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139
© 2020 Global Journal of Transfusion Medicine AATM | Published by Wolters Kluwer - Medknow
Background: NATSpert ID TripleH Detection test is a qualitative multiplex real
time PCR test for simultaneous screening of hepatitis B virus (HBV), hepatitis
C virus (HCV), and human immunodeciency virus type 1/2 in donated blood.
Objective of the study was to evaluate NATSpert ID TripleH Detection test,
developed indigenously for low resource countries for enhancing blood safety
without compromising on blood safety in terms of sensitivity and specicity.
Methods: The NATSpert ID TripleH Detection test consists of viral nucleic acid
extraction using magnetic beads and multiplex real-time PCR based on hydrolysis
probe technology for amplication and detection of the viral target. Quality
controls are included for validity of the test. The performance of the test was
evaluated for analytical sensitivity and specicity and precision using the World
Health Organization international standards. Results: Analytical sensitivity of
NATSpert ID TripleH Detection test is 2 IU/ml for HBV, 7 IU/ml for HCV, human
immunodeciency virus‑1(HIV‑1) M – 17.5 IU/ml, HIV‑1O – 18.5 copies/ml, and
HIV-2 8.17IU/ml using 1 ml sample input at 95% CI. The NATSpert ID TripleH
Detection test detected all three viruses and their genotypes with high repeatability
and reproducibility in validation studies. Conclusion: The NATSpert ID TripleH
Detection test developed as a cost‑eective solution was demonstrated to have the
capability to identify either individually or simultaneously the presence of viral
targets – HBV, HCV, and HIV in the donated blood. The NATSpert ID Triple H
Detection test serves as a highly sensitive, specic, and accurate test for screening
of donated blood for transfusion-transmitted viral infections.
Blood safety, individual‑donation‑NAT, real‑time polymerase chain
reaction
Evaluation of a NATSpert Test Developed for Low-resource Countries
for Enhancing Blood Safety
Minal Dakhave Bhosale, Shefali Desai
Access this article online
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Website: www.gjtmonline.com
DOI: 10.4103/GJTM.GJTM_17_20
Address for correspondence:
Ms. Minal Dakhave Bhosale,
E‑mail: minal@mylabdiscoverysolutions.com
seropositivity (window period [WP]). To address this, various
types of Nucleic Acid Test (NAT) were developed in the
1990s.[5] NAT in blood donor screening detects not only WP
donors but also those with chronic occult infections which are
negative by routine serological screening. It is based on direct
amplication and detection of viral nucleic acids, rather than
antibody production by the immune system of the infected
person. This allows for earlier detection of infection and
further decreases the possibility of TTI.[6]
Original Article
Blood and its components are transfused to save
innumerable lives, but the quality and safety of the
transfused blood and its products is a major public health
concern due to transfusion transmitted infections (TTIs).
With every unit of blood, there is a 1% chance of
transfusion-associated problems, including TTI.[1,2] Risk of
TTI is high in the patients receiving multiple transfusions
and undergoing invasive procedures with exposure of
circulating system.[3,4] Safety against three TTI, i.e., human
immunodeciency virus‑1 (HIV‑1), hepatitis B virus (HBV),
and hepatitis C virus (HCV) is a worldwide concern, in spite
of development of highly sensitive and specic serological
tests and other surrogate markers because of the time gap
between the time of acquiring infection and development of
R and D Department, Mylab
Discovery Solutions Pvt.
Ltd., Pune, India
This is an open access journal, and arcles are distributed under the terms of the Creave
Commons Aribuon‑NonCommercial‑ShareAlike 4.0 License, which allows others to
remix, tweak, and build upon the work non‑commercially, as long as appropriate credit is
given and the new creaons are licensed under the idencal terms.
For reprints contact: WKHLRPMedknow_reprints@wolterskluwer.com
How to cite this article: Bhosale MD, Desai S. Evaluation of a NATSpert
test developed for low-resource countries for enhancing blood safety.
Glob J Transfus Med 2020;5:139-45.
Submitted: 25-Feb-2020.
Revised: 07-Aug-2020.
Accepted: 14-Oct-2020.
Published: 13-Nov-2020.
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Bhosale and Desai: NAT testing using NATSpert ID tripleH test
140 Global Journal of Transfusion Medicine AATM ¦ Volume 5 ¦ Issue 2 ¦ July-December 2020
140 Global Journal of Transfusion Medicine AATM ¦ Volume 5 ¦ Issue 2 ¦ July-December 2020
Even in India, with its large population of around 1.23 billion
and high prevalence rate of HIV (0.29%), HBV (2%–8%), and
HCV (~2%), blood safety is a big challenge.[7] At present, all
blood banks follow the National Aids Control Organization
guidelines and use the fourth generation, advanced
antigen-antibody combined enzyme-linked immunosorbent
test (ELISA) to screen blood for HIV, HBV, and HCV. NAT
donors’ screening is not mandatory in India as of now, and
a review published in 2017 stated that NAT screening is
carried out in only 2% blood banks and covers only 7% of
all collected blood units in India[5] in spite of the fact that the
seroprevalence of infections in India is much higher than in
the developed world. Major barriers of implementing routine
NAT in India are its high cost and lack of technical expertise
in most of the blood centers.[8] NATSpert ID TripleH test is
our earnest attempt to provide solution to some key issues to
facilitate availability of high-quality NAT test at lower cost
compared to existing commercially available tests.
NAT is a highly sensitive and advanced technique which has
reduced the WP of HBV to 10.34 days, HCV to 1.34 days,
and HIV to 2.93 days,[9] but the implementation of NAT in
low-resource countries remains a challenge. While the World
Health Organization (WHO) recommends diagnostic devices
should be aordable, sensitive, and specic, in resource‑limited
countries, NAT remains highly technically demanding, incurs
high costs, and requires dedicated infrastructure facility,
equipment, consumables, and technical expertise.
NATSpert TripleH Detection test oers a statistically
signicant advantage over serology in ability to detect TTI in
blood donors. The NAT yield of 1:3829 was in line with other
Indian studies.[10]
A sensitive and specic multiplex NATSpert test is described
below for the simultaneous detection and discrimination of
HBV DNA, HCV RNA, and HIV RNA in individual blood
donor samples, blood products, and suspected individuals.
The NATSpert test provides viral nucleic acid extraction
using magnetic bead technology. The extracted viral nucleic
acid is amplied using multiplex real‑time polymerase chain
reaction (PCR) technology for detection and discrimination of
HIV RNA (HIV-1 Groups M, N, and O RNA and HIV-2 RNA),
HCV RNA (Genotype 1–6), and HBV DNA (Genotype A–H)
using real-time PCR system. The test does not discriminate
between HIV-1 Group M, HIV-1 Group O, and HIV-2. The
test also incorporates a heterologous Internal Control (IC)
to be processed during extraction and real-time PCR for
monitoring test performance for each individual sample.
Clinical standards
Sensitivity panels for HBV (WHO International Standard
3rd WHO International Standard for HBV for Nucleic Acid
Amplication Techniques NIBSC code: 10/264), HCV (WHO
International Standard 5th WHO International Standard for
HCV NAT NIBSC code: 14/150), HIV-1 (WHO International
Standard 3rd HIV-1 International Standard NIBSC code:
10/152), HIV-1 O (Internal calibrators), and HIV-2 (WHO
International Standard HIV-2 RNA International Standard
NIBSC Code: 08/150) were prepared by diluting standards
with certied negative plasma.
Nucleic acid isolation from plasma samples
Nucleic acid isolation procedure was developed using
paramagnetic bead technology and was performed on
semi-automated extraction machine.
The lysis buer, wash buer 1 and 2, and elution buer used
in this method are proprietary formulations of Mylab. They
have a unique formulation that increases the catalytic activities
of the enzymes and reduces the enzyme unit requirement,
resulting in reduced cost.
Buer component contains 2065 µl lysis buer
combo (including lysis buer, isopropanol, proteinase K,
and magnetic beads along with IC and carrier RNA), 1200
µl wash buer 1, 1800 µl wash buer two twice, and 90 µl
elution buer,. 1 ml K2EDTA plasma sample was added in
lysis buer combo. General steps include incubation of lysis
at 56°C for 20 min and washing using wash 1 and 2 buers,
followed by elution at 80°C.
Internal control
Internal Control (IC) was added to the lysis buer before
extraction as a control to determine the ecacy of extraction
and PCR amplication and absence of inhibitory compounds.
A noncompetitive and synthetic IC was chosen to avoid
competition with the primer/probe set for nucleic acid
detection for HIV, HBV, and HCV virus and therefore does
not compromise of test sensitivity.
Real‑time polymerase chain reaction preparation
and amplication/detection
The test was based on hydrolysis probe chemistry for the
amplication of conserved regions of HIV, HCV, and HBV
and IC using specic primers/probes. The primer/probe
combinations were indigenously designed (proprietary
formulations, Mylab) to cover all known genotypes and
were tested “in silico” as well. The amplied product was
detected by the generation of uorescent signals from
target‑specic uorescent probes. Four unique uorescent dyes
were used for HIV, HCV, HBV, and IC target, thus allowing
independent identication of all three viruses. All three HIV
targets (HIV‑M/N, HIV‑1O, and HIV‑2) were identied using
the same dye and were not discriminated from each other.
Reverse transcription (RT) and Taq polymerase enzymes in
PCR buer composition have unique formulation to enhance
the catalytic activities, thus allowing reduced use of the
enzymes resulting in reduced cost.
22 µl of extracted viral nucleic acid/positive control was
added to 28 µl of master mix constituting 12.5 µl of RT
and Taq polymerase in PCR buer components, 7.8 µl of
primer probe mix for all viral targets along with IC, and 7.7
µl of nuclease-free water. Testing is carried out by multiplex
real-time RT-PCR using QuantStudio 5 (Applied Biosystem)
real-time PCR machine. The thermal cycling conditions were
50°C, 15 min for reverse transcriptase, and 95°C, 20 s to
inactivate the RT enzyme, followed by 50 cycles of 5 sec at
95°C and 30 sec at 60°C.
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Bhosale and Desai: NAT testing using NATSpert ID tripleH test
141
Global Journal of Transfusion Medicine AATM ¦ Volume 5 ¦ Issue 2 ¦ July-December 2020
This complete protocol was also performed with closed fully
automated Compact system with a sample to result instrument
for NAT testing, using sealed prelled reagent cartridges.
Performance study on clinical samples
Type of the study
This was a prospective cohort study of blood donors at a
Blood Bank in Central India, which is a NABH-accredited
standalone blood bank to determine the NAT yield and also
determine the sensitivity and specicity of the NATSpert
assay.
Ethics
The ethical clearance for the study was taken from the board
of the trust that runs the blood bank, and blood donors were
informed, and consent was taken.
Sample size
All the serologically nonreactive blood donor samples during the
study period of June 2017-March 2019 and 77 randomly selected
and blinded serologically reactive blood donor samples, totaling
to 30,635, were screened on the NATSpert ID TripleH assay.
Statistics
The study was analyzed using Fisher exact test to nd any
statistically signicant dierence in ability to detect TTI using
NAT as compared to EIA.[10]
Data analysis and reporting
After completion of the real time PCR run, Ct values were
analyzed for positive controls and clinical samples using
QuantstudioTM Design and analysis software (V 1.3.1,
Thermo Fisher Scientic, Singapore, 2017), built into the
QuantstudioTM 5 Real time PCR machine (Thermo Fisher
Scientic, Singapore). Results were interpreted as reactive,
non- reactive and valid results.
Test validation
The validation was performed according to the recommended
guidelines for validating molecular tests for Infectious
Diseases and European guidelines.[11-13] The evaluation of
test was done for analytical sensitivity, analytical specicity,
precision, and clinical performance.
Analytical sensitivity
The analytical sensitivity of the NATSpert ID TripleH
Detection test was determined by testing ve independent
serial dilution series of the 3rd HIV-1 WHO International
Standard, WHO International Standard HIV-2 RNA, 5th
WHO International Standard for HCV NAT NIBSC, and
3rd WHO International Standard for HBV for Nucleic Acid
Amplication Techniques and internal calibrators for HIV‑1
O. Dilutions were prepared in HIV/HBV/HCV-negative EDTA
plasma and ranged from 107 to 1 IU/ml. Each dilution series
was tested in triplicate on dierent days with 1 ml plasma
input volume for extraction. Simultaneous target amplication
at low levels of vial loads was assessed using HBV, HCV,
and HIV Multiplex 14/198-XXX NIBSC code: 14/198-XXX.
Further, the conversion factor for IU/copies was estimated by
comparing the known copies/ml dilution series versus known
IU/ml NIBSC standards. Expected versus observed ratio was
calculated for the conversion factor.
Statistical analysis
The probit analysis module of SPSS (Statistics V 19, IBM,
US) was used to calculate the limit of detection (LOD). The
percent coecient of variation (% CV) is dened as “(standard
deviation/mean) × 100” and used for precision analysis.
Analytical specicity
The genotype/subtype inclusivity of all relevant genotypes
was ensured by testing the following international reference
standards: HIV-1 2nd WHO NIBSC Genotype panel #
12/224:041016, 3rd HCV genotype NIBSC WHO Panel (PN
12/172), and 1st HBV WHO NIBSC Genotype panel
PE5086/08 V2.
Cross-reactivity for each viral target was determined against
blood-borne viruses, bacteria, and fungi.
Accuracy and precision
The accuracy and precision of the NATSpert test were obtained
by the analysis of NIBSC standards using two dilutions. The
test was performed in replicates in independent runs, between
two dierent operators and using three dierent lots. The
data also were used to determine the intratest and intertest
precision. For intratest analysis, 10 replicates of each dilution
were tested, while for intertest analysis, three replicates of
each dilution in ve independent runs on dierent days were
performed. Accuracy was dened as the dierence between the
measured log concentration and the log nominal concentration.
The data were generated using semi-automated and automated
compact system and showed similar results.
Test sensitivity
The LOD of the NATSpert ID TripleH detection test for each
viral target was evaluated using sensitivity panels described
in Materials and Methods. The nal LOD was determined by
probit analysis module of SPSS. Overall, the LOD (with 95%
CI) of the NATSpert test was 2 IU/ml for HBV, 7 IU/ml for
HCV, 17.5 IU/ml for HIV-1M, 18.5 copies/ml for HIV-1O, and
8.17 IU/mL for HIV-2 [Table 1]. Conversion factors calculated
for HIV-1, HCV, and HBV are 1.57, 1.13 and 4.53 copies/IU,
respectively [Figures 1-3].
Test performance for simultaneous target
detection
The ability of NATSpert test to simultaneously detect HBV DNA,
HCV, and HIV-1 RNA in the same sample was evaluated using
the NIBSC multiplex standard. Studies have attributed the nominal
unitage is of <50 IU/ml HBV, <50 IU/ml HCV, and <200 IU/ml
HIV (package insert). The NIBSC standard was run in triplicate in
three dierent runs and specic amplication for each virus was
observed. The results are summarized in Table 2.
Analytical specicity
Genotype/subtype inclusivity of all relevant genotypes was
tested using international reference standards with 100%
detection rate. Known HBV clinical samples were tested to
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Bhosale and Desai: NAT testing using NATSpert ID tripleH test
142 Global Journal of Transfusion Medicine AATM ¦ Volume 5 ¦ Issue 2 ¦ July-December 2020
142 Global Journal of Transfusion Medicine AATM ¦ Volume 5 ¦ Issue 2 ¦ July-December 2020
verify the inclusivity of HBV Genotype H and pre-core mutant
[Tables 3-5].
Accuracy and precision
Coecient of Variation (CV) was determined for intratest and
intertest variation with < 10%.
The results are summarized in Table 6.
Performance study on clinical samples
Our study showed that 85 blood donor samples of 30,635
units were reactive on NAT screening, whereas only 77 of
these were reactive on serological screening. Signicantly,
there were eight reactive results on NAT screening that were
nonreactive on serologic testing (NAT yield of 1: 3829), of
which four were HCV reactive and two each were HBV and
HIV reactive [Table 8].
On statistical analysis using Fisher’s exact test, the NATSpert
ID TripleH assay was found to oer a statistically signicant
advantage over EIA in ability to detect TTI in blood
donors (P < 0.05, Fisher’s exact test) [Table 9].
Although advanced testing techniques are now becoming
available and are being adopted at many centers, the risk of
contracting transfusion-transmitted infections after transfusion
of blood or components still persists.
Reports from developed countries have shown the limited
value of NAT blood screening in improving blood safety. The
Scottish BTS reported a NAT yield rate for HIV and HCV of 1
per 1.9 and 0.77 million donations.[14] Reports on NAT yield of
screening 3.6 million blood donations from continental Europe
for HBV, HCV, and HIV-1 were 1 per 0.6 million donations
for HBV, HCV and 1 per 1.9 million for HIV-1[15,16] This is
primarily due to the low prevalence of HIV-1, HBV, and HCV
in these countries. In contrast to this, the prevalence of these
viral infections in resource-limited countries is generally high.
Most of the reports of NAT screening in these countries showed
NAT yield as high as 1:60 blood donation[17] for HBV and
1/3100 blood donations for HCV.[18] Also of importance in the
consideration of NAT blood screening in resource-limited areas
is to assessing infectivity for HCV and HBV.[18,19] India is in the
intermediate zone of HBV endemicity, with HBsAg prevalence
among the general population ranging from 2% to 8%.[20,21]
In India and around the world, fully automated multiplex
nucleic acid amplication technology (NAT) enabling ID
y = 0.9966x + 0.6728
R² = 0.9991
0
1
2
3
4
5
6
7
8
9
0246
81
0
log copies/ml
log IU/ml
Mylab HBV
Figure 2: IU/ml to copies/ml for hepatitis B virus
y = 0.998x - 0.0616
R² = 0.9984
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
log copies/ml
log IU/ml
My lab HCV
Figure 1: IU/ml to copies/ml for hepatitis C virus
y = 1.1738x - 0.4229
R² = 0.9962
0.00
2.00
4.00
6.00
8.00
10.00
02468
Log copies/ml
Log IU/ml
Mylab HIV-1M
Figure 3: IU/ml to copies/ml for human immunodeciency virus‑1M
or minipool of 6 (MP6) screening for HIV, HBV, and HCV
is provided by two major commercial players (Grifols
and Roche). Grifols oers three triplex NAT assays,
ProcleixUltrio, ProcleixUltrio Plus, and ProcleixUltrio
Elite and Roche oers Cobas TaqScreen MPX and Cobas
TaqScreen MPX v2.0.
Griols assays are based on transcription‑mediated
amplication (TMA), whereas Roche assays are based on PCR/
Table 1: Limit of detection or analytical sensitivity
Analyte Standard Units Average 95% LOD*
HIV-1 M NIBSC code: 10/152 IU/ml 17.5
HIV-1 O Indigenous standard Copies/ml 18.5
HIV-2 NIBSC Code: 08/150 IU/ml 8.17
HCV NIBSC code: 14/150 IU/ml 7
HBV NIBSC code: 10/264 IU/ml 2
*Calculated by probit analysis. LOD: Limit of detection,
HCV: Hepatitis C virus, HIV: Human immunodeciency virus,
HBV: Hepatitis B virus
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Bhosale and Desai: NAT testing using NATSpert ID tripleH test
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Global Journal of Transfusion Medicine AATM ¦ Volume 5 ¦ Issue 2 ¦ July-December 2020
RT-PCR technology.[22-27] These assays involve three main steps:
(a) specimen preparation, (b) amplication, and (c) detection.
Before the commercialization of NAT testing kits used for
blood screening, it is very important to determine whether the
manufactured kits meet the prescribed safety and eectiveness
standards. Analytical sensitivity, specicity, precision,
reproducibility, and repeatability are the most important
parameters of these standards.
The analytical sensitivity determines the ability of a test
method to dierentiate between two very close concentration
of any analyte and the LOD. The 95% LOD of Ultrio Elite
Assay, Cobas MPX V 2.0 assay and NATSpert ID test are
mentioned in Table 7. Taken together, it can be said that all
three tests have comparable 95% LOD.
The analytical specicity determines the ability of the test to
detect all the genetic variants and inability to detect closely
related or any other nonrelated analyte. The results of study
aimed at determining the ability to detect HIV 1, HIV 2,
HCV, and HBV genetic variants showed that NATSpert ID
test detected all major genotypes of HIV, HBV, and HCV
in genotype coverage, for HIV-1, genotype M, N, and O,
for HBV, genotype A–H, and for HCV, genotype 1–6 were
detected. Furthermore, it had no cross-reactivity with any of
the nonreactive viral and nonviral agents tested. These results
were similar to Grifols and Roche tests.[25,27] NATSpert ID
showed acceptable repeatability and reproducibility for the
results when tested for dierent lots, days, and operators.
In the rst multicentric study, a total of 12,224 samples
along with their serological results were obtained from
eight blood banks in India and were tested individually
manually by Procleix Ultrio test for HIV 1, HCV, and HBV.
The study observed eight NAT yield cases.[28] According
to a study from the western part of India, combined NAT
yield (NAT reactive/seronegative) for HIV, HCV, and HBV
was 0.034% (1 in 2972 donations)[29] which is high when
compared to studies from developed countries. In another
study conducted in North India, 18,354 donors were tested
by both ID-NAT and fourth-generation ELISA, 7 were found
to be NAT-positive but ELISA-negative (NAT yield) for
HBV and HCV. The prevalence of NAT yield cases among
routine donors was 1 in 2622 donations tested (0.038%)[30]
In our own study, during the study period of 2017–2019
at external evaluation site, all 70 seropositive specimens
detected positive, while of 30,558 prescreened seronegative
specimens, the NAT yield found was 2 each for HBV and
HIV and 4 of HCV.[10]
Commercially available NAT tests have their own limitations.
TMA-based test involves two step of detection and does
not discriminate between HIV, HCV, and HBV. It requires a
Table 2: Simultaneous target detection
HBV HCV HIV IC
Mean cT 36.68 38.47 37.44 35.08
SD 0.99 0.67 0.66 2.10
CV % 2.70 1.74 1.76 6.00
CV: C virus, HCV: Hepatitis C virus, HIV: Humanimmunodeciency
virus, HBV: Hepatitis B virus, SD: Standard deviation, IC: Internal
control, SD: Standard deviation
Table 3: Genotype coverage for human
HIV-1 2nd WHO NIBSC Genotype
panel # 12/224:041016
Cross reactivity
Genotype HIV IC HBV -
Genotype A + +
Genotype AA-GH + + HCV -
Genotype AE + +
Genotype B + + Chikungunya -
Genotype C + +
Genotype D + + Dengue -
Genotype F + +
Genotype G + + MTB -
Genotype Group N + +
Genotype Group O + + Bacterial cultures -
HCV: Hepatitis C virus, HIV: Human immunodeciency virus,
HBV: Hepatitis B virus, IC: Internal control, WHO: World Health
Organization, MTB: Mycobacterium tuberculosis, NIBSC: National
Institute for Biological Standards and Control
Table 4: Genotype coverage for hepatitis C virus
3rd HCV genotype NIBSC WHO
Panel (PN 12/172)
Cross-reactivity
Genotype HCV IC HBV -
1a + + HIV -
1b + + Chikungunya -
2i + + Dengue -
3a + + MTB -
4r + + Bacterial cultures -
5a + +
6i + +
HCV: Hepatitis C virus, HIV: Human immunodeciency virus,
HBV: Hepatitis B virus, IC: Internal control, WHO: World Health
Organization, MTB: Mycobacterium tuberculosis, NIBSC: National
Institute for Biological Standards and Control
Table 5: Genotype coverage for hepatitis B virus
1st HBV WHO NIBSC Genotype
panel PE5086/08 V2
Cross-reactivity
Genotype HBV IC HIV -
Genotype A1 + +
Genotype A2 + + HCV -
Genotype B2 + +
Genotype B4 + + Chikungunya -
Genotype C2 + +
Genotype D1 + + Dengue -
Genotype D3 + +
Genotype E + + MTB -
Genotype F2 + +
Genotype G + + Bacterial cultures -
HCV: Hepatitis C virus, HIV: Human immunodeciency virus,
HBV: Hepatitis B virus, IC: Internal control, WHO: World Health
Organization, MTB: Mycobacterium tuberculosis, NIBSC: National
Institute for Biological Standards and Control
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144 Global Journal of Transfusion Medicine AATM ¦ Volume 5 ¦ Issue 2 ¦ July-December 2020
144 Global Journal of Transfusion Medicine AATM ¦ Volume 5 ¦ Issue 2 ¦ July-December 2020
supplementary discriminatory viral test for the detection of
HIV RNA, HCV RNA, and HBV DNA. Thus, the turnaround
time is longer. In case of MP-NAT test, although it is based on
multi dye RT PCR technique, the sensitivity is compromised
due to pooled samples and requires the second test to conrm
positivity by ID testing, thus increasing the reporting time.
In contrast, NATSpert ID testing utilizes an aliquot of each
sample tested individually, with no dilution due to addition of
noninfectious sample. It provides faster results than MP-NAT
since it does not require balanced sample pooling and resolution
of positive samples.[30] Some recent data from Southeast Asia
highlight this advantage. With occult HBV infection frequency
of 1:40,000, ID-NAT detected HBV-DNA in blood samples at
levels of <100 IU/ml. If the same samples were tested in mini
pools of 6 with an analytical sensitivity of 5 IU/ml, 70.7% of
samples would have been not detected. Recent reports also
show that in case of HBV, apart from initial WP, a second WP
exists at 75–85 days post infection, when HBsAg is no longer
detectable and anti-HBsAg not yet detectable, but HBV NAT
is positive during this period. The ID-NAT test is important in
patients who receive multiple blood transfusions for diseases
such as thalassaemia and hemophilia. Such patients require
repeated lifelong blood transfusion and are at higher risk of
being infected with serious TTIs.[31]
Taken together, we present a NATSpert ID testing solution that
gives highly sensitive and specic real‑time PCR technology
for testing of individual donors, blood products, or suspected
individuals where multiple analytes can be detected and
identied in a single test, thus improving workow turnaround
time and sensitivity compared to the two currently available
commercial kits. Furthermore, Mylab’s proprietary buer
formulations oer a signicant cost advantage.
The NATSpert ID TripleH test demonstrated excellent
sensitivity and specicity in viral target detection and
discrimination. It is sensitive, specic, user‑friendly,
rapid, robust, and aordable. The test is suitable to screen
specimen in individual ID format and furthermore, pliable
for automation. The performance of NATSpert ID TripleH is
expected to be of great signicance to low‑resource countries,
as it resolves most of the hurdles currently faced by them in
screening blood donors for HIV, HBV, and HCV, given the
prevalence of HIV, HBV, and HCV in these countries.
Acknowledgment
The authors would like to thank Ms. Aditi Kavimandan
and Reshma Naiknaware for technical assistance and Mr.
Ranjit Desai and Ms. Guari Metkar for the execution of
experiments.
Table 6: Intra assay and inter assay variation
HIV-1, percentage CV HIV-2, percentage CV HCV, percentage CV HBV, percentage CV
Log 3.3 Log 2 Log2 Log 1.4 Log 3.3 Log 2 Log 2 Log 1.3
Intra assay (n=10) 1.73 2.28 1.41 1.38 1.73 2.28 1.11 1.4
Inter assay (n=5) 1.41 3.23 1.14 0.87 1.41 3.23 1.26 1.54
Inter operator (n=2) 1.25 3.46 1.56 1.51 1.61 2.56 0.86 1.41
Inter lot (n=3) 1.73 2.28 1.1 1.06 3.73 4.93 0.97 0.73
CV: Coecient of variation, HCV: Hepatitis C virus, HIV: Human immunodeciency virus, HBV: Hepatitis B virus
Total number of donations
TTI test
30,635
Total HBV, n (%) HCV, n (%) HIV, n (%)
Seroreactive samples 77 40 (0.13) 14 (0.45) 23 (0.075)
ID NAT reactive samples 85 42 (0.14) 18 (0.058) 25 (0.082)
NAT yield 8 2 (0.006) 4 (0.013) 2 (0.006)
HCV: Hepatitis C virus, HIV: Human immunodeciency virus, HBV: Hepatitis B virus, NAT: Nucleic acid test, ID: Individual donor,
TTI: Transfusion transmitted infection
Table 9: Fisher exact test 2 × 2 table
EIA (serology) NATSpert ID
Positive Negative
Positive 77 0
Negative 8 30,550
P<0.05, Fisher’s exact test. EIA: Enzyme immunoassay,
NAT: Nucleic acid test, ID: Individual donor
Table 7: Comparative LOD for NAT tests
95% LOD for various NAT tests
Ultrio Elite Grifols Cobas MPX Roche NATSpert ID
HIV-1 18 IU/ml 46.2 IU/ml 17.5 IU/ml
HIV-2 10.4 IU/ml 7.9 IU/ml 8.17 IU/ml
HBV 4.3 IU/ml 2.3 IU/ml 2.0 IU/ml
HCV 3.0 IU/ml 6.8 IU/ml 7.0 IU/ml
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Bhosale and Desai: NAT testing using NATSpert ID tripleH test
145
Global Journal of Transfusion Medicine AATM ¦ Volume 5 ¦ Issue 2 ¦ July-December 2020
Financial support and sponsorship
Nil.
Conicts of interest
The authors of this study are full time employees of Mylabs
Discovery Solutions Pvt Ltd.
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