New Approaches for Infectious Diseases Testing in Clinical Laboratories

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

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 AlgorithmIncludes 3 categories of tests: 4th generation HIV-1/2 Ab and Ag combo assayHIV-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 immunodominantepitope 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 immunodominantepitope of HIV-1 gp41 envelope glycoprotein

15. New CDC HIV-1/2 Testing Algorithm A1: GS HIV Combo Ag/Ab EIA A1(-) Negative for HIV-1 and HIV-2 antibodies and p24 Ag A1+ A2 Multispot * HIV-1&2 (-) HIV-2 +HIV-2 antibodies detected Initiate care HIV-1 + HIV-1 antibodies detected Initiate care (and viral load) 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 StudyMMWR 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

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

22. FDA-Approved Rapid HIV Antibody Screening Tests

23. Sensitivity for Early HIV Infection of Rapid HIV Tests Compared with 3rd and 4th Generation Assays J ClinVirol 2012;54:42-47

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 • Treponemapallidumcannot 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. pallidumproteins

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 • Treponemapallidumparticle agglutination (TP-PA) test

30. Serologic Diagnosis of Syphilis FTA-ABS T. pallidumParticle Agglutination (TP-PA) / T. pallidumHemAgglutination 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

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

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. gonorrhoeae and Chlamydia trachomatis

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 commensalNeisseria 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

48. NAATS for Detection f N. gonorrhoeaein 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

49. Use of NAATS for Oropharyngeal GCBachmann 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 GCBachmann et al, JCM 42:902-907, 2009