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ORIGINAL RESEARCH
HCV reflex testing: A single-sample, low-contamination method
that improves the diagnostic efficiency of HCV testing among
patients in Alberta, Canada
L Alexa Thompson BSc1, Jayne Fenton MLT2, Carmen L Charlton PhD1,2,3
BACKGROUND: Hepatitis C virus (HCV) can be cured with antiviral treatments. Diagnosis normally requires two blood samples,
one for serology screening and one for molecular confirmation. This multi-step process creates barriers in patient care and de-
creases testing for hard-to-reach populations. We used the cobas® 6800 to detect HCV RNA after antibody testing to investigate
whether a single-sample reflex testing method is effective and efficient for diagnosing HCV-positive patients. METHODS: HCV
RNA–positive clinical samples (n = 152) were interchangeably loaded on the ARCHITECT i2000SR with negative samples (n =
152) in a checkerboard fashion, tested for HCV antibodies using fixed probes, and directly transferred to the cobas 6800 for
molecular testing. Contamination rates, sensitivity, and specificity were determined by comparing Abbott m2000 and cobas
6800 viral loads. After implementing reflex testing, clinical data over a 6-month period were analyzed for diagnostic efficiency.
RESULTS: Contamination was present in 5 of 152 pairs (3.29%) after reflex testing. Sensitivity and specificity were 99.3% (95%
CI 95.1% to 99.9%) and 100% (95% CI 97.5% to 100%), respectively, using the cobas 6800 assay after serotesting. Approxima-
tely 97% of clinical patients received a conclusive test result with the reflex-testing algorithm. For HCV-positive patients, mean
diagnostic turnaround times were significantly lower using reflex testing versus the two-sample method (4 versus 39 days;
p < 0.0001). CONCLUSIONS: HCV reflex testing demonstrated low levels of contamination without compromising the integrity
of the molecular assay. Implementation in clinical laboratories would increase the efficiency of diagnosis and decrease steps in
the continuum of care for patients.
KEYWORDS: contamination, diagnosis, hepatitis C virus, public health, reflex testing
HISTORIQUE : Il est possible de vaincre le virus de l’hépatite C (VHC) par des traitements antirétroviraux. Pour poser un diagnostic,
il faut normalement deux prélèvements de sang : l’un pour le dépistage sérologique et l’autre pour la confirmation moléculaire.
Ce processus en plusieurs étapes crée des obstacles dans les soins aux patients et limite le dépistage auprès des populations dif-
ficiles à atteindre. Les auteurs ont utilisé la plateforme cobasMD 6800 pour déceler l’ARN du VHC après des tests de détection des
anticorps et pour explorer si une méthode de dépistage réflexe à échantillon unique est efficace et efficiente lors du diagnostic
des patients positifs au VHC. MÉTHODOLOGIE : Les chercheurs ont placé les échantillons cliniques positifs à l’ARN du VHC (n =
152) en échiquier avec des échantillons négatifs (n = 152) dans l’analyseur ARCHITECT i2000SR, ont dépisté les anticorps du VHC
à l’aide de sondes fixes, puis ont transféré les échantillons directement sur la plateforme cobas 6800 en vue du test moléculaire.
Ils ont établi les taux de contamination, la sensibilité et la spécificité en comparant les charges virales des plateformes Abbott
m2000 et cobas 6800. Après le dépistage réflexe, ils ont analysé les données cliniques sur une période de six mois pour en établir
l’efficience diagnostique. RÉSULTATS : Les chercheurs ont constaté une contamination dans cinq des 152 paires (3,29 %) après
le dépistage réflexe. Après l’examen sérologique, ils ont obtenu une sensibilité de 99,3 % (IC à 95 %, de 95,1 % à 99,9 %) et une
spécificité de 100 % (IC à 95 %, de 97,5 % à 100 %) au moyen de la plateforme cobas 6800. Environ 97 % des patients cliniques
ont reçu un test concluant selon l’algorithme du dépistage réflexe. Chez les patients positifs au VHC, le délai diagnostique moyen
était considérablement plus court après le dépistage réflexe qu’après la méthode à deux échantillons (quatre jours plutôt que
39; p < 0,0001). CONCLUSIONS : Le dépistage réflexe du VHC a démontré de faibles taux de contamination sans compromettre
l’intégrité du dosage moléculaire. Son adoption en laboratoire clinique accroîtrait l’efficience du diagnostic et réduirait le nombre
d’étapes dans le continuum des soins aux patients.
Official Journal of the Association of Medical Microbiology and Infectious Disease Canada
Journal officiel de l’Association pour la microbiologie médicale et l’infectiologie Canada
10.3138/jammi-2021-0028
e20210028
Official Journal of the Association of Medical Microbiology and Infectious Disease Canada 7.2, 2022 doi:10.3138/jammi-2021-0028
LA Thompson, J Fenton, CL Charlton
MOTS-CLÉS : contamination, dépistage réflexe, diagnostic, santé publique, virus de l’hépatite C
1Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada; 2Alberta Precision Laboratories
(ProvLab), University of Alberta Hospital, University of Alberta, Edmonton, Alberta, Canada; 3Li Ka Shing Institute of Virology, Edmonton,
Alberta, Canada
Correspondence: Carmen L Charlton, 2B3.07 Walter Mackenzie Centre, 8440-112 Street, University of Alberta, Edmonton, Alberta
T6G 2J2 Canada. Telephone: 780-407-8975. E-mail: Carmen.Charlton@albertaprecisionlabs.ca
INTRODUCTION
Hepatitis C virus (HCV) is a blood-borne pathogen that
preferentially replicates in the liver (1). According to the
World Health Organization (WHO), more than 71 million
people worldwide are infected with HCV (2). Infections are
spontaneously cleared in 25%–30% of individuals, whereas
70%–75% of infections become chronic and can cause liver
cirrhosis or hepatocellular carcinoma (3). Early diagnosis of
an HCV infection is therefore optimal to control and treat
the virus before liver damage can occur (4).
Upon infection with HCV, the immune system generates
antibodies against the virus (5). Serological testing identi-
fies previous exposure to the virus and is regularly used to
screen patients for infection (6). However, antibody test-
ing is unable to differentiate between exposure and active
infection, and those with spontaneous clearance or active,
chronic, or resolved infection will screen positive for HCV
antibodies. Subsequently, active infections need to be identi-
fied via molecular detection of HCV RNA (7). Because of
the higher sensitivity of molecular assays and the possibility
of upstream contamination on the serological instrument,
diagnosis typically requires two blood samples to be col-
lected (one for serology and one for molecular testing; the
two-sample method) (8).
Multiple studies have shown that patients are more likely
to be cured of the virus if HCV RNA molecular testing is
performed within 6 months of serology (9–11). Unfortunately,
the requirement for patients to submit a secondary blood
sample for HCV RNA testing means that patients are unable
to receive a conclusive diagnosis at the same time as their
serology result and are often lost to follow-up between tests.
One method to ensure molecular testing among seropositive
patients, and decrease those lost to follow-up, is to implement
HCV reflex testing, in which a single blood sample is used
for antibody testing and molecular confirmation if needed.
However, molecular reflex testing from the same tube used
for serology is often discouraged for the clinical diagnosis
of HCV because of contamination concerns, particularly
when fixed probes are used in upstream serology testing. In
addition, there is concern over the sensitivity and specific-
ity of molecular assays after antibody testing on the same
sample (12).
In this study, we emulated the molecular reflex testing
workflow on clinical samples by performing HCV antibody
testing on the ARCHITECT i2000SR (Abbott Laboratories,
Abbott Park, Illinois) and then transferring samples to the
cobas® 6800 (Roche Diagnostics, Rotkruez, Switzerland) for
HCV RNA nucleic acid testing (NAT). We analyzed rates
of contamination and calculated sensitivity and specificity
to examine the feasibility of HCV reflex testing in clinical
laboratories. Moreover, we assessed reflex testing results and
turnaround times in the public health laboratory system to
determine whether reflex testing implementation can improve
HCV diagnostic efficiencies for patients in Alberta.
MATERIALS AND METHODS
HCV testing in Alberta
All HCV testing in Alberta is performed at the provincial
public health laboratory (Alberta Precision Laboratories,
or ProvLab), which has two centralized locations: Calgary
and Edmonton. HCV antibody testing is carried out at
both locations, whereas HCV RNA confirmation testing is
performed at Edmonton. ProvLab uses a continuous com-
putational language (CCL) look-back method to identify
patients with a history of HCV antibody or RNA positivity.
Before December 12, 2019, all HCV antibody requests were
performed, but confirmation of previous positives was not
repeated. After December 12, 2019, all antibody requests for
patients who have previously been confirmed positive for
HCV are automatically cancelled, and physicians are guided
to monitor patients via molecular testing. Antibody testing is
performed using the Abbott Architect i2000SR and dedicated
molecular RNA testing is performed using the Abbott m2000.
Test results and patient demographics are uploaded to the
ProvLab Laboratory Information System (LIS), and samples
are kept for at least 2 years before discarding.
Sample selection
Clinical samples sent to ProvLab for routine HCV testing
between December 5, 2017, and December 5, 2019, were
used in this study. Selected samples were dedicated molecular
serum samples and were not previously run on any other
instruments besides the Abbott m2000. HCV-seropositive
Official Journal of the Association of Medical Microbiology and Infectious Disease Canada 7.2, 202298
Low contamination and efficiency of HCV reflex testing
samples (n = 170) with low-range (<1,000 IU/mL; n = 18),
mid-range (between 1,000 and 1,000,000 IU/mL; n = 12),
and high-range (>1,000,000 IU/mL; n = 140) viral loads
were selected for use and represented a cross-section of HCV
genotypes (1a, 1b, 2, 3, and 4; not typed; or mixed typed).
The mean values of HCV RNA viral loads across the low-
range, mid-range, and high-range groups were determined
from original viral load values (performed on the Abbott
m2000). HCV-seropositive samples negative for HCV RNA
(n = 147) were selected as negative controls.
HCV antibody testing
To evaluate possible contamination from the serology test-
ing instrument, mid- and high-range viral load samples
were run in pairs (n = 152) with negative samples on the
ARCHITECT i2000SR. All samples were initially stored at
–70°C and thawed at room temperature (20°C) before use.
Greater than or equal to 1.5 mL each of known HCV RNA–
positive samples followed by known HCV RNA–negative
samples were loaded in a checkerboard pattern and placed
on the ARCHITECT i2000SR. HCV antibody testing was
carried out on the ARCHITECT i2000SR using fixed probes
according to manufacturer instructions (13). Ten to 15 pairs
of HCV RNA–positive and RNA–negative samples were run
at a time until all samples were processed.
Comparing the cobas 6800 reflex testing with the
Abbott m2000 HCV RNA output
Once HCV antibody testing of samples was completed,
they were placed onto the cobas 6800 system for HCV
RNA detection (14). Low-range viral load samples (n =
13; the 5 contaminated samples were excluded) were also
run on the ARCHITECT i2000SR followed by the cobas
6800 to emulate the clinical workflow and to negate analyte
degradation resulting from increased freeze–thaw cycles.
Although the cobas 6800 platform has a diagnostic claim,
off-label technology access was used to extract the quanti-
tative viral load run in the background of every qualitative
test from the instrument software. Quantitative results from
the cobas 6800 were then compared with those originally
recorded from the Abbott m2000 to validate our approach
against a reference standard. Pairs were classified as either
concordant (Abbott m2000 detected–cobas 6800 detected
or Abbott m2000 not detected–cobas 6800 not detected)
or discordant (Abbott m2000 detected–cobas 6800 not
detected or Abbott m2000 not detected–cobas 6800 de-
tected). Abbott m2000 detected–cobas 6800 not detected
samples were assumed to be discordant as a result of viral
RNA degradation. Carry-over contamination was defined
as Abbott m2000 not detected–cobas 6800 detected. The
sensitivity and specificity of the cobas 6800 assay was
assessed after upstream serology testing. Finally, the reflex
testing workflow was validated using inter- and intra-assay
method validation, as previously described (15). Positive
and negative pooled samples were reflex tested in triplicates
for 3 days and as singles for 7 days, and a percent coef-
ficient of variation (%CV) threshold of less than 20% was
used for validation.
HCV reflex testing of clinical samples
HCV reflex testing was implemented in ProvLab on De-
cember 12, 2019. HCV antibody–positive samples with
RNA viral loads of more than 1,000 IU/mL were defined
as detected for HCV infection; those with undetected vi-
ral loads, as negative; and those with viral loads between
1 and 1,000 IU/mL were defined as indeterminate. The
cut-off for positive detection was chosen as 1,000 IU/mL
because it was 2 standard deviations above observed RNA
contamination during assay validation. HCV testing data
were extracted from the ProvLab LIS for 6 months before
(June 12, 2019–December 11, 2019) and 6 months after
(December 12, 2019–June 12, 2020) the implementation
of reflex testing. Data variables collected included test
results, patient identifiers, demographic information, speci-
men collection, submission, and result verification times.
Only presumed first-time HCV antibody–positive patients
were included in the analyses, which were identified by a
CCL look-back using Alberta personal health numbers
(PHNs) in the ProvLab software or manually in the LIS
using PHN (regardless of province), name, and date of
birth. Mean diagnostic turnaround times from specimen
collection for HCV antibody testing to completion of HCV
RNA molecular testing were calculated over the two time
periods. Follow-up HCV RNA testing for the two-sample
method was linked to initial HCV antibody testing using
overlapping PHNs, name, and date of birth. Turnaround
times were recorded in days.
Data analysis
All data were collated and graphed in Stata version 15.1
(StataCorp, College Station, Texas), and flowcharts were
created in Microsoft PowerPoint 2010 (Microsoft Corpo-
ration, Redmond, Washington). Paired t-tests and non-
parametric sign tests were used to compare mean viral
loads between the Abbott m2000 and the cobas 6800 HCV
RNA NATs. A two-sample t-test with unequal variances was
used to compare mean turnaround times before and after
the implementation of reflex testing (two-sample versus
reflex testing method). The threshold for a significant
p-value was set at α = 0.05, and 95% confidence intervals
(CIs) were constructed. Statistical analyses were performed
using Stata version 15.1.
7.2, 2022 Journal officiel de l’Association pour la microbiologie médicale et l’infectiologie Canada 99
LA Thompson, J Fenton, CL Charlton
from 165 of the 170 seropositive samples, including the
low-range viral load samples and excluding the 5 samples
with detected carry-over contamination, were compared
between the Abbott m2000 and the cobas 6800. Overall, there
was a difference in mean viral titres between the two testing
platforms, with original values on the Abbott reported to be
significantly lower than those detected on the cobas (Table 1;
mean 4.37 × 10
6
IU/mL, versus mean 5.14 × 10
6
IU/mL,
p < 0.0005, respectively). In 104 of 165 samples (63.0%), the
recorded viral load was higher on the cobas 6800 than on the
Abbott m2000. This was particularly seen with high-range viral
load samples, which were significantly higher on the cobas
6800 (high range: Abbott m2000 mean 4.55 × 106 IU/mL,
cobas 6800 mean 5.26 × 106 IU/mL, p = 0.0001; Figure 1).
However, there was no significant difference in mean viral
loads between low-range or mid-range samples when tested
RESULTS
A total of 152 pairs of serum samples were reflex tested from
the ARCHITECT serology instrument to the cobas 6800 plat
-
form (where a pair constituted two HCV antibody–positive
samples: one detected and one not detected by HCV viral
load testing). Of these, 5 previously not-detected samples
(Abbott m2000) had detectable viral loads on the cobas 6800
(Table 1; contamination event due to carry over). Four of the
five samples had detectable viral loads below the cobas 6800
limit of quantification (LOQ; titre minimum <15 IU/mL);
only 1 of the entire 152 samples (0.66%) that were initially
classified as not detected had a viral load above the LOQ
after reflex testing. In total, 147 of 152 pairs (96.7%) had no
detectable carry over contamination.
To ensure that the sensitivity of viral load testing was
maintained during upstream serology testing, viral loads
Table 1: Abbott m2000 HCV RNA viral titres compared with cobas® 6800 viral titres after HCV antibody testing on the Abbott
ARCHITECT to determine reflex testing contamination rates
Pairs*
m2000 initial
viral load†
cobas 6800
viral load‡Result Clinical interpretation
1
i 6.05 × 1061.02 × 107Carry over due to serology testing Not confirmed as a new HCV infection
ii Not detected 2.07 × 101
2
i 3.06 × 1063.74 × 106Possible carry over <15 IU/mL Not confirmed as a new HCV infection
ii Not detected < Titre min§
3
i 4.70 × 1063.55 × 106Possible carry over <15 IU/mL Not confirmed as a new HCV infection
ii Not detected < Titre min§
4
i 1.87 × 1062.32 × 106Possible carry over <15 IU/mL Not confirmed as a new HCV infection
ii Not detected < Titre min§
5
i 4.66 × 1062.72 × 106Possible carry over <15 IU/mL Not confirmed as a new HCV infection
ii Not detected < Titre min§
6–152, mean (SE),
p < 0.0005¶, **
4.37 × 106
(3.49 × 105)
5.14 × 106
(3.80 × 105)
No carry over detected Results consistent with m2000
* Pairs 1–5 are those with observed carry over and pairs 6–152 are those with no carry over detected
† Viral loads are depicted for the output from the i) positive and ii) negative samples initially run on the Abbott m2000
‡ Viral loads are depicted for the output from the cobas 6800 after being reflexed from the Abbott ARCHITECT
§ 15 IU/mL)
¶ Mean viral loads for positive samples without detectable carry over from reflex testing and standard error
** p-value compares mean viral loads between the Abbott m2000 and cobas 6800 (statistical analysis performed using a paired t-test.)
p < 0.05 is significant
HCV = Hepatitis C virus; Titre min = Titre minimum
Official Journal of the Association of Medical Microbiology and Infectious Disease Canada 7.2, 2022100
Low contamination and efficiency of HCV reflex testing
were RNA negative, 536 (36.4%) were RNA positive (viral
load >1,000 IU/ml), and 46 (3.13%) were indeterminate (viral
load ≤1,000 IU/mL). Of the 46 indeterminate specimens,
3 were patient duplicates and were removed from further
analysis, leaving 43 unique indeterminate patients with a mean
recorded viral load of 351.1 IU/mL (SD 43.7 IU/mL, range
26–973 IU/mL; Figure 2B). For patients with an indeterminate
result, 30 of 43 (69.8%) submitted a dedicated second blood
on the cobas 6800 compared with the Abbott m2000 (low-
range: Abbott m2000 mean 2.36 × 102 IU/mL, cobas 6800
mean 2.94 × 102 IU/mL, p = 0.27; mid-range: Abbott m2000
mean 5.06 × 105 IU/mL, cobas 6800 mean 5.46 × 105 IU/mL,
p = 0.07; Figure 1).
A total of 288 samples (141 low-, mid-, and high-range
RNA-positive and 147 RNA-negative samples) were used to
assess test characteristics of the cobas 6800 when used in a
reflex testing workflow (Table 2). When comparing results,
287 of 288 samples were concordant between the Abbott
m2000 and the cobas 6800 (agreement 99.7%; 95% CI 98.1%
to 100%). The one discordant sample had a viral load of 61
IU/mL on the Abbott m2000 but was not detected with the
cobas 6800 (sensitivity 99.3%, 95% CI 95.1% to 99.9%). The
negative predictive value was 99.3% (95% CI 95.4% to 99.9%).
All HCV RNA–negative samples were concordant between
the two assays (specificity 100%, 95% CI 97.5% to 100.0%)
with a positive predictive value of 100% (95% CI 97.5% to
100.0%). To validate the laboratory workflow in our study,
we measured the repeatability (intra-assay) and reproduc-
ibility (inter-assay) of the reflex testing method across two
different testing sites (Table 3). The %CV for repeatability and
reproducibility was below the accepted coefficient of varia-
tion threshold at 4.80% and 0.57%, respectively, validating
our reflex testing workflow for use in the clinical laboratory.
To evaluate the performance of HCV reflex testing in our
patient population, testing data were analyzed 6 months after
implementation of the reflex testing method in ProvLab. In
total, 105,845 specimens were tested for HCV antibodies
(anti-HCV); 1,478 specimens screened positive, and 1,472 of
those (99.6%) were of sufficient volume for molecular reflex
testing, corresponding to 1,414 unique patients presumed to
be first-time HCV positives on the basis of CCL look-back
(Figure 2A). Of the 1,472 specimens reflex tested, 890 (60.5%)
Figure 1: Comparison of low-, mid-, and high-range HCV viral load outputs from paired samples on the cobas® 6800 and Abbott
m2000 systems*
*p < 0.05 is significant
HCV = Hepatitis C virus; X
= mean
Table 2: Test characteristics of the cobas® 6800 system when
used within the HCV reflex testing workflow
Outcome or characteristic Result
Outcome, no.*
True positive 140
False negative 1†
False positive 0
True negative 147
Test characteristic, % (95% CI)‡
Sensitivity 99.3 (95.1 to 99.9)
Specificity 100 (97.5 to 100.0)
PPV 100 (97.5 to 100.0)
NPV 99.3 (95.4 to 99.9)
Agreement 99.7 (98.1 to 100)
*Results represent the number of samples from the cobas 6800
assay after reflex testing, compared with the Abbott m2000 gold
standard reference method, and categorized by outcome
†Abbott m2000 = 61 IU/mL; cobas 6800 = not detected
‡95% CIs are reported with upper and lower interval values
HCV = Hepatitis C virus; PPV = Positive predictive value;
NPV = Negative predictive value
7.2, 2022 Journal officiel de l’Association pour la microbiologie médicale et l’infectiologie Canada 101
LA Thompson, J Fenton, CL Charlton
first-time HCV antibody–positive patients were identified,
with an average turnaround time of 4 days (95% CI 3.8 to
4.1 days) from time of collection to confirmation of HCV
RNA results. Overall, the reflex testing method significantly
lowered mean diagnostic turnaround times for patients com-
pared with the standard two-sample method (p < 0.0001) and
decreased the minimum number of blood samples required
to receive an HCV diagnosis.
DISCUSSION
With the availability of highly effective direct-acting antivi-
rals against HCV (17), the WHO aims to eliminate global
viral hepatitis by 2030, with Canadian efforts for elimination
driven by the Canadian Network on Hepatitis C (18,19).
Improvements in HCV screening and linkage to care are
imperative to achieve this goal, particularly because the virus
often causes asymptomatic infection. The HCV diagnostic
protocol typically requires one blood sample for serology and
a second sample for HCV molecular testing. This two-sample
method, implemented in acute clinical and public health
laboratories around the world (20), is thought to be a major
limitation in diagnosing HCV because of the challenge of
maintaining patient engagement at the early screening stage
(21). Although using a single blood sample for both HCV
antibody and molecular testing would be clinically optimal
for patient engagement, there have previously been concerns
about sample contamination when using one sample across
two different testing platforms (12).
Because serology testing platforms using fixed probes
may result in upstream contamination, it is possible that
instruments with disposable probes may be better equipped
for HCV reflex testing strategies. Indeed, a study investigat-
ing HCV reflex testing contamination from the cobas 8000
analyzer (which uses single-use disposable tips for serology
testing) to the cobas 6800 showed no contamination in 120
samples that were reflex tested (22), suggesting that contami-
nation risk is lower on serology testing instruments without
fixed probes. When comparing contamination rates using
the same fixed probe serology platform from our study, a
sample and received follow-up HCV quantitative RNA test-
ing. Of those, 26 were HCV RNA positive (mean viral load
from the cobas 6800 400.9 IU/mL, SD 60.9 IU/mL, range
30–973 IU/mL) and 4 were HCV RNA negative (mean viral
load from the cobas 6800 79.3 IU/mL, SD 30.9 IU/mL, range
26–152 IU/mL). It is possible that the 4 patients with negative
follow-up testing could have initially screened indeterminate
because of contamination from upstream serology testing or
could represent a patient clearing the infection (low-range
viral load initially, which had cleared by the time the second
sample was collected). However, even if the results for all 4
of these indeterminate patients were caused by direct con-
tamination events, the reflex testing rate of contamination
from our clinical data would still be low (0.45%; 4 of 894 true
negatives). Accounting for indeterminate patients, only 3.04%
of all HCV antibody–positive patients (43 of 1,414; Figures
2A and 2B) whose samples were reflex tested were unable
to receive a positive or negative test result, indicating that
almost 97% of patients in our reflex testing population were
able to receive a conclusive HCV test result from submission
of a single blood sample.
Finally, we assessed the efficiency of HCV reflex testing for
patients by evaluating mean diagnostic turnaround times over
6-month periods before and after implementing reflex testing
in the public health laboratory. Mean turnaround times were
only analyzed for first-time HCV antibody–positive patients,
which we defined as not previously being positive in the
public health system on the basis of a manual look-back of
PHN, name, and date of birth. This was because patients with
a history of antibody positivity may only receive follow-up
testing every 6 months, whereas patients who are first-time
positive are immediately prioritized to stage infection and
be linked to specialty care (16). In the 6 months before reflex
testing implementation (where the two-sample method was
used), 983 first-time HCV antibody–positive patients were
identified, with an average turnaround time of 39 days (95%
CI 34.8 to 42.3 days) between specimen collection for HCV
antibody testing and confirmation of HCV RNA results (Table
4). In the 6 months after reflex testing implementation, 1,209
Table 3: Intra-assay repeatability and inter-assay reproducibility analyses of the reflex testing workflow in Alberta, Canada
Assay Sample size, nMethod
Viral load (Log10),
mean (SD [σ]) %CV
Intra-assay (repeatability) 20 Run in triplicate on 3 days and in single on
7 additional days
4.59 (0.22) 4.80
Inter-assay (reproducibility) 20 Run in triplicate on 3 days and in single on
7 additional days, across two sites
4.44 (0.014) 0.57
%CV = Percent coefficient of variation
Official Journal of the Association of Medical Microbiology and Infectious Disease Canada 7.2, 2022102
Low contamination and efficiency of HCV reflex testing
A
Specimens tested for anti-HCV
(n=105,845)
Anti-HCV Negative (n=104,367) Anti-HCV Positive (n=1,478)
RNA positive:
> 1000 IU/mL (n=536)
RNA indeterminate:
≤1000 IU/mL (n=46)
RNA negative:
0 IU/mL (n=890)
Reflex tested on cobas®6800
(n=1,472)
Insufficient sample volume for
reflex testing (n=6)
1,414
Figure 2: Six-month analysis of clinical samples reflex tested for HCV in public health laboratories across Alberta: stratification of
(A) HCV antibody specimen outcomes and (B) indeterminate specimens*
*Duplicate patient specimens: recorded patients with more than one sample tested for HCV in the public health system, corresponding to
different specimen collection dates
HCV = Hepatitis C virus; Anti-HCV = HCV antibodies; X
= Mean viral load from testing on the cobas® 6800
B
RNA indeterminate specimens
(n=46)
43
NO
(n=13)
YES
(n=30)
POSITIVE (n=26)
X=400.9 IU/mL ±60.9 IU/mL
Range: (30-973 IU/mL)
NEGATIVE (n=4)
X=79.3 IU/mL±30.9 IU/mL
Range: (26-152 IU/mL)
Duplicate patient specimens
(n=3)
Received follow-up HCV RNA testing?
X=351 IU/mL ±43.7 IU/mL
Range: (26-973 IU/mL)
Table 4: Comparison of mean diagnostic turnaround times for first-time HCV positive patients 6 months before and after
implementing reflex testing in public health laboratories across Alberta, Canada
Period HCV antibody–positive patients Turnaround time, days mean (SE),* 95% CI†, ‡
Before reflex testing implementation (2-sample
method)
983 39 (1.911), 34.8 to 42.3
After reflex testing implementation
(single-sample method)
1,209 4 (0.067), 3.8 to 4.1
*Turnaround time represents the period from first specimen collection date to HCV RNA test validation date
† 95% confidence intervals are reported with upper and lower interval values
‡ p < 0.0001 (statistical analysis performed using a two-sample t-test with unequal variances; p < 0.05 is significant)
HCV = Hepatitis C virus
7.2, 2022 Journal officiel de l’Association pour la microbiologie médicale et l’infectiologie Canada 103
LA Thompson, J Fenton, CL Charlton
testing were unable to get a diagnosis with a single blood
sample, we were able to show that approximately 97% of
patients were able to achieve a conclusive test result from
the single-sample reflex testing algorithm.
From a public health perspective, two targets of imple-
menting new HCV testing programs are (1) methods that are
efficient on a broad scale and (2) improved patient care and
outcomes (24). In our study, we showed that reflex testing
significantly improved mean diagnostic turnaround times
for first-time HCV antibody–positive patients in Alberta
(from 39 d with the two-sample method to 4 d with reflex
testing), demonstrating a direct improvement in HCV testing
efficiency and further emphasizing the benefits of using the
reflex testing algorithm in public health laboratories. A 2016
review from Cadieux et al (25) indicated that the majority
of all provinces and territories in Canada did not have any
form of reflex testing implemented. A more recent review
on HCV elimination from Action Hepatitis Canada (26)
showed an increase in the number of provinces and territories
with HCV reflex testing, although only half of all provinces
and territories currently have an HCV reflex algorithm in
place. The public health laboratory at the British Columbia
Centre for Disease Control (BCCDC) was among the first to
implement reflex testing in its population. It showed that in
the 28 years before implementation of reflex testing, 17% of
HCV antibody–positive patients in British Columbia never
received follow-up molecular testing; however, there are not
yet published data on whether reflex testing has improved
these numbers (27). In our study, we showed that reflex test-
ing improved molecular testing for HCV antibody–positive
patients in Alberta (70% receiving molecular confirmation
before reflex testing implementation (11) to almost 97% after
implementation). On the basis of these results, it is likely that
other provinces would also see an improvement in molecular
testing rates after implementation of reflex testing strategies.
Because HCV reflex testing is only used for patients
without a history of previous HCV positivity, it is important
to consider methods that would be effective at identifying
previous positives before undertaking testing. The labora-
tory in Alberta currently uses a CCL look-back method for
previous positivity that relies on in-province PHNs. However,
we found an additional 205 previously positive patients over
the 6-month period after implementation of reflex testing
using a manual look-back method in the LIS compared with
the CCL (1,209 patients versus 1,414 patients, respectively;
Table 4, Figure 2). This was because our manual look-back
method incorporated PHNs, regardless of province origin,
and a combination of name and date of birth could be used
to trace patients without recorded PHNs. Our data therefore
suggest that laboratory resources in Alberta are still being
overused for unnecessary antibody testing and that laboratories
Swedish team previously reported a contamination rate of
4% after HCV antibody testing on the Abbott ARCHITECT
followed by HCV RNA testing on the cobas AmpliPrep/
cobas Taq-Man48 Analyzer (23). Likewise, our HCV reflex
testing method (Abbott ARCHITECT to the cobas 6800)
showed a similar contamination rate (3.29%), suggesting
that contamination rates remain relatively low when using
serology testing platforms with fixed probes.
When samples were tested with the cobas 6800, the mean
recorded viral loads were overall significantly higher than
those tested on the Abbott m2000 (initially used for viral
load detection as a dedicated molecular sample), but this
was only significant for the samples with a high-range vi-
ral load. Regardless, none of our samples had a significant
reduction in measured viral load. This is in contrast to the
Swedish study, where 32% of HCV RNA–positive samples
were shown to have a one-third reduction in viral load after
reflex testing compared with original HCV viral load titres
(23), suggesting that the cobas 6800 platform may be better
adapted for molecular testing than the cobas AmpliPrep/
cobas Taq-Man48 Analyzer. In addition, we used samples that
represented all available HCV genotypes and ranges of RNA
viral loads, and we demonstrated a high sensitivity (99.3%)
and specificity (100.0%) of the cobas 6800 assay using reflex
testing, with a high level of agreement (99.7%) between the
cobas 6800 assay and the standard Abbott m2000 assay. This
surpassed the sensitivity and specificity thresholds (≥95%)
needed for assays to be implemented in clinical laboratories
(14), and as a result, we proceeded to implement this method
in public health laboratories across Alberta.
To ensure that reflex testing would be clinically successful,
we set the cut-off for an HCV-positive sample at 1,000 IU/mL,
2 standard deviations above our highest-seen contamination
value (20.7 IU/mL). Because our indeterminate range was
quite broad (1–1,000 IU/mL), we examined the number of
patients falling in this range who would subsequently have
to submit a second blood sample for follow-up; after imple-
menting reflex testing for 6 months, only 3.04% (43/1,414)
of all presumed first-time HCV antibody–positive patients
whose samples were reflex tested had indeterminate viral
loads. Because our previous data showed that only 70% of
patients screening positive for HCV antibodies had docu-
mented molecular follow-up testing (11), one concern was
that indeterminate patients from our study may likewise not
submit a second sample for follow-up testing. However, we
saw that the majority of our indeterminate patients (30 of
43; 69.8%) had a record of follow-up testing, and of those,
85% were HCV RNA positive, suggesting that our cut-off
was appropriate for our patient population, and follow-up
submission rates remained similar with the new process.
Although patients with indeterminate results from reflex
Official Journal of the Association of Medical Microbiology and Infectious Disease Canada 7.2, 2022104
Low contamination and efficiency of HCV reflex testing
where genotypes 1–4 are prevalent. This method could be
particularly beneficial in places where HCV prevalence is
high, such as Western Africa, Northeast Africa, Russia, and
Western Asia (32). Last, although our study showed a signifi-
cant improvement in turnaround times for diagnostic testing,
it is unclear whether this directly improves patient outcomes
downstream in the cascade of care, such as being referred
to specialists, being prescribed treatment, and achieving a
sustained viral response. Our study does, however, support
improved patient care at the screening and diagnostic stages,
because patients would require fewer health care visits to
submit blood samples and would receive test results faster
with HCV reflex testing.
Overall, we demonstrated that a single blood draw can be
used to diagnose HCV infections accurately and efficiently.
Our data support low contamination rates while maintaining
high sensitivity and specificity of the molecular assay. Most
notably, implementation of HCV reflex testing in the clini-
cal laboratory was shown to improve diagnostic turnaround
times for patients and resulted in fewer than 3.05% of patients
requiring a second blood sample for confirmation. Broad
implementation of single-sample reflex testing algorithms
in other public health and clinical laboratories could help
improve the screening and diagnosis of HCV within popula-
tions. To reach the goal of global hepatitis elimination in the
next decade, reflex testing should be considered an important
method for identifying HCV infections and improving the
efficiency of patient diagnosis.
ACKNOWLEDGEMENTS: The authors acknowledge the
support of the Alberta Precision Laboratories (ProvLab) staff
at ProvLab Northern Alberta and ProvLab Southern Alberta
for collecting, collating, and analyzing the clinical specimens
as we transitioned to HCV reflex testing.
CONTRIBUTORS: Conceptualization, LA Thompson,
J Fenton, CL Charlton; Methodology, LA Thompson, J Fen-
ton, CL Charlton; Validation, LA Thompson, J Fenton, CL
Charlton; Formal Analysis, LA Thompson; Investigation,
J Fenton, CL Charlton; Resources, CL Charlton; Supervision,
CL Charlton; Data Curation, LA Thompson, J Fenton, CL
Charlton; Writing – Original Draft, LA Thompson; Writing –
Review & Editing, LA Thompson, J Fenton, CL Charlton;
Visualization, LA Thompson, CL Charlton; Project Admin-
istration, CL Charlton; Funding Acquisition, CL Charlton.
ETHICS APPROVAL: This research was approved by the
University of Alberta Research Ethics Board (Pro00092635).
INFORMED CONSENT: N/A
should consider expanding their criteria for performing
look-backs of previous positives before undertaking reflex
testing strategies.
Although using a single sample across two testing plat-
forms has previously been a concern for contamination,
preventive steps can mitigate contamination risk. Because
fixed probes used on the Abbott ARCHITECT instrument
can be a source of HCV RNA carry over, laboratory staff
should try to minimize splashing when loading the blood
sample into the instrument carrier. In addition, laboratory
workflow can lead to sample contamination, and it is there-
fore imperative that molecular-level precautions for serology
samples be followed for specimen preparation and aliquot-
ing. Analysis of our laboratory workflow showed a %CV of
4.80% repeatability and 0.57% reproducibility, well below the
accepted threshold of variability, suggesting that a standard-
ized, validated workflow can be established to mitigate the
risk of contamination and sample variation.
One limitation of our study was the introduction of
a single freeze–thaw cycle between using the sample for
initial molecular testing and using it for our experiments,
which may have caused analyte degradation to occur and
could be a source of discordant results. Previous studies
have reported upward of 15% HCV viral titre loss after five
freeze–thaw cycles (28). Although our reflex viral titres for
samples with viral loads of more than 1,000,000 IU/mL were
higher than original titres from the Abbott m2000, sample
degradation may still be a factor in samples with lower viral
load (<1,000,000 IU/mL). However, we saw no significant
difference in low-range or mid-range titres from our reflex
testing results compared with original titres measured on the
Abbott m2000, suggesting that, overall, degradation did not
significantly affect the viral titres in our study.
Another limitation of our study was the range of HCV
genotypes that were available for use. We aimed to analyze
the reflex testing algorithm on all HCV genotypes; however,
only specimens with genotypes 1a, 1b, 2, 3, and 4; mixed type;
and not typed were available for testing. Genotypes 5 and 6,
which are most prevalent in Southeast Asia and South Africa
(29) and make up less than 5% of global HCV (30), were not
present among specimens collected for the study. Further
research encompassing reflex testing on HCV samples with
genotypes 5 and 6 should be done before implementation in
regions where those genotypes are most abundant. However,
99% of all samples tested at the public health laboratory in
2019 were genotypes 1–4 or mixed type, suggesting that
our sample selection was representative of the genotype
distribution in Alberta (data not shown). These genotypes
are exceedingly present in North America, South America,
Australia, and most of Africa and Europe (31); thus, our study
supports the use of the cobas 6800 platform for reflex testing
7.2, 2022 Journal officiel de l’Association pour la microbiologie médicale et l’infectiologie Canada 105
LA Thompson, J Fenton, CL Charlton
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REGISTRY AND THE REGISTRATION NO. OF THE
STUDY/TRIAL: N/A
FUNDING: Funding for this study was provided in part by the
Canadian Institutes of Health Research Frederick Banting and
Charles Best Canada Graduate Scholarship (LA Thompson),
University of Alberta Doctoral Recruitment Scholarship (LA
Thompson), and M.S.I. Foundation (CL Charlton).
DISCLOSURES: The authors have nothing to disclose.
PEER REVIEW: This manuscript has been peer reviewed.
ANIMAL STUDIES: N/A
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