rapid antibody tests, in the absence of virologic testing at the age of 18
months, in children receiving antiretroviral treatment can result in
interruption of treatment, and false-negative results occur with high
frequency, according to the results of a study carried out in Lesotho, published
in the advance online edition of AIDS.
per cent of children with negative or discordant post-18 month confirmatory
rapid HIV tests were definitively positive by HIV-DNA PCR while only 4% were definitively
negative, Garcia-Prats and colleagues report in this retrospective review of
routine programme data from an HIV clinic in Maseru, Lesotho.
ART at an early age (under nine months) was significantly associated with a
false-negative or discordant rapid test result (OR=4.25, p=0.002). 94% (46/49) of these children with a non-positive
result were on ART.
examining the challenges of post-18 month confirmatory HIV testing the authors
highlight this common occurrence of false-negative rapid tests which can lead
to the inappropriate interruption of treatment in children
with HIV, potentially leading to disease progression and the development of resistance.
settings of high HIV prevalence (over 5%) a two-test serial testing strategy is
recommended for diagnosis of HIV.
resource-poor settings, diagnosis of HIV in adults and children over
18 months of age is based on two positive HIV antibody tests and includes rapid
antibody tests (RT) or enzyme immunoassay (ELISA) antibody tests.
young infants, the risks of HIV transmission through breastfeeding, together
with the transfer during pregnancy of maternal antibodies that can persist
until 18 months of age further complicates HIV testing.
testing with HIV DNA polymerase chain reaction (PCR) is recommended to confirm
HIV infection in this group.
resource-poor settings, financial or other constraints restrict the use of PCR
either for a first test or for a second confirmatory one. In the first instance,
the World Health Organization (WHO) recommends clinical diagnosis to guide the
use of ART in infants under 18 months of age. In both circumstances WHO 2006
guidelines recommends HIV antibody testing after 18 months of age to confirm
the initial diagnosis.
spite of this recommendation, scant data exist assessing the outcomes of post-18
month confirmatory testing among infants starting ART immediately.
the authors chose to look at the challenges of such testing among infants
enrolled at the Baylor Children’s Clinical Center of Excellence (COE), a
paediatric and family HIV clinic in Maseru,
Lesotho has a 23.6% adult HIV prevalence, the third highest worldwide, an
estimated 28,000 children under the age of 15 living with HIV and 14,000
children are born to mothers who have HIV each year.
testing at the clinic follows national guidelines. In April 2006, a national
programme for early infant diagnosis using dried blood spot (DBS) HIV DNA PCR
began. Guidelines allowed for a single positive PCR test to be done for
children under 18 months of age and recommended antibody testing after 18
months of age for confirmation.
are based on three illustrative case studies and a retrospective chart
review. Children included those enrolled
from December 2005 until January 2009 with a documented positive HIV DNA PCR at
under 18 months of age and documented HIV rapid tests (RTs) after 18 months of
of a definitive HIV status was determined as follows:
- Definitively HIV positive – two positive PCRs, or one
positive PCR with either a positive post-18 month ELISA antibody test or two
positive post-18 month rapid antibody tests.
- Children were considered to be probably positive with one
positive PCR and discordant post-18 month rapid antibody tests.
- Children were considered definitively negative if they did
not meet the above criteria and after test results and clinical details were reviewed.
- Children not meeting any of the above criteria were
the 109 children included 49 (45%) had negative (22) or discordant (27)
confirmatory rapid tests.
of the 22 children with negative confirmatory rapid antibody tests were on ART. The mean age at
the start of ART was 8.0 months, standard deviation 3.6, range 2.4-15.7 months.
Of these 22, 60% (9/15) with follow-up PCRs were negative. Of these nine, four
were found to be positive after ELISA HIV antibody testing.
the 49, 29 (60%) were definitively positive, 17 (35%) probably positive while
only two were definitively negative and one of undetermined status.
young age at the start of ART was significantly associated with false-negative
results the authors note this is not just a problem among the youngest infants.
authors acknowledge that incorrect testing procedures – rapid antibody tests in field settings done
by non-laboratory personnel – may have contributed to their findings but cannot
explain the high rates of false-negatives.
Their findings, they add, contribute to
the phenomenon of seroreversion in children on effective ART but within the
context of confirmatory testing in resource-poor settings.
it has been hypothesized, among young children starting ART is due to the
suppression of viral replication during a time when immune responses are
impaired so decreasing antigenic stimulation.
reports, note the authors, show that rapid antibody tests may be less sensitive
than ELISA at lower antibody levels, so explaining positive ELISA after negative
rapid antibody tests. However, loss of detectable antibody also happens in
children on effective ART for both HIV ELISA and Western blot testing, they
authors note that ART should not affect DNA PCR results, yet their findings
showed children identified as HIV-infected through HIV DNA PCR having
subsequent negative PCRs after commencing antiretroviral treatment.
they add, have shown detectable proviral HIV DNA in spite of prolonged viral
suppression (under 50 copies/ml), yet in most of these a small percentage have
proviral DNA levels below the limits of detection. They also cite the example
of a child with known perinatal HIV infection who started ART at ten months of
age and after three years of suppressive ART had negative HIV ELISA, Western
blot and PCR testing.
copy number of proviral DNA is the most frequent explanation for false-negative
PCRs,” they state. They suggest that dried blood spot sample collection may
influence the detectability of HIV DNA in cases where HIV DNA copy numbers are
low due to early initiation of antiretroviral therapy.
of the study include a lack of standardisation of post-18-month testing. Viral
load testing is not routinely available so it was not possible to show
definitively that false-negatives happened in children on ART with suppressed
authors add that in line with WHO 2010 recommendations their findings support
two virologic tests, where available, to establish a definitive diagnosis
before a lengthy time on ART, so avoiding post-18-month confirmatory testing.
children over 18 months of age on ART and without a confirmed diagnosis, the
authors suggest that rapid antibody tests will provide a definitive diagnosis
in some, but ELISA testing may be better and HIV DNA PCR may be of use in
even if all confirmatory tests are negative, stopping ART needs to be done with
caution, they add, and the children closely followed up and repeat testing done
to establish a definitive diagnosis.
authors conclude “there is an urgent need for point of care HIV tests for use
in children under 18 months of age so increasing access to early infant
diagnosis, limiting the need for a presumptive clinical diagnosis and make
initial and confirmatory testing simpler and quicker…and ensure all children
receive accurate HIV tests for such a life-changing diagnosis.”