Clinical Microbiology Newsletter Vol. 21, No. 9 May 1,1999

Hepatitis C Virus Diagnosis and Therapeutic Methods and Interpretation Linda Cook, Ph.D. Department of Laboratory Medicine Luhey Clinic 41 Mall Road Burlington, MA 01805

Introduction Hepatitis C virus (HCV) has an

unusual history in that the virus was first identified not by traditional virus culture or immunoassay methods but through the use of molecular biology techniques developed by Dr. Michael Houghton and colleagues from Chiron Corporation (1). They used a high-titered plasma sample from a chimpanzee with hepatitis to extract nucleic acid and clone the first segment of the HCV genome. Further cloning and sequencing studies initially led to the identification of the first protein antigen, ClOO-3, and later to the sequencing of the entire HCV genome. HCV was found to have a pos- itive strand RNA genome with about 9,500 nucleotides. The overall structure is similar to that of pestiviruses and flaviviruses and has been classified as a member of the Flaviviridae family. The genome has been divided into seven areas, the core region that encodes the capsid C protein, the El and E2 regions that encode two envelope proteins, gp33 and gp72, and several nonstructural protein regions, NS2, NS3, NS4, and NS5, that encode six proteins. The non- structural proteins include NS3, a serine protease-helicase; NS2, a second pro- tease; NS4A, a proteinase cofactor pro- tein; NSSB an RNA polymerase, and NS2 and NS4B whose functions have

not been identified. The RNA sequence contains a single open reading frame that encodes a viral protein precursor of 3,011 amino acids, which is further pro- cessed into the six proteins. The genome contains a 5’ untranslated region of about 35 1 nucleotides and a small 3’ untrans- lated region of about 35 nucleotides.

HCV RNA sequences show signi- ficant genetic variability (2,3). Three major genotypes, 1,2, and 3, have been identified, with from three to eight other types identified depending on the classi- fication scheme used (la, lb, lc, 2a, 2b, 2c, 3a, 3b, 4a, 5a, and 6a according to the most detailed classification scheme). The relative prevalence of the HCV subtypes varies throughout the world; genotypes la and lb are most prevalent in the United States, lb is most common in Japan, northern Europe, and South America, type 4 in Africa and the Middle East, type 5 in South Africa, and type 6 in Singapore. Additional variation can be seen within the genotypes, in excess of 50 subtypes have also been identified. Further point mutations of the virus are common, leading to significant variation in the viral sequences within a single infected individual commonly referred to as quasispecies of virus. Genetic var- iation can be seen throughout the entire genome, with a significant focus of var- iation found in a hypervariable sequence region (HVRl) located in the 5’ portion of the E2 envelope protein region. The most conserved region of the genome is the 5’ untranslated region.

Traditional methods of virus isolation and culture have proven to be very dif-

Monitoring:

ficult and are not currently used in diag- nostic microbiology laboratories. In addition, traditional serological assays similar to the p24 antigen assay for HIV- 1 or the hepatitis B surface antigen assay that can be used to detect viral particles lack sensitivity for HCV and are not used for routine diagnosis. Cur- rently available assays for the diagnosis and monitoring of HCV infections are limited to antibody detection methods and molecular amplification assays.

HCV Diseases HCV is usually transmitted by expo-

sure to viral RNA in blood or blood products. Common exposures include blood tranfusions, blood products, organ

In This Issue

Hepatitis C Virus Diagnosis and Therapeutic Monitoring: Methods and Interpretation . . . . .67 The advent of speciJic antiviral therapies for hepatitis C viral infections has stimulated the need for better diagnostic and monitoting methods. The recent USFDA mandate of “look- back” testing for blood donors and recipients will undoubtedly increase the importance of this testing.

Optochin-resistant Streptococcus pneumoniue Isolated from a Blood Specimen . . . . . . . . . . . . . . . . . ...72 a case report

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transplantation, I.V. drug use, tattoos, and accidental needle sticks. Transmis- sion of the virus at low rates also can be seen for sexual and household contacts and maternal-fetal blood exchange. Acute hepatitis due to HCV is usually mild and produces symptoms of nausea, abdominal pain, and anorexia. Jaundice may or may not be present. Mild eleva- tions of liver enzymes and bilirubin can be seen. Only in rare cases does the dis- ease present with clinically severe ful- minant hepatitis.

The striking feature of HCV infection is the high rate of chronic infection that is established in the majority of exposed patients. Most patients recover from the acute infection and then maintain an asymptomatic chronic infection that may last up to 20 or more years. The chronic infection can then finally lead to clinically significant liver disease or a host of other HCV-related conditions. According to initial studies done in the United States by Alter et al. (4), chronic hepatitis developed in 62% of individu- als following an initial HCV infection and 33% of these individuals had liver biopsies consistent with chronic active hepatitis. A number of additional stud- ies have established that about 15% of primary HCV infections are self limit- ed, while the remaining 85% go on to establish a chronic infectious state. The chronic state is characterized by persis- tent viremia, which is intermittent in some individuals. Within the next 20 to 30 years, many of these patients will develop liver failure due to HCV infec- tion and about 20% will eventually pro- gress to liver cirrhosis. A l&s frequent manifestation seen an average of 30 years post infection in about 1 to 5% of patients is hepatocellular carcinoma. The chronic hepatitis caused by HCV can be exacerbated by co-infection with hepatitis B or HIV- 1, chronic alcohol

abuse, or genetic liver diseases such as alpha- 1 antitrypsin deficiency. Hepatitis C infections have also been shown to be highly associated with the development of autoimmune chronic hepatitis type 2. HCV infections have also been reported in patients with a variety of immune- mediated, non-hepatic diseases includ- ing Hashimoto’s thyroiditis, Types II and III cryoglobulinemia, aplastic ane- mia, glomerulonephritis, polyarteritis nodosa, pulmonary fibrosis, diabetes mellitis, rheumatoid arthritis, and a host of other immune-mediated diseases (5). HCV infections have been strongly associated with types II and III cryo- globulinemia but additional studies are necessary to determine whether HCV plays any role in the development of most of the remainder of these diseases.

Serological Methods Antibody assays used for documenta-

tion of exposure to the virus consist of screening enzyme immunoassays (EIA) and confirmatory immunoblots. The screening assay, developed in 1990, initially used a single protein antigen, the ClOO-3 fusion protein from the NS4AlB region of the genome, coated onto microdilution plate wells. This first-generation assay lacked sufficient specificity and sensitivity, especially during the first year following exposure to the virus. In 1992, a second-generation, multiple-antigen EIA was approved by the FDA for use in the U.S. The second- generation EIA used the CIOO-3 protein, and in addition, a C33c proteifi from the NS3 region, a C22-3 from the core region, and a C200 fusion protein fom the NS3 and NS4 regions of the viral genome. The sensitivity of the second generation assay is about 99% in blood donors with HCV viremia and in patients with acute and chronic HCV infections. A third-generation ETA was developed in 1994 in which an additional protein from

the NS5 region was added to the multi- antigen coat. Studies done to compare sensitivity and specificity for the sec- ond and third generation assays have shown no significant differences between the two tests.

Positive screening antibody assays should, in most cases, be followed by confirmatory recombinant immunoblot assays (RIBA) in which antibodies to each individual recombinant protein is detected. Three generations of RIBAs have been used, each generation of RIBA, i.e., RIBA-1, RIBA-2, and RIBA-3, corresponded to the three gen- erations of screening assays and the proteins they each contained. Second- and third-generation RIBA are considered positive if antibodies are present from two proteins from different regions of the genome. The confirmatory RIBAs are considered indeterminant if anti- bodies to only one protein are present. Inclusion of the core protein in the second- and third-generation assays decreased the average seroconversion time from about nine weeks to about six weeks because anti-core antibodies are the first to appear in most individuals. However, antibody reactivity to the core protein in normal individuals is the most common cause of false-positive antibody screening assays and indeterminant RIBA results. The percentage of samples with positive screening assays that are con- firmed as true-positives depends on the population being tested; most studies have shown >80% positive confirmatory test results when gastroenterology clin- ic patients with hepatitis are studied, whereas rates ~50% are usually found when normal blood donors are studied.

Antibody testing is much more useful in the detection of chronic HCV infections than of acute hepatitis. The first year after virus exposure, >99.5% of individuals with intact immune sys-

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, instructions 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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68 01964399/99 (see fronrmatter) 0 1999 Elsevier Science Inc. Clinical Microbiology Newsletter 21:9.1999

terns are antibody positive in both the screening and confirmatory assays. Rare infected individuals have been reported who are repetitively RIBA indeterminant or seronegative. The majority of infect- ed individuals who are seronegative are those with B-cell or antibody immuno- deficiency states or individuals with HIV infections. Once positive, the antibody persists in the serum indefinitely in essentially all untreated individuals. Only a few rare cases of spontaneous loss of antibody have been reported. However, effective therapy with inter- feron has been shown in some patients to result in decreases of antibody levels with a loss of detectable serum HCV antibody in a few patients (6).

Antibody testing is less reliable in acute hepatitis infections. With the tirst- generation antibody assay, more than six months was needed before IgG anti- bodies were detected in up to 50% of exposed individuals. With the newer assays, only about half of individuals are positive six weeks after initial expo- sure. Assays used to detect IgM anti- HCV antibodies have been developed but have not detected significant IgM antibody responses in most individuals (7). Because of this lack of IgM response and the slow appearance of IgG antibody in many individuals the serological diagnosis of acute hepatitis C infection lacks sensitivity and other methods must be used to document the infection.

Molecular Diagnostic Testing - Qualitative and Quantitative Assays

A variety of highly-sensitive molecular biology methods have been described to detect HCV RNA in plasma or serum. In the majority of diagnostic laboratories, HCV RNA testing is used either as an aid in the diagnosis of acute hepatitis or to determine the quantitative level of virus in the blood. Vii levels are used to monitor response to anti-viral therapies. Most of the molecular diagnostic methods detect and amplify the 5’ untranslated region where the viral RNA sequence most conserved. At present, there are no FDA-approved assays for diagnostic testing, however, there are at least seven different methods commercially avail- able produced either for research use only or currently under development. Currently available are the PCR method from Roche Diagnostics (both a qualita- tive and quantitative assay), the branched

chain DNA (bDNA) assay from Chiron Diagnostics, the nucleic acid sequenced- based amplification (NASBA) test from Organon Teknika, and the DNA ELISA assay from DiaSorin. Currently in devel- opment are the transcription mediated amplification (TMA) assay from Gen- Probe, the ligase chain reaction (LCX) test from Abbott, and the Invader (cleavase) method by Third Wave Technologies. In addition, there are a significant number of laboratories that have developed in-house nested or direct PCR tests based on a variety of published and unpublished methods. Within the last few years, instrumentation has been developed to detect the production of PCR amplicons during the thermocycling reaction, which has the potential of eliminating the post-PCR detection methods currently necessary. Real-time analysis of PCR products can be done with the Real Time reader (Idaho Technologies) or the 7700 Sequence Detector (Perkin-Elmer) instruments.

A wide variety of molecular diag- nostic methods have been developed because of the inherent difficulties of applying molecular biology techniques into routine diagnostic laboratories. Time consuming and inefficient extraction methods, long amplification times, the requirement for extremely precise pipetting, time consuming product detection methods, and contamination issues have all contributed to make these methods difficult to perform in routine testing laboratories. Currently, no single method has been shown to be superior to all others, each of the meth- ods has distinct technical features that have advantages or disadvantages depending on the circumstances and demands of the laboratory utilizing the assays. Instruments are also available or under development to automate some or most of the steps for the Roche, Chiron, Organon Teknika, Gen-Probe, and Abbott methods.

When different methods have been compared, one of the important charac- teristics is the assay sensitivity or the ability to measure low quantities of HCV in the serum. No universal stan- dard containing known quantities of virus was available when the first assays were developed by Roche and Chiron. The Roche PCR assay gener- ates results based on a viral standard expressed as viral copy number per mL.

The Chiron bDNA generates results based on the use of a viral standard expressed as milliequivalences per ml. Therefore, results from the PCR assay cannot be directly compared with the results from the bDNA assay. A large number of studies have now been pub- lished comparing results of the different methods, most of which compare either the Roche PCR with the Chiron bDNA assay or one of the other new assays with either the Roche or Chiron assay. Because of this lack of a single stan- dard, comparison of assay sensitivities between methods has proved difficult. For the commercially available assays, PCR assays that detect PCR amplicons by an EIA method have proven to be the most sensitive and the bDNA assay has been shown to be the least sensitive method. Manufacturers stated sensitivity claims vary from a low of 50 to 100 viral genome copies/ml for the DiaSorin DNA ELISA and National Genetics Institute in-house PCR to a high of 200,000 genome equivalents for the currently available bDNA assay.

Molecular Test for HCV vping Several methods are also available

to determine the HCV genotype of the virus. Genotyping has been used on a research basis for a number of years for epidemiological studies and has more recently been used during interferon therapy to predict response rates and to vary interferon treatment regimens. Although initially controversial, the majority of recently published studies have indicated that patients with HCV genotype 1 have significantly lower response rates to interferon therapy. Commercial genotyping kits currently available for research use only are the INNO-LiPA assay (Innogenetics), the GENE-TI-K assay (DiaSorin), type- specific PCR multiprimers (Genlab Diagnostics), and Cleavase Fragment Length Polymorphism (CFLP, Third Wave Technologies). The GENE-TI-K kit uses primers and probes for the core region. The LiPA kit uses the 5’ untrans- lated region for analysis where the dif- ference between the la and 1 b subtypes is a single nucleotide and the 2a and 2b subtypes have identical sequences. This leads to some difficulties with distin- guishing subtypes of 1 and 2 with this method. The CFLP assay generates PCR product from the 5’ untranslated region

Clinical Microbiology Newsletter 21:9,1999 Q 1999 Elsevier Science Inc. 0196-4399199 (see frontmatter) 69

(UTR) and then analysis of the product is done with the CFLP method. Direct DNA sequencing of PCR amplicons generated from either the 5’ UTR or the core region has also been described and has been the basis for the majority of genotyping classification schemes described. Reagent costs, performance time, and required instrumentation all vary widely with the different geno- typing methods, thus selection of the method must be done carefully to match the needs of the testing laboratories (8). HCV serotyping can also be done with an EIA assay kit from Murex or a RIBA HCV Serotyping SIA (Chiron) kit, but most studies have shown the serotyping method to be significantly less sensitive than the molecular typing methods.

HCV RNA Assay Standardization Issues

The first step in assay standardization is the preparation of standard materials that can be used by all reagent and instru- ment manufacturers and clinical testing laboratories. In 1997, the first WHO international standard for HCV designed for genomic amplification technology assays was made available by the National Institute for Biological Standards and Controls (United Kingdom). The stan- dard is WHO 96/790 and is available for purchase by writing to NIBSC,

Blanche Lane, South Mimms, Potters Bar, Hertfordshire, EN6 3QG, United Kingdom. A request for purchase must be accompanied by a U.S. customs per- mit for biological hazard material ship- ment that can be obtained from the Centers for Disease Control and Preven- tion (CDC). The 96/790 standard vial contains 50,000 International Units of HCV that is roughly equivalent to 50,000 copies of virus. The standard is designed to be titered in parallel with an in-house sample to determine the International Units of virus in the in- house sample that can then be used as the test system calibrator/reference material. This WHO standard material is now available to all assay manufac- turers and result values for the standard should now be available for most commercial assays. Further use of this standard by all manufacturers should ultimately lead to the ability to more directly compare results from different HCV RNA kits.

Additional secondary reference mate- rials are also available in the U.S. from Boston Biomedica. Available for use are series 100,200,300, and 400 Accurun HCV positive-control samples that can be used as quality control material. Package inserts for these materials con- tain HCV quantitative results for these controls used in the Roche PCR assay

Table 1. Summary of CAP proficiency surveys. HCV RNA testing - ID. survey

Year Shipment Samples

Average HCV

quantitation

Percent of laboratories with positive

test result HCV

aenotvne 1998 ID-C ID-11

ID-12 ID-13 ID-14 ID-15

Pending 46% 95% 83% 98% 92%

Pending

1998 ID-B ID-10 1 x 104 73% 2b

1997 ID-C ID-14 5.3 x 105 49% 2b

1997 ID-A ID-01 ID-02

5x105 5x104

99% 99%

la la

1996 ID-C ID-15 1.3 x 106 13% la

1996 ID-B ID-07 >1.2 x 106 77% ID-08 >1.2 x 108 73%

2b

1996 ID-A ID-05 Neg 8%

as determined by two calibration meth- ods: the Roche internal calibrators and the WHO standard. The currently avail- able series has a range of results from 1,000 to 100,000 IU/mL.

Direct comparison of results of dif- ferent methods and testing laboratories is available both from three years of HCV RNA samples sent as part of the College of American Pathology (CAP) ID surveys (1997 to 1999) and from studies done on a reference panel in Europe. A total of 14 samples have been sent to laboratories performing CAP proficiency survey testing within the ID testing set. This testing was performed by about 100 different laboratories with about two-thirds of the laboratories using the Roche Monitor and/or Amplicor kits. Table 1 contains a summary of some of the available data from the summary reports. The results demonstrate a num- ber of problems; sample 1996 ID-C contained degraded viral RNA, sample 1996 ID-A gave false positive results in 8% of labs, and samples with low titers of 2b genotype virus (1996 ID-B, 1997 ID-C, 1998 ID-B) have consistently given a significant number of false- negative results. These inconsistent results for identical samples tested in a large number of laboratories indicate that further improvements are necessary in both the available methods and in the technical performance of the assays by some laboratories. The final critique for the ID-C 1998 survey set, which will contain quantitative and genotype information, will be available soon.

A collaborative study of 86 labora- tories tested a second EUROHEP HCV RNA reference panel containing four HCV-RNA positive plasma samples, six HCV-RNA negative plasma samples, and two dilution series of HCV RNA genotype 1 and 3 plasma standards (9). The majority of laboratories performed in-house developed PCR methods, while about 30% used the Roche Amplicor kit. Results from this study showed signifi- cant variations in the results. Only 16% of laboratories had no errors, 29% of labs missed the low positive sample, and 55% of labs had either false-positive or false- negative results. Labs using the commer- cial PCR testing method had slightly better results than those using in-house developed PCR methods. Results for the dilution series for the genotype 1 sample showed up to five logs of differ-

70 01%4399/99 (see frontmatter) Q 1999 Elsevier Science Inc. Clinical Microbiology Newsletter 21:9,1999

ence in sensitivity. Results for the dilu- tion series for the genotype 3 sample showed very low detection efficiency with the commercial assay. Clearly the EUROHEP reference panel results and the CAP survey data have demonstrated the need for further improvements in the testing methods and better standard- ization of the existing methods.

Genotyping and Standardization The genetic variation seen within

the HCV also contributes to the current lack of standardization of HCV RNA testing. For the majority of amplifica- tion methods, the efficiency of replica- tion is affected by genotype-specific mutations present in the virus in the areas that bind the primer and probe sequences. It has been well documented that the Roche PCR assay has much lower efficiency of replication for dif- ferent HCV genotypes. This lower replication efficiency results in an underestimation of the quantity of HCV present for some genotypes (10). This technical problem has contributed to conflicting studies concerning the mean viral loads present in untreated patients with different HCV genotypes and to controversial data concerning better response to interferon therapy in patients with certain genotypes. The bDNA assay is the least sensitive to dif- ferences in genotype because of the large number of probes used over a broad area of the 5’ untranslated region. All of the amplification methods need further improvement to ensure that the HCV qualitative and quantitative assays are not influenced by the HCV genotype.

Monitoring HCV During Therapy Therapy protocols for treatment of

HCV hepatitis have changed rapidly since the discovery of the virus ten years ago. Recent consensus guidelines con- cerning prevention and management of HCV infections were issued by the CDC in October of 1998 (11) and by the NIH in March of 1997 (12). Although these documents are less than two years old, the therapy guidelines contained in these two documents are already out of date. In November of 1998, the FDA approved the combination ribavirin/interferon therapy based on the significantly improved results in several studies con- ducted by the manufacturer. Sustained response rates of 40 to 50% were seen

in studies of interferon/ribavirin com- pared with rates of 5 to 15% with inter- feron alone. However, combination therapy given to patients with HCV geno- type 1 resulted in sustained response rates of <30%, which was lower than observed with other genotypes studied. There are also several other drugs cur- rently being studied that have significant potential for therapy of HCV infections. Significant advances in the therapy of HCV will rapidly lead to changes in the recommended frequency and perhaps types of laboratory testing that may be performed. It could be anticipated that in an analagous manner to what happened to HIV- 1 RNA testing when effective drugs regimens became available, sig- nificantly improved therapy for HCV could lead to increased demand for more sensitive assays and for more frequent determinations of HCV viral load test- ing. A commonly accepted practice for the use of HCV quantitations to monitor HCV RNA serum levels during interfer- on therapy includes testing a sample for a baseline prior to initiation of therapy, at three months after therapy is begun, at the end of therapy (either 3,6, or 12 months), and then at one to three months following completion of interferon ther- apy. About 33% of patients will become HCV RNA negative at three months which is an indication of a good therapy response. At least half of these patients will relapse when therapy is stopped and HCV reappears in the serum. Partial responders and non-responders also can be seen when baseline HCV quantitations are compared to those done at three months. A partial response for the HCV RNA assays is defined as a drop in viral titer which is at least one- third of a log. Revised monitoring strate- gies for interferon/ribavirin therapy have yet to be established, but may be similar to the monitoring that was done in the research studies conducted by Shearing- Plough. The monitoring strategy used in the majority of the completed and ongoing ribavirin-containing protocols consisted of HCV RNA quantitations at baseline, 4, 8, 12, 24,28,36 and 48 wks (for a 24 wk course of therapy). Additional studies are necessary to determine the best monitoring strategy for the interferonfribavirin therapy and all other new emerging therapies.

According to published CDC guide-

lines, all HCV antibody positive patients should be evaluated for the presence and severity of chronic liver disease. Antiviral therapy should be given to individuals with consistently elevated ALT levels, detectable HCV RNA serum levels, and liver biopsies consistent with moderate inflammation and portal or bridging fibrosis. The 1998 guidelines suggested no strong indication for therapy in patients with less severe disease, but these guidelines may change now that more effective therapy is available. In addition, CDC guidelines specifically excluded from interferon therapy any patients with advanced cirrhosis, preg- nancy, individuals under 18 or over 60 years of age, and patients with a known history of drug or alcohol abuse until they have been substance free for at least six months.

The NIH consensus document con- tained several additional useful guide- lines for HCV testing. First, repeat testing for HCV RNA once viremia is documented is not helpful in disease management unless used to monitor therapy. Transient negative results dur- ing the course of the HCV infection in some patients was found to have no clinical relevance. Second, the liver biopsy is still the gold standard for the initial assessment of patients with chronic hepatitis, but the HCV RNA quantitation assay is better for monitoring therapy response. Third, single positive HCV RNA test is sufficient to confirm HCV infection, but a single negative HCV RNA test is not sufficient to exclude an HCV infection.

Regulatory Issues In November of 1998, the FDA

issued regulations concerning analyte- specific reagents (ASR) (13). ASRs, which include monoclonal antibodies and DNA probes and primers, are now exempt from submission to the FDA for clearance. With the exception of molec- ular testing for HIV and Mycobacter- ium tuberculosis, all other molecular diagnostic reagents now can be sold to clinical testing laboratories without FDA clearance. The manufacturers cannot sell the reagents with any information that makes claims of clinical usefulness or contains instructions for specific clinical methodologies. According to these new regulations, it is now the per- forming laboratory’s responsibility to

Clinical Microbiology Newsletter 21:9,1999 @ 1999 Elsevier Science Inc. Ol%-4399199 (see frontmatter) 71

validate the performance of the reagents with their own in-house quality control and quality assurance testing. Further information concerning these regulations can be found in reference 13. Additional changes to these regulations should be forthcoming because the FDA has recently convened its Microbiology Devices Panel to begin the process of determining how to evaluate HCV test- ing methods.

In March of 1998, the FDA issued a new policy concerning notification of “look-back” testing for HCV (14). This policy required the following steps: (i) identification of all donors who have tested positive for antibody to the HCV, (ii) identification of all blood and blood products donated by positive donors, especially those donated prior to the positive HCV antibody test, (iii) notiti- cation to all transfusion services of pos- sible HCV contamination of all blood and blood products from positive donors or subsequently positive donors, and (iv) notification to physicians by the tranfu- sion services of all patients exposed to blood or blood products from positive donors or subsequently positive donors. This notification requires that the physi- cian immediately notify each patient of the need to have HCV antibody testing done to screen for possible HCV infec- tion. This notification program should result in the identification of a large number of patients with subclinical HCV infections.

Summary HCV testing is an exciting area of

molecular diagnostics. As we better understand the clinical diseases caused by hepatitis, improve the therapy, and refine the laboratory testing performed for these patients, significant changes in existing procedures and protocols will take place.

References 1. Choo, Q.-L. et al. 1989. Isolation of

cDNA clone derived from a blood- borne non-A, non-B viral hepatitis genome. Science 2441359-362.

2. Brechot, C. 1996. Hepatitis C virus, molecular biology and genetic vari- ability. Dig. Dis. Sci. 41:6S-21s

3. Buch, J., R.H. Miller, and R.H. Purcell. 1995. Genetic heterogene- ity of hepatitis C virus: quasispecies and genotypes. Sem. Liver Dis. 15:441-463.

4. Alter, M.J. et al. 1992. The natural history of community-acquired hepatitis C in the United States. N. Eng. 3. Med. 327: 1899- 1905.

5. Hadziyannis, S.J. 1996. Nonhepatic manifestations and combined dis- eases in HCV infection. Dig. Dis. Sci. 41:63S-74s.

6. Yuki, N. et al. 1993. Quantitative analysis of antibodies to hepatitis C virus during interferon-a therapy. Hepatology 17:960-965.

7. Papatheodoridis, G.V. et al. 1997. Significance of IgM anti-HCV core level in chronic hepatitis C. J. Hepatol. 27:36-41. .

8. Lee, J.-H., W.K. Roth, and S.

Zeuzem. 1997. Evaluation and com- parison of different hepatitis C virus genotyping and serotyping assays. J. Hepatol. 26: 1001-1009.

9. Damen, M. et al. 1996. International collaborative study on the second EUROHEP HCV-RNA reference panel. J. Vir. Meth. 58:175-185.

10. Hawkins, A., E Davidson, and l?

11 Recommendations for prevention and control of Hepatitis C virus (HCV) infection and HCV related chronic disease. MMWR 47 (RR-19):1-39, October 16, 1998.

12. Management of Hepatitis C, NIH consensus statement 1997, March 24-26, 15(3).

Simmons. 1997. Comparison of plasma virus loads among individu- als infected with hepatitis C virus (HCV) genotypes 1,2, and 3 by Quantiplex HCV RNA assay ver- sion 1 and 2, Roche Monitor assay, and in-house limiting dilution method. J. Clin. Microbial. 35:187-192.

13. Medical devices; classification/ reclassification; restricted devices, analyte-specific reagents 2 ICFR 809. Final rule. Federal Register 1997,62:62243-62260.

14. FDA Regulations - Guidance for industry: supplemental testing and the notification of consignees of donor test results for antibody to Hepatitis C (Anti-HCV). Issued 20 March 1998.

Case Report

Optochin-resistant Streptococcus pneumoniae Isolated from a Blood Specimen Flavio Lejbkowicz, Ph.D. Magda Goldstein, M.S. Nehama Hashman, M.S. Lazar Cohn, M.S. Microbiology Laboratory Rambam Medical Center Haifa, Israel

One of the most common causes of pneumonia is the Sfreprococcus pneu-

moniae (pneumococcus). The pneumo- coccus inhabits the respiratory tract of more than two-thirds of the population as normal flora. In some circumstances, the organism may enter the bloodstream leading to septicemia, endocarditis, pericarditis, or meningitis (1).

The clinical laboratory has a pivotal function in distinguishing pneumococci

from other alpha-hemolytic streptococci to establish the need for patient treat- ment. Microscopically, pneumococci are gram-positive cocci appearing in pairs for a morphology commonly referred to as diplococcus. The organ- ism produces alpha-hemolysis on blood agar, and, in general, colonies are mucoid due to the production of a polysaccha-

72 01964399/99 (see ftmmnatter) Q 1999 Elsevier Science Inc. Clinical Microbiology Newsletter 21:9,1999