Transcript ChicagoPathSocietyPresentation2013FINAL

New Approaches for Infectious
Diseases Testing in Clinical
Laboratories
William M. Janda, Ph.D., D(ABMM)
Professor Emeritus of Pathology
University of Illinois College of Medicine
University of Illinois at Chicago
Director, Clinical Microbiology Laboratory
John H. Stroger, Jr. Hospital/Cook County Hospitals and Healthcare
System
Chicago, Illinois
Topics to be Addressed
• New CDC HIV-1/2 testing algorithm
• Reverse-sequence testing for syphilis
• Use of nucleic acid amplification tests (NAATs) for
diagnosis of extra-genital gonococcal/chlamydial
infections
• Use of NAATS for documentation of child sexual
abuse
Typical Course of HIV Infection
HIV Markers Early in HIV Infection: Window
Periods of Immunoassays (IA’s): First Through
Fourth Generation
HIV-1/2 Testing: The
Former Algorithm
• Performance of a HIV-1/2 antibody
immunoassay (IA) on a patient serum
specimen
• Reactive serum specimens are
retested in duplicate using the same IA
• Repeatedly reactive serum specimens
are tested by a confirmatory test
• HIV-1 Western immunoblot →
• Indirect fluorescent antibody test
• Cannot detect acute HIV-1 infection
CDC Criteria for
HIV-1 Western
Immunoblot
Interpretation
• Positive blot criteria
• 1 →→→ Ab to
gp160/120
and p24
• 2 →→→ Ab to gp41
and p24
• 3 →→→ Ab to
gp160/120
and gp41
• No bands → Negative
• Any other combination→
Indeterminant
HIV Antibody Immunoassays
Generation of HIV-1 assay
First generation
•Used either viral lysate antigens
•Detected only IgG-class antibodies
Second generation
•Used either recombinant or synthetic peptides as
antigens
•Detected only IgG-class antibodies
Third generation
Use synthetic peptides or recombinant antigens
Detect both IgG- and IgM-class antibodies
Fourth generation
Use synthetic peptides or recombinant antigens
Detect IgG- and IgM-class antibodies
Detect p24 (core) antigen
Third-Generation EIAs
• “Sandwich” technique with enzyme-coupled HIV antigens
• Take advantage of the bi-/multivalent nature of antibodies →
Improved specificity
• Only antibodies bound to ELISA wells that also bind HIV
antigens generate a signal
• Nonspecifically bound antibodies less likely to bind HIV
antigens
• Technology expands subtypes of detected antibodies
• In direct ELISAs the conjugate is directed against a specific
antibody subtype (e.g., IgG), whereas sandwich technology
permits detection of any antibody class, including IgM
• Sandwich ELISA thus increases the ability to detect HIV
antibodies early in HIV infection (e.g., IgM, IgA)
Comparison of First (A) and
Third-Generation (B) HIV EIA’s
• Assay to detect the presence
of the HIV viral core protein
p24 in serum and plasma
introduced in 1989
• Proved useful during early
infection and improved
detection of recent infection
• Used with antigen-antibody
dissociation techniques as
an aid to diagnosis of HIV
infection in infants (Women
Infants Transmission Study
[WITS])
• p24 antigen detection kits
approved by the FDA in
1989
p24 Antigen
Detection
Fourth Generation HIV-1/2 EIA
(courtesy, BioRad)
•New
HIV-1/2
Testing
Algorithm
Includes 3 categories
of tests:
4th generation HIV-1/2
Ab and Ag combo
assay
HIV-1/HIV-2
discrimination assay
Nucleic acid testing
• Approved by the FDA
in March, 2013 as a
supplemental test
• Recommended option
by CLSI
• Fast TAT of third- and
fourth-generation
immunoassays (<1
hour) and the Multispot
test enables definitive
same-day test results
Multispot HIV1/HIV-2 Rapid
Test (Bio-Rad)
Multi-Spot HIV-1/2 Rapid Test
• 1.
• 2.
•
• 3.
•
• 4.
•
Procedural control (goat anti-human IgG)
HIV-2 peptide
Peptide representing the immunodominant epitope of HIV-2 (gp36
envelope glycoprotein
Recombinant HIV-1 envelope glycoprotein
Recombinant gp41 expressed in E. coli (gp41 rDNA)
HIV-1 peptide
Peptide representing the immunodominant epitope of HIV-1 gp41
envelope glycoprotein
New CDC HIV-1/2 Testing Algorithm
A1: GS HIV Combo Ag/Ab EIA
A1(-)
A1+
A2
Negative for HIV-1 and HIV-2
antibodies and p24 Ag
Multispot *
HIV-1 +
HIV-1
antibodies
detected
Initiate care
(and viral
load)
HIV-1&2 (-)
HIV-2 +
HIV-2 antibodies
detected
Initiate care
*Multispot does not have confirmatory claim
but must be used as a differentiating test
NAAT
NAAT+
NAAT (-)
Acute HIV-1 infection Negative for HIV-1
Initiate care
Multispot - Interpretation
• Nonreactive →→
• HIV-2 Reactive→
• HIV-1 Reactive →
• HIV Reactive →→
• Undifferentiated
• INVALID →→→→
• RNA converted to DNA
by reverse transcription
• DNA used as a
transcription template
(DNA to RNA)
• RNA reverse
transcribed back to DNA
• RNA detected by probes
• 30 copy/ml sensitivity
• APTIMA HIV-1
qualitative assay (GenProbe) cleared for
diagnostic use
Confirmatory
Tests:TranscriptionMediated
Amplification
Arizona DOHS/Maricopa Integrated
Health Systems Study
MMWR 2013;62:489-494
• Screened all adult ED patients ages 18-64 years, July 2011-Feb
2013
• Fourth-generation EIA used for screening with reflex to
Multispot (MS and WB)
• Specimens negative by MS or WB tested for HIV-1 RNA
• Results
• Detected previously undiagnosed HIV infection in 37 patients
• 25 diagnoses were positive by MS, WB, or both
• For 12 of the 37 patients, infection established by negative
MS and/or WB results and positive for HIV-1 RNA
• Median HIV-1 viral loads in patients with acute infection was
3,636,176 copies/ml
Benefits to Identifying Acute
HIV-1 Infection
• Acute infection accounts for 5-10% of HIV infection among
those tested
• Risk of transmission from persons with early infection is higher
than from those with established infections
• Persons who have been infected for less than 6 months account
for almost 50% of all onwards transmission of HIV
• Enables intervention to interrupt transmission
• Persons with acute HIV infection named 2.5 times as many
partners
• Persons with acute HIV infection had nearly twice as many
partners with undiagnosed HIV infection as did persons with
established infections
Implications for Rapid Pointof-Care Testing
• Change in HIV testing algorithm applies to clinical
labs only
• Confirmation of HIV infection cannot be made at the
point-of-care using CLIA-waived tests
• Test providers should be aware of the new algorithm
and with the types of supplemental tests that may be
used to confirm preliminary positive results
FDA-Approved Rapid HIV Antibody Screening
Tests
Test
OraQuick
ADVANCE
Rapid HIV1/2 Antibody
Test
Uni-Gold
Recombigen
HIV
Reveal G-3
Rapid HIV-1
Antibody
Test
Specimen
Type
CLIA
Category
Sensitivity
Specificity
Manufacturer
Oral fluid
Waived
99.3%
99.8%
OraSure
Technologies, Inc.
Whole Blood
(f-stick,
venipunct)
Waived
99.6%
100%
Plasma
Moderate
Complexity
99.6%
99.9%
Whole blood
(f-stick,
venipunct)
Waived
100%
99.7%
Serum &
Plasma
Moderate
Complexity
100%
99.8%
Serum
Moderate
complexity
99.8%
99.1%
Plasma
Moderate
complexity
99.8%
98.6%
www.orasure.
com
Trinity Biotech
ww.unigoldhiv.com
MedMira, Inc.
www.medmira.com
FDA-Approved Rapid HIV Antibody Screening
Tests
Test
MultiSpotHI
V-1/HIV-2
Rapid Test
Clearview
HIV 1/2
STAT-PAK
Clearview
COMPLETE
HIV ½
Specimen
Type
CLIA
Category*
Sensitivity
(95% CI)
Specificity
(95% CI)
Manufacturer
Serum
Moderate
complexity
100%
99.93%
Plasma
Moderate
Complexity
100%
99.91%
BioRad
Laboratories
www.biorad.
com
Whole Blood
(f-stick,
venipunct)
Waived
99.7%
99.9%
Serum &
Plasma
Non-waived
99.7%
99.9%
Whole Blood
(f-stick,
venipunct)
Waived
99.7%
99.90%
Serum &
Plasma
Non-waived
99.7%
99.9%
Inverness
Medical
Professional
Diagnostics
www.inverness
medical
pd.com
Inverness
Medical
Professional
Diagnostics
www.inverness
medical
pd.com
Sensitivity for Early HIV Infection of Rapid HIV Tests
Compared with 3rd and 4th Generation Assays
J Clin Virol 2012;54:42-47
HIV Screening Assay
No. Specimens
Testing POS
Total No.
Tested
Sensitivity for Early
HIV Infection (%)
Architect HIV-1 Ag/Ab Combo
29
33
87.8
Determine HIV-1 Ag/Ab Rapid
Test (Alere, FDA-cleared
8/2013)
25
33
75.8
Genetic Systems HIV-1/2+O
19
11
33
33
57.5
33.3
8
8
27
33
29.6
24.2
7
7
31
32
22.6
21.9
Multispot HIV-1/2 Rapid Test
Clearview Complete HIV-1/2
Unigold Recombingen HIV
Clearview HIV-1/2 Stat-Pak
Oroquick Advance HIV-1/2
Use of Fourth Generation IA’s and
Supplemental Tests
• Improved HIV IA’s enhance ability to detect HIV infection earlier
• Acute infection, when substantial HIV transmission occurs
• Specimens with reactive IA’s and negative supplemental test
results must undergo further testing to differentiate acute HIV
infection from false-positive results
• Acute HIV infections detected with 3rd or 4th generation EIAs may
be misclassified as HIV-negative by WB/IFA →
• Adverse clinical outcomes for patients
• Further HIV transmission within the community
• With FDA-clearance of Multispot as a supplemental test, labs
can now adopt this algorithm
• Fast TAT enables delivery of same-day definitive test results
• 3rd and 4th generation EIA’s → <1 hour
• Multispot → 15 min
New HIV Testing Algorithm:
Conclusions
• Sensitive 3rd or 4th generation HIV-1/2 IA
• If REACTIVE, a supplemental test (i.e., Multispot) is used to
differentiate HIV-1 and HIV-2 antibodies
• If the supplemental test is REACTIVE for HIV-1 antibodies
• Confirmed HIV-1 Infection
• If the supplemental test is REACTIVE for HIV-2
• Confirmed HIV-2 Infection
• If the supplemental test is discrepant with the initial IA result, a
NAT test is recommended
• Distinguish acute early infection from false-positive IA
•
•
•
•
•
•
Treponema pallidum cannot be
cultured
Primary syphilis diagnosed by direct
detection methods (lesions)
• Darkfield microscopy
• Direct fluorescent antibody test for
T. pallidum (DFA-TP)
• Polymerase Chain reaction
Methods not widely available
Direct detection methods can miss up
to 30% of primary cases
Most patients present without
symptoms or signs of syphilis
• Healed early lesions
• Inapparent lesions
• Latent infections
Syphilis is usually diagnosed by
serologic tests
Diagnosis
of Syphilis
Lesions of Primary and Secondary
Syphilis
Serologic Diagnosis of Syphilis
• Serologic diagnosis always requires detection of two
types of antibodies
• Nontreponemal antibodies
• Antibodies directed against lipoidal antigens
• Damaged host cells
• Possibly from treponemes
• Treponemal antibodies
• Antibodies directed against T. pallidum proteins
• Nontreponemal tests
• Rapid plasma reagin
(RPR) test
• Venereal disease
research laboratory
(VDRL) test
• Toluidine red unheated
serum tests (TRUST)
• Treponemal tests
• Fluorescent treponemal
antibody absorbed
(FTA-ABS) test
• Treponema pallidum
particle agglutination
(TP-PA) test
Serologic
Diagnosis of
Syphilis
Serologic Diagnosis of Syphilis
FTA-ABS
T. pallidum Particle
Agglutination (TP-PA) /
T. pallidum
HemAgglutination assays
(TP-HA)
• Enzyme immunoassays
• Trep-Chek (Phoenix
Biotech)
• Trep-Sure (Phoenix
Biotech)
• Chemiluminescence
immunoassays (CIAs)
• Liaison
• Architect
• Microbead
immunoassays (MBIA)
• BioPlex 2200
Syphilis IgG →→→
• BioPlex 2200 IgM
Serologic
Diagnosis of
Syphilis
Serologic Reactivity in Syphilis
Traditional Testing Algorithm for
Diagnosis of Syphilis
Syphilis Serologic
Screening
Algorithms
– Reverse
Sequence
Syphilis Serology
• Traditional algorithm
• Detects active infection
• High rate of biological false-positives
• Confirmation with a treponemal test
• Use of both tests results in a high PPV
• Can miss early primary and treated infections
• Reverse sequence algorithm
• Detects early primary and treated infections that might be
missed with traditional screening
• Non-treponemal test needed to detect active infection
• Ideally, EIAs and CIAs should have perfect specificity
• False-positive results do occur
• Varies by risk group being tested
Syphilis Immunoassays - Timeline
1980’s
EIA is FDA-cleared for use as a confirmatory test and in blood
bank screening
2000
UK Public Health Lab Guidelines says EIA is an “appropriate
alternative” to VDRL/RPR and TPHA
2001
EIA is FDA-cleared for clinical diagnostic use
2008
EU Guidelines: EIA/TPPA recommended for screening; VDRL
and RPR no longer recommended
2009
CDC-APHL Report: Presents algorithm for screening with
treponemal EIA
Sensitivity and Specificity of
Serologic Tests for Syphilis
• Automated (high
throughput)
• 180 tests per hour
• Low cost in highvolume settings
• Less lab occupational
hazards
• No manual pipetting
• No false-negatives due
to prozone reaction
Why Switch to
EIA/CIA?
• Objective results
• Some EIA/CIA test
detect IgM
antibodies
• BioPlex 2200
assay
• Potentially useful
for diagnosis of
early syphilis
Why Switch to
EIA/CIA?
Challenges and Limitations of
EIA/CIA
• Cannot distinguish between active disease and old
disease (treated/untreated)
• Studies to compare test performance with other
serologic tests are lacking
• Studies evaluating performance of EIA/CIA to detect
IgM antibodies are lacking but ongoing
• Confusion regarding management of patients with
discrepant serology
• Positive EIA/CIA result and a negative RPR
Reasons for Discordant Test
Results: EIA/CIA+ → RPR• False-positive EIA/CIA
• Very sensitive
• Lower specificity
• Treated syphilis
• Treponemal antibodies are detected by sensitive
EIAs and CIAs
• Sero-reversion of non-treponemal antibodies
• Early primary syphilis
• Treponemal antibody titer rises before nontreponemal antibody titer
Interpretation of Serologic Tests
for Syphilis
• Reactive results in both treponemal and RPR tests →
• Untreated syphilis (unless ruled out by treatment history)
• Persons treated in the past are considered to have a new
infection if quantitative RPR testing reveals a four-fold or
greater increase in titer
• Reactive result in the treponemal test but non-reactive in the
RPR test →
• Those with a history of previous treatment require no further
management
• For those without a history of treatment, a second, different
treponemal test should be performed (i.e., TPPA)
• If the second treponemal test is non-reactive, no further
evaluation or treatment is indicated, or perform a third
treponemal test to resolve the discrepancy
Reverse Sequence Algorithm
for Syphilis
• Composite
results of
reverse
sequence
algorithm for
initial screening
Conclusions
• EIA/CIA have high sensitivity but lower specificity
• All reactive EIA/CIA must be reflexed to a quantitative RPR
• Confirms reactive EIA/CIA
• Detects active infection
• Although test performance varies by prevalence of syphilis in
the population, all discordant specimens (i.e., EIA+/RPR-) must
be confirmed with a confirmatory treponemal test
• Confirmatory treponemal test must have at least equivalent
sensitivity and a higher specificity compared to the screening
treponemal tests (EIA/CIA)
• TP-PA recommended
• FTA-ABS not recommended
Nucleic Acid Amplification Tests for N.
gonorrhoeae and Chlamydia trachomatis
Test
Manufacturer
Method of
Detection
FDA -Cleared
Not FDA-Cleared
Amplicor
Roche
Molecular
Diagnostics
PCR
Female endocervical;
Male urethral;
Male and female FV urine;
Self-collected vaginal swabs
Extragenital
sites;
Children (any
site)
Probe-Tec
BectonDickinson
Strand
displacement
amplification
Female endocervical;
Male urethral;
Male and female FV urine;
Self-collected vaginal swabs
Extragenital
sites;
Children (any
site)
APTIMA
Hologic/
Gen-Probe
Transcriptionmediated
amplification
Female endocervical;
Male urethral;
Male and female FV urine;
Self-collected vaginal swabs
Extragenital
sites;
Children (any
site)
Real-Time
m2000
Abbott
Molecular
Diagnostics
Real-time
PCR
Female endocervical;
Male urethral;
Male and female FV urine;
Self-collected vaginal swabs
Extragenital
sites;
Children (any
site)
NAATS for GC/CT
• Advantages over culture-based methods
• Greater sensitivity
• Use of non-invasive specimens
• Limitations (especially for GC)
• Genetic variation/recombination can affect gene targets for
amplification, leading to potentially false-negative results
• Horizontal interspecies exchange of genetic material
between Neisseria species may lead to false-positive results
when commensal Neisseria acquire gonococcal sequences
and vice versa
• PCR and SDA have both demonstrated cross-reactivity with
other Neisseria species
Gene Targets and CrossReactivity for Gonococcal NAATs
Test
Amplification Target
Positive Reactivity with other Neisseria
Species
Roche
Amplicor
Cytosine DNA
methyltransferase gene
(single copy)
N. meningitidis, N. lactamica, N.
subflava/sicca, N. cinerea, N. flavescens,
N. polysaccharea, M. catarrhalis
Gen-Probe 16S subunit of ribosomal
APTIMA 2 RNA gene (multicopy)
None reported
BD ProbeTec
Multi-copy pilin gene
inverting protein homolog
N. meningitidis, N. lactamica, N. cinerea,
N. mucosa, N. flavescens,
Abbott
Real-Time
PCR
Opa gene (multicopy)
None reported
NAATS for Detection f N. gonorrhoeae in
Oropharyngeal and Rectal Sites
• McNally et al, CID 47:e25-e27, 2008
• SDA had low positive predictive value for oral (30.4%) and
rectal (73.7%) specimens in an MSM population
• Schachter et al, STD 35:637-642, 2008
• Sensitivities of NAATS (PCR, SDA, TMA) were better than
culture for detection of oral/rectal GC in MSM
• Specificity of PCR 78.9% for oral swabs
• Specificity of SDA and TMA ≥99.4 for oral/rectal sites
• Bachmann et al, JCM 42:902-907, 2009
• PCR had specificity of 73% compared to 96.3% for SDA and
98.6% for TMA for oropharyngeal GC infection in population
with acknowledged oropharyngeal sexual contacts
Use of NAATS for Oropharyngeal GC
Bachmann et al, JCM 42:902-907, 2009
• Evaluated PCR (Roche), TMA (Gen-Probe) and SDA
(BD Probe-Tec)
• NAATS were compared with culture
• Males and females who acknowledged oral sexual
contacts in the previous two months recruited from 3
clinics in Birmingham, AL
• Data evaluated using a “rotating gold standard”
• Any positive results by two or three of the three
tests that excluded the test being evaluated
• 961 evaluable test sets obtained
Use of NAATS for Oropharyngeal GC
Bachmann et al, JCM 42:902-907, 2009
TEST
Sensitivity
Specificity
Positive results by two of three comparator tests
Culture
50%
99.4%
PCR
80.3%
73.0%
SDA
93.2%
96.3%
TMA
83.6%
98.5%
Positive results by three of three comparator tests
Culture
65.4%
99.0%
PCR
91.9%
71.8%
SDA
97.1%
94.2%
TMA
100%
96.2%
Use of NAATS for Oropharyngeal/Rectal GC/CT
Schachter et al STD 35:637-642, 2008
• Specimens from 1110 MSM
• Evaluated PCR, SDA, and TMA compared with
culture
• True-positive GC
• Culture positive, OR
• TMA/PCR positive or TMA/SDA positive, OR
• A single positive NAAT confirmed by an alternate target
NAAT
• True-positive CT
• Culture positive, OR
• Two positive NAATS, OR
• A single positive NAAT confirmed by an alternate target
NAAT
Use of NAATS for Oropharyngeal/Rectal
GC/CT
Schachter et al STD 35:637-642, 2008
• Based on initial findings with 205 MSM specimens, PCR had a
78.9% GC specificity with oropharyngeal swabs, so PCR
testing was discontinued
• Oropharyngeal GC infections (89 infections detected)
• Sensitivity of Culture →→41%
• Sensitivity of SDA →→→ 72%
• Sensitivity of TMA →→→ 93%
• Specificity of SDA/TMA → ≥99.4%
• Rectal GC infections (88 infections detected)
• Sensitivity of Culture →→43%
• Sensitivity of SDA →→→ 78%
• Sensitivity of TMA →→→ 93%
• Specificity of SDA/TMA → ≥99.4%
Use of NAATS for Oropharyngeal/Rectal
GC/CT
Schachter et al STD 35:637-642, 2008
• Oropharyngel CT infections (9 infections detected)
• Sensitivity of culture →→→ 44%
• Sensitivity of SDA →→→→ 78%
• Sensitivity of TMA →→→→ 100%
• Specificity of SDA/TMA →→≥99.4%
• Rectal CT infections (68 infections detected)
• Sensitivity of culture →→→ 27%
• Sensitivity of SDA →→→→ 63%
• Sensitivity of TMA →→→→ 93%
• Specificity of SDA/TMA →→≥99.4%
Use of NAATS in Children – 2010
CDC STD Treatment Guidelines
• Recommend initial culture of the pharynx, anal canal, and
genital tracts for GC for both boys and girls
• Recommend CT cultures from the anal canal in boys and girls
and from the vagina in girls
• NAATS can be used for detection of GC and CT in vaginal
swabs and urine from girls being evaluated for suspected
sexual abuse
• NAATS not recommended for use in boys or for extragenital
infections in children, as there are no supporting data
• For cases of suspected sexual abuse, confirmatory testing by a
second NAAT should be performed
• Laboratories should use newer “second generation” NAATs with
the highest sensitivity possible, preferably with a different target
• Specimens should be retained for further testing
Diagnostic Issues Raised by Using
NAATS for Non-FDA-Cleared Sites
• Validity of NAAT results may be legally challenged
• Address concerns, in part, by collection of
concurrent/follow-up cultures
• Reported results should include the caveat that the
site of the specimen is not FDA-cleared (off-label)
• Under U.S. law, laboratories may offer testing for
extra-genital gonococcal or chlamydial infection if
“internal validation of the method by a verification
study” is performed
• Acquire site-specific
specimens from:
• Asymptomatic children
who disclose contact
• Asymptomatic children
with known/highly
suspected contact with
an infected individual
• Those with an
allegation of abuse
and symptoms of
infection
• All positive NAATs
confirmed by a second
NAAT and/or culture
Prepubertal
Non-Acute
Sexual Abuse
Pubertal NonAcute Sexual
Abuse
• American Academy of
Pediatrics recommends
use of urine-based and
vaginal swab NAATS
(oral/anal NAAT testing
not addressed)
• Site-specific specimens
to detect STI’s by NAAT
obtained at first visit
• Repeated two weeks
later
• All positive tests
confirmed by an
additional NAAT and/or
culture
Thank You!