1. 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

2. 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

3. Typical Course of HIV Infection

4. HIV Markers Early in HIV Infection: Window Periods of Immunoassays (IA’s): First Through Fourth Generation

5. 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

6. 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

7. 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
Third generation
Use synthetic peptides or recombinant antigens
Detect both IgG- and IgM-class antibodies
Fourth generation
Detect IgG- and IgM-class antibodies
Detect p24 (core) antigen

8. 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)

9. Comparison of First (A) and Third-Generation (B) HIV EIA’s

10. p24 Antigen Detection
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

11. Fourth Generation HIV-1/2 EIA (courtesy, BioRad)

12. 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

13. Multispot HIV-1/HIV-2 Rapid Test (Bio-Rad)
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

14. Multi-Spot HIV-1/2 Rapid Test
1. Procedural control (goat anti-human IgG)
2. HIV-2 peptide
Peptide representing the immunodominant epitope of HIV-2 (gp36 envelope glycoprotein
3. Recombinant HIV-1 envelope glycoprotein
Recombinant gp41 expressed in E. coli (gp41 rDNA)
4. HIV-1 peptide
Peptide representing the immunodominant epitope of HIV-1 gp41 envelope glycoprotein

15. New CDC HIV-1/2 Testing Algorithm
A1: GS HIV Combo Ag/Ab EIA
A1+
A1(-)
Negative for HIV-1 and HIV-2 antibodies and p24 Ag A2
Multispot *
HIV-1 +
HIV-1 antibodies detected Initiate care (and viral load)
HIV HIV-2 antibodies detected Initiate care
HIV-1&2 (-)
NAAT
NAAT Acute HIV-1 infection Initiate care
NAAT (-) Negative for HIV-1
*Multispot does not have confirmatory claim
but must be used as a differentiating test

16. Multispot - Interpretation
Nonreactive →→
HIV-2 Reactive→
HIV-1 Reactive →
HIV Reactive →→
Undifferentiated
INVALID →→→→

17. Confirmatory Tests:Transcription-Mediated Amplification
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 (Gen-Probe) cleared for diagnostic use

18. Arizona DOHS/Maricopa Integrated Health Systems Study MMWR 2013;62:489-494
Screened all adult ED patients ages 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

19. 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

20. Implications for Rapid Point-of-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

21. FDA-Approved Rapid HIV Antibody Screening Tests
Specimen Type
CLIA Category
Sensitivity
Specificity
Manufacturer
OraQuick ADVANCE Rapid HIV-1/2 Antibody Test
Oral fluid
Waived
99.3%
99.8%
OraSure Technologies, Inc.
com
Whole Blood (f-stick, venipunct)
99.6%
100%
Plasma
Moderate Complexity
99.9%
Uni-Gold Recombigen HIV
Whole blood (f-stick, venipunct)
99.7%
Trinity Biotech
ww.unigoldhiv.com
Serum & Plasma
Reveal G-3 Rapid HIV-1 Antibody Test
Serum
Moderate complexity
99.1%
MedMira, Inc.
98.6%

22. FDA-Approved Rapid HIV Antibody Screening Tests
Specimen Type
CLIA Category*
Sensitivity
(95% CI)
Specificity
Manufacturer
MultiSpotHIV-1/HIV-2 Rapid Test
Serum
Moderate complexity
100%
99.93%
BioRad
Laboratories
com
Plasma
Moderate Complexity
99.91%
Clearview HIV 1/2 STAT-PAK
Whole Blood
(f-stick, venipunct)
Waived
99.7%
99.9%
Inverness Medical Professional Diagnostics
pd.com
Serum & Plasma
Non-waived
Clearview
COMPLETE HIV ½
Whole Blood (f-stick, venipunct)
99.90%

23. 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
75.8
Genetic Systems HIV-1/2+O 19
57.5
Multispot HIV-1/2 Rapid Test 11
33.3
Clearview Complete HIV-1/2 8 27
29.6
Unigold Recombingen HIV
24.2
Clearview HIV-1/2 Stat-Pak 7 31
22.6
Oroquick Advance HIV-1/2 32
21.9

24. 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

25. 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

26. Diagnosis of Syphilis 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

27. Lesions of Primary and Secondary Syphilis

28. 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

29. Serologic Diagnosis of Syphilis
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

30. Serologic Diagnosis of Syphilis
FTA-ABS
T. pallidum Particle Agglutination (TP-PA) /
T. pallidum HemAgglutination assays (TP-HA)

31. Serologic Diagnosis of Syphilis
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

32. Serologic Reactivity in Syphilis

33. Traditional Testing Algorithm for Diagnosis of Syphilis

34. Syphilis - Serologic Screening Algorithms – Reverse Sequence

35. 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

36. Syphilis Immunoassays - Timeline
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

37. Sensitivity and Specificity of Serologic Tests for Syphilis

38. Why Switch to EIA/CIA? Automated (high throughput) 180 tests per hour
Low cost in high-volume settings
Less lab occupational hazards
No manual pipetting
No false-negatives due to prozone reaction

39. Why Switch to EIA/CIA? Objective results
Some EIA/CIA test detect IgM antibodies
BioPlex 2200 assay
Potentially useful for diagnosis of early syphilis

40. 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

41. 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 non-treponemal antibody titer

42. 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

43. Reverse Sequence Algorithm for Syphilis
Composite results of reverse sequence algorithm for initial screening

44. 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

45. Nucleic Acid Amplification Tests for N
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
Becton-Dickinson
Strand displacement amplification
APTIMA
Hologic/
Gen-Probe
Transcription-mediated amplification
Real-Time m2000
Abbott Molecular Diagnostics
Real-time PCR

46. 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

47. Gene Targets and Cross-Reactivity 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 APTIMA 2
16S subunit of ribosomal RNA gene (multicopy)
None reported
BD Probe-Tec
Multi-copy pilin gene inverting protein homolog
N. meningitidis, N. lactamica, N. cinerea, N. mucosa, N. flavescens,
Abbott Real-Time PCR
Opa gene (multicopy)

48. 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: , 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: , 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

49. 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

50. 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
65.4%
99.0%
91.9%
71.8%
97.1%
94.2%
100%
96.2%

51. 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
Two positive NAATS, OR

52. 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%

53. 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%

54. 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

55. 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

56. Prepubertal Non-Acute Sexual Abuse
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

57. Pubertal Non-Acute 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

58. Thank You!