PREVENTION RESEARCH

True and False Positive HIV Point of Care Test Results in
a Prospective Multinational Study of At-Risk African

Women: Implications for Large-Scale Repeat HIV Testing in
HIV Prevention Programs

Susan Morrison, MD, MPHTM,a Joanne Batting, MBChB,b,c Valentine Wanga, PhD, MS,a,d

Ivana Beesham, MBChB, PhD,e Jennifer Deese, PhD, MPH,f,g G. Justus Hofmeyr, DSc,h,i

Margaret P. Kasaro, MBChB, MSc, MMed,j,k Cheryl Louw, MBChB,l Charles Morrison, PhD,fm

Nelly R. Mugo, MBChB, MMed, MPH,an Thesla Palanee-Phillips, M Med Sci, PhD, MSc,do

Melanie Pleaner, M Ed,o Krishnaveni Reddy, M Med Sci,o Caitlin W. Scoville, MPH,a

Jenni Smit, BPharm, MS, PhD,ep Jeffrey S.A. Stringer, MD,j,k Khatija Ahmed, MBBCH, FCPath(Micro),qr

Elizabeth Bukusi, MBChB, MMed (ObGyn), MPH, PhD, PGD(Research Ethics), MBE,a,st

Philip Kotze, MbChB, MMed,u and Jared M. Baeten, MD, PhD,av for the ECHO Trial Team

Background: Accurate HIV point of care testing is the cornerstone
of prevention and treatment efforts globally, although false (both
negative and positive) results are expected to occur.

Setting: We assessed the spectrum of true and false positive HIV
results in a large prospective study of HIV incidence in African
women using 3 contraceptive methods tested longitudinally in
Eswatini, Kenya, South Africa, and Zambia.

Methods: HIV serologic testing was conducted quarterly using 2
parallel rapid HIV tests. When one or both tests were positive,
additional confirmatory testing was conducted, including HIV
enzyme immunoassay (EIA) and RNA.

Results: A total of 7730 women contributed 48,234 visits: true
positive results occurred at 412 visits (0.9%) and false positives at 96
visits (0.2%). Of 412 women with HIV seroconversion, 10 had
discordant (ie, 1 negative and 1 positive) rapid tests and 13 had

Received for publication January 31, 2024; accepted July 8, 2024.
From the aDepartment of Global Health, University of Washington, Seattle; bEffective Care Research Unit, University of the Witwatersrand and Eastern Cape

Department of Health, East London, South Africa; cFoundation for Professional Development (FDP) Research Unit, East London, South Africa; dDepartment of
Epidemiology, University of Washington, Seattle; eDepartment of Obstetrics and Gynaecology, MatCH Research Unit (MRU), Commercial City, Faculty of
Health Sciences, University of the Witwatersrand, Durban, South Africa; fGlobal Health, Population and Nutrition, FHI 360, Durham, NC; gCurrently, Global
Respiratory Vaccines, Pfizer, Inc., Collegeville, PA; hEastern Cape Department of Health, Effective Care Research Unit, Universities of the Witwatersrand/Walter
Sisulu;East London, South Africa; iDepartment of Obstetrics and Gynecology, University of Botswana, Gaborone, Botswana; jUNC Global Projects Zambia,
Lusaka, Zambia; kDepartment of Obstetrics and Gynecology, University of North Carolina Chapel Hill, School of Medicine, North Carolina; lMadibeng Centre
for Research, Brits, South Africa; mBehavioral, Epidemiologic and Clinical Sciences, FHI 360, Durham, NC; nCentre for Clinical Research, Kenya Medical
Research Institute, Nairobi, Kenya; oWits Reproductive Health and HIV Institute, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg,
South Africa; pMatCH Research Unit Edendale- Department of Obstetrics and Gynaecology, Faculty of Health Sciences, University of the Witwatersrand,
Edendale, South Africa; qSetshaba Research Centre, Soshanguve, South Africa; rUniversity of Pretoria, Department of Medical Microbiology, Pretoria, South
Africa; sCentre for Microbiology Research, Kenya Medical Research Institute, Nairobi, Kenya; tDepartment of Obstetrics and Gynecology, University of
Washington, Seattle; uQhakaza Mbokodo Research Clinic, Ladysmith, South Africa; and vDepartment of Medicine, University of Washington, Seattle.

This work and the Evidence for Contraceptive Options and HIV Outcomes (ECHO) Study were made possible by the combined generous support of the Bill &
Melinda Gates Foundation (Grant OPP1032115), the American people through the United States Agency for International Development (Grant AID-OAA-A-
15–00045), the Swedish International Development Cooperation Agency (Grant 2017/762965–0), the South Africa Medical Research Council, and the
United Nations Population Fund. Contraceptive supplies were donated by the Government of South Africa and US Agency for International Development.
The contents of this paper are solely the responsibility of the authors and do not necessarily reflect the views, decisions, or policies of the institutions with
which they are affiliated, the ECHO trial funders, or the supporting governments.

J.M.B. is an employee of Gilead Sciences, outside of the present work. All other authors have no funding or conflicts of interest to disclose.
Access to data from this analysis of the ECHO Study may be requested through submission of a research concept to: icrc@uw.edu. The concept must include the

research question, data requested, analytic methods, and steps taken to ensure ethical use of the data. Access will be granted if the concept is evaluated to have
scientific merit and if sufficient data protections are in place. As of the time of publication, data access applications are in process with the governing
institutional review boards of the ECHO Study to make de-identified data from the full trial publicly available.

S.M., J.B., V.W., and J.M.B. designed the analysis and drafted the manuscript. V.W. conducted the data analysis. All authors critically reviewed and approved
the final manuscript.

Correspondence to: Susan Morrison, MD, MPHTM, Department of Global Health, International Clinical Research Center, University of Washington, HMC
#359927, 325 9th Avenue, Seattle, WA 98104 (e-mail: sam32@uw.edu).

Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. This is an open access article distributed under the Creative Commons Attribution
License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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undetectable HIV RNA levels. Of 62 women with false positive
rapid HIV results, most had discordant rapid testing, but 6 (9.7%)
had dually positive rapid results, and 4 (6.5%) had false positive or
indeterminate EIA results. The positive predictive value of dual
positive rapid results was 98.3%.

Conclusions: Although most rapid test results were accurate, false
positive results were expected and occurred in this population of
initially HIV seronegative individuals tested repeatedly and pro-
spectively. When HIV infection occurred, not all cases had textbook
laboratory results. Our findings highlight the importance of confir-
matory testing, particularly for individuals undergoing repeat testing
and in settings where the point prevalence is expected to be low.

Trial registration: ClinicalTrials.gov number NCT02550067.

Key Words: HIV testing, HIV point of care test, false positive, true
positive, African young women, prospective study

(J Acquir Immune Defic Syndr 2024;97:364–370)

INTRODUCTION
HIV testing is the cornerstone of prevention and

treatment efforts globally. For persons living with HIV,
testing is the first step to linkage to care and antiretroviral
therapy (ART) initiation. For persons who would benefit from
HIV prevention services, including preexposure prophylaxis
(PrEP), periodic testing is essential. In 2020, The Joint United
Nations Programme on HIV/AIDS proposed targets for 2025
that 95% of persons living with HIV know their HIV status as
part of a goal to end the global epidemic by 2030.1

Knowledge of HIV status is dependent on the avail-
ability and utilization of accurate HIV diagnostic testing. The
diagnostic accuracy of a test is defined by its specificity and
sensitivity. Historically, the emphasis has been placed on the
ability of an HIV test to correctly identify all those who have
the infection (sensitivity), as falsely negative tests potentially
delay lifesaving ART. However, the ability of an HIV test to
correctly identify all those without the condition (specificity)
is less frequently discussed. An erroneous HIV diagnosis may
lead to significant consequences for the individual2 such as
avoidable experience of stigma and unnecessary initiation of
lifelong ART.3,4 At a population level, cases of HIV
misdiagnoses could undermine confidence in HIV testing.

The World Health Organization (WHO) recommends
using an HIV testing strategy consisting of a combination of
rapid diagnostic tests and/or enzyme immunoassays
with $99% sensitivity and $98% specificity, which, when
used together, achieve at least a 99% positive predictive value
(PPV).5 However, PPV varies according to the prevalence of
HIV in the community where the testing is performed. As the
prevalence of HIV declines in a population, the fraction of
positive test results that are true positives also falls. The advent
of highly effective HIV prevention strategies such as PrEP
potentially creates a novel scenario of mandatory periodic HIV
testing among a population with low HIV incidence and thus
decreased likelihood of a truly positive result.

We investigated the rates of true and false positive rapid
HIV test results among women followed in a prospective

study conducted in 4 countries in eastern and southern Africa,
in which HIV acquisition was the primary end point. We
conducted confirmatory, and if needed, additional advanced
testing for all instances of positive HIV rapid test results,
allowing clear definition of true and false positive testing.

METHODS

Population and Procedures
Participants were women aged 16–35 years enrolled in

the Evidence for Contraceptive Options and HIV Outcomes
trial at 12 research sites in Eswatini, Kenya, South Africa, and
Zambia (ClinicalTrials.gov number NCT02550067).6 The
primary goal of the trial was to compare HIV incidence
among women randomized to 1 of 3 contraceptive methods
(intramuscular depot medroxyprogesterone acetate, copper
intrauterine device, or levonorgestrel implant). Overall HIV
incidence was 3.81 per 100 person-years, and there were not
statistically significant differences between randomized
groups. Enrollment began in December 2015 and concluded
in September 2017; follow-up concluded in October 2018.
Ethics review committees at each study site approved the
study protocol, and all participants provided written informed
consent.

Women were eligible for the trial if they were HIV
seronegative, sexually active, and desiring contraception. At
the enrollment visit, plasma was archived to allow retrospec-
tive, quantitative HIV RNA PCR testing to assess baseline
infection status for those who experienced subsequent HIV
seroconversion. After enrollment, participants attended sched-
uled study follow-up visits every 3 months for a period of 12–
18 months, including HIV testing with dual parallel rapid
tests. HIV pretest and post-test counseling was conducted
according to national guidelines. Participants received a com-
prehensive package of HIV preventive services at every study
visit including HIV risk reduction counselling, syndromic
sexually transmitted infection assessment and treatment, and
provision of condoms. Toward the end of the trial period,
PrEP was offered to participants either by referral or on-site
provision by trial staff, in accordance with national guide-
lines, as PrEP became the standard of care.7 Participants who
acquired HIV infection were referred for ART and supported
in linkage to care.

Laboratory Methods
HIV serologic testing consisted of 2 rapid HIV tests,

run in parallel and using 2 different brands of test kits,
conducted at each study site. If one or both of the rapid test
results was positive, additional confirmatory testing was
conducted with HIV enzyme immunoassay (EIA) (using
Abbott ARCHITECT, Abbott Murex or Roche Elecsys HIV
combi PT) and HIV RNA polymerase chain reaction (RNA
PCR) (Abbott RealTime, Roche COBAS TaqMan, or CO-
BAS AmpliPrep). HIV infection was considered confirmed if
HIV EIA was positive and HIV RNA PCR was .400 copies/
mL, and participants were considered uninfected if HIV EIA
was negative and HIV RNA PCR was not detected. If

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confirmatory testing was unable to establish or refute
infection conclusively, then additional supplemental testing,
including retesting of HIV RNA PCR, HIV western blot
(BIO-RAD GS HIV-1 Western Blot or Consort E455 with
UV transilluminator), and HIV DNA (DNA PCR) (Roche
COBAS TaqMan or COBAS AmpliPrep), and if indicated
retesting of HIV EIA and/or Geenius (BIO-RAD Geenius
HIV 1/2 Supplemental Assay) was conducted, as determined
by the trial medical monitor, and final HIV status was
determined by a study end points committee. Laboratories
followed Good Clinical Laboratory Practice, including regu-
lar internal quality control and external quality assurance for
the HIV rapid test kits and other HIV tests used throughout
the trial.

Statistical Analysis
We described the number and percentage of visits with

true and false positive HIV test results, including among visits
with discordant (1 positive and 1 negative) test results or 2
positive test results. Among women determined to have
acquired HIV, we described the HIV RNA PCR quantity
(copies/mL) using results from the first visit at which HIV
infection was detected.

Overall, 11 different HIV rapid test kits were used for
testing across the sites. For each test kit, we described the
total number of tests performed and the number of false
positive and true positive test results using site HIV status
determination as the gold standard. We also estimated the
false positive rates (FPRs) and the positive predictive values
(PPVs) for each test kit. To estimate the 95% confidence
intervals for the FPR and PPV, we used the bootstrap method
to account for correlation of tests within an individual.
Specifically, for each test kit, we first calculated the sampling
probability as the proportion of times the test kit was used
among all women. We then conducted 1000 simulations,
sampling with replacement from the number of times each
test kit was used, using the sampling probabilities. For each
simulation, we calculated the FPR and PPV, and among the
1000 estimates for each metric, we used the 50th quantile as
the point estimate, and the 2.5th and 97.5th quantiles as the
lower and upper confidence bounds, respectively. For some of
the test kits, the confidence limits for FPR or PPV were
inestimable because of zero or very low number of false
positive or true positive test results.

We also described advanced testing among women with
false positive rapid test results. All analyses were conducted
using SAS software (version 9.4; SAS Institute Inc., Cary,
NC)8 and R (Version 4.0.2; R Foundation for Statistical
Computing, Vienna, Austria).9

RESULTS
A total of 7830 women were enrolled in the study: 5769

(74%) in South Africa, 901 (12%) in Kenya, 658 (8%) in
Zambia, and 502 (6%) in Eswatini. Most women (4948, 63%)
were younger than 25 years of age.

Among the 7830 women enrolled, 7730 contributed
prospective follow-up visits with HIV testing. A total of

48,234 visits (96,468 tests), comprising 10,409 person-years
of prospective follow-up, were accrued. By study completion,
412 women became HIV seropositive: 397 acquired HIV
during study participation and 15 were retrospectively found
to have early, seronegative HIV infection at the time of
enrollment; 7318 remained HIV negative. Of the 48,234
visits, dual negative rapid test results occurred at 47,726
(98.9%) visits. Discordant (single positive tests) occurred at
99 (0.2%) visits and dual positive test results at 409 (0.9%)
visits. Among the 508 visits with 1 or 2 positive rapid test
results, true positive results (ie, confirmed by additional
testing) occurred in 412 (81.1%) visits and false positive
results occurred in 96 (18.9%) visits (Table 1). The PPV of
a positive rapid test result (either single or dual positive) was
81.1%, whereas the PPV was 10.1% for a single positive
(discordant) result. The PPV of HIV infection with a positive
result defined as dual positive rapid testing was 98.3%.

True Positive Results
Among the 412 women who were found to have

acquired HIV, 402 (97.6%) had dual positive rapid test
results, whereas 10 had discordant (1 positive and 1 negative)
results. Nearly all (410) had positive EIA results at the time
HIV infection was identified, with 2 results being indetermi-
nate. Although high HIV RNA levels often occur during
recent infections, we found that HIV RNA levels ranged from
undetectable to .1,000,000 copies/mL (Table 2). Twenty-
five participants (6.1%) had HIV RNA levels
of .1,000,000 copies/mL at the time of detection of infection
(with 4 of these having . 10,000,000 copies/mL). Most (356,
86.4%) of initial HIV RNA levels were between 400 and
1,000,000 copies/mL, and 18 (4.4%) had RNA levels that
were detectable but#400 copies/mL. Notably, 13 (3.2%) had
undetected HIV RNA levels at the visit at which HIV
seroconversion was detected: 5 reported ART use before
the visit (indicating diagnosis outside of the study), 2 did not
report ART use but had been lost to follow-up with .1 year
since the last negative testing at the study site (thus
seroconversion may have been detected after primary infec-
tion or ART use misreported), and 6 had no history of ART
use or missed visits (with infection diagnosed ;3 months

TABLE 1. True and False Positive Rapid Test Results

Description Number of Visits Percent

Among visits with 1 or 2 positive rapid test result (N = 508)

Positive confirmatory results (true positives) 412 81.1

Negative confirmatory results (false
positives)

96 18.9

Among visits with 1 positive and 1 negative rapid test result (N = 99)

Positive confirmatory results (true positives) 10 10.1

Negative confirmatory results (false
positives)

89 89.9

Among visits with 2 positive rapid test results (N = 409)

Positive confirmatory results (true positives) 402 98.3

Negative confirmatory results (false
positives)

7 1.7

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since last negative HIV testing). For 1 individual, the lower
limit of detection was 200 copies/mL for the first assay
performed and subsequent testing with a lower level of
quantification measured a viral load of 152 copies/mL.

Western blot testing was conducted for 36 individuals:
34 were positive and 2 were negative. Of the 2 women with
negative results, 1 went on to have a positive result 20 days
later and the other was lost to follow-up (EIA was indeter-
minate and HIV RNA was 256,451 copies/mL). HIV DNA
PCR was conducted for 28 individuals: 25 had positive results
and 3 were negative (1 had a positive result on follow-up
testing 24 days later, 1 had a second negative DNA PCR
result 8 days later, and 1 with persistently negative DNA PCR
later was suspected to have previously undisclosed ART use).
Two individuals had Geenius testing, both positive for HIV-1
and negative for HIV-2.

False Positive Rapid Test Results
Sixty-two women had false positive rapid HIV test

results, occurring at 96 visits; the cumulative probability of
a first false positive rapid test at 12 and 18 months were
0.06% and 0.08%, respectively. The proportion of false

positive results was higher in visits with discordant (ie, 1
positive and 1 negative) rapid test results, with false positives
occurring in 89 of 99 (89.9%) visits with single positive rapid
tests, compared with 7 of 409 (1.7%) visits with false positive
results when both rapid tests were positive. Fifty-one of the
62 women went on to have at least 1 subsequent visit with
HIV testing, and 15 (29.4%) had at least 1 additional
occurrence of a false positive result, with 11 of these being
at the next consecutive visit.

Three kit types had zero false positive and/or true
positive results, and we were not able to estimate their FPRs
or PPVs and respective confidence intervals. Of the remaining
8, the FPRs ranged from 0.02% to 0.3%, with PPVs ranging
from 75% to 98% (Table 3).

Of the 62 women with false positive rapid test results,
58 had negative EIA results, 2 had indeterminate EIA results,
and 2 had positive EIA testing. All 4 women (6.5%) with
positive or indeterminate EIA results had single positive rapid
test results (Table 4). One was pregnant and used ART for
2 months until confirmatory testing was complete, which
included repeat EIA with a different assay (fourth generation),
which was negative. Six women (9.7%) had dual false
positive rapid test results. One initiated and later stopped
ART at an outside facility.

All 62 women with false positive rapid results had
undetected RNA levels. Eleven of the 62 had western blot
testing, with 7 being negative and 4 indeterminate. Twelve
had HIV DNA PCR testing, all negative. In some cases,
several rounds of repeated rapid, EIA, and/or additional
confirmatory testing were needed to determine the HIV status.

DISCUSSION
In this large prospective study, involving nearly 50,000

visits with repeated HIV testing of previously HIV

TABLE 2. HIV RNA PCR Results Among HIV Seroconverters

Test Number (%)

HIV RNA PCR (copies/mL) (N = 412)

Not detected 13 (3.2)

.limit of detection—400 18 (4.4)

401–10,000 110 (26.7)

10,001–100,000 140 (34.0)

100,001–1,000,000 106 (25.7)

.1,000,000 25 (6.1)

TABLE 3. False Positive Rapid Test Results by Rapid Test Kit Type

Kit Type
Number of False
Positive Results

Number of True
Positive Results

Total Number of
Tests

Percent False Positive
(95% CI)

Positive Predictive Value
(95% CI)

Alere DetermineTM HIV-1/2 54 269 35,055 0.15 (0.08, 0.23) 83.3 (76.1, 90.6)

Advanced QualityTM Rapid
HIV Test

4 198 18,622 0.02 (0.00, 0.06) 98.0 (94.2, 100)

Uni-GoldTM Recombigen
�

HIV-1/2
17 131 17,147 0.10 (0.03, 0.20) 88.5 (78.1, 96.7)

Abon™ HIV 1/2/0 12 78 7261 0.17 (0.05, 0.49) 86.7 (65.5, 100)

Premier First Response HIV
1-2.O

7 57 6942 0.10 (0.05, 0.26) 89.1 (71.4, 100.0)

SD Bioline 2 6 4060 0.05 (0.00, 0.35) 75.0 (2, 2)†

OraQuick Advance� HIV-1/
2*

0 31 3229 0.00 (-, -)† 100.0 (2, 2)†

Advanced Quality One Step 2 27 2492 0.08 (0.00, 0.87) 93.1 (33.0, 100.0)

BIOTRACER 5 17 1652 0.30 (0.00, 2.15) 77.3 (0.0, 100.0)

Colloidal Gold HIV(1+2)
Antibody*

0 0 7 0.00 (2, 2)† Unable to calculate†

INSTI™ HIV-1 Antibody* 0 0 1 0.00 (2, 2)%† Unable to calculate†

All test kits 103 814 96,468 0.11 (0.07, 0.14) 88.8 (85.4, 91.9)

*FPR and positive predictive values could not be estimated because of zero false positive or zero true positive test results.
†Confidence limits for FPR or PPV were inestimable because of zero or very low number of false positive or true positive test results.

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seronegative women, we evaluated the frequency and nature
of true and false positive rapid HIV testing results. Our
findings highlight the challenge that false positive results,

which are expected and inherent in any screening test with
less than perfect specificity, will occur in programs conduct-
ing longitudinal HIV testing.

TABLE 4. Cases With Either Dual False Positive Rapid Tests or a Single False Positive Rapid Test and Positive/Indeterminate EIA
Results

HIV Rapid
Test Results

Initial Confirmatory
Testing (HIV EIA
and HIV RNA)

Additional Confirmatory
Testing (Repeat HIV RNA, HIV

DNA, HIV Western Blot,
Repeat HIV EIA as Needed) Subsequent HIV Rapid Testing Other Information

Determine
positive

First response
positive

HIV EIA negative

HIV RNA not detected

Repeat HIV RNA not detected

HIV DNA negative

HIV western blot indeterminate
with p31 (1+)

Dual negative HIV rapid tests at 3
subsequent visits including at final visit

Breastfeeding .1-year-old baby,
advised to stop (but did not)

Determine
positive

Unigold
positive

HIV EIA negative

HIV RNA not detected

Repeat HIV RNA not detected

HIV DNA negative

HIV western blot negative

Determine positive, First Response
negative at 3 subsequent quarterly visits,
dual negative HIV rapid tests at final

visit

Breastfeeding . 1-year-old baby,
advised to stop (but did not)

Determine
positive

First response
positive

HIV EIA negative

HIV RNA not detected

Repeat HIV RNA not detected

HIV DNA negative

HIV western blot indeterminate
with 6 p55

Dual negative HIV rapid testing at
subsequent 3 visits including final visit

Determine
positive

Unigold
positive

HIV EIA negative

HIV RNA not detected

Repeat HIV RNA not detected

HIV DNA negative

HIV western blot negative

Missed subsequent 2 visits, returned for
final visit at which dual HIV rapid tests

negative

Determine
positive

Advanced
quality
positive

HIV EIA negative

HIV RNA not detected

Repeat HIV RNA not detected

HIV DNA negative

Western blot negative

Missed next subsequent visit, then dual
negative HIV rapid tests at 3 visits

including final visit

Biotracer
positive

Advanced
quality
positive

HIV EIA negative

HIV RNA not detected

Repeat HIV RNA not detected

HIV DNA negative

HIV western blot negative

Dual positive HIV rapid tests at
subsequent (final) visit, all confirmatory

testing was repeated and negative

Initiated ART after dual positive HIV
rapid testing at an outside facility.
Results provided to care facility and
stopped ART. Reported negative
subsequent HIV rapid testing

Determine
positive

First response
negative

HIV EIA
indeterminate

HIV RNA not detected

Repeat HIV RNA not detected

HIV DNA negative

HIV western blot negative

Three subsequent visits with dual
negative HIV rapid testing including

final visit

ABON
positive

Advanced
quality
negative

HIV EIA
indeterminate

HIV RNA not detected

Repeat HIV rapid tests discordant
(ABON positive, advanced

quality negative)

Repeat HIV EIA negative

Repeat HIV RNA not detected
HIV DNA negative

HIV western blot negative

Four additional visits with single false
positive HIV rapids, all with negative
HIV EIA, including final visit. One

intervening visit with dual negative HIV
rapid testing

Reported negative testing at
a community clinic

Determine
negative

Unigold
positive

HIV EIA positive

HIV RNA not detected

Repeat HIV EIA positive, second
HIV EIA using different kit

negative

Repeat HIV RNA not detected

HIV DNA negative

Western blot indeterminate
(1+gp41, 1+p18)

HIV rapid tests negative at next visit and
at 2 subsequent visits including final

visit

Pregnant, took ART for 2 mo until
confirmatory testing and HIV status

concluded

Determine
positive

Unigold
negative

HIV EIA positive

HIV RNA not detected

Repeat HIV RNA not detected

HIV DNA negative

HIV western blot indeterminate
(6gp160, 1+gp41, 6p31)

Single false positive rapid HIV test at
next visit with negative HIV EIA,

followed by 3 visits with dual negative
HIV rapid testing. Final visit with single
false positive rapid testing, HIV EIA
indeterminate, HIV Western blot

indeterminate, HIV DNA negative, and
HIV RNA not detected

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Rapid testing and associated diagnostic algorithms have
made HIV testing accessible globally, resulting in substantial
individual and population-level benefits of persons living
with HIV knowing their status and initiating ART. To
optimize accurate diagnosis of true HIV infections, WHO
recommends serial rapid testing: first with a highly sensitive
($99%) test, with a positive test triggering a second highly
specific ($99%) test. Testing guidelines further include
strategies to safeguard against false HIV diagnoses, including
the use of approved tests, quality control, use of a third
confirmatory rapid or EIA, and the possibility of retesting
before ART initiation. For population-based HIV testing
programs in high prevalence settings, the public health
balance strongly favors obtaining HIV diagnoses over a rare
false positive result.

In lower prevalence populations, the relative fraction of
false positive results among total positive results will
inherently increase. Similarly, groups of recently HIV
seronegative persons tested prospectively, as in this study,
are effectively a lower-prevalence group, even when those
populations live in countries where HIV prevalence and
incidence are high. We found that at approximately 1 in 5
visits at which an individual had a positive rapid test, the
result was falsely positive, requiring additional confirmatory
testing. Notably, the PPV for 2 positive tests (98.3%) was
below the WHO goal of .99%, reflecting that, even in this
population with a high incidence of HIV, the point prevalence
of true positive results when retesting previously HIV
seronegative individuals is unlikely to generate a PPV.99%.
To maximize detection of incident HIV infections, the trial
algorithm conducted dual rapid testing in parallel, and a single
positive rapid test was more likely than dual positive tests to
be falsely positive, although there were a small number of
cases in which both rapids, or 1 rapid and EIA, were positive,
with further testing essential to determine a true negative
status. Of those with 2 positive tests, 1 initiated ART outside
of the study and 2 considered cessation of breastfeeding
a child (both over a year old). All 6 were identified by their
negative EIA and additional study-based results, and all
eventually reverted to negative rapid testing. Of the 66
women with single positive rapid tests, 56 were found to be
HIV negative. For some individuals, false positive rapid test
results recurred over multiple quarterly follow-up visits. False
positive rapid tests may occur because of the presence of
cross-reacting antibodies,10 which may explain both false
positive rapid and EIA results. National algorithms vary with
respect to confirmatory testing assays used, but our results
reinforce the importance of timely access to confirmatory
testing.

In the 4 countries where the Evidence for Contraceptive
Options and HIV Outcomes Study was conducted, and others
in the region, 2 rapid HIV tests conducted in sequence (a
positive screening rapid HIV test followed by a second
positive rapid test) is the usual algorithm to confer a diagnosis
of HIV infection.11–14 Most true positive results met this
pattern, although some had only 1 positive rapid or indeter-
minate EIA results, and an important fraction had low or even
undetected HIV RNA levels. Follow-up was quarterly in this
study, so infections were generally detected early. These

findings emphasize that the textbook pattern of very high HIV
RNA and rapid emergence of robustly positive serologic
results after HIV acquisition does not occur in all cases.

Our results have important implications for HIV testing
for persons taking HIV PrEP. Oral and injectable PrEP
agents, when used as prescribed, substantially reduce HIV
incidence,15–17 and thus the point prevalence of a truly
positive result should be very low among PrEP users. The
occurrence of false positive results, however, is inherent to
the tests used, and thus, it might be expected that most
positive rapid test results in consistent PrEP users will be
falsely positive.18 PrEP research has generally focused on the
possibility of false negative results—because of the risk of
generation of antiretroviral resistance if PrEP is initiated or
continued in someone acquiring HIV—but a false positive
result, especially if ART is wrongly initiated, can have
substantial and potentially lifelong consequences for individ-
uals. The lifelong consequences of HIV treatment after
a misdiagnosis may involve a range of burdensome aspects
such as wait time at treatment centers, transportation costs,
pill burden, and potential for stigma. Our results also have
implications for assessing true HIV seroconversion among
PrEP users, as we observed cases of low HIV RNA,
indeterminate EIA, and/or negative western blot even in the
absence of PrEP.

Limitations of this study include the fact that our results
are not directly comparable to most field settings, where rapid
tests are conducted in series, and testing is often self-initiated
based on perceived risk or known exposures. Rigorously
monitored laboratory procedures and test kit storage con-
ditions may also differ from community-based testing.

CONCLUSIONS
Accuracy in HIV testing is essential. In this prospective

study, results were accurate for most visits. However, for
individuals who acquired HIV, not all demonstrated the
textbook pattern of high HIV RNA levels and clear positive
serology. Moreover, although uncommon, false positive HIV
rapid tests, sometimes with 2 different tests, were observed.
False positive HIV results are more likely to occur with
periodic longitudinal testing such as in research settings and/
or with PrEP provision, and HIV prevention programs should
be aware of the potential for falsely positive results and
atypical patterns of positive results and be prepared to provide
additional confirmatory testing, particularly with discordant
point of care test results.

APPENDIX. ECHO Trial Consortium
ECHO Trial Consortium: Management Committee: Jared M. Baeten

(University of Washington, Seattle, WA), James Kiarie (WHO, Geneva,
Switzerland), Timothy D. Mastro (FHI 360, Durham, NC), Nelly R. Mugo
(Kenya Medical Research Institute, Nairobi, Kenya; University of Wash-
ington, Seattle, WA), Helen Rees (Wits Reproductive Health and HIV
Institute, Johannesburg, South Africa). Study Site Principal Investigators:
Manzini, Eswatini: Jessica Justman, Zelda Nhlabatsi (Family Life Associa-
tion of Eswatini & ICAP at Columbia University, New York, NY). Kisumu,
Kenya: Elizabeth A. Bukusi, Maricianah Onono (Kenya Medical Research
Institute, Nairobi, Kenya). Brits, South Africa: Cheryl Louw (Madibeng

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Centre for Research). Cape Town, South Africa: Linda-Gail Bekker,
Gonasagrie Nair (University of Cape Town and Desmond Tutu HIV Centre).
Durban, South Africa: Mags Beksinska, Jennifer Smit (MatCH Research
Unit, Faculty of Health Sciences, University of the Witwatersrand). East
London, South Africa: G. Justus Hofmeyr, Mandisa Singata-Madliki
(University of Fort Hare and University of the Witwatersrand). Edendale:
South Africa: Jennifer Smit (MatCH Research Unit, Faculty of Health
Sciences, University of the Witwatersrand). Johannesburg, South Africa:
Thesla Palanee-Phillips (Wits Reproductive Health and HIV Institute, Faculty
of Health Sciences, University of the Witwatersrand). Klerksdorp, South
Africa: Raesibe Agnes Pearl Selepe (The Aurum Institute). Ladysmith, South
Africa: Sydney Sibiya (Qhakaza Mbokodo Research Clinic). Soshanguve,
South Africa: Khatija Ahmed (Setshaba Research Centre). Lusaka, Zambia:
Margaret Phiri Kasaro, Jeffrey Stringer (UNC Global Projects Zambia and
University of North Carolina at Chapel Hill, Chapel Hill, NC). Other
members of the ECHO Trial Consortium: Deborah Baron (Wits Reproductive
Health and HIV Institute, Faculty of Health Sciences, University of the
Witwatersrand, Johannesburg, South Africa), Deborah Donnell (University of
Washington and Fred Hutchinson Cancer Research Center, Seattle, WA),
Peter B. Gichangi (International Centre for Reproductive Health—Kenya &
Technical University of Mombasa, Mombasa, Kenya), Kate B. Heller
(University of Washington, Seattle, WA), Nomthandazo Mbandazayo (Wits
Reproductive Health and HIV Institute, Johannesburg, South Africa), Charles
S. Morrison (FHI 360, Durham, NC), Kavita Nanda (FHI 360, Durham, NC),
Melanie Pleaner (Wits Reproductive Health and HIV Institute, Faculty of
Health Sciences, University of the Witwatersrand, Johannesburg, South
Africa), Caitlin W. Scoville (University of Washington, Seattle, WA),
Kathleen Shears (FHI 360, Washington, DC), Petrus S. Steyn (WHO,
Geneva, Switzerland), Douglas Taylor (FHI 360, Durham, NC), Katherine
K. Thomas (University of Washington, Seattle, WA), Julia D. Welch (FHI
360, Durham, NC).

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