Public Health Guidance for Community-Level Preparedness and Response to
Severe Acute Respiratory Syndrome (SARS) Version 2

Supplement F: Laboratory Guidance

#   {#section .unnumbered}

# Contents

I.  Rationale and Goals

II. Lessons Learned

III. Diagnostic Assays

IV. CDC's Laboratory Diagnostics Plan

References

Appendix F1: Proficiency Testing for Public Health Laboratories
Performing SARS-CoV EIA

and RT-PCR Diagnostics

Appendix F2: SARS-CoV Specimen Testing Guidelines: RT-PCR and Serology

Appendix F3: Guidelines for Clinicians: The Consent Process for SARS-CoV
RT-PCR and

EIA Testing at CDC and Public Health Laboratories

Appendix F4: Guidelines for Collecting Specimens from Potential SARS
Patients

Appendix F5: Laboratory Biosafety Guidelines for Handling and Processing
Specimens

Associated with SARS-CoV

Appendix F6: Guidelines for Medical Surveillance of Laboratory Personnel
Working with

SARS-CoV

Appendix F7: Fact Sheet for Clinicians: Interpreting SARS-CoV Test
Results from CDC

and Other Public Health Laboratories

Appendix F8: Guidelines for Laboratory Diagnosis of SARS-CoV Infection

# Laboratory Guidance

# I. Rationale and Goals

Laboratory diagnostics are essential for detecting and documenting a
reappearance of SARS-CoV, responding to and managing outbreaks, and
managing concerns about SARS in patients with other respiratory
illnesses. The identification of SARS-CoV led to the rapid development
of enzyme immunoassays (EIA) and immunofluorescence assays (IFA) for
serologic diagnosis and reverse-transcription PCR (RT-PCR) assays for
detection of SARS-CoV RNA in clinical samples. These assays can be very
sensitive and specific for detecting antibody and RNA, respectively, in
the later stages of SARS-CoV infection. However, both are less sensitive
for detecting infection early in illness.

As part of SARS preparedness, CDC is working to improve diagnostics by
developing new tools that should make definitive diagnosis early in
illness possible. In the interim, CDC is applying new knowledge about
the natural history of SARS-CoV disease to improving diagnostic yield by
optimizing the type, timing, and quantity of specimens collected. CDC's
laboratory diagnostics plan is designed to achieve two primary goals:

-   Provide ready access to high-quality SARS-CoV laboratory diagnostics
    for the public health community

-   Ensure that SARS-CoV laboratory diagnostics are used safely and
    appropriately and that results are interpreted appropriately

# II. Lessons Learned

The following lessons learned from the global and U.S. experience with
SARS-CoV laboratory diagnostics have been considered in developing this
Supplement:

-   High-quality SARS-CoV diagnostic assays have been developed, but
    they frequently do not provide a definitive diagnosis early in
    illness and need to be used and interpreted carefully.

-   Although the sensitivity of SARS-CoV PCR and antibody assays
    probably cannot be significantly improved, changes in the type,
    quality, and quantity of specimens and in procedures for processing
    specimens may improve the detection of SARS-CoV.

-   The majority of SARS-like illnesses will be caused by other
    respiratory pathogens. Diagnosis of these infections will often make
    it easier to manage community anxiety about SARS-CoV.

-   The possibility of false-positive and false-negative results with
    both PCR and serologic assays should always be considered when
    interpreting results. Clear strategies to minimize such
    possibilities and to confirm test results are essential.

# III. Diagnostic Assays

Among the several types of assays used to detect SARS-CoV, RT-PCR and
antibody assays are the most commonly used.

## A. Real-Time RT-PCR Assays

Many laboratories have developed SARS-CoV real-time RT-PCR assays
([www.cdc.gov/ncidod/sars/lab/rtpcr/](http://www.cdc.gov/ncidod/sars/lab/rtpcr/)),
which have several advantages over traditional RT-PCR assays. Because
real-time RT-PCR assays use internal probes as well as amplification
primers, they can be designed to be very specific for SARS-CoV RNA (or
cDNA). They can also be very sensitive, with consistent detection limits
of between 1 and 10 SARS-CoV RNA copies per reaction. Real-time PCR
assays can be performed faster than traditional RT-PCR assays and,
because they operate as closed systems, with reduced risk of
contamination in the laboratory. Finally, real-time RT-PCR assays can
give an accurate estimate of the quantity of virus present in a sample.

As with all PCR assays, interpretation of RT-PCR tests must account for
the possibility of false-negative and false-positive results.
False-negative results can arise from poor sample collection or
degradation of the viral RNA during shipping or storage. Application of
appropriate assay controls that identify poor-quality samples can help
avoid most false-negative results. A more difficult problem is the
apparently low titer of SARS-CoV shed in specimens collected early in
illness, which may make it difficult to confirm a diagnosis.

The most common cause of false-positive results is contamination with
previously amplified DNA. The use of real-time RT-PCR helps mitigate
this problem by operating as a contained system. A more difficult
problem is the cross-contamination that can occur between specimens
during collection, shipping, and aliquoting in the laboratory. Liberal
use of negative control samples in each assay and a well-designed plan
for confirmatory testing can help ensure that laboratory contamination
is detected and that specimens are not inappropriately labeled as
SARS-CoV positive.

In the absence of SARS-CoV transmission worldwide, the probability that
a positive test result will be a "false positive" is high. To decrease
the possibility of a false-positive result, testing should be limited to
patients with a high index of suspicion for having SARS-CoV disease. For
information on the indications for SARS-CoV testing, see *Clinical
Guidance on the Identification and Evaluation of Possible SARS-CoV
Disease among Persons Presenting with Community-Acquired Illness*
[www.cdc.gov/ncidod/sars/clinicalguidance.htm](http://www.cdc.gov/ncidod/sars/clinicalguidance.htm).

In addition, any positive specimen should be retested in a reference
laboratory to confirm that the specimen is positive. To be confident
that a positive PCR specimen indicates that the patient is infected with
SARS-CoV, a second specimen should also be confirmed positive. Finally,
all laboratory results should be interpreted in the context of the
clinical and epidemiologic information available for the patient.

## B. Antibody Assays

The most commonly used serologic assays
([www.cdc.gov/ncidod/sars/lab/eia/](http://www.cdc.gov/ncidod/sars/lab/eia/))
are based on cultured SARS-CoV antigen as either inactivated whole virus
lysate for EIA or inactivated virus in cells fixed for IFA. These assays
have proven to be highly specific, with no cross-reactivity with paired
serum specimens from patients infected with the other known human
coronaviruses (229E and OC43) or from healthy blood donors and other
persons without clinical or epidemiologic evidence of SARS-CoV disease.

Antibody assays have been the most reliable indicators of SARS-CoV
infection when applied to convalescent-phase serum specimens collected
\>28 days after onset of illness. Since previous SARS-CoV infection is
still rare in most populations, demonstration of SARS-CoV-specific
antibodies in a single serum specimen is sufficient for diagnosis.
However, demonstration of a four-fold or greater increase in antibody
titer or conversion from a negative to a positive result between acute-
and convalescent-phase serum specimens provides additional confidence
that SARS-CoV is linked to any corresponding illness. In some patients,
antibody becomes detectable within 8 to 10 days, and most have
detectable antibody by 2 weeks. However, some persons do not develop
detectable antibodies until 28 days after onset of illness. Although
false-positive SARS-CoV serology results are much less likely than
false-positive PCR results, guidelines for confirmatory testing similar
to those outlined for RT-PCR still apply. Neutralization antibody assays
can also be used to detect infection. IgM assays and assays using the S
or N proteins as antigens have been developed and are under evaluation.

## C. Other Assays

Among the other methods used to detect SARS-CoV are: isolation in cell
culture, electron microscopy for CoV-like particles, and
immunohistologic or in situ probe hybridization studies on tissue
specimens. These methods are less likely to detect SARS-CoV infection
than are RT-PCR or antibody assays. Although isolation of SARS-CoV in
cell culture represents a definitive diagnosis, it is not recommended
for routine detection as it lacks sensitivity compared to RT-PCR and
also requires more restrictive Biosafety Level (BSL) 3 conditions.

Diagnostic assays for other respiratory pathogens may be helpful in
differentiating SARS-CoV disease from other illnesses, but SARS patients
can sometimes be infected with SARS-CoV and another respiratory pathogen
simultaneously

# IV. CDC's Laboratory Diagnostics Plan

CDC is planning and embarking on a range of laboratory diagnostics
activities that will enhance the capacity to detect a reappearance of
SARS-CoV and respond to and manage outbreaks. Objectives and
descriptions of these activities are as follows.

> **Objective 1**: Expand public health access to high-quality SARS-CoV
> diagnostics.
>
> **Activities**

-   [Assay deployment]{.underline} \-- CDC has deployed the SARS-CoV
    RT-PCR diagnostic assay under an Investigational Device Exemption
    (IDE) from the Food and Drug Administration (FDA). Protocols for
    both the RT-PCR and the serologic assay have been approved by CDC's
    Institutional Review Board (IRB). RT-PCR assays were deployed
    through the Laboratory Response Network (LRN) to selected
    laboratories in nearly all states; serologic assays have been
    deployed to nearly all state public health laboratories.

-   [Proficiency testing]{.underline} \-- To assess the availability and
    quality of SARS-CoV diagnostics in laboratories that received CDC's
    RT-PCR and antibody assays, CDC will distribute a panel of positive
    and negative specimens for testing (proficiency panels). The
    receiving laboratories will test these specimens and send their
    results to CDC for analysis of findings and responses to a
    questionnaire. These data will provide information on the
    laboratory's readiness to perform SARS-CoV diagnostics (see Appendix
    F1).

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-   [Assessment of SARS-CoV diagnostics in non-public health
    laboratories]{.underline} \-- Determining the availability and
    quality of SARS-CoV testing in non-public health laboratories will
    provide an assessment of overall laboratory diagnostic preparedness.
    Several clinical pathology professional organizations conduct
    laboratory surveys and distribute proficiency panels. CDC will
    assist with SARS surveys and provide proficiency panels so that the
    professional organizations can assess the status of SARS-CoV
    diagnostics in their members' laboratories.

-   [Confirmatory testing]{.underline} \-- Positive RT-PCR test results
    should be confirmed in a reference laboratory. Confirmatory testing
    is particularly important in areas with a low prevalence of SARS-CoV
    disease, where the positive predictive value of the assay is likely
    to be quite low. CDC will conduct confirmatory testing during the
    early phases of an outbreak. Other laboratories that are proficient
    in SARS-CoV diagnostics will participate in confirmatory testing as
    outbreaks escalate. Early in an outbreak, positive serologic tests
    should also be confirmed; later tests conducted in a proficient
    laboratory do not required confirmation.

> A key factor in the value of confirmatory RT-PCR testing is specimen
> handling. To interpret confirmatory test results, the aliquot of the
> specimen submitted for testing should not have been at risk for
> template contamination or degradation. The approach for and
> interpretation of confirmatory testing must consider all potential
> sources and types of template contamination (e.g., whole viral genome;
> genome portions; PCR products). Guidelines for confirmatory testing
> are provided in Appendix F2.
>
> **Objective 2:** Improve the ability to detect SARS-CoV by optimizing
> the selection and timing of specimen collection and processing.
>
> Most patients in the early stages of SARS-CoV disease have a low titer
> of virus in respiratory and other secretions and require time to mount
> an antibody response. In one study (in patients treated with high-dose
> steroids and ribavirin), nasopharyngeal (NP) aspirates were found to
> be PCR positive in \<40% of patients during the first week of illness
> and in \>50% of patients during the second week of illness (Peiris
> 2003). During the second week of illness, stool specimens were found
> to be PCR positive in a higher percentage of patients than were NP
> aspirates. Limited data suggest that serum may be the best specimen
> for SARS-CoV PCR diagnostics during the first few days of illness.

**Activities**

-   [Specimen collection]{.underline} \-- CDC has developed guidance for
    health departments and laboratorians to maximize the efficiency and
    accuracy of diagnostic procedures. Clinicians and laboratorians are
    asked to:

    -   [Obtain informed consent]{.underline} \-- A signed consent form
        is recommended for RT-PCR and EIA testing because neither assay
        has been licensed by the FDA and the RT-PCR test is being used
        under an FDA-approved IDE. In addition, a signed consent form is
        required to store specimen remainders for future investigations
        (see Appendix F3).

    -   [Collect multiple specimens]{.underline} \-- The type and timing
        of specimen collection is important to maximize the probability
        of detecting evidence of SARS-CoV infection. Since it is not yet
        clear which specimen type is best for detecting viral RNA, it is
        important to collect different types of specimens and at
        multiple times during the illness. Appendix F4 provides guidance
        on the type and timing of specimens for SARS-CoV diagnostics.

    -   [Handle specimens correctly]{.underline} \-- CDC has developed
        guidance for specimen collection, handling, and shipping
        (Appendix F4). State and local health departments can use these
        guidelines to educate clinicians on appropriate methods of
        specimen management.

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-   [Assay sensitivity]{.underline} \-- CDC will evaluate ways to
    improve assay sensitivity, such as extracting RNA from a larger
    volume of the specimen and including a larger amount of template RNA
    in the RT-PCR reaction. CDC is developing IgG and IgM assays using
    expressed proteins as the antigens. Preliminary data suggest that
    antibody assays using the SARS-CoV S protein might detect an
    antibody response earlier in illness.

> **Objective 3:** Ensure that SARS-CoV specimens are handled safely and
> that SARS-CoV diagnostic tests are used and interpreted appropriately.

**Activities**

-   [Biosafety guidance]{.underline} \-- The laboratory-acquired
    SARS-CoV infection in Singapore (Singapore Ministry of Health 2003)
    and presumed laboratory-acquired SARS-CoV infection in Taiwan
    (Department of Health, Taiwan 2003) underscore the need to handle
    SARS-CoV specimens and SARS-CoV-infected tissue culture material
    safely. CDC has developed guidelines for handling these types of
    specimens and materials (Appendix F5) and for implementing a
    surveillance program in the event of a laboratory exposure (Appendix
    F6). State and local health departments can use these guidelines to
    educate personnel in viral diagnostic, research, and clinical
    laboratories about safe specimen handling and appropriate responses
    to a laboratory exposure.

-   [Test interpretation]{.underline} \-- Clinicians should interpret
    SARS-CoV test results in consultation with state or local health
    department officials and with consideration of data on the clinical
    and epidemiologic features of the illness and the type and timing of
    specimen collection. CDC has developed guidelines to guide state and
    local health department staff in their consultations with clinicians
    about test interpretation (Appendix F7). CDC, in cooperation with
    CSTE, has also developed criteria for laboratory diagnosis of
    SARS-CoV infection (Appendix F8).

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-   [Data reporting and integration]{.underline} \-- State and local
    health departments will collect clinical and epidemiologic data on
    potential cases of SARS-CoV disease and report cases to CDC through
    a web-based reporting system. CDC will send laboratory data back to
    state and local health departments daily. The clinical and
    epidemiologic information reported to CDC and downloaded back to the
    states can provide a source of patient information that can help
    laboratorians consider appropriate testing strategies and interpret
    test results. With guidance from state and local health departments,
    CDC will facilitate access to data as requested. In addition,
    results of laboratory testing on any specimens submitted to CDC will
    be integrated into the data provided to state and local health
    departments, allowing timely dissemination of this information.

-   [Training and education]{.underline} -- Diagnostic assays have an
    important role in detecting an introduction of SARS-CoV, managing a
    SARS outbreak, and addressing concerns about SARS. The healthcare
    and public health communities should be aware of the value,
    limitations, and appropriate use and interpretation of SARS-CoV
    diagnostics. CDC will provide training and educational materials
    that state and local health departments can use to educate
    clinicians and public health workers about SARS-CoV diagnostics.

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-   [Coordination]{.underline} \-- Coordinated information sharing among
    clinicians, laboratorians, and epidemiologists is central to
    efficient investigation of potential cases of SARS-CoV disease. CDC
    will assist public health laboratories and epidemiologists in
    developing rapid and coordinated strategies for: 1) collecting,
    tracking, and testing specimens, 2) interpreting test results, 3)
    reporting information to clinicians, and 4) communicating results to
    CDC, other public health officials, and the public.

> **Objective 4:** Ensure the availability of SARS-CoV diagnostic test
> kits and protocols for testing other respiratory pathogens.

**Activities**

-   [Diagnostic supplies]{.underline} \-- The supply of SARS-CoV RT-PCR
    and serologic test kits is limited. To ensure the availability of a
    sufficient number of kits to meet routine public health needs and
    the anticipated high demand associated with simultaneous outbreaks,
    CDC is monitoring both the deployment and number of kits. After
    patterns of use have been determined, CDC will plan the production
    of new kits to ensure that the supply can meet both projected
    baseline needs and the accelerated use associated with a SARS
    outbreak.

-   [Tests for alternative respiratory agents]{.underline} \-- CDC will
    complete the development and initial evaluation of real-time PCR
    assays for the most important common respiratory pathogens in the
    United States and make primer and probe sequences and protocols
    available to the LRN and other public health laboratories.

## References

Peiris JSM, Chu CM, Cheng VCC, et al. Clinical progression and viral
load in a community outbreak of coronavirus-associated SARS pneumonia: a
prospective study. Lancet 2003;361:1767-72.

Singapore Ministry of Health. Biosafety and SARS incident in Singapore,
September 2003: Report of the Review Panel on New SARS Case and
Biosafety. Singapore: Singapore Ministry of Health; 2003 Sep.
([www.moh.gov.sg/corp/sars/pdf/Report_SARS_Biosafety.pdf](http://www.moh.gov.sg/corp/sars/pdf/Report_SARS_Biosafety.pdf)).

Department of Health, Taiwan. Confirmed SARS case in research laboratory
in Taiwan, December 17, 2003 \[news release on the Internet\].Taiwan:
Department of Health; 2003.
([sars.doh.gov.tw/news/2003121701.html](http://sars.doh.gov.tw/news/2003121701.html))

** **

** **

# Appendix F1

# Proficiency Testing for Public Health Laboratories Providing

# SARS-CoV EIA and RT-PCR Diagnostics

CDC has developed and validated diagnostic assays for SARS-CoV,
including an enzyme immunoassay (EIA)
([www.cdc.gov/ncidod/sars/lab/eia/](http://www.cdc.gov/ncidod/sars/lab/eia/))
for detection of serum antibodies to SARS-CoV and a reverse
transcription-polymerase chain reaction (RT-PCR) assay
([www.cdc.gov/ncidod/sars/lab/rtpcr/](http://www.cdc.gov/ncidod/sars/lab/rtpcr/))
for detection of SARS-CoV RNA. Both the EIA and the RT-PCR tests are
sensitive and highly specific for diagnosis of SARS-CoV infection.
Testing with these assays is now available through state public health
laboratories and CDC's Laboratory Response Network (LRN).

CDC is in the process of developing and implementing a quality assurance
program to evaluate each laboratory's testing proficiency. To
demonstrate competence in performing these tests, public health and LRN
laboratories will be required to successfully identify positive and
negative specimens provided in the proficiency panels.

**Process for Proficiency Panel Testing**

1.  EIA proficiency panels will be shipped to designated public health
    laboratories, and RT-PCR proficiency panels will be distributed
    through the LRN.

2.  Each laboratory should complete testing promptly and return results
    by the designated date.

3.  Proficiency panel test results must be returned by electronic mail
    using a designated format.

4.  Each laboratory will receive a complete summary of its own results
    as well as an aggregate summary of performance from all laboratories
    completing the proficiency testing.

5.  CDC will provide a certificate of participation to the participating
    laboratory to help fulfill quality assurance requirements.

Results obtained from a laboratory's proficiency testing will initially
be considered "educational," and laboratories will not be required to
undergo additional training as remediation. However, successful
completion of the proficiency test will be required for approval of a
public health or LRN laboratory as a confirmatory testing site.

# Appendix F2

# 

# 

**T-PCR Confirmatory Testing**

**SARS-CoV Specimen Testing Guidelines: Serologic Testing**

# Appendix F3

# Guidelines for Clinicians: The Consent Process for SARS-CoV

# RT-PCR and EIA Testing at CDC and Public Health Laboratories

CDC has distributed a SARS-CoV RT-PCR assay
([www.cdc.gov/ncidod/sars/lab/rtpcr/](http://www.cdc.gov/ncidod/sars/lab/rtpcr/))
under an FDA investigational device exemption (IDE) and an EIA assay
([www.cdc.gov/ncidod/sars/lab/eia/](http://www.cdc.gov/ncidod/sars/lab/eia/))
to state and local public health laboratories to test specimens for
SARS-CoV. These tests are used to evaluate persons suspected of having
SARS-CoV disease. The RT-PCR assay is used to test for SARS-CoV viral
RNA in respiratory samples, stool, plasma, or serum. The EIA test is
used to detect SARS-CoV antibodies in blood or serum specimens. A signed
consent form for performance of each test is recommended because neither
test has been licensed by the FDA and the RT-PCR test is being used
under an FDA-approved IDE. A signed consent form is also required for
storage of specimen remainders for future investigations.

To submit specimens for SARS-CoV RT-PCR or EIA testing, healthcare
providers should follow these steps:

1\. **Consult the state or local health department** to determine if
SARS-CoV testing is indicated.

2\. **Seek informed consent** from the patient for testing.

> The RT-PCR consent form can be found at:
>
> [www.cdc.gov/ncidod/sars/lab/rtpcr/consent.htm](http://www.cdc.gov/ncidod/sars/lab/rtpcr/consent.htm).
>
> The EIA consent form can be found at:
>
> [www.cdc.gov/ncidod/sars/lab/eia/consent.htm](http://www.cdc.gov/ncidod/sars/lab/eia/consent.htm)

3\. **Collect specimens** for testing. Guidelines for specimen
collection are provided in Appendix F4.

4\. **Submit specimens**, with a signed consent form and completed
specimen submission form, to the state or local public health
laboratory.

> Specimens will be tested at the reference public health laboratory.
> Final results will be reported to you through the state or local
> health department. Information on interpreting these test results is
> provided in Appendix F7.

5\. **Deliver test results** to the patient. Provide a patient
information sheet/consent for long-term specimen storage to the patient
along with the test results. Specimen remainders stored long term may be
used for future investigations.

> The RT-PCR patient information sheet/consent for long-term specimen
> storage can be found at:
> [www.cdc.gov/ncidod/sars/lab/rtpcr/participant.htm](http://www.cdc.gov/ncidod/sars/lab/rtpcr/participant.htm).
>
> The EIA patient information sheet/consent for long-term specimen
> storage can be found at:
> [www.cdc.gov/ncidod/sars/lab/eia/participant.htm](http://www.cdc.gov/ncidod/sars/lab/eia/participant.htm).

6\. **If a signed patient information sheet/consent for long-term
storage is obtained, fax it** to the state or local public health
department. Contact information is provided at
[www.cdc.gov/other.htm#states](http://www.cdc.gov/other.htm#states).

## Frequently Asked Questions

**Where can I find information on how to report a possible SARS case and
submit specimens for SARS-CoV testing?**

This information is available through the state or local health
department. Contact information is provided at:
[www.cdc.gov/other.htm#states](http://www.cdc.gov/other.htm#states).

**Why is a signed informed consent form recommended for SARS-CoV
testing?**

A signed consent form is recommended because neither the RT-PCR test nor
the EIA test has been licensed by FDA and the RT-PCR test is being used
under an FDA-approved investigational device exemption (IDE). In
addition, consent is required to store specimen remainders for future
investigations.

**Why are there two different consent forms, one for RT-PCR and one for
EIA?**

Two forms are required because of differing IRB review requirements;
CDC's IRB reviewed and approved two separate protocols.

**What happens if I submit specimens for testing without a signed
consent form?**

Because of the potentially serious public health impact of SARS-CoV
transmission, specimens that are received by a state or local public
health laboratory without a signed consent form will still be tested.

**What is the patient information sheet, and when do I use it?**

The patient information sheet/consent for long-term specimen storage
will be sent to the physician along with the patient's test results. The
physician should provide this document to the patient. It explains to
the patient why SARS-CoV testing was performed on their specimens,
explains what the results mean, and asks the patient for permission to
store specimen remainders for future investigations.

**Why should a signed patient information sheet be returned?**

The patient information sheet asks the patient for permission to store
specimen remainders for future investigations. If a signed consent form
is not received before testing, it is necessary to receive the signed
patient information sheet to store any specimen remainders. Specimens
without a signed informed consent or signed patient information sheet
allowing long-term specimen storage must be destroyed.

# Appendix F4

# Guidelines for Collecting Specimens from Potential SARS Patients

Before collecting and shipping specimens for SARS-CoV testing, consult
with the state health department/state epidemiologist to determine
whether the patient meets the SARS case definition. Health department
contact information is available at:
[www.cste.org/members/state_and_territorial_epi.asp](http://www.cste.org/members/state_and_territorial_epi.asp).

When possible, collect multiple respiratory specimens for testing. For
example, collect specimens from two different sites on the same day
(e.g., one nasopharyngeal swab and a stool specimen or another
respiratory specimen) or from two different times during the illness.
The chart on the last page specifies recommended specimen types for
SARS-CoV diagnostics by stage of illness, including postmortem specimen
collection.

A signed consent form is recommended when collecting specimens for
SARS-CoV RT-PCR or antibody testing. Information on the consent process
for collection of respiratory specimens, blood or stool for RT-PCR
testing is provided at:
[www.cdc.gov/ncidod/sars/lab/rtpcr/index.htm](http://www.cdc.gov/ncidod/sars/lab/rtpcr/index.htm).
Information on the consent process for the collection of blood/serum for
antibody testing is provided at:
[www.cdc.gov/ncidod/sars/lab/eia/index.htm](http://www.cdc.gov/ncidod/sars/lab/eia/index.htm).

> **RT-PCR Diagnostics**
>
> Although studies to date have not definitively determined the best
> specimens for SARS RT-PCR diagnostics, it is reasonable to collect:
>
> *During the first week of illness:* Nasophyaryngeal (NP) swab plus
> oropharyngeal (OP) swab and a serum or plasma specimen
>
> *After the first week of illness:* NP swab plus OP swab and a stool
> specimen
>
> **Serologic Diagnostics**
>
> Serum specimens for SARS-CoV antibody testing should be collected when
> the diagnosis is first suspected and at later times if indicated. An
> antibody response is occasionally detected during the first week of
> illness, likely to be detected by the end of the second week of
> illness, and sometimes may not be detected until \>28 days after onset
> of symptoms.

## 

## I. Collecting Respiratory Specimens

Eight types of respiratory specimens may be collected for viral and/or
bacterial diagnostics: 1) nasopharyngeal wash/aspirates, 2)
nasopharyngeal swabs, 3) oropharyngeal swabs, 4) broncheoalveolar
lavage, 5) tracheal aspirate, 6) pleural fluid tap, 7) sputum; and 8)
post-mortem tissue. Nasopharyngeal wash/aspirates are the specimen of
choice for detection of most respiratory viruses and are the preferred
specimen type for children under age 2 years.

In contrast to most respiratory pathogens for which respiratory
specimens are optimally collected within 72 hours after the onset of
symptoms, levels of SARS-CoV may be higher later in the course of the
illness.

Before collecting specimens, review the infection control precautions in
Supplement I.

### 

### A. Collecting specimens from the upper respiratory tract 

> **1. Nasopharyngeal wash/aspirate**
>
> Have the patient sit with head tilted slightly backward. Instill 1
> ml-1.5 ml of nonbacteriostatic saline (pH 7.0) into one nostril. Flush
> a plastic catheter or tubing with 2 ml-3 ml of saline. Insert the
> tubing into the nostril parallel to the palate. Aspirate
> nasopharyngeal secretions. Repeat this procedure for the other
> nostril. Collect the specimens in sterile vials. Label each specimen
> container with the patient's ID number and the date collected. If
> shipping domestically, use cold packs to keep the sample at 4°C. If
> shipping internationally, pack in dry ice.
>
> **2. Nasopharyngeal or oropharyngeal swabs**[\
> ]{.underline}
>
> Use only sterile dacron or rayon swabs with plastic shafts. Do
> [not]{.underline} use calcium alginate swabs or swabs with wooden
> sticks, as they may contain substances that inactivate some viruses
> and inhibit PCR testing.
>
> **Nasopharyngeal swabs** \-- Insert a swab into the nostril parallel
> to the palate. Leave the swab in place for a few seconds to absorb
> secretions. Swab both nostrils.\
> **Oropharyngeal swabs** \-- Swab the posterior pharynx and tonsillar
> areas, avoiding the tongue.
>
> Place the swabs immediately into sterile vials containing 2 ml of
> viral transport media. Break the applicator sticks off near the tip to
> permit tightening of the cap. Label each specimen container with the
> patient's ID number and the date the sample was collected. If shipping
> domestically, use cold packs to keep sample at 4°C. If shipping
> internationally, pack in dry ice.

### B. Collecting specimens from the lower respiratory tract

> **1. Broncheoalveolar lavage, tracheal aspirate, pleural fluid tap**
>
> Centrifuge half of the specimen, and fix the cell pellet in formalin.
> Place the remaining unspun fluid in sterile vials with external caps
> and internal O-ring seals. If there is no internal O-ring seal, then
> seal tightly with the available cap and secure with Parafilm^®^. Label
> each specimen container with the patient's ID number and the date the
> sample was collected. If shipping domestically, use cold packs to keep
> sample at 4°C. If shipping internationally, ship fixed cells at room
> temperature and unfixed cells frozen.
>
> **2. Sputum**
>
> Educate the patient about the difference between sputum and oral
> secretions. Have the patient rinse the mouth with water and then
> expectorate deep cough sputum directly into a sterile screw-cap sputum
> collection cup or sterile dry container. If shipping domestically, use
> cold packs to keep sample at 4°C. If shipping internationally, pack in
> dry ice.

## 

## II. Collecting Blood Components

Serum and blood (plasma) should be collected early in the illness for
RT-PCR testing. The reliability of RT-PCR testing performed on blood
specimens decreases as the illness progresses.

Both acute and convalescent serum specimens should be collected for
antibody testing. To confirm or rule out SARS-CoV infection, it is
important to collect convalescent serum specimens \>28 days after the
onset of illness.

### 

### A. Collecting serum for antibody or RT-PCR testing

Collect 5 ml-10 ml of whole blood in a serum separator tube. Allow the
blood to clot, centrifuge briefly, and collect all resulting sera in
vials with external caps and internal O-ring seals. If there is no
internal O-ring seal, then seal tightly with the available cap and
secure with Parafilm^®^. The minimum amount of serum preferred for each
test is 200 microliters, which can easily be obtained from 5 mL of whole
blood.

A minimum of 1 cc of whole blood is needed for testing of pediatric
patients. If possible, collect 1 cc in an EDTA tube and in a clotting
tube. If only 1cc can be obtained, use a clotting tube.

Label each specimen container with the patient's ID number and the date
the specimen was collected. If unfrozen and transported domestically,
ship with cold packs to keep the sample at 4°C. If frozen or transported
internationally, ship on dry ice.

###  

### B. Collecting EDTA blood (plasma) for RT-PCR

Collect 5 ml-10 ml of blood in an EDTA (purple-top) tube. Transfer to
vials with external caps and internal O-ring seals. If there is no
internal O-ring seal, then seal tightly with the available cap and
secure with Parafilm^®^. Label each specimen container with patient's ID
number and date of collection. Store and ship blood specimens with cold
packs to keep the sample at 4°C.

## 

## III. Collecting Stool Specimens for PCR

Begin collecting stool specimens as soon as possible in the course of
the illness. Although collecting earlier specimens is ideal, SARS-CoV
has been detected in stool as late as one month after the onset of
symptoms.

Place each stool specimen \-- as large a quantity as can be obtained (at
least 10 cc) \-- in a leak-proof, clean, dry container, and refrigerate
at 4°C. Patients may drape plastic kitchen wrap across the back half of
the toilet, under the toilet seat, to facilitate collection of stool
specimens.

IMPORTANT: Refrigerate or freeze tubes after specimens are placed in
them. If specimens will be examined within 48 hours after collection,
they can be refrigerated. If specimens must be held longer than 48
hours, freeze them as soon as possible after collection. Although
storage in an ultra-low freezer (-70°C) is preferable, storage in a
home-type freezer (if properly set at -20°C) is acceptable for short
periods.

Specimens from possible and known SARS cases must be packaged, shipped,
and transported according to the current edition of the International
Air Transport Association (IATA) Dangerous Goods Regulations at
([www.iata.org/dangerousgoods/index](http://www.iata.org/dangerousgoods/index))
and US DOT 49 CFR Parts 171-180
([hazmat.dot.gov/rules.htm](http://hazmat.dot.gov/rules.htm)).

Step-by-step instructions on appropriate packaging and labeling are
available at:
[www.cdc.gov/ncidod/sars/pdf/packingspecimens-sars.pdf](http://www.cdc.gov/ncidod/sars/pdf/packingspecimens-sars.pdf).

![](media/image1.png)

+----------------------+-------------+-------------+------------------+
| **Specimens for      |             |             |                  |
| SARS-CoV Testing:**  |             |             |                  |
|                      |             |             |                  |
| **Priority Specimens |             |             |                  |
| and Timing for       |             |             |                  |
| Collection**         |             |             |                  |
|                      |             |             |                  |
| The likelihood of    |             |             |                  |
| detecting infection  |             |             |                  |
| is increased if      |             |             |                  |
| multiple specimens,  |             |             |                  |
| e.g., stool, serum,  |             |             |                  |
| and respiratory      |             |             |                  |
| tract specimens, are |             |             |                  |
| collected during the |             |             |                  |
| course of illness.   |             |             |                  |
+----------------------+-------------+-------------+------------------+
| **Specimen, by test  | **\<1 week  | **1-3 weeks | **\>3 weeks      |
| type**               | after       | after       | after symptom    |
|                      | symptom     | symptom     | onset**          |
|                      | onset**     | onset**     |                  |
+----------------------+-------------+-------------+------------------+
| RT-PCR^1^ for viral  |             |             |                  |
| RNA detection        |             |             |                  |
+----------------------+-------------+-------------+------------------+
| > Sputum^2^          | √^3^        | √√          | √                |
+----------------------+-------------+-------------+------------------+
| > Bronchoalveolar    | √           | √√          | √                |
| > lavage, tracheal   |             |             |                  |
| > aspirate, or       |             |             |                  |
| > pleural fluid      |             |             |                  |
| > tap^4^             |             |             |                  |
+----------------------+-------------+-------------+------------------+
| > Nasopharyngeal     | √           | √√          | √                |
| > wash/aspirate      |             |             |                  |
+----------------------+-------------+-------------+------------------+
| > Nasopharyngeal and | √           | √√          | √                |
| > oropharyngeal      |             |             |                  |
| > swabs              |             |             |                  |
+----------------------+-------------+-------------+------------------+
| > Serum (serum       | √√          | √           | not recommended  |
| > separator tube)    |             |             |                  |
+----------------------+-------------+-------------+------------------+
| > Blood (plasma)     | √√          | √           | not recommended  |
| >                    |             |             |                  |
| > (EDTA/purple top   |             |             |                  |
| > tube for plasma)   |             |             |                  |
+----------------------+-------------+-------------+------------------+
| > Stool (minimum 10  | √           | √√          | √√               |
| > cc specimen)       |             |             |                  |
+----------------------+-------------+-------------+------------------+
| EIA^1^ for antibody  |             |             |                  |
| detection            |             |             |                  |
+----------------------+-------------+-------------+------------------+
| > Serum^5^ (serum    | √√          | √√          | √√               |
| > separator tube)    |             |             |                  |
+----------------------+-------------+-------------+------------------+

^1^ Because of the investigational nature of both the SARS RT-PCR
(reverse transcription-polymerase chain reaction) and the SARS EIA
(enzyme immunoassay), it is recommended that the clinician obtain a
signed informed consent form from the patient. The consent form for the
RT-PCR test can be found at:
[www.cdc.gov/ncidod/sars/lab/rtpcr/consent.htm](http://www.cdc.gov/ncidod/sars/lab/rtpcr/consent.htm).
The consent form for the EIA test can be found at:
[www.cdc.gov/ncidod/sars/lab/eia/consent.htm](http://www.cdc.gov/ncidod/sars/lab/eia/consent.htm).

^2^ A sputum specimen is preferred if the patient has a productive
cough.

^3^ The more checks, the better the results from a particular specimen
at a specific point in the illness.

^4^ Consider these specimen types if sputum is not available.

^5^ Also collect a convalescent specimen \>28 days post onset.

# Appendix F5

# Laboratory Biosafety Guidelines for Handling and Processing

# Specimens Associated with SARS-CoV

Although routine clinical laboratories around the world have processed
an estimated several thousand diagnostic specimens from patients with
SARS, no cases of SARS-CoV disease among laboratory workers performing
diagnostic assays have been reported to date. However, there have been
two reported cases of SARS-CoV disease in workers in research
laboratories where SARS-CoV was being propagated. Until more information
about the transmission of SARS-CoV in the laboratory setting is known,
precautions should be taken in handling specimens (e.g., respiratory and
stool specimens, unfixed lung tissue, viral cultures) that might contain
large quantities of SARS-CoV.

Effective and timely communication between clinical and laboratory staff
is essential to minimize the risk incurred in handling specimens from
patients with possible SARS-CoV disease. Such specimens should be
labeled accordingly, and the laboratory should be alerted to ensure
proper specimen handling. Biosafety guidelines for handling SARS-CoV
specimens, by specimen type, are provided below. Guidelines on
implementing a medical surveillance system for laboratory workers are
provided in Appendix F6.

## A. Blood (blood, serum and plasma) and urine specimens

-   Handle these specimens using Universal Precautions, which includes
    use of gloves, gown, mask, and eye protection. For more information
    on Universal Precautions, see
    [www.osha.gov/SLTC/bloodbornepathogens/index.html#revised_standard](http://www.osha.gov/SLTC/bloodbornepathogens/index.html#revised_standard).

```{=html}
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```
-   Any procedure with the potential to generate fine-particulate
    aerosols (e.g., vortexing or sonication of specimens in an open
    tube) should be performed in a biological safety cabinet (BSC). Use
    sealed centrifuge rotors or sample cups, if available, for
    centrifugation. Ideally, rotors and cups should be loaded and
    unloaded in a BSC. Perform any procedures outside a BSC in a manner
    that minimizes the risk of exposure to an inadvertent sample
    release.

```{=html}
<!-- -->
```
-   After specimens are processed, decontaminate work surfaces and
    equipment. Use any EPA-registered hospital disinfectant. Follow
    manufacturer\'s recommendations for use-dilution (i.e.,
    concentration), contact time, and care in handling.

## B. Other specimens (e.g., respiratory secretions, stool, or tissue for procedures performed in microbiology or pathology laboratories)

-   The following activities may be performed in BSL-2 facilities with
    standard BSL-2 work practices:

```{=html}
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```
-   Pathologic examination and processing of formalin-fixed or otherwise
    inactivated tissues

-   Molecular analysis of extracted nucleic acid preparations

-   Electron microscopic studies with glutaraldehyde-fixed grids

-   Routine examination of bacterial and mycotic cultures

-   Routine staining and microscopic analysis of fixed smears

-   Final packaging of specimens for transport to diagnostic
    laboratories for additional testing. Specimens should already be in
    a sealed, decontaminated primary container.

```{=html}
<!-- -->
```
-   The following activities involving manipulation of untreated
    specimens should

> be performed in BSL-2 facilities and in a Class II BSC:

-   Aliquoting and/or diluting specimens

-   Inoculating bacterial or mycological culture media

-   Performing diagnostic tests that do not involve propagation of viral
    agents in vitro or in vivo

-   Nucleic acid extraction procedures involving untreated specimens

-   Preparation and chemical- or heat-fixing of smears for microscopic
    analysis

> Work surfaces should be decontaminated on completion of work with
> appropriate disinfectants. All disposable waste should be autoclaved.
>
> Laboratory workers should wear personal protective equipment (PPE),
> including disposable gloves and laboratory coats.
>
> Any procedure or process that cannot be conducted in a BSC should be
> performed while wearing gloves, gown, eye protection, and respiratory
> protection, (see Supplement I, Section III.D.5).
>
> Acceptable methods of respiratory protection include: a properly
> fit-tested, NIOSH-approved filter respirator (N-95 or higher level) or
> a powered air-purifying respirator (PAPR) equipped with
> high-efficiency particulate air (HEPA) filters. Accurate fit-testing
> is a key component of effective respirator use.[^1] Personnel who
> cannot wear fitted respirators because of facial hair or other fit
> limitations should wear loose-fitting hooded or helmeted PAPRs.
>
> Appropriate physical containment devices (e.g., centrifuge safety
> cups; sealed rotors) should also be used. Ideally, rotors and cups
> should be loaded and unloaded in a BSC.

-   The following activities must be performed in a BSL-3 facility using
    BSL-3 work practices:

```{=html}
<!-- -->
```
-   SARS-CoV propagation in cell culture

-   Initial characterization of viral agents recovered in cultures of
    SARS specimens

> Any procedure or process that cannot be conducted in a BSC should be
> performed while wearing gloves, gown, eye protection, and respiratory
> protection (see Supplement I, Section III.D.5).
>
> Acceptable methods of respiratory protection include: a properly
> fit-tested, NIOSH-approved filter respirator (N-95 or higher level) or
> PAPR equipped with HEPA filters. Accurate fit-testing is a key
> component of effective respirator use.[^2] Personnel who cannot wear
> fitted respirators because of facial hair or other fit limitations
> should wear loose-fitting hooded or helmeted PAPRs.
>
> Centrifugation should be carried out using sealed centrifuge cups or
> rotors that are unloaded in a BSC.

-   The following activities must be performed in Animal BSL-3
    facilities using Animal BSL-3 work practices:

    -   Inoculation of animals for potential recovery of SARS-CoV from
        SARS samples

    -   Protocols involving animal inoculation for characterization of
        putative SARS agents

Consideration may also be given to referral of specimens to a suitably
equipped reference laboratory.

**Note**: Packaging, shipping, and transport of specimens from possible
and known cases of SARS-CoV disease must follow the current edition of
the International Air Transport Association (IATA) Dangerous Goods
Regulations at
([www.iata.org/dangerousgoods/index](http://www.iata.org/dangerousgoods/index))
and US DOT 49 CFR Parts 171-180
([hazmat.dot.gov/rules.htm](http://hazmat.dot.gov/rules.htm)).
Step-by-step instructions on appropriate packaging and labeling are
provided at:
[www.cdc.gov/ncidod/sars/pdf/packingspecimens-sars.pdf](http://www.cdc.gov/ncidod/sars/pdf/packingspecimens-sars.pdf)

# Appendix F6

# Guidelines for Medical Surveillance of Laboratory Personnel

# Working with SARS-CoV

Medical surveillance of laboratory personnel can help to ensure that
workers who are at risk of occupational exposure to SARS-CoV and who
develop symptoms of illness receive appropriate medical evaluation and
treatment, both for the benefit of their health and to prevent further
transmission.

-   Laboratory workers should be provided training on the appropriate
    biosafety level and specific safety practices for the type of work
    being performed. Biosafety guidelines for laboratory work with
    SARS-CoV are available at:
    [www.cdc.gov/ncidod/sars/sarslabguide.htm](http://www.cdc.gov/ncidod/sars/sarslabguide.htm).

-   Before working with either live SARS-CoV or clinical specimens known
    to contain SARS-CoV, laboratory workers should have a baseline serum
    sample obtained and stored for future reference.

-   Laboratory workers should immediately contact their supervisor in
    the event of a recognized exposure or development of lower
    respiratory symptoms and/or fever. In addition, exposed laboratory
    workers should be monitored for the presence of any of the
    following: sore throat, rhinorrhea, chills, rigors, myalgia,
    headache, diarrhea. The supervisor should immediately contact
    appropriate healthcare personnel and facility contacts (e.g.,
    occupational health, infection control, or a designee); the local
    and/or state public health departments should be promptly notified
    as well.

## I. Management of a Break in Laboratory Procedure

In the event of an identifiable break in laboratory procedure (e.g.,
tear in a glove; spill of live virus), the laboratory worker should
immediately implement applicable laboratory procedures for emergency
exposure management and/or environmental decontamination and notify the
supervisor for further instructions. The worker and the supervisor, in
consultation with occupational health or infection control personnel,
should evaluate the break in procedure to determine if an exposure
occurred. If the break in procedure resulted in an exposure, the worker
should be managed as described below.

## II. Management of Exposed Laboratory Workers 

### A. Management of exposed laboratory workers who are asymptomatic

> 1\. Exposed workers should be instructed to be vigilant for the
> development of fever (i.e., measure and record body temperature twice
> daily for 10 days after the date of the last unprotected exposure),
> lower respiratory symptoms, or any of the following: sore throat,
> rhinorrhea, chills, rigors, myalgia, headache, diarrhea. Exposed
> workers should immediately notify the supervisor if symptoms develop.
>
> 2\. Exposed workers should be actively monitored for symptoms prior to
> reporting for duty.
>
> 3\. Decisions regarding activity restrictions (e.g., work) should be
> discussed with the health department, in accordance with the
> recommendations in Supplement D. Asmptomatic exposed workers generally
> do not need to be excluded from duty. However, a worker who has had a
> high-risk exposure may need to be furloughed.

### B. Management of exposed laboratory workers who develop symptoms within 10 days of exposure

1.  The exposed laboratory worker who develops fever, lower respiratory
    symptoms, sore throat, rhinorrhea, chills, rigors, myalgia,
    headache, or diarrhea should:

-   Immediately put on a surgical mask if at work, *and*

-   Immediately notify the appropriate facility contact (e.g., infection
    control, occupational health, or a designee in each facility where
    s/he works), *and*

-   Report to the designated location for clinical evaluation.

> 2\. Symptomatic laboratory workers should be managed in accordance
> with the recommendations in *Clinical Guidance on the Identification
> and Evaluation of Possible SARS-CoV Disease among Persons Presenting
> with Community-Acquired Illness*
> [www.cdc.gov/ncidod/sars/clinicalguidance.htm](http://www.cdc.gov/ncidod/sars/clinicalguidance.htm).
>
> 3\. Decisions on return to work should be guided by policies or
> regulations defined by the facility or health department.

## III. Management of Symptomatic Laboratory Workers with No Recognized Exposures

Laboratory workers who develop a fever or lower respiratory symptoms and
who have no recognized exposure should immediately contact the
supervisor. The supervisor should immediately contact the appropriate
healthcare personnel and facility contacts (e.g., occupational health,
infection control, or a designee), who should review the worker's
illness and potential laboratory exposures to determine if any SARS
precautions or additional consultations are necessary. If clinical or
exposure information suggests SARS-CoV infection, local or state public
health officials should be immediately be contacted and consulted about
managing the ill laboratory worker and contacts.

# Appendix F7

# Fact Sheet for Clinicians: Interpreting SARS-CoV Test Results

# from CDC and Other Public Health Laboratories

**Definitions**

+----------------+-----------------------------------------------------+
| SARS           | Severe acute respiratory syndrome                   |
+----------------+-----------------------------------------------------+
| SARS-CoV       | SARS-associated coronavirus; a newly described      |
|                | coronavirus that is genetically and antigenically   |
|                | distinct from other human coronaviruses             |
+----------------+-----------------------------------------------------+
| Labora         | -   Detection of any of the following by a          |
| tory-confirmed |     validated test, with confirmation in a          |
| SARS-CoV       |     reference laboratory:                           |
| infection      |                                                     |
|                |     -   Serum antibodies to SARS-CoV in a single    |
|                |         serum specimen, *or*                        |
|                |                                                     |
|                |     -   A four-fold or greater increase in SARS-CoV |
|                |         antibody titer between acute- and           |
|                |         convalescent-phase serum specimens tested   |
|                |         in parallel, *or*                           |
|                |                                                     |
|                |     -   Negative SARS-CoV antibody test result on   |
|                |         acute-phase serum and positive SARS-CoV     |
|                |         antibody test result on convalescent-phase  |
|                |         serum tested in parallel; **or**            |
|                |                                                     |
|                | ```{=html}                                          |
|                | <!-- -->                                            |
|                | ```                                                 |
|                | -   Isolation in cell culture of SARS-CoV from a    |
|                |     clinical specimen, with confirmation using a    |
|                |     test validated by CDC; **or**                   |
|                |                                                     |
|                | ```{=html}                                          |
|                | <!-- -->                                            |
|                | ```                                                 |
|                | -   Detection of SARS-CoV RNA by RT-PCR validated   |
|                |     by CDC, with confirmation in a reference        |
|                |     laboratory, from:                               |
|                |                                                     |
|                |     -   Two clinical specimens from different       |
|                |         sources, *or*                               |
|                |                                                     |
|                |     -   Two clinical specimens collected from the   |
|                |         same source on two different days           |
+----------------+-----------------------------------------------------+
| Confirmed case | A person with clinically compatible illness and     |
| of SARS-CoV    | laboratory-confirmed SARS-CoV infection             |
| disease        |                                                     |
+----------------+-----------------------------------------------------+

The Centers for Disease Control and Prevention (CDC) and other
institutions have been working to develop strategies to detect and
control the spread of severe acute respiratory syndrome (SARS). The
cause of SARS has been determined to be infection with a previously
unrecognized human coronavirus, SARS-associated coronavirus (SARS-CoV).
Information on SARS and SARS-CoV is provided on CDC's SARS website:
[www.cdc.gov/sars/](http://www.cdc.gov/sars/). All information and
guidelines, including those on SARS-CoV laboratory testing, may change
as we continue to learn more about this disease. Please check CDC's SARS
website regularly for the most current information.

Previous experience with SARS-CoV disease demonstrates that the best
guide to diagnosis is exposure to a person with SARS-CoV disease, a
setting where SARS-CoV transmission is occurring, or persons who are
part of a cluster of pneumonia without a known cause. Information in
diagnosing SARS-CoV disease is provided in *Clinical Guidance on the
Identification and Evaluation of Possible SARS-CoV Disease among Persons
Presenting with Community-Acquired Illness*
([www.cdc.gov/ncidod/sars/clinicalguidance.htm](http://www.cdc.gov/ncidod/sars/clinicalguidance.htm)).
Persons without a potential risk of exposure should usually not be
tested for SARS-CoV. Clinicians should seek guidance from the state or
local health department regarding current guidelines for SARS-CoV
testing.

Clinicians providing care for patients with possible SARS-CoV disease
may find the following information useful when interpreting SARS-CoV
test results.

**What tests for SARS-CoV are available?**\
CDC has developed and validated an enzyme immunoassay (EIA) for
detection of serum antibody
([www.cdc.gov/ncidod/sars/lab/eia/](http://www.cdc.gov/ncidod/sars/lab/eia/))
to SARS-CoV and a reverse transcription-polymerase chain reaction
(RT-PCR) assay
([www.cdc.gov/ncidod/sars/lab/rtpcr/](http://www.cdc.gov/ncidod/sars/lab/rtpcr/))
for detection of SARS-CoV RNA. The EIA has been distributed to most
state public health laboratories, and the RT-PCR has been distributed to
most laboratories in the Laboratory Response Network (LRN). Both the EIA
and the RT-PCR tests are sensitive and highly specific for SARS-CoV. The
ability to diagnose SARS-CoV infection in a patient is often limited,
however, by either the low concentration of virus in most clinical
specimens (RT-PCR assays) or the time it takes a person to mount a
measurable antibody response to SARS-CoV (serologic assays). The
likelihood of detecting infection is increased if multiple specimens
(e.g., stool, serum, respiratory tract specimens) are collected at
several times during the course of illness.

CDC considers detection of SARS-CoV antibody to be the most reliable
indicator of infection. Since previous infection is still rare in most
populations, seroconversion is not needed to diagnose infection.
Therefore, the presence of SARS-CoV antibody in someone without a
previous history of SARS is indicative of recent infection. A negative
serologic test can rule out SARS-CoV infection if the serum specimen is
collected \>28 days after onset of illness. Some persons do not mount an
antibody response (test positive) until more than 28 days after onset of
illness. Patients with a negative antibody test result whose specimens
were obtained 28 days before illness onset or before should have another
serum specimen collected \>28 days after onset of symptoms.

RT-PCR for SARS-CoV RNA is a very sensitive and specific assay when
performed appropriately. This test can detect SARS-CoV RNA in serum,
stool, upper and lower respiratory specimens, various tissues, and
occasionally urine specimens. Testing of multiple specimen types at
several times during the course of illness should increase the
likelihood of detecting infection.

Other tests for detection of SARS-CoV include immunofluorescence assay
(IFA) for SARS-CoV antibody, SARS-CoV isolation studies, electron
microscopic studies, and immunohistologic or in situ hybridization
studies on tissue specimens. The IFA for SARS-CoV antibody gives results
essentially identical to those for the EIA for SARS antibody. Cell
culture, electron microscopy, and histologic studies are less frequently
used and less sensitive than RT-PCR. Cell culture for SARS-CoV should be
done only in a BSL-3 laboratory using BSL-3 procedures (see Appendix
F5).

**What does it mean if a specimen tests positive for SARS-CoV?**\
Laboratory test results should always be considered with clinical
observations and epidemiologic data in making a final diagnosis. A
positive RT-PCR result should be confirmed by testing a second specimen
and confirming the result at a qualified second laboratory to ensure
that the result is not an artifact of laboratory contamination. A
positive serologic result is less likely to result from a laboratory
artifact but should also be subjected to confirmatory testing. If the
results are confirmed, then a positive RT-PCR or serologic test result
indicates that the patient has been recently infected with SARS-CoV
(unless the patient has a previous history of SARS-CoV disease).
Guidelines for managing patients with SARS-CoV disease are provided in
Supplement C and Supplement I.

**How is a SARS-CoV test confirmed?**

Positive antibody and RT-PCR test results should be confirmed by repeat
testing of the original specimen AND by testing of the same specimen in
an independent laboratory using a validated assay.

**What is difference between a laboratory-confirmed clinical specimen
and laboratory-confirmed SARS-CoV disease?**

This distinction is made for PCR test results because of concerns about
false-positive results. For serology, virus isolation, and
histopathologic studies, if a specimen is confirmed positive, the
patient is also considered to be confirmed positive. For PCR, a second
specimen is required to be confirmed positive to decrease the chance of
misclassifying a patient due to a false-positive result. In all
instances, laboratory results must be considered in the context of
clinical and epidemiologic information on the patient.

**What does it mean if a patient with an illness suggestive of SARS has
a negative SARS-CoV test result?**\
A negative antibody result on a serum specimen collected \>28 days after
onset of illness is sufficient to eliminate SARS-CoV as the cause of
illness. A negative antibody result on serum specimens collected ≤28
days after onset of illness or a negative RT-PCR test does not rule out
SARS-CoV infection. Clinical specimens do not always have sufficient
virus to be detected by RT-PCR. An antibody response may not be detected
in some patients until \>28 days after onset of illness.

**What does it mean if test results are positive for other respiratory
diseases?**\
A positive test result for another respiratory pathogen does not rule
out SARS-CoV disease. SARS patients can be co-infected with SARS-CoV and
other respiratory pathogens. Thus, detection of another respiratory
pathogen does not eliminate the possibility of SARS-CoV disease. In some
circumstances (e.g., another pathogen is detected in multiple patients
in a cluster of cases and can fully explain the severity of illness),
detection of another respiratory pathogen may make SARS-CoV disease less
likely. Factors that may be considered in assigning alternate diagnoses
include the strength of the epidemiologic exposure criteria for SARS-CoV
disease, the specificity of the diagnostic test, and the compatibility
of the clinical presentation and course of illness with the alternative
diagnosis.

**Does a negative SARS-CoV test result affect patient management?**\
As noted above, the interpretation of negative SARS-CoV test results
varies depending on the type of specimen, the timing of specimen
collection, and the test that was performed. With the exception of a
\>28-day negative serologic test result, a negative SARS-CoV test result
should not affect patient isolation or management decisions. The
clinical features of the illness and the type and risk of exposure are
the keys to making decisions on patient management and isolation.

**Has the identification of SARS-CoV as the etiologic agent changed the
recommendations for medical treatment of patients with SARS?**\
No. The discovery that SARS-CoV is the cause of SARS has not changed
treatment recommendations. Research on antiviral treatment for SARS-CoV
disease is currently under way.

**Should a person who may have been exposed to a location with
transmission of SARS-CoV or who had contact with a SARS patient be
tested even if not ill?**\
Persons who have potentially been exposed to SARS patients and are well
should be tested only as part of research studies. The exposed person
may contact their state health department or CDC about participating in
studies of persons exposed to SARS-CoV.

# Appendix F8

# Guidelines for Laboratory Diagnosis of SARS-CoV Infection

Laboratory confirmation of SARS-CoV infection is based on:

-   Detection of any of the following by a validated test, with
    confirmation in a reference laboratory:

    -   Serum antibodies to SARS-CoV in a single serum specimen, *or*

    -   A four-fold or greater increase in SARS-CoV antibody titer
        between acute- and convalescent-phase serum specimens tested in
        parallel, *or*

    -   Negative SARS-CoV antibody test result on acute-phase serum and
        positive SARS-CoV antibody test result on convalescent-phase
        serum tested in parallel; **or**

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-   Isolation in cell culture of SARS-CoV from a clinical specimen, with
    confirmation using a test validated by CDC; **or**

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-   Detection of SARS-CoV RNA by RT-PCR validated by CDC, with
    confirmation in a reference laboratory, from:

    -   Two clinical specimens from different sources, *or*

    -   Two clinical specimens collected from the same source on two
        different days

Guidelines for the collection of specimens from potential cases of SARS
are provided in Appendix F4.

For more information, visit
[www.cdc.gov/ncidod/sars](http://www.cdc.gov/ncidod/sars/) or call the
CDC public response hotline\
at (888) 246-2675 (English), (888) 246-2857 (Español), or (866) 874-2646
(TTY)

[^1]: Respirators should be used in the context of a complete
    respiratory protection program, as required by the Occupational
    Safety and Health Administration (OSHA). This includes training,
    fit-testing, and fit-checking to ensure appropriate respiratory
    selection and use. To be effective, respirators must provide a
    proper sealing surface on the wearer's face. Detailed information on
    a respiratory protection program can be found at:
    [www.osha.gov/SLTC/etools/respiratory/](http://www.osha.gov/SLTC/etools/respiratory/).

[^2]: ^1^ Respirators should be used in the context of a complete
    respiratory protection program, as required by the Occupational
    Safety and Health Administration (OSHA). This includes training,
    fit-testing, and fit-checking to ensure appropriate respiratory
    selection and use. To be effective, respirators must provide a
    proper sealing surface on the wearer's face. Detailed information on
    a respiratory protection program can be found at:
    [www.osha.gov/SLTC/etools/respiratory/](http://www.osha.gov/SLTC/etools/respiratory/).