be the areas of endemicity . However, the number of documented infections in industrialized countries seems to be increasing . Still, hepatitis E is regarded by many health care professionals as a typical travel-associated disease. A considerable proportion of autochthonous infections likely remain undiagnosed, and hepatitis of unknown etiology is in fact often caused by HEV .
Serological methods involving detection of HBsAg, anti-HBc IgM and anti-HCV are widely used for clinical diagnosis, while molecular detection of HBV and HCV are the gold standard for diagnosis. However, since viremia is transient, there is no equivalent test for HEV diagnosis. At present, RT-PCR is sensitive and specific method that is commonly used with success to accurately define the true burden of disease due to virus infections [14, 15]. Recently, RT-PCR with specific primers individually or in combination for detection of multiple human pathogens have proved to be comparable to or better than cell culture or other immuno-diagnostic methods for virus detection [16,17]. Conventional uniplex PCR with a single pair of primers that can detect only one target virus genome at a time is expensive. In contrast, multiplex RT-PCR with multiple pairs of specific primers for amplifying different viral genomes in one reaction tube enables to detect two or more targets in a single test. Thus the multiplex PCR becomes highly cost-effective method because of the reduction in labor and reagent and faster detection [18, 19].
Multiplex PCR has been used for detection and typing of hepatitis B virus , and co-infection of both HCV and GBV-C/HGV infection  earlier. The present study was designed with the aim of developing a reliable and specific multiplex PCR assay for the simultaneous detection of both DNA and RNA containing hepatitis viruses highly prevalent in India such as HBV, HCV and HEV in serum samples of patients with liver disease.
2. Material and methods
2.1 Study design
The present study has been designed for the diagnosis and detection of viral hepatitis in patients having super and co-infection of HBV and HCV with HEV, which is more common in Asian countries including India. A total of 54 acute as well as chronic liver disease (CLD) cases were included in study. Amongst them 30 cases were serologically positive for hepatitis B, C or E viruses, and were singly positive for either HBV DNA or HCV/HEV RNA. Therefore 'multiplex PCR' was standardized, using known PCR positive cases of HBV, HCV and HEV. The remaining 24 cases comprises patients with acute and chronic liver diseases, whose clinical, biochemical and serological profile were suggestive of either co-infection with HBV and HCV or superinfection with HEV. These 24 samples with evidence of dual infection were subjected to 'Multiplex PCR' in order to ascertain the clinical utility and reliability of multiplex PCR in serologically positive patients.
To further validate the results of multiplex PCR, the 30 known positive cases which were singly positive for either HBV DNA or HCV RNA or HEV RNA were subjected to cross evaluation by 'Multiplex PCR'. A few representative amplified PCR products of HBV, HCV and HEV were then subjected to direct nucleotide sequencing for validation. Informed consent was obtained from each participant before inclusion in the study. Sera from patients with different liver diseases were sampled once the diagnosis was made. Institutional review boards of Maulana Azad Medical College, New Delhi, have approved the study protocol. A complete aseptic protocol was taken for collecting, handling and storage of the serum samples. The serum was separated and aliquoted into eppendrof tubes and frozen at -70oC until further use.
2.2 Serological tests
Serological tests were performed using commercially available ELISA kits, according to the manufacturer's instructions. The various serological tests performed in all the study samples were HBsAg (SURASE B-96 kit; General Biological, Taiwan); IgM and IgG anti-HBc (anti-coarse MB-96 kit; General Biological, Taiwan); HBeAg (EASE BN-96 kit; General Biological Corp., Taiwan); Anti-HCV (Innotest HCV Ab III, Innogenetics N.V., Ghent, Belgium); IgM anti-HEV (IgM anti-HEV ELISA kit, Genelabs Diagnostics, Singapore).
2.3 Viral nucleic acid isolation
HBV DNA was extracted by QIAamp DNA Mini Kit (Qiagen Inc, Chatsworth, CA) according to manufacturer instructions. HCV or HEV RNA were extracted from serum samples using Trizol L.S reagent (GIBCO BRL, Life Technologies, Maryland, MD,USA) according to the manufacturer's protocol.
2.4 Multiplex RT-PCR for simultaneous amplification of HCV, HEV RNA AND HBV DNA
cDNA synthesis and first round of PCR (PCR-I):
The cDNA was synthesized by reverse transcription (mMuLV-RT, NEB) and then followed by amplification by polymerase chain reaction in 50 Âµl of the PCR master mix-I containing 10X Buffer, 1.5 mM MgCl2, 50 mM Kcl, BSA 50 ng/ml, 2.5 U of Taq DNA (Bangalore Genei, India), 25 mM each dNTPs and 20 pmol each of first round primers for HCV (HC-1and HC-2), HEV (#3043 and #3044), and HBV (HBMF1 and HBMR1) (Table 1), respectively. mMuLV-RT, RNasin, HBV DNA, HCV/HEV RNA were also added. To this master mix-I was added DEPC treated water to make a final volume of 50 Âµl. The cycling condition was 42°C for one hour followed by 10 min extension at 72°C to allow reverse transcription and cDNA synthesis. This is connected to the PCR profile where in the first cycle starts with initial denaturation for 5 min at 94° C, followed by denaturation for 1 min, annealing for 1.5 minute at 50°C and extension for 2 minutes at 72°C for 35 cycles. In the last cycle, the final step of extension at 72°C was carried out for 7 minutes.
2.5 Second round of PCR (PCR-II)
For the second round PCR another master mix-II was prepared, which contains the same constituents as described earlier for master mix-I except RNasin, mMuLV-RT and the outer primes. In this mix, 20 pmol each of second round primers for HCV (HC-3 and HC-4), HEV (HEV-1 and HEV-3) and HBV (HBMR2 and HBMF2) was added(Table1). To the 50 Âµl of the master mix II, 5 Âµl of the first PCR product was added and undergone 35 cycles of amplification using the same temperature and time profile as in the first PCR. The final amplified products were detected and confirmed by comparing with a marker of known molecular weight DNA digest (ØX 174 Hae III digest) by electrophoresis in 3% Nusieve-agarose gel containing ethidium bromide under a UV transilluminator. 251 bp amplicon was detected for HCV, a 343 bp amplicon for HEV while a 480 bp amplicon was detected for HBV . The primers for HBV DNA were designed from the surface region, primers for HCV RNA were selected from the UTR-core region and HEV RNA was obtained from the ORF-1 region. All these primer have been picked from regions which could detect major genotypes of the three viruses, respectively.
2.5 Direct nucleotide sequencing
Direct nucleotide sequencing of the few representative amplified PCR products of HBV, HCV and HEV infected cases was done after purification using QIA quick PCR purification kit (Qiagen Ltd) and directly sequenced from both direction by using an ABI PRISMTM Big DyeTM Terminator Cycle Sequencing Ready Reaction (Perkin Elmer Cetus, Norwalk, CT, USA) with an ABI 310 Genetic Analyzer (Perkin Elmer Cetus).
3.1 Multiplex RT-PCR Optimization and its comparison with uniplex PCR
Three previously developed individual/single RT-PCRs were integrated to generate a multiplex RT-PCR for simultaneous detection of HBV, HCV and HEV genomes. Optimum conditions for the multiplex RT-PCR assay were achieved by doubling the amount of reverse transcriptase (20 U per reaction) for cDNA preparation as well as the concentration of dNTPs (1.2 mM) in the amplification reaction. Increasing the concentration of polymerase did not significantly improve the amplification reaction. It was maintained at 2.5 U per reaction. Particular attention was paid to the concentration of MgCl2 as the optimal concentrations were different in the single virus PCR. The MgCl2 concentration was balanced to 5mM that provided optimal HBV and HCV amplification whereas the efficiency for HEV was a bit reduced. Concentrations of different primers were also adjusted accordingly. Increasing the concentration of HEV specific primers did not compensate totally for the slight loss of efficiency related to the suboptimal MgCl2 concentration.
HBV DNA was positive in 12 (22.2%) cases by uniplex PCR whereas 11 (20.4%) cases by multiplex PCR. Uniplex PCR showed 11 (20.3%) cases positive for HCV RNA but by multiplex PCR, 10 (18.5%) cases were positive for HCV RNA. Similarly, a higher percentage of HEV RNA positive cases were detected by uniplex PCR (33.3%) compared to multiplex PCR (25.9%). Co-infection of HBV and HCV was observed in 3 (5.6%) cases by uniplex as well as multiplex PCR. Co-infection of HBV and HEV was observed in 3 (5.6%) by uniplex PCR and 2 (3.7%) by multiplex PCR. Similarly, Co-infection of HEV and HCV was observed in 2 (3.7%) by uniplex PCR and 1 (1.9%) by multiplex PCR. The agreement value (Kappa) between multiplex PCR and single PCR is 0.75, which shows a full concurrence between multiplex PCR and individual PCR (Table 2). Three previously developed individual PCRs were combined to generate a multiplex PCR for simultaneous detection of HBV, HCV and HEV genomes. Our results show that HBV, HCV and HEV genomes were successfully amplified by the 'multiplex PCR' and were compared to their uniplex PCR.
3.2 Comparison of uniplex and multiplex PCR detection rates with Enzyme Immuno Assay
Out of 54 patients included in the study, 30 patients were singly positive for either HBV or HCV or HEV both in terms of serology and genomic PCR. 24 patients suspected to have multiple viral infections as detected by serology, individual PCR for HBV, HCV, HEV failed to detect infection in 20.8% (5/24) patients. Dual infections were confirmed in 33% (8/24) patients with no particular combination in predominance. Multiplex PCR could not detect any infection in 24% (13/54) patients in whom serology was suggestive of infection by at least one of the three viruses. The agreement value (kappa) between Multiplex PCR and serology is 0.4651 which shows that a moderate agreement exists between Multiplex PCR and serology.
3.3 Comparison of multiplex PCR results with Direct Nucleotide sequencing
The results of multiplex PCR analysis were confirmed by direct nucleotide sequencing of HBV, HCV and HEV gene PCR products, including randomly selected samples from the three viral groups. The multiplex PCR results were confirmed by analyzing the viral nucleotide sequences using online BLAST software. The results obtained by sequencing were similar to that observed by the multiplex PCR technique. The viral nucleotide sequence data reported in this paper were deposited in GenBank using the National Center for Biotechnology Information (Bethesda, MD) Sequin v. 5.26 submission tool under accession numbers EF015721 & EF015715 (HCV), EU370156 & EU370145 (HBV) and DQ318844 & DQ318838 (HEV), respectively.
In view of increasing multiple infections of hepatitis viruses and to diagnose these agents quickly and reliably in blood banking and for disease control, it is important to find simple and cost effective techniques for screening large numbers of samples at a time. Therefore, multiplex PCR amplification technique has been developed for the detection of HBV, HCV and HEV genomes and validated with higher specificity and sensitivity. Screening of infectious viral genomes represents a major step towards detecting infectious blood collected during the pre-seroconversion window period, as well as rare cases of immuno-silent latent infections and, possibly, a large spectrum of virus variants. The results of several studies suggest that the use of NATs could reduce the window period to 11 days for HCV, thereby diminishing further the risk of acquiring infections from blood transfusions [22,23]. However, a disadvantage is that the cost of NATs is 5-10 folds greater than that of the most expensive enzyme immunoassay. To reduce the cost of NATs, two approaches have been suggested, namely the use of pooled plasma testing so that fewer tests are required to screen large numbers of samples, and the use of multiplex assays that can detect several viruses simultaneously. The multiplex PCR has several advantages in that it reduces the number of steps, the cost of reagents and the time taken.
The commonly used ELISA test which detects antibody for the diagnosis of virus infection is applicable only one to two weeks after infection, but it can not explain the virus replication. PCR method could directly detect the viral genome and the status of virus replication. In this study, 20.8% (5/24) and 24% (13/54) of the patients who were positive by ELISA were undetectable by uniplex and multiplex PCR, respectively. This is partly because the patients were in the state of convalescence, and the virus was already cleaned up or was false positive by ELISA due to hyperimmunoglobulinemia, rheumatoid factor or superoxide dismutase.
Currently, the super-infection rate of HBV and HCV was very high, and it was 13.64% to 27.27% reported by Xu et al., . Konomi et al.,  reported a multiplex polymerase chain reaction (PCR) method for simultaneous detection of hepatitis B, C, and G viral genomes. The levels of concordance with the data obtained by conventional single PCR method were 100% for single infection, 98-100% for double infection, and 92% for triple infection. An automatic multiplex system for simultaneously screening HBV, HCV, and HIV-1 in blood donations has been established by Meng et al., . A Chinese group has designed a visual gene-detecting technique using nanoparticle-supported gene probes. With the aid of gold nanoparticle-supported 3'-end-mercapto-derivatized oligonucleotide serving as a detection probe, and 5'-end-amino-derivatized oligonucleotide immobilized on glass surface acting as a capturing probe, target DNA was detected visually by sandwich hybridization based on highly sensitive "nanoamplification" and silver staining. The present visual gene-detecting technique might avoid limitations of the reported methods due to for its high sensitivity, good specificity, simplicity, speed, and cost-effectiveness . The most advanced techniques are based on real-time detection, which essentially shortens the duration of analysis, improves its sensitivity, and at the same time allows quantitative determinations. Such systems do not require any post-amplification detection of the reaction products (electrophoresis and hybridization) and, thereby, substantially reduce the risk of contamination. There are several commercial systems developed for real-time NAT-analysis . Most of these real-time systems detect amplification products using either nonspecific DNA binding fluoroÂphores or specific fluorescently labeled oligonucleotide probes [28,29]. Unfortunately, the real-time PCR used in multiplex reactions has an essential limitation: the interference of absorption and fluorescence spectra of simultaneously used fluorophores allows reliable detection of no more than four reactions in one assay . The use of nonspecific DNA binding fluorophores, such as SYBR Green I, is also limited, allowing for real-time detection of the total amount of all amplification products synthesized in in-tube reactions . This type of dye is commonly used for melting curves measurements providing qualitative analysis .
The present study describes the development of a qualitative multiplex RT-PCR-based assay for the simultaneous extraction, amplification and detection of HBV DNA and HCV and HEV RNA in serum samples. The aims were to select target DNA sequences for HBV and RNA sequences for HCV and HEV in genome regions that show maximum sequence conservation, and to obtain primers capable of detecting simultaneously all genotypes of HBV, HCV and HEV. The development of multiplex NAT is made difficult by the different enzymes and ion optimal concentrations required by each target virus amplification and detection. Theoretically, there is no limit on the number of target sequences simultaneously amplified by multiplex PCR. But the stringency of specific conditions restricted the number of target sequences to be amplified. Hence, false amplifications happen frequently. One of the problems is the disparity of viral gene copy numbers, and genomes with a low copy number might not be amplified sufficiently. In the initial stage of this investigation, a false negative result occurred at 25th PCR cycle in the first round amplification of HEV positive blood sample. After increasing the number of cycles, all three samples gave visible bands in gel electrophoresis; therefore, a sufficient large cycle number is essential. In this experiment, all viral DNA bands were visible at 30th cycle and brighter bands were obtained at higher cycle numbers. Band brightness, related to the amount of the amplified viral DNA after the first round PCR, differed from each other at the same cycle number, e.g. due to different copy numbers in the viral DNA/RNA templates, HEV bands were weaker than those of other two viruses. There were no non-specific amplifications. The other considerations are different amplification efficiencies of distinct templates and cross interactions of the primers. In this experiment, primer pairs for the co-amplification reaction were carefully designed to obtain the similar amplification efficiency and reduce the cross interaction with the aid of the software.
The optimization of the multiplex RT-PCR amplification focused initially on the compatibility of the primer pairs during the RT-PCR amplification in order to avoid potential interactions among primers and between primer and amplicon . A decrease in sensitivity observed when the single RT-PCR reactions were combined was corrected by modifying the RT-PCR mixture to upgrade the limit of detection and decrease the mismatch between the sets of primers. The multiplex RT-PCR system was also tested for possible nonspecific bands, but no false-positive bands were detected with human or a range of virus genomes in a simultaneous amplification. For thermal cycler program, the annealing temperature was selected appropriately at 50°C in order to decrease nonspecific priming or other artifacts. Times for annealing and extension were minimized to reduce the possibility of nonspecific and unexpected amplification.
A multiplex reverse transcriptase polymerase chain reaction (RT-PCR) was also applied for the simultaneous detection of hepatitis A virus (HAV), poliovirus (PV) and simian rotavirus (RV-SA11), and compared with specific primers for each genome sequence. Three amplified DNA products representing HAV (192 bp), PV (394 bp) and RV (278 bp) were identified when positive controls were used . A multiplex RT-PCR method was described for the simultaneous detection of all four viruses in combination with a plant mRNA specific internal control which could be used as an indicator of the effectiveness of the extraction and RT- PCR. The upper detection limit for the four viruses was at an extract dilution of 1/200 . Multiplex method for HBV/HCV/HIV-1 has been used for screening 6,805,010 units of serologically negative donation and 112 HBV DNA-positives, 25 HCV RNA positives and 4 HIV-1 RNA positives were screened out and prevented transfusion of the positive blood [36,37].
The analysis of the 54 serologically characterized clinical serum samples showed almost perfect correlation with the expected status. For HBV, HCV and HEV concordance was perfect with the exception of two, two and six samples positive by uniplex PCR were found negative with multiplex PCR, respectively. The specificity of multiplex PCR for all three viruses was 100% but the sensitivity for HBV was 88.9% Â± 14%; for HCV was 87.5% Â± 18%, HEV was 73.9% Â± 18%, respectively. Wang et al., demonstrated the sensitivity of multiplex normalized PCR method was 78.6%, 75% and 83.3% for the detection of HBV DNA, HCV RNA, and super-infection of HBV and HCV, with specificity of 80%, 90% and 70%, respectively . Simultaneous amplification of HBV DNA and HEV RNA virus using multiplex PCR system developed by Singh et al., showed all samples of co-infection could be detected for HBV and or HEV successfully . These are good enough for a diagnostic assay. This implies that multiplex PCR designed by us can identify all the cases correctly which are negative for any of the viruses and does not give any false positive results when compared to results obtained by uniplex PCR. Although the sensitivity of multiplex PCR is less than 100% for all three viruses but its specificity of 100% ensures promising future, when used as a routine diagnostic procedure. It can detect both DNA and RNA simultaneously and the diagnosis can be completed in a few hours. It is not only suitable for clinical diagnosis, but also suitable for the screening of HBV and HCV from blood donors to prevent the transmission of these diseases.
The multiplex PCR described is an easy and cost-effective method for simultaneous screening out for any evidence of concomitant infection of HBV, HCV and HEV in patients with chronic liver diseases. HEV super-infection and HBV reactivation are major cause of acute on chronic liver failure in Asia and this test could be very important in Asian countries for simultaneous screening for evidence of super-infection with HEV as a cause of decompensation of chronic liver disease.
Acknowledgement: This work was supported by a research grant from Department of Science and Technology (DST).
Conflict of interest statement: None declared
Ethical Approval: Institutional review boards of Maulana Azad Medical College, New Delhi, have approved the study protocol.