The Roche cobas 4800 CT/NG assay is a commonly used commercial system for screening for Neisseria gonorrhoeae infection, and previous studies have shown the method to be highly sensitive and specific for urogenital samples. We present the first confirmed clinical N. gonorrhoeae false-positive result using the cobas 4800 NG assay, obtained from testing a pharyngeal swab sample and caused by cross-reaction with a commensal Neisseria strain

A. Upton

C. Bromhead

D. M. Whiley

Fairley

Hopkins

Parra-Sánchez

Relman

Rockett

Smith

Tabrizi

Taylor

Upton

Van Der Pol

Whiley

Upton, Arlo

Bromhead, Collette

Whiley, David M.

University of Queensland eSpace

Neisseria gonorrhoeae False-Positive Result Obtained from a
Pharyngeal Swab by Using the Roche cobas 4800 CT/NG Assay in
New Zealand in 2012
Arlo Upton,a Collette Bromhead,b David M. Whileyc
Labtests, Mt. Wellington, Auckland, New Zealanda; Aotea Pathology Ltd., Wellington, New Zealandb; Queensland Paediatric Infectious Diseases Laboratory, Queensland
Children’s Medical Research Institute, The University of Queensland, Brisbane, Queensland, Australiac
The Roche cobas 4800 CT/NG assay is a commonly used commercial system for screening forNeisseria gonorrhoeae infection,
and previous studies have shown the method to be highly sensitive and specific for urogenital samples. We present the first con-
firmed clinicalN. gonorrhoeae false-positive result using the cobas 4800 NG assay, obtained from testing a pharyngeal swab sam-
ple and caused by cross-reaction with a commensalNeisseria strain.
Nucleic acid amplification tests (NAATs) are widely used forthe detection of gonorrhea. However, the specificity of these
methods can be undermined by ongoing genetic exchange be-
tween species within the Neisseria genus, leading to commensal
Neisseria strains acquiringNeisseria gonorrhoeae (NG) NAAT tar-
get sequences. For these reasons, supplementary “confirmatory”
testing forN. gonorrhoeaeNAATs has been widely adopted (1, 2).
The Roche cobas 4800 CT/NG assay is a later-generation NAAT
method, and the NG component of the assay utilizes a dual-target
approach, using two assays to detect sequences within the direct-
repeat (DR-9) region (3). Performance data to date show excellent
sensitivity and specificity for urogenital specimens (3–7), and it
has been suggested that the assay does not require a second test to
confirm urogenital positive results (6). Also, to our knowledge,
there have been no definitive reports of the assay cross-reacting
with commensal Neisseria strains (3, 8); while initial testing in a
study by Tabrizi et al. (8) showed that the cobas 4800 NG assay
cross-reacted with two commensal Neisseria strains, both were
negative upon retesting using fresh cultures (8). Herein, we report
the first clinical demonstration of a Roche cobas 4800 NG false-
positive result obtained from a pharyngeal swab sample and
caused by a reproducible cross-reaction with a commensal Neis-
seria strain.
On 17 September 2012, as part of the heightened awareness
program sponsored by the New Zealand AIDS Foundation, self-
referred throat and rectal swabs and a first-void urine were re-
ceived from a 38-year-oldmale presenting to a sexual health clinic
in Auckland,NewZealand. The samples were tested by PCR forN.
gonorrhoeae and Chlamydia trachomatis (CT) on the Roche cobas
4800 CT/NG assay at Labtests, Auckland, New Zealand (Table 1).
The three specimens were all negative forC. trachomatisDNA; the
urine and rectal swabs were also negative forN. gonorrhoeaeDNA.
The throat swab was positive forN. gonorrhoeaeDNAwith a cycle
threshold value of 35.7, which was confirmed upon retesting at a
second laboratory (Aotea Pathology, Wellington; cobas 4800 NG
assay positive with a cycle threshold value of 35.3), and the patient
was treated with 500 mg intramuscular ceftriaxone. As part of an
ongoing study investigating the specificity of the cobas 4800 NG
assay in our population, the specimen was subsequently referred
for supplementary testing by the Abbott m2000 real-time PCR
(Canterbury Health Laboratories) and in-house PCR methods
targeting the gonococcal porA and opa genes (Aotea Pathology,
Wellington); the specimen was negative by all assays.
Approximately 7 weeks later, the patient presented to his gen-
eral practitioner with a sore throat and had a throat swab taken for
routine bacteriology and C. trachomatis/N. gonorrhoeae PCR. No
beta-hemolytic streptococci, Arcanobacterium haemolyticum, or
N. gonorrhoeae was isolated by routine culture. Again, the cobas
4800 assay detected N. gonorrhoeae DNA (cycle threshold value,
39.4) and did not detect C. trachomatis DNA. In response to this
result, the patient received another dose of ceftriaxone. After con-
cerns over the validity of the result were raised by laboratory staff
with the patient’s general practitioner (GP), the patient was then
contacted by his GP and he agreed to provide a further two throat
swabs for inoculation on sheep blood agar, chocolate agar, and
New York City agar. While no N. gonorrhoeae grew, three species
of commensal Neisseria grew and were identified as Neisseria
flavescens, Neisseria macacae, and Neisseria perflava by matrix-as-
sisted laser desorption ionization–time of flight mass spectrome-
try (MALDI-TOFMS) (Bruker Daltonics, Germany). Colonies of
each strain were tested in the cobas 4800 assay, and a positive
result was obtained for the N. macacae isolate (cycle threshold
value, 28.2) while the other two isolates were negative. Four indi-
vidual suspensions of the N. macacae isolate (prepared by inocu-
lating 2 colonies each into 1.0 ml sterile water) were subsequently
retested in the cobas 4800 NG assay at both the Labtests, Auck-
land, and Aotea Pathology laboratories; positive results were ob-
tained for all suspensions in both laboratories (cycle threshold
values ranged from 30.9 to 34.5 cycles; mean, 32.4 cycles).
The N. macacae isolate was further investigated by testing the
four above-mentioned suspensions by an in-houseN. gonorrhoeae
porA and opa PCR; negative results were obtained for all suspen-
sions. To further investigate species identification, a partial 16S
Received 21 February 2013 Returned for modification 28 February 2013
Accepted 5 March 2013
Published ahead of print 13 March 2013
Address correspondence to Arlo Upton, Arlo.Upton@labtests.co.nz.
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JCM.00485-13
May 2013 Volume 51 Number 5 Journal of Clinical Microbiology p. 1609–1610 jcm.asm.org 1609
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rRNA sequence of the isolate was subject to PCR and DNA se-
quencing using previously described primers (P515PPl and p13B
[9]). GenBank nucleotide blast analysis of a 787-bp 16S rRNA
sequence provided a 100% match with two Neisseria flava
sequences (GenBank accession numbers GU561419.1 and
AJ239301.1), two unidentified Neisseria species (GenBank acces-
sion numbers FJ976424.1 and EU663609.1), and one Neisseria
sicca sequence (GenBank accession number AJ239293.1). The
closest N. gonorrhoeae match was 98% (768/787 nucleotides;
GenBank accession number CP002440.1). The isolate was also
sent to the Environmental and Science Research (ESR) reference
laboratory (Porirua, New Zealand) and was identified asNeisseria
subflava biovar perflava by standard phenotypic testing.
The above-described data provide clear evidence that the N.
gonorrhoeae-positive results provided by the cobas 4800 assay for
the throat swabs from this patient were false-positive results and
that the problem arose through cross-reaction with a Neisseria
species strain present in the throat of this particular patient. Since
March 2012, we have had only eight other patients from whom
pharyngeal swabs have provided positive PCR results in the cobas
4800 NG assay, and all eight have been confirmed as N. gonor-
rhoeae positive by the Abbott m2000 real-time PCR. Based on
these limited data, the confirmation rate of the method for pha-
ryngeal swabs is 88.9%. This is lower than the overall positive
predictive value (PPV) of 97.1% (95% confidence interval, 95% to
98.5%) previously observed in our population based on testing of
over 40,000 mainly urogenital specimens, of which 361 were pos-
itive for N. gonorrhoeae (10). While the identifications of the iso-
late provided by the MALDI-TOF, 16S rRNA, and phenotypic
testingwere not in complete agreement, the results clearly indicate
that this was a commensalNeisseria strain and notN. gonorrhoeae
and that it had likely acquired the DR-9 sequence targeted by both
N. gonorrhoeae reactions of the cobas 4800 NG assay. We did not,
however, seek to derive theDR-9 sequence from this isolate, as the
precise region targeted by the cobas 4800 assay is not in the public
domain.
It should be noted thatmostN. gonorrhoeaeNAATs are neither
validated nor marketed for use on extragenital sites, including
pharyngeal swabs, but that NAAT testing of these sites is driven by
the relatively poor sensitivity of culture for nongenital specimens
(11). Overall, this case provides yet another example of specificity
problems faced with molecular detection of N. gonorrhoeae and
shows that false-positive results can be obtained from later-gen-
eration N. gonorrhoeae NAATs, including the cobas 4800 assay.
These data further highlight the ongoing need for supplementary
testing forN. gonorrhoeaeNAATs so as to avoid unnecessary treat-
ment and patient anxiety, particularly when they are applied to
pharyngeal samples in which commensal Neisseria strains are
prevalent.
ACKNOWLEDGMENT
We thank Kevin Barratt at Canterbury Health labs for Abbott m2000
real-time PCR testing.
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TABLE 1 Summary of NAAT and culture resultsa
Week collected
(day/mo/yr)
Anatomical site/type
of sample
Result with each N. gonorrhoeae diagnostic method
cobas 4800 NG
(threshold cycle value)
Abbott m2000
real-time PCR
In-house porA and
opa PCR assays
Bacterial
culture
1 (17/09/12) Urine Negative NP NP NP
Rectal swab Negative NP NP NP
Throat swab Positive (35.7, 35.3)* Negative Negative NP
7 (7/11/12) Throat swab Positive (39.4) NP Negative NP
8 (23/11/12) Throat swab ( 2) NP NP NP Negative
N. macacae isolate** Positive (28.2) NP Negative NA
a NA, not applicable; NP, not performed; *, tested at two separate laboratories; **, isolated from a throat swab taken in week 8.
Upton et al.
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Neisseria gonorrhoeae false-positive result obtained from a pharyngeal swab by using the roche cobas 4800 CT/NG assay in New Zealand in 2012

ABSTRACT
          
            The Roche cobas 4800 CT/NG assay is a commonly used commercial system for screening for
            Neisseria gonorrhoeae
            infection, and previous studies have shown the method to be highly sensitive and specific for urogenital samples. We present the first confirmed clinical
            N. gonorrhoeae
            false-positive result using the cobas 4800 NG assay, obtained from testing a pharyngeal swab sample and caused by cross-reaction with a commensal
            Neisseria
            strain.
          </jats:p

Arlo Upton

Collette Bromhead

David M. Whiley

Crossref

Journal of Clinical Microbiology

Neisseria gonorrhoeae False-Positive Result Obtained from a Pharyngeal Swab by Using the Roche cobas 4800 CT/NG Assay in New Zealand in 2012

http://espace.library.uq.edu.au/view/UQ:300944/UQ300944_OA.pdf

Neisseria gonorrhoeae false-positive result obtained from a pharyngeal swab by using the roche cobas 4800 CT/NG assay in New Zealand in 2012

Abstract

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