False-negative results in nucleic acid amplification tests-do we need to routinely use two genetic targets in all assays to overcome problems caused by sequence variation?

Abstract

Nucleic acid amplification tests (NAATs) have numerous advantages over traditional diagnostic techniques and so are now widely used by diagnostic laboratories for routine detection of infectious agents. However, there is some concern over the increasing numbers of reports of NAAT false-negative results caused by sequence variation. Highly conserved NAAT target sequences have been reported for many organisms, yet sequence-related problems continue to be observed in commercial and in-house assays targeting a broad range of microbial pathogens. In light of these ongoing problems, it may be time to consider the use of two genetic targets in NAAT methods to reduce the potential for sequence-related false-negative results.

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Critical Reviews in Microbiology, 34:71–76, 2008
Copyrightc ?Informa Healthcare USA, Inc.
ISSN: 1040-841X print / 1549-7828 online
DOI: 10.1080/10408410801960913
False-Negative Results in Nucleic Acid Amplification
Tests—Do We Need to Routinely Use Two Genetic
Targets in all Assays to Overcome Problems Caused
by Sequence Variation?
D. M. Whiley, S. B. Lambert, S. Bialasiewicz, and N. Goire
Queensland Paediatric Infectious Diseases Laboratory, Sir Albert Sakzewski Virus Research Centre,
Royal Children’s Hospital and Health Service District, Queensland, Australia; and
Clinical Medical Virology Centre, University of Queensland, Queensland, Australia
M. D. Nissen and T. P. Sloots
Queensland Paediatric Infectious Diseases Laboratory, Sir Albert Sakzewski Virus Research Centre,
Royal Children’s Hospital and Health Service District, Queensland, Australia; Clinical Medical Virology
Centre, University of Queensland, Queensland, Australia; Microbiology Division, Queensland Health
Pathology Service, Royal Brisbane Hospital Campus, Queensland, Australia; and Department of
Paediatric and Child Health, University of Queensland, Brisbane, Queensland, Australia
Nucleicacidamplificationtests(NAATs)havenumerousadvan-
tages over traditional diagnostic techniques and so are now widely
used by diagnostic laboratories for routine detection of infectious
agents.However,thereissomeconcernovertheincreasingnumbers
ofreportsofNAATfalse-negativeresultscausedbysequencevaria-
tion. Highly conserved NAAT target sequences have been reported
for many organisms, yet sequence-related problems continue to
be observed in commercial and in-house assays targeting a broad
range of microbial pathogens. In light of these ongoing problems,
it may be time to consider the use of two genetic targets in NAAT
methodstoreducethepotentialforsequence-relatedfalse-negative
results.
Keywords
NAAT; Nucleic Acid; Amplification; Sequence; Varia-
tion; False-Negative
INTRODUCTION
Nucleic acid amplification tests (NAATs) have revolution-
ized laboratory diagnosis of a wide range of infectious diseases,
from sexually transmitted diseases and respiratory pathogens
through to pathogens that affect individuals who are immuno-
compromised. NAATs offer several advantages over traditional
Received 29 September 2007; accepted 5 February 2008.
Address correspondence to David M. Whiley, Queensland Pae-
diatric Infectious Diseases Laboratory, Sir Albert Sakzewski Virus
Research Centre, Royal Children’s Hospital & Health Service Dis-
trict, Herston Road, Herston, Queensland, Australia 4029. E-mail:
d.whiley@uq.edu.au
methods. NAATs are highly sensitive and specific, only requir-
ing a few copies of nucleic acid from the target organism to
facilitate detection. In addition, specimens collected for NAATs
do not require viable organisms for detection. This has a several
impacts: NAATs can be used to detect microbial pathogens in
patients who have already received antibiotic treatment, it al-
lows for less invasive collection techniques, such as urine for
STI diagnosis or self-collection, and also requires less stringent
transport conditions compared to culture-based methods, which
depend on organism viability for isolation. Finally, NAATs are
rapid and can generally be performed within just a few hours,
whichisconsiderablyfasterthancultured-basedmethodswhich
cantakedaystoweeks.Withtheadventofreal-timetechnology,
NAATs have become more user-friendly and overcome many of
the initial laboratory-based limitations of conventional based
NAATs, including carry-over contamination and high work-
load requirements (Espy et al. 2006; Niesters 2004; Ratcliff
et al. 2007). Following these improvements, NAATs are now
making their way into diagnostic laboratories throughout the
world and are replacing traditional detection techniques, in-
cluding culture-based methods and direct fluorescent antibody
techniques.
No single method is ever perfect and NAATs do have some
ongoinglimitations.Failednucleicacidextractionandinhibition
of nucleic acid amplification by inhibitory substances present
withinthesamplematrixarewell-documentedproblemsthatare
theprimarycauseoffalse-negativeresultsinNAATs(Espyetal.
2006; Niesters 2004). However, an emerging concern is false-
negative results caused by sequence variation. The success of
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D. M. WHILEY ET AL.
anyNAATisinherentlydependentonthenucleicacidsequences
targeted by the assay. These sequences must be specific to the
target organism otherwise false-positive results may occur. Just
as important, the sequence targets must be conserved across dif-
ferent strains or types of the target organism, otherwise the sen-
sitivityoftheassaymaybecompromised.Extensivelyvalidated
andwidelyutilizedtargetsequenceshavebeenreportedforsome
organisms. However, for many other organisms it is proving in-
creasingly difficult to identify sufficiently conserved sequences
for diagnostic assays. Sequence variation occurs throughout the
genomes of many infectious agents and there are now frequent
reports of strain variability causing false-negative results. Table
1 provides a few examples of the problems observed to date.
Notably, sequence-related false-negative results have occurred
in NAATs targeting a broad range of organisms using both in-
house and commercial techniques.
The impact of sequence variation on real-time PCR assays
is not simply restricted to producing false-negative results, but
in some circumstances may have more subtle effects depending
on whether the variation falls within the primer or probe targets.
As a general rule, mismatches in the 3?end of a primer are more
likely to impact negatively on PCR amplification compared to
mismatches in the 5?end of the primer. However the extent of
TABLE 1
Examples of false-negative results caused by sequence variation.
OrganismAssay(s) affected Assay target
Basis of
false-negative resultsReference
Cytomegalovirusin-house PCR method glycoprotein B gene Up to three mismatches in
probe target
Oligonucleotide targets not
present due to a 377bp
deletion
Nye et al. 2005
Chlamydia trachomatis Cobas Amplicor
(Roche), TaqMan48
(Roche), Abbott
m2000 (Abbott)
cryptic plasmid
Unemo et al. 2007
Haemophilus influenzae in-house PCR method bexA geneUp to five mismatches in
probe target
Up to six mismatches in
primer targets
Oligonucleotide targets not
present due to absence of
cppB gene
Seven mismatches in
primer target
Mismatches in primer
targets
Sam and Smith 2005
HIV Type 1 Amplicor HIV-1
(Roche)
gag gene Barlow et al. 1997
Neisseria gonorrhoeae in-house PCR method cppB gene Lum et al. 2005
Plasmodium ovale
in-house PCR method 18SrRNA geneBialasiewicz et al. 2007
Parvovirus B19 LightCycler
parvovirus B19
Quantification kit
(Roche), RealArt
Parvo B19 PCR Kit
(Artus)
in-house PCR method N gene
NS gene Cohen et al. 2006
Rabies virusUp to six mismatches in
the probe target and up to
four in the primer targets
Hughes et al. 2004
any impact will further depend on the number of mismatches as
well as the particular nucleotides involved (Whiley and Sloots
2005a).Ratherthanpreventingamplificationaltogether,wehave
found thatoneortwomismatchestowards the3?endofaprimer
may simply delay PCR product amplification. In real-time PCR
assaysthismaybeobservedasanincreaseincyclethreshold(Ct)
value. Notably, these increases in Ct value can cause consider-
able problems for quantitative methods, underestimating organ-
ismloadsbynumerouslogs.Wehaveobservedthisphenomenon
for human polyomavirus BK (Whiley and Sloots 2005a). Simi-
lar observations have been made in the quantification of CMV
by real-time PCR (Lengerova et al. 2007). In purely qualitative
assays,theseminormismatchesintheprimertargetsmayreduce
the sensitivity of the assay to detect the variant strains. For ex-
ample, the assay may require at least 1000 copies of the variant
strain, rather than 10 copies of the wild type, to enable detec-
tion. Mismatches within a probe target can affect PCR product
detection by causing positive specimens to display decreased
fluorescent signal. For some positive specimens the fluores-
cent signal may deviate only slightly from the baseline fluores-
cence, making result interpretation difficult (Whiley and Sloots
2006). The use of hybridization probes for genotyping by melt-
ing curve analysis can also be impeded by sequence variation
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SEQUENCE-RELATED NAAT FALSE-NEGATIVE RESULTS
73
withintheprobetargets(Andersonetal.2003;WhileyandSloots
2005b).
The Need for a Solution
Accuratelaboratorydiagnosisispivotalinthesuccessfulcon-
trol and management of most infectious diseases. It ensures that
infections are both definitively diagnosed and treated optimally.
Hence,theincreasingnumberofreportsofNAATfalse-negative
results and other problems attributed to sequence variation do
raise concerns. For example, failure to detect individual cases
of CMV infection in immunocompromised patients may be life
threatening (Lengerova et al. 2007). Likewise, failing to accu-
rately quantify CMV may result in the significance of an infec-
tion being underestimated or otherwise may incorrectly suggest
successful treatment of disease. For sexually transmitted dis-
eases such as C. trachomatis or N. gonorrhoeae, failure to de-
tect certain strains may see unchecked spread of certain clones,
undermining public health efforts to control disease (Herrmann
2007; Unemo et al. 2007). The above are just a few examples,
nonetheless, they highlight the dependency of both clinicians
and public health services on the accuracy of laboratory results.
ThetraditionalapproachtosequencevariationinNAATshas
beentofindanothersuitablyconservedsinglegenetarget.How-
ever, the processes of finding and validating such targets can be
flawedandmayreproduceasimilarerror.Thisisbecausethereis
anincreasingbodyofevidencedemonstratingthattemporaland
geographicaldistributionofstrainssignificantlyimpactstheper-
formance of NAATs. Successful validation of one NAAT in one
patient population at one point in time may have little bearing
on how the assay may perform later or in different populations,
even within the same country, due to strain variability caused
by further evolutionary pressure or genetic drift. It is for these
reasons that many published assays, which have performed well
in the original evaluation, fail when applied to a patient pop-
ulation in a different geographical region. If another target is
chosen and successfully re-validated then it may again only be
representative of the genetic composition of strains within that
place and time.
Our laboratory identified sequence-related problems when
typing some of our local herpes simplex virus (HSV) 1 and 2
strainsusingapreviouslydescribedhybridizationprobemethod.
In response we developed another assay targeting the HSV gly-
coprotein D (gD) gene (Whiley et al. 2004). The new assay
performed well for our local HSV strains but similar typing
problems were experienced when our HSV gD assay was ap-
plied to other Australian populations (data not published). Sim-
ilarly, when originally evaluated in patient populations in the
Australian states of Queensland and Victoria, cppB based PCR
assays proved sensitive for the detection of N. gonorrhoeae
(Farrell 1999; Whiley et al. 2002; Tabrizi et al. 2004). Consid-
erable problems with false-negative results were observed when
the cppB based PCR was applied to patients living in the North-
ern Territory, Australia (Lum et al. 2005). Likewise, the Roche
Cobas Amplicor C. trachomatis assay has generally been con-
sideredverysensitiveforthedetectionofC.trachomatisandhas
been used successfully for a number of years in many countries.
A C. trachomatis variant possessing a deletion in the cryptic
plasmid sequence targeted by the Amplicor assay has now been
reported in Sweden (Unemo et al. 2007), and appears to have
spread to Norway (Moghaddam and Reinton 2007). Reports
haveshownthattemporalandgeographicalgeneticvariationcan
even impact the success of NAATs targeting parvovirus, which
is generally considered to be highly conserved (Hokynar et al.
2004; Cohen et al. 2006). Additionally, we have found that the
sequence polymorphism exhibited by respiratory viruses can be
problematic as each new season can bring a new variant, partic-
ularly for the rapidly evolving RNA viruses such as respiratory
syncytial virus (RSV) and the parainfluenza viruses.
Manyassaytargetsarechosenonthebasisofpreviousstudies
suggesting that a particular gene sequence may be highly con-
served and specific to a species. It is now evident that what may
beconsideredconservedinevolutionarygeneticstermsmaynot
necessarilybesufficientlyconservedfordiagnosticapplications,
particularly given that only a few nucleotide mismatches are re-
quiredtocauseafalse-negativeresult.Publicsequencedatabases
such as Genbank can also have limitations for assay design, as
there are often only limited data available for some organisms.
On the other hand, there may be a considerable amount of data
for some targets but these may have only been generated from
a single study concentrating on one geographic region and so
these data may be biased. In our opinion, such databases should
be used only as a general guide when designing assays and at-
tention should be given to the temporal and geographic origins
of the published sequences. Nevertheless, because we are still
only beginning to fully appreciate the extent of genetic diversity
of most species, we may be faced with the prospect that any one
genetic target used on its own may inevitability be insufficient
for diagnostic and epidemiological purposes.
The Use of Multi-Target Assays
To circumvent sequence-related problems, some assays have
been developed utilizing two sequence targets. The rationale for
using such assays is that by targeting two different regions there
is less chance of variation occurring in both sequences, and so
significantly reducing the potential for false-negative results. In
2003,ourlaboratorydescribedaduplexreal-timePCRforNeis-
seria meningitidis targeting two different genes, porA and ctrA,
to ensure detection of meningococcal DNA in patients with sus-
pected meningococcal disease. The duplex was developed in
response to concerns that insertion sequences or other muta-
tions may cause false-negative results in the singleplex porA
based method originally used in our laboratory (Whiley et al.
2003). Hermann and colleagues (2004) subsequently described
a duplex real-time PCR assay targeting the CMV polymerase
and glycoprotein B genes to minimize the risk of false-negative
results in the detection of CMV.
Rather than targeting two different gene targets, Geraats-
peters and colleagues (2005) recently described the use of a
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D. M. WHILEY ET AL.
real-time PCR targeting the gonococcal multi-copy opa genes,
which used an additional TaqMan probe. The assay had orig-
inally utilized two primers and only one TaqMan probe, but
an N. gonorrhoeae variant was found that contained mutations
in the original probe target and so produced false-negative re-
sults. A second TaqMan probe was subsequently designed and
successfully added to the assay to accommodate for the new
variation. This latter study is particularly interesting as by tar-
geting the multicopy opa genes, rather than a single copy target,
an assay should arguably be less susceptible to false-negative
results arising from sequence variation. This is because if one
opa gene copy mutates, there should still be other conserved
copies that can be detected. An added benefit of multi-copy
targets is that they can also improve the sensitivity of an as-
say. The results of the above study illustrate that even target-
ing multi-copy genes may not necessarily guarantee against
the occurrence of sequence-related false-negative results. It is
also worth noting that multi-copy targets are generally avoided
for quantitative NAATs as differences in copy number be-
tweenstrainscanintroduceerrorintotheestimationoforganism
load.
In the above examples, the duplex assays were designed to
circumvent known sequence variation problems. More recently
these duplex approaches have been utilized in the design of
assays targeting the Influenza A H5N1 subtype, for pandemic
preparedness (Ng et al. 2005). It is clear that we do not know
what the actual nucleotide sequence of a pandemic strain will
be, if such a pandemic were to occur. Therefore, the develop-
mentofassaysutilizingmultipletargetsdesignedonthebasisof
currently circulating H5N1 sequences offers the most effective
approach of detecting any newly emerging strains.
New or evolving organisms will of course continue to of-
fer considerable challenges for molecular diagnostics, given the
limitationsofsequencedata.Bywayofexample,ourlaboratory
experienced some difficulties when first investigating human
metapneumovirus (hMPV). Following the initial characteriza-
tion of the virus (van den Hoogen et al. 2001), our laboratory
developed a single target hMPV PCR assay and investigated the
incidence of hMPV in our local patient population (Mackay et
al. 2003). The assay was designed using limited sequence data
available at the time and, unfortunately, lead us to miss a second
lineage of hMPV in our population (Maertzdorf et al. 2004). It
is for this reason that our laboratory now routinely uses at least
twodifferentassaystargetingdifferentgenetargetstoovercome
potential problems with sequence variation when investigating
novel or newly described organisms.
Should Two Targets be Used for the Detection
of all Pathogens?
With an increasing number of variants being reported for a
wide range of organisms, it may be more prudent to use two
sequence targets in all NAATs for the diagnosis of infectious
diseases. Unemo and colleagues (2007) have called for C. tra-
chomatis NAATs to target two genetic sequences, including
at least one genomic sequence rather than purely plasmid se-
quences, in response to the appearance of the C. trachomatis
variant in Sweden.
The utilization of dual target assays, rather than single tar-
get assays, would require additional development time and re-
sources. Therefore, such an endeavor would need to be justi-
fied. Consideration would have to be given to whether such an
approach would consume unnecessary substrate and time for
many microorganisms that do not exhibit widespread sequence
variation.
In answering the above question, we have to consider that
most variants causing diagnostic problems are generally only
found by laboratories utilizing multiple methods for detection
of a particular organism, whether this be for assay validation or
for other research. For example, the C. trachomatis variant was
only found when a second assay utilizing an alternate genetic
target was implemented to investigate an unexpected 25% de-
creaseinC.trachomatisinfectionsinSwedenin2005(Ripaand
Nilsson 2006). Gonococcal strains lacking the cppB gene were
identifiedintheAustralianNorthernTerritoryonlybecausebac-
terialculturewasusedinparallelwiththecppBPCRassay(Lum
et al. 2005). It is possible that many variants remain undetected
by current testing strategies given screening with two different
NAAT assays, or parallel testing with culture or antigen identi-
fication, is certainly not routine practice for most laboratories.
Once a single target assay is implemented for routine use as the
sole means of diagnosis following successful validation, a lab-
oratory effectively becomes blind to new variants of the target
organism.
Of course, laboratories could opt to routinely perform both
a NAAT and a traditional method, such as culture, in parallel,
ratherthanusingaNAATalone.Thiswouldhavetheaddedben-
efit of using totally independent tests as a means of ensuring test
accuracy. However, this is not a feasible approach in a routine
diagnosticlaboratorygivenitwoulddramaticallyincreasework-
load and testing costs. The cost of performing a duplex NAAT
assaycomparedtoasingle-targetNAATisonlyincreasedbythe
additional primers and probes, which generally only comprise a
small percentage of NAAT reagent costs.
Other Options?
Other approaches could be adopted to help alleviate these
problems, such as well-planned quality assurance (QA) panels
or more extensive evaluations of NAATs. However, NAAT QA
panelsgenerallydonothavetheaimof,noraretheydesignedto,
sufficiently deal with these issues, and knowledge of a variant
would be required before it could be included in a panel. Even
if evaluations of NAATs could be conducted on a worldwide
scale, which would generally be unworkable, such evaluations
may still not be definitive: Do we really know how many differ-
ent strains need to be tested before a target could be considered
fullyconserved?Whenattemptingtoanswersuchaquestionone
would need to consider the known attributes of the target organ-
ism, including its genetic diversity, distribution, and population
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SEQUENCE-RELATED NAAT FALSE-NEGATIVE RESULTS
75
dynamics.Nevertheless,suchanapproachwouldnotaddressthe
emergence of new variants, such as the Swedish C. trachoma-
tis variant. In this manner, Nye and colleagues (2005) suggest
that assays targeting CMV should be tested against an extensive
panel of viral strains, but concedes that such an approach may
miss the existence of rare or newly arising variants.
Further Comments
OnecouldarguethateventhoughNAATsmayproducefalse-
negative results for a few strains, they detect significantly more
strains that may be missed by traditional methods. Hence, on
balance NAATs already provide enhanced detection of many
pathogens without the need for using multi-target protocols..
It is our opinion that this stance needs to be reconsidered as
NAATsnowformthenewgoldstandardforlaboratorydetection
ofmanyinfectiousagents.CliniciansexpectNAATstobehighly
sensitive, and expect a very high negative predictive value from
theseassaysgiventheyarepromotedasbeingabletodetectvery
low copy numbers of an organism’s nucleic acid. This increases
thepressureondiagnosticlaboratoriestominimizeanypotential
for false-negative results.
We emphasize that other factors can cause false-negative re-
sults in NAATs, including inadequate or failed extraction, in-
hibitory substances, or human error. In contrast to these factors,
however, the very best internal quality control measures cannot
prevent a NAAT false-negative result stemming from sequence
variation.Inthelastdecadetherehasbeenawealthofknowledge
gathered on the issues associated with successful implementa-
tion and performance of NAATs, and so we are now in a better
positiontoconsiderwhatconstitutesbestpractice.Inlightofthe
continuing problems observed with sequence variation, it may
be time to consider the use of two genetic targets in all NAAT
methods.
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