diagnostic tests for hepatitis c virus

Clinical Microbiology Newsletter Vol. 22, No. 19 October 1, 2000

Diagnostic Tests for Hepatitis C Virus Laura Chandler, Ph.D. Clinical Microbiology Division University of Texas Medical Branch Galveston, TX 77555-0740

Introduction/Background Hepatitis C virus (HCV) was iden-

tified and characterized in 1989, after a long and challenging search for this agent of non-A, non-B hepatitis. A portion of the genome was cloned (l), and nucleotide sequencing of the cloned fragments revealed that the agent was a virus related to members of the family Fluviviridae. The International Committee for the Taxonomy of Viruses (ICTV) has recently proposed that HCV, along with related agents, be assigned genus status within the Flaviviridae (2). HCV is remarkable in that not only the initial identification, but most of the subsequent characterization of this important disease agent have been achieved through the use of molecular, rather than classical, virological techniques. These molecular techniques have also provided the foun- dation for all of the diagnostic methods in use today, including both serological and molecular tests.

Virion and Genome Structure HCV virions are comprised of an

RNA genome, surrounded by an icosa- hedral capsid, or core, and an envelope containing two glycoproteins, El and E2. The viral particles are approximately 50 nm in diameter, and the core is approximately 30 nm (3). The genome of HCV is single-stranded, positive-sense RNA, approximately 9.5 kb in length. The structural genes (core, C; envelope, El and E2) are located in the 5’ portion of

the genome and the nonstructural genes (NS2,3,4a, 4b, 5a, 5b) in the 3’ portion. There are untranslated (UTR) sequences flanking the open reading frame at both the 5’ and the 3’ ends of the genome (3).

Genetic Variability of HCV An important feature of HCV is its

high level of genomic sequence heterogeneity. Genetic variability has implica- tions for pathogenesis and persistence of the virus (4). Genetic heterogeneity also impacts the design and interpreta- tion of diagnostic methods for HCV. Nucleotide sequence heterogeneity does not occur equally in all areas of the HCV genome. Certain parts of the genome are conserved, while other areas show various levels of genetic variation, with some areas showing extreme sequence variability. Within the coding region, the envelope genes, El and E2, exhibit the widest genetic variation (4), while the core protein gene is the most highly conserved (3). The 5’ UTR and portions of the 3’ UTR are highly conserved (3).

Based on nucleotide sequencing and subsequent phylogenetic analyses of four regions of the genome (core, El, NS4, and NS5 genes), six major groups of HCV virus, called genotypes have been defined (3). Genotypes are designated with numbers (genotypes 1 to 6). Within genotypes, subgenotypes also occur, and are designated with lowercase letters, Within an infected individual, high levels of genetic mutation occur during viral replication, in a hyper- variable region (HVRI) of the E2 gene. This extreme genomic variability results in a population of variant RNA genomes known as quasispecies. The HVRl region is probably the main neu-

tralization epitope of HCV and the mutations that accumulate in this region allow the virus to escape neutralization, contributing to the establishment of per- sistent infections (5).

The Role of Genotypes and Quasispecies in HCV Disease

The discovery of genetic heterogeneity of HCV and the subsequent assign- ment of genotypes led to a number of

In This Issue

Diagnostic Tests for Hepatitis C Virus . . . . . . . . . . . . .145

The inability to culture the Hepatitis C virus (HCV) has resulted in a varieo of new testing approaches for the diagnosis of HCV disease. The ability to interpret data from the classic serologic approach is complicated by the presence offalse-positive and -negative antibody results and the chronicity and asymptomatic nature of disease. The newer tests, developed with the information derived from the molecular structure of the virus, include expensive immunoblotting or molecular ampli-

fication techniques, none of which are approved by the Food and Drug Administration. In addition, tests to determine the genotype of HCV are being used as a means to direct anti-viral therapy. The use of these

supplemental tests in various clinical situations is discussed.

Massilia timonae: An Unusual Bacterium Causing Wound Infection Following Surgery . . . . 149 A case report

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studies designed to investigate the clinical significance of HCV genotypes. Conflicting results have been reported regarding the relationship between genotype and HCV disease. Several investigators have reported that genotype 1 b infections have a more aggres- sive clinical course and are more often associated with the development of hepatocellular carcinoma (6), while others have reported no correlation at all (7). A positive relationship between the genotype and level of viremia, and between viremia and disease (8) has been reported. One area in which investigators generally have agreed is the role of genotype with respect to response to antiviral therapy. There is a clear correlation between genotype lb and reduced responsiveness to interferon, while genotypes 2 and 3 respond more positively to therapy, and most investigators now agree that the viral genotype is an important predictor of response to antiviral therapy. The role of quasispecies in pathogenesis of HCV disease has also been investigated (9). Quasispecies evolution is associated with reduced response to interferon therapy (10).

General Concepts in HCV Diagnosis

There are two settings in which HCV diagnosis is performed: (i) screening low-risk populations such as blood donors and (ii) in clinical diagnostic laboratories, testing patients with risk factors for HCV or when otherwise clinically indicated (such as in symptomatic patients). Diagnosis of HCV infection is initially performed using a screening test, such as the enzyme immunoassay (EIA) for antibody, followed by supplemental testing to confirm HCV infection (11). Supple- mental tests for HCV include serological assays and molecular assays for detection, quantification, and genotyp-

ing of viral RNA. The choice of supplemental diagnostic tests used for HCV depends on the patient population and the clinical setting. Molecular techniques are now being used in many laboratories, but are not yet fully standardized. Only serological tests are approved by the U.S. Food and Drug Administration (FDA) for HCV diagnosis at this time (11); the supplemental molecular tests have not been approved and are considered research or investi- gational use only. If a testing laboratory develops and uses its own test, i.e., “home brew,” the performance charac- teristics must be developed in-house.

Screening Tests for HCV Testing The screening assay currently in use

for initial testing for HCV infection is antibody detection by EIA. EIAs are used for screening because they are rel- atively easy to perform, inexpensive, and are available in automated formats (12). HCV EIAs are all based on cloned recombinant or synthetic proteins, but the antigens incorporated into each test vary by generation. The first generation EIA (EIA- l), no longer in use, was based on a single recombinant antigen, ~100-3, derived from the NS4 gene. The EIA- 1 lacked sensitivity and specificity and was quickly replaced by the subsequent generations of tests containing different antigens. The EIAs in use today are the second generation (EIA-2) and third generation (EIA-3) formats (12). These EIAs have increased sensitivity and specificity compared to the EIA- 1. The EIA-2 test, which is more sensitive and specific than EIA- 1, contains HCV antigens derived from the core (C22-3) and NS3 (C33c) genes in addition to the NS4 (~100-3) gene.

The EIA-3 was developed to improve sensitivity and specificity. The EIA-3 format incorporates the same proteins as EIA-2 and an additional antigen,

derived from the NS5 region. The NS3 protein was also modified, resulting in increased sensitivity. In high prevalence populations, the EIA:3 is very sensitive (97 to 100%) (13). EIA-3 is primarily used in the setting of screening low- prevalence populations such as blood donors; in this population, specificity is 99.3 to 100% (13). While this test is improved compared to EIA-2, a small percentage of false positives in low- prevalence populations still occurs with EIA-3.

The recombinant antigens used in the serologic assays are derived from genotype la. This leaves the potential for false-negative results in patients who are infected with other genotypes. To preclude this type of problem, a genotype-specific, screening antibody test is under development. The test uses a multiple-epitope fusion antigen and incorporates proteins from the envelope region. This chimeric antigen contains the immunodominant.epitopes from the structural and genotype-specifi$ regions. Preliminary studies indicate that the genotype-specific envelope antigens could be used in EIAs to increase both the sensitivity and specificity (14). The EIAs incorporating genotype-specific antigens are not commercially available at this time.

Supplemental Tests for HCV Supplemental tests for HCV diag-

nosis fall into two major categories: antibody detection and RNA detection using molecular assays. The term “supplemental” rather than “confirmatory” is used to describe these tests because none have been approved by the F.D.A. as an intended use for confirmation of disease. There are several factors that are used when deciding when supplemental testing for HCV is necessary, and if so, which approach should be used. In populations with a low previ-

NOTE: No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, inshuctions or ideas contained in the material herein. No suggested test or procedure should be carried out unless, in the reader’s judgment. its risk is justified. Because of rapid advances in the medical sciences, we recommend that the independent verification of diagnoses and drug doses should be made. Discussions, views and recommendations as to medical procedures. choice of drugs and drug dosages are the responsibility of the authors.

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lence of infection (for example, in blood donors), EIAs are not 100% sensitive or specific and false-positive tests still occur; therefore, all positive EIA results must be confirmed with a supplemental test. In high prevalence populations, EIA tests are sensitive and specific, and in many cases, a positive EIA test is suffi- cient for diagnosis of HCV infection if other laboratory values such as liver function tests and clinical presentation are indicative of hepatitis (15). In this patient population, confirmation of the diagnosis with a second test is not necessary; rather, supplemental tests are often used for evaluation of HCV disease.

Several other factors are taken into consideration when evaluating the need for supplemental testing. EIAs do not distinguish between acute, chronic, or resolved infections; therefore, supplemental testing of antibody-positive persons may be indicated to determine if the infection has been resolved or is ongoing. The EIA screening tests exhibit a low sensitivity in the immunosuppressed patient population, and patients with immunodeficiency may be seronegative (16). Occasionally, there may be a prolonged interval between the onset of acute illness and seroconversion. In all of these cases, supplemental testing using molecular tests for detection of viral RNA may be indicated. Finally, in persons with confirmed HCV disease, supplemental testing using molecular techniques is becoming increasingly important for evaluation of disease, measuring viral loads, and to predict and evaluate response to therapy. Molecular tests are also used to identify sources of infection and transmission (6,17).

Supplemental Antibody Assays The recombinant immunoblot assay

(RIBA) is recommended for confirmation of EIA-positive sera in low-prevalence populations. The RIBA format uses HCV antigens immobilized onto nitrocellulose in a specific pattern of bands, allowing identification of anti- bodies to individual antigens (18). Positive (human IgG) and negative (human superoxide dismutase, SOD) controls are included in the RIBA test. The RIBA tests are interpreted by the intensity of the reactivity against the HCV antigens and controls. The inten-

sity of the reactive bands must be 2 l+ (the intensity of the weak IgG control) and non-reactive to SOD. Because of the format of individual antigen bands, RIBA tests are more specific than EIAs.

The first generation RIBA (RIBA-1) used two antigens representing NS3 and 4 genes, ClOO-3 and 5-l-l. Like the first generation EIA, the RIBA- 1 test lacked sensitivity and specificity, and was subsequently replaced with second and third generation tests. The second generation RIBA (RIBA-2) is similar to RIB A- l but contains two additional antigens representing a nonstructural gene (~33~) and the core gene (~22-3). These antigens are the same as in EIA-2. For RIBA-2, the test results are interpreted based on the number of bands with which the patient’s serum reacts. The test is considered positive if the serum reacts with two or more bands, without reactivity to SOD; indeterminate if the serum reacts with one band; and negative if it reacts with no bands. A serum must have < 1 + activity to all four HCV antigens to be scored as negative. Reactivity to only one antigen or to 21 antigen plus SOD is scored as an indeterminate result. When indeterminate results occur with the RIBA- assay, the serum almost always reacts with the nucleocapsid protein band (~22-3). In these cases, an HCV RNA assay and repeat RIBA at a later time are indicated. The RIBA- assay is more sensitive and specific than with RIBA- for confirmation of HCV antibody. Like RIBA-2, four proteins are incorporated into the test, but these are modified (~22~) or synthetic (~100~ and c22p) antigens. An additional recombinant protein, NS5, is also included. The RIBA- test results in far fewer indeterminate tests than RIBA-2.

Although the sensitivity and specificity of the supplemental RIBA antibody tests are high, a substantial number of specimens are still scored as indeterminate. It is important to note that positivity with a RIBA test does not necessarily indicate active HCV infection; patients with resolved HCV infections may remain seropositive for many years (12). In some patient populations, a high percentage of HCV-infected patients may be seronegative for HCV (19). In these cases, molecular techniques are indicated for definitive diagnosis and further work up of HCV disease.

Supplemental Molecular Assays for HCV

The second major category of supplemental assays for HCV relies on detection of viral RNA. Several formats for molecular detection of HCV are available, and the choice of tests depends on the information that is needed by the physician. Qualitative tests are used to confirm the diagnosis of active HCV infection, while quantitative tests and genotyping are used to evaluate HCV disease, to measure viral loads, to identify the genotype, and to monitor response to therapy.

Qualitative Molecular Assays for HCV Diagnosis

Qualitative molecular detection of HCV RNA is used to confirm the diagnosis of active disease in seropositive individuals and to test seronegative, immunosuppressed patients. Molecular detection of viral RNA ultimately repre- sentsthe gold standard for diagnosis of HCV. Qualitative assays for detection of HCV RNA in serum &e primarily based on reverse-transcription polymerase chain reaction (RT-PCR), but other methods, such as transcription- mediated amplification (TMA), are available. To reduce problems caused by genomic variability, the qualitaiive PCR incorporates primers that are based on the highly conserved 5’-UTR. Several manufacturers now offer kits for RT-PCR detection of HCV RNA, facilitating the inclusion of PCR assays into laboratory testing protocols. How- ever, these assays are still not fully standardized and remain problematic for laboratories not routinely performing molecular diagnostic tests. The sensitivity and specificity of qualitative RT- PCR are very high. The sensitivity of RT-PCR ranges from 50 to 700 copies of viral RNA/ml, depending on the kit that is used (12). Reports usually indicate the presence or absence of HCV RNA.

Quantitative Assays for HCV As more information concerning the

relationship between viremia and clinical course of disease has become available, viral load testing increasingly is being used to evaluate HCV disease and to guide therapeutic decisions (12). Patients with high viral loads respond poorly to interferon therapy when compared to patients with low viral loads (20); thus, the viral load may be used

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to predict response to therapy. Several different assays have been

developed for monitoring viral loads (21). These include: quantitative RT- PCR, nucleic acid sequence-based assay (NASBA), and branched DNA (bDNA) technology. The sensitivity of quantitative assays is from 50 to 500 copies of viral RNA/ml (12). Like qualitative assays, kits are commercially available for the quantitative assays, facilitating the incorporation of these methods into the diagnostic laboratory. Quantitative assays are characterized by a dynamic range. The dynamic range is used as a measure of the amount of viral RNA (range) within which quantitation will be linear. The quantitative’assays should reliably quantify HCV RNA within the ranges that are clinically relevant (21). Quantitative assays may be affected by the viral genotype. To preclude the possibility of inaccurate results due to sequence heterogeneity, efforts are now being directed toward development of tests that will accurately measure all genotypes equally. A second generation bDNA test has been developed to improve detection of all genotypes of HCV (22). Clinical evaluations of this test have shown high sensitivity and specificity for all genotypes (23).

Reporting of Quantitative Molecular Assay Results: Genome Equivalents, RNA Copies and International Units

As molecular detection methods have increased in importance, it has become evident that standardization of the results reported for these methods is needed. The sensitivities and specificities of the different testing methods vary between laboratories (24), and there is no single unit of measure by which results are reported. Quantitative molecular assay results are currently provided as either genome equivalents/ml or copies of RNA/ml of serum. In 1998, The World Health Organization Inter- national Working Group of the Stan- dardization of Gene Amplification Techniques for the Virological Safety Testing of Blood and Blood Products (SoGAT) recommended that an international standard for HCV be developed (25); this standard was established and recently approved by the SoGAT (24).

The HCV International Standard provides for a common standard unit of

measurement and will enable calibration of results obtained with the various types of quantitative assays. The International Standard describes an International Unit (IU). The JU is not a direct measure of viral particles or copies of RNA, and thus the relationship between IUs and actual number of virions is not known. When comparing copies/ml or genome equivalents/ml to IU/ml, there is currently no standard calculation to convert between the various reporting values. Rather, the IU provides a constant value by which other assays can be standardized. Eventually, a formula to convert between the values will be made available. Individual kit manufacturers should provide information for conversion which may vary with each lot number of kit.

The International Standard was instituted in the summer of 2000. Incor- poration of the International Standard mandates that all quantitative molecular assays for HCV be reported as IU/ml, rather than genome equivalents or copies of RNA/ml. These new reporting guide- lines should provide consistent quantitative data so that results from different laboratories can be interpreted correctly and meaningfully by everyone.

Genotyping Methods Although still somewhat controver-

sial, the genetic features of HCV are increasingly being recognized for their importance in assessment of HCV disease, and many physicians are now incorporating genotyping into their diagnostic work up. Genotyping information is used to predict response to therapy and is incorporated into molecular epidemiology studies. The gold standard for genotyping is determining the nucleotide sequence of an HCV isolate. This method is not practical for the clinical diagnostic laboratory, and most genotype analyses are performed using less labor-intensive techniques. Simpler genotyping methods include: restriction- fragment length polymorphism (REP), type-specific PCR, and the line probe assay (LiPA) (12). The LiPA uses genotype-specific oligonucleotide probes immobilized onto membrane strips. The products obtained from PCR amplification of the 5’ UTR region of the clinical isolate are hybridized to the oligonucleo- tides (26). The LiPA is the most practical genotyping method for the clinical

laboratory because it is standardized and available in kit format (13). Sero- logical genotyping methods also have been developed. These assays are based on binding of antiserum to type-specific peptides derived from the NS4 region. These peptides can distinguish anti- bodies to all six major genotypes (27). Serotyping assays are commercially available (12). Good correlation was seen when serotyping assays were compared to genotyping by PCR (28).

Summary Significant advances in the diagnosis

of HCV infection have been made since the virus was identified. Incorporation of both serological and molecular diagnostic techniques has vastly improved primary diagnosis of HCV. Now, emphasis is being placed on using HCV diagnostic assays, especially molecular assays, as tools to evaluate HCV disease and to guide therapy. In addition to serving as diagnostic and therapeutic tools, these &says can-provide valuable information for use in clinical ‘and epidemiological studies.

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Case Report

Massilia timonae: An Unusual Bacterium Causing Wound Infection Following Surgery V. Sintchenko, M.D. surgery on an immunocompetent P. Jelfs, B.S. patient with a knee injury. A. Shama, M.S. L. Hicks, B.S. (Hon) G.L. Gilbert, M.D. Centre for Infectious Diseases and

Microbiology Laboratory Services Institute of Clinical Pathology and

Case Report

Medical Research Westmead Hospital Westmead, New South Wales, Australia

C. Wailer, M.D. Orthosports Randwick, New South Wales, Australia

investigation at the time but a course of flucloxacillin was commenced. On review one week later, the patient had a seropurulent exudate draining from the wound and was readmitted to the hospital where his wound was surgically debrided, pus was sent for investigation, and suture material was removed: The patient was discharged home the following day on flucloxacillin. The wound leaked a small amount of hemo- serous fluid for approximately two weeks and then healed completely.

Massilia timonae is a newly characterized, fastidious, slow-growing, gram- negative bacillus belonging to the class Proteobacteria (1). We report here a case of wound infection with Massilia timonae following elective orthopedic

The patient was a 36-year-old, previ- ously healthy man. He was a keen foot- ball player who underwent an elective arthroscopic anterior cruciate ligament reconstruction on his left knee. He received prophylactic intravenous cefazolin perioperatively and made an uneventful recovery. However, approximately four weeks after his surgery, he developed a swelling at the site of the surgical incision over his upper medial tibia which was thought to be an infected hematoma. The lesion was aspirated under sterile conditions and a small amount of pus and loose Vicryl sutures in the wound were recovered. No speci- men was taken for microbiological

Microbiology Pus collected intraoperatively was

inoculated onto 5% horse blood agar and MacConkey agar and incubated aerobically in 5% CO, at 35°C. Micro- scopic examination of the pus showed polymorphonuclear leukocytes and a

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diagnostic tests for hepatitis c virus

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