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Routine HIV Screening in Health Care Settings. HIV Diagnostic Testing. David Spach, MD Clinical Director Northwest AIDS Education and Training Center Professor of Medicine, Division of Infectious Diseases University of Washington Seattle.

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Routine hiv screening in health care settings
Routine HIV Screening in Health Care Settings

HIVDiagnostic Testing

David Spach, MDClinical DirectorNorthwest AIDS Education and Training CenterProfessor of Medicine, Division of Infectious DiseasesUniversity of Washington Seattle

This project was funded under cooperative agreement number U65/PS000821from the Centers for Disease Control and Prevention (CDC). 


Hiv diagnostic testing
HIV Diagnostic Testing

  • Types of HIV Diagnostic Tests

  • Initial and Supplemental HIV Tests

  • Sensitivity and Specificity of HIV Tests

  • Laboratory-Based HIV Testing Algorithms

  • Point-of-Contact (Rapid) HIV Testing Algorithms

  • Diagnosis of Acute HIV


Hiv diagnostic testing1
HIV Diagnostic Testing

  • Types of HIV Diagnostic Tests

  • Initial and Supplemental HIV Tests

  • Sensitivity and Specificity of HIV Tests

  • Laboratory-Based HIV Testing Algorithms

  • Point-of-Contact (Rapid) HIV Testing Algorithms

  • Diagnosis of Acute HIV


Types of hiv diagnostic tests
Types of HIV Diagnostic Tests

HIV Antibodies

HIV-1 RNA

HIV p24 Antigen

Most Common Test for Established Infection

Used for Acute HIV and Indeterminate WB

Rarely UsedFuture use: 4th Generation EIA


Hiv diagnostic testing2
HIV Diagnostic Testing

  • Types of HIV Diagnostic Tests

  • Initial and Supplemental HIV Tests

  • Sensitivity and Specificity of HIV Tests

  • Laboratory-Based HIV Testing Algorithms

  • Point-of-Contact (Rapid) HIV Testing Algorithms

  • Diagnosis of Acute HIV


Initial and supplemental hiv tests
Initial and Supplemental HIV Tests

  • Initial Test - Enzyme Immunoassay (EIA) - Chemiluminescent Immunoassay (CIA)

  • Supplemental Tests - Western blot - Indirect Immunofluorescence Assay (IFA) - Qualitative HIV-1 RNA


Initial and supplemental hiv tests1
Initial and Supplemental HIV Tests

  • Initial Test - Enzyme Immunoassay (EIA) - Chemiluminescent Immunoassay (CIA)

  • Supplemental Tests - Western blot - Indirect Immunofluorescence Assay (IFA) - Qualitative HIV-1 RNA


Generation of eia tests
Generation of EIA Tests

First

Second

Third

*Fourth

*Not US FDA-approved as of 10/1/09

Uses crude viral lysate

Uses recombinant HIV antigens or peptides

Detects IgM and IgG in “Sandwich” EIA

Detects HIV antibodies and p24 antigen


Traditional hiv eias
Traditional HIV EIAs

96-Well Microtiter Plate EIA

Interpretation of EIAs

  • Based on color change/fluorescence

  • Change compared with standardized cut-off

  • Result positive or negative

  • No specific antibody reaction information

  • Multiple samples run with traditional EIA


Hiv 1 western blot antigens
HIV-1 Western Blot Antigens

p = protein

gp = glycoprotein

Number = molecular weight


Components used in hiv 1 western blot
Components Used in HIV-1 Western Blot

HIV Western blot Strip

Color Reagent

Enzyme Detector

Y

Y

Y

Antihuman IgG Antibodies

Y

Y

Y

Y

Y

Y

Human HIV Antibodies (from patient serum)

HIV Antigens (on Western blot)


Sample hiv 1 western blot
Sample HIV-1 Western Blot

Color Reagent

Enzyme Detector

Y

Anti-human IgG

Y

Y

Y

Antibodies to gp120

Y

Y

HIV gp120 antigen

Color Reagent

Enzyme Detector

Y

Y

Anti-human IgG

Y

Y

Y

Y

Antibodies to p24

HIV p24 antigen

Test Completed

gp120 & p24 bands Visible




Interpretive criteria for hiv 1 western blot
Interpretive Criteria for HIV-1 Western Blot

Positive Control

Source: CDC. MMWR. 1989:38(S-7):1-7.


Interpretive criteria for hiv 1 western blot1
Interpretive Criteria for HIV-1 Western Blot

Negative

No bands:

Source: CDC. MMWR. 1989:38(S-7):1-7.


Interpretive criteria for hiv 1 western blot2
Interpretive Criteria for HIV-1 Western Blot

Positive

At least two of the following bands:p24gp41gp120/160

Source: CDC. MMWR. 1989:38(S-7):1-7.


Interpretive criteria for hiv 1 western blot3
Interpretive Criteria for HIV-1 Western Blot

Examples

Indeterminate

One or more bands presentNot meeting positive criteria

Most common bands seen withindeterminate Western blot (IWB)p17, p24, p55

Source: CDC. MMWR. 1989:38(S-7):1-7.


Hiv diagnostic testing3
HIV Diagnostic Testing

  • Types of HIV Diagnostic Tests

  • Initial and Supplemental HIV Tests

  • Sensitivity and Specificity of HIV Tests

  • Laboratory-Based HIV Testing Algorithms

  • Point-of-Contact (Rapid) HIV Testing Algorithms

  • Diagnosis of Acute HIV


Hiv antibody test sensitivity
HIV Antibody Test: Sensitivity

Sensitivity

Probability that test is positive if person is infected


Hiv antibody test sensitivity1
HIV Antibody Test: Sensitivity

Persons Infected with HIV: n = 50

Example


Hiv antibody test sensitivity2
HIV Antibody Test: Sensitivity

Persons Infected with HIV: n = 50

HIV Antibody Testing: 49/50 Positive

Example


Hiv antibody test sensitivity3
HIV Antibody Test: Sensitivity

Persons Infected with HIV: n = 50

HIV Antibody Testing: 49/50 Positive

Sensitivity = 49/50 = 98%

Example


Sensitivity of hiv antibody tests used in us
Sensitivity of HIV Antibody Tests Used in US

HIV Antibody TestsSensitivity >> 99%


Hiv antibody test false negative
HIV Antibody Test: False Negative

HIV-Infected Persons

HIV Antibody Testing

Example


Hiv antibody test false negative1
HIV Antibody Test: False Negative

HIV-Infected Persons

HIV Antibody Testing

False Negative

Example


Some causes of false negative antibody tests
Some Causes of False-Negative Antibody Tests

  • Acute HIV Infection

  • Advanced HIV Infection

  • Antiretroviral Therapy


Hiv antibody test specificity
HIV Antibody Test: Specificity

SpecificityProbability that test is negative if person is not infected


Hiv antibody test specificity1
HIV Antibody Test: Specificity

Persons NOT Infected with HIV: n = 50

Example


Hiv antibody test specificity2
HIV Antibody Test: Specificity

Persons NOT Infected with HIV: n = 50

HIV Antibody Testing: 48/50 Negative

Example


Hiv antibody test specificity3
HIV Antibody Test: Specificity

Persons NOT Infected with HIV: n = 50

HIV Antibody Testing: 48/50 Negative

Specificity = 48/50 = 96%

Example


Hiv antibody test false positive
HIV Antibody Test: False Positive

Persons NOT Infected with HIV

HIV Antibody Testing

Example


Hiv antibody test false positive1
HIV Antibody Test: False Positive

Persons NOT Infected with HIV

HIV Antibody Testing

False Positive

Example


Specificity of hiv antibody tests used in us
Specificity of HIV Antibody Tests Used in US

Initial HIV Antibody TestsSpecificity > 98%

Supplemental HIV Test (if initial test positive)Specificity >> 99%


Hiv antibody test specificity4
HIV Antibody Test: Specificity

True Infection

False Positive

False Positives: proportion of false-positive tests increases in populations with low HIV prevalence


Causes of false positive hiv antibody tests
Causes of False-Positive HIV Antibody Tests

  • Other Viral Diseases

  • Hematologic Disorders

  • Liver Disease

  • Immunizations

  • Autoimmune Disorders


Interpreting test results
Interpreting Test Results

Sensitivity:Probability test = positive IF person = positive

Specificity:Probability test = negative IF person = negative

Positive Predictive Value:Probability person = positive IF test = positive

Negative Predictive Value:Probability person = negative IF test = negative


Positive predictive value of hiv tests in populations with differing hiv prevalence
Positive Predictive Value of HIV Tests in Populations with Differing HIV Prevalence

Example: HIV Testing 1,000 Persons


Hiv diagnostic testing4
HIV Diagnostic Testing Differing HIV Prevalence

  • Types of HIV Diagnostic Tests

  • Initial and Supplemental HIV Tests

  • Sensitivity and Specificity of HIV Tests

  • Laboratory-Based HIV Testing Algorithms

  • Point-of-Contact (Rapid) HIV Testing Algorithms

  • Diagnosis of Acute HIV


General approach to hiv diagnostic testing
General Approach to HIV Diagnostic Testing Differing HIV Prevalence

Initial

Supplemental

Optimized for Sensitivity

Optimized for Specificity


1989 cdc astphld hiv testing algorithm
1989 CDC/ASTPHLD HIV Testing Algorithm Differing HIV Prevalence

Initial

Supplemental

EIA

Western blotorIFA

Repeatedly Reactive

Source: CDC. MMWR. 1989;38(S-7):1-7.


Modern hiv testing algorithms
Modern HIV Testing Algorithms Differing HIV Prevalence

Initial

Point-of-Contact

Approach to HIV Testing

Laboratory-Based Approach to HIV Testing

Diagnosing Acute HIV

Source: CDC. MMWR. 2001;50(RR19):1-58.


Hiv testing algorithms
HIV Testing Algorithms Differing HIV Prevalence

http://www.aphl.org/aphlprograms/infectious/hiv/Pages/HIVStatusReport.aspx


Hiv testing algorithms1
HIV Testing Algorithms Differing HIV Prevalence

http://www.aphl.org/aphlprograms/infectious/hiv/Pages/HIVStatusReport.aspx


Laboratory based hiv testing algorithms
Laboratory-Based HIV Testing Algorithms Differing HIV Prevalence

Initial

HIV-1/HIV-2Immunoassay*

Nonreactive

HIV Negative

*Enzyme Immunoassay (EIA) or Chemiluminescent Immunoassay (CIA)


Laboratory based hiv testing algorithms1
Laboratory-Based HIV Testing Algorithms Differing HIV Prevalence

Initial

Repeat Testing (in duplicate)

HIV-1/HIV-2Immunoassay*

Reactive

*Enzyme Immunoassay (EIA) or Chemiluminescent Immunoassay (CIA)


Laboratory based hiv testing algorithms2
Laboratory-Based HIV Testing Algorithms Differing HIV Prevalence

Initial

Repeat Testing (in duplicate)

HIV-1/HIV-2Immunoassay*

Reactive

Both Nonreactive

HIV Negative

*Enzyme Immunoassay (EIA) or Chemiluminescent Immunoassay (CIA)


Laboratory based hiv testing algorithms3
Laboratory-Based HIV Testing Algorithms Differing HIV Prevalence

Initial

Supplemental

HIV-1 Western blotorHIV-1 IFAor

HIV-1 RNA

HIV-1/HIV-2Immunoassay*

Repeat Testing (in duplicate)

Reactive

Repeatedly Reactive

*Enzyme Immunoassay (EIA) or Chemiluminescent Immunoassay (CIA)


Laboratory based hiv testing algorithms4
Laboratory-Based HIV Testing Algorithms Differing HIV Prevalence

Initial

Supplemental

HIV-1 Western blotorHIV-1 IFAor

HIV-1 RNA

HIV-1/HIV-2Immunoassay*

Repeat Testing (in duplicate)

Reactive

Repeatedly Reactive

Negative

HIV Negative

*Enzyme Immunoassay (EIA) or Chemiluminescent Immunoassay (CIA)


Laboratory based hiv testing algorithms5
Laboratory-Based HIV Testing Algorithms Differing HIV Prevalence

  • Test for HIV-2 with any of Following:

  • Indicated by local HIV-2 prevalence

  • Indicated by travel or risk history

  • Indicated by clinical presentation

Initial

Supplemental

HIV-1 Western blotorHIV-1 IFAor

HIV-1 RNA

HIV-1/HIV-2Immunoassay*

Repeat Testing (in duplicate)

Reactive

Repeatedly Reactive

Negative

HIV Negative

*Enzyme Immunoassay (EIA) or Chemiluminescent Immunoassay (CIA)


Laboratory based hiv testing algorithms6
Laboratory-Based HIV Testing Algorithms Differing HIV Prevalence

Initial

Supplemental

HIV-1 Western blotorHIV-1 IFAor

HIV-1 RNA

HIV-1/HIV-2Immunoassay*

Repeat Testing (in duplicate)

Reactive

Repeatedly Reactive

Positive

HIV Infected

*Enzyme Immunoassay (EIA) or Chemiluminescent Immunoassay (CIA)


Laboratory based hiv testing algorithms7
Laboratory-Based HIV Testing Algorithms Differing HIV Prevalence

Initial

Supplemental

HIV-1 Western blot

HIV-1/HIV-2Immunoassay*

Repeat Testing (in duplicate)

Reactive

Repeatedly Reactive

Indeterminate

*Enzyme Immunoassay (EIA) or Chemiluminescent Immunoassay (CIA)


Approach to indeterminate western blot
Approach to Indeterminate Western blot Differing HIV Prevalence

HIV-1 Western blot

Indeterminate


Approach to indeterminate western blot1
Approach to Indeterminate Western blot Differing HIV Prevalence

HIV-1 Western blot

Indeterminate

Repeat HIV-1 Western blot after 2-4 Weeks

or

HIV RNA Test


Modern approach to indeterminate western blot
Modern Approach to Indeterminate Western blot Differing HIV Prevalence

HIV-1 Western blot

Indeterminate

Repeat HIV-1 Western blot after 2-4 Weeks

+

-

or

HIV RNA Test

Indeterminate(no recent exposure)

HIV Infected

Not HIV Infected

Source: CDC. MMWR. 2001;50(RR19):1-58.


Modern approach to indeterminate western blot1
Modern Approach to Indeterminate Western blot Differing HIV Prevalence

HIV-1 Western blot

Indeterminate

Repeat HIV-1 Western blot after 2-4 Weeks

+

-

or

HIV RNA Test

Indeterminate(no recent exposure)

HIV Infected

Not HIV Infected


Modern approach to indeterminate western blot2
Modern Approach to Indeterminate Western blot Differing HIV Prevalence

HIV-1 Western blot

Indeterminate

Repeat HIV-1 Western blot after 2-4 Weeks

+

-

or

HIV RNA Test

HIV Infected

Indeterminate(RECENT EXPOSURE )

Further Evaluation Needed

Not HIV Infected


Approach to indeterminate western blot2
Approach to Indeterminate Western blot Differing HIV Prevalence

HIV-1 Western blot

Indeterminate

Repeat HIV-1 Western blot after 2-4 Weeks

+

-

or

HIV RNA Test

Indeterminate(no recent exposure)

HIV Infected

Not HIV Infected


Hiv diagnostic testing5
HIV Diagnostic Testing Differing HIV Prevalence

  • Types of HIV Diagnostic Tests

  • Initial and Supplemental HIV Tests

  • Sensitivity and Specificity of HIV Tests

  • Laboratory-Based HIV Testing Algorithms

  • Point-of-Contact (Rapid) HIV Testing Algorithms

  • Diagnosis of Acute HIV


Point of contact rapid tests
Point of Contact (Rapid) Tests Differing HIV Prevalence

Multispot HIV-1/HIV-2

Clearview Complete HIV 1/2

Uni-Gold Recombigen

Reveal G3

Oraquick Advance HIV-1/2

Clearview HIV 1/2 Stat Pak


Point of contact rapid hiv tests fda approved tests in us august 2009
Point of Contact (Rapid) HIV Tests Differing HIV PrevalenceFDA-Approved Tests in US, August 2009


Point of contact rapid hiv testing algorithms
Point of Contact (Rapid) HIV Testing Algorithms Differing HIV Prevalence

Rapid Test

Nonreactive

Source: CDC. MMWR. 2004;53(10):221-2.


Point of contact rapid hiv testing algorithms1
Point of Contact (Rapid) HIV Testing Algorithms Differing HIV Prevalence

Rapid Test

Nonreactive

HIV Negative

Client considered HIV-negative unless recent exposure to HIV has occurred

Source: CDC. MMWR. 2004;53(10):221-2.


Point of contact rapid hiv testing algorithms2
Point of Contact (Rapid) HIV Testing Algorithms Differing HIV Prevalence

Rapid Test

ReactivePreliminary Positive

Counsel Client that HIV has not been ConfirmedFollow-Up Supplemental Testing is Necessary

Source: CDC. MMWR. 2004;53(10):221-2.


Point of contact rapid hiv testing algorithms3
Point of Contact (Rapid) HIV Testing Algorithms Differing HIV Prevalence

Supplemental

HIV-1 Western blotorHIV-1 IFAor

HIV-1 RNA

Rapid Test

ReactivePreliminary Positive

Source: CDC. MMWR. 2004;53(10):221-2.


Point of contact rapid hiv testing algorithms4
Point of Contact (Rapid) HIV Testing Algorithms Differing HIV Prevalence

Optional

Supplemental

HIV-1 Western blotorHIV-1 IFAor

HIV-1 RNA

Rapid Test

EIACIA

ReactivePreliminary Positive

EIA = Enzyme Immunoassay CIA= Chemiluminescent Immunoassay

EIA or CIA testing optionalPerform supplemental testing regardless of EIA or CIA result

Source: CDC. MMWR. 2004;53(10):221-2.


Point of contact rapid hiv testing algorithms5
Point of Contact (Rapid) HIV Testing Algorithms Differing HIV Prevalence

Initial

Supplemental

Optional

HIV-1 Western blotorHIV-1 IFAor

HIV-1 RNA

Rapid Test

EIACIA

ReactivePreliminary Positive

Positive

HIV Infected

Source: CDC. MMWR. 2004;53(10):221-2.


Point of contact rapid hiv testing algorithms6
Point of Contact (Rapid) HIV Testing Algorithms Differing HIV Prevalence

Supplemental

HIV-1 Western blotorHIV-1 IFAor

HIV-1 RNA

Rapid Test

ReactivePreliminary Positive

Non-Reactive

or

Indeterminate

Follow-up repeat supplemental testing 4 weeks after original reactive Rapid Test

Source: CDC. MMWR. 2004;53(10):221-2.


Point of contact rapid hiv testing algorithms7
Point of Contact (Rapid) HIV Testing Algorithms Differing HIV Prevalence

Supplemental

Repeat Supplemental

HIV-1 Western blotorHIV-1 IFAor

HIV-1 RNA

HIV-1 Western blotorHIV-1 IFAor

HIV-1 RNA

Rapid Test

4 weeks

ReactivePreliminary Positive

Non-Reactive

or

Indeterminate

Follow-up repeat supplemental testing 4 weeks after original reactive Rapid Test

Source: CDC. MMWR. 2004;53(10):221-2.


Point of contact rapid hiv testing algorithms8
Point of Contact (Rapid) HIV Testing Algorithms Differing HIV Prevalence

Supplemental

Initial

HIV-1 Western blotorHIV-1 IFAor

HIV-1 RNA

HIV-1/HIV-2Immunoassay*

Reactive


Point of contact rapid hiv testing algorithms9
Point of Contact (Rapid) HIV Testing Algorithms Differing HIV Prevalence

Initial

Repeat Testing (in duplicate)

HIV-1/HIV-2Immunoassay*

Reactive

Both Nonreactive

HIV Negative

*Enzyme Immunoassay (EIA) or Chemiluminescent Immunoassay (CIA)


Point of contact rapid hiv testing algorithms10
Point of Contact (Rapid) HIV Testing Algorithms Differing HIV Prevalence

Initial

Supplemental

HIV-1 Western blotorHIV-1 IFAor

HIV-1 RNA

HIV-1/HIV-2Immunoassay*

Repeat Testing (in duplicate)

Reactive

Repeatedly Reactive

*Enzyme Immunoassay (EIA) or Chemiluminescent Immunoassay (CIA)


Point of contact rapid hiv testing algorithms11
Point of Contact (Rapid) HIV Testing Algorithms Differing HIV Prevalence

Initial

Supplemental

HIV-1 Western blotorHIV-1 IFAor

HIV-1 RNA

HIV-1/HIV-2Immunoassay*

Repeat Testing (in duplicate)

Reactive

Repeatedly Reactive

Negative

HIV Negative

*Enzyme Immunoassay (EIA) or Chemiluminescent Immunoassay (CIA)


Hiv diagnostic testing6
HIV Diagnostic Testing Differing HIV Prevalence

  • Types of HIV Diagnostic Tests

  • Initial and Supplemental HIV Tests

  • Sensitivity and Specificity of HIV Tests

  • Laboratory-Based HIV Testing Algorithms

  • Point-of-Contact (Rapid) HIV Testing Algorithms

  • Diagnosis of Acute HIV


Diagnosing acute hiv
Diagnosing Acute HIV Differing HIV Prevalence

HIV RNA

Infection

HIV RNA levels abruptly rise within several weeks after acute infection


Diagnosing acute hiv1
Diagnosing Acute HIV Differing HIV Prevalence

Antibody Titer

HIV RNA

Detectable Antibody

Infection

HIV RNA “spike” precedes production of detectable HIV antibodies


Diagnosing acute hiv eclipse period
Diagnosing Acute HIV: Eclipse Period Differing HIV Prevalence

HIV RNA

Infection

Eclipse Period = Time between infection and detectable HIV RNA


Diagnosing acute hiv window period
Diagnosing Acute HIV: Window Period Differing HIV Prevalence

Window Period

Antibody Titer

Detectable Antibody

Infection

Window Period = Time between infection and detectable HIV antibodies


Timing of diagnostic tests after initial hiv infection
Timing of Diagnostic Tests after Initial HIV Infection Differing HIV Prevalence

HIV EIA*

3rd Generation, IgM-Sensitive EIA

2nd Generation EIA

Viral Lysate EIA

HIV EIA*

HIV RNA Detectable

HIV EIA*

Western blot

Weeks after HIV Infection

Modified from: Branson BM. Clin Infect Dis. 2007;5:S221-5.


Timing of diagnostic tests after initial hiv infection1
Timing of Diagnostic Tests after Initial HIV Infection Differing HIV Prevalence

Modified and Reproduced with permission from American Society of Microbiology

Source: Owen SM, et al. J ClinMicrobiol. 2008;46:1588-95.


Timing of diagnostic tests after initial hiv infection2
Timing of Diagnostic Tests after Initial HIV Infection Differing HIV Prevalence

Modified and Reproduced with permission from American Society of Microbiology

Source: Owen SM, et al. J ClinMicrobiol. 2008;46:1588-95.


Timing of diagnostic tests after initial hiv infection3
Timing of Diagnostic Tests after Initial HIV Infection Differing HIV Prevalence

Modified and Reproduced with permission from American Society of Microbiology

Source: Owen SM, et al. J ClinMicrobiol. 2008;46:1588-95.


Timing of diagnostic tests after initial hiv infection4
Timing of Diagnostic Tests after Initial HIV Infection Differing HIV Prevalence

Modified and Reproduced with permission from American Society of Microbiology

Source: Owen SM, et al. J ClinMicrobiol. 2008;46:1588-95.


Timing of diagnostic tests after initial hiv infection5
Timing of Diagnostic Tests after Initial HIV Infection Differing HIV Prevalence

Rapid HIV

HIV-1 RNA

Modified and Reproduced with permission from American Society of Microbiology

Source: Owen SM, et al. J ClinMicrobiol. 2008;46:1588-95.


Diagnosing acute hiv acute hiv
Diagnosing Acute HIV: Acute HIV Differing HIV Prevalence

Acute HIV

Antibody Titer

HIV RNA

Detectable Antibody

Infection

Acute HIV = patients may present with acute retroviral syndrome/illness


Diagnosing acute hiv clinical manifestations
Diagnosing Acute HIV: Clinical Manifestations Differing HIV Prevalence

Signs and Symptoms of 160 Patients with Acute HIV

Source: Vanhems P, et al. AIDS. 2000;14:375-81.


Laboratory diagnosis of acute hiv
Laboratory Diagnosis of Acute HIV Differing HIV Prevalence

  • Positive HIV-1 RNA Assay

  • Negative HIV Antibody Test

Acute HIV

Antibody Titer

HIV RNA

Detectable Antibody

Infection


Acknowledgement
Acknowledgement Differing HIV Prevalence

The project was funded under cooperative agreement number U65/PS000821 from the Centers for Disease Control and Prevention (CDC). 


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