RR5203-Front Cover.p65

Morbidity and Mortality Weekly Report

Recommendations and Reports February 7, 2003 / Vol. 52 / No. RR-3

INSIDE: Continuing Education Examination

depardepar

depar

tment of health and human sertment of health and human ser

tment of health and human ser

vicesvices

vices

Centers for Disease Control and PreventionCenters for Disease Control and Prevention

Centers for Disease Control and Prevention

Guidelines for Laboratory Testing and Result

Reporting of Antibody to Hepatitis C Virus

MMWR

SUGGESTED CITATION

Centers for Disease Control and Prevention.

Guidelines for laboratory testing and result reporting

of antibody to hepatitis C virus. MMWR

2003;52(No. RR-3):[inclusive page numbers].

The MMWR series of publications is published by the

Epidemiology Program Office, Centers for Disease

Control and Prevention (CDC), U.S. Department of

Health and Human Services, Atlanta, GA 30333.

Centers for Disease Control and Prevention

Julie L. Gerberding, M.D., M.P.H.

Director

David W. Fleming, M.D.

Deputy Director for Science and Public Health

Dixie E. Snider, Jr., M.D., M.P.H.

Associate Director for Science

Epidemiology Program Office

Stephen B. Thacker, M.D., M.Sc.

Director

Office of Scientific and Health Communications

John W. Ward, M.D.

Director

Editor, MMWR Series

Suzanne M. Hewitt, M.P.A.

Managing Editor

C. Kay Smith-Akin, M.Ed.

Project Editor

Beverly J. Holland

Lead Visual Information Specialist

Malbea A. Heilman

Visual Information Specialist

Quang M. Doan

Erica R. Shaver

Information Technology Specialists

On the Cover: Photographs by Greg H. Knoblock,

Public Health Practice Program Office. Testing methods

for diagnosis of hepatitis C virus (HCV) infection include

enzyme immunoassays for antibodies to HCV (anti-

HCV) upper-left), a more specific supplemental strip

immunoblot assay for anti-HCV (upper-right), and

nucleic acid tests for HCV RNA (bottom).

CONTENTS

Introduction ......................................................................... 1

Background ......................................................................... 2

Available Anti-HCV Screening Assays ................................ 2

Available Supplemental Tests ............................................ 2

Interpreting Anti-HCV Test Results ..................................... 2

Anti-HCV Testing Practices ................................................ 4

Using Screening-Test–Positive S/Co Ratios To Determine

Need for Reflex Supplemental Testing ............................ 5

Estimated Costs of Implementing Reflex Supplemental

Testing Based on Screening-Test–Positive S/Co Ratios ..... 7

Recommendations ............................................................... 8

Rationale .......................................................................... 8

Laboratory Algorithm for Anti-HCV Testing and Result

Reporting ...................................................................... 9

Implementation .............................................................. 10

Future Considerations ....................................................... 12

Acknowledgments ............................................................. 12

References ......................................................................... 12

Terms and Abbreviations Used in This Report ..................... 15

This report is available at http://www.cdc.gov/hepatitis. A limited

number of copies can be ordered at that website.

The preparers of this report have signed a conflict of interest

disclosure form that verifies no conflict of interest.

Vol. 52 / RR-3 Recommendations and Reports 1

The material in this report originated in the National Center for

Infectious Diseases, James M. Hughes, M.D., Director, and the Division

of Viral Hepatitis, Harold S. Margolis, M.D., Director.

Guidelines for Laboratory Testing and Result Reporting

of Antibody to Hepatitis C Virus*

Prepared by

Miriam J. Alter, Ph.D.

Wendi L. Kuhnert, Ph.D.

Lyn Finelli, Dr.P.H.

Division of Viral Hepatitis

National Center for Infectious Diseases

Summary

Testing for the presence of antibody to hepatitis C virus (anti-HCV) is recommended for initially identifying persons with

hepatitis C virus (HCV) infection (CDC. Recommendations for prevention and control of hepatitis C virus [HCV] infec-

tion and HCV-related chronic disease. MMWR 1998;47[No. RR-19]:1–33). Testing for anti-HCV should include use of an

antibody screening assay, and for screening test-positive results, a more specific supplemental assay. Verifying the presence of anti-

HCV minimizes unnecessary medical visits and psychological harm for persons who test falsely positive by screening assays and

ensures that counseling, medical referral, and evaluation are targeted for patients serologically confirmed as having been infected with

HCV. However, substantial variation in reflex supplemental testing practices exists among laboratories, and an anti-HCV–positive

laboratory report does not uniformly represent a confirmed positive result. These guidelines expand recommendations for anti-

HCV testing to include an option for reflex supplemental testing based on screening-test–positive signal-to-cut–off (s/co) ratios. Use

of s/co ratios minimizes the amount of supplemental testing that needs to be performed while improving the reliability of reported

test results. These guidelines were developed on the basis of available knowledge of CDC staff in consultation with representatives

from the Food and Drug Administration and public health, hospital, and independent laboratories. Adoption of these guidelines

by all public and private laboratories that perform in vitro diagnostic anti-HCV testing will improve the accuracy and utility of

reported anti-HCV test results for counseling and medical evaluation of patients by health-care professionals and for surveillance

by public health departments.

Introduction

Tests to detect antibody to hepatitis C virus (anti-HCV)

were first licensed by the Food and Drug Administration (FDA)

in 1990 (1). Since that time, new versions of these and other

FDA-approved anti-HCV tests have been used widely for clini-

cal diagnosis and screening of asymptomatic persons.

Persons being tested for anti-HCV are entitled to accurate

and correctly interpreted test results. CDC has recommended

that a person be considered to have serologic evidence of HCV

infection only after an anti-HCV screening-test–positive

result has been verified by a more specific serologic test (e.g.,

the recombinant immunoblot assay [RIBA

; Chiron

Corporation, Emeryville, California]) or a nucleic acid test

(NAT) (2). This recommendation is consistent with testing

practices for hepatitis B surface antigen and antibody to human

immunodeficiency virus (HIV), for which laboratories

routinely conduct more specific reflex testing before

reporting a result as positive (1,3). However, for anti-HCV,

the majority of laboratories report a positive result based on a

positive screening test result only, and do not verify these

results with more specific serologic or nucleic acid testing

unless ordered by the requesting physician. Unfortunately, cer-

tain health-care professionals lack an understanding of the

interpretation of anti-HCV screening test results, when more

specific testing should be performed, and which tests should

be considered for this purpose.

In certain clinical settings, false-positive anti-HCV results

are rare because the majority of persons being tested have evi-

dence of liver disease and the sensitivity and specificity of the

screening assays are high. However, among populations with

a low (<10%) prevalence of HCV infection, false-positive

results do occur (4–11). This is of concern when testing is

performed on asymptomatic persons for whom no clinical

information is available, when persons are being tested for

HCV infection for the first time, and when testing is

being used to determine the need for postexposure follow-up.

Without knowledge of the origin of the test sample or clinical

* These guidelines are not intended to be used for blood, plasma, organ, tissue,

or other donor screening or notification as provided for under FDA guidance

or applicable regulations. They also are not intended to change the

manufacturer’s labeling for performing a specific test.

2 MMWR February 7, 2003

information concerning the person being tested, the accuracy

of a screening-test–positive result for any given specimen can-

not be determined.

Multiple reasons exist regarding why laboratories do not

perform reflex supplemental testing for anti-HCV, including

lack of an established laboratory standard for such testing,

lack of understanding regarding the performance and inter-

pretation of the screening and supplemental HCV tests, and

the high cost of the supplemental HCV tests. To facilitate prac-

tice of reflex supplemental testing, the recommended anti-

HCV testing algorithm has been expanded to include an option

that uses the signal-to-cut–off (s/co) ratios of screening-

test–positive results to minimize the number of specimens that

require supplemental testing and provide a result that has

a high probability of reflecting the person’s true antibody

status.

Background

Available Anti-HCV Screening Assays

FDA-licensed or approved anti-HCV screening test kits

being used in the United States comprise three immunoas-

says; two enzyme immunoassays (EIA) (Abbott HCV EIA 2.0,

Abbott Laboratories, Abbott Park, Illinois, and ORTHO

HCV Version 3.0 ELISA, Ortho-Clinical Diagnostics, Raritan,

New Jersey) and one enhanced chemiluminescence

immunoassay (CIA) (VITROS

Anti-HCV assay, Ortho-

Clinical Diagnostics, Raritan, New Jersey). All of these

immunoassays use HCV-encoded recombinant antigens.

Available Supplemental Tests

Supplemental tests include a serologic anti-HCV assay and

NATs for HCV RNA. In the United States, the only FDA-

licensed supplemental anti-HCV test is the strip immunoblot

assay (Chiron RIBA

HCV 3.0 SIA, Chiron Corp.,

Emeryville, California). RIBA 3.0 uses both HCV-encoded

recombinant antigens and synthetic peptides. Because it is a

serologic assay, it can be performed on the same serum or

plasma sample collected for the screening anti-HCV assay.

FDA-approved diagnostic NATs for qualitative detection

of HCV RNA using reverse transcriptase polymerase chain

reaction (RT-PCR) amplification include AMPLICOR

Hepatitis C Virus (HCV) Test, version 2.0 and COBAS

AMPLICOR

Hepatitis C Virus Test, version 2.0 (Roche

Molecular Systems, Branchburg, New Jersey), which have a

lower limit of detection of approximately 50 IU/mL (12).

Detection of HCV RNA by these tests requires that the serum

or plasma sample be collected and handled in a manner suit-

able for NAT and that testing be performed in a laboratory

with facilities established for this purpose (see Recommenda-

tions). Other NATs for HCV RNA, both qualitative and quan-

titative, are available on a research-use basis from multiple

manufacturers of diagnostic reagents, and certain laboratories

perform NATs by using in-house laboratory methods and

reagents (12,13).

Interpreting Anti-HCV Test Results

Screening Immunoassay Test Results

Anti-HCV testing includes initial screening with an immu-

noassay. Criteria for interpretation of a reactive

†

anti-HCV

immunoassay result are based on data from clinical studies

performed under the auspices of each manufacturer. For EIAs

(e.g., HCV EIA 2.0 and HCV Version 3.0 ELISA), speci-

mens with a reactive result are retested in duplicate. If the

result of either duplicate test is reactive, the specimen is

defined as repeatedly reactive and is interpreted as screening-

test–positive. For CIAs, (e.g., VITROS Anti-HCV assay),

specimens with a single reactive result are considered screening-

test–positive and do not require retesting.

The specificity of the HCV EIA 2.0 and HCV Version 3.0

ELISA is

>99%. However, among a population with a low

prevalence of infection, even a specificity of 99% does not

provide the desired predictive value for a positive test. Among

immunocompetent populations with anti-HCV prevalences

<10% (e.g., volunteer blood donors, active duty and retired

military personnel, persons in the general population, health-

care workers, or clients attending sexually transmitted disease

[STD] clinics), the proportion of false-positive results with

HCV EIA 2.0 or HCV Version 3.0 ELISA averages approxi-

mately 35% (range: 15%–60%) (4–11; CDC, unpublished

data, 2002). Among immunocompromised populations (e.g.,

hemodialysis patients), the proportion of false-positive results

averages approximately 15% (14; CDC, unpublished data,

2002). For this reason, not relying exclusively on anti-HCV

screening-test–positive results to determine whether a person

has been infected with HCV is critical. Rather, screening-test–

positive results should be verified with an independent supple-

mental test with high specificity.

†

The terms reactive or nonreactive are used to describe serum or plasma

specimen test results from anti-HCV screening immunoassays before final

interpretation. The terms positive and negative are used to describe the final

interpretation of screening immunoassay test results (e.g., screening-test–

positive indicates that the specimen tested is repeatedly reactive by EIA or

reactive by CIA, or screening-test–negative indicates that the specimen tested

is nonreactive or not repeatedly reactive). The terms positive, indeterminate,

and negative are used to describe the interpretation of RIBA results based

on reactivity with a specific pattern of bands.

Vol. 52 / RR-3 Recommendations and Reports 3

anti-HCV test results are negative (screening-test–negative or

RIBA-negative) are considered uninfected (Box). However,

false-negative anti-HCV test results can occur during the first

weeks after infection (i.e., before antibody is detectable or

during seroconversion), although HCV RNA can be detected

as early as 1–2 weeks after exposure to the virus (16,17). Rarely,

antibody seroconversion might be delayed for months after

exposure (18,19). In certain persons whose HCV infection

has resolved, anti-HCV declines below detectable levels (20).

Occasionally, persons with chronic HCV infection, includ-

ing those who are immunocompromised, are persistently anti-

HCV–negative, and detection of HCV RNA might be the

only evidence of infection (14,21).

An indeterminate RIBA result indicates that the anti-HCV

result cannot be determined (Box). Indeterminate anti-HCV

supplemental test results have been observed in recently

infected persons who are in the process of seroconversion, and

occasionally in persons chronically infected with HCV (22).

Indeterminate results also might indicate a false-positive screen-

ing test result, which is the most common interpretation for

Supplemental Serologic Test Results

The strip immunoblot assay (RIBA), a supplemental anti-

HCV test with high specificity, is performed on screening-

test–positive samples and provides results that are interpreted

as positive, negative, or indeterminate. A positive RIBA result

is interpreted as anti-HCV–positive (Box). Although the pres-

ence of anti-HCV does not distinguish between current or

past infection, a confirmed anti-HCV–positive result indi-

cates the need for counseling and medical evaluation for HCV

infection, including additional testing for the presence of

virus (NAT for HCV RNA) and liver disease (e.g., alanine

aminotransferase [ALT]) (2,15). Anti-HCV testing usually

does not need to be repeated after the anti-HCV–positive re-

sult has been confirmed.

A negative RIBA result is interpreted as anti-HCV–negative

and indicates a false-positive screening test result. In this situ-

ation, the additional testing with RIBA minimizes unneces-

sary medical visits and psychological harm from reporting a

false-positive screening test result. Typically, persons whose

• An anti-HCV–negative person is considered uninfected.

• No further evaluation or follow-up for HCV is

required, unless recent infection is suspected or other

evidence exists to indicate HCV infection (e.g.,

abnormal liver enzyme levels in an immuno-

compromised person or a person with no other etiol-

ogy for their liver disease).

Anti-HCV–Indeterminate

An indeterminate anti-HCV result is defined as anti-

HCV screening-test–positive, RIBA-indeterminate.

• An indeterminate anti-HCV result indicates that the

HCV antibody status cannot be determined.

— Can indicate a false-positive anti-HCV screening

test result, the most likely interpretation among

those at low risk for HCV infection; such persons

are HCV RNA-negative.

— Can occur as a transient finding in a recently in-

fected person who is in the process of

seroconversion; such persons usually are HCV

RNA-positive.

— Can be a persistent finding among persons chroni-

cally infected with HCV; such persons usually are

HCV RNA-positive.

• If NAT is not performed, another sample should be

collected for repeat anti-HCV testing (

>1 month later).

BOX. Interpreting antibody to hepatitis C virus (anti-HCV) test results

Anti-HCV–Positive

An anti-HCV–positive result is defined as 1) anti-HCV

screening-test-positive* and recombinant immunoblot

assay (RIBA

)- or nucleic acid test (NAT)-positive; or

2) anti-HCV screening-test–positive, NAT-negative,

RIBA-positive.

• An anti-HCV–positive result indicates past or cur-

rent HCV infection.

— An HCV RNA-positive result indicates current

(active) infection, but the significance of a single

HCV RNA-negative result is unknown; it does not

differentiate intermittent viremia from resolved in-

fection.

• All anti-HCV–positive persons should receive coun-

seling and undergo medical evaluation, including ad-

ditional testing for the presence of virus and liver

disease.

— Anti-HCV testing usually does not need to be re-

peated after a positive anti-HCV result has been

confirmed.

Anti-HCV–Negative

An anti-HCV–negative result is defined as 1) anti-HCV

screening-test–negative*; or 2) anti-HCV screening-test–

positive, RIBA-negative; or 3) anti-HCV screening-test–

positive, NAT-negative, RIBA-negative.

* Interpretation of screening immunoassay test results based on criteria provided by the manufacturer.

4 MMWR February 7, 2003

these results among those at low risk for HCV infection

(23,24). Another sample should be collected for repeat anti-

HCV testing (

>1 month later) or for HCV RNA testing.

Supplemental NAT Results

NATs that detect HCV RNA also can be used as supple-

mental tests for anti-HCV. They are used commonly in clini-

cal practice for diagnosis of acute and chronic HCV infection

and for evaluating and managing patients with chronic

hepatitis C.

If the NAT result is positive in persons with a positive screen-

ing test result, NAT has the advantage of detecting the pres-

ence of active HCV infection as well as verifying the presence

of anti-HCV (Box). If the NAT result is negative in persons

with a positive screening test result, the HCV antibody or in-

fection status cannot be determined. Among persons with these

results, additional testing with RIBA is necessary to verify the

anti-HCV result and determine the need for counseling and

medical evaluation (Box); if the anti-HCV screening test

results are judged falsely positive (i.e., RIBA-negative), no fur-

ther evaluation of the person is needed; whereas if the anti-

HCV screening test results are verified as positive by RIBA,

the person should undergo medical evaluation, including

serial determinations of HCV RNA and ALT activity.

Certain situations exist in which the HCV RNA result can

be negative in persons with active HCV infection. As the titer

of anti-HCV increases during acute infection, the titer of HCV

RNA declines (17). Thus, HCV RNA is not detectable in cer-

tain persons during the acute phase of their hepatitis C, but

this finding can be transient and chronic infection can de-

velop (25). In addition, intermittent HCV RNA positivity has

been observed among persons with chronic HCV

infection (21,26,27). Therefore, in the absence of additional

clinical information, the significance of a single negative HCV

RNA result is unknown, and the need for further medical evalu-

ation is determined by verifying anti-HCV status.

A negative HCV RNA result also can indicate resolved

infection. Among anti-HCV–positive persons who acquired

their HCV infection as older adults (aged

>45 years), 15%–

25% apparently resolve their infection; this proportion is higher

(40%–45%) among anti-HCV–positive persons who acquired

their infection as children or younger adults (20). To deter-

mine if HCV infection has resolved, a negative HCV RNA

result should be demonstrated on multiple occasions; how-

ever, such follow-up testing is indicated only in persons with

serologically confirmed anti-HCV positive results.

Anti-HCV Testing Practices

Multiple commercial, hospital-based, and public health labo-

ratories that perform anti-HCV testing routinely report screen-

ing test results only. More specific testing (i.e., RIBA or NAT)

is performed only when ordered by a physician. Moreover, in

certain laboratories, more specific tests are not available.

During 2002, two surveys regarding anti-HCV testing prac-

tices were performed, one by the Association of Public Health

Laboratories (Barbara Werner, Ph.D., Association of Public

Health Laboratories, personal communication, September

2002) and one by a Veterans Affairs (VA) Medical Center

(D. Robert Dufour, M.D., VA Medical Center, Washington,

D.C., personal communication, October 2002). Forty-three

(80%) of 54 U.S. state and territorial public health laborato-

ries and 67 (39%) of 172 VA medical center laboratories

responded (Table 1).

Of the respondents, the public health laboratories were less

likely to offer screening or supplemental tests for HCV than

were the hospital-based VA laboratories. However, the public

health laboratories that did offer both types of testing were

more likely to perform reflex supplemental testing than were

the hospital-based laboratories, 75% of which performed

supplemental testing only by physician request. Regarding

the type of supplemental testing performed, the majority of

hospital-based laboratories performed only NATs, whereas the

public health laboratories most commonly performed either

RIBA alone or NAT followed by RIBA if the NAT result was

negative.

Although substantial differences existed in testing practices

between and among these two types of laboratories, the

majority of public and private sector laboratories depend on

the requesting physician to be knowledgeable concerning the

TABLE 1. Antibody to hepatitis C virus testing practices of

state and territorial public health laboratories and Veterans

Affairs Medical Center (VAMC) laboratories, 2002

Public health VAMC

Testing practices laboratories laboratories

Tests offered n = 43 n = 67

Screening test 65% 100%

Recombinant immunoblot assay (RIBA

) 38% 21%

Qualitative nucleic acid test (NAT) 29% 75%

Quantitative NAT 13% 98%

Supplemental testing performed n = 29 n = 67

All screening-test–positive results 35% 22%

Low-positive* screening-test results 10% 3%

Only by physician request 17% 75%

None offered 38% 0%

Type of supplemental test performed

†

n = 13 n = 16

RIBA only 31% 31%

NAT only 15% 63%

NAT followed by RIBA, if NAT-negative 39% 6%

RIBA followed by NAT, if RIBA-positive 15% 0%

* Signal-to-cut–off ratio below a specified value.

†

Includes only laboratories that routinely performed supplemental testing

on all specimens with screening-test–positive results or with low-positive

results.

Vol. 52 / RR-3 Recommendations and Reports 5

appropriate tests to order and the correct interpretation of

their results. However, a general lack of understanding exists

among health-care professionals regarding the interpretation

of screening test results, when more specific testing should be

performed, and which tests should be considered for this pur-

pose.

Using Screening-Test–Positive S/Co

Ratios To Determine Need for Reflex

Supplemental Testing

Analysis of early versions of anti-HCV EIA results from

volunteer blood donors indicated that average repeatedly

reactive s/co ratios could be used to predict supplemental test-

positive results (28). Similar data from volunteer blood

donors were generated by using HCV Version 3.0 ELISA, for

which the average s/co ratios of 24,700 samples repeatedly

reactive for anti-HCV were compared with their RIBA 3.0

results (Susan Stramer, Ph.D., American Red Cross, personal

communication, March 1999). Overall, 64.0% were RIBA-

positive. The proportion that tested RIBA-positive was 5.8%

for samples with an average s/co ratio 1.0–2.9; 37.1% for those

with average s/co ratio 3.0–3.4; 67% for those with average

s/co ratio 3.5–3.7; 88.1% for those with average s/co ratio

3.8–3.9; and 94.1% for those with average s/co ratio

>4.0.

Additional data from other populations were generated by

CDC to determine if a specific s/co ratio could be identified

that would predict a true antibody-positive result

>95% of

the time, regardless of the anti-HCV prevalence or character-

istics of the population being tested. The anti-HCV screening

tests evaluated were the two FDA-licensed EIAs, HCV EIA

2.0 and HCV Version 3.0 ELISA, and the one FDA-approved

CIA, VITROS Anti-HCV assay.

EIAs

All specimens with EIA screening-test–positive results were

tested by RIBA 3.0, and a sample of screening-test–positive

specimens were tested for HCV RNA by

>2 of the following

NAT methods: transcription-mediated amplification (TMA)

(Procleix™, Chiron Corporation, Emeryville, California);

AMPLICOR; and nested RT-PCR (13). Test results were used

from serum samples that had been collected as part of CDC-

sponsored anti-HCV seroprevalence studies that were con-

ducted among different groups of asymptomatic persons

(Robert Gunn, M.D., San Diego County Department of

Health and Human Services Agency; Steven Harris, M.D.,

Travis County, Texas Department of Health; Lu-Yu Hwang,

M.D., University of Texas — Houston School of Public

Health; Leslie Tobler, Ph.D., Blood Centers of the Pacific,

San Francisco; Gayle Shimokura, University of North Caro-

lina at Chapel Hill School of Public Health; Isaac Weisfuse,

M.D., New York City Department of Health and Mental

Hygiene, personal communications, 2001–2002; CDC,

unpublished data, 2002). Anti-HCV prevalences ranged from

0.8% to 25% (Table 2).

The proportion of screening-test–positive results that were

serologically confirmed as anti-HCV–positive (i.e., RIBA-

positive) increased as the anti-HCV prevalence in the popula-

tion increased (Table 2). Conversely, the proportion of

screening-test–positive results that were falsely antibody-

positive (RIBA-negative) or RIBA-indeterminate was inversely

related to prevalence (Table 2).

For each study group, the proportion of screening-test–

positive results that were RIBA-positive increased as the screen-

ing-test–positive average s/co ratios increased (Figure 1). On

the basis of these data, screening-test–positive average s/co

TABLE 2. Antibody to hepatitis C virus (anti-HCV) screening-test–positive average signal-to-cut–off (s/co) ratios by recombinant

immunoblot assay (RIBA

) 3.0 and nucleic acid test (NAT) results in groups with different anti-HCV prevalences

No. (%) HCV RNA-

Anti-HCV Total Average screening-

RIBA results (%)

positive

prevalence Study group tested* s/co ratio test–positive Negative Ind

†

Positive (%)

2% (average) Multiple groups

with prevalences 24,012 Total 689 (100.0) 26.9 7.3 65.9 41.6

ranging from 0.8% to 4.4% <3.8 231 (33.5) 78.8 16.9 4.3 3.7

>3.8 458 (66.5) 0.7 2.4 96.9 80.2

9.5% Hemodialysis patients 2,936 Total 351 (100.0) 9.7 7.3 83.0 80.3

<3.8 45 (12.8) 64.4 33.3 2.2 4.4

>3.8 306 (87.2) 1.6 3.4 95.0 91.5

24.9% STD clinic clients selected based 498 Total 124 (100.0) 1.6 4.0 94.4 NA

††

on risk** <3.8 3 (2.4) 66.7 33.3 0 NA

>3.8 121 (97.6) 0 3.3 96.7 NA

* HCV EIA 2.0 or HCV Version 3.0 ELISA.

†

Indeterminate.

College students, general population, health-care workers, and sexually transmitted disease (STD) clinic clients not selected by risk factor.

In the low (2%) prevalence group, only a sample of 214 were tested for HCV RNA.

** History of injection-drug use, blood transfusion before 1992, or incarceration.

††

Not available.

6 MMWR February 7, 2003

ratios >3.8 were highly predictive of RIBA positivity (>95%),

with limited variability (95%–97%) between groups with dif-

ferent prevalences (Table 2). Screening-test–positive average

s/co ratios

>3.8 also were highly predictive of HCV RNA posi-

tivity, although the proportions that were HCV RNA-posi-

tive were slightly lower than those for RIBA (Table 2).

These results indicate that for licensed EIAs, reporting anti-

HCV screening-test–positive results as anti-HCV positive for

samples with average s/co ratios

>3.8 would be highly predic-

tive of the true anti-HCV status. Reflex supplemental testing

before reporting the anti-HCV results could be limited to

screening-test–positive samples with average s/co ratios <3.8.

The feasibility of this approach is supported further by the

limited proportion (2.4%) of samples from persons at high

risk that have s/co ratios below this cut-off value. When test-

ing for anti-HCV is performed on persons at increased risk

for infection as recommended (2), a limited number of samples

will require additional testing.

CIA

The relation between s/co ratios and RIBA 3.0 results also

was evaluated for specimens that were screening-test–positive

by CIA (i.e., reactive by VITROS Anti-HCV) from four

groups. These included a group of 162 volunteer blood do-

nors with substantially low anti-HCV prevalence (Leslie Tobler,

Ph.D., Blood Centers of the Pacific, San Francisco, personal

communication, September 2002), a group of 163 persons

with low anti-HCV prevalence (college students, persons in

the general population, and health-care workers as described

previously), a group of 219 hemodialysis patients with inter-

mediate anti-HCV prevalence (as described previously), and

a group of 689 hospital-based patients with high anti-HCV

prevalence (signs or symptoms of liver disease or risk factors

for HCV infection) (D. Robert Dufour, M.D., VA Medical

Center, Washington, D.C., and Michael De Lucia, Ortho-

Clinical Diagnostics, personal communications, September

2002).

Overall, the proportion of CIA screening-test–positive

samples that tested RIBA-positive was 77.8% among the blood

donors, 74.2% among the low prevalence group, 86.3% among

the hemodialysis patients, and 94.5% among the high preva-

lence group. The direct relation between increasing s/co ratios

and RIBA positivity that was observed among samples tested

with the two EIAs evaluated by CDC also was observed among

the samples tested with the CIA (Figure 2). However, the range

of screening-test–positive s/co ratios obtained with VITROS

Anti-HCV was greater than that obtained with HCV EIA 2.0

or HCV Version 3.0 ELISA; thus, s/co ratios that were highly

predictive of RIBA positivity also were higher. Using VITROS

Anti-HCV, an s/co ratio of

>8 predicted RIBA positivity in

95%–98% of the screening-test–positive samples (Figure 2).

The proportion of CIA samples with low s/co ratios was in-

versely related to anti-HCV prevalence (i.e., 4.9% in the high

prevalence group, 8.7% in the intermediate prevalence group,

and 21.5% in the low prevalence group). These results indi-

cate that for the FDA-approved CIA, reflex supplemental test-

ing of screening-test–positive samples also could be limited to

those with a low (<8) s/co ratio; and among persons at in-

creased risk for infection,

<5% will have s/co ratios below the

cut-off value.

100

1.0–2.9 3.0 3.4– 3.5 3.7– >3.8

College students

General population

Health-care workers

STD clinic clients not selected by risk factor

†

Hemodialysis patients

STD clinic clients selected by risk factor

(n = 250) (n = 19) (n = 10) (n = 885)

Screening-test–positive average s/co ratio

Percentage RIBA-positive

FIGURE 1. Proportion of antibody to hepatitis C virus enzyme immunoassay* screening-

test–positive results that tested recombinant immunoblot assay (RIBA

) 3.0-positive

by average signal-to-cut–off (s/co) ratios and group tested

*HCV EIA 2.0 or HCV Version 3.0 ELISA.

†

Sexually transmitted diseases.

Vol. 52 / RR-3 Recommendations and Reports 7

Estimated Costs of Implementing

Reflex Supplemental Testing Based

on Screening-Test–Positive S/Co Ratios

To assist laboratories in assessing the potential financial

impact of implementing reflex supplemental testing for screening-

test–positive samples with low s/co ratios, the incremental costs

associated with such testing were estimated for three hypo-

thetical populations of 10,000 persons each, representing anti-

HCV prevalences of 2%, 10%, and 25%, respectively (similar

to those of the groups evaluated previously). For each popula-

tion, the costs of performing the screening test (by

using EIAs as the example) and each of two different supple-

mental testing schemes (schemes 1 and 2) were compared with

the cost of performing only the screening test (base scheme).

All schemes included performing a screening EIA on each

sample and repeating initially reactive specimens in duplicate.

Scheme 1 also included RIBA testing on all screening-test–

positive samples with average s/co ratios <3.8, and scheme 2

included NAT testing on all screening-test–positive samples

with average s/co ratios <3.8, followed by RIBA on those that

were NAT-negative.

The increased costs for schemes 1 and 2 were calculated per

sample tested compared with the base scheme. For RIBA and

NAT, minimum and maximum costs were estimated; mini-

mum costs were defined as costs for reagents only, and maxi-

mum costs were defined as costs incurred for tests performed

by a referral laboratory. The following assumptions were made:

• The percentage of initially reactive samples that were

repeatedly reactive (screening-test–positive) was assumed

to be 90% in the groups with anti-HCV prevalences of

2% and 10%, and 95% in the group with anti-HCV

prevalence of 25%.

• The proportion of screening-test–positive samples with

average s/co ratios <3.8 and the proportion of such samples

that tested RIBA-positive for each population was derived

(Table 2).

• The proportion of screening-test–positive samples with

average s/co ratios <3.8 that were NAT-positive was

derived (Table 2) for the populations with anti-HCV

prevalences of 2% and 10%. For the population with a

prevalence of 25%, this proportion was assumed to be

zero (on the basis of data from high-prevalence hospital-

based patients) (D. Robert Dufour, M.D., VA Medical

Center, Washington, D.C., personal communication,

September 2002).

Costs were estimated as follows and do not include person-

nel time or additional equipment:

• $5/sample for initial screening test;

• $15/sample for those testing initially reactive and repeated

in duplicate;

• $65–$158/sample tested with RIBA; and

• $50–$295/sample tested with a NAT.

Compared with performing only the screening test, perform-

ing reflex RIBA testing on all screening-test–positive samples

with average s/co ratios <3.8 (scheme 1) increases the cost of

testing per sample for immunocompetent populations from a

minimum of 5%–12% ($0.41–$0.66) to a maximum of 13%–

30% ($1.00–$1.60), depending on the anti-HCV prevalence

FIGURE 2. Proportion of antibody to hepatitis C virus (anti-HCV) enhanced

chemiluminescence immunoassay* screening-test–positive results that tested

recombinant immunoblot assay (RIBA

) 3.0-positive by signal-to-cut–off (s/co) ratios

and group tested

* VITROS

Anti-HCV assay.

†

College students, general population, and health-care workers.

Hospital-based patients.

100

1.0–7.9

(n = 142)

(n = 1,184)

Screening-test–positive s/co ratio

Volunteer blood donors

Low anti-HCV prevalence group

†

Hemodialysis patients

High anti-HCV prevalence group

Percentage RIBA-positive

8 MMWR February 7, 2003

of the population being tested (Figure 3). For hemodialysis

patients, the cost increases from a minimum of 16% ($1.00)

to a maximum of 38% ($2.44). Performing reflex NATs on

all screening-test–positive samples with average s/co ratios <3.8,

followed by RIBA on those that are NAT-negative (scheme

2), increases the cost of testing per sample for immunocom-

petent populations from a minimum of 9%–21% ($0.73–

$1.14) to a maximum of 37%–85% ($2.88–$4.54), compared

with performing only the screening test. For hemodialysis

patients, the cost increases from a minimum of 27% ($1.73)

to a maximum of 109% ($6.88). The higher incremental costs

of scheme 2 compared with scheme 1 are because virtually all

the screening-test–positive samples with s/co ratios <3.8 test

HCV RNA-negative and require follow-up testing with RIBA

to verify anti-HCV status.

Recommendations

Rationale

Testing for HCV infection by using anti-HCV is performed

for 1) clinical diagnosis of patients with signs or symptoms of

liver disease; 2) management of occupational and perinatal

exposures; and 3) screening asymptomatic persons to identify

HCV-infected persons who should receive counseling and

medical evaluation. Anti-HCV test results also are used for

public health surveillance to monitor incidence and preva-

lence and to target and evaluate HCV prevention efforts.

Anti-HCV testing is performed in multiple settings, includ-

ing hospitals and other health-care facilities, physicians’

offices, health department clinics, HIV or other freestanding

counseling and testing sites, employment sites, and health fairs.

The interpretation of anti-HCV screening-test–positive results

in these settings can be problematic. Clinical information

related to the persons tested often is lacking, and even persons

with risk factors for HCV infection might be at sufficiently

low enough risk for infection that their screening test results

could be falsely positive (e.g., health-care professionals are at

occupational risk for HCV infection, but their overall preva-

lence of infection is low) (29). Without knowledge of the ori-

gin of the test sample or clinical information related to the

person being tested, the accuracy of a screening-test–positive

result for any given specimen cannot be determined.

However, despite previous recommendations for reflex

supplemental testing of all anti-HCV screening-test–positive

results (2), the majority of laboratories report positive anti-

HCV results based only on a positive screening assay. To

facilitate and improve the practice of reflex supplemental test-

ing, the recommended anti-HCV testing algorithm has been

expanded to include an option for more specific testing based

FIGURE 3. Estimated increased cost per sample for reflex supplemental testing of

antibody to hepatitis C virus (anti-HCV) screening-test–positive results with low* average

signal-to-cut–off ratios compared with performing only the screening test among

populations with different anti-HCV prevalences

* <3.8 by HCV EIA 2.0 or HCV Version 3.0 ELISA.

†

Minimum costs based on reagents only; maximum costs based on testing performed by referral laboratory.

Includes only hemodialysis patients.

0510

15 20 25 30

Anti-HCV prevalence (%)

Minimum costs using recombinant immunoblot

assay (RIBA ) 3.0 only

Minimum costs using nucleic acid test (NAT),

followed by RIBA on NAT-negatives

Maximum costs using RIBA only

Maximum costs using NAT, followed by RIBA

on NAT-negatives

†

Incremental cost increase (dollars)

Vol. 52 / RR-3 Recommendations and Reports 9

on the s/co ratios of screening-test–positive results that can be

implemented without substantial increases in testing costs.

Implementation of these recommendations will provide

more reliable results for physicians and their patients, so that

further counseling and clinical evaluation are limited to those

confirmed to have been infected with HCV. This is critical

for persons being tested for HCV infection for the first time,

for persons being tested in nonclinical settings, and for those

being tested to determine the need for postexposure follow-

up. Implementation of these recommendations also will im-

prove public health surveillance systems for monitoring the

effect of HCV prevention and control activities.

Laboratory Algorithm for Anti-HCV

Testing and Result Reporting

All laboratories that provide anti-HCV testing should per-

form initial screening with an FDA-licensed or approved anti-

HCV test according to the manufacturer’s labeling.

• Screening-test–negative (i.e., nonreactive) samples require

no further testing and can be reported as anti-HCV–

negative (Figure 4).

• Screening-test–positive samples require reflex serologic or

nucleic acid supplemental testing according to the testing

algorithm (Figure 4). Laboratorians can choose to per-

form reflex supplemental testing 1) based on screening-

test–positive s/co ratios, or 2) on all specimens with

screening-test–positive results.

FIGURE 4. Laboratory algorithm for antibody to hepatitis C virus (anti-HCV) testing and result reporting

* Interpretation of screening immunoassay test results based on criteria provided by the manufacturer.

†

Signal-to-cut–off.

Screening-test–positive results are classified as having high s/co ratios if their ratios are at or above a predetermined value that predicts a supplemental-

test–positive result >95% of the time among all populations tested; screening-test–positive results are classified as having low s/co ratios if their ratios are

below this value.

Recombinant immunoblot assay.

Screening test for Anti-HCV

Positives* defined by s/co ratios

†

REPORT

Negative*

REPORT

REPORT REPORT REPORT

REPORT

Negative Positive

NegativePositive

NegativePositive Indeterminate

Indeterminate

All positives*

Positives with high s/co ratios

Positives with low s/co ratios

RIBA for anti-HCV

®¶

Nucleic acid test for HCV RNA

RIBA for anti-HCV

10 MMWR February 7, 2003

— For screening-test–positive samples that require reflex

supplemental testing (according to the testing option

chosen), the anti-HCV result should not be reported

until the results from the additional tests are available.

Reflex Supplemental Testing Based

on Screening-Test–Positive S/Co Ratios

• Laboratories should use only screening tests that have been

evaluated for this purpose

and for which high s/co ratios

have been demonstrated to predict a supplemental-test–

positive

>95% of the time among all populations tested.

• Screening-test–positive samples with high s/co ratios can

be reported as anti-HCV–positive without supplemental

testing (Figure 4).

• A comment should accompany the report indicating that

supplemental serologic testing was not performed, and it

should include a statement that samples with high s/co

ratios usually (

>95%) confirm positive, but <5 of every

100 samples with these results might be false-positives.

The ordering physician also should be informed that more

specific testing can be requested, if indicated.

• Screening-test–positive samples with low s/co ratios should

have reflex supplemental testing performed, preferably

RIBA (Figure 4).

Reflex Supplemental Testing on All Specimens

with Screening-Test–Positive Results

• RIBA only; or

• NAT, followed by RIBA for specimens with NAT-

negative results (Figure 4).

Considerations When Choosing a Reflex

Supplemental Testing Option

Serologic Supplemental Testing.

• RIBA can be performed on the same sample collected for

the screening test.

• RIBA is the most cost-effective supplemental test for veri-

fying anti-HCV status for screening-test–positive samples

with low s/co ratios.

• The RIBA result is used to report the anti-HCV result.

Nucleic Acid Supplemental Testing.

• NATs can be performed in laboratories that have facilities

specifically designed for that purpose.

• Serum or plasma samples must be collected, processed,

and stored in a manner suitable for NATs to minimize

false-negative results (30).

— Blood should be collected in sterile collection tubes

with no additives or in sterile tubes by using

ethylenediaminetetraacetic acid (EDTA).

— Serum or EDTA plasma must be separated from cel-

lular components within 2–6 hours after collection.

— Storage of serum or EDTA plasma at 2

C–5

C is

limited to 72 hours; for longer storage, freezing at

–20

C or –70

C is recommended. If shipping is

required, frozen samples should be protected from

thawing.

— Samples collected for serologic testing can be used only

if the previous conditions are met.

• Because of assay variability, rigorous quality assurance and

control should be standards of practice in clinical labora-

tories performing this assay; proficiency testing is recom-

mended, including monitoring for false-positive results.

— Technician proficiency can vary and increases in

direct relation to experience.

— Intra-assay contamination can occur, including aero-

solization, splashing, and carry-over.

• If the HCV RNA result is positive, the presence of active

HCV infection can be reported as well as a positive anti-

HCV result.

• An HCV RNA-negative result requires that RIBA be per-

formed and the RIBA result used to report the anti-HCV

result.

Other Reflex Supplemental Testing Options

Certain laboratories might choose to modify the recom-

mended supplemental testing options to provide additional

information before reporting results. One such modification

might include reflex NAT of screening-test–positive results

with high s/co ratios, which might be of interest to hospital-

based laboratories that usually test specimens from patients

being evaluated for liver disease. If the NAT result is positive,

the presence of active HCV infection can be reported as well

as a positive anti-HCV result. However, if the NAT result is

negative, reflex RIBA testing still is required before reporting

the results to verify the anti-HCV status. Certain specimens

will test RIBA-positive, indicating that the person should

receive further evaluation, including repeat testing for HCV

RNA (see Interpretation of Anti-HCV Test Results).

Implementation

To implement these recommendations for anti-HCV test-

ing and result reporting, laboratories should review their present

Data are available from three screening assays. For the two EIAs (HCV EIA

2.0 or HCV Version 3.0 ELISA), high s/co ratios are defined as screening-

test–positive results with average s/co ratios

>3.8, and low s/co ratios as

screening-test–positive results with average s/co ratios <3.8. For CIA (VITROS

Anti-HCV), high s/co ratios are defined as screening-test–positive results with

s/co ratios >8, and low s/co ratios as screening-test–positive results with s/co

ratios <8.

Vol. 52 / RR-3 Recommendations and Reports 11

testing and reporting methods and determine how those should

be modified. This process should include

• determining which reflex supplemental testing option will

be implemented;

• revising standard operating procedures to include the

reflex testing option selected (Figure 4), the procedure for

reporting results, and the interpretation of those results

(Table 3);

• educating the laboratory staff, physicians, and other end-

users; and

• modifying the laboratory requisition form, if necessary.

For purposes of reimbursement, the circumstances under

which reflex supplemental testing will be performed might

need to be included on the form to serve as documenta-

tion that the additional tests were ordered.

Laboratories that select a reflex supplemental testing option

based on screening-test–positive s/co ratios need to ensure that

their analyzers generate optical density (OD) values in a range

sufficient to calculate s/co ratios at or above the value defined

as a high s/co ratio for the screening test being used. The s/co

ratio is calculated by dividing the OD value of the sample

being tested by the OD value of the assay cut-off for that run.

Depending on the type of equipment in the laboratory, the

calculation of s/co ratios might be automatically performed

by the analyzer or require that the technician manually per-

form the calculation.

For screening tests that require only one reactive result to

indicate a screening-test–positive result (e.g., VITROS Anti-

HCV), the s/co ratio of the reactive result is used to deter-

mine the next step in the algorithm (i.e., reporting the result

or reflex supplemental testing). For screening tests that require

repeating initially reactive results in duplicate (e.g, HCV EIA

2.0 and HCV Version 3.0 ELISA), the s/co ratio of each of

the duplicate results is calculated. The average of the s/co

ratios of the reactive results is used to determine the next step

in the algorithm. If all three results are reactive for the sample,

the average s/co ratio can be determined either by averaging

the ratios of all three or by averaging only the ratios of the two

duplicate reactive results. If only one of the duplicate results is

reactive, the average s/co ratio is determined by averaging the

TABLE 3. Recommendations for reporting results of testing for antibody to hepatitis C virus (anti-HCV) by type of reflex

supplemental testing performed

Anti-HCV screening test results

Screening-test–negative*

Screening-test–positive*

with high signal-to-cut–off

(s/co) ratio

Screening-test–positive

Supplemental test results

Not applicable

Not done

Recombinant immunoblot

assay (RIBA

)-positive

RIBA-negative

RIBA-indeterminate

Nucleic acid test

(NAT)-positive

NAT-negative

RIBA-positive

NAT-negative

RIBA-negative

NAT-negative

RIBA-indeterminate

Interpretation

Anti-HCV–negative

Anti-HCV–positive

Anti-HCV–negative

Anti-HCV–indeterminate

Anti-HCV–positive,

HCV RNA-positive

Anti-HCV–positive,

HCV RNA-negative

Anti-HCV–negative,

HCV RNA-negative

Anti-HCV-indeterminate

HCV RNA-negative

Comments

Not infected with HCV, unless recent infection is

suspected or other evidence exists to indicate HCV

infection

Probably indicates past or present HCV infection;

supplemental serologic testing not performed.

Samples with high s/co ratios usually (>95%)

confirm positive, but <5 of every 100 might

represent false-positives; more specific testing can

be requested, if indicated

Indicates past or present HCV infection

Not infected with HCV, unless recent infection is

suspected or other evidence exists to indicate HCV

infection

HCV antibody and infection status cannot be

determined; another sample should be collected for

repeat anti-HCV testing (>1 month) or for HCV RNA

testing

Indicates active HCV infection

The presence of anti-HCV indicates past or present

HCV infection; a single negative HCV RNA result

does not rule out active infection

Not infected with HCV

Screening test anti-HCV result probably a false-

positive, which indicates no HCV infection

*Screening immunoassay test results interpreted as negative or positive on the basis of criteria provided by the manufacturer.

12 MMWR February 7, 2003

ratios from the initial reactive result and the one duplicate

reactive result.

For those screening-test–positive samples that undergo

reflex supplemental testing (according to the testing option

chosen), the screening test anti-HCV results should not be

reported before the results from the additional testing are avail-

able. If necessary, an interim report can be issued indicating

that the result is pending. This procedure should be followed

even if the laboratory does not perform the supplemental test-

ing in-house, but sends the sample to another reference labo-

ratory for such testing. After the results are received from the

reference laboratory, the final results can be reported on the

basis of the testing performed by both laboratories.

The reported results should be accompanied by interpretive

comments as determined by each laboratory (Table 3). The

content of these comments will vary on the basis of type of

supplemental testing option selected by the laboratory. These

comments are critical if screening-test–positive results are

reported as anti-HCV–positive on the basis of high s/co

ratios, because the health-care professional or other person

interpreting the results needs to understand the limitations of

the testing option used.

Before implementation, the laboratory staff should be edu-

cated regarding new methods of testing, calculating, and

reporting final results for the selected testing option. Labora-

tories also should inform and educate all customers regarding

the planned changes and what effects they will have on test

results generated. This information should be disseminated as

widely as possible (e.g., by laboratory bulletins, letters, Internet,

or continuing education programs).

Depending on the setting, reimbursement of clinical labo-

ratory tests used for reflex supplemental testing might depend

on documentation that the physician ordered the tests. This

documentation can be achieved through a printed requisition

form that clearly identifies for anti-HCV the specified level of

results of the screening test that will trigger additional supple-

mental testing and what type(s) of supplemental testing will

be performed. In addition, each of the supplemental tests (e.g.,

RIBA or NAT) that are offered by the laboratory should be

listed separately, because physicians should be able to order

these as they deem necessary for further medical evaluation.

Future Considerations

As new anti-HCV screening assays are approved or licensed

for use, each will need to be evaluated for its specificity among

populations with different anti-HCV prevalences. In addition,

before using a new assay to perform reflex supplemental test-

ing based on screening-test–positive s/co ratios, the s/co ratio

value at or above which supplemental test results are positive

>95% of the time in populations in which the test will be

used, should be determined. Such documentation also should

be required for approved screening assays if any modifications

are made to the testing procedures that might affect the s/co

ratio values. Similarly, the relation between screening-test–

positive results and the results of newly available supplemen-

tal tests will need to be evaluated.

Acknowledgments

We acknowledge Garth Austin, M.D., Ph.D., Veterans Affairs

Medical Center, Atlanta, Georgia, for performing serologic testing,

and D. Robert Dufour, M.D., Veterans Affairs Medical Center,

Washington, D.C., Leslie Tobler, Ph.D., and Michael Busch, M.D.,

Ph.D., Blood Centers of the Pacific, San Francisco, California, and

Susan Stramer, Ph.D., American Red Cross, Gaithersburg, Maryland,

for sharing their data and expertise and for performing serologic and

nucleic acid testing to generate additional information that was

needed to develop these guidelines.

References

1. CDC. Public Health Service inter-agency guidelines for screening

donors of blood, plasma, organs, tissues, and semen for evidence of

hepatitis B and hepatitis C. MMWR 1991;40(No. RR-4):1–17.

2. CDC. Recommendations for prevention and control of hepatitis C

virus (HCV) infection and HCV-related chronic disease. MMWR

1998;47(No. RR-19):1–33.

3. CDC. Revised guidelines for HIV counseling, testing, and referral.

MMWR 2001;50(No. RR-19):1–53.

4. Kleinman S, Alter H, Busch M, et al. Increased detection of hepatitis C

virus (HCV)-infected blood donors by a multiple-antigen HCV

enzyme immunoassay. Transfusion 1992;32:805–13.

5. Conry-Cantilena C, VanRaden M, Gibble J, et al. Routes of infection,

viremia, and liver disease in blood donors found to have hepatitis C

virus infection. N Engl J Med 1996;334:1691–6.

6. Hyams KC, Riddle J, Rubertone M, et al. Prevalence and incidence of

hepatitis C virus infection in the US military: a seroepidemiologic sur-

vey of 21,000 troops. Am J Epidemiol 2001;153:764–70.

7. Alter MJ, Kruszon-Moran D, Nainan OV, et al. Prevalence of hepatitis

C virus infection in the United States, 1988 through 1994. N Engl J

Med 1999;341:556–62.

8. Montecalvo MA, Lee S, DePalma H, et al. Seroprevalence of human

immunodeficiency virus-1, hepatitis B virus, and hepatitis C virus in

patients having major surgery. Infect Control Hosp Epidemiol

1995;16:627–32.

9. Goetz AM, Ndimbie OK, Wagener MM, Muder RR. Prevalence of

hepatitis C infection in health care workers affiliated with a liver trans-

plant center. Transplantation 1995;59:990–4.

10. CDC. Hepatitis C virus infection among firefighters, emergency medi-

cal technicians, and paramedics—selected locations, United States, 1991–

2000. MMWR 2000;49:660–5.

11. Gunn, RA, Murray PJ, Ackers ML, Hardison WGM, Margolis HS.

Screening for chronic hepatitis B and C virus infections in an urban

sexually transmitted disease clinic: rationale for integrating services. Sex

Transm Dis 2001;28:166–70.

Vol. 52 / RR-3 Recommendations and Reports 13

12. Pawlotsky JM. Use and interpretation of virological tests for hepatitis

C. Hepatology 2002;36:S65–S73.

13. Nainan OV, Cromeans TL, Margolis HS. Sequence-specific, single-

primer amplification and detection of PCR products for identification

of hepatitis viruses. J Virol Methods 1996;61:127–34.

14. CDC. Recommendations for preventing transmission of infections

among chronic hemodialysis patients. MMWR 2001;50(No. RR-5):1–43.

15. National Institutes of Health. National Institutes of Health Consensus

Development Conference Statement: management of hepatitis C:

2002—June 10–12, 2002. Hepatology 2002;36:S3–S20.

16. Alter HJ, Jett BW, Polito AJ, et al. Analysis of the role of hepatitis C

virus in transfusion-associated hepatitis. In: Hollinger FB, Lemon SM,

Margolis H.S., eds. Viral hepatitis and liver disease. Baltimore, MD:

Williams and Wilkins, 1991;396–402.

17. Busch MP, Kleinman SH, Jackson B, Stramer SL, Hewlett I, Preston S.

Committee report. Nucleic acid amplification testing of blood donors

for transfusion-transmitted infectious diseases: report of the

Interorganizational Task Force on Nucleic Acid Amplification of Blood

Donors. Transfusion 2000;40:143–59.

18. Ridzon R, Gallagher K, Ciesielski C, et al. Simultaneous transmission

of human immunodeficiency virus and hepatitis C virus from a needle-

stick injury. N Engl J Med 1997;336:919–22.

19. Bux-Gewehr I, Schmandt S, Zotz RB, Scharf RE, Roggendorf M. Long-

term hepatitis C seroconversion in a blood donor. Transfusion

2001;41:427.

20. Alter HJ, Seeff LB. Recovery, persistence, and sequelae in hepatitis C

virus infection: a perspective on long-term outcome. Semin Liver Dis

2000;20:17–35.

21. Alter MJ, Margolis HS, Krawczynski K, et al. Natural history of com-

munity-acquired hepatitis C in the United States. N Engl J Med

1992;327:1899–905.

22. Tobler LH, Stramer SL, Kleinman SH, Brodsky JP, Todd DS, Busch

MP. Misclassification of HCV-viremic blood donors as indeterminate

by RIBA 3.0 because of human superoxide dismutase reactivity. Trans-

fusion 2001;41:1625–6.

23. Vernelen K, Claeys H, Verhaert AH, Volckaerts A, Vermylen C. Signifi-

cance of NS3 and NS5 antigens in screening for HCV antibody. Lancet

1994;343:853.

24. Damen M, Zaaijer HL, Cuypers HT, Vrielink H, van der Poel CL,

Reesink HW, Lelie PN. Reliability of the third-generation recombinant

immunoblot assay for hepatitis C virus. Transfusion 1995;35:745–9.

25. Williams IT, Gretch D, Fleenor M, et al. Hepatitis C virus RNA con-

centration and chronic hepatitis in a cohort of patients followed after

developing acute hepatitis C. In: Margolis HS, Alter MJ, Liang TJ,

Dienstag JL, eds. Viral hepatitis and liver disease. Atlanta, GA: Interna-

tional Medical Press, 2002;341–4.

26. Thomas DL, Astemborski J, Rai RM, et al. Natural history of hepatitis

C virus infection: host, viral, and environmental factors. JAMA

2000;284:450–6.

27. Larghi A, Zuin M, Crosignani A, et al. Outcome of an outbreak of

acute hepatitis C among healthy volunteers participating in pharmaco-

kinetics studies. Hepatology 2002;36:993–1000.

28. Tobler LH, Tegtmeier G, Stramer SL, et al. Lookback on donors who

are repeatedly reactive on first-generation hepatitis C virus assays: justi-

fication and rational implementation. Transfusion 2000;40:15–24.

29. Alter MJ. Prevention of spread of hepatitis C. Hepatology 2002;36:S93–

S98.

30. Davis GL, Lau JY, Urdea MS, et al. Quantitative detection of hepatitis

C virus RNA with a solid-phase signal amplification method: definition

of optimal conditions for specimen collection and clinical application

in interferon-treated patients. Hepatology 1994;19:1337–41.

14 MMWR February 7, 2003

Vol. 52 / RR-3 Recommendations and Reports 15

Terms and Abbreviations Used in This Report

ALT Alanine aminotransferase

Anti-HCV Antibody to hepatitis C virus

CIA Chemiluminescence immunoassay, a screening test format for anti-HCV (e.g., VITROS

Anti-HCV assay)

EIA Enzyme immunoassay, a screening test format for anti-HCV (e.g., Abbott HCV EIA 2.0 and

ORTHO

HCV Version 3.0 ELISA)

FDA Food and Drug Administration

FDA-approved Assays intended only for in vitro diagnostic use; approved by the Center for Devices and Radiological

Health, FDA

FDA-licensed Assays intended primarily for donor screening; licensed by the Center for Biologics Evaluation and

Research, FDA

HCV Hepatitis C virus

HCV RNA Hepatitis C virus ribonucleic acid

NAT Nucleic acid test, detects HCV RNA by amplification of viral genetic sequences

OD Optical density

Reflex testing Additional testing automatically performed in response to a screening-test–positive result

RIBA

Recombinant immunoblot assay, a more specific serological anti-HCV assay

RT-PCR Reverse transcriptase-polymerase chain reaction amplification, a nucleic acid testing method for

detection of HCV RNA

S/Co ratio Signal to cut-off ratio, calculated by dividing the OD value of the sample being tested by the OD

value of the assay cut-off for that run

Screening tests Serologic immunoassays for detection of anti-HCV (e.g., Abbott HCV EIA 2.0, ORTHO HCV

Version 3.0 ELISA, and VITROS Anti-HCV)

Screening-test–negative Screening immunoassay test result interpreted as negative on the basis of criteria provided by the

manufacturer

Screening-test–positive Screening immunoassay test result interpreted as positive on the basis of criteria provided by the

manufacturer

STD Sexually transmitted disease

Supplemental test More specific test (e.g., RIBA or NAT) used to verify a positive anti-HCV screening test result

TMA Transcription-mediated amplification, a nucleic acid testing method for detection of HCV RNA

VA Veterans Affairs

16 MMWR February 7, 2003

Recommendations and Reports February 7, 2003 / Vol. 52 / No. RR-3

Morbidity and Mortality Weekly Report

depardepar

depar

tment of health and human sertment of health and human ser

tment of health and human ser

vicesvices

vices

Centers for Disease Control and PreventionCenters for Disease Control and Prevention

Centers for Disease Control and Prevention

Guidelines for Laboratory Testing and Result Reporting

of Antibody to Hepatitis C Virus

ACCREDITATION

Continuing Medical Education (CME). CDC is accredited by the Accreditation Council for Continuing Medical Education (ACCME) to provide

continuing medical education for physicians. CDC designates this educational activity for a maximum of 1.5 hours in category 1 credit toward the AMA

Physician’s Recognition Award. Each physician should claim only those hours of credit that he/she actually spent in the educational activity.

Continuing Education Unit (CEU). CDC has been approved as an authorized provider of continuing education and training programs by the

International Association for Continuing Education and Training and awards 0.15 Continuing Education Units (CEUs).

Continuing Nursing Education (CNE). This activity for 1.8 contact hours is provided by CDC, which is accredited as a provider of continuing education

in nursing by the American Nurses Credentialing Center’s Commission on Accreditation.

You must complete and return the response form electronically or by mail by

February 7, 2006, to receive continuing education credit. If you answer all

of the questions, you will receive an award letter for 1.5 hours Continuing

Medical Education (CME) credit; 0.15 Continuing Education Units (CEUs);

EXPIRATION — February 7, 2006

or 1.8 contact hours Continuing Nursing Education (CNE) credit. If you

return the form electronically, you will receive educational credit immediately.

If you mail the form, you will receive educational credit in approximately 30

days. No fees are charged for participating in this continuing education activity.

By Internet

1. Read this MMWR (Vol. 52, RR-3), which contains the correct answers to

the questions beginning on the next page.

2. Go to the MMWR Continuing Education Internet site at <http://

www.cdc.gov/mmwr/cme/conted.html>.

3. Select which exam you want to take and select whether you want to register

for CME, CEU, or CNE credit.

4. Fill out and submit the registration form.

5. Select exam questions. To receive continuing education credit, you must

answer all of the questions. Questions with more than one correct answer

will instruct you to “Indicate all that apply.”

6. Submit your answers no later than February 7, 2006.

7. Immediately print your Certificate of Completion for your records.

By Mail or Fax

1. Read this MMWR (Vol. 52, RR-3), which contains the correct answers to

the questions beginning on the next page.

2. Complete all registration information on the response form, including your

name, mailing address, phone number, and e-mail address, if available.

3. Indicate whether you are registering for CME, CEU, or CNE credit.

4. Select your answers to the questions, and mark the corresponding letters on

the response form. To receive continuing education credit, you must

answer all of the questions. Questions with more than one correct answer

will instruct you to “Indicate all that apply.”

5. Sign and date the response form or a photocopy of the form and send no

later than February 7, 2006, to

Fax: 404-639-4198 Mail: MMWR CE Credit

Office of Scientific and Health Communications

Epidemiology Program Office, MS C-08

Centers for Disease Control and Prevention

1600 Clifton Rd, N.E.

Atlanta, GA 30333

6. Your Certificate of Completion will be mailed to you within 30 days.

INSTRUCTIONS

CE-2 MMWR February 7, 2003

1. Why should laboratories verify anti-HCV screening-test–positive

results with a more specific supplemental assay before reporting the

results? (Indicate all that apply.)

A. To minimize unnecessary medical visits and psychological harm for

persons who test falsely positive.

B. To ensure that counseling, medical referral, and evaluation are targeted

to persons serologically confirmed as having been infected with HCV.

C. Because the accuracy of a screening-test–positive result for any given

specimen cannot be determined without clinical or risk information

regarding the person being tested.

D. Because the ordering physician might not understand the

interpretation of anti-HCV screening test results, when more specific

testing should be performed, and which tests should be considered for

this purpose.

2. Which of the following verifies a false-positive anti-HCV screening test

result? (Indicate all that apply.)

A. Screening-test–positive, recombinant immunoblot assay (RIBA

negative.

B. Screening-test–positive, RIBA-indeterminate.

C. Screening-test–positive, nucleic acid test (NAT)-negative.

D. Screening-test–positive, NAT-negative, RIBA-negative.

3. Which of the following verifies a true positive anti-HCV screening test

result? (Indicate all that apply.)

A. Screening-test–positive, RIBA-positive.

B. Screening-test–positive, RIBA-indeterminate.

C. Screening-test–positive, NAT-positive.

D. Screening-test–positive, NAT-negative, RIBA-positive.

4. Which of the following is true regarding a RIBA indeterminate anti-

HCV test result? (Indicate all that apply.)

A. Might indicate recent infection in persons who are in the process of

seroconversion.

B. Might indicate a false-positive anti-HCV screening test result.

C. Occasionally is observed in persons with chronic HCV infection.

D. Further testing needs to be done.

5. Which of the following actions should be taken for specimens that are

anti-HCV screening-test–positive and NAT-negative. (Choose the one

correct answer.)

A. No further testing is required.

B. NAT should be repeated.

C. The screening immunoassay should be repeated.

D. RIBA should be performed.

6. Which of these statements concerning NAT for HCV RNA is not true?

(Choose the one correct answer.)

A. NAT can be used as a reflex supplemental assay to verify the presence

of anti-HCV.

B. NAT can detect the presence of active HCV infection.

C. In persons with a positive anti-HCV screening test, a single negative

NAT result indicates that the person is not infected with HCV.

D. In persons with a positive anti-HCV screening test, a single negative

NAT result cannot determine the HCV antibody or infection status.

Goal and Objectives

This MMWR provides guidelines for laboratory testing and result reporting of antibody to hepatitis C virus (anti-HCV). These guidelines were prepared by CDC

staff after consultation with staff members from other federal agencies and specialists in the field and are intended for laboratorians and other health-care professionals

who request, interpret, or perform anti-HCV tests. The goal of this report is to improve the accuracy and utility of reported anti-HCV test results for counseling and

medical evaluation of patients by health-care professionals and for surveillance by public health departments. Upon completion of this educational activity, the reader

should be able to describe 1) the importance of reflex supplemental testing to verify the presence of anti-HCV; 2) anti-HCV screening and supplemental test results;

and 3) when more specific anti-HCV testing should be performed and which tests should be used for this purpose.

To receive continuing education credit, please answer all of the following questions.

7. In which situations might HCV RNA be negative in persons with

active HCV infection? (Indicate all that apply.)

A. During the period of anti-HCV seroconversion in persons with acute

HCV infection.

B. During chronic HCV infection, if HCV RNA is intermittently positive.

C. None; persons with active HCV infection always test HCV RNA-positive.

D. Laboratory testing error resulting in a false-negative HCV RNA result.

8. Which of the following statements is true regarding procedures for

performing NAT for HCV RNA? (Indicate all that apply.)

A. Laboratories performing the test must have facilities specifically

designed for this purpose.

B. Whole blood can be stored at 2

C–5

C for <72 hours before separation

of serum or plasma.

C. Serum or plasma must be separated from cellular components within

2–6 hours after collection.

D. For storage >72 hours, serum or plasma should be frozen.

9. Which of the following are included in the recommendations for

laboratory testing and result reporting for anti-HCV? (Indicate all that

apply.)

A. Perform initial screening with an FDA-licensed or approved anti-

HCV test and report the final results on the basis of the screening test.

B. Only screening-test–negative samples can be reported as anti-HCV–

negative without further testing.

C. Reflex supplemental testing can be performed on the basis of

screening-test–positive signal-to-cut–off (s/co) ratios or on all

specimens with screening-test–positive results.

D. For screening-test–positive samples that require reflex supplemental

testing, the anti-HCV result should not be reported until the results

from the additional tests are available.

10. Which of the following statements is true regarding reflex

supplemental testing based on screening-test–positive s/co ratios?

(Indicate all that apply.)

A. Laboratories should use only screening tests for which high s/co ratios

have been demonstrated to predict a supplemental test-positive

>95%

of the time among all populations tested.

B. Screening-test–positive samples with high s/co ratios can be reported

as anti-HCV–positive without supplemental testing, along with a

comment explaining the results.

C. Screening-test–positive samples with low s/co ratios require reflex

supplemental testing.

D. Screening-test–positive samples with low s/co ratios that test RIBA-

negative can be reported as anti-HCV–negative.

E. Screening-test–positive samples with low s/co ratios that are NAT-

negative require additional testing by RIBA to determine anti-HCV status.

11. For the currently approved screening immunoassays, what are the s/co

ratio values below which all screening-test–positive samples require

supplemental testing. (Choose the one correct answer.)

A. <3.8 for the enzyme immunoassays (EIA) and the chemiluminescence

assay (CIA).

B. <3.8 for the EIA and <8.0 for the CIA.

C. <4.0 for the EIA and <12.0 for the CIA.

D. None of the above.

Vol. 52 / No. RR-3 Recommendations and Reports CE-3

Detach or photocopy.

MMWR Response Form for Continuing Education Credit

February 7, 2003/Vol. 52/No. RR-3

Guidelines for Laboratory Testing and Result Reporting

of Antibody to Hepatitis C Virus

To receive continuing education credit, you must

1. provide your contact information;

2. indicate your choice of CME, CEU, or CNE credit;

3. answer

all of the test questions;

4. sign and date this form or a photocopy;

5. submit your answer form by February 7, 2006.

Failure to complete these items can result in a delay or

rejection of your application for continuing education credit.

Last Name First Name

Street Address or P.O. Box

Apartment or Suite

City State ZIP Code

Phone Number Fax Number

E-Mail Address

Signature Date I Completed Exam

Fill in the appropriate blocks to indicate your answers. Remember, you must answer all

of the questions to receive continuing education credit!

1. [ ] A [ ] B [ ] C [ ] D 14. [ ] A [ ] B [ ] C [ ] D [ ] E

2. [ ] A [ ] B [ ] C [ ] D 15. [ ] A [ ] B [ ] C [ ] D [ ] E

3. [ ] A [ ] B [ ] C [ ] D 16. [ ] A [ ] B [ ] C [ ] D

4. [ ] A [ ] B [ ] C [ ] D 17. [ ] A [ ] B [ ] C [ ] D [ ] E

5. [ ] A [ ] B [ ] C [ ] D 18. [ ] A [ ] B [ ] C [ ] D [ ] E

6. [ ] A [ ] B [ ] C [ ] D 19. [ ] A [ ] B [ ] C [ ] D [ ] E

7. [ ] A [ ] B [ ] C [ ] D 20. [ ] A [ ] B [ ] C [ ] D [ ] E

8. [ ] A [ ] B [ ] C [ ] D 21. [ ] A [ ] B [ ] C [ ] D [ ] E

9. [ ] A [ ] B [ ] C [ ] D 22. [ ] A [ ] B [ ] C [ ] D [ ] E

10. [ ] A [ ] B [ ] C [ ] D [ ] E 23. [ ] A [ ] B [ ] C [ ] D [ ] E

11. [ ] A [ ] B [ ] C [ ] D 24. [ ] A [ ] B [ ] C [ ] D [ ] E

12. [ ] A [ ] B [ ] C [ ] D [ ] E [ ] F 25. [ ] A [ ] B [ ] C [ ] D [ ] E [ ] F

13. [ ] A [ ] B [ ] C [ ] D [ ] E [ ] F

Check One

CME Credit

CNE Credit

CEU Credit

12. Indicate your work setting.

A. State/local health department.

B. Other public health setting.

C. Hospital clinic/private practice.

D. Managed care organization.

E. Academic institution.

F. Other.

13. Which best describes your professional activities?

A. Patient care — emergency/urgent care department.

B. Patient care — inpatient.

C. Patient care — primary-care clinic or office.

D. Laboratory/pharmacy.

E. Public health.

F. Other.

14. I plan to use these recommendations as the basis for . . . (Indicate all

that apply.)

A. health education materials.

B. insurance reimbursement policies.

C. local practice guidelines.

D. public policy.

E. other.

15. Each month, approximately how many patients do you test for

hepatitis C?

A. None.

B. 1–10.

C. 11–100.

D. 101–1,000.

E. >1,000.

16. How much time did you spend reading this report and completing the

exam?

A. <1.0 hour.

>1.0 hour but <2.0 hours.

>2.0 but <3.0 hours.

>3.0 hours.

17. After reading this report, I am confident I can describe the importance

of reflex supplemental testing to verify the presence of anti-HCV.

A. Strongly agree.

B. Agree.

C. Neither agree nor disagree.

D. Disagree.

E. Strongly disagree.

18. After reading this report, I am confident I can describe anti-HCV

screening and supplemental test results.

A. Strongly agree.

B. Agree.

C. Neither agree nor disagree.

D. Disagree.

E. Strongly disagree.

19. After reading this report, I am confident I can describe when more

specific anti-HCV testing should be performed and which tests should

be used for this purpose.

A. Strongly agree.

B. Agree.

C. Neither agree nor disagree.

D. Disagree.

E. Strongly disagree.

CE-4 MMWR February 7, 2003

20. The objectives are relevant to the goal of this report.

A. Strongly agree.

B. Agree.

C. Neither agree nor disagree.

D. Disagree.

E. Strongly disagree.

21. The box, tables, and figures are useful.

A. Strongly agree.

B. Agree.

C. Neither agree nor disagree.

D. Disagree.

E. Strongly disagree.

22. Overall, the presentation of the report enhanced my ability to

understand the material.

A. Strongly agree.

B. Agree.

C. Neither agree nor disagree.

D. Disagree.

E. Strongly disagree.

23. These recommendations will affect my practice.

A. Strongly agree.

B. Agree.

C. Neither agree nor disagree.

D. Disagree.

E. Strongly disagree.

24. The availability of continuing education credit influenced my decision

to read this report.

A. Strongly agree.

B. Agree.

C. Neither agree nor disagree.

D. Disagree.

E. Strongly disagree.

25. How did you learn about this continuing education activity?

A. Internet.

B. Advertisement (e.g., fact sheet, MMWR cover, newsletter, or journal).

C. Coworker/supervisor.

D. Conference presentation.

E. MMWR subscription.

F. Other.

Correct answers for questions 1–11.

1. A, B, C, and D; 2. A and D; 3. A, C, and D; 4. A, B, C, and D; 5. D; 6. C;

7. A, B, and D; 8. A, C, and D; 9. B, C, and D; 10. A, B, C, D, and E; 11. B.

All MMWR references are available on the Internet at http://www.cdc.gov/mmwr. Use the search function to find specific articles.

——————

Use of trade names and commercial sources is for identification only and does not imply endorsement by the U.S. Department of Health

and Human Services.

——————

References to non-CDC sites on the Internet are provided as a service to MMWR readers and do not constitute or imply endorsement of

these organizations or their programs by CDC or the U.S. Department of Health and Human Services. CDC is not responsible for the

content of these sites. URL addresses listed in MMWR were current as of the date of publication.

Laboratory Testing and Result Reporting of Antibody to Hepatitis C Virus

Working Group Members

Miriam J. Alter, Ph.D., Wendi L. Kuhnert, Ph.D., Lyn Finelli, Dr.P.H., Division of Viral Hepatitis, National Center for Infectious Diseases, CDC; William

Schalla, M.S., Ana Stankovic, M.D., Ph.D., Robert Martin, Dr.P.H., Division of Laboratory Systems, Public Health Program Practice Office, CDC; Robin

Biswas, M.D., Brian Harvey, M.D., Ph.D., Food and Drug Administration, Bethesda, Maryland; John Bryan, M.D., American Society for Clinical Pathology,

Chicago, Illinois; D. Robert Dufour, M.D., National Academy of Clinical Biochemistry, Washington, D.C.; Mark Jandreski, Ph.D., American Association for

Clinical Chemistry, Washington, D.C.; Amy Leber, Ph.D., American Clinical Laboratory Association, Washington, D.C.; Frederick Nolte, Ph.D., Emory

University School of Medicine, Atlanta, Georgia; Robin Stombler, American Society for Clinical Pathology, Washington, D.C.; David Sundwall, M.D.,

American Clinical Laboratory Association, Washington, D.C.; James Versalovic, M.D., Ph.D., College of American Pathologists, Northfield, Illinois; Judith

Wethers, M.S., New York State Public Health Laboratory, Albany, New York; Barbara Werner, Ph.D., Association of Public Health Laboratories, Washington,

D.C.; and Susanne Zanto, Association of Public Health Laboratories, Washington, D.C.

MMWR

The Morbidity and Mortality Weekly Report (MMWR) series is prepared by the Centers for Disease Control and Prevention (CDC) and

is available free of charge in electronic format and on a paid subscription basis for paper copy. To receive an electronic copy each week, send

an e-mail message to listserv@listserv.cdc.gov. The body content should read SUBscribe mmwr-toc. Electronic copy also is available from

CDC’s Internet server at http://www.cdc.gov/mmwr or from CDC’s file transfer protocol server at ftp://ftp.cdc.gov/pub/publications/mmwr. To

subscribe for paper copy, contact Superintendent of Documents, U.S. Government Printing Office, Washington, DC 20402; telephone

202-512-1800.

Data in the weekly MMWR are provisional, based on weekly reports to CDC by state health departments. The reporting week

concludes at close of business on Friday; compiled data on a national basis are officially released to the public on the following Friday.

Address inquiries about the MMWR series, including material to be considered for publication, to Editor, MMWR Series, Mailstop C-08,

CDC, 1600 Clifton Rd., N.E., Atlanta, GA 30333; telephone 888-232-3228.

All material in the MMWR series is in the public domain and may be used and reprinted without permission; however, citation of the

source is appreciated.

✩U.S. Government Printing Office: 2003-533-155/69089 Region IV