HIV Diagnostics: New Developments and Challenges

Orlando, Florida

Feb 28 - Mar 1, 2005

Dried Blood Spots in HIV and HCV Epidemiology and Drug Resistance Testing

National HIV & Retrovirology Labs

Public Health Agency of Canada

Ottawa, Ontario

CANADA

Dried Blood Spots (DBS)

• use in HIV serological and nucleic acid testing well-documented & validated

• simple, robust, inexpensive

• two approved EIA tests (GSrLAV & OT Vironostika)

• surveillance, diagnostic, clinical care management

DBS - Collection

Good ! Baaad !

Dried Blood Spots (DBS)(antibody elution-GSrLAV EIA)

1. 1/4" disk is punched into an uncoated plate.

2. 200uL of specimen diluent (normal bovine serum / 0.1% Proclin added and mixed well.

3. eluted o/n at 40 C.

4. next day samples are brought to RT and mixed well.

5. Mix 40uLsample into 60uL sp.diluent into coated plate.

Rest of assay as per norm.

CBC HIV-1 Western Blot INNO-LIA HIV

Figure 1. Performance of Cameroon DBS on Confirmatory Tests

HIV testing AlgorithmDBS

HIV-1 screen test(bioMerieux Vironostika HIV-1 EIA)

Non-reactive Low Reactive

s/co<3

Report Negative Repeat test

Reactive

HIV-1 Western Blot(Bio-Rad GS WB)

Non-Reactive

Reactive

Positive

Negative

Report Positive

Hepatitis C testing Algorithm(DBS)

HCV screen test(Ortho HCV EIA)

Non-reactive Low Reactive

s/co<3

Report Negative Repeat screen test

Reactive

Supplementary screen Test(Innogenetics HCV Inno-test)

Reactive

Non-reactive

Report Positive

Applications of DBS in NHRL

• Diagnostics

• Surveillance (Prevalence/Incidence)

• V.I.D.U.S. (Vancouver Intrav Drug User Study)

• I-Trak

• M-Trak (MSM in Montreal)

• International

- Kosovo

- Pakistan

Molecular Analysis of HIV & HCV from DBS

Wash/EluteDBS

Purified RNA

Elute

Bind to silica

RT-PCR Sequencing

HIV

HCV

Sample ID NLHRS# cp/ml Subtype

CN008 00-0781 73 000 A (HIV-1)CN009 00-0782 210 000 A (HIV-1)CN010 00-0783 110 000 A (HIV-1)CN016 00-0785 220 000 A (HIV-1)CN014 00-0794 1 100 000 D (HIV-1)

Table 3. HIV-1 Subtyping Data(C2-V3 env gene) Cameroon DBS

Figure 2. HIV-1 Drug Resistance Genotyping on Cameroon DBS (Visible Genetics) and Subtyping (inset)

HCV-RT-PCR/Sequencing

Core5’NCR

PCRRT

Phylogenetic comparison(LANL HCV Sequence Database)

Sequencing

HCV-Genotype Distribution(I-TRAK)

I-Track HCV Core Genotypes

72%

21%

3%

4%0%

1a

3a

1b

2a

2b

HCV 5'NCR Genotype

42%

25%

17%

4% 2% 1% 1%2%

2%

4%

1a

3a

1b

1b/1a

2a/1a2b

1c/1a

3b

5a

6b/h/k

• high HCV 3a

• high-risk vs low risk populations ?

(26,500) (154,300) (31,000) (16,600) (461,200) Viral Load

+ + + + + + + - - + - + + - +

In House (Nested PCR)

Trugene

(Bayer)

ViroSeq

(ABI)

Performance of HIV DR Methods(DBS-RNA)

+ RNase+ DNase

Nature of Nucleic Acid from DBS Extraction

FTA

903WEEK2 WEEK3 WEEK4 WEEK5

20°C 4 °C -20°C -80°C

WEEK2 WEEK3 WEEK4 WEEK5 20°C 4 °C -20°C -80°C

DBS – Viral Load 490,000

FTA

903

WEEK2 WEEK3 WEEK4 WEEK5 20°C 4 °C -20°C -80°C

DBS – Viral Load 20,000

WEEK2 WEEK3 WEEK4 WEEK5 20°C 4 °C -20°C -80°C

Effect of Storage Temperature -DBS

DBS2DBS100

1

2

3

PCR AMP

TIME

Sample.3 .6 .9 1 2Time (weeks)

PCRAmplification

Effect of HIGH Humidity/Temp – 903 Paper (45% Relative Humidity and 37°C)

DBS2

DBS1000.5

11.5

2

2.5

3

PCR AMP

TIME

Sample

PCRAmplification

.3 .6 .9 1 2Time (weeks)

Effect of HIGH Humidity/Temp – FTA Paper (45% Relative Humidity and 37°C)

# S

ucc

essf

ul A

mp

lific

ati

on

s

(n=

5)

Day 1Day 3

Day 6Day 9

Day 14

0

1

2

3

4

5

Time

Stabilization of DBS-RNA by RNAlater(85% Relative Humidity and 37°C)

903 untreated/no desiccant 903 untreated/dessicant

903 treated/no desiccant 903 treated/desiccant

Conclusion/Future Activities - DBS

• HIV and HCV serology and NA testing• Clinical monitoring – viral load/DR

testing• Venipuncture vs DBS sequences• Limits of DBS to ‘extreme’ conditions

“DBS are economical, easy to collect, transport and store. Their ease of use and versatility make DBS an ideal tool for large scale surveillance studies, both domestically and abroad”

I-TrakEnhanced surveillance to track HIV- and HCV- associated risk

behaviours in injecting drug users (IDU).

• Cross-sectional design• Interviewer administered questionnaire• Information collected on:

Demographics Injecting and non-injecting substance use Injecting, sexual, testing behaviours

• DBS collected for HIV & HCV serological testing To describe changing patterns in the prevalence of HIV

and HCV at the national and local level.

Conclusions1. I-Track found 21% of infections involving genotype 3, whereas the

database estimates for Canada and the US were 3% and 4% respectively, and Bernier et al. found 14% in Montreal. Bernier et al. also found about 30% each 1a and 1b, whereas I-Track was 72% 1a. This suggests that the observed HCV genotype distribution may be unique to IDU when compared to the general population.

2. Although the numbers are still too low to permit complete statistical interpretation, preliminary univariate analysis suggests that the emergence of genotypes other than 1a in IDU is a relatively new phenomenon.

3. Linking surveillance data to findings of molecular analysis (regional clusters, genotype distribution) will lead to more targeted approaches to prevention, helping those who are most susceptible.

4. DBS are economical, easy to collect, transport and store, and produced quality HCV sequence data for this study. Their ease of use and versatility make DBS an ideal tool for large scale surveillance studies, both here and abroad.

5. HIV sent in about 10% HCV infected.

HIV RNA appears to be preferentially amplified (consistent with plasma)

Commercial sequencing kits are compatible although lack of secondary PCR may be problematic for low viral loads

Similar performance between FTA and 903 under “ideal” conditions

Poorer performance for FTA under elevated temperatures and humidity

Humidity is detrimental to recovery (desiccant & suitable storage pouches)

Improved recovery by pre-treatment of membrane with RNA stabilizer

Conclusions

Future WorkQuantitate HIV RNA on DBS by real time PCR

Establish consensus between DBS and plasma derived sequences

Genotype Distribution

• Genotype 1a is the most prevalent genotype across all sites, followed by 3a (based on core region).

• Other genotypes (non-1a/3a) make up less than 8% of the HCV prevalence among I-Track participants.

• There was a large regional variation in genotype distribution, although the numbers are still too low to permit complete statistical interpretation.

What Next?

• Need to address differences observed between the core and 5’NCR genetic regions through analysis of other genetic regions (E1, NS5B).

• The phylogenetic information obtained was often insufficient to achieve statistical significance of clusters. Phylogenetic analysis will be repeated with the inclusion of additional regions as they become available.

• About 10% of HCV I-Track participants were co-infected with HIV. HIV sequencing (from HCV extracts) is currently underway to characterize the co-infections using phylogenetic methods.

Study Design

A laboratory investigation of factors influencing the durability of DBS

particularly under extreme conditions.

Type of membraneViral LoadStorage TemperatureDuration of StorageHumiditySequencing TechnologyNature of viral target found on DBS

(RNA/DNA)

Methods

• Blood samples were collected from HIV +ve patients (with established viral loads) in 10 ml EDTA vacutainers

• DBS were prepared by spotting 50 l of blood (903 cards) or 200 l of blood (FTA cards)

• Viral RNA was extracted using Nuclisens extraction system

• DBS (903) or ¼ DBS (FTA) were cut into four equal strips and lysis buffer added.

Methods

• RT-PCR was performed using 10 l of isolated viral RNA with “One Step RT-PCR” (Qiagen)

• Secondary PCR was performed using 4 l of RT-PCR amplicon with “Platinum PCR Supermix” (Invitrogen)

• Secondary amplicons were sequenced on a LiCor 4200 sequencer using simultaneous bidirectional sequencing following manufactures suggested protocol.

• Primers (two sets)• PR1Forward/RT Protease Inner Reverse (2171-2188 to 2901-

2925)• RT Outer Forward/3’ Half (2813-2836 to 3506-3536)