# Presentation: 438691

## New Technologies in HIV and Related Diagnostics

- CDC/UVRI Research Collaboration

## De-tuned assays

- Prevalence and incidence
- Affinity maturation
- Normal and ‘de-tuned’ mode
- Incident and long-term infection
- Uganda’s experience

- CDC/UVRI Research Collaboration

## Monitoring Patients on ARVs – the Current Situation

- Cost for triple therapy  ~$800 pa
- CD4/VL costs ~ $500 pa
- Reduce drug costs still further
- Reduce costs of monitoring
- reduce frequency
- clinical monitoring
- new, cheaper technologies

- CDC/UVRI Research Collaboration

## CD4+ T Cell Counting– Available Methodologies

- Microscopy
- Dynabeads
- Cytospheres
- EIA
- Capcellia
- Flow Cytometry
- FACSCount
- FACScan

- CDC/UVRI Research Collaboration

## Dynabeads Protocol

- Sample preparation
- Monocyte depletion
- CD4/8+ T cell isolation
- Cell lysis
- Staining
- Counting under UV

- CDC/UVRI Research Collaboration

## Cytospheres Protocol

- Monocytes blocked with anti-CD14-coated latex spheres
- CD4+ lymphocytes bound to large latex spheres forming rosettes
- Red cell lysis and nuclear staining
- Rosettes counted in a haemocytometer under normal light

- CDC/UVRI Research Collaboration

## Capcellia Protocol

- CDC/UVRI Research Collaboration

## Flow Cytometry – Cost Constraints

**FACSCount**
- Instrument costs; US$ 33,000
- Reagent costs; ~$15 per test including controls, buffers, maintenance, peripherals and repeats
**FACScan**
- Instrument costs; ~US$ 75,000
- Reagent costs with calibration beads, inclusive ; ~$13

- CDC/UVRI Research Collaboration

## CD4+ T Cell Counts Technical Constraints

**FACSCount**
- Dedicated, reliable instrument
- Minimal infrastructure, training, maintenance
- Requires reliable electrical power
- Maximum of 30 specimens per day
**FACScan**
- Good infrastructure, trained staff, regular maintenance
- Suitable only for well-developed labs
- Maximum of ~100 specimens per day

- CDC/UVRI Research Collaboration

## Absolute CD4+ T cell Count by Flow Cytometry

- Flow cytometers count cells
- Flow cytometry gives only a relative count
- For absolute count, need volume of blood
- Volumetric flow cytometry
- Calibration beads
- Reference cell population

- CDC/UVRI Research Collaboration

## Dual-Platform Flow Cytometry

- Total number of lymphocytes from CBC
- %lymphs x WCC
- CD3+ cells on flow cytometer
- Total number of leucocytes from CBC
-  Panleucogating [PLG]
- WCC
- CD45+ cells on flow cytometer

- CDC/UVRI Research Collaboration

## Why DP – Flow Cytometry?

- Volumetric flow cytometers
- Expensive
- Cheaper versions not yet validated
- Calibration beads
- Double the cost/test
- Fixed amounts of reagents and blood
- DP – Flow cytometry
- Flexible amounts of blood and reagents

- CDC/UVRI Research Collaboration

## TriTEST: Reagent-Sparing Protocols

- CD4a: R2=0.974
- CD4b: R2=0.965
- CD4c: R2=0.962

- CDC/UVRI Research Collaboration

## Comparison of FACSCount Results with DP Lymphocyte or Panleucocyte Gating

- Pan*leuco*cyte:
- R2=0.889
- Lymphocyte:
- R2=0.796

- CDC/UVRI Research Collaboration

**Notes:**
2 spx with CD4>2000 removed

## Paired % Differences in Absolute CD4: FACSCount - DP Lymphocyte or Panleucogated

- CDC/UVRI Research Collaboration

**Notes:**
2 spx with WBC=750 – normal is>4000 FACSCount consistent with previous results
Other errors likely to be due to pipettingb

## CD4+ T Cell Monitoring: Reagent Costs

- FACSCount
- TriTEST with TRUCount
- TriTEST without TRUCount
- [full-volume]
- [half-volume]
- [quarter-volume]
- Pan-*leuco*gating
- * requires WBC/differential

- US$ 13.6
- US$ 10.8
- US$   7.2*
- US$   3.6*
- US$   1.8*
- US$    1.8*
- US$   0.8

- CDC/UVRI Research Collaboration

## Viral Load Monitoring

- Quantitative RT-PCR, bDNA, NASBA
- Real-Time PCR – Taqman
- Enhanced HIV p24 antigen assay
- Reverse transcriptase viral load assay
- AMP-RT
- PERT

- CDC/UVRI Research Collaboration

## Quantitative RT-PCR Cost and Technical Constraints

- COST
- Amplicor HIV-1 Monitor reagent costs for both manual and automatic versions ~US$ 100 per test including buffers, maintenance, peripherals and repeats
- New price structure in place shortly
- COMPLEXITY
- Requires highly trained staff, reliable power, excellent infrastructure and regular maintenance

- CDC/UVRI Research Collaboration

## Real-Time PCR – Taqman

- One-step rxn with modified RT & polymerase
- 90 specimens per assay
- 2-3 hour reaction
- Amplification and detection simultaneous
- RNA preparation is rate limiting step
- High initial investment
- Calibrator needed for absolute quantitation

- CDC/UVRI Research Collaboration

## Enhanced HIV p24 Antigen Assay

- Old assay
- Immune complexes/RF factors
- Acid-dissociation
- Enhanced assay
- Serum boiled
- Enhanced detection compensates for reduced sensitivity
- VL>500,000 copies/ml:	4/14 detected VL<500,000 copies/ml:	0/22 detected

- CDC/UVRI Research Collaboration

## eHIV p24 Ag Assay Results with Subtypes ‘A’ & ‘D’

- CDC/UVRI Research Collaboration

## Reverse Transcriptase Viral Load Assay 

- Viral particles purified by gel filtration
- Particles lysed in the gel and RT eluted
- RT activity measured by incorporation of BrdUTP into immobilized poly(rA).oligo(dT) using conjugated antibody to BrdU
- Excellent reproducibility [Pearson: 0.986]

- CDC/UVRI Research Collaboration

## Reverse Transcriptase AssayResults 1

- CDC/UVRI Research Collaboration

## Reverse Transcriptase AssayResults 2

- CDC/UVRI Research Collaboration

## Reverse Transcriptase AssayResults 3

- CDC/UVRI Research Collaboration