Indian J Med Res 121, April 2005, pp 345-355
345
The cost of ART (antiretroviral therapy) has
reduced dramatically due to the production by generic
manufactures and this has led to the increased usage
of highly active antiretroviral therapy (HAART)  in
developing countries1-5. But the cost of monitoring is
prohibitively high. As antiretroviral therapy is
becoming more affordable and accessible, inexpensive
laboratory tests are also needed to monitor the
progression of disease in HIV infected individuals,
living in resource-limited environments most heavily
impacted by the epidemic6.
Though clinical assessment remains  the most
essential basis for monitoring HIV infection, it lacks
sensitivity in  determining both disease stage  and
progression and, is  thus used in conjunction  with
laboratory measures7. These include total lymphocyte
counts(TLC), CD4+ T lymphocyte counts (CD4+ T
cell),  HIV plasma viral load  (PVL), and other
surrogate markers, such as  ? 2-microglobulin levels8.
Ideally, a surrogate marker should be biologically
plausible, predictive of disease progression and
measurable by standardized assays. Historically, a
number of candidate markers have been explored for
monitoring the course of HIV infection and response
to treatment. While the level of absolute numbers of
peripheral CD4+ T cells and the PVL have eventually
become the reference markers in clinical practice for
monitoring HIV infected subjects, several additional
parameters are still being evaluated. These indicators
are used to determine disease stage and progression,
assist in decisions regarding when to start or change
antiretroviral therapy, and assess treatment responses9.
In addition, access to HIV genotypic or phenotypic
Key words CD4 T-lymphocyte count  - HIV monitoring - low-cost assays - plasma viral load - resource-limited countries
Low-cost monitoring of HIV infected individuals on highly active
antiretroviral therapy (HAART) in developing countries
Pachamuthu Balakrishnan, Suniti Solomon, Nagalingeswaran Kumarasamy & Kenneth H. Mayer*
YRG  Centre for AIDS Research & Education, Chennai, India & *Brown University,
RI, Providence, United States
Accepted February 4, 2005
The standard methods  to monitor HIV infection are flow cytometry-based for CD4+ T lymphocyte
count and molecular assays to quantify plasma viral load of HIV. Few laboratories in resource-
limited countries can  run these  tests as a majority of the HIV infected individuals are poor. A
number of currently available  low-cost assays which require less  expensive equipment and
reagents, may be well-suited to such countries. These include manual and ELISA based CD4 cell
assays, and ultrasensitive reverse transcriptase quantitation (Cavidi) and p24 (ELAST) assays
to monitor virus load. But better internal quality assurance and   quality control (QA/QC)
programmes are essential. This review discusses the low-cost assays and their role in clinical
monitoring of HIV infected individuals in resource-limited countries like as India.
346 INDIAN J MED RES, APRIL  2005
analysis is increasingly available and used to guide
the choice of antiretroviral  treatment. The recent
reduction  in cost and thus  availability of generic
antiretroviral  drugs within many resource-limited
countries has focused attention   on the lack of
infrastructure for effective monitoring of HIV infection
in  these countries. It is  unlikely that the cost  of
laboratory assays to  monitor HIV infection will  be
swayed to the same extent by similar pressure brought
to bear on manufacturers of antiretroviral drugs.
The reference standard for CD4+ T cell testing is
flow cytometric enumeration of T cell subsets. For
PVL, a number of molecularly based assays that use
reverse transcriptase-polymerase chain reaction (RT-
PCR), branched DNA (b-DNA), and  nucleic acid
sequence based amplification (NASBA) technology
are approved in  many countries. Such reference
assays are expensive, require sophisticated equipment
as well  as trained personnel, and  are not widely
available in resource-limited countries. Geographic
difficulties, such as lack of ready access to technical
support and quality control and quality assurance (QA/
QC) programmes, also limit their potential use in such
settings.
Over the past decade, low-cost, less technically
demanding assays, that quantify CD4+ T cell  and PVL
have  been developed and assessed  but have not
become generally established in resource-poor settings
due to several reasons (Tables I, II). There is now a
resurgence of interest in implementing these tests in
regions in which  flow cytometric and molecular
facilities are not affordable or practical. The possibility
of extending potent antiretroviral therapies to
developing countries has raised the need of simple,
reliable and cost-effective tests to measure prognostic
markers for disease evolution and assessment of
therapy efficacy. This review summarizes the
benefits and limits of reference and candidate
surrogate markers and their integration for optimal
antiretroviral therapy.
The reference standard  and low-cost monitoring
of CD4+ T-lymphocyte
CD4+ T cell monitoring in conjunction with clinical
status will  be of benefit in   terms of assessing
prognosis, guiding the treatment, and subsequently
monitoring the response. Also, CD4+ T cell count
allows timely institution of  primary prophylaxis
against specific  opportunistic pathogens and  an
associated reduction in hospital admissions, with use
of affordable drugs such   as trimethoprim-
sulphamethoxazole10,11.  The precise CD4+ T cell
count at which the  risk of specific opportunistic
infections justifies prophylaxis has   been well
established in  the United States and Europe12. At
the population level, CD4+ T cell monitoring is
necessary for regional studies of the natural history
of HIV infection and assessing interactions of HIV
with locally prevalent infectious diseases.
The reference standard for CD4+ T lymphocyte
count: Multiparameter flow cytometric quantification
of CD4+  T cells with microprocessor -assisted
analysis is the reference standard. A wide range of
instruments is available commercially from different
companies, including  Becton Dickinson, Coulter
Corporation and Partec as well as newer instruments
such as FlowCare (PointCare Technologies).  The
high initial cost of equipment and problems associated
with equipment routine preventive maintenance, lack
of technical support, and lack of access to QA/QC
programmes have prevented widespread use of flow
cytometric monitoring of  CD4+ T cell counts  in
resource-limited countries.  The cost of reagents to
perform full lymphocyte  subset multiplatform
analysis, in which CD45+ T cells are identified by
staining with  8 monoclonal antibodies(MAbs)   by
means of 3-colour  fluorescence to identify CD3+/
CD4+ and CD3+/CD8+ T cells, is also prohibitive in
many countries. However, new approaches include
primary gating, in which CD4+ T cells are defined
by an autogate in a single histogram of CD4+ T cell
fluorescence intensity versus light scatter by use of
a single-platform volumetric flow cytometer. This is
considered relatively economical compared with use
of the  full test panel of  MAbs13.   Among the Flow
cytometers available, the  FACSCount (Becton
Dickinson) instrument is  a simpler, relatively less
expensive and automated  for clinical laboratories that
quantifies CD4+/CD8+ T cell counts   by
simultaneous fluorescent MAb staining of CD3, CD4,
and CD8 surface molecules  by use of different
fluorochromes attached to each MAb.
347
Table I. Standard and low-cost assays to enumerate CD4+ T cells
Assay Key features Advantages Disadvantages Cost/test in INR
(Approximate)
FACSCount  (Becton Dedicated (single bead Fully automated, no red No CD4 % , closed 1200.00
Dickinson): 2 color platform) flow cytometry cell lysis, no flow system, specific reagents,
Flow cytometer *  instrument for CD4, CD8 cytometric experience long processing time, require
and CD3 absolute counts required accurate reverse pipetting skill,
and CD4/CD8 ratio high cost per test
Manual CD4 kit, Latex bead-based kit Absolute CD4 result, No CD4 % value, low 600.00
Cytosphere assay for light microscope, simple, low cost microscope, throughput, high cost
(Beckman Coulter) pipette and haemocytometer, per test, labour intensive.
Manual count of can be performed at The samples should be
rosetted cells  remote sites processed within 6 h
Dynabeads CD4/CD8 Kit based Absolute CD4 result, No CD4 % value, low 700.00
kit (Dynal, Biotech) simple, low-cost microscope, throughput, high cost
Magnetic bead cell isolation, pipette, can be performed per test, labour intensive,
requires fluorescent at remote sites long process time, specific
microscopy  or  ordinary magnet and mixer required
microscope from the company. The
samples should be processed
within 24 h
Capcellia (BioRad) ELISA based using CD2 CD4/CD8 absolute from Labour intensive, requires 400.00
magnetic bead isolation, standard curve calculation, microplate reader, magnet,
color reaction detection for 40 tests/ELISA plate, 15 multichannel pipette, calculation
CD4/CD8 samples/h based on standard curve with
controls, Batch analysis required.
The samples should be processed
within 6 h
Easy CD4/CD8 Micro-capillary flow Additional applications, No CD4%, daily bead 300.00
(Guava Technologies) cytometry  (single  platform require small volume of calibration, computer literacy,
volumetric), absolute blood,  minimal waste requires red cell lysis and
CD4/CD3 and/or disposal, require minimal specific antibody reagents,
CD8/CD3, training for the operator. ~20 samples/day
Allows 48 h  samples
Partec CyFlow Dedicated (single No  lyse system, no beads, Volumetric counting 200.00
volumetric platform) 15 min turnaround, can stability is questionable.
flow  cytometer. Absolute give percentage CD4 no independent validation
CD4/CD8 depends on of performance, computer
choice of reagent software  literacy
*Standard assay
348 INDIAN J MED RES, APRIL  2005
Total lymphocyte count as a surrogate marker for
absolute CD4+ T cell count: In April 2002, World
Health Organization (WHO) released provisional
guidelines for implementation and scaling up of
antiretroviral treatment in resource-limited settings14.
Recognizing the urgent need for ART in developing
countries, where sophisticated and expensive
laboratory monitoring is not widely available, WHO
is advocating the use of simple and inexpensive
laboratory measures in determining response to
treatment. The total leucocyte count (TLC) is
available to most clinics worldwide at very little cost
and it requires just the leucocyte count plus the
differential i.e., the total leucocyte count is multiplied
by the percentage of lymphocytes to obtain the TLC.
Where CD4 count is unavailable, TLC use has already
been recommended for the timing of opportunistic
infection prophylaxis in Latin America, Africa, and
Asia15-18.  For example, in a cohort of HIV-positive
south Indians, a TLC <1400 cells/µl is a good predictor
of CD4+ T count <200 cells/µl and thus, an
appropriate surrogate marker for initiating co-
trimoxazole prophylaxis in resource-limited settings17.
In an analysis of patients initiating a triple
antiretroviral drug regimen, change in TLC had a
direct positive association with change in CD4+ T
cell count and an indirect association with change in
PVL18. Another study suggests that either the TLC
alone or in combination with the change in
haemoglobin19 may serve as adequate tools to monitor
antiretroviral treatment response. However, though
there is a positive correlation between CD4+ T cell
Table II. Standard and low-cost assays to quantify HIV
Assay Key features Parameters Advantages Disadvantages Cost/test in INR
measured (Approximate)
Roche Amplicor Cut-off 50 copies/ HIV RNA ?  Can be used for other ?  Requires PCR set-up. 4000.00
(version 1.5)* ml with standard  by RT-PCR diseases such as HBV, ?  COBAS for automated
assay and 400 copies/ HCV and CMV. and PCR thermocycler
ml with ultrasensitive ?  Can be used for for Manual.
assay. Semi-automated all clades ?  Contamination risk
(manual) or automated ?  High throughput ?  Skill.
(COBAS) ?  Higher cost.
?  Infrastructure
?  Need good technical
support
Perkin Elmer Clinical cut-off HIV P24 ?  Equipment can be ?  May not be effective 700.00
Ultrasensitive around 30,000 antigen shared (ELISA:reader/ for all clades
p24 copies/ml washer) ?  Batch analysis alone
?  Uses same consumables reduce substantial cost
as ELISA testing
?  Easy training
?  1 day turn around time
?  Easy to perform assay
?  High throughput
Cavidi ExaVir Cut-off HIV reverse ?  Easy training ?  32°C dedicated 900.00
approximately transcriptase ?  Can be used for incubator needed
5,000 copies/ml. activity all clades? ?  performance time
?  Easy to perform  about 3 days
assay ?  Batch analysis alone
reduce substantial cost
?  Needs more evaluation
?  Positive and negative
control not supplied
*Standard assay
349
and TLC, the latter is not sufficiently useful to predict
CD4 cell counts in many settings. In a study of 2777
HIV-seropositive persons in South Africa20, the overall
correlation between CD4 cell and total lymphocyte
count was only modest (R=0.70). This is supported by
a prospective study in the United Kingdom21, in which
the  correlation between 1535 paired   absolute
lymphocyte and CD4 cell counts was 0.64 for persons
with asymptomatic HIV infection, and a study of 32
Nigerian adults22 with HIV infection who commenced
treatment with antiretroviral drugs, in which there was
only a very weak correlation between total lymphocyte
and CD4 cell count (R=0.25).  These studies indicate
that TLC is an imperfect predictor of CD4 cell count.
Therefore, the larger longitudinal studies involving
long term cohort follow up may answer the question.
Low-cost alternative assays for CD4+ T cell
count: Non-cytofluorographic methods are largely of
the ELISA format (Capcellia CD4/CD8 Whole Blood
Assay;BioRad  and TRAx CD4 Test Kit assays;T Cell
Diagnostics, which is currently unavailable), or manual
methods,  using magnetic beads (Dynabeads T4-T8
System; Dynal Biotech ASA) or cytospheres (Coulter
Manual  CD4 Count kit). The assays with modified
flow cytometry technologies are EasyCD4/CD8
(Guava Technologies) and CyFlow (Partec).
Extensive evaluation studies have been carried out in
different countries on the efficiency of the each assay
in providing accurate and reproducible results.
The Capcellia CD4/CD8 whole blood assay is an
ELISA that  uses MAb-coated paramagnetic
microparticles to identify T cells (by use of anti CD2
MAb) followed by  anti-CD4 (or anti-CD8) MAb-
peroxidase conjugate. The sample should be collected
in EDTA anticoagulant and processed within 6 h. The
assay is in a 96-well format, and up to 43 samples can
be assayed in a single run per kit (plus standards). A
magnetic frame, plate reader, manual washing manifold,
and pipettes (including a multichannel pipette) are used
for the assay. The Capcellia assay provides an absolute
CD4+ T cell count calculated from a 4-point standard
curve. This measurement of CD4+ T cells has been
found to  correlate well with flow   cytometry in
European laboratories  (R=0.81)23,  although its
performance was lower in West Africa in an evaluation
of HIV infected and uninfected persons (R=0.61)24.
The studies from India also reveal the significant
correlation with the standard methods25,26.
The Dynabeads T4-T8 system uses “dynabeads”,
which are magnetic particles coated with antibodies
to CD4  or CD8 molecules. The sample should  be
collected in EDTA  or acid-citrate-dextrose
anticoagulant and processed within 24 h. Only 250 µl
of  blood is required for  the assay. Monocytes are
removed by using additional beads coated with anti-
CD14, thus avoiding potential confusion when counting
cells under the microscope. CD4+ T cells are isolated
by use of the magnet and lysed, and nuclei are counted
after staining  with Sternheimer-Malbin staining
solution  (containing crystal violet, safranin   O,
ammonium oxalate, and ethanol). Alternatively, nuclei
are  stained with acridine orange  and counted by
fluorescence microscopy. The absolute CD4 cell count
is obtained by counting all the cells in one side of a 0.1
mm haemocytometer and dividing the number by 0.9.
Because counts must be done within 1 h after addition
of lysis reagent, the batch size needs to be limited to
<6  samples. In addition to  a microscope, a
haemocytometer, a manual counter, a rotating wheel
to mix samples, and a magnet are required. Several
investigators  have reported good correlation with flow
cytometry (R= 0.9)27,28. Recently the study from West
Africa has shown that the implementation of this low-
cost method was easy and successful in the West
African context29.
The  Coulter Manual CD4 Count  kit uses
“cytospheres” (latex beads coated with anti-CD4) that
bind to CD4+ T cells to form a cell sphere rosette,
detected by light microscopy. Monocytes (which also
express CD4 on their surface) are identified by binding
to smaller spheres  coated with anti-CD14. Blood
should be processed within  6 h, according to  the
manufacturer’s instructions. Only  100 µl of blood,
collected in EDTA anticoagulant,  is required. Very
little equipment is needed for this assay: a microscope
with a 40X objective, haemocytometer,  a manual
counter, test  tubes, and calibrated pipettes.
Erythrocytes are lysed, and  crystal violet stained
CD4+ T cells with beads attached are identified by
counting in a haemocytometer. Cells in all squares on
both sides of a 0.1mm haemocytometer are counted
BALAKRISHNAN et al: LOW-COST HIV MONITORING
350 INDIAN J MED RES, APRIL  2005
and the number multiplied by a dilution factor of 7.3 to
obtain the absolute CD4+ T cell count. The sample is
stable for only approximately 15 min after addition of
lysis/stain reagent, which  limits the batch size  to
approximately 2-4 samples. Several investigators have
found a good correlation between CD4+ T cell counts
measured by  the cytospheres method and   flow
cytometry (R > 0.91)30-32. The recent study from our
laboratory has shown excellent correlation with the
standard assay33. The correlation coefficient of the
cytosphere assay compared with that of flow
cytometry for CD4+ T cells was 0.97 (P< 0.0001),
with a confidence interval (CI) of 0.95 to 0.98. The
sensitivity, specificity, positive predictive value, and
negative predictive value of the cytosphere assay in
enumerating absolute CD4+ T cell counts of less than
200/µl were 94.9, 96.4, 92.5 and 97.6 per cent,
respectively. Overall this manual assay yielded CD4+
T cell counts greater than flow cytometry by a mean
of 10 cells/ml (95% CI,  -152 to 132 cells/ml); as
assessed by Bland-Altman analysis. However, another
study27, which analyzed only 10 samples, has found
poor correlation with flow cytometry (R=0.45) and the
poor correlation could be due to sample bias. In
general, this is a simple inexpensive method and does
not require special equipment.     
CyFlow is a portable flow cytometry system. The
machine is less expensive and more robust than
conventional FACS systems, uses less expensive
reagents, and is able to produce an absolute CD4+ T
cell count without additional instrumentation. A mobile
CD4+ T cell  counting laboratory can be established
for the field level use. CyFlow is able to produce an
absolute CD4+ T-cell count using two monoclonal
reagents. Investigators from Cambodia, Thailand,
Malawi, Negeria and Rwanda have evaluated the
precision of the CyFlow system and observed good
correlation with the standard assay34.
Guava EasyCD4/CD8 assay is a micro-capillary
flow cytometry technology based system. The reagent
kit contains three direct immunofluorescence reagents
for enumeration of mature CD4+ and CD8+ T cells.
The kit consists of a murine monoclonal anti-human
CD3 antibody conjugated to the tandem dye
phycoerythrin (PE)-Cy5 (PECy5), a murine
monoclonal anti-human CD4 antibody conjugated to
PE and a murine monoclonal anti-human CD8
antibody conjugated to PE. The CD3 antibody
uniquely identifies T cells and recognizes an epitope
expressed on the epsilon chain of the CD3/T cell
antigen receptor (TcR) complex. This assay provides
a method for enumeration of CD4+ and CD8+ T cells.
Whole blood is stained with the antibodies from the
EasyCD4/CD8 Reagent Kit, which bind to antigens
on surface of lymphocytes; then erythrocytes are
removed by lysis by treating with Lysing Reagent to
lyse and finally analyzed on the Guava PCA
instrument to obtain the absolute CD4+ and CD8+ T
cell counts. This assay requires EDTA blood  and
the sample should be processed within 48 h of
collection for optimal results. Our study (unpublished)
has shown that it is easy to perform, generate reliable
results (correlation coefficient 0.99) and the
technicians needs substantially less training than the
standard flow cytometry. This assay requires micro-
volume of specimen and generates very minimal
biowaste. Also the studies from America and South
Africa have shown the good correlation35,36.
Flow cytometry versus low-cost assays to
enumerate CD4+T cell count: There are advantages
and disadvantages  of each system (Table I). Flow
cytometry is considered  the reference standard.
However,  disadvantages include the fact  that, it is
expensive in terms of initial  cost of equipment and
requires a haematologic analyzer  to provide the
absolute CD4+ T cell count. The use of the equipment
is technically complicated, and highly trained personnel
are generally needed to run these instruments. There
are reports that lysis of RBCs during flow cytometric
analysis may be problematic37; resistance to lysis
(possibly associated with elevated levels of nucleated
erythrocytes  in blood) has been  reported in African
patients28. Further, lysis may result in cell loss (up to
20%), with  significant donor and lysing  solution
variability38. The advantages of flow cytometry include
provision of percentage and absolute CD4+ T cell
count.  Whereas most clinicians  generally use the
absolute CD4+ T cell count in the clinic, the CD4+ T
cell percentage is very useful as a built-in control to
determine whether the CD4+ T cell count is changing
or stable, needed because of often large variation in
absolute CD4+ T cell counts despite clinical stability39.
Also biological variables, including diurnal variation,
351
exercise or rest, and smoking, can significantly alter
CD4+ T cell count40.
For major cities in regions, where there is a high
incidence of  HIV infection and a  large number of
samples  need to be analyzed per day, manual methods
are not practical. Provided there is access to company
technical support and QA/QC programmes,
automated evaluation of CD4+ T cells (perhaps by use
of an instrument such as the FACSCount) may be the
most economical method on a long-term basis. The
FACSCount is simple; the equipment, although still
costing significantly (approximately Rs.22 lakhs), it
still is less  expensive than other standard flow
cytometry instruments. It does  not require a highly
trained personnel and lysis of blood samples is also
not required. A disadvantage of the method is that only
the absolute  CD4+ T cell count is   provided and
haematology analyzer has been used for percentage
of CD4+ calculation.
The manual methods such as  Dynal and Coulter
assays are suitable for smaller regional centres where
<10 samples are requested per day and where there
is no ready access, including convenient and reliable
transport, to laboratories with flow cytometry facilities.
Both of these methods are simple and inexpensive and
require  very little investment for the equipment
(ordinary light microscope). Both provide only absolute
CD4+ T cell counts. Counting under the microscope
is both time consuming and laborious (approximately
10 min/sample). The studies show that higher cell
counts are more prone to inaccuracy compared with
values obtained by  flow cytometry8. The Capcellia
assay is also appealing because, it is an ELISA-based
assay and as such it is readily established in most small
laboratories and Voluntary Counselling and Testing
(VCT) centres25,26.
Though the manufacturers of these manual assays
state that blood should be assayed within the storage
period (6 h for Coulter) after the collection, this is
not always possible in regions, where transport may
be delayed, and thus the robustness of these assays
for use in the  field requires scrutiny. Analysis of
specimen transport condition and performance of these
assays with the aged samples require further studies
and currently the work is in progress in our laboratory.
Low-cost monitoring of HIV load
Molecular assays, including  the Amplicor HIV
Monitor RT-PCR assay (Roche Diagnostic Systems)
are now available in many countries (this can be done
manually or with fully automated using   COBAS
equipment). Early version (1.0) of the Amplicor RT-
PCR assay was insensitive in detecting certain HIV
subtypes41,42. In an evaluation of the   Amplicor assay,
detection  of subtypes A and  E was considerably
improved with version 1.5 compared with version 1.0.
The Quantiplex HIV RNA assay, which uses b-DNA
technology (Bayer  Diagnostics), and NASBA
(Organon Teknika), are the other reference standards
for monitoring plasma HIV load. The NASBA assay
has also been reported   to fail detection of   or
inadequately quantify, HIV RNA in plasma of persons
infected with non-B subtypes43-45, whereas the b-DNA
assay has generally been reported to perform well with
non-B  subtypes41,44.
There are very few low-cost assays (with no need
to purchase any special equipments) that may provide
an alternative for virus load measurement. Although
several low-cost assays for plasma HIV load are at
the research level, there are two assays (Table II),
which have been evaluated in different countries.
These include a  p24 Ultrasensitive Assay; ELAST
(Perkin  Elmer Life Sciences) and an  ultrasensitive
reverse-transcriptase (RT) assay (RT Viral Load Kit;
Cavidi Tech).
The HIV p24 Ultrasensitive Assay provided a 3-log
increase in sensitivity above the standard p24 assay by
means of a signal amplification-boosted ELISA method
(enzyme-linked amplified sorbent test; ELAST)
following heat denaturation of the plasma sample to
release immunecomplexed HIV p24 antigen. The assay
provides results comparable to those of RT-PCR, is in
a  microplate ELISA format and  is significantly
inexpensive  than the standard46-49.  The plasma p24
levels appeared to be correlated significantly with the
plasma RNA viral loads (R=0.75, P<0.0001). The heat-
denatured p24 antigen assay was capable of measuring
the plasma level of p24 derived from all the HIV-1
subtypes and recombinants selected for this study, in
contrast to the RNA viral load test which lacked
sensitivity towards HIV-1 group O48.
BALAKRISHNAN et al: LOW-COST HIV MONITORING
352 INDIAN J MED RES, APRIL  2005
Cavidi RT assay (ExaVir Load): The procedure
has only 2 major steps (purification of virions and
recovery of RT, including  removal of potential
inhibitors, followed by quantification of RT activity in
a 96-well microtiter plate format). Very little equipment
is necessary (provided by the company as a start-up
kit at free of cost); mainly a vacuum pump, waste
bottle, some rubber tubing and an ELISA reader or a
fluorometer and some buckets for the wash procedure.
The company reports excellent correlations between
standard RT-PCR assay and the  Cavidi RT assay
(R=0.9, P <0.0001)50. The fluorometer appears to be
more sensitive than the  ELISA reader. The assay is
not affected by the presence of RT inhibitors (drugs)
in plasma of  HIV infected persons.  However, this
assay needs more evaluation with different subtypes
before using it for the ARV monitoring. Another
advantage of this low-cost viral load assay is that
samples can be stored and batch testing can be done.
Currently our centre is evaluating this assay for the
suitability of the kit in monitoring HIV infected
individuals, who are on generic HAART. These low-
cost viral load  assays have their greatest value  in
smaller laboratories in  resource-limited  countries,
where the cost is a major issue and, where setting up
of RT-PCR or similar molecular assays is difficult and
these assays are unavailable.
Other surrogate markers for HIV monitoring
Extensive studies have been carried out to
evaluate whether B2-microglobulin and neopterin
could be used as markers of disease progression and
also as prognostic factors51-55. In general, these have
not  been found to be   reliable. These factors in
combination with CD4+ T cell counts, have been
shown to have good predictive value52,53. One potential
reason for lack of correlation may be the fact that a
number of other infectious agents may also increase
? 2-microglobulin and neopterin levels. Thus, B2-
microglobulin and neopterin may be an  unreliable
indicator of HIV infection or disease stage in those
co-infected with other pathogens.
HIV monitoring in resource-limited settings
The HIV/AIDS statistics are well known, with
resource-limited countries bearing more than 90 per
cent of the global disease burden56. Added to the cost
of ARV therapy are substantial costs associated with
laboratory monitoring57, which also include liver
function tests (LFT) and CBC (complete blood count).
The inclusion of some important parameters of the
ART toxicity-related assays such as platelet,
haemoglobin, erythrocyte sedimentation rate (ESR),
bilirubin (direct and indirect) and serum glutamate
pyruvic transaminase (SGPT) could also be a cost-
saving strategy in the developing nations.
Although the introduction of antiretroviral therapy
to resource-limited countries should not be prevented
because of  a lack of monitoring  tests, every effort
should be made to introduce appropriate laboratory
monitoring assays. Sustainability of assays (recognizing
issues such as ongoing costs, delivery of reagents and
kits, kit shelf-life, access to instrument service, and
QA/QC programmes) must also be considered at the
time of technology transfer.  As a minimum, a low-
cost CD4+ T cell count should be monitored  as an
adjunct to clinical examination. Establishing CD4+ T
cell monitoring facilities must include implementation
of  QA/QC program. Since there are some CD4+
count difference between standard assay and
alternative technologies, the normal reference ranges
should be established with the local population for any
particular low-cost assay8. Implementation of low-
cost viral load assays awaits rigorous evaluation of
their performance against standard assays.
The incidence of HIV infection and clinical
disease continue to increase rapidly in the developing
nations14. As antiretroviral therapy is becoming more
affordable and accessible in India, inexpensive
laboratory tests are very much needed to monitor
the disease progression and ART. Hence the
implementation of HIV monitoring by low-cost
assays would reduce the economic burdon of the
patients in this country.
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tertiary referral  centre. J Assoc Physicians India 2000; 48 :
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Reprint requests: Dr P. Balakrishnan, YRG Centre for AIDS Research and Education (YRG CARE)
Voluntary Health Services Campus, Taramani, Chennai 600113, India
e-mail: bala@yrgcare.org
BALAKRISHNAN et al: LOW-COST HIV MONITORING