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Connecting clusters of COVID-19: an epidemiological and serological investigation


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Background Elucidation of the chain of disease transmission and identification of the source of coronavirus disease 2019 (COVID-19) infections are crucial for effective disease containment. We describe an epidemiological investigation that, with use of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological assays, established links between three clusters of COVID-19. Methods In Singapore, active case-finding and contact tracing were undertaken for all COVID-19 cases. Diagnosis for acute disease was confirmed with RT-PCR testing. When epidemiological information suggested that people might have been nodes of disease transmission but had recovered from illness, SARS-CoV-2 IgG serology testing was used to establish past infection. Findings Three clusters of COVID-19, comprising 28 locally transmitted cases, were identified in Singapore; these clusters were from two churches (Church A and Church B) and a family gathering. The clusters in Church A and Church B were linked by an individual from Church A (A2), who transmitted SARS-CoV-2 infection to the primary case from Church B (F1) at a family gathering they both attended on Jan 25, 2020. All cases were confirmed by RT-PCR testing because they had active disease, except for A2, who at the time of testing had recovered from their illness and tested negative. This individual was eventually diagnosed with past infection by serological testing. ELISA assays showed an optical density of more than 1·4 for SARS-CoV-2 nucleoprotein and receptor binding domain antigens in titres up to 1/400, and viral neutralisation was noted in titres up to 1/320. Interpretation Development and application of a serological assay has helped to establish connections between COVID-19 clusters in Singapore. Serological testing can have a crucial role in identifying convalescent cases or people with milder disease who might have been missed by other surveillance methods. Funding National Research Foundation (Singapore), National Natural Science Foundation (China), and National Medical Research Council (Singapore).
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Connecting clusters of COVID-19: an epidemiological and
serological investigation
Sarah Ee Fang Yong*, Danielle Elizabeth Anderson*, Wycliffe E Wei, Junxiong Pang, Wan Ni Chia, Chee Wah Tan, Yee Leong Teoh,
Priyanka Rajendram, Matthias Paul Han Sim Toh, Cuiqin Poh, Valerie T J Koh, Joshua Lum, Nur-Afidah Md Suhaimi, Po Ying Chia,
Mark I-Cheng Chen, Shawn Vasoo, Benjamin Ong, Yee Sin Leo, Linfa Wang*, Vernon J M Lee*
Background Elucidation of the chain of disease transmission and identification of the source of coronavirus disease
2019 (COVID-19) infections are crucial for eective disease containment. We describe an epidemiological investigation
that, with use of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological assays, established links
between three clusters of COVID-19.
Methods In Singapore, active case-finding and contact tracing were undertaken for all COVID-19 cases. Diagnosis for
acute disease was confirmed with RT-PCR testing. When epidemiological information suggested that people might
have been nodes of disease transmission but had recovered from illness, SARS-CoV-2 IgG serology testing was used
to establish past infection.
Findings Three clusters of COVID-19, comprising 28 locally transmitted cases, were identified in Singapore; these
clusters were from two churches (Church A and Church B) and a family gathering. The clusters in Church A and
Church B were linked by an individual from Church A (A2), who transmitted SARS-CoV-2 infection to the primary
case from Church B (F1) at a family gathering they both attended on Jan 25, 2020. All cases were confirmed by
RT-PCR testing because they had active disease, except for A2, who at the time of testing had recovered from their
illness and tested negative. This individual was eventually diagnosed with past infection by serological testing. ELISA
assays showed an optical density of more than 1·4 for SARS-CoV-2 nucleoprotein and receptor binding domain
antigens in titres up to 1/400, and viral neutralisation was noted in titres up to 1/320.
Interpretation Development and application of a serological assay has helped to establish connections between
COVID-19 clusters in Singapore. Serological testing can have a crucial role in identifying convalescent cases or people
with milder disease who might have been missed by other surveillance methods.
Funding National Research Foundation (Singapore), National Natural Science Foundation (China), and National
Medical Research Council (Singapore).
Copyright © 2020 Elsevier Ltd. All rights reserved.
As of April 15, 2020, more than 1·9 million cases of
coronavirus disease 2019 (COVID-19), and more than
120 000 deaths from the disease, have been recorded
worldwide.1 Many initial cases reported outside of China
were imported or were linked to travellers from China.2,3
However, as community transmission has become
widespread, the source of cases of COVID-19 in several
countries has not been established.
In Singapore, a globally connected city-state in southeast
Asia, health ocials have attempted to contain the spread
of COVID-19 through intensive epidemiological investi-
gations coupled with isolation of cases and quarantine of
close contacts. However, establishing the source of infec-
tion to ascertain the possible extent of spread can be
dicult, because scant epidemiological data might be
available. Even when possible nodes of transmission are
retrospectively identified through epidemiological investi-
gations, nucleic acid-based tests would not be diag-
nostically useful if these infected individuals have
recovered and no longer shed the virus. Hence, serological
tests are needed to identify convalescent cases and aid
investigations and containment eorts.
We present findings of investigations from Jan 29 to
Feb 24, 2020, that linked two people with COVID-19 from
Wuhan, China, to three clusters of COVID-19 cases in
Singapore. Serological testing had a crucial role in estab-
lishing a link between clusters, showing its use in
identifying convalescent COVID-19 cases and supporting
epidemiological investigations.
Surveillance methods and identification of cases
In Singapore, several surveillance methods are used to
identify people with COVID-19. On Jan 2, 2020, a suspect
case-definition of COVID-19 was circulated to all doctors
in Singapore;4 doctors are legally required to notify
the Ministry of Health of cases of COVID-19.5 From
Jan 31, 2020, Singapore began testing all patients
with pneumonia in hospital for severe acute respiratory
Lancet Infect Dis 2020
Published Online
April 21, 2020
See Online/Comment
*Contributed equally
Ministry of Health, Singapore
(S E F Yong MMed, W E Wei MPH,
P Rajendram MPH, C Poh MPH,
V T J Koh MPH, J Lum BA,
N-A M Suhaimi PhD, B Ong FRCP,
V J M Lee PhD); National
University Health System,
Singapore (S E F Yong, B Ong);
Programme in Emerging
Infectious Diseases,
Duke-National University
of Singapore Medical School,
Singapore (D E Anderson PhD,
W N Chia PhD, C W Tan PhD,
Prof L Wang PhD); National
Centre for Infectious Diseases,
Singapore (W E Wei,
M P H S Toh MMed,
P Y Chia MRCP, M I-C Chen PhD,
S Vasoo MRCP,
Prof Y S Leo FRCP); Centre for
Infectious Disease
Epidemiology and Research
(J Pang PhD), and
Saw Swee Hock School of
Public Health (J Pang,
Y L Teoh MMed, M P H S Toh,
M I-C Chen, V J M Lee), National
University of Singapore,
Singapore (B Ong); Singapore
Clinical Research Institute,
Singapore (Y L Teoh); Ministry
of Health Office for Healthcare
Transformation, Singapore
(Y L Teoh); and Tan Tock Seng
Hospital, Singapore (P Y Chia,
S Vasoo, Prof Y S Leo)
Correspondence to:
Dr Vernon J M Lee,
Communicable Diseases
Division, Ministry of Health,
Singapore 169854
2 Published online April 21, 2020
syndrome coronavirus 2 (SARS-CoV-2); this testing was
later expanded to include people with pneumonia in
primary care. The diagnosis of COVID-19 is confirmed
either by a respiratory sample testing positive for
SARS-CoV-2 using a laboratory-based RT-PCR6 or by a
serum sample testing positive for SARS-CoV-2 on sero-
logical analysis.7
Once individuals with COVID-19 were identified, their
activities from 14 days before symptom onset until they
were isolated were mapped and their close contacts traced.
Contact tracing before symptom onset was done to identify
the source of exposure that led to the case being infected,
allowing for further active case-finding around the source.
Contact tracing after symptom onset until isolation was
done to identify exposed individuals for quarantine to
break the transmission chain. Both these approaches were
part of the containment strategy. A close contact was
defined as anyone who had prolonged contact within 2 m
of the case. All close contacts with active or recent
symptoms were tested, whereas those who were asymp-
tomatic and exposed while the case was symptomatic were
quarantined. Activity maps were reviewed and cross-
checked to establish potential expo sures and identify
possible epidemiological links between cases and clusters.
All epidemiological investigations and outbreak con-
tain ment measures were implemented under the
Infectious Diseases Act,5 which allows use of data for
analysis to control outbreaks.
Laboratory techniques
For laboratory confirmation of COVID-19, we did RT-PCR
testing for SARS-CoV-2, using previously published
methods.6 Two serological platforms were developed for
confirmation of specific antibody responses to SARS-CoV-2
in people with suspected infection or individuals with
PCR-confirmed disease. A virus neutralisation test (VNT)
was established at the Duke-National University of
Singapore Medical School ABSL3 facility using a
SARS-CoV-2 virus isolate (BetaCoV/Singapore/2/2020;
GISAID accession number EPI_ISL_407987) cultured
from a patient in Singapore; VNT was done using protocols
previously published for severe acute respiratory syndrome
coronavirus (SARS-CoV).7 For ELISA assays, we used
recombinant nucleocapsid protein from SARS-CoV and
SARS-CoV-2 expressed in mammalian cell culture using
the pcDNA3.1 vector (ThermoFisher Scientific, Carlsbad,
CA, USA), according to previously published methods,8
and a recombinant receptor binding domain (RBD) of
the SARS-CoV-2 spike protein custom-produced by a
commercial provider (GenScript, Piscataway, NJ, USA).
ELISA wells were coated with 100 ng of the respective
protein per well and serum samples were used at dilutions
from 1/50 to 1/400, followed by horseradish peroxidase-
conjugated goat anti-human IgG (Santa Cruz, Dallas, TX,
USA) used at a dilution of 1/2000.
Role of the funding source
The funders had no role in study design, data collection,
data analysis, data interpretation, or writing of the report.
The corresponding author had full access to all data
in the study and had final responsibility for the decision
to submit for publication.
As of April 6, 2020, Singapore had recorded 1375 cases of
COVID-19, of which 554 were imported and 821 locally
transmitted. Three clusters were identified that involved
two churches (Church A and Church B) and one family
gathering. These clusters comprised 28 locally transmitted
cases. The clusters were linked to two travellers (W1 and
W2) from Wuhan, China, who attended a church service
at Church A on Jan 19, 2020 (figure 1). The clusters at
Research in context
Evidence before this study
We searched PubMed on March 3, 2020, for reports on
serological testing in individuals with coronavirus disease 2019
(COVID-19). We used the keywords (“COVID-19”, OR
“2019-nCoV”, OR “SARS-CoV-2”) AND (“serology” OR
“serologic testing”). Our search did not identify any reports
of the epidemiological application of serological tests in
COVID-19. In one report, researchers described serological
characteristics of COVID-19, and in other publications,
researchers have commented on the potential importance
of COVID-19 serological tests. In another study, epidemiological
investigations were reported of the epidemic in Singapore,
but serological methods had not been used.
Added value of this study
In our epidemiological investigation, we used RT-PCR and
serological testing to diagnose cases of COVID-19 and
establish links between clusters. RT-PCR testing alone
is limited by its ability to detect convalescent cases of
COVID-19, because RT-PCR can only detect severe acute
respiratory syndrome coronavirus 2 during the period of viral
shedding, which is the acute phase of infection. Serological
testing can be useful in detecting previous infection in people
with suspected infection who have recovered, assisting in
epidemiological investigation and containment efforts.
Implications of all the available evidence
COVID-19 laboratory testing is focused on use of quantitative
RT-PCR for diagnosis, and serological testing can be
overlooked. We have highlighted the importance of serological
testing for epidemiological investigation of COVID-19 cases,
and we urge further development of serological testing
Articles Published online April 21, 2020
Church A and Church B were linked by one individual
from Church A (A2), who probably transmitted the
infection to the primary case of the Church B cluster (F1)
at a family gathering on Jan 25, 2020. All cases were
confirmed by RT-PCR testing, except for A2, who was
diagnosed by serological testing.
The clusters at Church A and Church B were detected
in early February and mid-February, respectively.
Although W1 and W2 were diagnosed with COVID-19 at
the end of January, their possible link to Church A was
only discovered after the Church A cluster was identified,
through investigation and repeat interviews.5 By that
time, A2 had recovered from COVID-19 and was not
immediately linked to either cluster. Family members
who had been infected at the family gathering on
Jan 25, 2020, were first linked to F1 and were initially
regarded as part of the cluster at Church B. However,
subsequent investigations into case histories indicated
that the family cluster was a distinct cluster and that A2
was most probably the missing link between the two
church clusters; this idea was substantiated when A2’s
serological results were confirmed to be positive.
Five locally transmitted cases of COVID-19 (A1–A5)
were linked to Church A. These people attended a church
service on Jan 19, 2020, the same day W1 and W2 visited
the church. Although all five people had developed
symptoms by Feb 2, 2020 (figure 2), only A1, A4, and A5
were diagnosed (between Feb 6 and Feb 8, 2020), because
they had been hospitalised for pneumonia and tested for
SARS-CoV-2 as part of enhanced surveillance measures
to test all patients admitted to hospital with pneumonia.
A2 and A3 were not diagnosed when symptomatic in late
January because their symptoms were mild and they did
not meet the suspect case-definition at that time. A2 and
A3 were tested only after mapping of activities and
movements of other cases suggested that A2 could be the
missing link between the clusters at Church A and
Church B. An RT-PCR test of a nasopharyngeal specimen
taken from A3 on Feb 18, 2020, was positive, although
this individual had clinically recovered from the illness,
which persisted from Jan 28 to Feb 10, 2020. Serological
analysis of a serum sample obtained on the same day as
the nasopharyngeal specimen was also positive. Although
A2 had two negative RT-PCR tests, the serological result
was positive, indicating past infection.
A2 and A3 attended a Chinese New Year family
gathering on Jan 25, 2020, at the home of F1. Nine cases
(A2, A3, and F1–F7) were linked to this family gathering.
A2, whose symptoms started on Jan 23, 2020, was
unwell at this event and most probably was the primary
Figure 1: Transmission map of COVID-19
Map shows how COVID-19 was linked to two travellers from Wuhan, China, and two church clusters and a family gathering in Singapore. COVID-19=coronavirus
disease 2019.
Primary case of a cluster
Church A service Jan 19, 2020
Wuhan travellers
Church A
Family gathering
Church B
Possible transmission
Indirect link
Attended Church A service
B2 B3
January February
20 21 22 23 24 25 26 27 28 29 30 31 1234567891011121314151617
4 Published online April 21, 2020
Figure 2: Incubation period,
duration of symptoms, and
length of admission, from
Jan 14 to Feb 26, 2020
Data for 30 people with
coronavirus disease 2019 are
shown. Individuals are
labelled with cluster letter and
number, age (years) and sex
(M=male; F=female). Median
age of affected individuals
was 50·5 (IQR 25–79) years
and half the cohort (15 of 30)
were female.
Incubation period
Possible incubation period
Symptom onset until admission
Duration of admission
Length of current admission
Symptom onset until laboratory confirmation (not admitted)
Isolation date
Laboratory confirmation date
W1, 56F
Travellers from Wuhan
Church A cluster
Family gathering cluster
Church B cluster
W2, 56M
A1, 53M
A2, 58F
A3, 54M
A4, 39F
A5, 52F
F1, 28M
F2, 79F
F3, 27F
F4, 35F
F5, 38F
F6, 25M
F7, 30F
B1, 34M
B2, 46M
B3, 48M
B4, 55M
B5, 57M
B6, 51F
B7, 44F
B8, 56F
B9, 43M
B10, 54M
B11, 55F
B12, 54F
B13, 26M
B14, 57F
B15, 29M
B16 50M
14 19 24 29 3813 18 23 28
January February
Articles Published online April 21, 2020
source of transmission. A3 developed symptoms later,
on Jan 28, 2020, and was, therefore, unlikely to be the
source of infection at this gathering.
17 locally transmitted cases were linked to Church B
(B1–B16 and F1). Thorough review of activity maps
ascertained that F1, who had developed symptoms on
Jan 29, 2020, and continued to work at Church B while
ill, was the primary case of the Church B cluster.
In mid-February, epidemiological and clinical evidence
strongly suggested that A2 was the missing link between
the clusters at Church A and Church B through attendance
at the family gathering on Jan 25, 2020, while symptomatic.
However, by the time this link was ascertained, more than
3 weeks had passed from symptom onset on Jan 23, 2020,
and symptoms had resolved completely a week previously,
on Feb 8–10, 2020. A2 had two negative RT-PCR test
results from samples taken from the nasopharynx, with
testing done 1 day apart on Feb 18 and Feb 19, 2020. The
sample for serological testing was taken on Feb 18, 2020,
and a positive result was confirmed on Feb 22, 2020.
ELISA results for A2 and A3 on Feb 20, 2020 (figure 3A)
showed a strong antibody response to the SARS-CoV-2
RBD protein, which was not seen in serum samples
from SARS-CoV patients. The results were further
confirmed by VNT on Feb 22, 2020 (figure 3B).
This investigation shows how SARS-CoV-2 serological
analysis (ELISA detecting IgG and VNT detecting
neutralising antibodies), in addition to use of traditional
epidemiological methods, was important in establishing
links among locally transmitted COVID-19 cases and
tracing the transmission chain to an imported source.
Detection of COVID-19 can be dicult because of the non-
specific mild respiratory symptoms in many aected
individuals and because some people might recover
without being diagnosed.9 Although PCR tests oer a rapid
diagnostic solution, they can only detect SARS-CoV-2
during the period of viral shedding, which is the acute
phase of infection. The duration of viral shedding for
COVID-19 is not certain,10 but SARS-CoV data indicate that
21 days after symptom onset, 53% of cases achieved viral
clearance in nasopharyngeal aspirate samples.11 As such,
PCR testing alone is limited by its ability to detect
convalescent cases.
Serological testing can be especially useful in detecting
a previous suspected infection in people who have
recovered. For seroconversion kinetics, past coronavirus
studies indicate that all patients with Middle East
respiratory syndrome (MERS) seroconverted 3 weeks
after symptoms started,12 and 93% of patients with severe
acute respiratory syndrome (SARS) seroconverted at
an average of 20 days from symptom onset.11 The first
preliminary analysis of SARS-CoV-2 IgM and IgG
indicated that the antibody response in COVID-19
patients is similar to, if not earlier than, these times.13
Cross-reactivity of immunoglobulins to closely related
viruses such as SARS-CoV is a potential issue,14 but our
Figure 3: Serological testing of two patients
(A) ELISA testing using SARS-CoV-2 NP and RBD antigens. In addition to serum samples from the two suspected cases (A2 and A3), known positive samples for
COVID-19 and SARS, and negative samples, were included in the testing, and serial dilutions were done. (B) VNT results at the highest dilution that could effectively
neutralise the SARS-CoV-2 infection. COVID-19=coronavirus disease 2019. NP=nucleocapsid protein. RBD=receptor binding domain. SARS=severe acute respiratory
syndrome. SARS-CoV-2=severe acute respiratory syndrome coronavirus 2. VNT=virus neutralisation test.
Optical density
Optical density
1/50 1/100 1/200 1/400
SARSCOVID-19 Control A2 A3
1/80 1/160 1/80
1/160 1/320 1/320
1/160 1/160 1/80
6 Published online April 21, 2020
RBD-based ELISA showed sucient dierentiation
power, a finding further supported by VNT results.
IgM serological testing might hold promise as
a diagnostic method, although for SARS and MERS, limi-
tations for its use have been noted.12,13 For MERS, IgM was
not detected earlier than IgG, and IgM against prevalent
human coronaviruses showed cross-reactivity.12 Pre-
liminary data for SARS-CoV-2 IgM are promising,13 but
more work is needed to assess the feasibility of IgM
serological analysis as a rapid diagnostic method to
enhance COVID-19 detection capabilities.
For most people in the three clusters we report here,
transmission of infection was accounted for by close
contact with a symptomatic case. Our findings suggest
that COVID-19 is largely transmitted by close contact,
particularly when contact occurs over a prolonged period
and in close congregation. Two churches were the
setting for COVID-19 transmission in our report.
A church cluster has also been reported in South Korea.15
Churches host prolonged repeated activities, during
which close contact occurs, thus providing the oppor-
tunity for disease spread through droplets or fomites.
Singing (a common practice in churches) can generate
droplets in a similar quantity to coughing.16,17 Repeated
social interactions of church groups has also facilitated
discovery of transmission, compared with other settings
in which people might not know each other. The inter-
actions are similar in nature to large family gatherings,
which other cases in our report were linked to. Other
similar settings include school and workplaces, in which
respiratory disease transmission is not uncommon and
should be the focus for prepared ness, surveillance, and
containment measures.
Risk of transmission could be reduced if symptomatic
people do not attend events in which prolonged social
interactions take place (eg, at the family gathering when
A2 was unwell, and F1 who continued to work at
Church B while unwell). Other risk reduction measures
could include having smaller group activities and
prevention of interactions across these groups. Moreover,
there may be common touchpoints within each setting
that could result in contact transmission. To prevent
transmission, people should practice increased personal
hygiene and reduce physical contact to minimise indirect
transmission risks.
Linking disease transmission to an imported source
and contact tracing for each identified case has facilitated
a high capture of cases in Singapore. This successful
linking of a large proportion of cases to imported sources
provides encouraging evidence that the intense contain-
ment measures undertaken in Singapore have been
eective. The three clusters we report here occurred
relatively early in the emergence of COVID-19 in
Singapore, within 4 weeks of the first imported case.
As the epidemic continues, it might be progressively
dicult to establish linkages by relying on traditional
epidemiological methods alone. Challenges include
diculty in obtaining information from cases and
contacts, which could be inaccurate because of recall and
other biases. In our investigation, information for
symptom onset of W1 and W2 was based on case history
alone and could not be independently corroborated.5
In such instances, determining the transmission chain
would have to account for possible inconsistencies in
case history and rely on triangulation with other methods.
The development and adoption of additional laboratory
techniques, such as serological tests and phylogenetic
analysis by whole-genome sequencing, could help
identify possible links between cases.
Serological testing is a key method in the response to
the COVID-19 epidemic. As shown in our study, it enabled
detection of a convalescent case, which could be key in
initial containment eorts to discover transmission links
to support containment eorts. Serological testing also
detects people with mild or asymptomatic disease who
have recovered, allowing for more accurate deter mination
of the number of people probably infected in a cluster or
the population. Identifying people who were probably
infected in household or school clusters could help
ascertain attack rates by age, particularly among children
who mostly manifest less severe disease.18 Calculating
population-level attack rates is also important to estimate
disease incidence and the case-fatality rate (CFR). Thus
far, the CFR for COVID-19 has been based on PCR-
diagnosed symptomatic cases. Serological surveys would
be important to estimate CFR more accurately and would
better inform calibrated responses to COVID-19. As the
pandemic progresses, monitoring seroprevalence would
enable countries to track transmission dynamics and
population immunity levels and to inform disease
control policies. Such monitoring would necessitate the
development of ready serological testing solutions that are
cost-eective and the establishment of population-level
surveillance prog rammes to obtain blood samples.
Development and application of serological assays
has helped to unravel connections between three
clusters of COVID-19 in Singapore, linking the disease
to two travellers from China. Serological assays should
be considered to identify mild or subclinical infections
in the community.
VJML and YLT had the idea for the study and contributed to study design.
CP, VTJK, JL, N-AMS, and PYC contributed to the epidemiological
investigation and data collection. VJML, YLT, SEFY, PR, MPHST, JP, and
WEW contributed to analysis and interpretation of epidemiological data.
LW, DEA, WNC, and CWT developed the serological methods and
contributed to the analysis and interpretation. SEFY, WEW, and JP wrote
the report, and VJML, MI-CC, SV, BO, YSL, LW, and DEA contributed to
critical revision.
Declaration of interests
We declare no competing interests.
The development of serological tests was funded in part by a joint grant
under the National Research Foundation (Singapore) and the National
Natural Science Foundation of China (NRF2016NRFNSFC002-013).
The study was partly funded by the National Medical Research Council
Articles Published online April 21, 2020
(Singapore) COVID19 research fund (COVID19RF-001). We thank our
colleagues who contributed to the investigations, including the
COVID-19 epidemiology workgroup, contact tracing team, and hospital
epidemiology teams; and Viji Vijayan, Benson Ng, and Velraj Sivalingam,
(Duke-National University of Singapore ABSL3 facility) for logistics
management and assistance.
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2019 (COVID-19) in Zhejiang, China: an observational cohort study.
Lancet Infect Dis 2020; published online March 25.
... The key-like paradigm as the same as antibody-antigen is passed by angiotensin-converting enzyme -2 (ACE2), through which the coronavirus is hooked into and entered in the lung's cells [10]. The onset of the disease causes respiratory failure due to alveolar injury [10,11]. Generally, there are two subunits in the coronavirus's structure through which the virus could be attached to the cell membrane and internalized. ...
... Swabs must be refrigerated to be safely transferred to a microbiology laboratory. RT-PCR is an accurate diagnostic method that diagnoses infection to SARS-CoV-2, at the acute stage of infection [11]. COVID-19 diagnosis kits target regions on a gene that codes for the protein that makes the virus's nucleocapsid, an envelope that houses its RNA [34,35]. ...
... On the contrary, serological assays detecting antibodies against SARS-CoV-2 not only indicate the previous infection but also help to track the immune response of the host 66,67 . Furthermore, serological analysis is an effective supplement to nucleic acid testing in COVID-19 J o u r n a l P r e -p r o o f epidemiological studies and vaccine development 68,69 . Traditional methods for COVID-19 management, including enzyme-linked immunosorbent assays (ELISA) and chemiluminescent immunoassays (CLIA), are currently available. ...
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The ongoing coronavirus disease 2019 (COVID-19) pandemic has boosted the development of antiviral research. Microfluidic technologies offer powerful platforms for diagnosis and drug discovery for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnosis and drug discovery. In this review, we introduce the structure of SARS-CoV-2 and the basic knowledge of microfluidic design. We discuss the application of microfluidic devices in SARS-CoV-2 diagnosis based on detecting viral nucleic acid, antibodies, and antigens. We highlight the contribution of lab-on-a-chip to manufacturing point-of-care equipment of accurate, sensitive, low-cost, and user-friendly virus-detection devices. We then investigate the efforts in organ-on-a-chip and lipid nanoparticles (LNPs) synthesizing chips in antiviral drug screening and mRNA vaccine preparation. Microfluidic technologies contribute to the ongoing SARS-CoV-2 research efforts and provide tools for future viral outbreaks.
... For example, regarding actions focusing on global scenarios, the data sharing of genomes and genotypes from the different countries has become essential in establishing policies for defining travel bans [11], confinement measures and border closures. On the national level, genetic data is an important tool when applied to trace transmission within borders [12], to identify super-spreading events through molecular epidemiology [13], to test the level of efficiency of the vaccination on local spreading [14] and even to propose measures leading to disease eradication [15]. On a global level, complete genomics applied to the populations' genetics and phylogeography enabled the understanding of different stages of the pandemic and also enabled people to follow the spread of the virus locally and worldwide [16,17]. ...
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Following the emergence of COVID-19 in December 2019, caused by the coronavirus SARS-CoV-2, the disease spread dramatically worldwide. The use of genomics to trace the dissemination of the virus and the identification of novel variants was essential in defining measures for containing the disease. We aim to evaluate the global effort to genomically characterize the circulating lineages of SARS-CoV-2, considering the data deposited in GISAID, the major platform for data sharing in a massive worldwide collaborative undertaking. We contextualize data for nearly three years (January 2020-October 2022) for the major contributing countries, percentage of characterized isolates and time for data processing in the context of the global pandemic. Within this collaborative effort, we also evaluated the early detection of seven major SARS-CoV-2 lineages, G, GR, GH, GK, GV, GRY and GRA. While Europe and the USA, following an initial period, showed positive results across time in terms of cases sequenced and time for data deposition, this effort is heterogeneous worldwide. Given the current immunization the major threat is the appearance of variants that evade the acquired immunity. In that scenario, the monitoring of those hypothetical variants will still play an essential role.
... In general, the sources of an infectious disease spread in a given reservoir indicates with their subsequent clusters the possible existence of an outbreak, but this search is in general very long and difficult as for HIV in North America [4]. Concerning these initial clusters, from observations made during investigations of the start of the outbreak in some countries [5,6,7,8,9,10,11,12,13,14,15,16,17,18,19], it is sometime possible to get spatial and temporal information on the start of the epidemy, but rarely these studies allows estimating the parameters S(0) and τ (0) in the concerned population and worse, they give no indication of how long they remain constant. This is the reason why we assumed that they remained constant only during the period of exponential growth of new cases observed. ...
This thesis is devoted to the mathematical and statistical modeling of epidemic data andIt is divided into two broad parts, which are subdivided into different sections. The modeling of infectious diseases has been a subject of interest to researchers, policy makers, andmedical practitioners, most especially during the recent global COVID-19 pandemic, whichIt has been devastating to the health infrastructure and socio-economic status of many nations.It has affected mobility and interaction among citizens due to the many daily new cases and deaths.Hence, the need to contribute to understanding the mechanisms of virulence and spread using different mathematical and statistical modeling approaches. The first part is dedicated to the mathematical modeling aspect, which consists of the deterministic and discrete approaches to epidemiology modeling, which in this case is mainly focused on the COVID-19 pandemic. The daily reproduction number of the COVID-19 outbreak calculation is approached by discretization using the idea of deconvolution and a unique biphasic pattern is observed that is more prevalent during the contagiousness period across various countries. Furthermore, a discrete model is formulated from Usher’s model in order to calculate the life span loss due to COVID-19 disease and to also explain the role of comorbidities, which are very essential in the disease spread and its dynamics at an individual level. Also, the formulation of Susceptible-Infectious-Geneanewsusceptible-Recovered (SIGR) age-dependent modelling is proposed in order to perform some mathematical analysis and present the role of different epidemiology parameters, most especially vaccination, and finally, a new technique to identify the point of inflection on the smoothed curves of the new infected pandemic cases using the Bernoulli equation is presented. This procedure is important because not all countries have reached the turning point (maximum number of daily cases) in the epidemic curve. The approach is used to calculate the transmission rate and the maximum reproduction number for various countries.The statistical modelling of the COVID-19 pandemic using various data analysis models (namely machine and deep learning models) is presented in the second part in order to understand the dynamics of the pandemic in different countries and also predict and forecast the daily new cases and deaths due to the disease alongside some socio-economic parameters. It is observed that the prediction and forecasting are consistent with the disease evolution at different waves in these countries and that there are socio-economic determinants of the disease depending on whether the country is developed or developing. Also, the study of the shapes and peaks of the COVID-19 disease is presented. The peaks of the curves of the daily new cases and deaths are identified using the spectral analysis method, which enables the weekly peak patterns to be visible. Finally, the clustering of different regions in France due to the spread of the disease is modeled using functional data analysis. The study shows clear differences between the periods when vaccination has not been introduced (but only non-pharmaceutical mitigation measures) and when it was introduced. The results presented in this thesis are useful to better understand the modeling of a viral disease, the COVID-19 virus.
... The vaccine induces a cellular and humoral response mediated by T cells and antibodies. Most current COVID-19 vaccine candidates concentrate on spike proteins [100], which are also prime neutralizing antibody targets [101,102]. ...
In the biological immune process, the major histocompatibility complex (MHC) plays an indispensable role in the expression of HLA molecules in the human body when viral infection activates the T-cell response to remove the virus. Since the first case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in 2019, how to address and prevent SARS-CoV-2 has become a common problem facing all mankind. The T-cell immune response activated by MHC peptides is a way to construct a defense line and reduce the transmission and harm of the virus. Presentation of SARS-CoV-2 antigen is associated with different types of HLA phenotypes, and different HLA phenotypes induce different immune responses. The prediction of SARS-CoV-2 mutation information and the design of vaccines based on HLAs can effectively activate autoimmunity and cope with virus mutations, which can provide some references for the prevention and treatment of SARS-CoV-2.
... Detection of antibodies to SARS-CoV-2-specific protein in serum plays a very important role in the detection of prior infection with the new coronavirus [14]. Zhang et al. developed a rapid and sensitive magnetofluidic immuno-PCR technique [15], which preloads a programmable magnetic arm in the analytical reagent to attract and transport magnetically captured specific antibodies, followed by the use of a microthermal immuno-PCR performed with a circulator and a fluorescence detector to detect specific antibodies. ...
In 2019, a new coronavirus was identified that has caused significant morbidity and mortality worldwide. Like all RNA viruses, severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) evolves over time through random mutation resulting in genetic variations in the population. Although the currently approved coronavirus disease 2019 vaccines can be given to those over 5 years of age and older in most countries, strikingly, the number of people diagnosed positive for SARS-Cov-2 is still increasing. Therefore, to prevent and control this epidemic, early diagnosis of infected individuals is of great importance. The current detection of SARS-Cov-2 coronavirus variants are mainly based on reverse transcription-polymerase chain reaction. Although the sensitivity of reverse transcription-polymerase chain reaction is high, it has some disadvantages, for example, multiple temperature changes, long detection time, complicated operation, expensive instruments, and the need for professional personnel, which brings considerable inconvenience to the early diagnosis of this virus. This review comprehensively summarizes the development and application of various current detection technologies for novel coronaviruses, including isothermal amplification, CRISPR-Cas detection, serological detection, biosensor, ensemble, and microfluidic technology, along with next-generation sequencing. Those findings offer us a great potential to replace or combine with reverse transcription-polymerase chain reaction detection to achieve the purpose of allowing predictive diagnostics and targeted prevention of SARS-Cov-2 in the future.
... The situation surrounding the COVID-19 pandemic has changed drastically over the last two years. Multiple serologic tests for SARS-CoV-2 have been developed and used for various indications such as: diagnosing recent or past infections, performing sero-prevalence studies assessing herd immunity, sero-epidemiologic tracing of outbreak clusters, and risk assessment of healthcare workers; preparing convalescence plasma (CP) therapy, assessing neutralizing antibodies in COVID-19 patients, and evaluating protective immunity from past infections and/or vaccinations; according to the status of the COVID-19 pandemic, the significance of each clinical implication has differed (Ko et al., 2017a;Ko et al., 2017b;Ko et al., 2017c;Ko et al., 2018;Ahn et al., 2020;Ko et al., 2020a;Ko et al., 2020b;Yong et al., 2020;van Kampen et al., 2021). Among these clinical implications, while the presence of binding antibodies is important for seroprevalence studies to distinguish previous infections, the detection and quantification of neutralizing antibodies are crucial for several indications, including the preparation of CP therapy, assessment of neutralizing antibodies in COVID-19 patients, and evaluation of protective immunity. ...
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Contact tracing is a non-pharmaceutical intervention (NPI) widely used in the control of the COVID-19 pandemic. Its effectiveness may depend on a number of factors including the proportion of contacts traced, delays in tracing, the mode of contact tracing (e.g. forward, backward or bidirectional contact training), the types of contacts who are traced (e.g. contacts of index cases or contacts of contacts of index cases), or the setting where contacts are traced (e.g. the household or the workplace). We performed a systematic review of the evidence regarding the comparative effectiveness of contact tracing interventions. 78 studies were included in the review, 12 observational (ten ecological studies, one retrospective cohort study and one pre-post study with two patient cohorts) and 66 mathematical modelling studies. Based on the results from six of the 12 observational studies, contact tracing can be effective at controlling COVID-19. Two high quality ecological studies showed the incremental effectiveness of adding digital contact tracing to manual contact tracing. One ecological study of intermediate quality showed that increases in contact tracing were associated with a drop in COVID-19 mortality, and a pre-post study of acceptable quality showed that prompt contact tracing of contacts of COVID-19 case clusters / symptomatic individuals led to a reduction in the reproduction number R. Within the seven observational studies exploring the effectiveness of contact tracing in the context of the implementation of other non-pharmaceutical interventions, contact tracing was found to have an effect on COVID-19 epidemic control in two studies and not in the remaining five studies. However, a limitation in many of these studies is the lack of description of the extent of implementation of contact tracing interventions. Based on the results from the mathematical modelling studies, we identified the following highly effective policies: (1) manual contact tracing with high tracing coverage and either medium-term immunity, highly efficacious isolation/quarantine and/ or physical distancing (2) hybrid manual and digital contact tracing with high app adoption with highly effective isolation/ quarantine and social distancing, (3) secondary contact tracing, (4) eliminating contact tracing delays, (5) bidirectional contact tracing, (6) contact tracing with high coverage in reopening educational institutions. We also highlighted the role of social distancing to enhance the effectiveness of some of these interventions in the context of 2020 lockdown reopening. While limited, the evidence from observational studies shows a role for manual and digital contact tracing in controlling the COVID-19 epidemic. More empirical studies accounting for the extent of contact tracing implementation are required.
Enquadramento: a pandemia por COVID-19 implicou na indústria fortes medidas ocupacionais e individuais. Objetivos: avaliar a incidência de SARS-CoV-2 e cadeias de transmissão, controlar a propagação da doença e avaliar a presença de anticorpos anti SARS-CoV-2. Metodologia: estudo descritivo longitudinal prospetivo (ensaio laboratorial) conduzido após aparecimento de um caso de SARS-CoV-2 numa indústria de madeira da região Norte de Portugal. Participaram no estudo 873 trabalhadores (idade média: 41,77 anos, 50,4% homens). Foram implementadas medidas de isolamento e desinfeção dos espaços e rastreio à população trabalhadora, e uma combinação de testes serológicos e de RT-PCR. Resultados: o caso-índice tem link epidemiológico fora da referida indústria. 31 trabalhadores (3,55%) apresentaram resultado positivo nos testes serológicos, tendo sido sujeitos a teste RT-PCR, de que resultou um novo caso. Posteriormente, 31 trabalhadores, foram re-testados com testes serológicos, verificando-se 10 testes positivos para IgM e 2 para IgG. Conclusão: o teste serológico cujo resultado é positivo ou negativo, por si só, não deve constituir prova (exclusão) infeção. Para limitar a disseminação do vírus é crucial garantir o seu diagnóstico com teste RT-PCR. A associação promissora entre a IgG e a imunidade carece de melhor evidencia. Com as medidas implementadas foi possível controlar a disseminação da doença e promover o regresso ao trabalho.
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In December 2019, a novel coronavirus (2019-nCoV) caused an outbreak in Wuhan, China, and soon spread to other parts of the world. It was believed that 2019-nCoV was transmitted through respiratory tract and then induced pneumonia, thus molecular diagnosis based on oral swabs was used for confirmation of this disease. Likewise, patient will be released upon two times of negative detection from oral swabs. However, many coronaviruses can also be transmitted through oral–fecal route by infecting intestines. Whether 2019-nCoV infected patients also carry virus in other organs like intestine need to be tested. We conducted investigation on patients in a local hospital who were infected with this virus. We found the presence of 2019-nCoV in anal swabs and blood as well, and more anal swab positives than oral swab positives in a later stage of infection, suggesting shedding and thereby transmitted through oral–fecal route. We also showed serology test can improve detection positive rate thus should be used in future epidemiology. Our report provides a cautionary warning that 2019-nCoV may be shed through multiple routes.
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Mechanistic hypotheses about airborne infectious disease transmission have traditionally emphasized the role of coughing and sneezing, which are dramatic expiratory events that yield both easily visible droplets and large quantities of particles too small to see by eye. Nonetheless, it has long been known that normal speech also yields large quantities of particles that are too small to see by eye, but are large enough to carry a variety of communicable respiratory pathogens. Here we show that the rate of particle emission during normal human speech is positively correlated with the loudness (amplitude) of vocalization, ranging from approximately 1 to 50 particles per second (0.06 to 3 particles per cm3) for low to high amplitudes, regardless of the language spoken (English, Spanish, Mandarin, or Arabic). Furthermore, a small fraction of individuals behaves as “speech superemitters,” consistently releasing an order of magnitude more particles than their peers. Our data demonstrate that the phenomenon of speech superemission cannot be fully explained either by the phonic structures or the amplitude of the speech. These results suggest that other unknown physiological factors, varying dramatically among individuals, could affect the probability of respiratory infectious disease transmission, and also help explain the existence of superspreaders who are disproportionately responsible for outbreaks of airborne infectious disease.
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Background: The MERS coronavirus causes isolated cases and outbreaks of severe respiratory disease. Essential features of the natural history of disease are poorly understood. Methods: We studied 37 adult patients infected with MERS-CoV for viral load in the lower and upper respiratory tract (LRT and URT), blood, stool, and urine. Antibodies and serum neutralizing activities were determined over the course of disease. Results: 199 LRT samples collected during the 3 weeks following diagnosis yielded virus RNA in 93% of tests. Average (maximum) viral loads were 5x10(6) (6x10(10)) copies per mL. Viral loads (positive detection frequencies) in 84 URT samples were 1.9x10(4) cop/mL (47.6%). 33% of all 108 sera tested yielded viral RNA. Only 14.6% of stool and 2.4% of urine samples yielded viral RNA. All seroconversions occurred during the first 2 weeks after diagnosis, which corresponds to the 2nd and 3rd week after symptoms onset. IgM detection provided no advantage in sensitivity over IgG detection. All surviving patients, but only slightly more than half of all fatal cases, produced IgG and neutralizing antibodies. The levels of IgG and neutralizing antibodies were weakly and inversely correlated with LRT viral loads. Presence of antibodies did not lead to the elimination of virus from LRT. Conclusions: The timing and intensity of respiratory viral shedding in MERS patients closely matches that of Severe Acute Respiratory Syndrome (SARS) patients. Blood viral RNA does not seem to be infectious. Extra-pulmonary loci of virus replication seem possible. Neutralizing antibodies do not suffice to clear the infection.
目的: 新型冠状病毒肺炎在武汉暴发流行以来,已在全国范围内蔓延。对截至2020年2月11日中国内地报告所有病例的流行病学特征进行描述和分析。 方法: 选取截至2020年2月11日中国内地传染病报告信息系统中上报所有新型冠状病毒肺炎病例。分析包括:①患者特征;②病死率;③年龄分布和性别比例;④疾病传播的时空特点;⑤所有病例、湖北省以外病例和医务人员病例的流行病学曲线。 结果: 中国内地共报告72 314例病例,其中确诊病例44 672例(61.8%),疑似病例16 186例(22.4%),临床诊断病例10 567例(14.6%),无症状感染者889例(1.2%)。在确诊病例中,大多数年龄在30~79岁(86.6%),湖北省(74.7%),轻/中症病例为主(80.9%)。确诊病例中,死亡1 023例,粗病死率为2.3%。个案调查结果提示,疫情在2019年12月从湖北向外传播,截至2020年2月11日,全国31个省的1 386个县区受到了影响。流行曲线显示在1月23-26日达到峰值,并且观察到发病数下降趋势。截至2月11日,共有1 716名医务工作者感染,其中5人死亡,粗病死率为0.3%。 结论: 新型冠状病毒肺炎传播流行迅速,从首次报告病例日后30 d蔓延至31个省(区/市),疫情在1月24-26日达到首个流行峰,2月1日出现单日发病异常高值,而后逐渐下降。随着人们返回工作岗位,需积极应对可能出现的疫情反弹。.
Background Since December, 2019, an outbreak of coronavirus disease 2019 (COVID-19) has spread globally. Little is known about the epidemiological and clinical features of paediatric patients with COVID-19. Methods We retrospectively retrieved data for paediatric patients (aged 0–16 years) with confirmed COVID-19 from electronic medical records in three hospitals in Zhejiang, China. We recorded patients' epidemiological and clinical features. Findings From Jan 17 to March 1, 2020, 36 children (mean age 8·3 [SD 3·5] years) were identified to be infected with severe acute respiratory syndrome coronavirus 2. The route of transmission was by close contact with family members (32 [89%]) or a history of exposure to the epidemic area (12 [33%]); eight (22%) patients had both exposures. 19 (53%) patients had moderate clinical type with pneumonia; 17 (47%) had mild clinical type and either were asymptomatic (ten [28%]) or had acute upper respiratory symptoms (seven [19%]). Common symptoms on admission were fever (13 [36%]) and dry cough (seven [19%]). Of those with fever, four (11%) had a body temperature of 38·5°C or higher, and nine (25%) had a body temperature of 37·5–38·5°C. Typical abnormal laboratory findings were elevated creatine kinase MB (11 [31%]), decreased lymphocytes (11 [31%]), leucopenia (seven [19%]), and elevated procalcitonin (six [17%]). Besides radiographic presentations, variables that were associated significantly with severity of COVID-19 were decreased lymphocytes, elevated body temperature, and high levels of procalcitonin, D-dimer, and creatine kinase MB. All children received interferon alfa by aerosolisation twice a day, 14 (39%) received lopinavir–ritonavir syrup twice a day, and six (17%) needed oxygen inhalation. Mean time in hospital was 14 (SD 3) days. By Feb 28, 2020, all patients were cured. Interpretation Although all paediatric patients in our cohort had mild or moderate type of COVID-19, the large proportion of asymptomatic children indicates the difficulty in identifying paediatric patients who do not have clear epidemiological information, leading to a dangerous situation in community-acquired infections. Funding Ningbo Clinical Research Center for Children's Health and Diseases, Ningbo Reproductive Medicine Centre, and Key Scientific and Technological Innovation Projects of Wenzhou.
Background Three clusters of coronavirus disease 2019 (COVID-19) linked to a tour group from China, a company conference, and a church were identified in Singapore in February, 2020. Methods We gathered epidemiological and clinical data from individuals with confirmed COVID-19, via interviews and inpatient medical records, and we did field investigations to assess interactions and possible modes of transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Open source reports were obtained for overseas cases. We reported the median (IQR) incubation period of SARS-CoV-2. Findings As of Feb 15, 2020, 36 cases of COVID-19 were linked epidemiologically to the first three clusters of circumscribed local transmission in Singapore. 425 close contacts were quarantined. Direct or prolonged close contact was reported among affected individuals, although indirect transmission (eg, via fomites and shared food) could not be excluded. The median incubation period of SARS-CoV-2 was 4 days (IQR 3–6). The serial interval between transmission pairs ranged between 3 days and 8 days. Interpretation SARS-CoV-2 is transmissible in community settings, and local clusters of COVID-19 are expected in countries with high travel volume from China before the lockdown of Wuhan and institution of travel restrictions. Enhanced surveillance and contact tracing is essential to minimise the risk of widespread transmission in the community. Funding None.