Journal of Virological Methods 194 (2013) 132–137

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Journal of Virological Methods

journal homepage: www.elsevier.com/locate/jviromet

Evaluation of five simple rapid HIV assays for potential use in the
Brazilian national HIV testing algorithm
Leonardo Rapone da Motta a,∗ , Andréa Cristina Vanni a , Sérgio Kakuta Kato a,b,c ,
Luiz Gustavo dos Anjos Borges a , Rosa Dea Sperhacke a , Rosangela Maria M. Ribeiro d ,
Lilian Amaral Inocêncio e , the HIV Rapid Test Evaluation Group
a
Laboratório de Pesquisa em HIV/AIDS, Centro de Ciências da Saúde, Universidade de Caxias do Sul, Rua Francisco Getúlio Vargas, 1130 – Bloco S – Sala
315, Caxias do Sul, RS 95070-560, Brazil
b
Faculdade de Matemática, Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Av. Ipiranga, 6681 – Predio 30 – Bloco C – FAMAT, Porto Alegre,
RS 90619-900, Brazil
c
Departamento de Saúde Coletiva, Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA), Rua Sarmento Leite, 245, Porto Alegre, RS CEP
90050-170, Brazil
d
Coordenação do Cuidado e Qualidade de Vida, Departamento de DST, AIDS e Hepatites Virais, Secretária da Vigilância em Saúde, Ministério da Saúde, SAF
Sul, Trecho 2, Bloco F, Torre 1, Edifício Premium, Auditório, Sala 03, Brasília, DF 70070-600, Brazil
e
Instituto Tecnológico de Imunobiológicos – Bio-Manguinhos, Fundação Oswaldo Cruz, Av. Brasil, 4365 – Manguinhos, RJ 21040-900, Brazil

a b s t r a c t

Article history: Since 2005, the Department of Sexually Transmitted Diseases (STDs), Acquired Immunodeficiency Syn-
Received 19 March 2013 drome (AIDS) and Viral Hepatitis under the Health Surveillance Secretariat in Brazil’s Ministry of Health
Received in revised form 8 August 2013 has approved a testing algorithm for using rapid human immunodeficiency virus (HIV) tests in the coun-
Accepted 15 August 2013
try. Given the constant emergence of new rapid HIV tests in the market, it is necessary to maintain an
Available online 28 August 2013
evaluation program for them. Conscious of this need, this multicenter study was conducted to evaluate
five commercially available rapid HIV tests used to detect anti-HIV antibodies in Brazil.
Keywords:
The five commercial rapid tests under assessment were the VIKIA HIV-1/2 (bioMérieux, Rio de Janeiro,
HIV-1
HIV-2
Brazil), the Rapid Check HIV 1 & 2 (Center of Infectious Diseases, Federal University of Espírito Santo,
HIV diagnosis Vitória, Brazil), the HIV-1/2 3.0 Strip Test Bioeasy (S.D., Kyonggi-do, South Korea), the Labtest HIV (Labtest
Immune assays Diagnóstica, Lagoa Santa, Brazil) and the HIV-1/2 Rapid Test Bio-Manguinhos (Oswaldo Cruz Foundation,
Rio de Janeiro, Brazil). A total of 972 whole-blood samples were collected from HIV-infected patients,
pregnant women and individuals seeking voluntary counselling and testing who were recruited from five
centers in different regions of the country. Informed consent was obtained from the study participants.
The results were compared with those obtained using the HIV algorithm used currently in Brazil, which
includes two enzyme immunoassays and one Western blot test. The operational performance of each
assay was also compared to the defined criteria.
A total of 972 samples were tested using reference assays, and the results indicated 143 (14.7%) reactive
samples and 829 (85.3%) nonreactive samples. Sensitivity values ranged from 99.3 to 100%, and specificity
was 100% for all five rapid tests. All of the rapid tests performed well, were easy to perform and yielded
high scores in the operational performance analysis. Three tests, however, fulfilled all of the prerequisites
established previously by the Department of STDs, AIDS and Viral Hepatitis: the HIV-1/2 3.0 Strip Test
Bioeasy, the Rapid Check HIV 1 & 2 and the VIKIA HIV-1/2.
Three of the five tests evaluated (the HIV-1/2 3.0 Strip Test Bioeasy, the Rapid Check HIV 1 and 2 and
the VIKIA HIV-1/2) performed as well as the reference assays and fulfilled the requirements for use in the
Brazilian national algorithm.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Since the beginning of the Human Immunodeficiency Virus

∗ Corresponding author. Tel.: +55 54 32182737; fax: +55 54 32182056.
E-mail addresses: lrmotta@ucs.br (L.R. da Motta), acvanni@ucs.br
(A.C. Vanni), klmucs@terra.com.br (S.K. Kato), luizgaborges@gmail.com
(L.G.d.A. Borges), rdsperha@ucs.br (R.D. Sperhacke), rosangelammr@gmail.com
(R.M.M. Ribeiro), innozencio@gmail.com (L.A. Inocêncio).
(HIV) epidemic, 608,230 HIV infection cases and 241,469 Acquired
Immunodeficiency Syndrome (AIDS) deaths have been reported
in Brazil. Over the last 12 years, the HIV/AIDS epidemic in Brazil
has been relatively stable (with an incidence rate of 12.9/100,000
cases), although there has been an increase in the northern and

0166-0934/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jviromet.2013.08.016
 L.R. da Motta et al. / Journal of Virological Methods 194 (2013) 132–137 133

Table 1
Characteristics of the five rapid HIV tests evaluated.

VIKIA HIV-1/2 Rapid Check HIV 1 & 2 HIV-1/2 3.0 Strip Test Labtest HIV HIV-1/2 Rapid Test –
Bioeasy Bio-Manguinhos

Manufacturer bioMérieux, Rio de Janeiro, Center of Infectious Standard Diagnostics, Labtest Diagnóstica, Oswaldo Cruz
Brazil Diseases, Federal Kyonggi-do, South Lagoa Santa, Brazil Foundation, Rio de
University of Espírito Korea Janeiro, Brazil
Santo, Vitória, Brazil
Assay type Immunochromatographic Immunochromatographic Immunochromatographic Immunochromatographic Immunochromatographic
assay, lateral flow assay, lateral flow assay, lateral flow assay, lateral flow assay, lateral flow
Specimen type Whole blood, plasma, Whole blood, plasma, Whole blood, plasma, Whole blood, plasma, Whole blood, plasma,
serum serum serum serum serum
Required specimen 75 ␮L 10 ␮L (serum or 10 ␮L (serum or 10 ␮L 5 ␮L
volume plasma) plasma)
20 ␮L (whole blood) 20 ␮L (whole blood)
Storage conditions 4–30 ◦ C 2–27 ◦ C 2–30 ◦ C 2–30 ◦ C 8–30 ◦ C

northeastern region s of the country (Brazilian Ministry of Health,
2012).
To overcome the lack of infrastructure and human resources in
remote regions, reduce laboratory turnaround times and expand
the coverage of HIV testing, the Department of Sexually Trans-
mitted Diseases (STDs), AIDS and Viral Hepatitis under the Health
Surveillance Secretariat, Ministry of Health, has developed a testing
algorithm in Brazil that is based solely on using rapid anti-HIV tests
for patients aged 18 months and older in certain situations (e.g.,
for occupational exposures, in inaccessible areas and for pregnant
women who did not receive prenatal care) (Brazilian Ministry of
Health, 2007, 2005).
The testing algorithm using rapid tests was reviewed and
amended four years after its publication (Brazilian Ministry of
Health, 2009). A single rapid test can produce a negative result,
whereas a positive result requires the use of two rapid tests of dif-
ferent brands or methodologies. Discordant results between the
tests require blood samples collected by venipuncture to be sub-
jected to the testing algorithm using conventional assays.
There are currently several rapid testing options for detecting
anti-HIV antibodies in Brazil, and new tests and technologies are
emerging constantly. Department of STDs, AIDS and Viral Hepati-
tis maintains a program for evaluating rapid HIV tests. Eleven tests
were assessed in the last two evaluations, and six were approved:
Determine HIV-1/2 (Abbott Laboratories, Abbott Park, IL, USA); HIV
Rapid Check (Center of Infectious Diseases, Federal University of
Espirito Santo, Vitória, Brazil); HIV 1/2 STAT PAK (Chembio Diag-
nostic Systems, Medford, NY, USA); Uni-Gold HIV (Trinity Biotech
plc, Wicklow, Ireland); BD Check HIV Multi-test (Becton Dickin-
son, Franklin Lakes, NJ, USA) and HIV 1/2 Colloidal Gold (Shanghai
Kehua Bio-Engineering, Beijing, China). To be approved, the tests
must have a sensitivity of 99.5% or greater and a specificity of 99.0%
or greater when compared with an enzyme-linked immunosorbent
assay (ELISA) in combination with a confirmatory test; good oper-
ational assay performance is also required for approval (Brazilian
Ministry of Health, 2009; Ferreira Junior et al., 2005).
This multicenter study was conducted as part of a program to
evaluate rapid tests for detecting anti-HIV antibodies in the Brazil-
ian population. The analytical and operational performances of five
rapid tests were evaluated.
2. Materials and methods
2.1. Selection criteria
be donated by the manufacturer or distributor in sufficient quanti-
ties to perform this evaluation.
2.2. Rapid test assays
The five rapid tests that were assessed were the VIKIA HIV-1/2
(bioMérieux, Rio de Janeiro, Brazil), the Rapid Check HIV 1 & 2
(Center of Infectious Diseases, Federal University of Espírito Santo,
Vitória, Brazil), the HIV-1/2 3.0 Strip Test Bioeasy (S.D., Kyonggi-do,
South Korea), the Labtest HIV (Labtest Diagnóstica, Lagoa Santa,
Brazil) and the HIV-1/2 Rapid Test – Bio-Manguinhos (Oswaldo
Cruz Foundation, Rio de Janeiro, Brazil) (Table 1). The tests were
used according to the manufacturer’s instructions by a trained lab-
oratory technologist.
2.3. Reference assays
The diagnostic kits used as reference assays were the Vidas HIV
Duo Ultra (bioMérieux, Marcy l’Etoile, France) and the Vironos-
tika Uni-Form II plus O Microelisa System (bioMérieux, Boxtel, The
Netherlands). The Vidas HIV Duo Ultra is an automated enzyme-
linked fluorescent assay that enables simultaneous detection of
p24 antigen and HIV types 1 and 2 IgM and IgG antibodies using a
double-sandwich EIA, as described previously (Weber et al., 2002).
The Vironostika Uni-Form II plus O is a third-generation ELISA that
uses a one-step sandwich principle to detect antibodies for HIV-
1, HIV-2 and HIV-1 group O (Van Binsbergen et al., 1996). The
samples that were nonreactive in both reference assays were clas-
sified as negative, and no additional tests were performed on these
samples. Specimens with discordant EIA results were subjected to
a confirmatory test using the Cambridge Biotech HIV-1 Western
Blot diagnostic kit (Calypte Biomedical Corporation, Rockville, MD,
USA). The Cambridge Biotech HIV-1 Western Blot is an enzyme-
linked immunosorbent blot capable of detecting antibodies for any
of the major HIV-1 antigens, including one or more of the following
HIV-1 proteins (p) or glycoproteins (gp): p17, p24, p31, gp41, p51,
p55, p66, gp120 and gp160. The Western blot was considered pos-
itive when the p24 gag protein was present in combination with
any two other viral envelope proteins (e.g., gp41, gp120 or gp160),
regardless of the band intensity observed, as recommended by the
algorithm used currently in Brazil (Brazilian Ministry of Health,
2009). The samples with negative Western Blot results were consid-
ered negative, and samples with indeterminate results on Western
Blot were excluded from the analysis.

Participation in this third round of evaluation of rapid HIV tests
was voluntary; however, the tests must be approved for bulk pro-
curement by the Department of STDs, AIDS and Viral Hepatitis. To
be eligible for participation, the test kits must (a) be registered in
Brazil, (b) be able to detect HIV-1 and HIV-2 simultaneously and (c)
2.4. Reference panels
Highly characterized samples from commercial performance
and seroconversion panels produced by Sera Care Life Sciences
(Milford, MA, USA) were tested using the rapid tests under
134 L.R. da Motta et al. / Journal of Virological Methods 194 (2013) 132–137

Table 2
Evaluation of the sensitivity and specificity of the five rapid tests using whole-blood samples.

Assay n Sensitivity, % Specificity, % (95% CI) Positive predictive Negative

(95% CI) value, % (95% CI) predictive value,
% (95% CI)

VIKIA HIV-1/2 884 100 (97.2–100) 100 (99.5–100) 100 (97.9–100) 100 (99.6–100)
Rapid Check HIV 1 & 2 964 100 (97.3–100) 100 (99.5–100) 100 (98.0–100) 100 (99.7–100)
HIV-1/2 3.0 Strip Test Bioeasy 966 100 (97.4–100) 100 (99.5–100) 100 (98.0–100) 100 (99.7–100)
Labtest HIV 965 99.3 100 (99.5–100) 100 (97.8–100) 99.9 (99.5–99.9)
(96.1–99.9)
HIV-1/2 Rapid test – Bio-Manguinhos 965 99.3 100 (99.5–100) 100 (97.8–100) 99.9 (99.5–99.9)
(96.1–99.9)

evaluation. Two seroconversion panels, each containing undiluted
samples collected from a single donor during the development
of HIV-1 infection and the subsequent immune response, were
used: PRB965 (six specimens) and PRB914 (five specimens). Two
performance panels containing undiluted samples collected from
multiple donors with various antibody concentrations, serotypes
and ethnic/demographic backgrounds were used: PRB204(M) Anti-
HIV 1 Mixed Titer Performance Panel (21 specimens) and PRZ207
Anti-HIV-1/2 Combo Performance Panel (15 specimens). The pres-
ence of HIV serotypes (type 1 and type 2) was assessed using
the PRZ207, which includes seven HIV-1-positive samples, seven
HIV-2-positive samples and one negative sample. Because of their
high cost, the specimens belonging to the four commercial panels
were tested only once with each assay under evaluation.
2.5. Sample collection
A total of 966 individuals over 18 years of age were recruited
consecutively between July 20, 2009, and January 28, 2010. Of
the total sample, 575 patients were referred from voluntary coun-
selling and testing (VCT) centers, 298 patients were pregnant
women receiving screening, and 93 patients were confirmed to

Table 3
Results from commercial performance panels PRB204(M) and PRZ207 (Sera Care Life Sciences, Milford, MA, USA).

Performance panels Rapid test under assessment Reference assays

Sample Expected result VIKIA® Rapid HIV-1/2 3.0 Labtest HIV HIV-1/2 Vidas HIV Vironostika Cambridge
identification HIV-1/2 Check HIV 1 Strip Test Rapid Test – Duo Ultra HIV Biotech
&2 Bioeasy Bio- Ab Uni-Form HIV-1 WB
Manguinhos Plus O
Microelisa
System

PRZ207-01 POS POS POS POS POS POS POS POS POS
PRZ207-02 POS POS POS POS POS POS POS POS POS
PRZ207-03 POS POS POS POS POS POS POS POS POS
PRZ207-04 POS POS POS POS POS POS POS POS POS
PRZ207-05 POS POS POS POS POS NEG NEG POS POS
PRZ207-06 POS POS POS POS POS POS POS POS POS
PRZ207-07 POS POS POS POS POS POS POS POS POS
PRZ207-08 POS POS POS POS POS POS POS POS IND
PRZ207-09 POS POS POS POS POS POS POS POS POS
PRZ207-10 POS POS POS POS POS POS POS POS POS
PRZ207-11 POS POS POS POS POS POS POS POS POS
PRZ207-12 POS POS POS POS POS POS POS POS POS
PRZ207-13 POS POS POS POS POS POS POS POS IND
PRZ207-14 POS POS POS POS POS POS POS POS POS
PRZ207-15 NEG NEG NEG NEG NEG NEG NEG NEG NEG

PRB204(M)-01 IND NEG POS POS NEG NEG NEG NEG NEG
PRB204(M)-02 POS POS POS POS POS POS POS POS POS
PRB204(M)-03 NEG NEG NEG NEG NEG NEG NEG NEG NEG
PRB204(M)-04 POS POS POS POS POS POS POS POS POS
PRB204(M)-05 POS POS POS POS POS POS POS POS POS
PRB204(M)-09 IND POS POS POS POS NEG POS POS NEG
PRB204(M)-10a POS POS POS POS POS POS POS POS POS
PRB204(M)-11 POS POS POS POS POS POS POS POS POS
PRB204(M)-12 POS POS POS POS POS POS POS POS POS
PRB204(M)-13 POS POS POS POS POS POS POS POS IND
PRB204(M)-14 POS POS POS POS POS POS POS POS POS
PRB204(M)-15 POS POS POS POS POS POS POS POS POS
PRB204(M)-16 POS POS POS POS POS POS POS POS POS
PRB204(M)-17 POS POS POS POS POS POS POS POS POS
PRB204(M)-18 POS POS POS POS POS POS POS POS IND
PRB204(M)-19 POS POS POS POS POS POS POS POS POS
PRB204(M)-20 POS POS POS POS POS POS POS POS POS
PRB204(M)-21 POS POS POS POS POS POS POS POS POS
PRB204(M)-23 NEG NEG NEG NEG NEG NEG NEG POS NEG
PRB204(M)-24 POS POS NEG NEG POS NEG POS POS NEG
PRB204(M)-25 IND POS POS POS POS NEG POS POS NEG

Abbreviations: POS – positive; NEG – negative; IND – indeterminate.

a
PRB204-10 is a bleed from the same donor as PRB204-24, drawn 8 days later.
 L.R. da Motta et al. / Journal of Virological Methods 194 (2013) 132–137 135

Table 4
Results from commercial seroconversion panels PRB914 and PRB965 (Sera Care Life Sciences, Milford, MA, USA).

Seroconversion Days VIKIA® Rapid Check HIV-1/2 3.0 Labtest HIV HIV-1/2 Rapid Test – Vidas HIV Vironostika HIV Cambridge
panels HIV-1/2 HIV 1 & 2 Strip Test Bio-Manguinhos Duo Ultra Uni-Form Plus O Biotech HIV-1
Bioeasy Ab Microelisa WB
System

PRB914-01 0 POS POS POS POS NEG POS POS POS

PRB914-02 4 POS POS POS POS NEG POS POS POS
PRB914-03 7 POS POS POS POS NEG POS POS POS
PRB914-04 25 POS POS POS POS NEG POS POS POS
PRB914-05 31 POS POS POS POS NEG POS POS POS

PRB965-01 0 NEG NEG NEG NEG NEG NEG NEG NEG

PRB965-02 5 NEG NEG NEG NEG NEG NEG NEG NEG
PRB965-03 7 NEG NEG NEG NEG NEG NEG NEG NEG
PRB965-04 12 POS POS POS POS NEG POS POS IND
PRB965-05 14 POS POS POS POS NEG POS POS IND
PRB965-06 21 POS POS POS POS POS POS POS POS

Abbreviations: POS – positive; NEG – negative; IND – indeterminate.

be positive for HIV antibodies but were treatment-naïve. The
study included patients from three different regions of Brazil:
422 from the south (Florianópolis, Porto Alegre and Viamão), 246
from the midwest (Brasília) and 298 from the northeastern region
(Natal).
The patients provided written informed consent, and 4 mL of
whole blood was collected from each subject into tubes contain-
ing ethylenediaminetetraacetic acid (EDTA) (BD Vacutainer EDTA
K2 4 mL, BD Diagnostics, Juiz de Fora, Brazil) as an anticoagu-
lant. The rapid tests were performed using whole blood at the
five study reference centers: the Central Health Laboratory of the
Federal District (Brasília, Distrito Federal), the Central Health Lab-
oratory of Rio Grande do Norte (Natal, Rio Grande do Norte), the
HIV/AIDS Research Laboratory at the University of Caxias do Sul
(Caxias do Sul, Rio Grande do Sul), the Hospital Nossa Senhora da
Conceição Central Laboratory (Porto Alegre, Rio Grande do Sul)
and the University Hospital of the Federal University of Santa
Catarina (Florianópolis, Santa Catarina). After the samples were
tested using the rapid tests, the blood samples were centrifuged
for 10 min at a speed of 1300 g, and 1-mL plasma aliquots were
placed in sterile microtubes using filter pipette tips to minimize
cross-contamination. Next, the samples were stored at −20 ◦ C (or
less) and forwarded to the HIV/AIDS Research Laboratory of the
University of Caxias do Sul to be tested using the reference assays.
2.6. Operational performance
storage temperature (1, ambient temperature; 0, under refriger-
ation); number of reagents needed to run the assay (1, only one
reagent; 0, more than one reagent needed); total number of assay
steps (1, four steps or less; 0, more than four steps); required
expertise (1, no laboratory experience; 0, laboratory experience
recommended); and total execution time (1, 20 min or less; 0, more
than 20 min).
2.7. Criteria for inclusion in the national HIV testing algorithm
The Department of STDs, AIDS and Viral Hepatitis established
the following criteria for approving rapid tests for detecting anti-
HIV antibodies. The assay must (a) have a sensitivity of 99.5%
or greater, (b) have a specificity of 100% and (c) have an opera-
tional performance that meets at least four of the five criteria listed
above.
2.8. Statistical analysis
The results obtained from the rapid tests under evaluation
were compared to the results obtained from the reference assays.
Sensitivity, specificity and positive and negative predictive values
were calculated using previously described methods (CLSI, 2008)
and were expressed with a binomial confidence interval (CI) of
95%.

The operational assay performance described by Ferreira Junior
(Ferreira Junior et al., 2005) was used to assess the operational
characteristics of the rapid tests under evaluation. Briefly, each
operational characteristic was assigned a score from 0 (negative)
to 1 (positive). To achieve good operational performance, the rapid
tests had to meet at least four of the five following criteria: reagent
2.9. Ethical considerations
This unlinked, anonymous study was approved by the National
Bioethics Commission of Brazil (Conselho Nacional de Ética em
Pesquisa – CONEP). Informed consent was obtained from all of the
participants prior to enrollment.

Table 5
Evaluation of the operational assay performance based on five characteristics of the rapid test.

Assay Score for the evaluation of the operational assay performance Assay performance
a
No. of reagents Reagent storage temperature No. of steps Total execution time Technical skills
(hh:mm) of operator

VIKIA HIV-1/2 1 (1) 1 (4–30 ◦ C) 1 (3) 0 (00:31) 1 Good (4/5)

Rapid Check HIV 1 & 2 1 (1) 1 (2–27 ◦ C) 1 (3) 1 (00:16) 1 Good (5/5)
HIV-1/2 3.0 Strip Test 1 (1) 1 (2–30 ◦ C) 1 (3) 1 (00:16) 1 Good (5/5)
Bioeasy
Labtest HIV 1 (1) 1 (2–30 ◦ C) 1 (3) 1 (00:11) 1 Good (5/5)
HIV-1/2 Rapid test – 1 (1) 1 (8–30 ◦ C) 1 (3) 1 (00:11) 1 Good (5/5)
Bio-Manguinhos
a
The steps include all activities performed by the operator (plus incubation time) before reading the test.
136 L.R. da Motta et al. / Journal of Virological Methods 194 (2013) 132–137
3. Results
3.1. Clinical samples
A total of 966 samples (excluding the seroconversion and per-
formance panels) were tested with the reference assays; 142 were
classified as reactive samples (14.7%), and 824 were classified as
nonreactive samples (85.3%).
Table 2 shows the sensitivity and specificity of the five rapid
tests that were assessed. All of the clinical samples were classified
correctly by the VIKIA HIV-1/2, the Rapid Check HIV 1 & 2 and the
HIV-1/2 3.0 Strip Test Bioeasy. The Labtest HIV and the HIV-1/2
Rapid Test – Bio-Manguinhos each presented one false-negative
result. The positive predictive values (PPV) were 100% for all of the
rapid tests. The negative predictive values (NPV) were 100%, except
for the Labtest HIV and the HIV-1/2 Rapid Test – Bio-Manguinhos
(99.9%, 95% CI; 99.5–99.9).
A total of 82 specimens were not assessed using the VIKIA HIV-
1/2 for the following reasons: 66 tests were stored incorrectly at
−20 ◦ C or below because of a clinical technical error; 15 specimens
were not tested because the kit expired during the evaluation, and
the clinical site did not report the result for one specimen; in addi-
tion, two specimens were not assessed using the Rapid Check HIV
1 & 2, and one specimen was not assessed using the Labtest HIV or
the HIV-1/2 Rapid Test – Bio-Manguinhos because the clinical site
did not report their results.
3.2. Reference panels
The results obtained from the performance panels are shown
in Table 3. One sample from panel PRZ207 (PRZ207-05) was clas-
sified incorrectly by the HIV-1/2 Rapid Test – Bio-Manguinhos.
Sample PRB204(M)-24 was classified incorrectly by three tests
(Rapid Check HIV 1 & 2, Labtest HIV and HIV-1/2 Rapid Test –
Bio-Manguinhos). Note that although this sample was positive
according to the reference assays, repeated Western blot analysis
did not reveal any positive bands.
The results obtained from the seroconversion panels are pre-
sented in Table 4. The HIV-1/2 Rapid Test – Bio-Manguinhos
classified seven of 11 samples (7/11, 64%) incorrectly. The other
tests produced the same results as the reference assays.
3.3. Operational performance
All of the rapid tests obtained maximum scores (5/5), except
for the VIKIA HIV-1/2, which performed well (4/5), but the total
execution time of the assay was greater than 20 min (31 min).
Overall, all of the tests that were evaluated operate in a similar
manner: the reagents did not require refrigeration, and the steps
included applying the sample, adding a running buffer (one to three
drops) and reading the results visually (between five and 30 min).
Data obtained from the operational assay performance analysis are
shown in Table 5.
4. Discussion
The evaluation criteria were reviewed before the third eval-
uation, and stricter guidelines were applied beginning with this
evaluation. Previous evaluations required the test sensitivity to be
99.5% or greater, the specificity to be 99.0% or greater and the oper-
ational performance to meet four or five criteria (Ferreira Junior
et al., 2005). Because rapid HIV testing technology has improved,
there has been a large increase in the number of tests and brands
available in the Brazilian market, and an increasing number of rapid
tests are being performed each year. Thus, the evaluation criteria
were revised. To receive approval for use in the national algorithm
for diagnosing HIV infection in patients aged 18 months and older,
sensitivity must be 99.5% or greater, and specificity must be 100%.
There were no changes made to the operational performance crite-
ria.
Overall, all five tests demonstrated good performance with
regard to all of the criteria; however, only three assays fulfilled all of
the previously established prerequisites: the HIV-1/2 3.0 Strip Test
Bioeasy, the Rapid Check HIV 1 & 2 and the VIKIA HIV-1/2. The per-
formance data obtained in this study were consistent with other
studies (Gomes and Azevedo-Pereira, 2011; Santos et al., 2011;
Chaillet et al., 2010; Pavie et al., 2010; Lyamuya et al., 2009; Ferreira
Junior et al., 2005; Ribeiro-Rodrigues et al., 2003).
The Labtest HIV and the HIV-1/2 Rapid Test – Bio-Manguinhos
both produced false-negative results for the same sample. This
sample produced a positive result with the Vidas HIV Duo
Ultra (OD/CO ratio: 11.66), the Vironostika HIV Uni-Form Plus O
Microelisa System (OD/CO: 21.11) and the Cambridge Biotech HIV-
1 WB (bands detected: gp160, p55, p51 and p24) tests. A potentially
important consideration regarding these findings is the difference
in the volume of blood specimens required for each test. Whereas
the Labtest HIV and the HIV-1/2 Rapid Test – Bio-Manguinhos tests
use 10 ␮L and 5 ␮L of whole blood, respectively, the HIV-1/2 3.0
Strip Test Bioeasy, Rapid Check HIV 1 & 2 and VIKIA HIV-1/2 tests
require 20, 20 and 75 ␮L of specimen, respectively. Hence, it is
conceivable that a larger amount of specimen improves the test
sensitivity. It is also possible that the different rapid tests use differ-
ent antigens/epitopes and that these differences may render certain
tests more or less sensitive to detecting the antibody response.
The VIKIA HIV-1/2 and the Labtest HIV classified all of the
samples in the performance and seroconversion reference panels
correctly. The sample PRB204(M)-24 was the only sample that was
classified incorrectly by the HIV-1/2 3.0 Strip Test Bioeasy and the
Rapid Check HIV 1 & 2 tests. The Vidas HIV Duo Ultra (OD/CO:
2,06) and the Vironostika HIV Uni-Form Plus O Microelisa Sys-
tem (OD/CO: 3.2) produced positive results for this sample, but the
Western blot classified it as negative. However, the performance
panel package insert provides results obtained using a United States
(U.S.) Food and Drug Administration (FDA)-licensed Western blot
assay (GS HIV-1 Western Blot, Bio-Rad Laboratories, Redmond,
WA, USA), and it revealed four bands (p24, p51, p55 and gp160),
although the last three bands were described as faint bands. In addi-
tion, four rapid tests and one U.S. FDA-licensed HIV test (Organon
Teknika EIA HIV-1, Organon Teknika, Durham, NC, USA) yielded
negative results in the performance panel manufacturer’s assess-
ment. The HIV-1/2 Rapid Test – Bio-Manguinhos performed poorly,
classifying nine panel samples (9/46, 19.6%) incorrectly.
Regarding operational performance, all of the tests were
performed easily, and none required personnel with advanced
laboratory expertise or additional equipment. The only test that
failed to obtain a maximum score was the VIKIA HIV-1/2 (for
exceeding the 20-min threshold for executing the test and read-
ing the results). Although the manufacturer suggests that the
reading should be performed 30 min after adding the sample,
a positive result was identified after one minute. This find-
ing is in agreement with study results published elsewhere
(Gomes and Azevedo-Pereira, 2011).
The HIV-1/2 3.0 Strip Test Bioeasy allows for the differentiation
of virus subtypes (Type 1 or Type 2). Although it was not the aim of
the study, the PRZ207 performance panel allowed us to evaluate the
virus type in a small number of samples. Of the seven samples that
were positive for HIV-1, the test identified four samples correctly
as HIV-1-positive and identified three samples incorrectly as pos-
itive for both subtypes. Of the seven samples that were positive
for HIV-2, three were identified correctly and four were identi-
fied incorrectly as positive for both subtypes. Similar results were
obtained in a study conducted by Chaillet et al. (2010).
 L.R. da Motta et al. / Journal of Virological Methods 194 (2013) 132–137
The availability of a regulated algorithm and the use of approved
rapid tests performed by trained technicians have proven to be
powerful tools for diagnosing HIV in special situations in Brazil.
5. Conclusion
In conclusion, the HIV-1/2 3.0 Strip Test Bioeasy, the Rapid Check
HIV 1 & 2 and the VIKIA HIV-1/2 performed as well as the refer-
ence assays and fulfilled the requirements for use in the national
algorithm for diagnosing HIV infection in patients aged 18 months
and older. It is recommended that these three tests be included
on the list of tests that are approved for bulk procurement by the
Department of STDs, AIDS and Viral Hepatitis.
Acknowledgements
This study was funded by the Department of STDs, AIDS and Viral
Hepatitis under the Health Surveillance Secretariat in Brazil’s Min-
istry of Health, and the United Nations Office on Drugs and Crime
(UNODC).
Appendix.
The other members of the HIV Rapid Test Evaluation Group (in
alphabetical order) are Adriana Borges de Lemos Carlos, Agapito
Souza Martins, Ana Carla Weiss, Andréa Cauduro de Castro, Bárbara
Machado de Almeida, Daniele Sporck Gonçalves da Silva, Fátima
Silveira Fanfa, Fernanda Neves Woisikoski, Flávia Fornara Nohatto,
Grasiela Araújo da Silva, Haíra Maria Slobodianuk Andreossi,
Heloísa Peregrino da Silva, Isabel Cristina D. Martins, Karina Sal-
vador, Louise Ramos da Silva, Luciana Brasil Canto, Luiz Alberto
Peregrino Ferreira, Maria Luiza Bazzo, Mara Liane Rieck Silveira,
Maria de Fátima de Carvalho Pires, Maria Goretti Lins de Queiroz,
Mariana Krys, Mavis Wash G. Ribeiro Dantas, Mayra Sotero Sala,
Morgana Oliveira Gomes, Najla Adel Saleh, Paola Costa Bandinelli,
Patrícia Colombo Corrêa, Raquel Arrieche Fernandes, Ricardo da
137
Silva de Souza (in memoriam), Ruiter Roberto Silva, Samira Santos
Charara and Tayná Falquievvicz de Lima.
References
Brazilian Ministry of Health, 2007. HIV Rapid Tests: Why Not?, pp. 133.
Brazilian Ministry of Health, 2012. Epidemiological Bulletin. Year VIII N◦ 01., pp. 162.
Brazilian Ministry of Health, 2005. Health Surveillance Secretariat. Decree No. 34 of
July 28, 2005. In: Official Gazette, July 29, 2005., pp. 77–78.
Brazilian Ministry of Health, 2009. Health Surveillance Secretariat. Decree No. 151
of October 14, In: Official Gazette, October 16, 2009., pp. 40–44.
Chaillet, P., Tayler-Smith, K., Zachariah, R., Duclos, N., Moctar, D., Beelaert, G.,
Fransen, K., 2010. Evaluation of four rapid tests for diagnosis and differentia-
tion of HIV-1 and HIV-2 infections in Guinea-Conakry West Africa. Trans. R. Soc.
Trop. Med. Hyg. 104, 571–576.
C.L.S.I, 2008. User Protocol for Evaluation of Qualitative Test Performance; Approved
Guideline – Second Edition. CLSI document EP12-A2. Clinical and Laboratory
Standards Institute, Wayne, PA.
Ferreira Junior, O.C., Ferreira, C., Riedel, M., Widolin, M.R., Barbosa-Júnior, A.,
2005. Evaluation of rapid tests for anti-HIV detection in Brazil. AIDS 19,
S70–S75.
Gomes, C., Azevedo-Pereira, J.M., 2011. The performance of the VIKIA® HIV1/2 rapid
test – Evaluation of the reliability and sensitivity. J. Virol. Methods 173, 353–356.
Lyamuya, E.F., Aboud, S., Urassa, W.K., Sufi, J., Mbwana, J., Ndugulile, F., Massambu,
C., 2009. Evaluation of simple rapid HIV assays and development of national
rapid HIV test algorithms in Dar es Salaam, Tanzania. BMC Infect. Dis. 9, 19.
Pavie, J., Rachline, A., Loze, B., Niedbalski, L., Delaugerre, C., Laforgerie, E., Plantier,
J.C., Rozenbaum, W., Chevret, S., Molina, J.M., Simon, F., 2010. Sensitivity of five
rapid HIV tests on oral fluid or finger-stick whole blood: a real-time comparison
in a healthcare setting. PLoS ONE 5, e11581.
Ribeiro-Rodrigues, R., Neto, L.F., Cunha, C.B., Cabral, V.P., Dietze, R., 2003. Perfor-
mance characteristics of a rapid new immunochromatographic test for detection
of antibodies to human immunodeficiency virus. Clin. Diagn. Lab. Immunol. 10,
303–307.
Santos, A., Clemente, S., Bártolo, I., Palladino, C., Cavaco-Silva, P., Franco, V., Epalanga,
M., Pinto, R., Taveira, N., 2011. Evaluation of the diagnostic performance of the
rapid test VIKIA HIV1/2 in a highly complex HIV-1 epidemic. Diagn. Microbiol.
Infect. Dis. 71, 90–92.
Van Binsbergen, J., De Rijka, D., Peelsa, H., Driesa, C., Scherdersa, J., Koolena, M.,
Zekengb, I., Gürtlerb, L.G., 1996. Evaluation of a new third generation anti-HIV-
1/anti-HIV-2 assay with increased sensitivity for HIV-1 group O. J. Virol. Methods
60, 131–137.
Weber, B., Berger, A., Rabenau, H., Doerr, H.W., 2002. Evaluation of a new combined
antigen and antibody human immunodeficiency virus screening assay VIDAS
DUO Ultra. J. Clin. Microbiol. 40, 1420–1426.