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Journal of Clinical Virology 96 (2017) 60–63

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Journal of Clinical Virology


journal homepage: www.elsevier.com/locate/jcv

Full length article

Evaluation of clinical sensitivity and specificity of hepatitis B virus (HBV), T


hepatitis C virus, and human immunodeficiency Virus-1 by cobas MPX:
Detection of occult HBV infection in an HBV-endemic area

Jihye Ha, Younhee Park, Hyon-Suk Kim
Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea

A R T I C L E I N F O A B S T R A C T

Keywords: Background: Transfusion-transmitted infectious diseases remain a major concern for blood safety, particularly
Cobas MPX with hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV). Nucleic acid
Blood donor screening testing (NAT) in donor screening shortens the serologically negative window period and reduces virus trans-
Occult HBV infection mission. The cobas MPX (Roche Molecular Systems, Inc., Branchburg, New Jersey) is a recently developed
Multiplex PCR
multiplex qualitative PCR system that enables the simultaneous detection of HBV, HCV, and HIV with improved
sensitivity and throughput using cobas 6800 and 8800 instruments.
Objectives: The aim of this study was to conduct an evaluation of the clinical sensitivity and specificity of cobas
MPX detection of HBV, HCV, and HIV in clinical specimens.
Study design: Among samples referred for HBV, HCV, and HIV-1 quantification at Severance Hospital, Yonsei
University College of Medicine, positive samples were selected to evaluate sensitivity. A total of 843 samples was
tested using both cobas MPX and COBAS AmpliPrep/COBAS TaqMan Tests for HBV, HCV, and HIV-1 using the
cobas 8800 system and a COBAS TaqMan 96 analyzer, respectively. Samples that showed discrepancies were
confirmed by nested PCR.
Conclusions: The cobas MPX achieved excellent sensitivity and specificity for the detection of HBV, HCV, and
HIV-1 in clinical samples. We found that the lower limit of detection (LOD) of blood screening by NAT actually
improves clinical sensitivity, and occult HBV infection prevalence among healthy employees of the hospital was
rather high.

1. Background multi-dye probe that eliminated additional discriminatory testing [5].


Recently, Roche released the new cobas MPX, a multiplex qualitative
Transfusion-transmitted infectious diseases remain a major concern PCR system detecting HBV, HCV, and HIV (HIV-1 Group M, HIV-1
for blood safety, particularly with hepatitis B virus (HBV), hepatitis C Group O, and HIV-2), with improved throughput using cobas 6800 and
virus (HCV), and human immunodeficiency virus (HIV). To reduce the 8800 systems and a low limit of detection (LOD) [6] (Table 1).
risk of transfusion-transmitted viral infection, serologic screening was
commonly performed in the past, although infectious blood units from 2. Objective
donors in the window period could still be transfused [1]. Nucleic acid
testing (NAT) in donor screening has shortened the serologically ne- HBV, in particular, is widespread on the Asian continent, but until
gative window period and reduced virus transmission [2,3]. Blood now, the cobas MPX test has not been evaluated for clinical sensitivity
screening laboratories handle large quantities of blood products that and specificity in HBV-endemic areas, which could have a great clinical
require timely release, demanding automation and high-throughput. impact. We therefore aimed to evaluate the clinical sensitivity and
Roche Molecular Systems, Inc. (RMS) (Branchburg, New Jersey) de- specificity of the cobas MPX test using clinical samples derived from
veloped a fully automated multiplex NAT system for HBV, HCV, and Korean patients and healthy volunteers.
HIV detection, the cobas TaqScreen MPX test, which used a single-dye
probe for the target viruses necessitating additional discriminatory
testing [4]. The next version, the cobas TaqScreen MPX v2.0 test, used a


Corresponding author.
E-mail addresses: KIMHS54@yuhs.ac, kimhs54@gmail.com (H.-S. Kim).

http://dx.doi.org/10.1016/j.jcv.2017.09.010
Received 6 March 2017; Received in revised form 13 August 2017; Accepted 18 September 2017
1386-6532/ © 2017 Elsevier B.V. All rights reserved.
J. Ha et al. Journal of Clinical Virology 96 (2017) 60–63

Table 1 (Plasmid #65459); this was a gift from Wang-Shick Ryu and had a
Claimed limit of detection by Probit analysis at a 95% hit rate for HIV, HCV, and HBV for known concentration [7]. The LOD of the assay was determined by
COBAS AmpliPrep/COBAS TaqMan Test, cobas MPX, and previous versions.
serially diluting the standard plasmid. DNA for the nested PCR was re-
Limit of detection, IU/mL extracted using a QIAamp Viral-RNA Mini Kit (Qiagen Inc., Valencia,
CA) according to the manufacturer's instructions.
HIV-1 HCV HBV

a 3.3. Serological assays for HBV infection


COBAS AmpliPrep/COBAS TaqMan v2.0 [15–17] 27.5 11 9
cobas Taqscreen MPX Test [4] 49 11 3.8
cobas Taqscreen MPX Test, v2.0 [5] 50.3 6.8 2.3 Serum samples stored at −70 °C after performing the molecular
cobas MPX [6] 25.7 7 1.4 assays were used for serologic evaluation. HBsAg, anti-HBs, and anti-
a
HBc were detected using ARCHITECT HBsAg Qualitative II,
Numbers in parentheses indicate reference numbers.
ARCHITECT anti-HBs, and ARCHITECT anti-HBc II assays (Abbott
Laboratories, IL, USA), respectively. Serological assays were performed
3. Study design following the manufacturer’s recommendations without any modifica-
tions.
3.1. Subjects and samples
3.4. Statistical analyses
Among samples referred for HBV, HCV, and HIV-1 quantification at
Severance Hospital, Yonsei University College of Medicine, positive Analysis of the data was performed with analyze-it, version 3.80
samples were selected to evaluate sensitivity. In total, 117, 111, and (Analyze-it Software Ltd., Leeds, UK). A P value of less than 0.05 was
105 positive samples by COBAS AmpliPrep/COBAS TaqMan tests for considered significant.
HBV, HCV, and HIV-1 [14–16] using a COBAS TaqMan 96 analyzer
were collected to evaluate HBV, HCV, and HIV-1 sensitivity, respec- 4. Results
tively. A further 510 samples collected during annual check-ups of
hospital employees with no clinical history of HBV, HCV, or HIV-1 in- 4.1. Clinical sensitivity
fection were used to evaluate specificity. The median age of 510 sub-
jects enrolled was 40 years (range 23–67). A total of 121 subjects were All positive samples by COBAS AmpliPrep/COBAS TaqMan showed
male and 389 were female. This study was performed with the per- positive results by cobas MPX. The sensitivity of cobas MPX was 100%
mission of our hospital Institutional Review Board. The enrolled sub- (333/333, 95% CI 98.90-100.00).
jects were anonymized for the study.
4.2. Clinical specificity
3.2. Molecular assays
All HIV-1- and HCV-negative samples by COBAS AmpliPrep/COBAS
In total, 843 samples were tested using both cobas MPX and COBAS TaqMan were also shown to be negative by cobas MPX (510/510).
AmpliPrep/COBAS TaqMan tests for HBV, HCV, and HIV-1 [14–16] Therefore, specificities for HIV-1 and HCV were 100% (510/510, 95%
using the cobas 8800 system and a COBAS TaqMan 96 analyzer, re- CI 99.28-100.00) (Table 2). However, for HBV, 17 samples from
spectively, following the manufacturer’s instructions. The limits of de- healthcare workers that were HBV negative by COBAS AmpliPrep/
tection of reagents used in this study are summarized in Table 1. COBAS TaqMan showed positive results by cobas MPX. These dis-
To calculate clinical sensitivity and specificity, concordant results crepant samples were tested by nested PCR and direct sequencing,
from both tests were assumed to be true results. Samples that showed which showed 15 of the 17 samples as positive. Assuming that the two
discrepant HBV results between cobas MPX and COBAS AmpliPrep/ samples that were positive by cobas MPX and negative by nested PCR
COBAS TaqMan tests were confirmed by nested PCR (primers for first with direct sequencing were false positives, the specificity was 99.59%
round of PCR: BRB1, GATCGGATCCAGACTCGTGGACTTCTC; BRB2, (508/510, 95% CI 98.54-99.95) (Table 2). The LOD of nested PCR we
GATCTCTAGACCAAAAGACCCACAATT, primers for second round of used for confirmation was within 100–101 copies/mL
PCR: BRB1;BRB3, GATCTCTAGATGACATACTTTCCAATCAAT). Direct
sequencing of PCR products (primer: BRB1) of positive samples was 4.3. Concordance
performed using a 3500xL Genetic Analyzer (Life Technologies,
Carlsbad, CA) to confirm the pathogen. To estimate the LOD of the The overall concordance rate and kappa coefficient between COBAS
nested PCR used in this study, an HBV 1.3-mer WT replicon was used AmpliPrep/COBAS TaqMan and MPX were 98.0% (826/843, 95% CI

Table 2
Clinical sensitivity and specificity of cobas MPX.

Resultsa Total tested MPX results Clinical Sensitivity (%) 95% CI Clinical Specificity (%) 95% CI

HIV-1 Positive 117 Positive: 117 100 96.9–100.0 100 99.5–100.0


Negativeb 726 Negative: 726

HCV Positive 111 Positive: 111 100 96.7–100.0 100 99.5–100.0


Negativeb 732 Negative: 732

HBV Positivec 122 Positive: 122 100 97.2–100.0 99.7 99.0–99.9


Negativec 721 Positive: 2, Negative: 721

CI: confidence interval.


a
Concordant results by COBAS AmpliPrep/COBAS TaqMan Test v2.0, cobas MPX, and the nested PCR and sequencing results of discordant samples were assumed to be true.
b
Negative samples are consist of 510 of hospital employees with no history of HBV, HCV or HIV-1 and predefined positive samples for other targets.
c
15 samples of hospital employees showed positive in MPX and confirmed by nested PCR and sequencing was included in positive samples.

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J. Ha et al. Journal of Clinical Virology 96 (2017) 60–63

Table 3
Serologic status of the subjects with suspected occult HBV infection.

Case No. Sex Age HBsAg Anti-HBc Anti-HBs Cobas MPX Nested PCR and Sequencing

1 M 47 Negative Positive Positive HBV HBV


2 M 52 Negative Positive Positive HBV HBV
3 F 38 Negative Positive Positive HBV HBV
4 F 35 Negative Positive Positive HBV HBV
5 F 51 Negative Positive Positive HBV HBV
6 M 63 Negative Positive Negative HBV HBV
7 F 45 Negative Negative Positive HBV Negative
8 F 32 Negative Negative Positive HBV Negative
9 F 41 Negative Negative Positive HBV HBV
10 F 46 Negative Negative Positive HBV HBV
11 F 33 Negative Negative Positive HBV HBV
12 F 36 Negative Negative Positive HBV HBV
13 M 67 Negative Negative Negative HBV HBV
14 F 30 Negative Negative Negative HBV HBV
15 F 34 Negative Negative Negative HBV HBV
16 F 43 Negative Negative Negative HBV HBV
17 F 51 Negative Negative Negative HBV HBV

96.7–98.8) and 0.96 (95% CI, 0.94–0.98, P < 0.0001), respectively. absence of liver disease in Korea [9–12] (Table 4). Because anti-HBc has
HCV and HIV-1 testing showed a concordance rate of 100% (843/843, been considered as a marker of previous HBV exposure, many studies
95% CI 99.6–100.00) with kappa coefficient of 1.0. HBV testing showed performed HBV DNA tests on anti-HBc positive subjects to investigate
a concordance rate of 98.0% (826/843, 95% CI 96.8–98.8) with kappa the prevalence of OBI. However, Song et al. reported that anti-HBc was
coefficient of 0.91 (95% CI, 0.87–0.95, P < 0.0001). positive in only 27% of OBI cases in Korean adult subjects [11]. In this
study, anti-HBc was detected in almost half of the samples (6/15,
4.4. Serological assays for occult HBV infection samples 40.0%). Therefore, we reviewed literature that investigated OBI pre-
valence regardless of anti-HBc. Overall, 17–67% of OBI cases from the
Among the 510 samples from healthy employees, anti-HBs were literature were anti-HBc positive, not counting the extremes. Interest-
detected in 307 (60.2%). Among 17 HBV positive samples by the cobas ingly, the prevalence of OBI varied considerably from study to study.
MPX, HBsAg was negative for all samples. Anti-HBc and anti-HBs were Kim et al. detected 31 (16%) OBI cases from 195 outpatients of a gas-
detected in 6 (35.3%) and 11 (64.7%) samples, respectively (Table 3). trointestinal clinic with normal ALT levels. However, Song et al. re-
ported only 0.7% (7/1047) OBI prevalence for medical check-up sam-
5. Discussion ples. Various OBI prevalences were reported in Korea (Table 4). These
considerable differences in OBI prevalences could be explained mainly
This study found that the lower LOD of blood screening by NAT by the characteristics of the subjects. The different OBI prevalences of
actually improves clinical sensitivity. Molecular study of healthy em- same population in a study were resulted from the analytical sensitivity
ployee samples using the COBAS AmpliPrep/COBAS TaqMan test de- of HBV DNA detection methods [10,12]. Despite similar analytical
tected no positive samples. On the contrary, using cobas MPX, 17 po- sensitivity, OBI prevalence in this study was four-times higher than that
sitive samples were detected. This implies that occult HBV infection was reported by Song et al. (2.9 vs. 0.7%) [11]. This difference is
(OBI) could be better screened using cobas MPX by lowering the LOD in consistent with other studies, which concluded that the risk of con-
endemic areas. Among 17 positive samples by cobas MPX, 15 were tracting HBV by health care workers is four-times greater than that of
confirmed to be HBV positive by nested PCR and subsequent sequen- the general adult population [13,14].
cing. The remaining two samples showed no bands in nested PCR In conclusion, the cobas MPX test achieved excellent sensitivity and
analysis. However, although we used nested PCR as a confirmatory test, specificity for the detection of HBV, HCV, and HIV-1. We found that the
there is a possibility that the cobas MPX test has a lower LOD than lower LOD of NAT blood screening actually improves clinical sensi-
nested PCR. In our study, LOD of the nested PCR performed in the study tivity. Using the cobas MPX test, OBI was detected in 2.9% (15/510) of
was between 100-101 copies/mL and the claimed LOD of cobas MPX healthy hospital employees. The excellent clinical sensitivity and spe-
was 1.4 IU/mL (8.1 copies/mL under conversion factor of 5.82) [6]. cificity was accompanied by improved efficiency and reduction of the
Considering that the true results of the two discrepant samples showing costs and turn-around time of NAT for donor screening.
discrepancy remain unclear, the cobas MPX showed excellent clinical
sensitivity and specificity.
Whereas OBI infectivity depends on recipient immune status, blood Competing interests
taken from donors with OBI was shown to be potentially infectious, and
the risk is increased when large volumes of plasma are transfused [8]. No potential conflicts of interest relevant to this article were re-
In HBV endemic areas, the residual risk of transfusion-transmitted HBV ported.
infection originates from HBsAg-negative donors with OBI [1]. In this
study, the cobas MPX test detected HBV in 17 samples from healthy
workers, of which 15 samples were also confirmed by nested PCR and Funding
subsequent sequencing. When these 15 positive samples were con-
sidered as OBIs, the prevalence of OBI among the tested subjects was This research received no specific grant from any funding agency in
2.9% (15/510). the public, commercial, or not-for-profit sectors.
We reviewed literature that investigated OBI prevalence in the

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J. Ha et al. Journal of Clinical Virology 96 (2017) 60–63

Ethical approval

55% (17/31)
70% (16/23)

65% (11/17)
100% (1/1)
75% (6/8)
86% (6/7)
67% (2/3)
Anti-HBs
This study was approved by Severance Hospital IRB (IRB No. 1-
2015-0047, Protocol No. RSS-MD-15-01).

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52% (16/31)
17% (4/23)

53% (6/17)
100% (1/1)
29% (2/7)
67% (2/3)
0% (0/8)
Anti-HBc

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Outpatient of a GI clinic with normal ALT

Healthy employees of our hospital


Previous reports of prevalence of occult HBV infection without overt liver disease in Korea.

Hemodialysis patients
Hemodialysis patients
Medical check-up
Pregnant women
Pregnant women
Subjects

11.4% (23/202)
OBI prevalence

0.7% (7/1047)

2.9% (15/510)
16% (31/195)

3.2% (3/94)
1.1% (1/94)
4% (8/202)

Yoo, Hwang et al, 2013 [12]


Yoo, Hwang et al, 2013 [12]
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Kwon et al, 2008 [10]


Kwon et al, 2008 [10]
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Present study
Table 4

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