Validation of a direct-to-PCR COVID-19

detection protocol utilizing mechanical

homogenization: A model for reducing
resources needed for accurate testing

Morehouse ZP, Samikwa L, Proctor CM, Meleke H, Kamdolozi M, Ryan

GL, et al. (2021) Validation of a direct-to-PCR COVID-19 detection
protocol utilizing mechanical homogenization: A model for reducing
resources needed for accurate testing. PLoS ONE 16(8): e0256316.
https://doi. org/10.1371/journal.pone.0256316

Journal Reading
Assignment
by Rokhmatul Asiyah
Introduction
The COVID-19 global pandemic caused by SARS-CoV-2 since March of 2020  activation of public health efforts
geared towards disease surveillance and prevention practices Efficient and effective SARS-CoV-2 detection

Reverse transcriptase polymerase chain reaction (RT-PCR) testing quickly emerged as the global standard for
accurate SARS-CoV-2

In the traditional model of RT-PCR samples collected, stored in viral transport media, and sent to the laboratory
for processing including viral RNA extraction and purification utilizing a series of chemical reagents  consumable
plastics and chemical reagents

One way to reduce cost and increase access to SARS-CoV-2 testing reduce the resources needed for detection,
without reducing the sensitivity.

The authors are utilizing a mechanical homogenization based direct-to-PCR viral detection method for SARS-CoV-2
detection to screen for both symptomatic and asymptomatic COVID-19 in Blantyre, Malawi

Based direct-to-PCR viral detection in vitro simulated nasopharyngeal swab samples spiked with clinically
significant levels of human coronavirus 229E (HCoV-229E) revealed a limit of detection of 1.2x10 3 viral copies/mL
with 96.30% sensitivity

• Examine the clinical feasibility and efficiency of this methodology

• Compare of the direct-to-PCR testing and the traditional extraction-based PCR testing
Objective >>> methods in asymptomatic and mild to moderately symptomatic patients were screened
with both methods for SARS-CoV-2 detection.
Materials and methods

• All samples were obtained with informed patient consent

approved by the Institutional Review Board of the University of
Malawi College of Medicine (Blantyre, Malawi).
• Samples were obtained from a prospective cohort study to
compare the risk of SARS-CoV-2 infection and exposure in
healthcare workers compared to community members in
Blantyre, Malawi between June and August 2020. 15 SARS-CoV-2
Sample PCR positive and 15 SARS-CoV-2 PCR negative were randomly
collection selected from the cohort of 300 adult participants.
• Suspected severe COVID-19 in respiratory distress were excluded
from this study.
• Each patient provided two swab specimens, one nasopharyngeal
swab and one oropharyngeal swab, which were stored at 4 0 C for
up to 24 hr, if the sample was not processed within 24 hr it was
stored at -800 C
Materials and methods

• One mL of viral transport media was taken from the tube at room
temperature and utilized in the Omega Bio-Tek Mag-Bind Viral
Extraction-based DNA/RNA Kit in accordance with manufactures guidelines (Omega Bio-
sample preparation Tek, Cat. No.M6246). Final product of purified viral RNA was held on
ice in preparation for PCR testing

• One mL of viral transport media was transferred from the original

sample collection tube to a 2 mL screw cap tube (Omni International
Homogenization- Inc, Cat. No. 19–647) and sealed. Sample tubes were then loaded
based sample into the Omni Bead Ruptor Elite for processing.
preparation • The samples were run at 4.2 m/s for 30 s, removed from the device,
and allowed to sit for 60s to allow for any forth in the tubes to settle
Materials and methods
RT-qPCR detection of SARS-CoV-2
• Following sample processing with either the extraction-based or homogenization-based methods, Real-Time
RT-PCR Diagnostic Panel (Cat. No. 2019-nCoVEUA-01 1000 reactions) was used.
• 5μL of purified RNA or lysate, transferred into 15 μL reactions of Quantabio qScript XLT One-Step RT-qPCR
ToughMix comprising of 3.5μL Nuclease-free Water, 1.5μL Combined Primer/Probe Mix, and 10μL qScript XLT
One-Step RT-qPCR ToughMix (2X).
• The RT-qPCR reaction was premixed and then loaded into the QuantiStudio 7 Flex Real Time PCR System
(ThermoFisher Scientific, Cat. No. 4485698) and run for 45 amplification cycles.
• Cycle threshold (Ct) values were recorded less than or equal to 40 was labeled as positive for COVID-19
detection

Statistical comparison of extraction and homogenization

methodologies
• The final Ct values resulting from each method were paired under the original patient identification number
for direct comparison.
• Negative samples where no amplification was detected in the presence of confirmed internal controls, were
assigned a Ct value of 45.0.
• The positive predictive value (PPV) and negative predictive value (NPV) for the homogenization-based
workflow were calculated using the extraction-based workflow as the gold standard method to compare
against.
• the two resultant Ct values linked to the same patient were plotted for correlative visualization and a standard
best fit line was produced to evaluate the R2 value.
Re
sul
ts

Overall 80% agreeability between both methods. A strong positive

correlation was observed with an R2 of 0.763 (Fig 2 ).

Except 1 patient, a Ct value following homogenization-based

greater than extraction-based.

6 positive samples that demonstrated disagreement under both

methodologies, where a sample was Ct positive one method and
not the other (Table 2).
These disagreements were observed in Ct values greater than 37,
indicating a low viral load. When comparing Ct values below 37,
there was a 100% agreement for both methodologies
Discussion
Efficient and effective diagnostic testing has proven paramount to the global response, with PCR based
viral detection off swabs as gold standard for testing. However, the traditional extraction-based
methodology detailed in this manuscript requires many chemical reagents  become financially
burdensome

The total run time for each sample can take up to 3 hours from extraction to PCR When receiving
thousands of samples per day causing delays, relaying critical positive or negative results

Developing approaches while maintaining accuracy also emerged in response to the COVID-19
pandemic antigen tests offer solutions on either time or cost associated with testing  do not
maintaining accuracy to the current extraction-based PCR workflow

The validation of the proposed direct-to-PCR, homogenization-based methodology offers a solution

through reducing the time and reagents while maintaining an acceptable level of accuracy when
compared to the extraction based method.
Discussion

An R2 of 0.763 and positive predictive value of 80% demonstrate a moderate level of acceptability for implementing
the proposed direct-toPCR method into clinical practice.

The demonstrated agreeability between the direct-to-PCR and traditional methods displayed a lower Ct value
following the extraction protocol versus the homogenization protocol. This is representative of the PCR
inhibitors present in the lysate following homogenization that are not removed due to the lack of chemical
purification steps when compared to the extraction protocols.

The high agreeability in detecting SARS-CoV-2 between the methods demonstrates that these inhibitors
remaining in the lysate transferred to the RT-qPCR in the homogenization method do not prohibit
adequate RNA amplification for detection  confirmed with a strong R2 value

• Inconsistent detection were greater than 37, the 100% positive
and 100% negative predictive value at Ct values less than 37
qualifies this methodology for further large-scale evaluations. It
has been documented that between commercially available kits
there is decreasing agreement on SARS-CoV-2 detection as Ct
values grow past 36
• The authors acknowledge that this manuscript has its limitations
in statistical robustness and the limited data it provides, such as
the lack of technical replicates or viral load quantifications
Further notes
Conclusion
• This novel process for viral detection in an effort to assist in the
current international need for COVID-19 testing innovation and
improvement efficient and cost-effective testing can work to
fill the void in many areas of the world.
• Using the small mechanical footprint of a bench top homogenizer
and a standard thermocycler, and without multitude of chemical
reagents, the direct-to-PCR workflow can be set up almost
anywhere. The ease of system configuration and simple
operational steps to support the adaptability of this novel method
• Given the high levels of agreeability between the observed
workflows, in conjunction with the significant reduction in cost
and processing time by utilizing a 30 second mechanical
homogenization step versus an hour-long reagent heavy
extraction procedure, this direct-toPCR methodology has
potential to be further incorporation into global COVID-19
detection strategies.
 CRITICAL APPRAISAL - PICO
Patient or Problem:
Population and
Problem Metodologi deteksi virus yang efisien dan efektif menjadi sangat penting dalam menghadapi pandemi COVID-19. Pengujian swab
nasofaring dan orofaringeal berbasis RT-PCR menjadi standar emas saat ini. Metode direct-to-PCR yang didasarkan pada homogenisasi
mekanis dapat digunakan untuk mengurangi sumber daya yang diperlukan dengan mempertahankan sensitivitas yang sebanding. Diperlukan
uji kelayakan klinis dan efisiensi metodologi ini. Dengan membandingkan langsung dari metode direct-to-PCR dan metode pengujian PCR
berbasis ekstraksi tradisional.

Populasi:

Populasinya adalah seluruh penduduk Blantyre, Malawi. Sampel diperoleh dari studi kohort untuk membandingkan risiko infeksi dan
paparan SARS-CoV-2 pada petugas kesehatan dibandingkan penduduk di Blantyre, Malawi. Sampel diambil antara Juni dan Agustus 2020. 15
partisipan positif dan 15 partisipan negatif RT-PCR SARS-CoV-2 dipilih secara acak dari kelompok 300 partisipan.

Intervention Sampel dari 300 orang diambil berupa nasofaring dan orofaring swab pada Juni sampai Agustus 2020. Dengan kriteria eksklusi dengan gejala
parah yang mengarah pasti COVID-19. Kemudian dilakukan 2 metode RT-PCR yang berbeda yaitu metode direct-to-PCR dan metode pengujian
PCR berbasis ekstraksi tradisional. Kemudian 15 partisipan positif dan 15 partisipan negatif RT-PCR SARS-CoV-2 dipilih secara acak. Data yang
didapatkan dibandingkan dan diuji statistik.

Comparation Perbandingan langsung dari Metode direct-to-PCR dan metode pengujian PCR berbasis ekstraksi tradisional pada partisipan yang tanpa gejala
dan gejala ringan hingga sedang.
Outcome Didapatkan tingkat kesesuaian yang tinggi dari metode direct-to-PCR dan metode pengujian PCR berbasis ekstraksi tradisional. Dengan
pengurangan biaya dan waktu pemrosesan singkat, maka metode ini berpotensi berperan lebih masif dalam strategi deteksi COVID-19 global.
CRITICAL APPRAISAL
Apakah fokus penelitian ini YA
- VIA
sesuai dengan tujuan
penelitian?
Apakah subjek penelitian ini
diambil dengan cara yang
tepat dan jelas?
Apakah penelitian ini TIDAK
mempunyai jumlah subjek Karena dari dari 300 orang partisipan hanya 30 orang dilakukan pengolahan data yaitu 15 partisipan positif dan 15 partisipan
yang cukup untuk negatif RT-PCR SARS-CoV-2 yang dipilih secara acak. Hal ini juga disebukan penulis sebagai kekurangan pada penilitian ini.
meminimalisirkan kebetulan?
Apakah data yang
dikumpulkan sesuai dengan
tujuan penelitian?
Apakah analisa data
Validity
dilakukan baik?
Importanc
Apakah penelitian ini
penting?
e
Apakah penelitian ini dapat
diterapkan?
Applicable
Tujuan penelitian ini adalah menguji kelayakan klinis dan efisiensi metode direct-to-PCR maka dilakukan dengan
membandingkan langsung dari metode direct-to-PCR dan metode pengujian PCR berbasis ekstraksi tradisional.
YA
Semua sampel diperoleh dengan informed consent partisipan yang disetujui oleh Institutional Review Board of the University
of Malawi College of Medicine (Blantyre, Malawi). Sampel dari 300 orang diambil berupa nasofaring dan orofaring swab pada
Juni sampai Agustus 2020. Dengan kriteria eksklusi dengan gejala parah yang mengarah pasti COVID-19. Kemudian 15
partisipan positif dan 15 partisipan negatif RT-PCR SARS-CoV-2 dipilih secara acak.
YA
Sampel dari 300 orang diambil berupa nasofaring dan orofaring swab. Dengan kriteria eksklusi dengan gejala parah yang
mengarah pasti COVID-19. Kemudian dilakukan 2 metode RT-PCR yang berbeda yaitu metode direct-to-PCR dan metode
pengujian PCR berbasis ekstraksi tradisional. Kemudian 15 partisipan positif dan 15 partisipan negatif RT-PCR SARS-CoV-2
dipilih secara acak. Data yang didapatkan dibandingkan antara kedua metode. Sehingga sudah sesuai untuk menguji
kelayakan klinis dan efisiensi metode direct-to-PCR.
TIDAK
Karena uji statistik hanya analisa korelasi dari 30 data yang didapatkan. Hal ini juga disebukan penulis sebagai kekurang
dalam studi ini.
YA
Kondisi COVID-19 yang masih menjadi pandemic di seluruh dunia. Sehingga metode deteksi virus yang efisien dan efektif
menjadi sangat penting untuk meningkatkan sasaran demi pecegahan penyebaran. Juga metode direct-to-PCR ini berpotensi
mengurangi kebutuhan akan reagen dan plastik yang banyak digunakan dalam protokol sebelumnya.
YA
Metode direct-to-PCR ini menggunakan alat homogenizer yang kecil, thermocycler standar, dan tanpa banyak reagen kimia,
dengan kemudahan konfigurasi sistem serta langkah operasional sederhana sehingga mendukung kemampuan beradaptasi di
mana saja. Hasil pemeriksaan dengan tingkat kesesuaian yang tinggi, juga hemat biaya dan waktu pemrosesan yang
signifikan, maka metode ini berpotensi berperan lebih masif dalam strategi deteksi COVID-19 global.
THANK YOU
FOR YOUR ATTENTION
 Viral transport media formulation and
production
Viral transport media (VTM) was produced
following
the US CDC (Atlanta, GA, USA) guidelines,
found freely
available on their website for formulation of
VTM in a
Materials and methods
laboratory as an alternative to commercial
Cell culture and virus growth
VTM purchases. 500 mL of Hanks Balanced
Human coronavirus 229E (HcoV-229E)
Salt Solution (HBSS)
(ATCC, Cat.No. VR-740) was added at a
1X with calcium and magnesium ions (no
multiplicity of infection(MOI) of 1.0 to an
phenol red)
approximately 85% confluent T75 flask of
(Fisher Science, Cat. No. SH3058801) was
MRC-5 cells (ATCC, Cat. No. CCL-171), 48 h
supplemented
after plating. The flask was maintained with
with 2% heat inactivated fetal bovine serum
DMEM (Fisher Scientific, Cat. No. 11–965-
(Gemini
118) supplemented
Bioproducts, Cat. No. 100–500). 100 mg of
with 5% heat inactivated fetal bovine serum
Gentamicin
(Gemini Bioproducts, Cat. No. 100–500) and
(Gemini Bioproducts, Cat. No. 400-100P) and
1% L-Glutamine (Gemini Bioproducts, Cat.
500 μg of
No. 400–106), incubated at 37 °C with 5%
Amphotericin B (Gemini Bioproducts, Cat.
CO2 [15]. The cell culture supernatant
No. 400-
was harvested at 72 h post infection when
104P) was added to the mixture and mixed
80% cytopathic effect (CPE) was observed.
thoroughly
to create a final product of VIRAL
TRANSPOT MEDIA,
2% FBS, 100 μg/mL Gentamicin, 0.5 μg/mL
Amphotericin B. This viral transport media
was used for storage
and processing of all swab samples in this
manuscript.
Swab viral spike
Sterile cotton swabs (Fisher Science, Cat. No.
22–029-
488) were submerged for 5 s in viral solutions
ranging
Shaker-mill swab processing for viral lysis
from 1.2 × 107 to 1.2 × 101 viral copies/mL
To maintain optimal levels of biosafety, the
[16]. The
following
swabs were exposed in a serial dilution
shaker-mill processing was completed in a
pattern, with
biosafety cabinet to protect the user from any
three swabs being exposed at each
potential aerosol production during
concentration log to
processing. Twenty-four 2 mL screw cap
evaluate the detection capabilities of this
tubes containing the virally spiked swabs were
method. The
processed
saturated swabs were then placed in a 2 mL
on the Omni Bead Ruptor Elite (Omni
screw
International,
capped tube (Omni International, Cat. No. 19–
Cat. No. 19-040E) for 30 s at 4.2 m/s. This
648) prefilled with 1 mL of viral transfer
processing
buffer [17]. The stem of
generated froth within the tube which was
the swab was then broken off at a level even
allowed to
with the
settle prior to removal of 1 μL of lysate for
top of the tube to allow for the cap to be
RT-qPCR
screwed on for
(Fig. 1).
transporting and processing. The samples
were prepared
at 23 °C and then incubated for 1 h at 23 °C
prior to
processing.