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Bonino F, Brunetto MR, Negro F. Diagnostic tools from molecular biology. Liver 1992 (Spec. issue) 12: 213-216.

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Ferruccio Bonino, Maurizia Rossana Brunetto and Francesco Negro Laboratory of Hepatology, Department of Gastroenterology, Molinette Hospital, Torino, Italy

Key words: polymerase chain reaction; ligase chain reaction; self-sustdned sequence replication; virus heterogeneity; hepatitis B virus; hepatitis C virus Bonino Ferrucdo MD, Divisione di Gastroenterologia, Ospedale Molinette. Corso Bramante 88, 10126 Torino, Italy, tel. 01 1-6626732, Fax: 01 1-3290726 or 595588. Portable Phone 39 9337 221762

I Exploiting the natural properties of nucleic acids and the enzymes which regulate their synthesis and replication, molecular biology has progressively become a major research tool. More than a decade ago, molecular hybridization techniques began to be used in the diagnostic laboratory, for instance to detect hepatitis B virus (HBV) DNA in routine clinical specimens (1, 2). The exquisite specificity of detecting viremia by means of these techniques has contributed immeasurably to hasten the growth of knowledge in the pathobiology of HBV infection and liver disease. Today, molecular biology techniques have a profound impact on diagnostics, contributing to the etiologic definition of genetic and infectious diseases and, in research, identifying new genes and viruses and unraveling the mysteries of genetic heterogeneity and its impact in disease variability and drug resistance. We present a brief review of principles, potentials and pitfalls of some of the molecular biology techniques that are now being introduced in the routine laboratory and discuss some of the diagnostic concepts that these techniques have changed. Methods for the amplification of nucleic acids: polymerase chain reaction and related techniques

The recent scientific literature is replete with valuable reviews which describe in details the nucleic acid amplification techniques and discuss their advantages and problems (3, 4). A major advantage is the absolute sensitivity, while problems are essentially false-positive results due to contamination with the amplification products and falsenegative results due to genetic heterogeneity of tar-

Accepted for publication 21 February 1992

get nucleic acids at the site that hybridizes to the oligonucleotide primers. The improvement of sensitivity has led to the detection of minute amounts of viruses in unexpected circumstances and in individuals who were negative for conventional virus markers. This has raised medical scepticism and contributed to overemphasis on the risk of falsepositive results. However, many of the controversial results have been confirmed to be specific by the introduction of contamination control systems. As the problems of contamination are being handled, for instance, by the introduction of PCR carryover prevention methods (5, 6), a major pitfall of PCR application to diagnostics remains the unsuitability of this technique to quantitative analysis. The efficiency of oligonucleotides to prime all appropriate target sequences at each cycle of amplification depends on a series of factors: number of oligonucleotides that prime target sequences before rehybridizing to themselves, quantity of enzymes, and dNTPs to engage primed sequences and furnish materials for polymerization. A major problem is variability of these factors. However, a consistent improvement in RNA detection can be achieved with the introduction of recombinant Tth DNA polymerase, a thermostable reverse transcriptase (7). This allows reverse transcription and cDNA amplification in a single tube with the same enzyme. Terminal dilutions of each biological sample are currently used for quantitative analysis but they are costly and impractical. An elegant alternative method is the addition of definite amounts of a target nucleic acid of unusual size (inluding an insert that makes it distinguishable from the typical target sequence because of a 213

Bonino et al. different migration length in gel electrophoresis) to each test sample (8). In spite of a slightly lower sensitivity, this method allows a quantitative evaluation by the analysis of the competitive amplification of artificially added versus naturally occurring target sequences. Finally, another possibility is the quantitation of the PCR product by immunometric assays, for instance using an enzyme-labeled monoclonal antibody that specifically recognizes the stechiometric bonds of double-stranded DNA hybrids (9). A series of other nucleic acid amplification methods are being proposed as valid alternatives to PCR (3); all these techniques maintain three essential steps of PCR; namely, sample denaturation, addition of primers, and hybridization of target sequence. They differ by target amplification methods or because they amplify the detection signal rather than the target sequence. An interesting example of nucleic acid amplification for diagnostics is ligase chain reaction (LCR) that, like PCR, is based on a thermostable enzyme, namely Thermophilus aquaticus DNA ligase (Taq ligase). Four specific primers are used to hybridize the target sequence; two adjacent oligonucleotides hybridize

to one strand and a complementary set of adjacent oligonucleotides hybridizes the opposite strand (Fig. 1). Thermostable ligase will covalently attach only adjacent oligonucleotides that are perfectly complementary and annealed to the target sequence. The products of one cycle of ligation become the targets for the next cycle and the "signal" of the first hybridization is amplified exponentially. Tagging the two adjacent oligonucleotides with different molecules (e.g. biotin and fluorescein in Fig. 1 ) allows a quantitative detection of LCR products by an automated system (e.g. IMx, Abbott Laboratories, Ill. USA) (3). This immuno microparticle assay has an extremely high capturing capacity for double-labeled signals and quantitation of ligated oligonucleotides is not influenced by the number of free unligated labeled primers. Thermocycling is required for both LCR and PCR, but LCR differs from PCR because it amplifies the signal instead of the target and has a superior quantitative potential. An alternative method that can be used for either diagnosis, cloning or sequencing is Self-sustained Sequence Replication or 3SR (Fig. 1). One interesting feature of 3SR is the isothermal transcriptionbased amplification that does not need a thermocy-

LCR

3SR

5-t

A+

5 1

--

--

11'

13'

-L-a)

-r

-a

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Fig. 1. Strategy of nucleic acid amplification by ligase chain reaction (LCR, left) and self-sustained sequence replication (3SR, right). TCS= target complementary sequence, RT = reverse transcriptase, A and B = oligonucleotide primers.

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Diagnostic tools from molecular biology

cler. The strategy of this technique is to produce continuous cycles of reverse transcription and RNA transcription to generate double-stranded cDNA intermediates containing functional T7 promoters. The synthesis of ds cDNA is achieved by using two oligonucleotide primers, one of which is ligated to the single-stranded sequence of T7 promoter. Transcription-competent double-stranded cDNAs are used to produce multiple (50-1000) copies of antisense RNA transcripts of the original target sequence. Antisense transcripts are then converted to T7 promoter containing ds cDNA copies which are used again as transcription templates. The reaction continues in a self-sustained manner under isothermal conditions (42°C) until the components of the reactions become limiting or inactivated (Fig. 1). An interesting advantage of this technique is that the amplification product is a labile RNA rather than a highly resistant DNA; this reduces the contamination problems. However, major disadvantages of 3SR appear to be the difficulty in sizing the RNA products and a poor quantitation potential. At present, unlike PCR, no contamination control system is yet available for the LCR and 3SR methods. Other amplification methods like QBeta replicase and Ampliprobe can be used for the exponential or linear amplification of the signal of molecular hybridization without the need of thermocycling (3). Unlike the automated LCR system, which has the best quantitative potential, all these techniques have limitations to being applied in diagnostic routine (10, 1 1). Therefore, we may foresee that other amplification methods will have to be developed before definite standard procedures will be commonly applied to diagnostics, as has happened for solid-phase enzyme immunoassays. Changing concepts after the introduction of molecular biology techniques: the problem of quantitation

Before the PCR revolution, viruses were detected by isolation and growth in tissue cultures or cell lines or by molecular hybridization using nucleic acid probes, and the sensitivity of these techniques was limited to > 100000 virions. PCR can detect a few molecules of viral nucleic acids and has led to detection of minute amounts of viruses in individuals who are negative by conventional virus replication markers. Now we have to apply to viruses the same diagnostic criteria used for bacteria that are undetectable in biologic specimens but can be amplified in culture (Table 1). This allows an absolute sensitivity and a precise quantitative analysis, as the number of growing colonies in the culture corresponds to the number of bacteria in the original material. According to this, in bacter-

Table 1. Quantitative diagnosis in microbiology Bacteria

Viruses

Quantitative amplification in culture with number of colonies corresponding to number of bacteria in the biological sample

Exponential amplification of nucleic acid by PCR (poorly quantitative)

Definition of clinically meaningful cut-off values YES NOT YET AVAILABLE

iology it is well-known that “sterile” does not mean absence of bacteria, but a number below a welldefined cut-off value. As for bacteria, viruses can also now be detected to absolute sensitivity after PCR amplification; therefore, also for viruses, diagnostic and therapeutic decisions should rely on quantitative cut-offs (Table 1). Unfortunately, at variance with bacterial cultures, most nucleic acid amplification techniques are hampered by quantitation problems. To date, the most promising quantitative assay appears to be LCR automated systems. The quantitative capacity of similar systems has allowed us to overcome the quantitation problems of other assays, e.g. IgM anti-HBc. The improved sensitivity of IgM antiHBc detection has changed the diagnostic concept of this assay. Serum IgM anti-HBc has become detectable in any form of liver disease caused by HBV, independent of the duration of the virus infection. The most elevated serum anti-HBc IgM levels are detected after acute flare-ups of alanine aminotransferases (2-4 weeks) that occur in primary hepatitis B following recent HBV infection or in acutely relapsing chronic hepatitis B where ALT flare-ups can be interspersed by periods of apparent asymptomatic HBV carriage. Therefore, only the characterization of the sedimentation rate of IgM anti-HBc can help to distinguish primary from relapsing acute hepatitis B as 19s IgM prevails in the former and 7s IgM is detected in the latter. PCR has been applied to the study of genetic heterogeneity of hepatitis viruses and allowed the identification of mutants, important in pathobiology of viral hepatitis (14-16). One example are HBV variants that are unable to secrete hepatitis B “e” antigen (HBeAg) which explain, from a virological point of view, the serologicalpattern of patients with chronic anti-HBe-positive hepatitis B who have a more severe liver disease than classic HBeAg-positive hepatitis, with uncommon spontaneous remissions and a low rate of response to interferon (15). In these patients persistence of viral replication (serum HBV-DNA and intrahepatic Hepatitis B core Antigen, HBcAg) is associated with the infection of HBV mutants, defective in the expression of the pre-core region of the C gene that is necessary 215

Bonino et al. for secretion of HBeAg, but does not influence the virus viability. A mutation from guanosine (G) to adenosine (A) at nucleotide 1896 of pre-core region, resulting in a translational stop codon, was found as the most frequent cause (in more than 90% of cases) of defective expression of pre-core. HBeAg-minus mutants have been found in unrelated patients worldwide, suggesting that it is not segregated in restricted geographical areas as originally believed. It prevails during the HBeAg/anti-HBe seroconversion phase and in fulminant hepatitis B. These findings suggest that immunoelimination determines a positive selection of HBeAg-minus HBV; however, the early appearance of these mutants during the immunotolerance phase indicates that their resurgence can be independent of the pressure of immunoelimination. Consistent with this hypothesis is the finding that HBeAg-minus viremia increases significantly over that of wild-type HBV after ALT flare-ups, while wild-type virus always increased before episodes of liver cell necrosis. According to these studies, HBV heterogeneity appears to condition the efficiency and extent of infected hepatocytes’ immunoelimination and variations of the relative prevalence of HBeAg-minus viremia are associated with important events in the natural history of HBV infection. In conclusion, these studies have shown that the HBeAg/anti-HBe serological status of HBV carriers is determined not only by the extent of virus replication and integration of HBV-DNA into cellular DNA, but also by heterogeneity of HBV. A baseline HBeAg-minus viremia higher than 20% is associated, in both HBeAg- and anti-HBepositive patients, with an unfavorable outcome of the hepatitis and a lack of response to therapy (15). A comparative analysis of different hepatitis C virus (HCV) isolates by the same techniques has indicated that a high degree of heterogeneity is present in one region of the HCV genome corresponding at the N-terminus (amino-acids 384-414) to E2/NS1 glycoprotein (gp72) (16). Considerable similarities were found between this hypervariable domain and the gp 120 V3 sequence of human immunodeficiency virus type 1 (HIV-1) RNA both in terms of degree of amino-acid variation and predictive adverse effects of amino-acid substitutions and putative antibody bindings. One of our patients with chronic HCV infection and hepatitis was found to have two E2/NS1 hypervariable region variants, each of which associated with different ALT flare-ups. This patient was treated with interferon and showed a temporary response with normalization of biochemical signs of liver disease, but he experienced a relapse of hepatitis after discontinuation of therapy. During treatment the patient developed antibodies against one of the two E2/NSl hypervariable region synthetic peptides 216

but not against the peptide corresponding to the variant that was found to prevail at the time of hepatitis relapse. These observations suggest that HCV heterogeneity plays an important role in HCV pathogenicity and the N-terminal E2 region of the HCV genome may encode protective epitopes subjected to immune selection. Molecular hybridization techniques have been used also for histopathological studies and detection of viral nucleic acid in liver tissue: normal, cirrhotic or neoplastic (4).Unfortunately PCR is unsuitable for these studies because of the easy contamination of tissues with serum or secretions at the time of sample collection. These problems are overcome by in situ hybridization whose principles, potentials and pitfalls are extensively discussed in the other chapters of this supplement. References 1. BONINO F, HOVERB, NELSON J et al. Hepatitis B virus DNA

in the sera of HBsAg carriers, a marker of active hepatitis B virus replication in the liver. Hepatology 1981: 1: 386391. 2. BONINO F. The importance of Hepatitis B viral DNA in serum and liver. H Heparol 1986; 3: 136141. L G, MUSHAHWAR I K. DNA probe amplifi3. B~RKENMEYER cation methods. J Virol Meth 1991: 35: 117-126. 4. BRECHOT C. Polymerase chain reaction, a new tool for the study of viral infections in hepatology. J Hepntol 1991: 11: 124-129. 5. HICUCHIR, KWOKS. Avoiding false positives with PCR. Nature 1989: 339: 237-238. 6. KWOKS. Procedures to minimize PCR-product carry-over.

In: INNIS M A, GELFAND D H, SNINSKY J J, WHITET J, eds. PCR Protocols. A guide to Methods and Applications Academic Press Inc., San Diego, CA 1990; 17: 142-145. 7. MYERST. Biochemistry 1991: 30: 7661-7666. 8. CHANCR T, DIENSTAG J L, KAPLAN L M. Precise quantitation of hepatitis C RNA using competitive polymerase chain reaction: correlation of clinical course with levels of circulating RNA. Hepatology 1991: 14: 65A, 69. 9. MANTERO G , ZDNARO A, ALBERTINI A, BERTOLO P, PRIMI D. DNA enzyme immunoassay (DEIA): a general method for detecting products of polymerase chain reaction. Clin. Chem 1991: 37: 422429. 10. BARANY F. Genetic disease detection and DNA amplification using cloned thermostable ligase. Proc Narl Acad Sci 1991: 88: 189-193. 11. BARANY F. The ligase chain reaction in a PCR world. P C R Merh App 1991: 1: 5-16. 12. COMPTOM J. Nucleic acid sequence-based amplification. Science 1991: 350: 91-92. 13. FAHYE, KWOHD H, GINCERAS T R. Self-sustained sequence replication (3SR): an isothermal transcription-based amplification system alternative to PCR. P C R Meth App 1991: 1: 25-33. M R, PURCELL R H, ZUCKERMAN 14. BONINOF, BRUNETTO

A J. Genetic heterogeneity of hepatitis viruses: Clinical implications. Supplement 1991: 13: 1-174. 15. BRUNETTO M R, GIARIN M, OLIVERIF, et al. “e” Antigen defective hepatitis B virus and course of chronic infection. J Hepatol 1991: 13: S82-86. A J, CHRISTOPHERSON C, HALLJ E, et al. Sequence 16. WEINER variation in hepatitis C viral isolates. J Heparol 1991: 13: Sd-14.

Diagnostic tools from molecular biology.

Diamostic tools from molecular biolom U U Bonino F, Brunetto MR, Negro F. Diagnostic tools from molecular biology. Liver 1992 (Spec. issue) 12: 213-...
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