Application of serology VsPCR in infectious disease diagnosis ,eg COVID 19

Abstract

The word infection stands for invasion and multiplication of one or more pathogens. They cause infectious diseases in humans after establishing an entry into the human body through routes such as skin, mouth, nose, and genitourinary tract. These infections can escalate into a level of a pandemic such as the current COVID-19 pandemic resulting in severe implications on health, economy, and social life worldwide. Therefore, early diagnosis has become vital to identifying, control, and eradicate these infections. Serological and molecular assays such as Polymerase chain reaction (PCR) have an advantage over conventional diagnostic methods such as culturing, microscopy due to higher specificity and sensitivity exhibited over detecting a large number of infectious diseases.PCR is classified as a direct diagnostic assay. It is a popular diagnostic assay in infectious disease diagnosis due to its multiple advantages such as high sensitivity, specificity, and rapid results. The arrival of real-time PCR is a great achievement in PCR technology and it increased the scope of infectious disease diagnosis. In COVID-19 caused by SARS-COV 2 virus, diagnosis is carried out mainly by Reverse transcriptase PCR coupled with real-time quantification. Serology is the protein (Antibody) identification and is an indirect diagnosis method of infectious diseases that consists of conventional serological tests such as complement fixation, enzyme immunoassay tests, latex agglutination tests which are widely used and rapid tests include point of care tests. In this review, more emphasis will be made on the applications of serological assays and PCR in infectious disease diagnosis.

Full text

International Journal of Medical Science in Clinical Research and Review
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Review article
Application of serology VsPCR in infectious disease
diagnosis ,eg COVID 19
Achini Udeshika Maha Gamage & Chamindri Witharana
Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of
Colombo
Corresponding Author: Chamindri Witharana
Official Address: Department of Biochemistry and Molecular Biology, Faculty of
Medicine, University of Colombo, Kensey Road, Borella 008, Colombo.
Text - Word count ; 3033Abstract - 249
Article Received 10-10-2020 , Accepted 10-11-2020 , Published 20-11-2020
Abstract
The word infection stands for invasion and multiplication of one or more pathogens. They
cause infectious diseases in humans after establishing an entry into the human body through
routes such as skin, mouth, nose, and genitourinary tract. These infections can escalate into a
level of a pandemic such as the current COVID-19 pandemic resulting in severe implications
on health, economy, and social life worldwide. Therefore, early diagnosis has become vital to
identifying, control, and eradicate these infections. Serological and molecular assays such as
Polymerase chain reaction (PCR) have an advantage over conventional diagnostic methods
such as culturing, microscopy due to higher specificity and sensitivity exhibited over
detecting a large number of infectious diseases.PCR is classified as a direct diagnostic assay.
It is a popular diagnostic assay in infectious disease diagnosis due to its multiple advantages
such as high sensitivity, specificity, and rapid results. The arrival of real-time PCR is a great
achievement in PCR technology and it increased the scope of infectious disease diagnosis. In
COVID – 19 caused by SARS-COV 2 virus, diagnosis is carried out mainly by Reverse
transcriptase PCR coupled with real-time quantification. Serology is the protein (Antibody)
identification and is an indirect diagnosis method of infectious diseases that consists of
conventional serological tests such as complement fixation, enzyme immunoassay tests, latex
agglutination tests which are widely used and rapid tests include point of care tests. In this
review, more emphasis will be made on the applications of serological assays and PCR in
infectious disease diagnosis.
Keywords – COVID-19, RT-PCR, infectious diseases , Serology

@2020 IJMSCRR All Rights Reserved (Vol 03, Issue 06 November – December) 2020 Page | 491
Introduction.
The Infectious diseases have become a
major cause of morbidity and mortality
worldwide. Appropriate and timely
diagnosis is the basis for treatments,
disease control and prevention. [1].
Availability of rapid, reliable diagnostic
assays are important in early and
accurate identification of infected
individuals and informed early therapeutic
interventions [2]. The evolution of
infectious disease diagnostics are possible
due to advances in chemistry, molecular
biology, immunology, automation, and
nucleic acid amplification majorly. New
technologies are important in bringing
advancements in infectious diseases
diagnostics [3]. The two tiers of infectious
disease diagnosis are direct pathogen
detection and specific antibody detection
where serological tests involved in the
detection of specific antibodies provide a
longer diagnostic window than direct
methods [4]. Serology is the study of
proteins mainly antibodies in body fluids
majorly in blood following cerebrospinal
fluid and/or saliva and classified as direct
or indirect [5]. Classical serological assays
widely applicable in total antibody tests
are ELISA, agglutination, precipitation,
complement fixation, fluorescent
antibodies test, and Chemiluminescence
tests which do not provide a rapid
diagnosis, where as rapid diagnosis can be
achieved via indirect assay that binds the
antibodies to the immobilized antigens [6].
Examples of rapid tests done for antigen
detection with a specific antibody are
detection of the antigens of Streptococcus
pneumoniae, Legionella
pneumophila serogroup 1, and
Histoplasma capsulatum in urine and
cryptococcal antigen in blood and CSF [3].
Polymerase chain reaction (PCR) comes
under direct diagnostic assays and the
application of PCR in infectious disease
diagnosis was witnessed for the last two
decades [3,4]. This facilitates the rapid
diagnosis of microorganisms, which was
difficult or not possible with traditional
microbiological techniques [7].
The principle of PCR is based on in vitro
generation of a large number of copies of
the particular DNA fragment, available in
small quantities in a DNA extract[8]. This
DNA extract can be genomic DNA or
complementary DNA obtained by RT-
PCR from a messenger RNA extract [9].
This is an enzyme driven process, the
oligonucleotide primers that specifically
bound to targets are designed from target
DNA at the beginning. The targets are then
amplified in a thermocycler by undergoing
target DNA denaturation, primer
hybridization, and primer extension,
leading to exponential amplification using

@2020 IJMSCRR All Rights Reserved (Vol 03, Issue 06 November – December) 2020 Page | 492
a DNA polymerase, nucleotides, and
designed primers. [2]
Application of Serological assays and
PCR for infectious diseases diagnosis
Serological tests are used in detecting a
vast number of infectious diseases and if
the viral diagnosis is considered, it is
based on either the demonstration of viral-
specific IgM antibodies or detection of a
significant increase in specific IgG
antibodies. The method used for
demonstrating specific antibody response
is the evaluation of antibody response via
serological methods and most commonly
experienced serological assays are
immunoassays and point of care (POC)
tests done for antigens and antibodies [10].
Applications of serology in the
identification of global viral diseases, for
example, Human immunodeficiency virus
(HIV) which has been infected more than
40 million people and it is of two types as
HIV 1/HIV 2[6]. Serological tests such as
enzyme immunoassays (EIA) are sensitive
after antibodies formed in patients and this
takes around one to two weeks the period
after contacting the infection [11]. The EIA
can detect IgG or IgG/IgM Combination of
specimens [12]. The positive results of EIA
tests should be confirmed by a Western
blot test to detect antibodies for viral
antigens [11]. In certain instances, such as
diagnosing HIV positive donors during
organ or blood transfusion, rapid
serological diagnosis is done by detecting
HIV p 24 antigen whereas this antigen is
detectable before antibodies in early
diagnosis of the infection [12].
Application of serological tests in the
detection of antibodies of patients having
fungal infections is advantageous over non
culture-based diagnosis since the middle of
the last century. Serology gains
importance in diagnosing endemic
mycoses where diagnostic methods
include immuno diffusion (ID),
complement fixation (CF), and an enzyme
immunoassay (EIA). Histoplasmosis is a
fungal infection that is more commonly
diagnosed by CF and immuno diffusion.
Immuno diffusion is capable of detecting
precipitating antibodies for H and M
antigens of Histoplasma and also these
tests involved in diagnosing
coccidioidomycosis, where immuno
diffusion can be used to determine
coccidioidal IgM and CF detects IgG
antibodies [13].
Serological tests apply in detecting many
bacterial infections whereas Yersinia
enterocolitica, Campylobacter jejuni,
Salmonella, Shigella, and Chlamydia
trachomatis are associated with reactive
arthritis and disease diagnosis is performed

@2020 IJMSCRR All Rights Reserved (Vol 03, Issue 06 November – December) 2020 Page | 493
by indirect heamagglutination test and
complement fixation test [14].
Serological assays are useful in providing
information about etiology after the acute
stage during primary infection and can
result in epidemiological data also involve
in determining vaccine induces immunity
[10]. Also advantageous in detecting
invasive fungal infections since it reduces
the necessity of culturing of hazardous
fungi such as Coccidioides spp. if
serological results are positive[13]. Also,
early diagnosis of acute infections such as
Epstein–Barr virus and diagnosis of
chronic infections such as HIV (human
immunodeficiency virus) or viral hepatitis
can be done and further benefits include
enhanced performance, traceability, and
cost-effectiveness [6].
Not only advantages but there are
disadvantages also since serology depends
on the immunological response of the
patient that may take weeks to a month to
develop antibodies [15]. False-negative
results and diminished specificity may
result when already available or chosen
serological antigens for assays become too
specific or mismatched to the pathogens
present. Antibody cross-reactivity where
certain viruses in a group share antigenic
determinants and serological tests cannot
differentiate on past and current infections
are limitations [12]. It encounters limitations
as in some infections strong antibody
response is not witnessed or the specificity
of the antigens become decreased therefore
distinct interpretation of the results does
not happen and when comes to the
serological response of
immunocompromised patients it is often
too weak to allow the demonstration of
specific responses [10]. Same time certain
assays are time-consuming, for example,
complement fixation, and also it is difficult
to distinguish between current or previous
infection [16].
The role of serology is diminished in the
early stage of infection due to the
difficulty in obtaining specific antisera
required for serological tests and with the
arrival of PCR technology, for example,
multiplex PCR, real-time PCR with
improved efficiency the role of molecular
methods in infectious disease diagnosis
was further increased [7].
Antibody detection for certain viruses
becomes relatively insensitive but an
improved detection of a number of these
viruses can be obtained by performing the
PCR technique. Diagnosis of Herpes
simplex virus (HSV) encephalitis termed
as a serious infection was carried out
before, requiring a brain biopsy in certain
cases where low sensitivity was reported
for serology and cerebrospinal fluid
culture (CSF) and With PCR brain biopsy

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was replaced with HSV DNA detection
from CSF with high sensitivity. Compared
to culturing PCR can relatively detect viral
meningitis caused by enteroviruses or
HSV. Genital ulceration caused by HSV
type 2 infection, can be routinely detected
by PCR due to its increased sensitivity
over viral culture, and the detection of
blood-borne virus infection is improved
with PCR. Intrauterine infection of a fetus
caused due to cytomegalovirus (CMV),
rubella, and varicella zoster virus is
detected by carrying out a PCR in
amniocentesis fluid [7].
The infectious disease diagnosis caused by
fastidious bacteria has benefited greatly
from molecular detection where PCR,
which has a higher sensitivity replaced
direct fluorescent-antibody and culture
methods in the detection of Bordetella
pertussis. Laboratory detection of
Bordetella pertussis during an outbreak
resulted in a 48% PCR detection and only
5% by culturing.Neuro-Whipple’s disease
and endocarditis can be diagnosed after
carrying out PCR to detecting Tropheryma
whipplei, the causative agent [16].
PCR is used to detect fungal pathogens
causing diseases to humans and offer
several features that may aid in
overcoming current shortcomings for the
diagnosis of fungal infections
[17]. Pneumocystis jiroveci is fungi involve
in causing severe pneumonia in immuno
compromised and HIV-infected patients
but the pathogen detection is limited to the
microscopy of respiratory tract specimens
but PCR is useful in detecting
Pneumocystis jiroveci in HIV-non-infected
individuals and this microbe, a ubiquitous
commensal can be detected by PCR in the
absence of pneumonia [16].
There are many advantages of PCR. It is
highly sensitive, specific, and produces
rapid results [8]. Advantageous over
serological testings in early diagnosis of
infection and different PCR primers can be
designed to specifically target disease-
causing microbes at their species or strain
levels [9]. PCR can make many copies, a
billion number of the particular genetic
sequence for further analysis [8].
Application of PCR in infectious disease
diagnosis is advantageous since rapid
multiplication of viral sequences can be
obtained by using PCR with specific
primers and it replaces viral isolation also
PCR provides a diagnostic advantage for
viruses that cannot be cultivated such as
papilloma viruses, parvoviruses, and
hepatitis viruses also capable of detecting
emerging viruses [10].
Limitations include, more expensive than
other detection methods since expensive

@2020 IJMSCRR All Rights Reserved (Vol 03, Issue 06 November – December) 2020 Page | 495
instruments, chemicals, and trained staff is
needed [9]. Multiple pathogens or species
detected in multiplex PCR assays but
achieving optimal sensitivity for all
pathogens is difficult and also co-infecting
pathogens may cause competition and
cross-reaction in the PCR reaction process
[18]. Reduced specificity is observed when
primers bind nonspecifically and to design
primers, prior knowledge of target
sequence is necessary [8].
Applications of PCR and serological
assays in the diagnosis of a novel
coronavirus, cause for Covid -19
COVID-19 (coronavirus disease 2019) is
caused by a novel enveloped RNA
betacoronavirus severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2)
and this has been revealed as an
international public health concern due to
its global spread, molecular methods and
serological assays are used for clinical
diagnosis of the disease [19].
Better control over Covid -19 can be
achieved by rapid and accurate diagnostic
methods and this has two categories
serological tests for detection of anti-
SARS-CoV-2 immunoglobulins and
molecular methods for viral RNA
detection [20].
Direct Diagnosis of SARS-CoV-2
Infection
Direct diagnosis of infection of SARS-
CoV-2 is by identification of viral RNA in
lower respiratory tract specimens and
nasopharyngeal swabs, but nasopharyngeal
swabs are the gold standard [21]. RT-PCR
involves in the detection of SARS-CoV-2
infection[22]. It is reverse transcription of
viral RNA into complementary DNA
(cDNA) and amplification of a particular
region of the cDNA takes place and it is
the most widely used method in detection
of COVID-19 samples [23]. The genome of
this SARS-CoV-2 virus consists of around
30 000 nucleotides and 15 genes and many
of these genes are used as primer/probe for
diagnosis of infected individuals in
reverse-transcription polymerase chain
reaction (RT-PCR) assays[21].
Real-time rt-PCR is the most used direct
diagnosis method of SARS-CoV-2 viral
RNA detection due to the specificity and
simplicity as a one-step assay and higher
sensitivity obtained in early detection but
there is a risk of exhibiting false positive
and false negative results yet a
combination of Real-time rt-PCR and
clinical features has given an improvement
in managing SARS-COV -2 pandemic [22].

@2020 IJMSCRR All Rights Reserved (Vol 03, Issue 06 November – December) 2020 Page | 496
Indirect Diagnosis of SARS-CoV-2
Infection
This method of diagnosis of SARS-CoV-2
infection depends on the detection of
specific IgG and/or IgM antibodies
whereas WHO (World Health
Organization) has announced the strong
requirement of serological IgM and IgG
testing in detecting SARS-CoV-2,
therefore, it has been developed many
serological assays to diagnose SARS-
CoV-2 viral proteins and antibodies in
serum or plasma samples. Most widely
used are rapid lateral flow immunoassay
(LFIA), automated chemiluminescence
immunoassay (CLIA), and enzyme-linked
immune assay (ELISA) [21].
Enzyme-Linked Immune Assay
(ELISA)
Enzyme-linked immune assay (ELISA)
was developed to detect anti-SARS-CoV-2
IgG and IgM responses by detecting
antibodies produced against the
nucleocapsid protein and spike proteins[21].
The sensitivity of ELISA is high in total
antibody detection and it is advantageous
to measure antibody titers and determining
selective isotypes but can be used for point
of care testing [24].
Chemiluminescent Immunoassay
(CLIA)
Chemiluminescent Immunoassay is
advantages in the quantification of
antibodies over traditional assay detection
methods since the light-producing
chemical reactions occur as the substrate
reacts with the Avidin-Horseradish
Peroxidase allowed estimating the titers of
IgG and IgM considering the emitted
luminous signal [21].
Lateral Flow Immunoassay (LFIA)
Lateral Flow Immunoassay (LFIA) is
productive in SARS-CoV-2 testing can be
used for point-of-care testing and LFIA
provide results for both IgM and IgG
where it demonstrated higher sensitivity
and specificity in detection of both
isotypes, but a higher specificity was
observed with the detection of the IgG
isotype over the detection of the IgM
isotype alone [24].
Serology is important in detecting people
who have been subjected to this disease
and recovered but antibody cross-
reactivity can be a disadvantage yet can
overcome to a greater extent by using
RBD–based ELISA and rapid tests done
for IgG have given higher sensitivity and
specificity as recently performed ELISA,
indicating better results with higher
specificity and sensitivity in detecting
COVID-19 infection [25].

@2020 IJMSCRR All Rights Reserved (Vol 03, Issue 06 November – December) 2020 Page | 497
Summary
Infectious diseases caused by pathogens
can be severe or mild depending on the
pathogen virulence, environmental factors,
and host immunity. Serological tests and
PCR are widely used due to higher
specificity and sensitivity than other
diagnostic assays. Conventional
serological tests applicable to infectious
disease diagnosis mainly include
complement fixation, heamagglutination,
enzyme immunoassays, and immuno
diffusion. Rapid diagnostic tests include
point of care tests. Serological methods are
advantages but disadvantageous to a
certain extent whereas most of these
disadvantages can overcome by molecular
assays such as PCR. PCR assays are more
versatile in disease diagnosis and there are
many types according to the function
required. COVID -19 caused by SARS-
COV 2 virus, diagnosis carried out mainly
by real-time rt-PCR assay which is
sensitive, specific yet results in false
positive and negative. The use of real-
timert-PCR with clinical features aids in
managing the COVID-19 pandemic.
Serological tests for the identification of
specific IgG and/or IgM antibodies are
used for the diagnosis of COVID -19. This
includes mainly enzyme immune assay,
chemiluminescence immunoassay, and
lateral flow immune assay.
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