Sample collection and transport strategies to enhance yield, accessibility, and biosafety of COVID-19 RT-PCR testing

Abstract

Introduction. Non-invasive sample collection and viral sterilizing buffers have independently enabled workflows for more widespread COVID-19 testing by reverse-transcriptase polymerase chain reaction (RT-PCR).
Gap statement. The combined use of sterilizing buffers across non-invasive sample types to optimize sensitive, accessible, and biosafe sampling methods has not been directly and systematically compared.
Aim. We aimed to evaluate diagnostic yield across different non-invasive samples with standard viral transport media (VTM) versus a sterilizing buffer eNAT- (Copan diagnostics Murrieta, CA) in a point-of-care diagnostic assay system.
Methods. We prospectively collected 84 sets of nasal swabs, oral swabs, and saliva, from 52 COVID-19 RT-PCR-confirmed patients, and nasopharyngeal (NP) swabs from 37 patients. Nasal swabs, oral swabs, and saliva were placed in either VTM or eNAT, prior to testing with the Xpert Xpress SARS-CoV-2 (Xpert). The sensitivity of each sampling strategy was compared using a composite positive standard.
Results. Swab specimens collected in eNAT showed an overall superior sensitivity compared to swabs in VTM (70 % vs 57 %, P =0.0022). Direct saliva 90.5 %, (95 % CI: 82 %, 95 %), followed by NP swabs in VTM and saliva in eNAT, was significantly more sensitive than nasal swabs in VTM (50 %, P <0.001) or eNAT (67.8 %, P =0.0012) and oral swabs in VTM (50 %, P <0.0001) or eNAT (58 %, P <0.0001). Saliva and use of eNAT buffer each increased detection of SARS-CoV-2 with the Xpert; however, no single sample matrix identified all positive cases.
Conclusion. Saliva and eNAT sterilizing buffer can enhance safe and sensitive detection of COVID-19 using point-of-care GeneXpert instruments.

Full text

1
Sample collection and transport strategies to enhance yield,
accessibility, and biosafety of COVID-19 RT- PCRtesting
PadmapriyaBanada1, DavidElson1†, NaranjargalDaivaa1†, ClairePark1, SamuelDesind1, IbsenMontalvan2,
RobertKwiatkowski3, SoumiteshChakravorty1,3, DavidAlland1,* and Yingda L.Xie1,*
RESEARCH ARTICLE
Banada etal., Journal of Medical Microbiology 2021;70:001380
DOI 10.1099/jmm.0.001380
Received 11 March 2021; Accepted 15 May 2021; Published 06 September 2021
Author aliations: 1The Public Health Research Institute and Department of Medicine, Rutgers New Jersey Medical School, Newark, NJ 07103, USA;
2University Hospital, Newark, NJ 07103, USA; 3Cepheid, Sunnyvale, CA, USA.
*Correspondence: Yingda L. Xie, ylx1@ njms. rutgers. edu; David Alland, allandda@ njms. rutgers. edu
Keywords: Saliva; Nasal; Oral; eNAT; Inactivation.
Abbreviations: CI, confidence interval; CLIA, Clinical Laboratory improvement Amendment; CT, cycle threshold; eNAT, eNAT™ commercial transport
medium; EUA, Emergency use authorization; FDA, United States Food and Drug Administration; NP, Nasopharyngeal; RT- PCR, real- time polymerase
chain reaction; SD, standard deviation; VTM, universal viral transport medium; Xpert, Xpert Xpress SARS- CoV-2 test.
†These authors contributed equally to this work
One supplementary table and two supplementary figures are available with the online version of this article.
001380 © 2021 The Authors
This is an open- access article distributed under the terms of the Creative Commons Attribution License. The Microbiology Society waived the open access fees for this article.
Abstract
Introduction. Non- invasive sample collection and viral sterilizing buers have independently enabled workflows for more
widespread COVID-19 testing by reverse- transcriptase polymerase chain reaction (RT- PCR).
Gap statement. The combined use of sterilizing buers across non- invasive sample types to optimize sensitive, accessible, and
biosafe sampling methods has not been directly and systematically compared.
Aim. We aimed to evaluate diagnostic yield across dierent non- invasive samples with standard viral transport media (VTM)
versus a sterilizing buer eNAT- (Copan diagnostics Murrieta, CA) in a point- of- care diagnostic assay system.
Methods. We prospectively collected 84 sets of nasal swabs, oral swabs, and saliva, from 52 COVID-19 RT- PCR- confirmed
patients, and nasopharyngeal (NP) swabs from 37 patients. Nasal swabs, oral swabs, and saliva were placed in either VTM or
eNAT, prior to testing with the Xpert Xpress SARS- CoV-2 (Xpert). The sensitivity of each sampling strategy was compared using
a composite positive standard.
Results. Swab specimens collected in eNAT showed an overall superior sensitivity compared to swabs in VTM (70 % vs 57 %,
P=0.0022). Direct saliva 90.5 %, (95 % CI: 82 %, 95 %), followed by NP swabs in VTM and saliva in eNAT, was significantly more
sensitive than nasal swabs in VTM (50 %, P<0.001) or eNAT (67.8 %, P=0.0012) and oral swabs in VTM (50 %, P<0.0001) or eNAT
(58 %, P<0.0001). Saliva and use of eNAT buer each increased detection of SARS- CoV-2 with the Xpert; however, no single
sample matrix identified all positive cases.
Conclusion. Saliva and eNAT sterilizing buer can enhance safe and sensitive detection of COVID-19 using point- of- care Gen-
eXpert instruments.
INTRODUCTION
Accurate, ecient, and biosafe detection of SARS- CoV-2
in both symptomatic and asymptomatic individuals with
active COVID-19 infection is an essential public health
strategy for preventing transmission and controlling the
COVID-19 pandemic. Although nasopharyngeal (NP)
swabs are a preferred specimen type, the invasiveness of
this procedure, potential for variable collection quality,
and need for supervised collection with biohazard risks
have hindered the scalability of this testing method [1–4].
Non- invasive sampling methods such as saliva [5–9] or
self- collected nasal or oral swabs [10, 11] combined with
rapid, CLIA- waived COVID-19 assays [12, 13] have shown
promise to enable broader testing of at- risk populations
and increase public access to COVID-19 testing. Steri-
lizing buers such as the guanidine- thiocyanate transport
buer eNAT (Copan diagnostics Murrieta, CA) have also
been found to enable more biosafe NP swab transport and
testing by reverse- transcriptase polymerase chain reaction
OPEN
ACCESS

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Banada etal., Journal of Medical Microbiology 2021;70:001380
(RT- PCR) platforms outside of carefully controlled envi-
ronments [14; 15]. As a combined strategy, non- invasive
sampling and sterilizing buers such as eNAT have the
potential to further enhance yield, biosafety, and acces-
sibility of COVID-19 RT- PCR testing in broad settings.
However, to date, this has not been systematically evaluated.
We recently demonstrated that in saliva samples, eNAT
buer leads to viral inactivation, with at least 5- log reduc-
tion in viable SARS- COV-2, and stabilization of viral RNA
[16]. With the premise that eNAT could optimize yield
and simplify transport and handling of saliva and other
non- invasive samples, we evaluated and compared the
yield of eNAT versus VTM across dierent non- invasive
samples using the Cepheid Xpert Xpress SARS- COV-2 test
(‘Xpert’). Xpert is an FDA- EUA approved rapid, integrated,
cartridge- based RT- PCR test that can be run on widely
existing GeneXpert instruments used in over 130 countries.
To our knowledge, this is the rst study to demonstrate
the use of a viral inactivating buer across various non-
invasive samples in a point- of- care test to deliver a complete
and scalable workow for biosafe handling and testing of
COVID-19 samples.
METHODS
Study population and sample collection
To collect SARS- CoV-2 positive samples from COVID-19
PCR conrmed participants, we conducted a sub- study to
an observational cohort study of hospitalized and emer
-
gency room COVID-19 patients at University Hospital
(UH) aliated with the Rutgers New Jersey Medical
School in Newark, NJ, USA. All patients presenting to
UH were routinely screened by a SARS- CoV-2 RT- PCR
test. is study was approved by the Rutgers Institutional
Review Board for human subject research (Rutgers IRB #
Pro2020001138). Eligible patients included adults (age≥18)
who tested SARS- CoV-2 PCR- positive by the in- hospital
NP swab PCR tests (most commonly Simplexa COVID-19
Direct EUA (Diasorin Molecular LLC, Cypress, CA).
Patients that could not or did not consent, were pregnant
or breastfeeding, prisoners, or who were unable to provide
any respiratory specimens were excluded. Trained study
personnel collected one NP swab (baseline only), two
oral swabs, two nasal swabs, and a saliva sample from all
participants who consented to all sample types. A subset
of participants being evaluated for hospital outcomes in
the parent study continued to be sampled longitudinally
by oral swabs, nasal swabs, and saliva every 2–3 days until
discharge. All swab types were immediately placed into
3 ml of sterile Universal Viral Transport Medium (VTM;
Labscoop, Little Rock, AR) whereas a second nasal and
oral swab was collected and immediately placed into 3 ml
of eNAT (COPAN Diagnostics, Murrieta, CA, USA). A
thinner nylon tip swab designed for nasopharyngeal (NP)
sampling was used to obtain the NP swab (baseline only),
and a thicker nylon tip swab designed for oral and nasal
samples (Copan diagnostic, Murrieta, CA) was used for
these sample types. NP swab collection was performed in
accordance with CDC guidelines [17]; oral swab collec-
tion was performed by swabbing both buccal surfaces
and tongue with an alternating order of collection for
each media; nasal (anterior nares) swab collection was
performed by rotating the swab 1 cm inside the nostril for
10–15 s, alternating nostrils for each media. Additionally,
participants were instructed to self- collect a posterior saliva
sample by clearing the back of their throat, then collecting
4 ml of saliva into a marked, empty, sterile wide- mouth cup
(though any volume over 0.5 ml was accepted). All speci-
mens were transported at room temperature and stored
in a 2–4 °C prior to testing, which occurred within 48 h of
sample collection.
Testing by xpert xpress SARS-Cov-2 (‘Xpert’)
NP, nasal, and oral swabs were tested by adding 300 µl of
the sample (either in VTM or eNAT) directly to the Xpert
SARS- Cov-2 test cartridges and the test was run in the
GeneXpert system as per the manufacturer’s instructions.
e saliva sample was tested using three dierent methods.
First, 300 µl of the saliva sample was directly added into
the Xpert test cartridge (‘saliva direct’ sample). Addition-
ally, the same saliva sample was swabbed with two separate
swabs (thicker nylon tip swabs) for ten twirls followed by
incubating each swab in the saliva for~10–20 s (‘saliva
swab’ sample). Each saliva swab sample was then trans-
ferred into test tubes containing 3 ml of either VTM or
2 ml of eNAT buer and mixed well. From each of these
mixtures, 300 µl were added directly to the sample chamber
of Xpert cartridges. Saliva samples <300 µl were tested only
by swabbing in eNAT and VTM. We also compared saliva
samples directly diluted in 1 : 1, 1 : 2 and 1 : 4 ratios of saliva
to eNAT. A minimum volume of 700 µl of saliva was needed
to test all saliva processing methods: ‘saliva direct’, saliva
swab in eNAT and all three dilutions. For saliva samples
with volumes less than 700 µl, we prioritized saliva direct
and saliva swab testing. Out of the 44 saliva direct posi-
tive samples tested with eNAT ratios, 1 : 1 dilution was
not performed for one saliva sample due to insucient
volume. One each of the sample types had an error either
due to pressure aborts (Error 2008) or probe check error
(Error 5017) or instrument hardware error (Error 2025)
and were not repeat tested. e saliva:eNAT mixtures were
then tested using the GeneXpert system by adding 300 µl
of the mixture to the Xpert SARS- CoV-2 test cartridge. e
eect on the assay inhibition, N2 gene cycle threshold (Ct),
percent positive rate and cartridge pressure values were
evaluated.
Definitions
We compared samples that were collected contemporane-
ously (sample comparison set) and applied a composite
SARS- CoV-2 positive reference standard, dened as at least
one sample type being positive in the sample comparison
set. We did not compare sample sets in which no samples
were positive, as we reasoned that the PCR- negative samples

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Banada etal., Journal of Medical Microbiology 2021;70:001380
from these individuals could not be considered false nega-
tive due to biological variability in sampling over time, but
they were also not suitable as true negative comparators
due to known COVID-19 status of these individuals. To
conrm the discordancy that negative comparison sets was
not due to dierence in the in- hospital versus Xpert PCR
tests, we obtained leover media from positive NP swabs
of a random subset of six participants. We then tested this
archived sample as validation samples on Xpert. Xpert
correctly detected SARS- CoV-2 in all six of these archived
samples.
Statistical analyses
Standard statistical analyses (average, standard deviation,
and t- test) and proportion of positive tests by each sampling
method were compared by Chi- square, t- test or z- test as
appropriate, using Microso Excel 365 for Windows,
GraphPad Prism version eight or online soware (http://
vassarstats. net/ propdi_ ind. html). Scatter plots for Ct
values showing the mean and SD were included for the
positive samples.
RESULTS
Participant enrollment and characteristics
Between 12 June 2020 and 23 October 2020, 70 subjects were
enrolled into the study (Fig.1). From these 70 enrollees, a
total of 116 sample comparison sets were collected - 70 at
baseline and 46 at follow- up time- points. Of note, some
participants consented to all sample types except for NP
swabs. Of the 116 comparison sets, 84 sample sets from
52 participants were complete with all specimen types
and had at least one sample positive (by the composite
reference standard) and were thus included in the sample
comparison analysis (Fig. 1). Characteristics of the 52
participants in the analysis population (participants with
at least one study sample positive for SARS- CoV-2) are
shown in Table1 and characteristics of the 13 participants
with all negative samples are shown in Table S1 (available
in the online version of this article). Among the 52 partici-
pants in the analysis population, 41 (79 %) had symptoms
potentially consistent with COVID-19 whereas 11 (21 %) of
these participants presented to the hospital for non- COVID
indications, had no respiratory symptoms (asymptomatic),
and were incidentally found to be COVID-19 positive by
screening. Among the 41 symptomatic COVID-19 patients,
nine (22 %) did not require oxygen and had mild- moderate
infection. Average participant age was 55, 37 % were female,
and the most common comorbidities were hypertension
and diabetes.
On average, the baseline collection took place 2 days aer
the last positive in- hospital NP swab PCR test for partici-
pants in the analysis group, and 3 days for participants
Fig. 1. Study flowchart.

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Banada etal., Journal of Medical Microbiology 2021;70:001380
with no positive samples. e biological variability of
PCR positivity from samples collected several days apart
was evident in the discordancy of longitudinal in- hospital
NP swab PCR testing results even when the same test was
used. Nineteen (38%) of the 52 participants in the analysis
group had at least one subsequent negative in- hospital NP
swab PCR test during their hospital admission (Table1).
Additionally, we validated 100 % agreement of the in- house
test with Xpert (all the original samples were Xpert positive)
from the original le- over positive NP swab specimen of
six participants. ese observations support that positive-
negative discordancy across time was likely biological
or sampling variability and unlikely due to discordancy
between the in- hospital and Cepheid tests, and is consistent
with previous comparative performance of Xpert Xpress
SARS- CoV-2 with other SARS- CoV-2 RT- PCR platforms
[12, 18, 19].
Comparative testing of dierent respiratory
specimens in Xpert Xpress SARS-COV-2
A total of 84 sample comparison sets from 52 patients were
included in the sample comparison analysis based on the
composite reference, where at least one specimen in the
comparison set was positive. Seventeen of these patients
had follow- up samples collected on alternative days during
their hospital stay. us, a total of 84 sets (49 baseline and
35 follow- up sets) of all specimen types were included in
the analysis. Of the 49 completed baseline collections, 12
participants declined NP swab, leaving a total of 37 sample
sets that could be analysed with NP swab.
As shown in Fig.2a, undiluted saliva added directly to the
cartridge (‘direct saliva’) gave the highest detection rate at
90.5 % (76/84), followed by NP- VTM (86.5 %, 32/37) and
saliva in eNAT buffer (84.5 %; 71/84), which were signifi-
cantly higher compared to nasal or oral swabs (P<0.0001).
Saliva in VTM (71.4 %; 60/84) also performed better
than oral swabs in VTM (50 %; 42/84) or eNAT (58 %;
49/84), as well as nasal swabs in VTM (50 %; 42/84) or
eNAT (67.8 %; 57/84). We further analysed N2- gene cycle
threshold (Ct) values for all positive samples as shown
in Fig. 2b. Average N2 gene Ct values were the earliest
for NP- VTM (32±5.4) and saliva direct (Ct=34.2±5.8)
and most delayed for oral- VTM (37.5±4.9). The Ct range
difference was statistically significant between saliva direct
and oral- VTM (P<0.0001), oral- eNAT (P=0.0003) and
saliva- VTM (P=0.0026). However, there was no significant
difference of N2- Ct range for NP- VTM (P=0.28), nasal-
VTM (P=0.09), nasal- eNAT (P=0.82) and saliva- eNAT
(P=0.26) compared to saliva direct (Fig.2b). There were
three negative NP specimens that were detected in saliva,
which we observed to have N2 Ct values of 39.4, 40.3 and
36.1 (Fig. S1c), indicating below LOD level viral loads [20]
possibly contributing to the discrepancy. Only one of the
sample sets was positive by NP swab (Ct=35.4) but negative
in saliva direct and both saliva swabs (VTM and eNAT).
Overall, we found that saliva performed better or equal
to NP swabs in detecting COVID-19 positive patients.
Similarly, the samples that were negative by other respira-
tory specimens (nasal or oral swab) but detected by saliva
swab in VTM or eNAT had an overall delayed N2- Ct values
of>37, indicating better performance in saliva for samples
with low viral load (sub- LoD) or less variability in saliva
collection.
Table 1. Characteristics of participants in the analysis population
(participants with at least one study sample positive for SARS- CoV-2)
Analysis
population(N=52)
Mean Age in years (SD) 55 (15.1)
# of Men (%) 33 (63 %)
# of Women (%) 19 (37 %)
Ethnicity (%)
Hispanic 35 (67 %)
Black 15 (29 %)
White 2 (4 %)
Comorbidities
Hypertension 27 (52 %)
Diabetes mellitus 16 (31 %)
Coronary artery disease 7 (13 %)
Chronic kidney disease 4 (8 %)
Lung disease (e.g. COPD) 8 (15 %)
No chronic disease 19 (36 %)
COVID symptoms (%)
Cough 33 (64 %)
Shortness of bre ath 32 (62 %)
Fever 31 (60 %)
Diarrhoea 13 (25 %)
Chest pain 10 (19 %)
No COVID symptoms 11 (21 %)
Oxygen Support Required (%)
None 20 (38 %)
Nasal canula 29 (56 %)
Non- invasive mechanical ventilation 2 (4 %)
Intubation 1 (2 %)
Symptom duration prior to baseline collection: mean (range) 7 days (1–23 days)
Days between in- hospital NP swab PCR and baseline
collection: mean (range)
2 days (0–10 days)
Number of follow- up time- points per participant: mean
(range)
1.5 (0–10)
Number of follow- up time- points per participant: mean
(range) 1.5 (0–10)
Participants with negative NP swab PCR collected in routine
clinical follow- up during hospitalization 19 (38 %)

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Banada etal., Journal of Medical Microbiology 2021;70:001380
Influence of transport media on detection across all
sample types
We also evaluated if the composition of dierent transport
media, specically VTM and eNAT, had any inuence
on the detection sensitivity. As described, nasal and oral
swabs were collected in both VTM and eNAT whereas
saliva was collected from patients in an empty sterile cup,
then subsequently swabbed and stored in VTM and eNAT.
As shown before in Fig. 2A, compared to VTM, eNAT
increased the positivity rate by about 20 % (40/84 vs 57/84)
for nasal swabs (P=0.008), followed by 12 % for saliva (60/84
vs 70/84, P=0.065) and 6 % for oral swabs (42/84 vs 47/84,
P=0.43). When data from all sample types were combined
to compare the two media, eNAT oered over 12 % advan-
tage (142 vs 174 out of 252 samples) in overall detection rate
compared to VTM (P=0.003).
Optimizing the use of eNAT buer for saliva
Compared to saliva swabbed into eNAT, direct saliva
yielded an overall delayed SPC- Ct values in the Xpert test,
indicating possible PCR inhibition and increased (>60 PSI)
in- cartridge pressure values (Fig. S2b). Saliva diluted into
eNAT at a ratio of 1 : 2 (N=43) yielded the second highest
PCR positive rate (97.7 %, 42/43) aer saliva direct (100 %,
43/43) (Fig.3). Dilutions of 1 : 1 and 1 : 4 yielded 95 % (40/42)
and 93 % (40/43) positive rate, respectively. Saliva swabs
in eNAT showed the lowest sensitivity at 86 % (37/43,
P>0.05; Fig.3a). A sample missed by 1 : 2 and 1 : 4 dilutions
and another by 1 : 1 and 1 : 4 dilutions, had delayed N2- Ct
values of 44.3 and 41.7 with saliva direct, respectively, indi-
cating the inuence of Poisson distribution for viral loads
considerably below the limit of detection. Whereas the
average N2- Ct values were similar (ca. 33–34) for all saliva
Fig. 3. eNAT as a transport media for saliva. Saliva diluted with eNAT at 1 : 1, 1 : 2, and 1 : 4 ratio showing (A) Percent positive rate and (B)
N2- Ct values from patient saliva samples tested directly (N=44), as a swab in eNAT (N=44), diluted 1 : 1 (N=44), 1 : 2 (N=42), and 1 : 4 (N=42)
in eNAT transport media. ns=not statistically significant.
Fig. 2. Comparative testing of dierent respiratory specimens using the Xpert Xpress SARS- CoV-2 test. (A) Percent positive rate and (B)
N2 gene cycle threshold (Ct) values of samples from all participants with at least one SARS- CoV-2 positive sample (N=84 for all samples
and N=37 for NP swab). NP=Nasopharyngeal: VTM=Viral transport medium; eNAT=eNAT transport media, Copan diagnostics. ns=not
statistically dierent. ****P<0.0001; ***P<0.001, **P=0.02.

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Banada etal., Journal of Medical Microbiology 2021;70:001380
conditions tested (P>0.05), the SPC- Ct values were earlier
with saliva in eNAT compared to saliva direct (31.25±1.74,
P<0.001, Fig. S2c), suggesting that PCR inhibition was
mitigated by the addition of eNAT to an appreciable extent.
Operational characteristics of processing saliva in
GeneXpert cartridges
To evaluate saliva processing proles in the GeneXpert
cartridges, we analysed the sample processing control (SPC)
Ct values and in- cartridge pressure values. All respiratory
samples collected in either VTM or eNAT did not have any
signicant dierence either with SPC Ct or the max pres-
sure values (Fig. S3A and B). Saliva direct, the only sample
type analysed as is without dilution, yielded slightly delayed
SPC Ct and higher cartridge pressure values with an average
of 58±13.48, with one sample aborting the run due to pres-
sure exceeding 100 psi (vs NP- VTM, P<0.0001). However,
when the saliva was swabbed and transferred to VTM or
eNAT, average pressure values fell to 53.2±6.06 (P<0.0001)
and 52.2±5.4 (P<0.0001), respectively. Dilution with eNAT
at 1 : 1, 1 : 2 and 1 : 4 ratio reduced the inhibition from saliva
direct (P<0.0001) by lowering the average SPC- Ct values
by~2 Ct values (Ct 29.1 in 1 : 2 vs 31.2 in saliva direct). ere
was no signicant dierence in maximum in- cartridge
pressure values with saliva dilution in eNAT (P>0.05),
except for swab in eNAT (P=0.02). ese results suggest
that particles or mucus present in direct saliva samples can
occasionally interfere with assay function, and that swab
testing may be considered when these situations occur.
DISCUSSION
We found that saliva is an excellent test matrix for the
Xpert Xpress SARS- CoV-2 test, providing a sensitivity
(90.5 % in VTM, 84.5 % in eNAT) that is comparable
to that of NP swabs (86.5 % in VTM) and better than
nasal (50 % in VTM, 67.8 % in eNAT) and oral swabs
(50 % in VTM, 58 % in eNAT). This finding is consistent
with previously published studies using other RT- qPCR
modalities [21–26]. Although a handful of previous
studies have looked at saliva tested in the Xpert SARS-
CoV-2 [12, 27, 28], to our knowledge this study is the
first study to comprehensively test multiple non- invasive
sampling methods, in the setting of both symptomatic
and asymptomatic SARS- CoV-2 infection, with and
without the use of a sterilizing sample/transport buffer.
By applying a composite reference standard for a positive
sample, we observed that saliva enhanced the detection
of SARS- CoV-2 compared all other sampling types,
consistent with similar observations from other studies
[1, 5–7]. It is worth noting that no sample matrix was
100 % sensitive compared to the composite reference
standard. Discordancy between sample matrices was most
pronounced in samples that had a delayed cycle threshold
indicating low viral load. This suggests that for patients
with high- risk or severe disease, testing with multiple
samples and perhaps multiple sample types when clinical
suspicion is high may provide the highest sensitivity and
negative predictive value for SARS- CoV-2 to guide treat
-
ment decisions.
We additionally found that eNAT, a buer we have previously
determined to be eective at inactivating SARS- CoV-2 in- vitro
[16], increased the test positivity rates across all non- invasive
sample types compared to VTM (P=0.0032), with a saliva to
eNAT ratio of 1 : 2 being optimal in our sample set. We also
found that adding eNAT to saliva possibly mitigates the PCR
interference from saliva with lower pressure values and recovery
of otherwise delayed SPC Ct values seen with direct saliva. ese
ndings suggest that the application of eNAT as a sample buer
may be advantageous not only in safe handling and transport,
but also in improving yield and processing capability of non-
invasive samples on the Cepheid system.
ere were several limitations in this study. First, there were
less contemporaneous NP swabs collected with saliva, thereby
reducing the number of direct comparisons between these two
sample types, although they were found to be comparable. An
underlying reason for this – participants declining NP swab
collection due to its discomfort – also demonstrates the real-
world limitations that would be magnied with larger scale
testing such as in schools or the workplace. Secondly, we added
eNAT to saliva in the laboratory, whereas the benet of eNAT
would be to sterilize samples immediately aer collection and
before transport and test set up. However, this allowed us to
evaluate the combination of eNAT and saliva under dierent
conditions and inform optimal design of kits to add eNAT
immediately to saliva upon collection. Finally, our participants
were patients who had either been admitted to the hospital or
seen in the emergency department. is population may not
be generalizable to ambulatory individuals who would benet
the most from self- collection. However, we captured a diverse
patient group in our cohort including those who were never
admitted, as well as patients who were detected by universal
screening but reported no COVID-19 symptoms.
Altogether, our ndings support the use of saliva and eNAT
sterilizing buer with non- invasive samples to enhance eec-
tive, safe, and accessible COVID-19 testing and screening in the
many health care systems worldwide already using GeneXpert
instruments.
Funding information
This study was partially funded by the National Institute of Allergy and
Infectious Diseases of the National Institutes of Health under award
number R01 AI131617 and Rutgers University Institutional Support.
Acknowledgements
We thank Dr Jason H. Yang (Rutgers New Jersey Medical School) for
supporting DE’s and CP’s role in the studies, Cepheid for in- kind dona-
tion of cartridges, and Copan for donation of eNAT media and swabs.
Conflicts of interest
The authors declare that there are no conflicts of interest.
Ethical statement
Written consent was obtained from all study participants under
a Rutgers Institutional Review Board for human subject research
(Rutgers IRB # Pro2020001138).

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Banada etal., Journal of Medical Microbiology 2021;70:001380
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