Advances of Basic

Sciences – Diagnostic &

Prognostic Application

Kamini Walia, PhD, MPH

Director, Research and Development
PATH-India kwalia@path.org
PATH Mission Statement

Our mission is to improve the health of

people around the world by:
 Advancing technologies
 Strengthening systems
 Encouraging healthy behaviors
PATH focus

 Solutions for emerging and epidemic diseases,

like AIDS, tuberculosis, and malaria.
 Safer childbirth and healthy children.
 Health technologies designed for low-resource
settings, by the people who will use them.
 The basic protection of vaccines for women
and children around the world.
 Development of diagnostics: lower cost, ease
of usage in field, neglected diseases
Importance of diagnostics

 Laboratory diagnosis of HIV infection is

fundamental for detecting and monitoring
infection, qualitative and quantitative
 Understanding of the virus and host response,
led to development of better diagnostic tools
 Limitations to implement these tests through
program, unmet need for new diagnostics,
Needs careful validation
HIV/AIDS situation in India: NACO

 Total no. of HIV-infected patients in India: 2.31

million
 Total no. of HIV-infected on ART: 2,52,336
 Total no. of ART centers: 228
 Regular clinical and lab-based monitoring of
the patient’s treatment status: CD4 counts,
viral load, drug resistance
Virological and serological markers during the first
weeks following infection with HIV-1.

HHRF 2011
Time of detection of specific markers of HIV infection, according
standardised, commercially available kits.

 1st generation:1985, employed

whole virus
 2nd generation: 1987,
employed recombinant
antigens and peptides
 3rd generation: 1994, addition
of new antigens, also HIV-2
 4th generation: antibodies, p24,
second diagnostic window,
 3rd & 4th:high sensitivity(99.5-
99.9%), low specificity

HHRF 2011
Serologic Tests

 Human immunodeficiency virus (HIV-1) antibody screening assays

• Enzyme-linked immunoabsorbent assays (ELISA)
• Rapid tests : based principles of 2nd or 3rd generation
– OraQuick ADVANCE (OraSure Technologies)
– Reveal Rapid HIV-1 Antibody Test (MedMira Laboratories)
– Uni-Gold Recombigen HIV Test (Trinity Biotech)
 HIV-1 confirmatory antibody assays
• Western blot (WB); detects envelope glycoproteins , Gag or pol
• Indirect immunofluorescence assay (IFA)
 HIV-2 antibody screening assays
• Combination ELISA

HHRF 2011
 Serologic testing algorithms for recent HIV
seroconversion(STARSH)
 Monitoring transmission patterns and guiding the interventions
to curb spread
 BED, Avidity, IDE-V3, Detuned assays
 Accuracy of STARSH

HHRF 2011
Serologic testing algorithms for recent HIV
seroconversion(STARSH)
 BED: a commercial enzyme immunoassay (EIA) anti-HIV IgG
present in a specimen relative to the total amount of IgG, which is
an indicator of disease progression and provides an estimate of the
time period since seroconversion.
 Uses synthetic oligopeptide derived from immunodominant region
of gp41 of HIV-1 subtypes B, CRF_01AE and D
 Avidity: modified ELISAs designed to investigate the maturity of
the anti-HIV antibody response by using a chaotropic agent
 IDE-V3 : uses two highly conserved immunogenic epitopes of Env
Glycoprotein
 Detuned assays: involve increasing the dilution of a known anti-
HIV antibody-positive sample and reducing the incubation time to
deliberately lower the sensitivity of an EIA.

HHRF 2011
Accuracy of STARSH

 Panels of specimens that may not be representative of the

population, have approximated seroconversion dates, and/or are
taken from patients who exhibit very different immunological
responses to HIV , may lead to lower sensitivities
 Sensitivity estimates ranged from 42 to 100% (89% overall
median) for incident infections and specificity estimates ranged
from 35 to 100% (98% overall median) for long-standing,
nonincident infections
 Need to establish standards for calculating cut off values,
algorithms for using multiple assays, framework for
comparing different assays

HHRF 2011
Challenges for diagnosis

 Quality assurance programs for evaluation

and monitoring in RLS
 Tests detect anti-HIV antibodies and are
suboptimal for infant and adult acute infection
testing.
 Accurately distinguishing between recent and
long-standing infections is important for
monitoring HIV transmission patterns, but
current incidence assays are prone to error.
Technologies for achieving sustained viral
load suppression

Patient
Treatment Advanced
monitoring Newer
Adherence diagnostics
antiretroviral
Improved s
patient
outcomes

HHRF 2011
Monitoring Tests
 Lymphocyte analysis (CD4 percentage)
 Viral load assays
• Reverse transcription-polymerase chain reaction (RT-PCR) (Roche Amplicor HIV-
1 Monitor and Roche Amplicor HIV-1 Monitor Ultrasensitive)
• Branched chain DNA (bDNA) (Versant HIV-1 RNA 3.0 assay)
• Nucleic acid sequence-based assays (NucliSens HIV-1 QT assay [bioMérieux])
 Drug resistance tests
• Genotypic assays
– INNO-LiPA HIV-1 RT (Innogenetic/Bayer Diagnostics)
– Trugene HIV-1 genotyping test (Visible Genetics)
– ViroSeq HIV-1 genotyping system (Applied Biosystems)
• Phenotypic assays
– Antivirogram (Tibotec-Vicro)
– PhenoSense (ViroLogics

HHRF 2011
CD4 and viral load estimations

 Important for staging, initiating and monitoring

treatment
 Current cost of CD4 count monitoring is $25 per test,
and the cost of viral load monitoring is $100 per test
 20 per cent of clients in the public sector reported
receiving CD4 count monitoring, and 40 per cent of
clients in the private sector
 239 labs for CD4 counts
 7 viral load labs,10 second line centres for the 3,000
patients

HHRF 2011
CD4 counting assays currently undergoing
evaluation, or already in use
Manual bead based technologies
 Dynal Manual Dynabeads Dynal® T4 Quant Kit
 Beckman Coulter cytosphere kit (Coulter® Manual CD4 Count Kit1-4)
Flow Cytometry systems that require minimal technical input
 Guava Easy Cd4223
 BD FACSCount
 • Point Care (no peer reviewed publications)
 • CD4 Select (uses same equipment as machinery for complete blood counts)224
Flow Cytometry based systems that require operator input/flow cytometry skill
 • Beckman Coluter flow cytometry(FL, USA) (FlowCare, PLG CD4)
 • Becton Dickinson, (Multiset, SP Tetraone)
 • Partec (CyFlow™, CyFlow SL Blue , CyFlow Counter®/ CyFLOW(Green)

HHRF 2011
Limitations of existing CD4 testing systems
 High capital and running costs
 unreliable electricity supply, lack of staff with sufficient education to
receive training, distance of health facilities from laboratories and lack of
reliable transport for materials
• A microchip-based assay: currently under development by LabNow.
• Zyomyx: quantitative biochip-based assay due for external validation early in
2009.
• Inverness/ClonDiag assay, uses a CCD imager to image CD4 cells isolated
in an array.
• BeckmanCoulter: semi-quantitative assay, three potential technology
strategies currently under investigation.
• Burnet Institute / Duke and Rush University: semi-quantitative lateral flow
device, final prototype due to enter clinical studies in early 2009.
• Cornell University: liposomal-based lateral flow assay; dye-carrying
liposomes that are stable at high temperatures will bind to CD4 receptors
within the assay..

HHRF 20 11
 A Microchip CD4 Counting Method for HIV
Monitoring in Resource-Poor Settings

 Based on a novel microchip detection system for measuring

various analytes in very small volumes.
 A series of chemical and immunological reactions carried out on
microspheres are visualized and captured on a charge-coupled
device (developed for digital camera technology).
 Allows for accurate measurement of CD4, CD8, and CD4/CD8.
 Shows extremely good agreement with currently used flow
cytometry.
 Most importantly, the investigators claim that the cost of each
assay is much lower than that for flow cytometry.

Rodriguez WR et al. A microchip CD4 counting method for HIV monitoring in resource-poor settings. PloS Medicine 2: e182,
2005.
POC CD4+ T-lymphocyte test developed
by PATH
 Minimally instrumented CD4+ purification technologies
and proprietary flow-through cassettes to measure cell
count status.
 Semiquantitatively assesses CD4+ lymphocyte cell
numbers as insufficient (0–250), borderline (250–350),
and sufficient (350 and above) using colorimetric
changes in a flow-through membrane.
 Completed in less than two hours and can be performed
by laboratory technicians with low levels of training in
developing countries.
 Next steps for this test include fabrication of a capillary
tube collection device, a card-based purification system,
and further optimization of the membrane-based signal
system.

HHRF 2011
POC for CD4 counts

 Low sensitivity;
 Unable to quantify moderate changes in CD4 count indicative of
treatment failure,
 Need to determine what sort of assays will be required in order to
support treatment switch decisions.
Further questions:
 How sensitive do point of care CD4 assays need to be? Are they
only needed for staging individuals for treatment initiation, or for
monitoring for treatment failure too?
 What is the relative need for point of care versus more sensitive
assays likely to be in the medium term

HHRF 2011
Viral load estimation

 Polymerase chain reaction(PCR): RNA or

DNA, targets gag
 Branched DNA (bDNA): targets pol
 Nucleic acid sequence based
amplification(NASBA)
 Ligase chain recation
 Real-time PCR
 Limitations: CRFs, HIV-2 infections

HHRF 2011
Current FDA-licensed assays
•Current assays require a high
level of training and laboratory
facilities, and are expensive,
making them unsuitable for use
in most resource-limited settings.
•WHO does not currently
recommend viral load testing as
a priority for implementation
• May be cost-effective in the
short term, it will be impossible to
detect early virologic failure
resulting in a future epidemic of
drug resistant HIV
 Roche COBASR Amplicor PCR
version 1.5 (using 3 different
formats: manual, manual extraction
and COBAS (comprehensive
bioanalytical system) amplification
and detection , COBAS
Ampliprep/COBAS Amplicor
(Roche Molecular Systems,NJ)
 Roche COBASR TaqmanR
 Bayer/Siemens Versant bDNA
 bioMerieux Nuclisens HIV-1 QT
assay (bioMerieux Inc,
Boxtel,Netherlands)
 Abbott RealTime
Alternatives to PCR are currently being used
or tested
 Ultrasensitive p24 heat-denatured antigen assay: can be
used for paediatric diagnosis, but still not validated for adult use;
runs on same platform as ELISA, high throughput.
 Reverse transcriptase measurement: Cavidi’s ExaVir assay is
already available at a discount through the Clinton Foundation;
takes three days to run a test, but simple equipment. Detection
limit of 2000 copies/ml231.
 Real-time PCR: Real-time assays are faster, have higher
throuputs, larger dynamic ranges and fully automated extraction
steps.

 Fiscus SA, Cheng B, Crowe SA. HIV-1 viral load assays for resource-limited settings. PLoS Medicine 3 (10): e107, 2006.
 Greengrass V., et al. Evaluation of the Cavidi ExaVir™ Load quantitative HIV RT load kit as an alternative HIV viral load monitoring
test for use in Melbourne. Fourth International AIDS Society Conference on HIV Treatment and Pathogenesis, Sydney, abstract
TUPEB019, 2007.
An ideal viral load test
 No more than a fingerstick’s worth of blood (about a tenth of a millilitre),
a single cartridge into which a sample of blood could be inserted and
which would give a result, that it should not require refrigeration, be able
to be run on batteries,
 should not cost more than $1000 per instrument and $8 per test, should
give a result within two hours
 would be able to be done by a field health worker with 1-2 days’ training.
Approaches to delivering a viable alternative include:
 SAMBA – nucleic acid-based test kit being developed by the University
of Cambridge and Diagnostics for the Real World. Being field tested in
Kenya
 Siemens (now owner of Bayer): battery operated, hand-held nucleic
acid-based testing device, being developed in collaboration with IAVI
and NIAID.
 University of Maryland: anti-p24 antibody capture with an amplification
step to improve on current ELISA-based p24 tests.
 Product concept
Sample processing device
Patient Specimen RNA Specimen
collection extraction preservation

Cold chain

Diagnostic Test

Central Laboratories and others

HHRF 2011
PATH sample processing technology

 Low cost, ease of use

 Efficient RNA extraction and elution
 Quantitative RNA stabilisation
 Eliminates need for cold storage reduces
cost of transport of specimens to the lab
 Detection at 500 copies/ml and above
with 200 l of sample
 Direct cost savings to health care system
 Staff requires additional training

HHRF 2011
Path sample processing technology

10 Specimen Batch PATH technology Current practices

Reagents (Home Brew):
Sample Preparation $3.16 $6.60
vRNA Stabilization $2.00 -
Avg Specimen Transportation Cost* - $3.13
Disposables
Sample Preparation $2.50 $6.95
Total Costs per Specimen $7.66 $16.68
Direct Material Cost Savings 54%
*Assumes all 10 specimens are shipped together
 LAMP based low cost viral load test
3).

Bioluminescent Assay in Real-Time

 BART quantitative detection of nucleic
acids during isothermal DNA
amplification without the need for
fluorescent reporters. 
 Generation of pyrophosphate coupled
to the emission of light from a highly
thermostable version of firefly
luciferase
 Light outputs that give both a
rapid increase and decrease in light
only when a target sequence is
amplified
 BART reagents have no adverse
affects on amplification

HHRF 2011
Monitoring Drug resistance

 Genotyping assays
 Ultra Deep Sequencing technologies
 Phenotyping assays: DR and Tropism
 Virtual phenotyping

HHRF 2011
HHRF 2011
Genotypic Assays

 Involve sequence analysis of the HIV-1 pol gene, which

contains the HIV-1 reverse-transcription and protease gene
sequences,
 Detect dominant mutants but not low frequency mutants
 Commercial assays incorporate software to analyze viral
sequences and algorithms that have been formulated by a
panel of experts
 1-2 years may elapse between the time of new data
becoming available and the information being incorporated
into the test interpretation
 complex mutation patterns of multidrug-resistant patients
difficult to interpret

Johnson VA, et al. Top HIV Med. 2008 Dec;16(5):138-45

HHRF 2011
Cell based Phenotypic assays
 Determine dug resistance and tropism
 Patient’s viral reverse-transcription and protease genes
incorporated into a laboratory virus strain grown in the presence of
varying concentrations of ARVs.
 Viral susceptibility are presented in the context of either biological
cutoffs or clinical cutoffs
 More useful for complex mutation patterns where genotypic
interpretation may be difficult.
 Phenotypic assays cannot specifically account for the combined
activity or lack of antiretroviral combinations and less predicitive
when mixed strains present

Qari Shet al.. J Clin Microbiol. 2002 Jan;40(1):31-5.

HHRF 2011
Virtual Phenotype

 A genotypic test for the HIV-1 that determines phenotype by using a

bioinformatics engine to match the patient’s viral sequence to sequences in
a database by linear regression modeling.
 The virtual phenotype provides clinical cutoffs 1 and 2 that determine a 20%
and 80% loss of antiretroviral activity
 Virtual phenotype results are highly concordant true phenotype results, and
randomized clinical trials have found similar virologic outcomes

 Winters B, Montaner J, Harrigan PR, Gazzard B, Pozniak A, Miller MD, Emery S, van Leth F, Robinson P,
Baxter JD, Perez-Elias M, Castor D, Hammer S, Rinehart A, Vermeiren H, Van Craenenbroeck E, Bacheler L. J
Acquir Immune Defic Syndr. 2008 May 1;48(1):26-34.
 Mazzotta F, Lo Caputo S, Torti C, Tinelli C, Pierotti P, Castelli F, Lazzarin A, Angarano G, Maserati R, Gianotti
N, Ladisa N, Quiros-Roldan E, Rinehart AR, Carosi G. J Acquir Immune Defic Syndr. 2003 Mar 1;32(3):268-80.
Hales G, Birch C, Crowe S, Workman C, Hoy JF, Law MG, Kelleher AD, Lincoln D, Emery S.PLoS Clin Trials.
2006 Jul 28;1(3):e18
Ultra deep sequencing

 Pyrosequencing, polymerase-based sequencing-by-synthesis and

ligase-based sequencing,
 Can generate data on hundreds of millions of base pairs
 Characterize genotypic diversity, analyze tropisms, and accurately
discern rare and drug-resistant strains circulating in the population
 Can detect mutations in 1-2% of populations
Tropism Testing
Standard Trofile assay[1]
 Proven negative predictive value
 Detects X4 virus with 100% accuracy
when X4 comprises ≥ 10% of viral
population Virus uses CCR5
coreceptors to enter the CD4+ cell
 Activity of CCR5 antagonist anticipated
Enhanced Trofile assay[2]
 Increased sensitivity by 10- to 100-fold
over standard Trofile assay
 Retrospective analysis of ACTG 5211
study of VCV: identified more patients
with D/M virus at screening vs
standardtropism assay

HHRF 2011
Resistance testing : a cornerstone of
antiretroviral therapy selection
Improving patient outcomes:
 Selection of the appropriate resistance test is based on the patient’s clinical
history and drug exposure
 Early use of tropism assays may select patients who are candidates for
CCR5 entry inhibitors before virus populations change to mixed tropism
Strengthening surveillance
 Ultra-deep sequencing technologies have the ability to detect drug-resistant
and low-frequency HIV strains.
 The WHO is leading an initiative to genotype and monitor drug resistance
in RLS
 Develop genotype panels, data bases testing protocols and research
agendas
 60 nations adopted this initiative hence presents an opportunity for
capturing data and minimize DR

HHRF 2011
Challenges

 Expanding genetic diversity raises concerns over the ability of

diagnostic assays to detect unique CRFs, target highly
conserved regions
 Development of an affordable point mutation assay which
screens only for specific mutations be a priority, and the
evaluation of affordable sequencing techniques
 OIs reduce the accuracy of HIV tests, creating a need for
diagnostics that can quickly and reliably account for these co-
infections
 Diagnostic tests that can distinguish between vaccine- and HIV-
induced anti-HIV antibodies will be needed in communities
where large-scale vaccine trials take place.
 Screening tests need to be more sensitive and have shorter
turnaround times to ensure organ and tissue transplants are free
of HIV.
HHRF 2011
Opportunities & future perspectives
 As the amount of sequence information and size of HIV repositories
increase, stakeholders will have better tools from which to develop
improved and more accurate diagnostic tests.
 Microfluidic lab-on-a-chip, label-free and antibody/antigen combination
platforms hold promise as the next generation of diagnostic tools.
 Nanoparticles used in place of fluorescence-based modalities can
improve detection sensitivities.
 Microarrays can address the need for multiplexed diagnostic tests, but
high-quality capture agents will be required.
 Developers and health officials should communicate to ensure
diagnostics have optimal usefulness in real-world settings and
researchers should focus on how to bring their novel discoveries to
market for actual use.