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Application of Polymerase Chain Reaction to Detect HIV-1 DNA in Pools of Dried Blood Spots

Vemu Lakshmi • Talasila Sudha • Dandona Rakhi •

G. Anilkumar • Lalit Dandona

Received: 26 November 2009 / Accepted: 29 September 2010 / Published online: 28 January 2011

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� Association of Microbiologists of India 2011

Abstract Nucleic acid tests that detect HIV infection at

an early phase are available and have been applied on

individual dried blood spot (DBS). The present study was

undertaken with an aim to evaluate the feasibility of per-

forming PCR for HIV-1 DNA on pools of DBS as an

alternative to individual testing. Standardization of PCR by

a modified Amplicor HIV-1 DNA assay version 1.5 (Roche

molecular diagnostics, USA), on pooled DBS was per-

formed using five confirmed HIV reactive samples with

known low viral load of HIV-1 and HIV non-reactive

samples in pools of 5, 10 and 20 DBS. After successful

standardization of pooling procedure, a total of 183 pools

(of 10 DBS each) were prepared from 1,823 DBS samples,

collected from a population-based study that tested nega-

tive for HIV antibodies and p24 antigen. All these pools

were screened for HIV-1 DNA by the Amplicor assay.

Standardization of pooling procedure indicated that pool-

ing of 10 DBS gave an optimum result. Out of 183 pools

tested, one pool of 10 samples was positive and of these ten

DBS that were tested individually to identify the positive

DBS, one sample was detected to be positive for HIV-1

DNA. Our study demonstrates that PCR for HIV-1 DNA

can be successfully performed on pools of DBS. However,

this may be needed only on specialized studies of HIV and

not for routine epidemiology studies as only a very small

fraction of cases would be missed if only antibody/antigen

testing were done.

Keywords Dried blood spots (DBS) � Polymerase chain reaction (PCR) � Pooling strategy � HIV screening

Introduction

Whole blood dried onto filter paper constitutes a potentially

useful material for molecular testing of viruses, including

HIV [1–3]. Though HIV is a RNA virus, once inside the

host CD4 cell following infection, HIV RNA is converted

to DNA. This viral DNA, after combining with host DNA,

exists as Proviral DNA. Dried blood spots (DBS) represent

a valuable and comprehensive genetic repository because

genomic DNA can be isolated from blood samples pro-

cessed on these filter papers for use in polymerase chain

reaction (PCR) [4]. Besides the extraction of DNA from

DBS, amplification from punches of DBS directly taken

into the PCR reaction mixture has been reported [5].

In spite of continuous improvements of the serological

assays, a residual risk that is extremely small still exists

attributed largely during the antibody-negative, pre sero-

conversion window period, the period between infection

and detectable seroconversion [1, 6]. Direct detection of

the virus can be achieved by nucleic acid test (NAT),

which enables detection of the viral nucleic acid. Previous

studies reported the detection of HIV DNA approximately

V. Lakshmi (&) � T. Sudha Nizam’s Institute of Medical Sciences, Hyderabad, AP, India

e-mail: vemulakshmigorthi@gmail.com

T. Sudha

e-mail: s_talasila@hotmail.com

D. Rakhi � G. Anilkumar � L. Dandona Public Health Foundation of India, New Delhi, India

e-mail: rakhi.dandona@phfi.org

L. Dandona

e-mail: lalit.dandona@phfi.org

L. Dandona

Institute for Health Metrics and Evaluation, University of

Washington, Seattle, Washington, USA

123

Indian J Microbiol (Apr–June 2011) 51(2):147–152

DOI 10.1007/s12088-011-0135-0

1 week earlier than detection of p24 antigen and about

2 weeks earlier than antibody detection [6, 7].

PCR technique was introduced in 1983 and the use of

DBS for PCR testing was first shown to have utility in 1987

[2] using in-house technique. Later, DBS PCR was adapted

to a standardized, commercially available microwell plate

amplification and detection kit, Amplicor HIV-1 produced

by Roche molecular systems [8]. Though several studies

have extracted DNA and RNA from a single DBS and

performed PCR for HIV DNA, there have been no reports

of performing HIV PCR on DBS that have been pooled

together. The principle of pooling is simply to mix a pre-

determined number of samples in a pool and test the pool

as one sample. Since pooling is a cost effective and time

saving way of performing nucleic acid tests, PCR on

pooled DBS is especially suited for samples that require

testing on large scale (e.g. samples from general

population).

With the absence of availability of any publications on

HIV PCR on pooled DBS, there has been a need of stan-

dardization and evaluation of the feasibility of performing

PCR for HIV-1 DNA from pooled DBS. The present study

was undertaken with an aim to evaluate the feasibility of

performing PCR for HIV-1 DNA on pools of DBS as an

alternative to individual testing.

Materials and Methods

Standardization of HIV-1 DNA PCR on Pooled DBS

DBS from five confirmed HIV reactives with known HIV-1

viral load (ranging between 500 and 600 copies/ml) in the

corresponding plasma sample and 32 HIV ELISA non-

reactive samples were used for standardization. All the five

HIV reactive DBS were positive for HIV-1 DNA when

tested individually to confirm that DNA could be detected

from DBS. Earlier reports have shown a good correlation

of HIV-1 viral load (quantitative test) in paired DBS and

plasma samples [3, 9]. Initially, the HIV status was deter-

mined on DBS collected from all these individuals using a

4th generation HIV ELISA [10].

Three groups of pools with 5, 10 and 20 DBS in each

were prepared as shown in Table 1. In each pool only one

known HIV reactive sample was included whereas the rest

of the DBS were from confirmed HIV non-reactive sam-

ples. Thus using five confirmed HIV reactive DBS a total

of 15 pools were prepared that included five pools of 5

DBS in each pool, five pools of 10 DBS in each pool and

five pools of 15 DBS in each pool. Viral load assay was

performed with Roche Amplicor version 1.5 monitor assay,

Roche molecular diagnostics, Branchburg, USA. For

obtaining DNA from DBS, a 6 mm punch was taken by

following the protocol as mentioned in several other

studies [8, 11, 12].

Preparation of DNA Pools from 5 DBS

For the 5 DBS pooling process, only one confirmed HIV-1

positive DBS sample with known viral load copies (S1)

was taken along with rest four confirmed HIV negative

samples (S2, S3, S4, S5). A 6 mm punch from each DBS

was extracted separately with 200 ll chelex reagent pro- vided in the Amplicor HIV-1 DNA test, version 1.5 by

following all the steps for extraction as mentioned in kit

insert [13]. After extracting all the 5 DBS separately, 10 ll from each extracted DNA sample was pooled together into

one PCR amplification tube, thus making the final volume

as 50 ll. This pooled extract was further processed by a qualitative PCR.

Preparation of DNA Pools from 10 DBS

For the 10 DBS pooling process, only one confirmed HIV

positive DBS sample with known viral load copies (S1)

was taken along with rest nine confirmed HIV negative

samples (S2, S3, S4, S5, S6, S7, S8, S9, S10). Instead of a

single DBS, two 6 mm punches, each taken from 2 sepa-

rate samples were extracted with 200 ll chelex reagent provided in the Amplicor HIV-1 DNA test, version 1.5 by

following all the steps for extraction as mentioned in kit

insert [13]. Thus five smaller pools of 2 DBS each were

prepared for extraction. After extracting all the five pools

of 2 DBS each, 10 ll from each was pooled together into one PCR amplification tube, thus making the final volume

Table 1 Pooling procedure for qualitative DNA PCR

standardisation

Number of DBS

pooled together

in each pool

Number of pools

prepared with

each positive DBS

DBS extracted

in 200 ll chelex using 6 mm punch

Amount of

DNA taken in

each test pool (ll)

5 5 1 10

10 5 2 10

20 5 2 5

148 Indian J Microbiol (Apr–June 2011) 51(2):147–152

123

as 50 ll. This pooled extract was further processed by a qualitative PCR.

Preparation of DNA Pool from 20 DBS

For the 20 DBS pooling process, only one confirmed positive

DBS sample with known viral load copies (S1) was taken

long with rest 19 confirmed HIV negative samples (S2–S20).

Instead of a single punch, two 6 mm punches, each taken

from two separate DBS were extracted with 200 ll chelex reagent provided in the Amplicor HIV-1 DNA test, version

1.5 by following all the steps for extraction as mentioned in

kit insert [13]. Thus ten smaller pools of 2 DBS each were

prepared for extraction. After extracting all the ten pools of 2

DBS each, 5 ll from each was pooled together into one PCR amplification tube, thus making the final volume as 50 ll. This pooled extract was further processed by a qualitative

PCR. Table 1 presents the detailed pooling procedures.

HIV-1 DNA PCR on Pooled DBS from Known HIV-1

Negative Individuals

Dried blood spots (DBS) were collected as part of population-

based HIV study of 12,617 respondents (aged 15–49 years)

from south India [14] where a blood sample by the finger-prick

method was obtained from these respondents on filter paper

(Whatman No. 3; Whatman International Ltd, Maidstone,

Kent, UK), preferably six drops of blood, without any anti-

coagulant on it. These blood drops were allowed to dry and

were stored in sealed polythene bags with desiccant in the field

office at room temperature and within 1 week were trans-

ported to the testing laboratory. In the laboratory, DBS were

stored under refrigeration at 2–8�C until testing for HIV-1 DNA was performed, maximum being within 1 year of col-

lection. Of these 12,617 respondents tested for HIV infection,

240 were found to be HIV positive. Of the samples negative

for both antibody and antigen, a subset of 585 samples

belonging to people who were considered at relatively high

risk of HIV and another set of 10% of all samples (1238)

negative for HIV antibody or antigen underwent qualitative

PCR (Amplicor 1.5; Roche molecular diagnostics, Branch-

burg, USA) testing for HIV viral nucleic acid to detect very

recent infections. After the successful standardisation of

pooled PCR, DBS from these 1,823 individuals who were

negative for both HIV antigen and antibodies were tested for

HIV-1 DNA by PCR by the optimized pooling procedure

(i.e. using 10 DBS pooling strategy).

DNA from 10 DBS each was pooled together, except for

a single pool that had only 3 DBS pooled together as

described above. To identify the positive DBS from the

positive pool, the DNA extracted from all the ten indi-

vidual DBS that were included in the positive pool were

processed again by HIV-1 PCR.

PCR Procedure

PCR for detecting the presence of HIV-1 proviral DNA in

all the above cases was performed by using Amplicor HIV-

1 DNA version 1.5 (Roche molecular diagnostics,

Branchburg, USA). The Amlicor HIV-1 DNA test, version

1.5 is based on four major processes: sample preparation;

PCR amplification of target DNA using HIV-1 specific

complementary primers; hybridization of the amplified

products to oligonucleotide probes specific to the target;

and detection of the probe-bound amplified products by

colorimetric determination [13].

Primers

Primers SK145 and SKCC1B supplied in the commercial kit

of Amlicor HIV-1 DNA test, version 1.5 are used to amplify a

155-nucleotide sequence of the HIV-1 gag gene [13].

RT and downstream PCR primer SKCC1B complemen-

tary to nucleotides 1485–1512 of HIV-1HXB2 is (5 0-TAC

TAGTAGTTCCTGCTATGTCACTTCC-30) and upstream primer SK145 (50-AGTGGGGGGACATCAAGCAGCCAT GCAAAT-30).

Preparation of Samples from DBS [2, 13]

A 6 mm punch of DBS was taken into a 1.5 ml screw-cap

tube containing 1.0 ml of blood wash solution (BLD WS)

and was incubated for 30 min at room temperature. After

micro-centrifugation for 3 min at maximum speed, super-

natant was aspirated; 1.0 ml of BLD WS was added to each

tube, recapped and vortexed. Tubes were microcentrifuged

for 3 min at maximum speed. All this was repeated again.

Supernatant was aspirated, being careful to avoid disturb-

ing the DBS. The dry DBS was extracted immediately

(alternatively it can be stored at -70�C until extraction). 200 ll working Extraction Reagent was added to each DBS and vortexed. Tubes were incubated for 30 min at 60�C ± 2�C in a dry heat block containing sand and then for 30 min at 100�C ± 2�C in a dry heat block containing sand. Samples were vortexed briefly and microcentrifuged for 3 s. 50 ll of samples was added to appropriate PCR reaction tubes con-

taining the working master mix. Rest of the steps for PCR test

was performed as mentioned in the kit insert [13].

Results

Standardization of HIV-1 DNA PCR on Pooled DBS

All the negative and positive controls in the HIV-1 DNA

PCR runs were valid. Results of all the 15 DNA pools from

the DBS are presented in Fig. 1 and Table 2. Internal

Indian J Microbiol (Apr–June 2011) 51(2):147–152 149

123

control (IC) that serves as an extraction and amplification

control was observed to have sufficiently high OD values

for all 15 pools, indicating that the control DNA in all the

pools was properly amplified.

Specificity, sensitivity, positive and negative predictive

values of the assay were all observed to be 100% when

tested on pools of 5 and 10 DBS as all the pools of 5 and 10

DBS pooled together were positive for HIV-1 PCR. It was

noted that DNA could be detected from only 2/5 pools

from the 20 DBS pooled together thus indicating a sensi-

tivity of only 60% and a negative predictive value of 98%

though both specificity and positive predictive values

remained to be 100%. Thus pooling of 20 DBS that indi-

cated 98.06% overall accuracy of the assay was ruled out.

Standardization of pooling procedure accordingly indicated

that pooling of 10 DBS gave an optimum result due to its

cost effectiveness compared to 5 DBS pooled together and

hence was then adopted for pooling the study samples.

HIV-1 DNA PCR on Pooled DBS from Known HIV-1

Negative Individuals

Only one pool from the 183 pools was positive by PCR.

From the single PCR positive study DBS pool, after per-

forming PCR on ten individual samples, 1 DBS was found

to be positive for HIV-1 DNA PCR. The results of PCR on

pooled DBS are presented in Fig. 2. By including this one

HIV-1 positive sample that was detected by PCR, total HIV

positive samples increased from 240 to 241. This single

positive sample was 0.4% of the total 241 HIV positive

cases from this population-based study. Pooling strategy

also resulted in 88% reduction in overall cost compared to

PCR testing on individual sample. It also resulted in a

considerable reduction in testing time; ten times less than

individual testing.

Discussion

In spite of a person screened as negative by a 4th genera-

tion HIV assay, only direct monitoring by sensitive NAT

would provide accurate data of HIV infection in the pop-

ulation being tested. However, NAT is too expensive and

labor-intensive for routine screening of single specimens.

Because of the high cost of PCR on individual samples,

costs saving strategies have been devised for large numbers

of samples. One such strategy is to mix a predetermined

number of the samples to be assayed to make a testing pool

and process this pool as one sample. A positive result will

3.21

2.38

2.912.8

2.16

0.13

2.13

0.16

1.89

0.11

3.27 3.133.24 3.02

2.56

NC =0.2

PC =0.8

0

0.5

1

1.5

2

2.5

3

3.5

0 1 2 3 4 5 6

Pools of DBS

O D

o f P

C R

5 DBS POOL 10 DBS POOL 20 DBS POOL NC PC NC

Fig. 1 Individual results of DBS pooled together for standardization [Arrowheads indicate the pools (20 DBS pooled together) that were reported as negative in spite of a positive sample among each of them]

Table 2 Standardization results of HIV-1 DNA PCR on pooled DBS

No. of DBS

pooled together

Range of PCR OD

for HIV-1 DNA

Range of OD

for Internal Control

No. of pools

tested

No. of positive

pools

No. of negative

pools

5 DBS 2.38–3.0 2.35–3.0 5 5 0

10 DBS 2.16–3.0 2.45–3.0 5 5 0

20 DBS 0.16–2.13 2.48–3.0 5 2 3

4th gen Elisa for HIV Ag/ab on DBS collected from a South Indian Study (N=12,617)

HIV-1 DNA PCR on total 183 pools (One pool had 3

DBS) (N = 1813)

Pools were prepared by taking 10 DBS in each pool (59 pools) last pool included 5

samples from subset -2 (N=580)

Positive pools = 1 pool (N= 10)

Negative pools = 124 pools (N=1233)

HIV-1 DNA PCR on individual 10 DBS from the positive pool

Positive pool was opened up into 10 DBS

Negative DBS (N=9) Positive DBS (N= 1) Total Negative DBS

(N=1,813)

Subset 1: DBS belonging to people at relatively high risk of

HIV (N=585)

Subset 2: 10% of all samples negative for HIV antibody or

antigen (N=1238)

HIV Positive (N=240)

Pools were prepared by taking 10 DBS in each pool (124 pools) (last pool had

3 DBS) (N=1233)

Negative pools = 58 pools (N=580)

Fig. 2 Results of HIV-1 DNA PCR on pooled DBS detected as negative for HIV antibody and antigen

150 Indian J Microbiol (Apr–June 2011) 51(2):147–152

123

indicate the presence of the viral DNA in one or more

samples within the pool and will necessitate retesting of

each of the samples that constituted the pool, so as to

identify the truly positive sample(s). On the other hand, a

negative result would indicate that none of the samples in

the pool contain the viral DNA.

The issue of sensitivity of PCR on pooled samples pri-

marily depends on dilution of the viral DNA. Hence, the

number of samples in the pool (pool size) has to be

optimized. In our study, the optimum pool size was

determined by using varying pool sizes, each containing

one known HIV-1 positive sample. The viral DNA could

be detected in all the pools with 5 and 10 DBS. However,

the 3/5 pools with 20 DBS were recorded false negative,

due to the dilution of the DNA below undetectable levels.

Since pooling of 10 DBS was more cost effective com-

pared to pools of 5 DBS, it was considered as the optimum

pool size and hence was used in our study.

Adopting the pooling strategy on 1,823 DBS from our

study samples resulted in detection of an additional one

positive sample by HIV-1 DNA PCR, making the total

number of HIV positive DBS to 241 out of 12,617 total

study samples. This PCR positive sample was actually

from the subset of 585 samples belonging to people who

were considered at relatively high risk of HIV and probably

was from an individual who was either in the early sero-

conversion phase of HIV infection or was on anti retroviral

therapy, the details of which were not available. Due to

insufficient sample, a quantitative PCR could not be per-

formed that would have indicated the viral load of the

positive sample and the probable time since sero-conver-

sion. Another set of 10% of all samples (1238) negative for

HIV antibody or antigen that underwent qualitative PCR in

pools of ten samples remained negative for HIV-1 DNA.

Pooling strategy also resulted in a considerable reduction

in overall cost and testing time compared to PCR testing on

individual testing. In spite of several advantages of PCR, one

limitation of the PCR test is that though theoretically 100%

sensitivity and specificity can be achieved, the observed

sensitivity of HIV-1 DNA PCR assays in clinical practice is

reported to be 96–99% [15–17] due to the undetectable levels

of viral DNA in the eclipse phase of infection i.e. 3–7 days

from exposure and infection [6].

Pooling strategy can be more cost effective for testing

HIV prevalence in research studies conducted on special-

ized population where the prevalence rates are low. How-

ever, such pooling strategy for PCR if applied to screen

high risk populations, the number of HIV case detections

can be increased cost effectively, only if all samples that

are negative for HIV Ag or Ab are pooled together, similar

to the strategy adapted in our study. But if all samples from

high risk population studies are pooled together, as an

initial step, then the pooling strategy may lose its cost

effectiveness as due to high prevalence of HIV infection,

quite a lot of of the pools will be positive and all samples in

the positive pool will need individual testing for detection

of HIV-1 DNA in the sample that may lead to higher cost

and also will be more time consuming.

Our study has demonstrated that PCR for HIV-1 DNA

can be successfully performed on pools of DBS with time

and cost savings without compromising the sensitivity of

the PCR.

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152 Indian J Microbiol (Apr–June 2011) 51(2):147–152

123

  • Application of Polymerase Chain Reaction to Detect HIV-1 DNA in Pools of Dried Blood Spots
    • Abstract
    • Introduction
    • Materials and Methods
      • Standardization of HIV-1 DNA PCR on Pooled DBS
        • Preparation of DNA Pools from 5 DBS
        • Preparation of DNA Pools from 10 DBS
        • Preparation of DNA Pool from 20 DBS
      • HIV-1 DNA PCR on Pooled DBS from Known HIV-1 Negative Individuals
      • PCR Procedure
        • Primers
        • Preparation of Samples from DBS [2, 13]
    • Results
      • Standardization of HIV-1 DNA PCR on Pooled DBS
      • HIV-1 DNA PCR on Pooled DBS from Known HIV-1 Negative Individuals
    • Discussion
    • References

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