UNITAID-HIV Diagnostic Landscape-3rd Edition PDF
UNITAID-HIV Diagnostic Landscape-3rd Edition PDF
UNITAID-HIV Diagnostic Landscape-3rd Edition PDF
hiv/aids
Diagnostic Technology Landscape
3rd Edition
june 2013
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The designations employed and the presentation of the material in this publication do not imply the expression of any
opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory,
city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. The mention of specific
companies or of certain manufacturers’ products does not imply that they are endorsed or recommended by the World
Health Organization in preference to others of a similar nature that are not mentioned. All reasonable precautions have
been taken by the World Health Organization to verify the information contained in this publication. However, the
published material is being distributed without warranty of any kind either expressed or implied. The responsibility and
use of the material lies with the reader. In no event shall the World Health Organization be liable for damages arising from
its use.
This report was prepared by Maurine Murtagh with support from UNITAID. All reasonable precautions have been taken
by the author to verify the information contained in this publication. However, the published material is being distributed
without warranty of any kind, either expressed or implied. The responsibility for the interpretation and use of the material
lies with the reader. In no event shall UNITAID or the World Health Organization be liable for damages arising from its use.
This printing (August 2013) includes revisions from an earlier printing of this report (June 2013) as noted on pages 121-126.
ii
TABLE OF CONTENTS
List of Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iv
Executive Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
CD4+ T-Cell Counting Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Existing CD4 Technologies/Platforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Conclusions—CD4 Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Viral Load Technologies and Future Directions for Viral Load Testing . . . . . . . . . . . . . . . . . . . 71
What Should the HIV Diagnostic Landscape Look Like Going Forward? . . . . . . . . . . . . . . . . . . 72
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
List of Abbreviations
AIDS a cquired immunodeficiency MRSA ethicillin-resistant staphylococcus
m
syndrome aureus
ALT alanine aminotransferase MSF Médicens Sans Frontières
ART antiretroviral therapy MTB mycobacterium tuberculosis
AZT zidovudine NASBA nucleic acid sequence-based
BART Bioluminescent Assay in Real-Time amplification
bDNA branched chain deoxyribonucleic NAT nucleic acid-based test
acid NIAID National Institute of Allergy and
CAP COBAS® AmpliPrep Infectious Diseases
CDC Centers for Disease Control and NIH National Institutes of Health
Prevention NWGHF Northwestern Global Health
CRF circulating recombinant forms Foundation
CROI Conference on Retroviruses and PARC Palo Alto Research Center
Opportunistic Infections PCR polymerase chain reaction
CTM COBAS® TaqMan® PEPFAR President’s Emergency Plan for AIDS
DBS dried blood spot Relief
DDU diagnostics development unit PMT photo-multiplying tubes
DNA deoxyribonucleic acid PMTCT prevention of mother-to-child
transmission
DSP digital signal processing
POC point of care
EDTA ethylenediaminetetraacetic acid
QC quality control
EID early infant diagnosis
RIF resistance to rifampicin
ELISA enzyme-linked immunosorbent
assay RNA ribonucleic acid
EQA external quality assurance RPA recombinase polymerase
amplification
FDA Food and Drug Administration
RT reverse transcriptase
FIND Foundation for Innovative New
Diagnostics RUO research use only
FSC forward scatter channel SAMBA simple amplification based assay
GBS group B streptococcus SBIR Small Business Innovation Research
GSK GlaxoSmithKline SMS short message service
GSM Global System for Mobile SSC side scatter channel
Communications TDF tenofovir
HBDC high-burden developing countries UPS uninterruptible power supply
HDA helicase-dependent amplification URS unitized reagent strip
HIV human immunodeficiency virus USAID United States Agency for
HSV herpes simplex virus International Development
iNAAT isothermal nucleic acid amplification USB universal serial bus
test VCT voluntary counseling and testing
IVD in vitro diagnostic WBC white blood cell
LAMP loop-mediated amplification WHO World Health Organization
LTR long terminal repeat
iv
HIV/AIDS DIAGNOSTIC LANDSCAPE
Executive Summary
There is growing demand within the global health community to find ways to simplify and improve the effi-
ciency of diagnostics for HIV/AIDS without diminishing the quality of patient care. At the same time, there is a
need to significantly increase the level of access to robust, high-quality diagnostics in resource-limited settings
in order to facilitate early detection and treatment of HIV/AIDS.
Of the various tests required for initial diagnosis, staging, and ongoing monitoring of HIV, those that present
the most persistent challenges to improved access and efficiency are CD4, viral load, and early infant diagnosis
(EID). This report reviews both current diagnostic platforms and pipeline technologies for these three key tests.
For each, the great majority of testing options available today are laboratory-based platforms performed on
sophisticated instrumentation requiring dedicated laboratory space and trained laboratory technicians. In many
cases, laboratory-based testing is expensive; in almost all cases, it requires sample transport networks to enable
access for patients in peri-urban and rural settings.
Given the limitations of laboratory-based testing, it is generally accepted that in order to improve access to,
and reduce the cost of, CD4, viral load, and EID testing in resource-limited settings, such testing needs to be
brought closer to the point of patient care. This report therefore examines the new diagnostic technologies in
the pipeline—most of which are designed for use at or near the point of patient care—and considers to what
degree they meet the World Health Organization’s (WHO’s) “ASSURED” criteria, meaning that they are (or will
be): Affordable, Sensitive, Specific, User-friendly, Robust/Rapid, Equipment-free, and Deliverable to those who
need the test.
With respect to CD4 testing, which is used for staging and monitoring HIV patients prior to initiation onto
antiretroviral therapy (ART), the general conclusion is that currently there are a number of good laboratory-
based platforms using proven flow cytometry technology. These tests can be efficient and cost-effective when
performed by well-trained laboratory technicians and when combined with good sample transport systems.
However, in order to improve access, especially for rural patients, and to reduce patient loss to follow-up, there
remains a need for high-quality, cost-effective point of care (POC) CD4 testing options. Three such options are
already on the market, and several others are under development with anticipated release over the coming two
years. At least one of these will be a disposable POC CD4 test. Assuming that the performance of these POC
tests stands up to robust evaluation, the pipeline presents real promise.
With respect to viral load testing, which is primarily used for monitoring HIV patients following initiation onto
ART, there are also a good number of sophisticated laboratory-based platforms on the market. However, despite
the clinical consensus on the importance of viral load testing for detecting virological failure, access is very
limited in resource-limited settings, with a few exceptions, including South Africa and Brazil. Factors restricting
access include the need for sophisticated laboratory capacity and instrumentation, along with training for labo-
ratory technicians and well-functioning sample transport networks. In addition, the cost of viral load testing is
considerably higher than CD4. Viral load testing that could be conducted at the point of patient care with assays
meeting the ASSURED criteria would reduce the need for infrastructure and training, and could also lower the
Technical Report 1
2013 HIV/AIDS Diagnostic Technology Landscape
cost of testing. Although there is currently only one POC viral load assay on the market in limited release, there
are a number of platforms and assays in development, at least one of which may come to market in 2013.
Finally, with respect to testing for infants under 18 months of age, the most widely-used test for EID is a DNA
PCR molecular test, which is also performed on sophisticated laboratory-based instruments. Alternatively, EID
can be performed on viral load platforms. The DNA PCR test is subject to some of the same drawbacks and limi-
tations as viral load testing with respect to implementation in resource-limited settings. However, the cost of EID
testing has come down, sample transport networks have been developed, and EID training has been implement-
ed with funding from UNITAID and support from its implementing partners. As a result of these improvements
and the urgent need for infant testing, there has been considerable uptake of EID. Access is far from universal,
however, and the availability of EID at or near the point of care could improve access in harder-to-reach areas,
decrease patient loss to follow-up, and bring down the cost of testing. Because viral load platforms can be used
for EID, the new technologies in this testing area are viable options as well. In addition, there are at least two
POC assays being developed specifically for EID. At least one of these may be launched in 2013.
Advances in access to tests for infant diagnosis, as well as for ART staging and monitoring are needed in
resource-limited settings, and new technologies in the pipeline are likely to bring about significant changes
in how these tests are delivered. At the same time, new platforms for high-volume testing are also becoming
available, allowing cost-effective consolidation of testing in high volume centers (e.g., super-labs). Innovation is
ongoing as well to facilitate the use of open polyvalent platforms, which can be more adapted to medium-size
facilities in resource-limited settings.
The level of CD4, viral load, and EID testing required in resource-limited settings over the coming years will
likely necessitate scale-up in centralized testing facilities, including, in some cases, super-labs. At the same
time, increased demand will require POC testing to improve access, especially for hard-to-reach populations.
The appropriate mix of high-volume laboratories and POC testing will be country-specific, and will depend on
such factors as the urban/rural split of the country, the expected volume of each category of testing, and the
ability to effectively transport samples between collection sites and laboratories and ensure the efficient return
of laboratory results back to collection sites. Realistically, it will also depend on the comparative all-in cost of
centralized versus decentralized testing. Ultimately, the landscape for HIV/AIDS diagnostics in resource-limited
settings is unlikely to be either all laboratory-based or all POC.
Determining the optimal mix of centralized, high-volume diagnostics and POC diagnostics based on each coun-
try’s unique needs is a challenge, but is central to ensuring efficient access to quality HIV diagnostic services in
resource-limited settings. Strategic funding from UNITAID and others can help countries meet these challenges
and accelerate the introduction of new diagnostic technologies, especially those designed for use at or near the
point of care.
Introduction
In the interest of improving the accessibility and affordability of high-quality antiretroviral therapy (ART), there
is a growing demand for simple, affordable, reliable, and quality-assured POC diagnostics for use in resource-
limited settings. Many contend that POC diagnostics can make ART more scalable and will allow ART service
delivery to be significantly decentralized to the community level. At the same time, simplifying diagnostic tech-
nologies may reduce the cost of diagnosing and monitoring HIV/AIDS patients without diminishing the quality
of care.
In order to understand the benefits POC diagnostics may offer, it is necessary to understand the current diag-
nostic technology landscape. With an eye to maintaining high standards of patient care, it is also important to
consider the future landscape of HIV diagnostics and what efficiencies might be achieved with respect to test
algorithms, the cost of testing and decentralized service delivery, especially with respect to the introduction of
diagnostics performed at the point of patient care.
The initial hypothesis is that there is a need to significantly increase the level of access to robust, high-quality
diagnostics in resource-limited settings because access to testing is crucial in facilitating early detection and
treatment of HIV/AIDS. This, in turn, will maximize the preventive impact of ART, and will help to ensure
an appropriate and rapid response to drug resistance—a problem likely to grow substantially over the com-
2
Overview
countries that combines sophisticated, high-volume, low unit-cost laboratories in high-density areas, and lower-
volume, simpler, POC or near-POC platforms in less densely populated regions. However, the best technology
mix is unclear in most countries and new models for delivery may also emerge. For now, it is essential that
stakeholders, including ministries of health, UNITAID and other funders, trying to determine the most appro-
priate mix of investments to improve access to HIV diagnostics in resource-limited settings understand current
diagnostic technologies and the pipeline for new products.
This report reviews the current technology landscape for HIV diagnostics, including (i) the algorithms and tests
required in HIV/AIDS care and treatment, both before and after treatment initiation; (ii) the platforms used and
price points of that testing; and (iii) the ways in which testing is delivered. With this information as background,
the report then reviews the current technologies and diagnostic platforms in three key testing areas: CD4 and
viral load testing for adults and children, as well as EID (including EID run on viral load platforms)—all of
which are today typically accessed through sophisticated laboratory-based testing platforms, even in resource-
limited settings. The report describes the POC and near-POC CD4, viral load, and EID platforms on the market
and in the development pipeline, and considers the implications of the landscape, including what efficiencies
might be achieved with respect to test algorithms, the cost of testing and decentralized service delivery.
Methodology
The HIV/AIDS Diagnostic Landscape is compiled by Maurine M. Murtagh with support from UNITAID. The ma-
terial in this landscape was gathered by the author from publicly available information, published and unpub-
lished reports and prospectuses, and interviews with developers and manufacturers. The prices for diagnostic
equipment and reagents cited in this report were obtained directly from manufacturers and are ex works prices,
meaning that they are the prices at the manufacturer’s factory, and do not include any delivery, distribution or
commission charges. The material is current through April 30, 2013.
Overview1
Diagnostics for HIV/AIDS can generally be divided into three test categories: (i) tests to facilitate initial diagno-
sis, (ii) tests to stage the patient, and (iii) tests to monitor the patient, both before and after initiation of ART.
There are generally accepted algorithms and tests used at each stage as discussed below [1].
HIV disease involves a continuum of progressive damage to the immune system from the time of infection
to the manifestation of significant immunologic damage by various opportunistic infections, wasting, or CD4
lymphocyte count that marks the development of full-blown AIDS [2]. The period of time from infection to
the development of AIDS, known as the incubation period, can vary significantly from person to person. It is
generally quite long (i.e., a number of years) as compared to the short period (i.e., days or weeks) common to
many other viral infections (e.g., the common cold or influenza) [3]. A typical, but approximate, clinical disease
progression showing the relationship between the levels of HIV (viral load) and CD4+ T cell counts over the
usual course of untreated HIV infection is presented below [4].
1 This section owes much of its content to data originally gathered by the author and the laboratory services team of the Clinton Health Access Initiative (CHAI) in 2010 and subsequently
updated by the author. Portions of this overview are drawn from an unpublished report, entitled “ART 2.0 – Implications for Diagnostics in Resource-Limited Settings,” co-authored with Dr. Trevor
F. Peter of CHAI. Additional sources of information are referenced in the text.
Technical Report 3
2013 HIV/AIDS Diagnostic Technology Landscape
Adapted from Pantaleo G, Graziosi C and Fauci AS. New concepts in the immunopathogenesis of human immunodeficiency virus infection [4].
HIV infection is generally characterized by a spike in HIV antigens during the first few weeks after infection.
Subsequent to that early period of acute infection, antibodies produced as a result of HIV infection appear and
are then present throughout the course of the disease. The detection of these antibodies to HIV is the most com-
mon means to identify the infection, and HIV rapid tests for initial diagnosis of infection target this antibody
response.
The extended incubation period of AIDS means that laboratory tests are required to identify persons at high risk
of disease progression in order to guide clinical decision-making in asymptomatic seropositive patients, such as
when to initiate ART. Because depletion of CD4+ T lymphocytes is the hallmark and the apparent source of the
central immune defect of HIV disease, determination of the CD4 lymphocyte count (or percentage) has been the
most important laboratory marker of disease progression [1].
Tracking the course of the HIV virus itself by accurate measurement of the quantity of viral ribonucleic acid
(RNA) in the patient’s plasma has become as important a laboratory marker as CD4 lymphocyte count and is
considered the best marker to use for ART decision-making after initiation of therapy [1]. The measurement of
the number of viral copies per milliliter of plasma (commonly known as “viral load”) provides a clinically use-
ful range of values that can indicate the effectiveness of ART in HIV disease.
2 Some countries run the screening tests in parallel and all patients will therefore get two tests; most countries run the screening tests serially as described. In addition, some countries only use
ELISA tests for initiation screening (e.g., Vietnam) and, as indicated, some still use an ELISA test for confirmatory testing.
3 Some countries use an ELISA test in the case of discordant results.
4
Overview
HIV rapid tests generally come in the form of lateral flow strips or cassettes, which are convenient, self-con-
tained tools for HIV serologic testing. They are relatively easy-to-use, can usually be performed on fingerstick
blood, contain built-in quality controls, and can be administered by technicians and non-technicians alike,
including community health workers. Further, as a rule, tests can be completed in less than 10 - 25 minutes. The
cost of these HIV rapid antibody tests in resource-limited settings, excluding any distributor mark-ups, ranges
from about $0.50 per test to about $1.60 per test4 for blood-based tests, but can be as much as $5.00 per test
for saliva-based tests. ELISA testing is laboratory-based and generally costs $1.50 to $2.00 per test, including
consumables, but is no longer widely used for HIV screening.
Because of the persistence of maternal antibodies in infants under the age of 18 months, the use of antibody
tests, like commercially-available HIV rapid disposable tests, cannot be used to accurately screen infants for
HIV/AIDS. Instead, DNA PCR or RNA PCR testing (i.e., virological testing), which detects the genetic material
of HIV, should be used to determine the HIV status of infants in that age group.5
The most widely-used test for EID is the DNA PCR molecular test. It is also possible to use RNA detection
methods (e.g., viral load) or p24 testing6 for this purpose, but these methods are used in very few settings. In
either case, the test itself is laboratory-based and requires relatively sophisticated instrumentation and a trained
laboratory technician. In order to reach the broader population, blood collection for the DNA PCR test has been
decentralized to clinics, prevention of mother-to-child transmission (PMTCT) centers and the like. The infant’s
blood is collected on filter paper (known as dried blood spots [DBS]), which is transferred via couriers to the
laboratory for testing, and test results are then returned to the clinic or other collection site for dissemination
to caregivers. Because this process can sometimes be slow, especially the return of results from laboratories,
some countries have introduced short message service (SMS) printers (or other mobile technologies) in order to
achieve markedly improved turnaround time for return of results from laboratory to collection sites.
DNA PCR testing can be run on either low-throughput or high-throughput instruments according to the needs
in a given setting. The cost of a single instrument platform and related equipment (e.g., centrifuge, bio-safety
cabinet, freezer, etc.) can range from about $100,000 to more than $200,000, depending on the throughput
of the platform. The cost of the test itself ranges from about $10.00 per test on low-throughput platforms to
about $12.00 to $20.00 per test on high-throughput platforms. This cost covers the test reagents and associated
supplier-provided, non-commodity consumables only and does not include DBS collection supplies, which cost
from about $1.40 per test to about $2.75 per test depending on bundle configuration. It also does not include
more general laboratory consumables (e.g., gloves, pipettes, etc.), which cost from about $0.35 per test to $4.00
per test depending on the instrument platform chosen.
Patient Staging
Once an adult is diagnosed as HIV positive, CD4 testing is used together with clinical staging to determine
whether the patient is eligible for treatment [6]. After a primary HIV infection, the virus directly attacks CD4 T
lymphocyte cells (which effectively coordinate the body’s immune response), and begins to destroy them while
at the same time using them as host cells for replication. Billions of CD4 T lymphocytes may be destroyed each
day, eventually overwhelming the immune system’s ability to regenerate such cells. In HIV-infected adults, the
measure of an individual’s CD4 T lymphocytes, or absolute CD4 count, is the most robust surrogate marker for
immune competence [7]; for children under 5 years of age, the %CD4 measure is considered more reliable.7
Clinicians therefore seek to routinely test an individual’s CD4 count in order to monitor disease progression and
to determine when an individual should be initiated onto ART. Per the WHO 2010 Guidelines on ART initiation,8
if the absolute CD4 count of an adult or a child over 5 years of age is below a defined threshold (currently
4 In this report, the fully-loaded cost of testing, including the cost of human resources and overhead associated with testing, is not considered. These costs can vary considerably from
country-to-country. Also, none of the cost data discussed includes distributor mark-ups, which can range from a low of 5% of the cost of the test to as much as 30% of the cost of the test or more,
nor does the data include freight, insurance, taxes or other such ancillary charges, again which vary country-to-country. As indicated earlier herein, costs for instruments and reagents in this
document are ex works pricing, unless otherwise noted. With respect to distributor costs, it is important to keep in mind that for platforms based on laboratory instruments, distributors play an
important role in service and maintenance of the instruments, and in managing the supply chain. The distributor margin covers most of this cost. However, for disposable tests (e.g., HIV rapid
tests and some POC tests being developed), there is no instrument and the margin is used to cover the costs of importation, storage and handling.
5 Per the World Health Organization (WHO) 2010 Guidelines on ART initiation for infants and children (the “WHO 2010 Guidelines”): “It is strongly recommended that HIV virological testing be
used to diagnose HIV infection in infants and children less than 18 months of age.”
6 Viral load testing is discussed in more detail below in connection with monitoring the HIV+ patient who is on treatment.
7 The absolute CD4 cell count of healthy infants who are not infected with HIV is considerably higher than in adults who are HIV negative. These cells slowly decline to adult levels by the age of
about 6 years. Percentage CD4+ T-cell values vary less with age. Per the WHO 2010 Guidelines, relative to the measurement of absolute CD4 count, “the measurement of %CD4+ T-cell is thought
to be more valuable in children under 5 years of age.” [5]
8 It should be noted that the WHO is currently in the process of updating its guidelines on ART initiation and monitoring. The WHO 2013 guidelines are expected to be announced at the
meeting of the International AIDS Society in Kuala Lumpur in June 2013.
Technical Report 5
2013 HIV/AIDS Diagnostic Technology Landscape
less than or equal to 350 cells/mm³), ART should be initiated [5].9 For children between the ages of 24 and 59
months, the guideline is to initiate ART at an absolute CD4 count of ≤750 cells/mm³ or at a %CD4+ T-cell ≤25,
whichever is lower, irrespective of the clinical stage of HIV infection [5].
Whether in low- or high-throughput settings, CD4 testing is primarily conducted on laboratory-based instru-
ments, although there are three POC CD4 test platforms currently available on the market. In rural settings,
peri-urban settings and even in some urban settings, blood collection is done at clinics and blood samples are
transported (via courier, post or other services, including motorcycle services) to labs for testing; results are
then returned, generally via the same mechanism, although mobile technologies (e.g., SMS) have been intro-
duced at some sites for this purpose. For CD4 testing, it is not currently recommended to use DBS for sample
collection.10
The cost of laboratory-based CD4 testing varies based on testing volumes, reagents used and whether testing is
conducted on high- or low-throughput instruments. Generally speaking, the cost of CD4 reagents varies from a
low of about $2.00 per test to approximately $14.00 per test, excluding collection and laboratory consumables.
The cost of consumables will add between $1.00 and $2.00 per test to the cost. Instruments range in price from
about $25,000 for low-throughput devices to $90,000 for high-throughput instruments.
The cost of currently-available POC CD4 testing ranges from just under $4.00 per test to about $12.00 per test
for the test reagents alone, with associated sample collection consumables adding approximately $1.00 per test.
The instruments cost from $6,500 to $25,000 per device. As additional POC CD4 products enter the market,
including at least one disposable test, prices will likely fall. It is possible that a disposable CD4 test could ulti-
mately cost between $2.00 and $3.00 per test, but early pricing will be higher.
It is important to note that for infants no staging is required following an initial HIV-positive diagnosis. Per the
WHO, infants are to be initiated onto ART immediately [5]. If a follow-up diagnosis proves negative, ART would
be ceased. Countries are currently at various stages of adopting this recommendation.
Patient Monitoring
Prior to initiation onto ART, the current WHO recommendations are to repeat CD4 testing approximately every 6
months (and more frequently as patients approach the threshold to initiate ART), or as needed based on clinical
symptoms [6]. WHO guidance indicates that CD4 testing is required to identify whether patients with HIV and
WHO clinical stage 1 or 2 disease need to start ART. Similarly, following initiation onto ART, the WHO recom-
mends CD4 testing every six months if the patient is stable, but more frequently if needed for deciding when to
initiate or switch ART [6]. It is worth noting that CD4 testing, along with clinical symptoms, is also being used
to diagnose treatment failure in many resource-limited settings.
9 It is likely that the WHO 2013 guidelines will recommend earlier initiation of ART, at CD4 counts of ≤500 cells/mm³, but will prioritize treatment initiation at ≤350 cells/mm³. Further, it is likely
that the WHO will recommend ART initiation independent of CD4 counts in certain categories of patients, e.g., pregnant women, sero-discordant couples, and children under 5 years of age. In
the absence of CD4 testing, the WHO currently recommends ART initiation for all patients with WHO clinical stage 3 or 4 disease.
10 Recently, the use of DBS as a possible alternative for CD4 testing in resource-limited settings has been investigated (Redd et al), but the variability in the results and the failure to detect
immature lymphocytes suggest the need for more research before the use of DBS in connection with CD4 testing should be considered a viable alternative to extant methods [7, 8].
6
Overview
analysers designed for low-end laboratories are widely available and are becoming a standard option. Similarly,
semi-automated spectrophotometers for chemistry analysis have been traditionally placed in low-end laborato-
ries and remain in widespread use today.
In addition, for high-volume settings, high-throughput chemistry and hematology instruments (large bench-
top or floor-standing models) are available. Significant dedicated laboratory space is required, typically with
features such as large reagent storage capacity and air-conditioning, dedicated uninterruptible power supply
(UPS), and well trained, computer-literate technicians.
The technology options available for POC chemistry or hematology are not widely available in resource-limited
settings. Nevertheless, simple hand-held instruments exist for tests such as blood glucose and haemoglobin, as
well as for fixed ranges of 3 to 6 chemistry parameters. These are mobile units, which cost approximately $1,000
to $5,000, and were designed for doctors’ offices, home-use or bedside testing in patient wards. There are also
a limited number of POC chemistry and hematology platforms that are less mobile, larger in size and capable of
running a wider range of tests. With price ranges of approximately $3,000 to $10,000 depending on the features
available, these are designed to be placed in a clinical care setting, such as a patient ward, outpatient clinic, or
doctor’s office, and can be operated by non-laboratory healthcare workers after minimal training.
The average cost of the basic full blood count is approximately $1.15 per test, while consumables average
approximately $2.00 per test. For chemistry testing, the costs vary per test run and on average range from $0.10
per test to $0.45 per test. Consumables average approximately $1.50 per test.
Technical Report 7
2013 HIV/AIDS Diagnostic Technology Landscape
do not include the large upfront investment required to establish viral load-ready laboratories and purchase
instruments for testing. Instruments themselves generally cost from about $100,000 to $225,000, including
installation and training. In addition, collection consumables and laboratory consumables for viral load testing
currently are not bundled and must be purchased separately by users. These items add approximately $2.75 per
test and $1.50 per test, respectively, to the cost of viral load testing.
These operational characteristics are set out in Appendix 1 for each of the platforms currently available for
CD4, viral load, and EID testing, and, where sufficient information is available from the developer, for each such
platform in the pipeline.
In addition to the operational characteristics of the various platforms/devices, it is also important to consider
the performance of the platform, i.e., the ability of the technology to give accurate and reproducible results.
Both the accuracy and precision of a quantitative test should be evaluated.16
16 Note, however, that for a qualitative test—e.g., HIV rapid tests and DNA PCR—accuracy and precision are not the relevant measures. Rather, sensitivity and specificity, as well as negative/
positive predictive values are needed.
8
CD4+ T-Cell Counting Technologies
The accuracy of a technology is a measure of the degree of closeness of the reported value to the true value,
and is evaluated by comparing results obtained by the test under evaluation with those obtained for the same
samples using a reference technology. Although correlation of those results is one measure of accuracy, it is
generally not a sufficient measure. It is important to measure bias and misclassification of the test results as
well. Bias, which may be reported using Bland-Altman analysis, reflects the average/mean difference between
the results of the technology under evaluation and the comparator or reference technology [17]. Misclassifica-
tion probabilities, which may be upward misclassification probability or downward misclassification probabil-
ity, describe the likelihood that a test will incorrectly categorize a result as higher or lower than a given cut-off
value, respectively.
The precision of a test is determined by the closeness of results when testing is repeated using a single technol-
ogy. It is a particularly important measure when used in the context of following a patient’s serial measure-
ments using the same technology—e.g., the level of a patient’s absolute CD4 count or viral load from test to
test. Data on precision are often reported as the coefficient of variation (CV), which is a measure of dispersion.
A lower CV indicates less variation and greater assay reproducibility.
17 Glover [16] notes that a more important measure might be the probability that a patient with an absolute CD4 count well below the ART initiation threshold might be incorrectly classified
as above the threshold, but that such data are rarely available in the published literature.
Technical Report 9
2013 HIV/AIDS Diagnostic Technology Landscape
• G
iven the potential for error described above, access to QC reagents and participation in EQA programs
are very important.
Diagnostic manufacturers routinely publish information on their technology’s accuracy and precision. However,
this is often self-reported data. Independent, peer-reviewed evaluations are a more reliable source of perfor-
mance information for diagnostics. For each platform/device considered in this report, an indication of perfor-
mance and/or performance data availability is provided.
10
CD4+ T-Cell Counting Technologies
Each of the three main component systems of a flow cytometer—fluidics, optics, and electronics—is discussed
in more detail below.
Fluidics System
The fluidics system (an example of which is pictured below) transports particles/cells in a fluid stream to a
laser beam for interrogation. The fluid, called sheath fluid, is usually a saline solution. The portion of the fluid
stream where particles are located is called the sample core. The flow of sheath fluid accelerates the cells and
constrains them to the center of the sample core where the laser beam then interacts with the cells. Typically,
cells are ejected through the flow chamber at a rate of about 1,000 cells per second [24].
Technical Report 11
2013 HIV/AIDS Diagnostic Technology Landscape
Optics System
Flow cytometry optics consist of a complex system of lenses made up of excitation/illumination options and
collection components. The excitation components include lasers, lenses, and filters to route the laser beams
to the flow cell, while the collection components consist of a special lens to amass light signals emitted from
the cells.
When particles pass through the laser intercepts (or interrogation points), they scatter light (both in a forward
direction and in a side direction). Light that is scattered in the forward direction (along the same axis the laser
is traveling) is detected in the forward scatter channel (FSC). Light scattered at 90 degrees to the axis of the
laser path is detected in the side scatter channel (SSC) (see diagram below). The intensity of the FSC depends
on the size of the cell and not its refractive index. The intensity of the SSC is proportional to cell granularity or
12
CD4+ T-Cell Counting Technologies
complexity. Because FSC is related to cell size and SSC is related to its internal structure, a correlated measure
between the two can allow for differentiation of cell types in a heterogeneous cell population. For example,
larger and more granular granulocyte cells produce a large population with high SSC and FSC. Monocytes, on
the other hand, are large cells, but with less granularity, and they produce a separate population with high
FSC and lower SSC. Therefore, these cells can be separated into different populations based on their FSC and
SSC alone.
Schematic courtesy of Dorothy Kratochwil-Otto, Flow Cytometry Lab, University of Alberta, Canada (http://www.flowcytometry.ualberta.ca).
Finally, as the laser interrogates the cell, fluorochromes on or in the cell (either intrinsic or extrinsic) may
absorb some of the light and become excited. As those fluorochromes leave their excited state, they release
energy in the form of a photon with a specific wavelength, longer than the excitation wavelength. These fluo-
rescent stained particles or cells can be detected individually.
Forward and side-scattered light and fluorescence from stained cells are split into defined wavelengths and
channeled by a set of filters (e.g., dichroic) and mirrors within the flow cytometer. The fluorescent light is
filtered so each sensor will detect fluorescence only at a specified wavelength. These sensors are called photo-
multiplying tubes (PMT’s).
Electronics System
In a flow cytometer, as the fluorescing cells pass through the laser beam, they create a peak or pulse over time
in the number of photons. The PMTs detect and collect these photons of light and convert them to current
(voltage). The electronics system then processes that light signal and converts the current to a digitized value
or number that a computer can graph. This is done by using a series of linear and log amplifiers. Linear ampli-
fication is frequently used to amplify FSC and SSC light signals of cells; logarithmic amplification is most often
used to measure fluorescence in cells.
Electronic signals are then further processed (by an analog to digital converter) and sent to a computer so that
the results can be interpreted. These profiles of cells may be displayed in a number of formats, including dot
plots, contour plots and density plots. Below is an example of a dot-plot quadrant analysis for human blood
lymphocytes [25].
Technical Report 13
2013 HIV/AIDS Diagnostic Technology Landscape
18 Unless otherwise noted, information on each of the CD4 technologies described below has been taken from company materials generally available on the respective company websites
and/or from direct discussions with each of the manufacturers/developers of such technologies. Images used herein have been reproduced with the permission of each of the respective
companies/developers.
14
Existing CD4 Technologies/Platforms
hematology analyser from Sysmex, and a team from Chiang Mai University has developed reagents, called CD4
Select, that can be used to enumerate CD4 cells on a hematology analyser alone. Moderate training is required
for this method of analysis, and there are currently no peer-reviewed, independent evaluations of these tech-
nologies available.
In resource-limited settings, single platform methods for CD4 cell enumeration have become the methodology of
choice. Single platform methods provide absolute CD4 (and in most cases, %CD4) measurements using a single
instrument. In these assays, CD4 T lymphocytes can be counted in a precisely-determined volume of blood or
by using known numbers of fluorescent microbeads “admixed” to a known volume of CD4-stained blood [25].
There are several single-platform technologies, including the platforms from BD and Coulter, each of which is a
bead-based technology, and those from Millipore and Partec, each of which uses volumetric methods.
Some of these single platform systems, including the BD FACSCalibur and the Coulter Cytomics FC 500, are
open platforms. This means that the platforms will accept a variety of reagents. For example, TruCount reagents
from BD can be used on the Cytomics platform. Cytognos beads (from Cytognos SL) can be used on Coulter
Cytomics FC 500 or BD FACSCalibur. However, each time different reagents are used on any of these platforms,
the instrument must be re-calibrated. The remaining single platform systems commonly used in resource-limit-
ed settings are closed systems, including the FACSCount, Millipore-Guava Auto CD4/CD4% and PointCare NOW
platforms. This means that they can only use reagents manufactured by the platform manufacturer; reagents
from other manufacturers are not inter-changeable.
Each of these laboratory-based, single platform CD4 testing systems is discussed in some detail below. They are
presented in order of their throughput capability, which also influences the level of the healthcare system in
which the instruments can and should be used.
Technical Report 15
2013 HIV/AIDS Diagnostic Technology Landscape
While the FACSCalibur system is relatively easy to use, with walk away automation via a loader option or a
high-throughput sampler that can handle assays in 96 or even 384 microtiter plates, it is a sophisticated, high-
performance system engineered for use both for in vitro diagnostics and for research laboratories. It is especially
useful in settings that can take advantage of its capabilities for assay development, verification, and identifica-
tion of cellular populations of interest.
As discussed earlier, although most experts agree that there is no true “gold standard” for CD4 testing, many
consider the FACSCalibur system to be the reference standard for CD4 counting. It is the platform against which
the performance of other CD4 systems is most frequently compared and there is at least one published, peer-
reviewed evaluation of the platform using TruCount reagents [31]. It is in use in resource-limited settings, but is
generally only appropriate for central/national reference laboratories where its high throughput (approximately
200 samples per day or 40 samples per hour) and sophisticated capabilities can be used appropriately.
The cost of the FACSCalibur instrument is about $75,000, but can be higher depending on the country/region,
options chosen and whether there are any special negotiated prices available. For the basic three-color reagent
test (TruCount) used by most laboratories in resource-limited settings, the cost of reagents is volume-dependent
and ranges from about $3.00 per test at volumes of more than 75,000 tests per instrument per annum to as
much as $7.00 per test at significantly lower annual volumes.
Cytomics FC 500™ MCL or Cytomics FC 500 MPL™ System (Beckman Coulter, Inc.)
Like the BD FACSCalibur, the Cytomics FC 500 MCL and Cytomics FC 500 MPL Systems19 (pictured below),
manufactured by Beckman Coulter, are large, bench-top flow cytometers. These systems are automated and can
simultaneously analyse up to 4 colors of immunofluorescence from a single laser. The Cytomics FC 500 series
platform (with either MCL or MPL sample loading capability) is a bead-based system that can perform absolute
and percentage CD4 counts (using FlowCARE™ PLG reagents), but can also perform multi-parametric DNA
analysis, platelet studies, reticulocyte enumeration, cell biology/functional studies as well as a broad range of
research applications. The instrument is self-contained and biohazard safe.
19 The Epics XL and XL-MCL are being slowly phased out by Beckman Coulter over the next 4-5 years; the company will fully support these platforms during that period. The Cytomics FC 500
MCL (Multi Carriage Loader) replaces the Epics XL-MCL. In addition, the Cytomics FC 500 MPL contains a multi-platform automated loading system, which allows the platform to serve ultra high
volume laboratories doing more than 500 samples per day.
16
Existing CD4 Technologies/Platforms
The Cytomics FC 500 system automates many of the steps involved in quality control and flow cytometric
analysis, which were previously required to be done manually. In addition, the system contains 2 lasers (an
air-cooled Argon ion laser and an air-cooled Helium-Neon ion laser) and can measure 5-color antibody com-
binations from a single or dual laser excitation in a single tube, which enables laboratories using the system
to reduce the number of tubes and overall costs. In addition, the system offers state-of-the-art Digital Signal
Processing (DSP) for reliable linearity and drift-free amplification and compensation.
Like the FACSCalibur system, the Cytomics FC 500 system is relatively easy to use and provides walk away auto-
mation. The MCL system has a carousel that may be loaded with up to 32 tubes, each to be run automatically;
while the MPL cytometer loads a 40-tube rack and plate loader (i.e., it has the ability to process samples using
either 96-well microtiter plates or tubes, depending on the application or workflow). Like the Epics system,
the Cytomics FC 500 system is a high-volume (on average, 47 samples per hour, or about 375 samples per day,
with the MCL, and more than 500 samples per day with the MPL and the Coulter CellMek automated prepara-
tion system), high-performance system that is geared for use in busy reference laboratories where, in addition
to CD4 counting, it can be employed for other analyses, including diagnosis of acute and chronic leukemias,
lymphomas and platelet disorders, among others.
Assuming certain test volume commitments, the cost of the Cytomics FC 500 MCL instrument is about $90,000;
with the addition of the CellMek system, the cost is about $100,000. For the basic FlowCare PLG reagents used
by most laboratories in resource-limited settings, the cost of reagents is volume-dependent and ranges from
about $2.50 to $4.50 per test at volumes of more than 75,000 tests per instrument per annum to about $5.00 to
$8.00 per test at volumes under 11,000 tests per instrument per annum.
Currently, thirty-five CellMek/Cytomics FC 500 MPL system instruments have been placed in Namibia, Zambia,
and South Africa. Although no independent published peer reviewed articles were found evaluating the Cytom-
ics FC 500 system against comparable systems for CD4 testing, there is an article looking at the positive impact
of the system as used in a clinical research laboratory in Canada [32].
20 Note that Partec also manufactures another device, the CyFlow® SL_3, which performs volumetric absolute counting of CD4 and CD4% for pediatric patients, total lymphocyte count and
WBC. The instrument costs about €22,000 (~$30,000) and uses the same reagents as the CyFlow Counter. The SL_3 operates on the same principles as the CyFlow counter, which is a newer
generation device from Partec.
Technical Report 17
2013 HIV/AIDS Diagnostic Technology Landscape
The CyFlow Counter can be combined with a CyFlow sample preparation and autoloading system (pictured
above on the right). This station is intended for use with Partec dry CD4/CD4% reagents (Partec also offers
liquid CD4/CD4% reagents for use without the loading system). The system allows 10, 20, 30 or 40 samples
at a time to be loaded on a tray; alternatively, 96 well plates can be used. Whereas typical CyFlow Counter
throughput is about 250 samples per day, the company indicates that this added capability allows for acquisi-
tion of up to 400 samples per day, making the system a compact, but high-throughput option. Further, because
the reagents are available in a dry/lyophilized form in ready-to-use test tubes, there is no need for cold chain
and refrigeration of reagents.
Since 2012, Partec has made available on the CyFlow Counter a detailed, on-screen video operations manual
that covers set-up, instrument operation, instructions on how to perform the CD4 and CD4% assays, basic
maintenance instructions, etc.
Because the CyFlow Counter is relatively compact but has high throughput, it can be used not only at national
and reference laboratories, but also in hospitals and laboratories at the provincial and district level [33]. The
device is also small enough to be used in mobile laboratories. Further, the instrument can be run off of a car
battery or solar panels, if needed. The company has placed more than 1,800 instruments in-country, which
provided more than 3.9 million patient tests (both CD4 and %CD4) in 2012.
The cost of the CyFlow Counter instrument alone is about €16,850 (~$22,220), but the total cost will be higher
with the addition of the sample preparation and auto-loading system. Reagents are available both in dry and
liquid form. Absolute CD4 reagents cost approximately €1.75 (~$2.30) per test, while %CD4 reagents for
pediatric use cost approximately €2.50 (~$3.30 per test). Discounts on reagent pricing are available with bulk
procurement.
Published, peer-reviewed literature is available on performance of CyFlow [34,35].
18
Existing CD4 Technologies/Platforms
The FACSCount system uses a whole blood sample, eliminating lyse and wash steps, which, in turn, simplifies
sample preparation for the operator. Fluorescence reference beads, included in a reagent tube, ensure accurate
enumeration of the lymphocyte populations of interest; no operator intervention is required. The software in the
instrument can calculate automatically both absolute CD4 counts and CD4 percentages (important for use on
children under 5 years of age, as discussed earlier in this report) using a single-tube assay (pictured below).
Technical Report 19
2013 HIV/AIDS Diagnostic Technology Landscape
The FACSCount platform is a closed system, although BD has a strategic collaboration with ReaMetrix, a pri-
vately-owned biotechnology company based in Bangalore, India, to develop dried reagents for the FACSCount
system. The cost of the FACSCount instrument is about $30,000. Pricing for reagents depends on test reagents
chosen (single tube absolute CD4 only, single tube absolute CD4 and percentage CD4, or double tube) as well
as volume of testing per annum per instrument. The pricing for the reagents alone ranges from approximately
$3.50 per test for test volumes of more than 10,000 tests per instrument per annum up to $10.00 per test for test
volumes up to 4,500 tests per instrument per annum.
The BD FACSClearCount system includes the instrument, integrated software, a sample prep workstation, dedi-
cated reagents, and whole blood controls. Dedicated reagents are provided in a new dried format, in ready-
to-use cartridges. Reagents include both fluorescently-labeled antibodies for the identification of CD4 T-cell
populations, and counting beads for simultaneous CD4 T-cell enumeration. The counting beads are also used
for daily instrument quality control. Dried-down reagent technology eliminates the need for a cold chain, sim-
plifying storage and reducing costs. The reagents have been designed to meet the requirements of a wide variety
of temperature settings.
To simplify the workflow, a carousel holds 20 innovative two-tube reagent cartridges with reagent and beads in
one tube and patient sample in the other. In standard operation mode, the instrument automatically prepares
and acquires the sample—the precise sample volume is pipetted into the cartridge tube containing the air-dried
reagent and beads. Following incubation, the lysing solution is added and the sample is acquired. Manual steps
are eliminated to improve workflow. Test results including absolute and percentage CD4 counts, are provided
on-screen, and can be printed using the on-board thermal printer. They can also be exported using the front
access universal serial bus (USB) port and provided USB flash drive.
The BD FACSClearCount uses an integrated software and touchscreen interface to further simplify use and
reduce operator training time. The interface is straightforward—users simply need to touch a button to navigate
to and execute a function. All actions are run from the touchscreen. Touchscreen software is available in the
following six languages: English, French, Portuguese, Russian, Simplified Chinese and Spanish.
20
Existing CD4 Technologies/Platforms
The cost of the FACSClearCount instrument is expected to be about $38,000. Pricing for reagents depends on
volume of testing per annum per instrument. The general range of pricing for the reagents alone ranges from
approximately $4.50 per test for test volumes of more than 10,000 tests per instrument per annum up to $12.00
per test for test volumes up to 4,500 tests per instrument per annum. The FACSClearCount is now expected to
launch in early 2014.
The Aquios CL processes samples continuously; batch processing of samples is not required. The sample loader
holds up to 8 cassettes at a time with up to 5 sample tubes each (i.e., total capacity is 40 sample tubes), and
allows for continuous loading and unloading. The first test results are available approximately 20 minutes after
loading the sample. In addition, the Aquios CL is preloaded with a range of barcoded reagents and consumables
and automatically scans barcodes to track reagents, lot numbers, open and closed vial expiration dates, etc.
There is continuous tracking of reagent usage by product. This tracking means that there is no need for manual
QC or reagent logs, and if QC fails, the operator is notified via text message or email.
The Aquios CL system, which is a bench-top platform with a relatively small footprint, features an all-in-one
computer and monitor with touch-screen operation. There is also an alternative keyboard and mouse. Data anal-
ysis is performed via advanced automated algorithms with the option of user-adjustable gates and regions.
Future applications that aid in the diagnosis, monitoring and treatment of diseases are pending.
Technical Report 21
2013 HIV/AIDS Diagnostic Technology Landscape
The Guava software module provides automated data acquisition, gating and analysis, which increases ease of
use and simplicity. The company estimates that the system can be learned in about a day’s training. In addition,
the Guava system is generally rugged because of its simplified fluidics, self-aligning lasers and user-changeable
microcapillaries. In turn, this means that the Guava system is relatively easy to maintain.
The Guava system, which is a closed system, is a medium- to low-throughput platform, allowing for up to 100
samples per day to be processed. Like the FACSCount, the Guava instrument can be used in a wide range of set-
tings. In recent years, the company has expanded its ability to provide service and maintenance on the instru-
ments through a network of local distributors. On average, the cost of the Guava instrument is approximately
$20,000. The pricing for the reagents (combined CD4 cell count, CD4% and total lymphocyte count) is $2.50
per test (including the distribution margin), regardless of volume.
With respect to the performance of the Guava system, the WHO concludes that it is difficult to place it in the
platform hierarchy. Although there were studies on the earlier version of Guava reagents (Easy CD4) [38,39],
there is a dearth of evidence on the performance of the Guava Auto CD4/CD4% reagents, with no peer-reviewed
studies having been published to date [7].
22
Point of Care CD4 Testing Platforms
The Apogee system was designed for both military environments and resource-limited settings. Accordingly,
the instrument is rugged. Sample preparation is similar to that for FACSCalibur and requires vortexing as well
as 25-minute incubation in a dark room. Sample run time is approximately 90 seconds, but can be longer for
samples with low CD4+ cells. Data is stored in the Apogee’s internal hard drive for immediate or later analysis
by the operator.
The Apogee Auto40 is a medium-throughput system that can run a maximum of 20 samples per hour. Although
it is an automatic instrument, it also offers an option to manually analyse difficult or damaged samples. The
cost of the Apogee Auto40 is about $27,000. The pricing for reagents is approximately $2.50 per test for absolute
CD4 counts and $3.50 per test for percentage CD4.
Several peer-reviewed studies of the Apogee Auto40 platform have been published [40,41].
Technical Report 23
2013 HIV/AIDS Diagnostic Technology Landscape
staining, followed by capture or count by digital photography, measuring CD4 molecules instead of cells, or
measuring proxy molecules of CD4. Point-of-care CD4 testing is likely to require new, simpler technologies.
Both instrument-based and disposable tests are in the CD4 development pipeline. Generally speaking, such
POC CD4 tests would preferably meet the ASSURED criteria for the ideal rapid test, which was developed by
the WHO [42]. The ASSURED criteria are as follows:
A = Affordable
S = Sensitive
S = Specific
U = User-friendly (simple to perform in a few steps with minimal training)
R = Robust and rapid (results available in less than 30 minutes)
E = Equipment-free
D = Deliverable to those who need the test
Below, POC diagnostics for CD4 testing that are either on the market or in development are discussed in
some detail, including technical specifications. Three of these technologies are already on the market: Point-
Care NOW™, the Pima™ CD4 Analyser and the CyFlow™ CD4 miniPOC. The remaining technologies discussed,
including those from Daktari, Omega, Zyomyx, MBio and others, are not yet available on the market.
The current CD4 POC pipeline is presented in Appendix 2. It is interesting to note that since this report was
first published in 2011, the companies in the CD4 POC pipeline have not changed. However, the expected date of
market introduction for each of the platforms has been delayed. While in 2011, several platforms were expected
to be introduced in 2012, these same platforms are now expected to be launched in mid- to late-2013 and early
2014. These delays generally reflect the technical challenges of developing CD4 platforms for use at the point of
care, and in some cases, reflect the difficulty of obtaining funding for developing these products.
21 The label consists of anti-CD4 antibodies coupled with nano-sized gold particles.
24
Point of Care CD4 Testing Platforms
PointCare reports that by the summer of 2013 it expects to introduce a validated system of internal QC that
entirely does away with the requirement for EQA controls, materials that are both perishable and impractical
in remote settings. PointCare also expects to introduce a new version of the PointCare NOW instrument that
provides a printed-out warning to clinicians and care-givers when patients have “out-of-range” hematology
results and urgent clinical action is required. This upgrade also will provide simplified on-screen instructions
to the instrument operator when the sample-run shows abnormalities or when the clinician should be warned
about urgent concurrent conditions.
The PointCare NOW system is a medium- to low-throughput platform that can handle about 50 samples per day
and is appropriate in settings with that level of volume. The system is closed and requires the use of PointCare
reagents. The cost of the PointCare NOW instrument is about $25,000. The pricing for reagents, which includes
PointCare’s heat stable Daily Check controls, is approximately $10 per test.
A peer reviewed evaluation of the PointCare NOW™ platform was published in 2012. The review found that the
instrument had low sensitivity in adults, misclassifying 53% and 61% of patients at the 350 and 200 cells/μL
thresholds, respectively; while sensitivity was better for children, the authors concluded that the sample size
was not large enough to draw a conclusion [43].22 The company concluded a method-comparison with BD’s
FACSCalibur in March 2011 at the National Microbiology Reference Laboratory (NRL), Harare, Zimbabwe. The
results from this evaluation in Zimbabwe as well as the results of an evaluation conducted at military clinics in
Uganda are expected to be published in the near future.
22 Sensitivity to identify children in need of ART using a 25% CD4 threshold was 90% and sensitivity was 100% using a 750 CD4 cells/mm3 threshold.
Technical Report 25
2013 HIV/AIDS Diagnostic Technology Landscape
The Alere Pima CD4 system is made up of the Analyser and a disposable CD4 test cartridge (pictured below)
which contains dried reagents. As such, it is a closed system with no compatible third party reagents available.
The system is capable of measuring absolute CD4 counts in whole blood, but it cannot currently determine
%CD4 counts. This capability could be added to the system, along with other cell type counts.
Venous blood or capillary blood derived from a fingerprick are both acceptable samples. There is no require-
ment to measure the volume of blood used in the test; the cartridge is designed to take up 25 µL of blood in a
self-regulated manner, eliminating the need for calibrated volumetric pipettes. Once the sample is applied to
the cartridge it is irreversibly capped and inserted into the analyser. The dried reagents, including fluorescently
labeled anti-CD3 and anti-CD4 antibodies, are re-dissolved in the sample and allowed to incubate before the
sample is passed into an optical imaging chamber. Once capped, all test steps are actually performed within the
sealed cartridge and no part of the Pima Analyser comes into contact with the blood sample during processing,
thus minimising the risk of analyser contamination.
26
Point of Care CD4 Testing Platforms
The Analyser is equipped with miniaturized, multi-color fluorescence imaging optics. Fluorescence images
are collected by an on-board camera and analysed using proprietary software algorithms on the embedded
computer to derive absolute CD4 counts. Up to 1,000 test results are stored in an on-board archive. Operator
ID, sample ID, date, time, CD4 count and the outcome of numerous internal controls are stored with every test
result. Data can be viewed on the on-board display, printed onto archival thermal paper with the accessory
Pima printer, or exported by the operator at any time after the test has been completed. Export can be to a
USB memory stick, and Alere has also launched an optional USB connectivity module for sending data to cen-
tral servers via mobile telephone networks. A LAN connectivity solution is also available. A power extender,
including an extended life battery and adaptors for charging sources, including solar panels and mains, has
been added to the product family.
The system can perform approximately 20 tests per day (3 tests per hour) with minimal operator interaction –
walk away testing. As a simplified, low-throughput POC system, Pima can be used appropriately at all levels
of the healthcare system where high throughput is either not required or for use in situations where same-day
results are particularly important, even in high-volume settings.
The CD4 Analyser/Pima has been pre-qualified by the WHO, is CE-IVD marked, and has performed well in an
evaluation done by the United States Centers for Disease Control and Prevention (CDC). Alere is in the process
of submitting a 510k for the product with the FDA and approval is expected in 2013. At least ten peer-reviewed,
independent evaluations of the Pima system have been published since product launch (see, for example,
references 44-48). In these studies the Pima CD4 system was tested in laboratory as well as non-laboratory set-
tings such as rural voluntary counseling and testing (VCT) sites and mobile healthcare units. The studies also
incorporate diverse geographies, including Asia and sub-Saharan Africa, as well as diverse operators, including
physicians, laboratory technologists, nurses and lay healthcare workers. Despite the different settings and study
objectives, the results all demonstrated very good correlation with the predicate flow cytometry technologies,
even when performed at the point of care on capillary blood obtained by fingerstick, although one study did
find that the coefficients of variation were slightly larger with fingerstick than venous blood and the authors
noted that this may have been related to insufficient training of operators [47]. One of the published studies
demonstrated, for the first time, the positive impact point of care CD4 testing can have on patient retention and
ART initiation. The study authors concluded that “point of care CD4 testing enabled clinics to stage patients
rapidly on-site after enrolment, which reduced opportunities for pretreatment loss to follow-up. As a result,
more patients were identified as eligible for and initiated antiretroviral treatment.” [48]
The cost of the Pima Analyser varies among countries and regions, with prices ranging from approximately
$6,500 to $12,000, and the cost per test ranging from approximately $6.00 to $12.00. The instrument requires
Technical Report 27
2013 HIV/AIDS Diagnostic Technology Landscape
no routine preventive maintenance, and Alere has established a global technical support network to ensure fast,
reliable and consistent service.
The CD4 miniPOC requires only 20µL of blood, which is added to a Partec reagent-filled tube and incubated for
15 minutes. Buffer is added, and ultimately the sample blood is drawn up into a syringe to a precise fill line. The
operator then places that syringe onto the POC device and the instrument slowly injects the processed sample
into the instrument, where CD4 detection takes place. Sample processing, which is automated in some systems,
is stripped from the Partec device. Sample processing takes place outside of the device.
Results can be displayed in routine or expert modes (illustrated below on the left and right, respectively).
The expert mode features a histogram with the display of cell clusters thus offering an additional built-in
quality control.
28
Point of Care CD4 Testing Platforms
As it did for the CyFlow Counter, since 2012, Partec has made available on the CD4 miniPOC instrument a
detailed, on-screen video operation manual that covers set-up, instrument operation, instructions on how to
perform the CD4 and CD4% assays, basic maintenance instructions, etc.
The cost of the CD4 miniPOC instrument is approximately €7,100 (~$9,380). The system uses the same dried
reagents as its larger sibling, the CyFlow Counter, but in different packing that includes all required consum-
ables at a total cost of €3.00 (~$3.96) per test kit, which yields both absolute CD4 and %CD4 results. On occa-
sion, the company also offers special point-of-care packages at price savings for the instrument and reagents.
To date, no peer-reviewed, independent performance evaluations of the Partec CD4 miniPOC device were found
in a literature review.
Technical Report 29
2013 HIV/AIDS Diagnostic Technology Landscape
Intended for use at the point of patient care, the Daktari system eliminates sample preparation through the
use of a technology known as “microfluidic cell chromatography,” which isolates cells and other particles in a
miniature sensing chamber. No pipetting, labels or reagents are required; the only user step is to apply a drop
of whole blood to the cartridge. Similarly, the Daktari device does not require fragile and expensive optical
sensors, but rather uses a second innovation, “lysate impedance spectroscopy,” which employs a simple sen-
sor to count captured CD4 cells by measuring their internal contents electrically. The Daktari instrument then
interprets the electrical signal and reports the CD4 count in 10 minutes.
The Daktari CD4 system will include a data management system with a keypad user interface and a back-end
data package that will come built into the device.
The anticipated cost of the Daktari CD4 counter is less than $5,000 for the device. Per test cost is anticipated to
be approximately $8.00, but volume discounts are expected to drive the price lower. If the device is damaged,
the low cost and portability of the instrument would allow it to be swapped out with a replacement device
rather than being repaired on-site.
There is currently no published performance data available for the Daktari CD4 system.
30
Point of Care CD4 Testing Platforms
The System includes an on-board computer for sample analysis, results management, internal QC and event logs
that can be exported in common and viewable file formats for data review. The user interface is an intuitive touch-
screen with administrator-configurable settings such as user lockout/validation and QC scheduling. Cartridge
Technical Report 31
2013 HIV/AIDS Diagnostic Technology Landscape
barcodes will be read automatically, and the instrument will have multiple USB ports to support printers, external
barcode readers, and other peripherals. The System will include integrated GSM/GPRS connectivity.
Pre-market field evaluations in sub-Saharan Africa were initiated in 2012 and will continue during the first half
of 2013. In-country clinical trials are scheduled for the second half of 2013. A CE-IVD mark on the MBio CD4
System is anticipated in 2013 followed by product launch.
The sample is collected from the patient using a fingerstick or an ethylenediaminetetraacetic acid (EDTA) tube.
The cartridge is self-contained and is inserted by the operator into the device. After a short incubation period,
detection takes place automatically and the result can be read immediately in a single, easy step. The new and
innovative cartridge technology contains dried reagents and requires no-cold chain, which enables longer shelf
life over a wide range of environmental conditions. Market launch is expected in late 2013.
32
Point of Care CD4 Testing Platforms
The anticipated cost of the Zyomyx assay is estimated to be less than $8.00 per test. The company expects that,
with a minimum purchase volume of cartridges (still be to be determined), there will not be a separate cost for
the mixer/spinner. The mixer/spinner is capable of performing a test preparation in less than 10 minutes and
will support at least 10,000 tests. If additional throughput is required, it can be increased by adding an addi-
tional mixer/spinner at nominal cost.
There is currently no performance data available for the Zyomyx system; clinical trials are in process.
Technical Report 33
2013 HIV/AIDS Diagnostic Technology Landscape
Because of some concerns about the ability of users to read the results of the test, which requires operators to
identify the result line and compare it with the reference and controls lines on the strip (pictured below, top),
Burnet developed a reader for the device (pictured below, bottom), which also provides data storage and con-
nectivity options, as well as real-time operating instructions for the test devices. The reader, which has been
developed in collaboration with Axxin Ltd. (Australia), is expected initially to cost about $3,000, but may decline
to about $2,000 over time. The reader will be provided free of charge dependent on committed volumes.
34
Point of Care CD4 Testing Platforms
Evaluation of the prototype version of the test at the 350 CD4/µL cutoff at the Burnet and Alfred Hospital, Mel-
bourne, has shown 97% sensitivity for samples below 350 CD4/µL and 80% specificity for samples above 350
CD4/µL (total n=126). Clinical validation trials of the Visitect CD4 are planned to follow in the UK, the United
States and Southern Africa in the first half of 2013.
Technical Report 35
2013 HIV/AIDS Diagnostic Technology Landscape
Conclusions—CD4 Testing
Technologies
Currently, there are a good number of technology choices for CD4 testing in resource-limited settings. Most
of these are laboratory-based platforms using proven flow cytometry methodologies. In reference laboratory
settings with well-trained technicians, these technologies function well and can be cost-effective. Many, but
not all, of these CD4 testing platforms, including BD FACSCalibur and FACSCount, have been the subject of
independent evaluations and have performed well, within the recognized limitations, both physiological and
technical, of CD4 performance.
However, in order to reach patients in peri-urban and rural settings with these laboratory-based CD4 tools, it is
necessary to set up sample transport networks to transfer patient blood samples to the reference laboratory for
testing and to set up a results return system, involving the same transport used for inbound samples (generally
courier services of some sort) or mobile technologies, including SMS. This is made more difficult by the fact
that the transport of samples for CD4 testing generally requires the transport of whole blood, which has limited
stability, as opposed to DBS, which extends the life of samples. Moreover, sample transport is an additional cost
to the provision of CD4 testing and prevents the availability of same day results to patients, which can result in
loss to follow-up.
Therefore, in order to improve access to CD4 testing in resource-limited settings, there is a need for good and
cost-effective POC CD4 testing options. Several such options are already on the market, and others are under
development, more than one of which are likely to become available in 2013. The current options available for
POC CD4 testing are device-based, but disposable CD4 testing is on the near-term horizon. To date, with the
exception of extensive evaluations of the Pima Analyser™, the performance data for POC CD4 platforms are
limited. It is anticipated that as more POC CD4 testing devices are introduced, the results of independent clini-
cal evaluations by the CDC (United States), the National Health Laboratory Service (South Africa) and others,
as well as evaluations performed in-country, both in laboratory settings and in the field, will become available.
Indeed, it is important that this data become accessible.
36
Viral Load Testing Technologies
testing overall, and the ability to effectively transport samples between collection sites and laboratories. Ulti-
mately, the market for CD4 testing and viral load testing (discussed below) between the two extremes of super
labs and POC may be relatively small, but in any event, the landscape will neither be all laboratory-based or
all POC-based.
Technical Report 37
2013 HIV/AIDS Diagnostic Technology Landscape
and platforms and that should inform the choice of platforms for a given setting. These include HIV diversity
and certain practical challenges, including laboratory infrastructure and transport of samples.
HIV Diversity
In 1985, several years after HIV was recognized as an infectious agent, a genetically similar virus causing AIDS
was discovered in West Africa. As a result, two types of HIV have been classified and characterized: HIV-1,
the original virus, and HIV-2, the strain of virus discovered in West Africa. Of the two types of HIV, HIV-1 is
predominant and has been most responsible for the HIV pandemic that exists today [52]. Further complicating
matters, HIV-1 is divided into four groups, designated M, N, O, and P, the main group of which is group M. And,
there are also multiple clades, and within each clade, there are sub-clusters of individual strains of the virus that
have been isolated around the world. Finally, mutation of the virus and different evolutionary rates have led to
extensive genetic diversity, which in turn has contributed to the divergence of the distinct clades. When viruses
from two or more strains exchange their genetic material and become established, they are called recombinant
viruses. In all, there are at least 43 circulating recombinant forms (CRF) or inter-subtype recombinant HIV-1.
The high level of genetic heterogeneity of HIV-1 and the emergence of recombinant strains of the virus com-
plicate viral load assay development [56,57]. In an ideal world, viral load assays would detect and quantify all
known HIV-1 subtypes (as the Cavidi ExaVir assay can do today), as well as inter-subtype recombinants and
emerging variations thereon. But, currently, that is not the case, although the assays are able to recognize most
HIV-1 subtypes. Therefore, it is important to consider the prevalence of HIV-1 and HIV-2 groups and subtypes
in a particular geographical region when choosing a viral load assay.
Laboratory Infrastructure
Currently available viral load platforms are laboratory-based and require significant infrastructure, including
continuous power, clean running water and air conditioning. For example, the typical, non-POC viral load plat-
form based on nucleic acid technology (discussed below) will require two to three dedicated rooms in a labo-
ratory.23 Each room should have minimal dust and preferably will be temperature controlled (air conditioned in
hot climates). The rooms are needed to accommodate the different stages of the testing process: Room 1 would
be dedicated to receipt of the patient sample and sample extraction (most of which is done in a bio-safety cabi-
net). Room 2 (which could be reduced to a Clean-Air Box in Room 1 if space is limited) would be used to pre-
pare the reagents, which are prone to contamination. Finally, Room 3, which will become highly contaminated
through the test process, would be dedicated to amplification and detection of the virus and results processing.
In order to avoid contamination, work flow must proceed from Room 1 to Room 2 to Room 3. Each room needs
to have 3 to 4 meters (approximately 10 to 13 feet) of bench space. Further, test reagents generally will have
to be stored at between 4º and 8º C. And, as mentioned above, steady current is required so that the electrical
test equipment is not damaged.
Sample Transport
Most methods of viral load determination require venous blood collection, processing (centrifuging) of that
blood to obtain plasma within a certain timeframe, cold chain and storage of specimens by trained personnel.
In resource limited settings where viral load testing will generally take place only in a national reference, or
comparable, laboratory, this means that patient samples will have to be transported from urban, peri-urban
and rural settings to the laboratory for processing. This is done using sample transport networks in-country,
taking advantage of courier or similar services to take samples to the laboratory and to return results at a later
date. But, frequently, these services are not well developed, leading to long delays in returning sample results
to patients and loss to follow-up.
Therefore, the ability to use DBS samples for viral load is an important consideration in the implementation of
the testing because it greatly simplifies the transport of samples, providing enhanced stability and ease of use
for healthcare workers. The use of DBS is also cost effective. There has been some concern about the correla-
tion of viral load measures using DBS as opposed to plasma. But, recently, several studies have demonstrated
good correlation between the two using different viral-load methodologies, with sensitivity ranges close to 3 log
23 Two exceptions to this are the Siemens kPCR Molecular System and the Siemens VERSANT 440 Molecular System, each of which requires only a single room.
38
Existing Viral Load Technologies
HIV-RNA copies/mL [58,59].24 In a review of viral load monitoring technologies, MSF notes that: “given that
the DBS technique is currently the only means of sample transport over long distances and without the need
for cold storage, it will be important for manufacturers of laboratory-based tests to validate their platforms for
use with DBS.” [63]
NAT-Based Technologies
NAT-based assays have become the core viral load monitoring technology used in both developed countries and
resource-limited settings. The NAT-based systems manufactured by Abbott, bioMérieux, Roche and Siemens
currently dominate the market.
All such technologies incorporate amplification techniques because levels of nucleic acids are otherwise too low
to be detected directly. Amplification methods are either aimed at increasing the number of target molecules
(viral nucleic acids) to a level that permits detection (target amplification methods) or are aimed at increasing
the signal generated by the method (signal amplification methods) [52]. Currently, the bulk of commercially
available viral load assays are based on target amplification.
24 Note that although the correlation between plasma and DBS viral load is generally good, for some platforms the correlation falls away at low cp/mL because of interference from non-
plasma-associated virus. However, this occurs below 5,000 cp/mL, which is the level which the WHO currently considers to be the measure of virological failure. Therefore, for diagnosing
virological failure, the poor correlation may not be a problem [60,61]. It might mean, though, that DBS viral load should not be used as an adherence monitoring tool where being able to detect
1,000 cp/mL is important [62].
Technical Report 39
2013 HIV/AIDS Diagnostic Technology Landscape
Whether an assay is based on target amplification or signal amplification, the assay will consist of the follow-
ing common steps: (i) sample preparation and/or viral nucleic acid extraction; (ii) the actual amplification step
that is either target amplification- or signal amplification-based; and (iii) detection and/or quantification of the
amplified viral nucleic acids.
Pre-amplification methods (sample preparation and/or viral nucleic acid extraction) are critical to the viral
load testing process. For each sample to be analysed correctly and to achieve an accurate result, the nucleic
acid must be both available for the reaction and purified. Protocols for the pre-amplification steps include the
use of purification methods for cells, and virion centrifugation or a capture step for RNA in plasma, followed
by an extraction step to free the target viral nucleic acid [52]. Although HIV nucleic acids are relatively stable,
molecular detection methods require prompt processing of samples (generally within 6 hours of collection), a
rapid extraction method and appropriate storage of plasma or cells prior to assessing.
There are several amplification methods used to detect viral RNA or DNA after preparation of samples. In target
amplification, many copies of a portion of the viral nucleic acid are synthesized via an amplification reaction;
in effect, this method enhances the ability to detect very low levels of nucleic acids that occur naturally in
the blood. These techniques include the reverse transcriptase polymerase chain reaction (RT-PCR) used in the
Roche, Abbott and QIAGEN assays and nucleic acid sequence-based amplification (NASBA) used in the bio-
Mérieux assay. In signal and probe amplification methods, a probe or a reporter molecule attached to a probe
is detected and the signal generated by this reaction is amplified/increased; in effect, these methods increase
the “marker” that shows that the target is present. Signal amplification techniques include branched chain DNA
(bDNA), which is used in the VERSANT™ HIV-1 3.0 assay by Siemens.
Finally, post-amplification methods require the detection and/or quantification of either the amplification prod-
ucts (in target amplification methods) or the increased detection of signals that have been amplified (in signal
amplification methods) [52]. Detection can be achieved using any one of a number of reagents – e.g., colori-
metric, radioactive, fluorescence. Detection can either be done at the endpoint of the process (completion of the
run) or in “real time” (during the production of results as they occur). Real-time techniques, in which amplifi-
cation and detection occur simultaneously, are now commonly used. For example, the Roche Taqman platform
uses real-time detection, which is achieved via specific, fluorescently-labeled probes that bind to the DNA that
is generated via the amplification process (called amplicons).
In general, the advantages of NAT-based approaches include that many of the assays using these approaches
have been evaluated and are well-validated; the assays are available in quality-assured kits, and clinicians are
comfortable interpreting the results. The assays vary in terms of sample preparation and amplification/detec-
tion methodologies, among other things. The major NAT-based assays and platforms are discussed below.25
25 Unless otherwise noted, technical information on the various platforms has been obtained from the online resources provided by manufacturers and/or directly from company
representatives. The images used below to illustrate the platforms are being used with the permission of the respective companies/developers.
26 One example is the Generic HIV Viral Load assay from Bio-Centric (France), which is for research use only. This assay can be run on a real-time thermocycler and requires other basic
consumables that would cost about $40,000. Time to result is about 4 hours, including RNA isolation. The cost per test ranges from approximately $10.00 to $20.00.
27 Roche has globally discontinued manufacture of version 1 of the COBAS® AmpliPrep/COBAS® TaqMan® assay.
40
Existing Viral Load Technologies
The COBAS AmpliPrep/COBAS TaqMan version 2 test was designed specifically to address HIV-1 mutations.
In order to do this, a dual-target approach is used. The dual-target technology provides additional confidence
in results in the event of mutation. The assay is able to co-amplify two target regions of HIV-1 (known as the
gag and long terminal repeat [LTR] regions), which were specifically chosen as they are not current HIV drug
targets. By targeting both regions of the genome simultaneously, the test increases the probability of detection
of virus particles.
The COBAS AmpliPrep/COBAS TaqMan HIV-1 Test version 2 is intended for use in conjunction with clinical
presentation and other laboratory markers of disease progress for the clinical management of HIV patients. The
assay can be run using DBS in addition to plasma specimens, which is an advantage for resource-limited set-
tings. It is able to quantify HIV-1 group M (subtypes A through H) and HIV-1 group O, and has a limit of detec-
tion as low as 20 copies per mL. At the other end of the spectrum, it can also quantify the amount of HIV-1 in
a patient sample up to 10 million copies/mL.
The COBAS AmpliPrep/COBAS TaqMan HIV-1 Test version 2 for TaqMan 48 and TaqMan 96 is prequalified by
the WHO. The test is also FDA approved for plasma, but is “research use only” (RUO)28 for use with DBS. Per-
formance of the test has proven to have good correlation with the AMPLICOR HIV-1 MONITOR™ v1.5 assay (the
MONITOR assay), which has generally been considered to be the gold standard [64].
The cost per test for the least developed countries and certain high-burden middle income countries is about
$11 to $25. Actual pricing is dependent on variables such as outright instrument purchase, reagent rental and
volume-based, tiered pricing arrangements.
The COBAS® AmpliPrep System—The COBAS AmpliPrep instrument is an automated sample preparation tech-
nology (pictured below) for use in conjunction with the Roche COBAS TaqMan analysers discussed below. The
company considers the AmpliPrep to provide “walk-away” sample preparation/extraction capability, which can
significantly reduce hands-on time of laboratory technicians.
The instrument is large, weighing over 680 pounds. The run size for the instrument is 24 specimens, but it can
process up to 72 samples at any given time. The first 24 samples take 2 hours to process. However, because the
instrument allows for parallel processing, subsequent batches of 24 can be completed every hour as one rack of
specimens will begin processing before the previous rack processing has been completed. The system is closed
and requires the use of test-specific, bar coded, ready-to-use COBAS AmpliPrep kits. The cost of the instrument
is approximately $80,000 to $100,000 (with the lowest pricing reserved for lower income countries).
Roche TaqMan Analysers—Roche manufactures two versions of its TaqMan Analyser, the COBAS® TaqMan
48 Analyser and the COBAS® TaqMan 96 Analyser. Each of the analysers is a fully automated, closed-tube
system. The TaqMan 48 (pictured below) is relatively compact and can run from 6 to 48 samples at a time.
The instrument is equipped with two thermal cyclers that operate independently and provide run times of 90
to 120 minutes.
28 The RUO (Research Use Only) designation is required by the FDA for non-FDA approved in vitro diagnostic products that are manufactured in the United States and exported for sale and use
outside the United States.
Technical Report 41
2013 HIV/AIDS Diagnostic Technology Landscape
The cost of the COBAS TaqMan 96 Analyser is approximately $100,000 to $110,000. This price includes a dock-
ing station.
29 In addition, Roche provides the COBAS p630 instrument for use with the COBAS AmpliPrep/COBAS TaqMan System, which provides a fully automated pre-analytical solution for primary
tube handling. The instrument will de-cap and cap sample tubes, pipette Roche controls from control tubes to sample tubes, and pipette samples from primary tubes to sample tubes. The
COBAS p630 also provides sample traceability (using bar-code tracking from primary tube to result) and process surveillance (through liquid handling monitoring). In addition, the device
transfers samples, controls and order information to AMPLILINK Software.
42
Existing Viral Load Technologies
The Abbott RealTime assay can be automated using the Abbott m2000sp (or m24sp) for sample preparation
and the m2000rt for amplification and detection. The assay introduces an RNA sequence that is unrelated to the
HIV-1 target into each specimen at the beginning of sample preparation. This unrelated RNA sequence is simul-
taneously amplified by RT-PCR, and serves as an internal control to demonstrate that the sample has proceeded
correctly through the process. The amount of HIV-1 target sequence that is present at each amplification cycle is
measured through the use of fluorescent-labeled oligonucleotide probes on the m2000rt instrument. The probes
do not generate a signal unless they are specifically bound to the amplified product. The amplification cycle at
which the fluorescent signal is detected by the m2000rt is proportional to the log of the HIV-1 RNA concentra-
tion present in the original sample.
The RealTime assay has a linear range of 40 copies/mL to 10 million copies/mL and can detect HIV-1 group
M (subtypes A-H), group O, and group N. The sensitivity of the assay is dependent on specimen volume. The
limit of detection is 40 copies/mL for 0.6mL input and 150 copies/mL for 0.2mL input. Performance has been
assessed with good results [65]. Like the other assays discussed in this report, it is intended for use in conjunc-
tion with clinical presentation and other laboratory markers for HIV disease prognosis and for use as an aid in
assessing viral response to ART as measured by changes in plasma HIV-1 RNA levels.
The Abbott RealTime HIV-1 assay has been pre-qualified by the WHO. The price per test of the assay ranges
from $25 to $40 and is dependent on volumes as well as any negotiations with Abbott.
Sample Preparation with the m2000 System—The Abbott RealTime assay is designed to be used with the
m2000rt amplification and detection instrument as well as with one of three methods of sample preparation:
(i) manual (for laboratories with low throughput requirements); (ii) the m24sp instrument, which automates
sample purification steps; or (iii) the m2000sp instrument, which fully automates sample preparation.
The m24sp (pictured below) is a bench-top sample preparation and extraction device with a small footprint that
is generally appropriate for facilities with medium throughput requirements. It provides a variable extraction
system (extraction output can be stored either in deepwell trays or 1.5ml tubes) with ready-to-use and re-usable
reagents as well as flexible batch size capabilities.
Technical Report 43
2013 HIV/AIDS Diagnostic Technology Landscape
44
Existing Viral Load Technologies
The m2000rt—The Abbott m2000rt is the amplification and detection platform for use with the m24sp and the
m2000sp instruments, as described above. It is a high-performance system, but is relatively compact, weighing
in at just over 75 pounds. The m2000rt (pictured below) can run 96 samples at one time in about 3 hours of
cycling time (not including time for sample preparation). The system will run both quantitative and qualitative
analyses and contains internal controls. Like other laboratory-based viral load systems, the operator must have
a thorough knowledge of the applications run on the instrument (and on the sample preparation instrument)
and must follow good laboratory practices when operating them.
The cost of the m2000rt is approximately $38,000 when purchased with the m24sp or m2000sp, but about
$44,000 if manual extraction is used.
Technical Report 45
2013 HIV/AIDS Diagnostic Technology Landscape
The Sample Preparation module along with the VERSANT Sample Preparation 1.0 Reagents Kit are used to
extract RNA from plasma. The reagents kit includes proprietary magnetic silica beads that provide for efficient
and high-quality extraction of nucleic acids. Extraction consists of a lysis step that utilizes proteinase K and
a chaotropic buffer, several washes to remove non-nucleic acid components of the sample and elution. In the
Amplification Detection Module, the purified RNA is eluted and added to a PCR plate containing an HIV-1
primer/probe mix and the HIV-1 enzyme mix. The wells are then sealed. At this point, HIV and internal control
RNA molecules are reverse transcribed to make cDNA and then simultaneously amplified and detected using
the kPCR technique. The RT-PCR step uses primers and probes that target a highly conserved region of the pol
integrase gene. A schematic representation of the assay principle is shown below.
46
Existing Viral Load Technologies
The VERSANT kPCR Molecular System processes samples in batch mode in a 96-well format. The HIV assay
provides patient results for up to 89 samples per run. Total time to result is less than 6 hours. The linear range
of the assay is between 37 HIV-RNA copies/mL and 11,000,000 copies/mL. The assay can detect HIV-1 Group M
(subtypes A - G) and Group O variants [66]. Performance of the assay is comparable to its competitors [67].
The VERSANT HIV-1 RNA 1.0 assay has been pre-qualified by the WHO.
The artus HIV-1 QS-RGQ assay has a linear range of 45 HIV-1 RNA copies/mL to 45 million copies/mL (using
automated extraction) and can detect HIV-1 group M (subtypes A-H) down to a limit of detection of approxi-
mately 35 copies/mL. The time to result is about 5 to 6 hours for 24 samples. Performance of the artus assay
has been evaluated and is comparable to that of the Abbott RealTime system [68].
QIAsymphony® SP/AS
Sample preparation for the artus HIV assay may be conducted manually using the CE-IVD marked QIAGEN
QIAamp® DSP Virus Kit, which provides silica-membrane-based RNA purification using a vacuum process.
Fully-integrated automated sample preparation and assay setup is also available using the QIAsymphony SP/
AS instruments. The QIAsymphony SP can process 1 to 96 samples (in batches of 24) with sample volumes up
to 1 mL. It is a ready-to-run instrument that requires minimal installation. The SP can be combined with the
Technical Report 47
2013 HIV/AIDS Diagnostic Technology Landscape
QIAsymphony AS device in a fully-integrated system that can automate the entire workflow. To reduce manual
handling and minimize the risk of sample contamination, samples processed on the SP can be transferred au-
tomatically to the AS, or the two instruments can be operated independently.
The SP/AS system includes touchscreen controls, bar code-labeled sample tubes containing pre-filled reagents,
and allows for continuous loading in batches of up to 24 samples plus internal controls. The QIAsymphony
SP/AS instruments can also be integrated in laboratory information management systems. In addition to HIV,
the artus panels for QIAsymphony Rotor-Gene Q (RGQ) include assays for the hepatitis B and C viruses, plus
a transplantation/immunosuppressed panel, with assays for detection and quantification of cytomegalovirus,
Epstein-Barr virus, herpes simplex virus 1 and 2, varicella-zoster virus, and BK virus.
Despite its relatively small size, the miniMAG has reasonably high through-
put – with 12 extractions in 45 minutes (using 1 miniMAG system) and 24
extractions in 60 minutes (using 2 miniMAG systems). The instrument has
one standardized extraction protocol for multiple downstream applications
and is considered to have an easy workflow for operators.
The price of the miniMAG extraction device is about €6,800 (~$9,000).
48
Existing Viral Load Technologies
For higher throughput needs, the easyMAG is an automated benchtop nucleic acid extraction device that is able
to perform 24 extractions in as little as 40 minutes (and offers the possibility to extract different samples types,
to be used in several applications, in the same run). The instrument (pictured below) has one generic extraction
protocol (DNA/RNA) and one set of reagents for all applications, which together with touch screen technology,
makes the process relatively simple.
The extraction process uses magnetic silica based beads and is based on Boom® technology. The average price
of the easyMAG instrument is approximately €72,000 (~$95,000).
NucliSENS EasyQ® Amplification and Detection—The NucliSENS EasyQ® is a closed system made up of a
real-time NASBA amplification step with automated data analysis; the instrument is pictured below. No post-
amplification steps are required. The risk of contamination is decreased in the system as the tubes containing
the amplification product remain sealed throughout the analysis. The viral load of each sample is calculated
automatically and displayed on a computer.
The EasyQ analyser is compact, weighing only about 45 pounds, and can fit easily onto the average laboratory
workbench. Further, amplification and real-time detection of 48 samples require only 60 minutes.
The average price of the analyser is approximately €37,100 (~$49,000).
Technical Report 49
2013 HIV/AIDS Diagnostic Technology Landscape
NucliSENS Connectivity—bioMérieux also provides NucliSENtral™, which is an integrated software system that
can be used to link NucliSENS® easyMAG® and NucliSENS EasyQ® with a Laboratory Information System.
bDNA Technology
Versant™ 440 Molecular System (Siemens Healthcare Diagnostics, Inc.)
Siemens Healthcare Diagnostics, Inc. manufactures the VERSANT® HIV-1 RNA 3.0 Assay, which is a bDNA
sandwich nucleic acid hybridization method that targets a well-conserved region of the gag gene and quantifies
plasma HIV-1 by amplifying the signal rather than the target RNA. A phosphorescent chemical that binds to
the HIV particles is added to the sample. The amount of light is measured and is converted into a viral count.
This assay does not require viral RNA purification/extraction or PCR amplification steps. The bDNA assay is
performed on the VERSANT 440 analyser and has a linear range of 50 to 500,000 copies/mL; it can detect HIV-1
Group M (subtypes A through G). The performance of the assay correlates well with that of the Roche AMPLI-
COR assay [72, 73].
As indicated above, the Siemens VERSANT™ 440 Molecular system, pictured below, uses bDNA technology,
which eliminates the need for nucleic acid extraction steps. Compared to PCR methods, this lowers the risk of
contamination. Like the VERSANT kPCR Molecular System, this technology can be set up in a single room; no
separate clean room is required. The technology is also a walk-away system with samples being run in a 96 well
format, with automated reagent preparation and delivery that allows processing of up to 168 samples per run.
However, the time to result is about 24 hours, including 2.5 hours of hands-on time by the test operator.
The VERSANT 440 analyser has a relatively compact footprint.
50
Existing Viral Load Technologies
The ExaVir Load assay is more manual than most of the other viral load assays described herein, but it is gener-
ally less expensive than other current molecular detection methods. Samples are processed in batches of 30. A
total of 180 samples can be run during a five-day week. The total time to result for 30 tests is 48 hours, which
Technical Report 51
2013 HIV/AIDS Diagnostic Technology Landscape
includes 5 hours of hands-on time for the operator. The remaining time is used for incubations. The hands-on
time per test is comparable to running some of the automated NAT-technologies.
An advantage of the assay is that because the ExaVir Load determines viral load based on quantification of RT
activity and does not target a specific nucleic acid sequence, it can measure any HIV type or subtype with high
accuracy, including O and N groups. The measuring range of the assay is the equivalent of about 200 to 600,000
copies/mL (or 1 to 3,000 femtograms/mL). There is performance data available on the ExaVir Load showing
good correlation with the AMPLICOR assay [74,75].
The ExaVir Load assay requires a vacuum pump (supplied with the first order), a standard ELISA plate reader,
a vortex, a 33ºC incubator and a freezer, in addition to other basic lab commodities. Further, in order to analyse
results, the ExaVir Load Analyser software is required (supplied with the first order) as well as a computer with
Microsoft Excel® and Adobe® Reader®.
The cost of the ExaVir equipment supplied by Cavidi is about $9,000 to $10,000, and the cost per test, which
varies according to volume, ranges from about $13 to $15. Despite its reasonable cost and the ability to use the
assay in district hospitals and other second tier settings, the ExaVir Load has not gained significant traction,
likely because of its manual nature and relatively long time to result.
52
Viral Load Technologies in the Pipeline
The current viral load POC pipeline is presented in Appendix 2. Since this report was first published in 2011,
additional platforms for POC viral load have been added to the pipeline. However, like the CD4 POC pipeline,
there have been delays in the introduction of POC viral load platforms. In 2011, it was expected that at least two
POC viral load platforms would be introduced into the market that year, but in fact, only one platform has been
introduced to date. It is now anticipated that at least 1 to 2 additional products will be launched in 2013. Similar
to POC CD4 platforms, these delays can be attributed primarily to the technical challenges of product develop-
ment, and in some cases, difficulty in obtaining sufficient funding to complete such development.
The Alere Q HIV viral load test is comprised of a disposable cartridge that contains all reagents required for the
assay in a stabilized form. The cartridge provides for sample collection, cell lysis, amplification target capture,
reverse transcription, PCR amplification and real time fluorescence detection based on competitive reporter
probe hybridization on an integrated micro probe array. The company expects sensitivity and specificity will be
comparable to current virological testing reference technologies (e.g., COBAS® AmpliPrep/COBAS® TaqMan).
The system detects HIV-1 Groups M, N, and O, and HIV-2.
The Alere Q platform is designed to require no manual sample preparation or pre-treatment. The required 25 µL
of blood can be collected via fingerstick, heelprick or venipuncture. In the case of either fingerstick or heelstick,
blood is applied directly into the test cartridge’s sample collection capillary as shown below. When using venous
blood, the sample is transferred to the cartridge capillary with a transfer pipette. Although a volumetric pipette
can also be used, it is not necessary as there is no need to apply a precise volume of blood to the cartridge. The
disposable assay cartridge is fully self-contained, and once capped, cannot be reopened; the cartridge remains
completely sealed. At no time does the sample or the reagent actually come into contact with the analyser, thus
greatly reducing any possibility for cross contamination. The actual hands-on time for the device is expected to
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be less than 3 minutes (i.e., sample collection and loading of the cartridge onto the analyser and subsequent
reading of result and cartridge disposal).
Test workflow for the operator is straightforward and consists of: (i) lancing the patient’s finger/heel (or col-
lecting blood via venipuncture) and wicking whole blood directly into the cartridge sample collection capillary;
(ii) manually capping the cartridge; (iii) inserting the cartridge into the analyser; (iv) entering the operator and
sample IDs on the analyser; and (v) selecting “run.” When the assay is complete, audible and visual prompts
alert the operator to remove the cartridge from the instrument and the viral load results are displayed on a built-
in screen. The result can be printed immediately, but results are also stored in an on-board archive and can
be viewed and printed at a later date, exported to a USB memory stick or exported to a remote server via the
use of an optional USB connectivity package that makes use of GSM mobile telephone network infrastructure.
Additionally, the system will be fully compatible with existing EQA programs.
It is anticipated that the product will be commercially available in 2013. The company will be seeking CE-IVD
marking of the Alere Q system in the EU and FDA clearance throughout 2013. Pricing for the instrument and
disposable test cartridges has not yet been determined.
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Viral Load Technologies in the Pipeline
To aid the operator and provide reliable results, the Liat Analyser incorporates a variety of intelligent and
advanced features: barcode data entry avoids errors in sample or assay coding and on-screen prompts provide
easy-to-follow directions to guide the operator through sample loading and tube insertion. Sample metering
capabilities ensure that the correct volume of sample is used for the test, or outputs a warning if the sample
volume is insufficient. A comprehensive set of sensors further monitors system operations in real time and auto-
matically recovers from errors or aborts the assay to prevent incorrect results from being reported. An internal
control contained in each Liat tube is processed and detected with the sample to ensure the proper function of
each step of the assay process. PCR curve pattern recognition and automated data interpretation provide results
in plain English. The developer states that, collectively, these sophisticated features ensure the quality of results
when testing is performed by minimally-trained operators.
The analyser is small and portable and it executes all required assay steps and reports a quantitative test result
within 30 minutes to just under 1 hour, depending on the limit of detection required by the user. For example,
if the user wants to measure viral load down to 500 to 1,000 copies per mL, the device takes about 30 minutes
to produce a result; if the user wants to measure viral load at 50 copies per mL, the device will take about 55
minutes to arrive at the result.
The Liat Analyser has an internal optical system that provides six independent optical detection channels for
real-time monitoring and quantification, allowing for the detection of multiple targets in each test and providing
future expandability for detection of multiple diseases at lower per test cost. It can be powered by AC mains or
by battery, allowing mobile use.
The company expects that the list price for the Liat Analyser, which is currently $25,000, may decrease for
resource-limited settings. Susan A. Fiscus at the University of North Carolina at Chapel Hill has completed an
evaluation similar to that previously conducted by Robert Coombs at the University of Washington, comparing
the Liat Analyser’s viral load detection capabilities against the Roche COBAS® and the Abbott m2000 system. In
both evaluations, the performance of the Liat device compared favorably to the predicate devices. The HIV viral
load assay for the Liat platform is ready for market launch, but actual launch will depend on the availability of
financing for in-country clinical evaluations and implementation.
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The system (pictured below) has three components: (i) the disposable cartridge, which contains integrated
sample preparation and assay modules; (ii) a small, low cost, battery-powered processing unit; and (iii) a small,
portable reader, with touchscreen display, that can run on a rechargeable 8 hour battery or mains power. The
system is easy to use and will require at most 1-day training for operators.
EOSCAPE System:
single-use, all reagents Deposit fingerstick blood into Scan results in 2 minutes; intuitive
on-board cartridge, 45 minute runtime touchscreen with data connectivity
The testing process is straightforward. The operator inserts a disposable cartridge into the small processing
unit. Using a fingerstick lancet, 50 µL of whole blood is applied directly into the cartridge; no external sample
preparation is required. The sample is automatically processed in 45 minutes; the operator then inserts the pro-
cessing unit into the reader for a quick 2 minute scan. Equipped with a simple touchscreen interface, the reader
is capable of transmitting test results through wired and wireless connectivity. For higher patient loads, multiple
processing units can be used for parallel processing, ~50 samples per day per analyser.
Two HIV NAT cartridge models will be available as part of the EOSCAPE-HIV system. The EOSCAPE-HIV-D car-
tridge will provide a qualitative HIV-1 RNA test result with a threshold above 1,000 copies/mL. The EOSCAPE-
HIV-Q cartridge will return a fully quantitative viral load result. Full scale validation and clinical testing of the
Wave 80 EOSCAPE HIV-1 RNA rapid test is expected to begin in late 2013, followed by in-country testing for
market launch.
Truelab™ Real Time micro PCR System (Molbio Diagnostics Pvt. Ltd. [A Tulip Group – Bigtec labs
partnership])
Molbio Diagnostics has developed a comprehensive, rapid, near-patient RT PCR platform, called the Truelab™
Real Time micro PCR System. The system is portable and includes all instrumentation, reagents and essential
accessories that are required for the operator to conduct a real time, quantitative PCR assay, from sample prepa-
ration through to final result reporting, all within one hour. A Truelab™ micro PCR printer is also available. The
system works on ready-to-use Truenat™ disease-specific assays that are stable at room temperature. Assays for
MTB, HBV, dengue fever, Chikungunya, H1N1 and malaria (both p. falciparum and p. vivax) are currently avail-
able, and assays for HIV viral load, among others, are in development.
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Viral Load Technologies in the Pipeline
The testing process begins with sample collection (blood, serum or plasma) followed by extraction, which uses
the Trueprep™ MAG Sample Prep Device and Trueprep Mag sample prep kits. The extraction process takes about
20 to 25 minutes per sample. From there, 6 µL of the extracted nucleic acid is dispensed into the reaction well
of the disease-specific Truenat™ micro PCR chip. The chip, which contains all of the chemistry required to com-
plete an assay, is then inserted into the Truelab™ Uno Real Time micro PCR Analyser, pictured below. Thermal
cycling takes place automatically within the analyser.
During amplification, the Truenat micro PCR chip exponentially releases flurophores. These signals are captured
by sensors and are displayed as an amplification curve on the Truelab screen. Test results are compared to lot-
specific standard values preset into the Truenat chip, which enables quantitative estimation of the test analyte
and display as RT PCR results in approximately 30 minutes. An internal control is provided from the extraction
stage for a complete validation of the test results.
Test results are automatically stored in the analyser memory (up to 5,000 results), can be printed, transported
wirelessly to any server/compatible device by Wi-Fi, GPRS, Bluetooth or even SMS.
The HIV viral load assay is expected to launch in the third quarter of 2013.
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The SAMBA HIV test uses 200 µL of plasma for the semi-quantitative viral load assay, 500 µL of plasma for the
qualitative acute infection assay and 100µL of whole blood for the EID assay. The sample preparation process is
an aqueous-based method involving cell lysis and nucleic acid extraction using a solid phase. The amplification
and detection process is integrated into a closed cartridge to prevent amplicon contamination and targets the
LTR region of the genome. Amplification is based on both target and signal amplification (see below).
A capture probe is used to capture the target sequence, and a detection probe with multiple hapten labels is sub-
sequently attached to the target sequence, enabling amplification of the signal to improve sensitivity and allow
visual reading. The lattice structures, shown above, ensure visual detection of the RNA or DNA target, which
can be visually read off of a test strip within 25 minutes. The test strip is based on a nitrocellulose membrane
in a lateral flow format.
Based on an assessment with the WHO international standard HIV RNA genotype panel containing 400 cp/mL,
the SAMBA assay was able to detect all HIV-1 subtypes. Several evaluations have taken place:
• S
AMBA semi-quantitative viral load test: evaluated in clinical samples from St. Thomas Hospital, Royal
London Hospital and two MSF sites (Chiradzulu, Malawi and Arua, Uganda).
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Additional Viral Load Technologies in the Pipeline
• S AMBA EID assay: evaluated in HIV-positive or negative adult whole blood clinical samples in comparison
with DBS testing using the Roche AMPLICOR assay and the COBAS AmpliPrep/COBAS TaqMan carried
out by laboratories in Zambia and Uganda.
• Field evaluation in infant blood will take place in Uganda and Malawi during second quarter of 2013 upon
receiving ethical approvals.
Currently, the total assay time is 2 hours for the SAMBA EID and 90 minutes for the semi-quantitative viral load
assay. SAMBA is suitable for use at the district hospital level or in large clinics in sub-Saharan Africa where
electricity is available.
Diagnostics for the Real World, Ltd, the spinout company of DDU located in California, is the manufacturer
of the SAMBA system. The SAMBA viral load assay was released in Malawi in the second quarter of 2013 and
is expected to be released shortly in Uganda. The expected market release date for the EID assay is the fourth
quarter of 2013. There is currently no pricing information available from the company.
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The GeneXpert® System integrates and automates sample preparation, amplification, and detection in a single-
use, self-contained cartridge, pictured below. Most liquids and dry reagents along with enzymes are prefilled
so that pre-analytical steps are minimized, greatly reducing opportunities for sample mix-ups and operational
errors. GeneXpert cartridges can handle a variety of sample volumes (milliliter range) within macrofluidic
chambers and then concentrate the target material down to microfluidic volumes, which can increase the sen-
sitivity of the assays, if needed. Currently, the HIV viral load assay involves a single transfer of 1 mL of plasma
directly into the open sample port of the cartridge by using a single disposable Pasteur transfer pipette. By car-
rying out all dilution and extraction steps inside the chambers of the cartridge, plastic disposables are kept to
a minimum.
Further, the GeneXpert® System is modular. Individual modules contain solid state circuitry that control tem-
perature, pressure, rotation of the valve that moves the liquid between reservoirs, and the detection software.
These individual modules are packaged in units of 1, 2, 4, 16, 48, or 80, and the latter two systems are fully
automated, walk-away robotic instruments developed for high-throughput laboratory applications. Addition-
ally, the modules can be removed and replaced individually so that the entire system is not incapacitated if one
module fails.
The GeneXpert® System is sufficiently simple that training can usually be completed within half a day. Further,
although the system was designed to use AC power, its low wattage requirements allow it to be powered by a
12VDC/120VAC voltage converter in mobile laboratories, and it has also been installed in remote clinic sites
powered by solar panels. The GeneXpert software comes pre-installed on a desktop or laptop computer and
results can be displayed for each module in real time or uploaded via an Internet connection to a central data-
base. Wireless data connections via satellite phone networks are in development, as is a cloud-based system for
remote access, online system calibration, and interfacing with laboratory information systems.
NWGHF LYNX Viral Load Test and Platform (Northwestern Global Health Foundation)
The Northwestern Global Health Foundation (NWGHF) in collaboration with Quidel Corporation is developing
a POC rapid RT-PCR testing platform that will be both easy-to-use and low cost. The product design calls for a
small device (pictured below) that can process multiple samples at a time with test results in 60 to 90 minutes.
The proposed viral load assay will achieve a limit of detection of 1,000 copies/mL of plasma, using ~150 µL of
whole blood that is converted into plasma with simple sample preparation materials provided by NWGHF.
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Additional Viral Load Technologies in the Pipeline
The processor is powered by an external power transformer that connects to either an AC or DC power cable
that, in turn, connects to an AC or DC power socket in the clinic or laboratory. A fully-charged battery will
complete the cartridges in the processor.
NWGHF/Quidel expect to launch this product in late 2014 or early 2015.
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Lumora’s assay system and technology is flexible enough to be utilized in high-throughput applications as well
as in highly decentralized settings. The hardware (pictured below) is also portable and powered by mains or
a battery, culminating in a low-cost unit with a small footprint that can be used in challenging environments,
including non-laboratory settings.
The successful launch of a food pathogen test in sixty countries that includes Lumora’s technology demonstrates
that BART is a proven technology for low resource settings because it is easy-to-use and relatively low cost. Wider
adoption of this technology could be expected in laboratories where currently available methodologies are not
being used, due to ongoing high costs or the practical limitations created by the accessibility of consumables,
power, and a laboratory environment that is suitable for highly sensitive preparation and testing procedures.
Lumora believes that BART will offer a simple and effective method for monitoring viral load in developing coun-
tries and could support current efforts to increase the effectiveness of, and adherence to, ART regimens.
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Additional Viral Load Technologies in the Pipeline
available Genie®, a portable instrument developed by OptiGene Ltd. In order to accomplish this, Ustar plans
to: (i) develop a modified CPA reaction for amplification from direct plasma or minimally-processed whole
blood sample (i.e., diluted/buffered); (ii) develop a quantitative RT CPA viral load assay with internal control
using a novel RT-active DNA polymerase; and (iii) develop new user interface software for automatic viral
load calculations.
The final Ustar diagnostic test kit is expected to comprise only reagents and a portable device for amplification
and detection. Reagents will consist of glassified enzymes for ambient temperature transport and storage with
a reconstitution buffer.
The testing process will require the user to: (i) take a finger/heel prick and place a drop of blood directly onto
a plasma separating filter; (ii) add the reconstitution buffer using a preloaded pipette to the glassified enzyme
contained in the amplification tube; (iii) punch a fixed volume disk out of the plasma containing a portion of
the filter, and place it directly into the amplification tube; and (iv) close the tube and place it in the Genie instru-
ment for amplification and detection.
A fully-quantitative viral load measure will be available in as little as 20 minutes (depending on the limit of
detection required), and the sample can be run for 45 minutes to ensure a viral load measure of <1,000 cp/mL.
On-board software will calculate an offset value based on any delay in the amplification of the internal control
caused by inhibition and a simple readout – “number of RNA cp/mL”, “not detectable”, or “invalid” – will be
available to the user and will be automatically uploaded to an external server (e.g., a national HIV program),
along with detailed information regarding each run.
The development of the Ustar viral load assay is in an early stage, and its completion is dependent on funding.
No market launch date has yet been determined.
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The system will combine the strengths of RT technology with the advantages of an automated walk-away plat-
form. This should provide fast and robust viral load monitoring for all HIV types and subtypes. The company’s
planned launch date for the AMP system is the end 2014.
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Additional Viral Load Technologies in the Pipeline
All reagents and consumables required for lysis, extraction and PCR, which is optional, are loaded into a unit-
ized reagent strip (URS), which simplifies instrument set-up.
The BD MAX uses microfluidic-based real-time PCR, with PCR reactions being performed individually in dispos-
able microfluidic cartridges. The company indicates that small reaction volumes together with microthermal
circuits allow for short thermocycling times. Detection is based on multi-wavelength fluorescence detection.
Currently, the BD MAX System is cleared for use with the BD MAX GBS (group B streptococcus) Assay.
Other assays are being developed, but there is no current indication from the company that a viral load assay
is planned.
The Enigma system includes: automated sample preparation, real-time PCR instrumentation, unique direct heat-
ing thermal cycling, novel real-time PCR chemistries and freeze-dried PCR reagents. Sample analysis consists of
three steps: (i) dispensing the sample into a single-use cartridge; (ii) loading the cartridge into the instrument
and selecting test (at which point the operator can walk away); and (iii) reading the result.
Although Enigma has plans to develop assays across blood-borne infectious diseases, at this point in time, there
is no indication that a viral load assay is planned for the ML platform in the near future.
BioHelix Corporation
BioHelix Corporation, which was recently acquired by Quidel Corporation, is developing assays based on the
company’s iNAAT platform for infectious diseases. BioHelix has developed an “instrument-free” molecular di-
agnostic platform (the IsoAmp® Molecular Analyser) for these assays. The platform consists of the company’s
proprietary helicase-dependent amplification (HDA) technology (which uses a helicase enzyme to unwind
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2013 HIV/AIDS Diagnostic Technology Landscape
double-stranded DNA into single strands, thus eliminating the need for a thermocycler and providing a method
for assay development) and an enclosed disposable detection device called the BESt™ (biohelix express strip)
Cassette, which minimizes cross-contamination. Workflow on the analyser consists of simple sample prep, iso-
thermal amplification at 65ºC followed by amplicon detection via the BESt Cassette. Per the company, the test
results on the strip can be available in as little as 10 minutes. The company is currently developing integrated
sample prep, dry reagents and a faster HDA platform.
The first assay being developed by BioHelix, with funding from the National Institutes of Health (NIH), is for
genital herpes. The company recently received FDA 510(k) clearance on the IsoAmp HSV Assay for detecting
genital and oral herpes. Additional assays are in development, one of which is a qualitative test for HIV, which
is also funded by NIH. The project involves the development of a primer than can detect different HIV subtypes
and, per the company, this could be converted into a quantitative assay with the addition of a fluorescence
label probe.
BioHelix has received a two-year SBIR grant from the NIAID to develop a quantitative HIV RNA assay based
on its isothermal HDA amplification technology and a portable fluorescence analyser that is capable of
monitoring fluorescent signals in real time. Pursuant to the SBIR grant, BioHelix will collaborate with Dr.
Jeanne Jordan at George Washington University to develop a low-cost HIV viral load test for use at the POC
in resource-limited settings.
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Additional Viral Load Technologies in the Pipeline
RPA is a nucleic amplification system that uses prokaryotic enzymes (recombinases) to guide synthetic oli-
gonucleotide primers to target sites in sample nucleic acids. Similar to PCR, the process involves exponential
amplification of the target by reiteration of oligonucleotide-primed DNA synthesis. But, unlike PCR, RPA does
not require a thermocycler. Instead, RPA will operate at low and constant ambient temperatures (from 24ºC to
as high as 45ºC). This means that less power is demanded than with PCR. In addition, RPA begins operating
the moment a sample comes into contact with reagents; no melting of DNA or heating of RNA is required first.
This cuts the time for amplification.
Although the TwistDx system does not yet include any sort of integrated sample preparation technology, the
company has introduced the Twista™ portable real-time fluorometer for monitoring/detection. The Twista con-
tains a heated incubation chamber for a strip of eight reagent tubes. It has a small footprint and can be used
with a rechargeable battery pack. Detection data can be analysed by PC-link or the device can function as a
stand-alone unit capable of storing and running user-defined data. The basic kit contains all enzymes and
reagents necessary for the amplification of DNA.
Currently, TwistDx is producing two of the building blocks for an integrated, portable system that could be
applied to viral load testing, but at this stage, the products are for research use only. Therefore, while the tech-
nology is promising, any HIV assay using this chemistry is only in very early stage development.
ALL does not currently have a viral load assay, but in 2009 was awarded a four-year, $5.2 million contract from
the National Institute of Allergy and Infectious Diseases (NIAID) for the development of a rapid, POC diagnostic
device for the detection of HIV in low-resource settings. The development of a viral load assay may flow from
this work.
31 Note that this summary of the ALL platform has not been updated by the company since September 2012.
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To date, Epistem has developed a TB assay, which is CE-IVD marked, for the Genedrive. In addition to detecting
TB, the platform also detects Rifampicin resistance mutations. Test results are available in less than 60 minutes.
The Genedrive platform is integrated with a simple extraction cartridge based on composite paper that allows
extraction in a single step. The sample is manually transferred with one pipetting step into the Genedrive reac-
tion cartridge. Analysis and diagnosis are integrated. Epistem expects to launch the TB assay in India in 2013
and has entered into an agreement with BD for the supply and distribution of the platform in the rest of the
world (excluding India and the Indian sub-continent).
Epistem also plans to develop Genedrive as a diagnostic platform for a number of additional infectious diseases,
including HCV and HIV (viral panels).
32 The RUO (Research Use Only) designation is required by the FDA for non-FDA approved in vitro diagnostic products that are manufactured in the United States and exported for sale and use
outside the United States.
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New Technologies for EID in the Pipeline
bring the advantages of RT technology to EID, like subtype independence, cost-efficiency and accessibility,
while producing results that should be at least as sensitive and specific as DNA PCR testing.
The DNA PCR qualitative tests, like the RNA PCR quantitative tests discussed earlier, require sophisticated labo-
ratory infrastructure, including clean rooms and trained laboratory technicians, and are subject to some of the
same drawbacks and limitations as RNA PCR tests for implementation in resource-limited settings. Nonetheless,
the Roche AMPLICOR Qualitative test, which is considered the gold standard for DNA PCR testing, has had
considerable uptake in resource-limited settings. One reason for this is that the cost of the assay is lower than
that of quantitative assays and another reason is that the use of DBS with this test is well established and the
performance of the test is well-accepted with DBS samples. The ability to use the test with DBS samples, which
have greater stability than fresh whole blood or plasma, has made it possible for countries to expand access to
testing into peri-urban and rural settings with the use of sample transport networks. In addition, the generic
equipment used with the AMPLICOR Qualitative test is quite a bit less expensive (approximately $25,000) than
either the TaqMan or the RealTime amplification platform (the cost of which can range from about $45,000 to
$150,000). Therefore, cost has not been as big an issue as it has been for the introduction of viral load testing.
The assay procedure involves collecting about 80µL of heel-stick blood from the infant using a blood collection
tube; separating plasma from the sample; adding buffer to the sample and “heat shocking” it in a small, battery-
powered processor device; inserting the rapid test strip into the device; and waiting approximately 30 to 40 min-
utes to read the result. The total assay duration is about 45 to 50 minutes. The procedure is illustrated below.
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Note that, similar to other rapid tests, if only the top line appears (the control line only), the test is negative
and the infant has not been infected with HIV. If both lines appear (the control line and the test line), the test
is positive and the infant has been infected with HIV. If the top (control) line does not appear, the test is invalid
and must be re-run.
In early testing, the assay has shown about 95% sensitivity and 99% specificity. The price of the processor
device is expected to be between $700 and $900, and the per-test cost is expected to range from $7 to $15. Clini-
cal and field trials on the assay commenced in 2012, with availability expected in 2013.
The cartridge incorporate probes, primers, enzymes, buffers and controls for sample purification, amplifica-
tion and detection, and because it is a closed cartridge system, there is no PCR product cross-contamination.
Cartridge design permits storage at ambient temperatures for prolonged periods. All waste is captured in the
cartridge for safe disposal.
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Viral Load Technologies and Future Directions for Viral Load Testing
Micronics has a number of tests in development, including an assay for Shiga toxin-producing E-coli, as well as
other infectious disease diagnostics. Commercial launch for a first test and system is targeted for 2013. Micron-
ics has also been funded to develop qualitative assays for each of HIV, HBV and HCV; however, the company
has no current plans for a quantitative viral load assay.
Viral Load Technologies and Future Directions for Viral Load Testing
The Technologies
Unlike CD4 testing where even laboratory-based systems have become the norm and are well-established in
resource-limited settings, the same cannot be said of viral load testing. With the exception of South Africa and
Brazil, where viral load testing is routinely conducted on a large scale, with more than 1 million viral load
tests done in each country annually on systems from Siemens (Brazil) and Roche/Abbott (South Africa). Other
countries that have established viral load testing on a relatively large scale include Botswana and Thailand.
Beyond that, there is very little viral load testing done in the public health sector in resource-limited settings.
There are a few countries, including China, Kenya and Lesotho, that are increasingly using viral load, but still
on a small scale As indicated earlier, the reasons for this include cost, infrastructure requirements, the need for
trained laboratory technicians and WHO guidance on the use of viral load testing that stops short of calling for
its routine use.
Analogous to CD4 testing, in order to reach patients in peri-urban and rural settings with laboratory-based viral
load platforms only, it is necessary to set up sample transport networks to transfer patient blood samples to
the reference laboratory for testing and to return results to the patient. Since viral load tests generally require
plasma for extraction, there is a requirement to centrifuge the whole blood samples from patients, usually
within 6 hours of the blood draw. In addition, plasma must be transported and stored under refrigeration.
These demands put pressure on the sample transport system and add costs to the process. The introduction of
the use of DBS with some of the laboratory-based viral load platforms (Roche Taqman, Abbot RealTime, and
bioMérieux EasyQ) and its use for EID testing, help to make the sample transport process more manageable,
removing some of the time pressure.
Future Directions for Viral Load Testing and Implications for Viral Load Technologies
Given the growing consensus of the importance of viral load testing for detection of virological failure for
patients on ART, it is possible that there will be a movement of testing algorithms towards routine viral load
testing. The frequency of testing remains to be determined, but if ease of testing and cost allow, in the future
it might be as frequent as every 1 to 2 months or more often (analogous to glucose testing for diabetics). The
purpose of global ART should be the effective, long-term management of chronic patients so as to ensure the
successful treatment of as many people for as long as possible. Early detection of viral resistance and reduc-
tions in treatment efficacy on an individual basis, followed by improvements in adherence to save the existing
treatment regimen or early diagnosis of treatment failure requiring a switch, is essential for this goal. Patient
management algorithms will need to be upgraded to accommodate the effective use of viral load information.
As discussed in connection with the scale-up of CD4 testing, the level of access required for viral load testing
will likely necessitate centralized testing facilities, including the so-called super labs that carry out very high-
volume testing, and at the same time, a drive towards POC testing. As indicated above, there has been limited
launch of the SAMBA viral load technology in 2013 and additional viral load and EID POC technologies are in
development, with possible launch of additional products in 2013. It is too early to predict the exact pricing of
the POC devices and tests, but it is anticipated that the price per test will be at or below $10.00 per test. Com-
petition among POC and non-POC platforms could eventually lead to pricing similar to CD4 pricing levels.
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What Should the HIV Diagnostic Landscape Look Like Going Forward?
This report has detailed the current HIV diagnostic landscape from detection of the virus through staging and
monitoring of the disease for the HIV-positive patient. Given the emphasis of Treatment 2.0 on ART efficiency and
simplification, consideration should be given to how the diagnostic landscape must adapt and change over the
next few years in order to achieve robust, high-quality, efficient, cost-effective and accessible diagnostic services
for the necessary complement of testing required to diagnose, stage and monitor the HIV patient effectively.
Arguably, diagnostic services should be delivered strategically, whether centrally or at the point of care, using
the most effective, robust and efficient technologies available. A significant increase in the level of access to
such robust, high quality diagnostics will play a critical role in: (i) detecting and treating HIV/AIDS early, there-
by maximizing the preventive impact of treatment; (ii) detecting drug resistance early, thereby reducing the
spread of drug-resistant strains of the virus, and (iii) preserving drug regimens, thereby increasing the period of
successful treatment for each patient.
While considerable advances have been made in expanding access to tests for initial diagnosis of HIV, similar
advances in access to tests for infant diagnosis and ART staging and monitoring are needed, and new technolo-
gies in the pipeline are likely to bring about significant changes to how these tests are delivered. At the same
time, new platforms for high-volume testing are also becoming available, allowing cost-effective consolidation
of testing in high-volume centers. The pace at which countries implement an optimized mix of high-volume
centralized and low-volume POC diagnostic services tailored to suit their individual needs will determine the
impact these improved technologies have on access, efficiency and quality over the next decade.
There are a number of important areas for future work to improve diagnostics for HIV/AIDS. These include:
• Focus on quality improvements at all levels of diagnostic testing for HIV/AIDS;
• Analysis of the optimal mix of monitoring technologies relative to country characteristics;
• Mapping barriers to, and fostering acceleration of, new technology introduction, especially for POC
technologies; and
• Improving systems for sample referral and results distribution for central labs.
Strategic funding on the part of UNITAID and other funders could make a difference in a number of these areas,
including in the acceleration of new POC diagnostic technology introduction. Recently, UNITAID committed to
funding several projects to facilitate and support the commercialization of POC diagnostics, including a project
that will fund procurement of quality POC diagnostics and a project that will fund pre-market evaluation of new
POC diagnostics.
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APPENDIX 1: Operational Characteristics of Diagnostic Platforms
APPENDIX 1:
Operational Characteristics of Diagnostic Platforms
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APPENDIX 1: Operational Characteristics of Diagnostic Platforms
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APPENDIX 1: Operational Characteristics of Diagnostic Platforms
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BD FACSCount™ System
Type of Technology Bench-top, bead-based Flow Cytometer
Single tube reagents measure absolute and percentage CD4 (FACSCount CD4 Reagents);
Output Single tube CD4/CD3 reagents measure CD4 and CD3 T-cells; Paired tubes of CD4/CD3
and CD8/CD3 reagents for enumeration of CD4, CD3 and CD8 T-cells
Turnaround time 60 – 90 minute incubation, 2 – 3 minutes per test
Capacity Approximately 30 – 80 samples per day
Throughput per technician/ per day 20 per hour, after initial 60 – 90 minute incubation
0.5 – 5 mL whole blood collected in EDTA anticoagulant; staining to take place w/n 24
Sample needed and stability
hours of blood draw; analysis to take place w/n 48 hours of blood draw
Required. Process: (i) blood is collected and added to tube; (ii) sample is vortexed and
Sample preparation and protocol
incubated; (iii) fixative is added to the tube, which is vortexed and incubated; and (iv)
complexity
sample is vortexed and run on the instrument.
Reagents are shipped to customers with an expiration date of 5 months or longer;
Reagent stability
reagents must be stored at 2º – 8º C (36º – 46º F)
Cost/test Volume based; ranges from approximately $3.50 – $10.00 per test
Cost/instrument Approximately $30,000
FACSCount and paired tube reagents, FDA approved and CE-marked; CD4/CD3 reagents
Regulatory Status
neither FDA approved or CE-marked.
Width: 43.2 cm
Physical dimensions
Height: 38.1 cm
(W x H x D)
Depth: 55.9 cm
Weight 25.9 kg (57.1 lb), fluid reservoirs empty
3rd party supplies Refrigerator, vortex and pipettor; cost: approximately $1,500 – $2,500
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BD FACSClearCount™ System
Type of Technology Bench-top, bead-based Flow Cytometer
Output Single tube reagents measure absolute and percentage CD4 (BD CD4 Assay Kit)
Standard sample preparation mode: 30 – 180 minutes, including incubation and run
Turnaround time (time dependent on number of samples loaded on carousel).
Manual sample preparation mode: 30 – 40 minute incubation time; 1 – 2 minutes per test
Standard sample preparation mode: approximately 30 – 60 samples per day
Capacity
Manual sample prep mode: approximately 60 – 120 samples per day
Standard sample preparation mode: 30 – 180 minutes depending on number of samples
Throughput per technician/ per day loaded on carousel.
Manual sample preparation mode: 30 per hour, after initial 30 – 40 minute incubation
0.5 – 5 mL whole blood collected in EDTA anticoagulant; staining to take place w/n 24
Sample needed and stability
hours of blood draw; analysis to take place w/n 48 hours of blood draw
Required. Standard sample prep process: (i) blood is collected and added to tube; (ii)
instrument performs remainder of sample preparation
Sample preparation and protocol
Manual sample prep process: (i) blood is collected and added to tube; (ii) sample
complexity
is vortexed and incubated; (iii) fixative is added to the tube, which is vortexed and
incubated; and (iv) sample is vortexed and run on the instrument
Reagents are shipped to customers with an expiration date of 5 months or longer;
Reagent stability
reagents must be stored at 4º – 35º C (39º – 95º F) in sealed pouches
Cost/test Volume based; ranges from approximately $4.50 – $12.00 per test
Cost/instrument Approximately $38,000
Regulatory Status FACSClearCount Systems and BD CD4 Assay Kit, FDA approved and CE-marked
Width: 59.44 cm
Physical dimensions
Height: 57.24 cm
(W x H x D)
Depth: 60.78 cm
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PointCare NOW™
Type of Technology Desk top, flow cytometer
Absolute and percentage CD4 counts, WBC, hemoglobin concentration, total and
Output percentage lymphocytes, monocyte count and monocyte %, neutrophil count and
neutrophil %, eosinophil count and eosinophil %
Turnaround time 8 minutes
Capacity 50 samples per day
Throughput per technician/ per day ~40 – 50 samples per technician per day; no batching capabilities; walk-away operation.
40 µL whole blood collected in 2 mL or 4 mL vacuum K2 EDTA anticoagulant tubes
Sample needed and stability
provided by PointCare. Sample is stable for 8 hours from time of draw.
No sample preparation steps. (i) Draw venous blood into PointCare-supplied tube; (ii)
Sample preparation and protocol
scan sample ID with barcode reader; (iii) insert unopened sample tube into instrument
complexity
slot and press “run” button.
Reagents are stable for 12 months from date of manufacture when stored at 2º – 30º C
Reagent stability (36º – 86º F); transient exposure (shipping delay or temperature excursion) of 10 days at
50ºC (122º F).
Cost/test About $10.00 per test, including Daily Check™ controls
Cost/instrument Approximately $25,000
Regulatory Status FDA cleared (CLIA moderate-complexity rating); CE-IVD marked
Physical dimensions Width: 25 cm
(cytometer only) Height: 35cm
(W x H x D) Depth: 34 cm
Weight 12 kg (~26.5 lbs) (cytometer only)
3rd party supplies All phlebotomy supplies provided in CD4NOW™ Reagent Kit 100
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Pima™ Analyser
Type of Technology Portable bench-top, fixed volume cytometer
Output Absolute CD4 counts only
Turnaround time 18 – 20 minutes
Capacity Maximum of ~20 samples per day
Throughput per technician/ per day ~20 samples per technician per day; no batching capabilities; walk-away operation.
25 µL of capillary (fingerstick) blood wicked directly into the sample collector contained
in the Pima cartridge or 25 µL of venous blood collected in EDTA anti-coagulant tube.
Sample needed and stability
Cartridge must be inserted and tested within 5 minutes of sample application. When
using venous blood, sample is stable for 36 hours from time of draw.
No sample preparation required. For capillary blood: (i) lancet finger; (ii) wipe away
Sample preparation and protocol first drops and apply following blood drops to cartridge; (iii) close cartridge; (iv) insert
complexity cartridge into analyser; (v) analysis starts automatically; (vi) enter patient ID data; (vii) read
result from LED screen; (viii) print result
Reagent stability Freeze-dried reagents require no refrigeration. Stable for 12 months at 2º – 30°C
Cost/test Between $6 and $12 per test
Cost/instrument From $6,500 – $12,000
Regulatory Status CE-IVD marked; WHO pre-qualified
Physical dimensions Length: 22 cm (8.7”)
(cytometer only) Height: 16 cm (6.3”)
(L x H x D) Depth: 13 cm (5.1”)
Weight 2.54 kg (~5.6 lbs) (instrument only)
For venous samples: volumetric or transfer pipette
3rd party supplies
For capillary samples: sterile lancets, alcohol swabs, dry swabs (also available from Alere)
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Pima™ Analyser
100 – 240 V (A/C) at 47 – 63 Hz mains power
Analyser contains on-board rechargeable battery with sufficient capacity to run
Electric Power Requirements approximately 17 tests (actual duration will depend on conditions of use). Power
extender is available (module with an extended battery life and adaptors for charging
sources, including solar panels, car batteries, mains power).
• Operating Temperature: 10º – 40º C (50º – 104º F)
• Humidity: 10% – 95%; no direct sunlight; keep dry
Environmental Requirements
• Maximum altitude: Tested to 2,000 meters (~6,500 feet); actual maximum operating
altitude not evaluated.
Dedicated CPU integrated into instrument; approximately 1,000 test results can be
stored on the instrument archive; results can be downloaded via USB. Supports wired
Data Station
connectivity via LAN and wireless connectivity via an optional UBS GPRS modem to
connect to remote servers over mobile telephone networks.
Monitor LED mono-color screen integrated into instrument
Separate printer (prints on thermal paper); powered by the instrument (with
Printer rechargeable batteries on board)
L 95mm x W 93mm x H 66mm, weight: ~350 grams, including paper roll.
Bar-code Scanner Integrated into instrument for test cartridges only
Minimal training required. Lay person can be trained in less than half a day. Primary skill
Training
required is for correct lancet blood draw.
Analyser contains an integrated camera and computer that might be susceptible to
Maintenance damage if dropped. If damaged, low cost and portability of device allows for direct swap-
out replacement rather than on-site repair.
Extensive internal controls: sample volume control; reagent control; automatic control of
Internal QC
cartridge expiry date; internal process controls; automatic test identification
External QA Known to be compatible with Pima: QASI and UK-NEQAS
Infrastructure Requirements Can be used at all levels of health facility, including health centers or in mobile facilities.
User interface 16 button keypad
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BD FACSPresto™
Type of Technology Small, bench-top, fixed volume cytometer
Output Absolute CD4, CD4% and Hb
Turnaround time 2 – 5 minutes reading; plus incubation of cartridge (20 minutes)
Capacity Maximum of ~40 – 50 samples per day
Throughput per technician/ per day ~40 – 50 samples per technician per day; [batching] capabilities; walk-away operation.
~ 20 µL of capillary (fingerstick) blood wicked directly into BD cartridge or ~ 20µL of
Sample needed and stability venous blood collected in EDTA anti-coagulant tube. Cartridge must be inserted and
tested within a few hours of sample application.
No sample preparation required. For capillary blood: (i) lancet finger; (ii) apply blood
Sample preparation and protocol
drops to cartridge; (iii) close cartridge; (iv) incubate cartridge; (v) insert cartridge into
complexity
analyser; (vi) press “start”; (vii) read result from LED screen; (viii) print result
Reagent stability Dried reagents require no refrigeration. Stable for 12 months at 10º – 40°C
Cost/test TBD
Cost/instrument TBD
Regulatory Status Will be CE-IVD marked, FDA-approval will follow.
Physical dimensions Length: ~ 26 cm (10.2”)
(cytometer only) Height: ~ 28.5 cm (11.2”)
(L x H x D) Depth: ~ 25 cm (9.8”)
Weight ~ 5 kg (~ 11lbs) (instrument only)
For venous samples: volumetric or transfer pipette
3rd party supplies
For capillary samples: sterile lancets, alcohol swabs, cotton gauze, Band Aid
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BD FACSPresto™ (2)
100 – 240 V (A/C) at 45 – 65 Hz mains power
Electric Power Requirements
Analyser contains on-board rechargeable battery. Can be charged with cigarette lighter.
• Operating Temperature: 10º – 40º C (50º – 104º F) (ongoing validation)
Environmental Requirements • Humidity: 5% – 95% (ongoing validation)
• Maximum altitude: 2500 meters (8200 feet) (ongoing validation)
Dedicated CPU integrated into instrument; approximately 1,000 test results can be stored
on the instrument archive; results can be downloaded via USB. The USB port also can be
used to support an external blue tooth or GPRS/GSM module to communicate with SMS
Data Station
printer or the port would be developed but not enabled, providing an option for wireless
to be enabled post launch. Potential to install an SMS chip to transmit results or internal
calibration data.
Monitor LED multi-color screen integrated into instrument
Printer On board printer (prints on thermal paper);
Bar-code Scanner Integrated into instrument for test cartridges only
Minimal training required. Lay person can be trained in less than half a day. Primary skill
Training
required is for correct lancet blood draw.
Analyser contains an integrated camera and microscope that might be susceptible to
Maintenance damage if dropped. If damaged, low cost and portability of device allows for direct swap-
out replacement rather than on-site repair.
Internal QC Yes. Instrument will check itself each day and each cartridge will have onboard QC.
External QA Will e compatible with CD4 EQA programs (ongoing validation)
Infrastructure Requirements Can be used at all levels of health facility, including health centers or in mobile facilities.
User interface Touch screen keyboard on the device
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Visitect CD4
Disposable cartridge containing test strip (lateral flow) that measures CD4 proteins on T
Type of Technology
cells qualitatively (above and below 350 cells/µL).
Output Absolute CD4 counts only
Turnaround time ~40 minutes, including incubation
Capacity
Throughput per technician/ per day ~120 samples per technician per day; batching capabilities (up to ≈10/technician).
Sample needed and stability 30 µL of capillary (fingerstick) blood, or peripheral blood into EDTA anticoagulant
Protocol: (i) lancet finger; (ii) add whole blood to Well A of test strip using MicroSafe
Sample preparation and protocol pipette; (iii) wait 3 minutes; (iv) add 1 drop of supplied buffer to Well A and allow sample
complexity to run for 17 minutes; (v) add 3 drops of buffer to Well B of test strip; (vi) wait for 20
minutes; (vii) read results.
Reagent stability > 6 months at 40°C
Cost/test $5 per test (estimated)
$3,000 for reader (eventual price estimated to be $2,000). Reader will be provided free of
Cost/reader
charge dependent on committed volumes. Note that tests can also be read by eye.
Regulatory Status TBD
Physical dimensions Width: 12 cm (4.7”)
of reader Height: 8.5 cm (3.3”)
(W x H x D) Depth: 7.7 cm (3.0”)
Weight of reader 390 g (~14 oz)
None required. Sterile lancets (for capillary blood samples) and alcohol swabs are
3rd party supplies
provided in the test kit.
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RT-PCR: QIAsymphony® SP/AS: Sample Preparation and Assay Set-up Instruments (2)
Electric Power Requirements 100 – 240 VAC, 50 – 60 HZ
• Temperature: 15ºC – 32º C (59ºF – 89ºF)
• Humidity: 15% – 75% (for temperatures up to 31ºC, decreasing linearly to 50% humidity
Environmental Requirements
at 32°C) (noncondensing)
• Maximum altitude: 2,000 meters (6,500 feet)
Data Station
Monitor
Printer
Bar-code Scanner Supplied with instrument
Training Fully-trained lab tech required; dedicated training on instrument
Routine preventative maintenance required. In case of breakdown, vendor-trained
Maintenance
technician required to repair.
Internal QC
Infrastructure Requirements Technology can be used at national reference (or comparable) laboratories
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RT-PCR: Rotor-Gene Q
Automated amplification/detection instrument
Type of Technology Automated amplification and detection
Output RNA HIV-1 quantification
Turnaround time Including sample preparation, 5 – 6 hours per 24 reactions
Capacity (per run) 67 samples
Throughput per technician/ per day
Sample needed and stability PCR-ready set-up samples from QIAsymphony AS or QIAamp DSP Virus Kit
Specimen preparation and protocol
complexity
Reagent stability Varies by reagent, but most must be stored at 2º – 8º C (36º – 46º F)
Cost/test
Cost/instrument
Regulatory Status of Assays CE-IVD marked
Physical dimensions Width: 37 cm (14.6”)
(cytometer only) Depth: 42 cm (16.5”); door open: 56 cm (22.0”)
(W x D x H) Height: 27.5 cm (41”)
Weight 14 kg (31 lbs)
3rd party supplies Centrifuge, refrigerator, laboratory freezer and various additional laboratory consumables
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Internal QC Yes, a synthetic calibrator added in a known concentration at the extraction stage,
functions as an internal control for the isolation, amplification and detection procedure.
Infrastructure Requirements Technology can be used at the regional/central level or national reference (or comparable)
laboratories. Access to decentralized settings via DBS.
*Note: some details in above table revised August 2013 following first printing of this report (June 2013)
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Internal QC Yes, a synthetic calibrator added in a known concentration at the extraction stage,
functions as an internal control for the isolation, amplification and detection procedure.
Infrastructure Requirements Technology can be used at the regional/central level or national reference (or comparable)
laboratories. Access to decentralized settings via DBS.
*Note: some details in above table revised August 2013 following first printing of this report (June 2013)
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Sample needed and stability Eluates extracted with miniMAG or easyMAG. Can be stored at 2º C – 8º C; all reagents are
stable until expiration date.
Sample preparation and protocol
Moderate complexity. Dehydrated reagents are quickly reconstituted.
complexity
NucliSENS EasyQ HIV-1 v 2.0 assay storage at 2º C – 8º C; all reagents are stable until
Reagent stability
expiration date.
The average price per test of EasyQ HIV assay v 2.0, including extraction and detection/
Cost/test
amplification is about €18.00 ($23.75).
Cost/instrument Approximately €37,100 ($49,000)
Regulatory Status NUCLISENS EasyQ® HIV-1 v 2.0 is WHO prequalified and CE-IVD marked
42 cm (16.5 ins)
Physical dimensions
42 cm (16.5 ins)
(W x D x H)
22 cm ( 8.7 ins)
Weight 20.5 kg (45 lbs)
*Note: some details in above table revised August 2013 following first printing of this report (June 2013)
Internal QC Yes, a synthetic calibrator added in a known concentration at the extraction stage,
functions as an internal control for the isolation, amplification and detection procedure
Infrastructure Requirements Technology can be used at central/national reference (or comparable) laboratories.
Access to decentralized settings via DBS.
*Note: some details in above table revised August 2013 following first printing of this report (June 2013)
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Alere Q
Portable bench-top, NAT-based purification, amplification and detection system for total
Type of Technology
HIV RNA
Output Quantitative HIV-1, Groups M, N and O, and HIV-2 RNA viral load measurement
Turnaround time About 30 – 60 minutes
Capacity Maximum of ~10 samples per day
Throughput per technician/ per day ~10 samples per technician per day; no batching capabilities; walk-away operation.
25 µL of capillary (fingerstick) blood wicked directly into the sample collector contained
in the dedicated cartridge. Alternatively, 25 µL of heelprick or venous blood can be used
Sample needed and stability in the device. For venous blood, no metering is required onto the cartridge. Cartridge
containing sample can be stored and shipped if needed as sample is expected to be
stable for weeks.
No sample preparation required. For capillary blood: (i) lancet finger; (ii) wick whole
blood directly into cartridge; (iii) close cartridge; (iv) insert cartridge into analyser; (v)
Sample preparation and protocol
enter operator and sample ID; (vi) analysis starts automatically; (vii) remove cartridge
complexity
from analyser and dispose of it; and (viii) read result from screen. Hands-on time
expected to be ~10 minutes.
Reagent stability Freeze-dried reagents require no refrigeration. Stable for 12 months at 2 – 30°C
Cost/test TBD
Cost/instrument TBD
Alere will seek regulatory approval for CE-IVD marking and FDA approval in 2012 and
Regulatory Status
2013.
Physical dimensions Length: 28 cm (11”)
(analyser only) Height: 17 cm (6.7”)
(L x H x D) Depth: 17 cm (6.7”)
Weight <5 kg (< 11 lbs)
3rd party supplies For capillary samples: sterile lancets, alcohol swabs, dry swabs (also available from Alere)
Alere Q (2)
Analyser contains on-board rechargeable battery that provides a full work day (at least 8
Electric Power Requirements
hours) of operation.
• Operating Temperature: 15º – 40º C (59º – 104º F)
Environmental Requirements • Humidity: < 90% relative humidity
• Maximum altitude: N/A (permissible atmospheric pressure: 850 – 1100 hPa)
Dedicated CPU integrated into instrument; approximately 5,000 test results can be stored
on the instrument archive; results can be downloaded via USB. Potential to install an SMS
Data Station
chip to transmit results or internal calibration data. Will support wireless connectivity and
device can be attached to a USB port for sample tracking, if desired by user.
Monitor Color touch screen integrated into instrument
Separate printer (prints on thermal paper); battery powered
Printer
L 95mm x W 93mm x H 66mm, weight: ~350 grams, including paper roll.
Bar-code Scanner Integrated into instrument for test cartridges only
Minimal training required. Lay person can be trained in less than half a day. Primary skill
Training
required is for correct lancet blood draw.
If damaged, low cost and portability of device allows for direct swap-out replacement
Maintenance
rather than on-site repair.
Internal QC Yes
External QA Will be fully-compatible with existing EQA programs.
Infrastructure Requirements Can be used at all levels of health facility, including health centers or in mobile facilities.
Touch-screen color display to enter patient information, view results, adjust settings,
User interface
download results and navigate system software
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Liat™ Analyser
Type of Technology Portable bench-top, sample preparation and real time PCR
Output Qualitative or quantitative (viral load) (limit of detection ~50 cp/mL)
Turnaround time 30 – 55 minutes depending on limit of detection set (30 minutes for 500/1000 cp/mL)
Capacity ~8 – 15 samples per 8 hour day, depending on limit of detection
Throughput per technician/ per day ~8 – 15 samples per technician per 8 hour day; no batching capabilities on device.
Sample needed and stability 200 µL of plasma or 10 – 50 µL of finger-stick blood wicked directly into Liat tube.
No manual sample preparation required, even using capillary blood. Operation only
Sample preparation and protocol requires: (i) apply blood drops from finger lancet or plasma to Liat tube; (ii) scan the tube’s
complexity bar code on the device; (iii) insert tube into Liat analyser; the analyser will start assay and
the result will be reported in ~30 minutes automatically.
Reagents expected to be shipped with an expiration date of at least 6 months;
Reagent stability reagents should be stored at approximately 4ºC (39.2°F); but 37ºC (98.6°F) storage allowed
for 3 weeks.
Cost/test TBD
Cost/instrument ~$25,000, but may be priced lower for resource-limited settings
Regulatory Status US FDA 510 (k) clearance for Liat Influenza A/B Assay, TBD for Liat HIV Quant Assay
Width: 4.5 inches
Physical dimensions
Height: 7.5 inches
(W x H x D)
Depth: 9.5 inches
Weight 3.75 kg (~8.3 lbs)
3rd party supplies For capillary samples: sterile lancets, alcohol swabs, dry swabs
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SAMBA
Type of Technology Isothermal target/signal amplification and visual detection; separate extraction
Qualitative for EID and semi-quantitative for viral load (limit of detection ~400 cp/mL
RNA with 100µL of whole blood, also detects DNA); detection of acute infection (limit
Output
of detection ~100 cp/mL with 500µL of plasma); and semi-quantitative viral load test for
monitoring of patients on ART (1,000 cp/mL cutoff with 200µL of plasma).
Turnaround time 90 – 120 minutes, depending on the assay
Capacity 6 samples per run
24 samples per day for EID or acute infection tests; 36 viral lad tests, assuming a 6.5 hour
Throughput per technician/ per day
work day.
200µL (plasma) for viral load test; 500µL (plasma) for acute infection test; 100µL (whole
Sample needed and stability
blood) for EID test. Sample is stable at room temperature for 6 – 8 hours.
Sample preparation and protocol
Simple, pre-loaded, disposable cartridges containing all required liquid or dry reagents
complexity
Transport stability up to 55°C for one month. Reagents do not require cold-chain storage
Reagent stability
and are stable up to 37° for 1 year.
Cost/test TBD
Cost/instrument TBD
Regulatory Status Regulatory approval obtained in Malawi for viral load assay
Width: TBD
Physical dimensions
Height: TBD
(W x H x D)
Depth: TBD
Weight TBD
3rd party supplies
SAMBA (2)
Electric Power Requirements AC powered (100 – 240V); can be battery powered to compare the run
• Operating Temperature: 10° – 37°C
Environmental Requirements • Humidity: up to 95%
• Maximum altitude: N/A
Data Station None
Monitor Small screen integrated into instrument
Printer No printer provided
Bar-code Scanner None
Training Minimal training required
Maintenance No maintenance required; swap-out of instrument if needed.
Internal QC Synthetic, non-target nucleic acid internal controls
Freeze-dried EQA panel provided consisting of a negative sample and a range of positive
External QA
samples.
Can be used at various levels of health facility, including health centers or in mobile
Infrastructure Requirements
facilities. Electricity is required.
User interface Touch screen
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GeneXpert® System
Type of Technology PCR-based NAAT test
Output Quantitative HIV-1 (viral load) and Qualitative HIV-1
Turnaround time < 90 min
Dependent on GeneXpert system and number of modules ranging from 1 – 80 per
Capacity
system, Comparable to GeneXpert MTB
Dependent on GeneXpert system and number of modules. For example, 397 results per 8
Throughput per technician/ per day
hour shift with an Infinity-80 *(80 modules)
Sample needed and stability 1 ml plasma for Quantitative HIV-1; Qualitative whole blood and DBS under development.
Sample preparation and protocol
Automated within cartridge
complexity
Reagent stability No refrigeration required. In development.
Cost/test TBD
Cost/instrument Comparable to GeneXpert MTB
Regulatory Status Regulatory submissions for CE IVD and FDA expected in 2014.
Please see www.cepheid.com for brochure on systems available.
Below are specifications for a GX-IV Processing Unit
Physical dimensions
Length: 11.00”
(W x H x D)
Height: 12.00”
Depth: 13.25”
Weight For GX-IV Processing Unit: 25 lbs
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APPENDIX 2: Pipeline for POC Diagnostics
APPENDIX 2:
Pipeline for POC Diagnostics
APPENDIX 3:
Technical Specifications for HIV Qualitative Assays
Company Roche Roche Abbott
COBAS® AmpliPrep / COBAS®
AMPLICOR® HIV-1 DNA Test Abbott RealTime RUO
Assay Name TaqMan® (CAP/CTM) HIV-1
v1.5 (RUO) Qualitative HIV-1
Qualitative (RUO)
Type of assay PCR, qualitative Real-time PCR, qualitative Real-time PCR, qualitative
Dynamic Range
N/A N/A N/A
(copies/ml)
Contamination
Amperase Amperase Not reported
Control
Run-in (neg., pos) Run-in (neg., pos)
Controls Not reported
Internal control Internal control
Whole blood,
Specimen Type Whole blood / EDTA plasma / DBS EDTA and ACD plasma3
DBS
100 µl whole blood (Infants)
100 µl whole blood
Specimen volume 500 µl whole blood (adults) 200µL plasma or DBS
60-70 µl DBS
60-70 µl DBS
Area of HIV genome
Gag Gag Pol/INT
amplified
Group M, subtypes A, B, C, D,
HIV-1 subtypes
Group M, subtypes A-H Group M, subtypes A-H CRF01-AE, F, CRF02-AG, G, and H,
amplified
Group O and Group N
$10 – $15 per test in resource- $12 – $16 per test in resource-
Cost/test2 limited settings; $15 – $30 per limited settings; $16 – $30 per $15 – $20 per test
test elsewhere test elsewhere
22-66 batch loading
Number of samples/ 21-93 patient samples
9-21 (176/8 hour day continuous
run (+3 external controls)
loading)
Thermocycler,
ELISA reader / washer, COBAS® AmpliPrep with COBAS® m2000rt; m2000sp, m24sp or
Equipment required3
microcentrifuge TaqMan® 96 COBAS® TaqMan® 48 manual sample preparation
not supplied by Roche
COBAS TaqMan 48:
$45,000 – $100,000 m24sp: $90,000, m2000sp:
COBAS TaqMan 96: $120,000; or manual (magnetic
Equipment Cost ($US) ~$25,000
$80,000 – $150,000 racks, plate cooler): $500 and
COBAS AmpliPrep: m2000rt: $38,000
$80,000 – $150,000
Regulatory Status WHO Prequalified
1 Note that the COBAS® AMPLICOR HIV-1 MONITOR™ v1.5 (the MONITOR assay) from Roche Diagnostics is no longer being sold by Roche except to current customers using the COBAS
AMPLICOR Analyser, which is still being supported by Roche, but is no longer available for sale from the company.
2 Prices will vary considerably depending on quantities, infrastructure and support required plus special negotiations.
3 All assays require pipettes, vortex mixers, and a refrigerator.
148
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