himpunan riset covid19

Fig. 2. Clinical picture and disease progression of case. ROSC, return of spontaneous circulation; iv,
intravenous; †, died. Y-axis scale: SBP, systolic blood pressure (mmHg, violet triangle); HR, heart rate
(times/min, red circle); RR, respiratory rate (times/min, green circle); T, temperature (◦C, blue
circle);SpO2, oxygen saturation (%, yellow circle). (For interpretation of the references to colour in this
figure legend, the reader is referred to the Web version of this article.)

Pneumonia analysis of COVID-19 shows that damage, thickening of the vascular walls, and
the air sacs in the lungs of patients are not filled formation of microthrombi that clog capillaries.
with fluid or pus as in pneumonia infections in Hypercoagulable conditions accelerate the
general but instead the virus causes the water worsening of V/Q mismatch and lung tissue
sacs to collapse, thereby reducing the oxygen damage [16].
level and causing hypoxia in the patient, but the Risk factors for silent hypoxemia are old age
reaction still enhances the normal lung ability to and having diabetes [9] and these factors are
expel carbon dioxide. Since carbon dioxide known to blunt the body’s regulatory response to
removal is still effective, patients do not feel hypoxia [17], as in our case: a 60-year-old-male
shortness of breath [15]. Another mechanism with comorbidity of diabetes mellitus. Therefore,
proposed is intra- pulmonary shunting. Infection early detection of silent hypoxemia such as by
causes interstitial edema, loss of surfac- tant using prehospital pulse oximetry [5], or radiology
and superimposed pressure, which induces imaging [18,19] might provide some red flag
alveolar collapse and substantial fraction of signs of impending danger of eminent cardiac
cardiac output perfusing non-aerated lung arrest or sudden respiratory failure.
tissue. Over time, increased edema will increase
lung weight, alveolar collapse, and dependent 4. Conclusions
atelectasis, leading to increased shunting [16]. Silent hypoxemia might be considered as an
Endothelial injury as a central hallmark in the early clinical sign of deterioration of patients with
pathogenesis of COVID-19 is thought to also COVID-19, thus, the physician may be able to
play a role in the mechanism of silent intervene early and decrease its morbidity and
hypoxemia. SARS-CoV-2 can directly infect lung mortality.
capillary endothelial cells expressing ACE2.
Endothelial injury and acute inflammation pro- Consent Of Patient
voke the formation of intravascular Written informed consent was obtained from the
microthrombi. Lung autopsies in patients after patient for publi- cation of this case report and
severe disease have shown diffuse alveolar accompanying images. A copy of the written

consent is available for review by the Editor-in- of up-regulated proteins in human
Chief of this journal on request. promonocyte cells expressing severe acute
respiratory syndrome coronavirus 3C-like
Provenance And Peer Review protease, Proteomics 7 (2007) 1446–1460.
Not commissioned, externally peer reviewed. 7. W.J. Guan, Z.Y. Ni, Y. Hu, W.H. Liang, C.Q.
Ou, J.X. He, L. Liu, H. Shan, C.L. Lei, D.
Acknowledgement 8. S. Hui, B. Du, Clinical characteristics of
This study was funded by Indonesia Ministry of coronavirus disease 2019 in China, N. Engl.
Research and Technology/National Agency for J. Med. 382 (2020) 1708–1720.
Research and Innovation. 9. M.J. Tobin, F. Laghi, A. Jubran, Why COVID-
19 silent hypoxemia is baffling to physicians,
List of Abbreviations Am. J. Respir. Crit. Care Med. 202 (3) (2020)
• SARS-CoV-2 Severe Acute Respiratory 356–360.
10.A.R. Bourgonje, A.E. Abdulle, W. Timens,
Syndrome Coronavirus 2 COVID-19 J.L. Hillebrands, G.J. Navis, S. J. Gordijn,
Coronavirus Disease 2019 M.C. Bolling, G. Dijkstra, A.A. Voors, A.D.
• RR-Respiratory rate Osterhaus, P.H. van der Voort, Angiotensin-
• ACE2-Angiotensin-converting-enzyme 2 converting enzyme-2 (ACE2), SARS-CoV-2
• RAAS-renin-angiotensin-aldosterone system and pathophysiology of coronavirus disease
2019 (COVID-19), J. Pathol. 251 (3) (2020)
Appendix A. Supplementary Data 228–248.
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PROLONGED NUCLEIC ACID CONVERSION AND FALSE-NEGATIVE RT-PCR
RESULTS INPATIENTS WITH COVID-19: A CASE SERIES

Ika Trisnawatia,**, Riat El Khair b, Dyah Ayu Puspitaranic, Aditya Rifqi Fauzic, Gunadi c,*

aPulmonology Division, Department of Internal Medicine, Faculty of Medicine, Public Health and
Nursing, Universitas Gadjah Mada/Dr. Sardjito Hospital, Yogyakarta, 55281, Indonesia
bDepartment of Clinical Pathology and Laboratory Medicine, Faculty of Medicine, Public Health and
Nursing, Universitas Gadjah Mada/Dr. Sardjito Hospital,Yogyakarta, 55281, Indonesia
cGenetics Working Group, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah
Mada/Dr. Sardjito Hospital, Yogyakarta, 55281, Indonesia

ABSTRACT
Background: Prolonged nucleic acid conversion and false-negative real-time polymerase chain
reaction (RT-PCR) results might occur in COVID-19 patients rather than infection recurrence
Presentation of cases: We reported four cases who had negative RT-PCR results, in addition to
the last two consecutive negative results. Patient-1 had negative RT-PCR results twice (the 6th and
8th) from a total of 11 swabs. Patient-2 had negative RT-PCR results once (the 5th) from a total of 8
swabs. Patient-3 showed negative results of RT-PCR twice (the 4th and 6th) from a total of 11
swabs. Patient-4 had negative RT-PCR results twice (the 2nd and 10th) from a total of 14 swabs.
Discussion: The fluctuating trend of our RT-PCR results in our cases might be due to insufficient
viral material in the specimen, laboratory errors during sampling, restrictions on sample
transportation, or mutations in the primary and probe target regions in the SARS-CoV-2 genome.
Several factors might affect the occurrence of prolonged nucleic acid conversion, including older
age, comorbidities, such as diabetes and hypertension, and impaired immune function.
Conclusion: Here, we confirmed the occurrence of prolonged nucleic acid conversion and the
possibility of false negative RT-PCR results in COVID-19 patients.

Keywords: COVID-19, Case series, False negative of RT-PCR Prolonged nucleic acidconversion
SARS-Cov-2

1. Introduction Nucleic acid conversion is defined as the
In December 2019, an outbreak of infection period from the date of symptom onset to
of severe acute respi- ratory syndrome- the date of the first negative real-time
coronavirus-2 (SARS-CoV-2) was detected reverse transcription polymerase chain
in Wuhan, China. This virus has the reaction (RT-PCR) test result [7]. It has
etiology of Coronavirus disease 2019 been hypothesized that prolonged nucleic
(COVID- 19), first announced by the World acid conversion and false negative results
Health Organization (WHO) on January 12, of RT-PCR occur in some patients with
2020, and now becomes a global pandemic COVID-19 rather than recurrence of
[1,2]. The length of the virus incubation infection [7]. Here, we report four cases of
period is approximately five days [3]. COVID-19 with the possibility of prolonged
Several studies [4,5] reported a median nucleic acid conversion and false negative
length of viral shedding between 12 and 20 results of RT-PCR in our institution,
days from the onset of symptoms. Indonesia. This case series has been
Previously, cases of prolonged nucleic acid reported in line with the PROCESS criteria
conversion have also been reported, with [8].
the longest reported being 60 days [6].

2. Presentation Of Cases antibiotics and antiviral therapy based on
2.1 Case 1
the COVID-19 Prevention and Control
A 36-year-old male patient complained of
coughing up phlegm for one week before guidelines by the Indonesian Ministry of
admission. He had a history of contact with
a positive confirmed case of COVID-19 on Health, namely, azithromycin,
March 13, 2020, and swab tests were
conducted on March 24 and 25, 2020, with hydroxychloroquine, oseltamivir,
positive results. The physical examination
recorded a blood pressure of 187/94 lopinavir-ritonavir, and umifenovir. SARS-
mmHg, with normal results on his
remaining vital signs. Lung auscultation CoV-2 retesting with nasopharyngeal and
revealed no apparent abnormality. Chest
X-rays showed the appearance of mild oropharyngeal swabs was performed with
pneumonia in the right lung (Fig. 1). Blood
tests showed an increase in the positive results in the 3rd until 5th test. In
neutrophil-lymphocyte ratio (NLR) of 2.03
and C-reactive protein (CRP) of 10 mg/L. the 6th swab, the results were negative but
After admission, the patient received
were positive in the 7th swab. In the 8th

swab, the results returned negative but

were positive in the 9th swab. Two

consecutive negative results were found on

the 10th and 11th swabs. The patient was

discharged from the hospital on April 25,

2020, 31 days after the onset of illness

(Table 1).

Tabel 1
RT-PCR findings in COVID-19 patients treated in Dr. Sardjito Hospital, Indonesia.

2.2 Case 2 examination recorded blood pressure of
A 54-year-old male patient complained of 131/72 mmHg, pulse of 96 per minute,
shortness of breath that worsened with respiratory rate of 24 per minute, body
activity. The patient began experiencing temperature of 38 ◦C, and oxy gen
fever nine days before admission. Three saturation of 97% with oxygenation of 2 L
days before admission, he experienced a per minute using nasal cannula. Lung
cough with phlegm. He had a history of auscultation revealed no apparent
contact with a confirmed case of COVID-19 abnormality. Rapid diagnostic tests using
two weeks before admission. He had a SARS-CoV-2 antibody were performed and
comorbidity of diabetes controlled with showed non-reactive results. Chest X-rays
routine medication. The physical showed inhomogeneous

Fig. 1. Chest X-ray indicated mild pneumonia in the right lung.

opacity on bilateral paracardial and lateral limits. Lung auscultation revealed crackles
aspects, typical of viral pneumonia caused in both lungs. Rapid diagnostic tests using
by COVID-19 infection (Fig. 2). We found SARS-CoV-2 antibodies were performed
increases in the NLR and CRP of 2.94 and and showed reactive results. Chest X-rays
107 mg/L, respectively. After admission, the showed bilateral pneumonia (Fig. 3). We
patient received antibiotics and antiviral found increases in the NLR and CRP of
therapy based on the COVID-19 Prevention 20.90 and 32 mg/L, respectively. A blood
and Control guidelines by the Indonesian culture test was performed and showed
Ministry of Health, namely, azithromycin, negative bacterial growth. After admission,
hydroxychloroquine, and lopinavir- ritonavir. the patient received antibiotics and antiviral
On the following day, naso/oropharyngeal therapy based on the COVID-19 Prevention
swabs were per- formed with positive and Control guidelines by the Indonesian
results. On the fifth day of treatment, naso/ Ministry of Health, namely, azithromycin,
oropharyngeal swabs were performed hydroxychloroquine, umife- novir and
again, and the results were still positive, lopinavir-ritonavir. On the 15th day of
even though his symptoms were relieved. treatment, he felt chest throbbing and
Positive PCR results were found until the shortness of breath.
4th test but were negative on the 5th test, During treatment, the patient often
turned positive again on the 6th, and then complained of tightness in the chest and
two consecutive results were found to be fever that rose suddenly, so we suspected
negative on the 7th and 8th tests (Table 1). pneumonia. The patient received antibiotic
The patient was un- eventfully discharged escalation therapy with meropenem and
after 29 days of treatment. levofloxacin, but his condition did not
improve. Naso/oropharyngeal swab tests
2.3 Case 3 were performed 11 times during the course
A 47-year-old man presented with of the treatment, and positive results of
complaints of fever, cough, sore throat, and SARS-CoV-2 infection were obtained and
diarrhea that were experienced from 10 remained positive, except for the negative
days before admission. The patient has results on the 4th and 6th tests. Two
comorbidities of asthma and heart rhythm consecutive negative results were obtained
disorders in the form of ventricular extra on the 10th and 11th swabs (Table 1). The
systole. He had a history of penicillin patient was uneventfully dis- charged after
allergy. His vital signs are within normal 52 days of treatment.

Fig. 2. Chest X-ray revealed inhomogeneous opacity on bilateral paracardial and lateral aspects.

Fig. 3. Chest X-ray revealed bilateral pneumonia.

2.4 Case 4 abnormality in either lung. Rapid diagnostic
A 56-year-old woman came to the tests using SARS-CoV-2 antibody were
emergency department with complaints of performed and showed non-reactive
lethargy that worsened in the last 6 days results. Chest X-rays showed bilateral
before admission. Her complaints included pneumonia (Fig. 4), while routine blood
diarrhea more than 10 times daily, without tests revealed pancytopenia. On the ninth
mucus or blood. The patient also day of admis- sion, her husband was
experienced fever for 4 days before known to have flu symptoms, had a rapid
admission. The patient went to a private diag- nostic test for SARS-CoV-2 antibodies
hospital, was hospitalized for 4 days, and with reactive results, and had been
was referred to our hospital for further examined by naso/oropharyngeal swab
tracking related to her neutropenia. She has test with positive results. The patient’s
a history of hyperthyroidism that she had husband is known to have a history of
suffered from for the past 1.5 months and contact with people traveling from the local
has received 100 mg twice daily PTU COVID-19 transmission area. After
therapy and thiamazole 5 mg once daily. admission,
She had a history of penicillin allergy. Her
vital signs were within normal limits. Lung
auscultation revealed no apparent

Fig. 4. Chest X-ray showed bilateral pneumonia.

our patient received antibiotics and antiviral there is a potential for transmission from
therapy based on the COVID-19 Prevention patients without or with mild symptoms
and Control guidelines by the Indonesian [9,10]. Our cases recovered from mild
Ministry of Health, namely, azithromycin, symptoms of COVID-19 but experienced
hydroxychloroquine, umifenovir and an extended duration of viral shedding.
lopinavir-ritonavir. Nasal and oropharyngeal Moreover, during treatment in the hospital,
swab tests were performed 14 times during our cases showed negative results between
the course of the treatment, and positive the positive results of RT-PCR. This finding
results of SARS-CoV-2 infection were is similar to previous reports [7, 11,12],
obtained and remained positive, except for which reported the high false negative
the negative results on the 2nd and 10th rate of RT-PCR for COVID-19 detection.
tests (Table 1). Two consecutive negative The fluctuating trend of our RT-PCR results
results were obtained on the 13th and 14th in our cases might be due to insufficient
swabs (Table 1). The patient was viral material in the specimen, labo- ratory
discharged uneventfully after 70 days of errors during sampling, restrictions on
treatment. sample transportation [11], or mutations
in the primary and probe target regions in
3. Discussion the SARS-CoV-2 genome [13].
We report the occurrence of prolonged RT-PCR is indeed a “gold standard”
nucleic acid conversion and the possibility diagnosis of COVID-19 because it detects
of false negative RT-PCR test results of RNA but not the active infectious virus. A
SARS-Cov-2 from Indonesian patients with virus culture study by Bullard et al. [14]
COVID-19. To the best of our knowledge, reported that virus infectivity decreased
our case is the longest prolonged nucleic significantly when RT-PCR cycle
acid conversion reported being 70 days. threshold (CT) values were >24. CT
Prolonged nucleic acid conversion is values of RT-PCR tests are inversely
defined as conversion for more than 24 related to viral load. For every 1 unit increase
days from the onset of the typical in CT, the odds ratio for infectivity
symptoms of COVID-19 [7]. Interestingly, decreases by 32%. Some studies also
previous studies have reported that reported that viral infectivity in patients
symptomatic and asymptomatic patients with a symptom duration >8 days may be
have the same viral loads, showing that low [14,15]. Several factors might affect the

occurrence of prolonged nucleic acid possibility of false negative RT-PCR results in
conversion, including older age, pa- tients with COVID-19 instead of recurrence
comorbidities, such as diabetes and of infection.
hypertension, and impaired immune
function [16]. Our cases, except patient #1, Consent
had comorbidities, such as diabetes, hy- Written informed consent was obtained from the
perthyroid, asthma and arrhythmia. patient for publi- cation of this case series and
Previously, on January 12, 2020, the WHO accompanying images. A copy of the written
recommended that COVID-19 patients be consent forms is available for review by the
discharged from isolation when the patients Editor-in-Chief of this journal on reasonable
are clinically recovered and show two request.
negative RT-PCR results on sequential
samples taken at least 24 h apart [17]. On Provenance And Peer Review
May 27, 2020, WHO revised the Not commissioned, externally peer reviewed.
recommendation for discharging COVID-19
patients from isolation without requiring Declaration Of Competing Interest
retesting of RT-PCR: a) for symptomatic No potential conflict of interest relevant to this
patients: 10 days after symptom onset, plus article was reported.
at least 3 additional days without symptoms
(including without fever and without Acknowledgements
respiratory symptoms); b) for asymptomatic We thank patient family and the nursing staff
cases: 10 days after positive test of RT- who were involved in the patient care. This study
PCR for SARS-CoV-2 [18]. Since August 1, was funded by Indonesian Ministry of Research
2020, our government has already adopted and Technology/National Agency for Research
these recommendations for the and Innovation.
management of patients with COVID-19 in
Indonesia, except for patients with severe Appendix A. Supplementary Data
and critical COVID-19: the criteria for Supplementary data to this article can be found
discharging patients from isolation needs a online at https://doi.
negative RT-PCR result [19]. org/10.1016/j.amsu.2020.09.040.
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GLOBAL PEDIATRIC PULMONOLOGY ALLIANCE RECOMMENDATION TO
STRENGTHEN PREVENTION OF PEDIATRIC SEASONAL INFLUENZA UNDER

COVID‑19 PANDEMIC

Kun‑Ling Shen1. Leyla Namazova‑Baranova2. Yong‑Hong Yang3. Gary Wing Kin Wong4. Lanny J. Rosenwasser5. Lance E.
Rodewald6. Anne Eng Neo Goh7. Eitan Kerem8. Chris O’Callaghan9. T. Bernard Kinane10. Basil Elnazir11. Rina Triasih12.
Rosemary Horne13. Anne B. Chang14. Jim Buttery15. Ruth A. Etzel16. Kazunobu Ouchi17. Hilary Hoey18. Varinder Singh19.
Genesis C. Rivera20. Spencer S. Li21. Yu Guan21 on behalf of the Global Pediatric Pulmonology Alliance (GPPA) Council ·
Ling Cao22. Yue‑Jie Zheng3. Lu‑Zhao Feng23. Wu Zhong24. Zheng‑De Xie25. Bao‑Ping Xu1. Rong‑Jun Lin26. Gen Lu27. Qiang
Qin1. Chun‑Mei Zhu22. Su‑Yun Qian28. Gang Liu29. Cheng‑Song Zhao29. Zhuang Wei30. Yu‑Hong Zhao31 on behalf of the
Global Pediatric Pulmonology Alliance (GPPA) Expert Panel on Infectious Diseases & COVID‑19

The World Health Organization (WHO) alert [8], as immense pressure will be placed on
announced COVID‑ 19 as a global pandemic in hos‑ pitals’ overall diagnosis and treatment
March of 2020 [1]. The COVID‑ 19 pandemic capacity and on their critical care capacity.
may persist for a long period of time. Global Co‑infection of influenza and COVID‑ 19 can
prevention and control becomes a complex and occur in patients and present with severe
challenging task, and such efforts should be symptoms. This will further increase the
sustained. Although general measures, such as complexity of clinical diagno‑ sis and treatment
social distancing, face masks, respiratory [6]. Hospitals usually have concentrated patient
hygiene and hand sanitization, will bear fruits for flows, and the risk of cross infection is
decreas‑ ing spread of other respiratory illnesses increased, which threatens the health of all
including influenza, the specific prevention patients, their caregivers, and medical staff
through vaccination is a key focus especially in members.
the upcoming winter and spring seasons [2].
The WHO, the United Nations International
Winter and spring are the peak seasons for Children’s Emergency Fund (UNICEF), and
influenza and other common respiratory various nations have called for strengthening of
infectious diseases. Children are a high‑risk prevention and control of seasonal influ‑ enza
group for influenza. The prevalence of influenza while making every effort to cope with the
in children is 20–30% every year [3], and it often COVID‑19 pandemic. These organizations
enhances outbreaks in the community. The vigorously advocate influenza vaccination as the
pediatric population in child care centers, primary measure for pandemic prevention and
kindergartens and primary schools is closely control, so as to minimize the interference of an
confined and is vulnerable to mass outbreaks influenza epidemic during the prevention and
[4]. The high infection rate of influenza in control of the COVID‑19 pandemic situation.
children is also one of the important reasons for According to the WHO recommendation,
the spread of influenza virus in the community influenza vaccination is an effective
[5]. If an outbreak of influenza coincides with supplementary measure against the COVID‑19
that of COVID‑19, the impact on the pediatric pandemic for countries that are about to enter
population will be even more severe [6]. the winter. All eligible individuals should receive
the influenza vaccine [2]. UNICEF calls for
According to WHO, the COVID‑19 outbreak in influenza vaccination for children during the
early 2020 occurred at the same time as the COVID‑19 pandemic to protect them from other
influenza outbreak [7], and the primary clinical diseases [9]. The US Centers for Disease
manifestations of influenza and COVID‑19 Control (CDC) stated that in the coming winter
overlap considerably. An epidemic of influenza influenza and COVID‑ 19 may spread
would challenge the diagnosis and treatment in simultaneously and recommended vaccination for
fever clinics and the supplies of personal all children older than 6 months [10]. The
protective equipment (PPE). Therefore, the Australian government strongly recommends that
upcoming winter and spring seasons are of high people, especially children and other high risk

groups, should be vaccinated with influenza STRATEGIES IN INFLUENZA VACCINATION
vaccine when actively coping with the possible
overlap of COVID‑19 and influenza [11]. The 1. Infants under 6 months
British public health agency also stated that owing
to the COVID‑19 outbreak, influ‑ enza vaccination Influenza vaccine cannot be given to infants
is more important than ever before [12]. New
Zealand called for vaccination for high‑risk target under 6 months. We recommend parents,
populations [13] (such as children, seniors and
pregnant women) to protect communities and to caregivers, and family members of the
utilize resources most effectively. Brazil initiated
early vaccination to reduce influenza cases in the infants to be vaccinated [4], and if included
midst of COVID‑19, with children from 6 months
to 6 years of age being the priority target in national recommendations, expectant
population [14]. Thus, preschool‑age and younger
children are the key vaccination population, and mothers should receive influenza
influenza vaccination is the best preventive
measure. vaccination during pregnancy to protect

RECOMMENDATIONS themselves and protect newborns from
To control the pandemic situation more effectively,
the Global Pediatric Pulmonology Alliance influenza infection during their first six
(GPPA), based on best‑prac‑ tices and evidence
from various countries, recommends that all month of life.
global and regional organizations, medical
societies, and health agencies join hands in the 2. Children over 6 months and school‑age
prevention of seasonal influ‑ enza in children.
Measures to improve influenza vaccination children
include promoting public awareness about
infection control measures and thorough This is the priority population for influenza
education of the benefit of influenza vaccination.
The ultimate goal is to achieve early notification, vaccination. All children over 6 months of
early appointment and early vaccination, which will
reduce the likelihood of coincident epidemics of age are eligible, except for those who are
influenza and COVID‑19 this winter. It is
recommended that vaccination policy, vaccine allergic to vaccine substances/components
safety, and medical advice be promoted and
reiterated in child care centers, kindergartens, or who are experiencing fever or an acute
and schools [15]. Countries and regions with
ample resources should implement free influenza infection. Children with a his‑ tory of egg
vaccination in child care centers, kindergartens
and schools, so as to increase the coverage rate of allergy of any severity can receive any
influenza vaccination among the pediatric
population [16]. It is also advised that in coun‑ licensed, recommended, and
tries or regions, where influenza vaccination may
not be part of national immunization program age‑appropriate influenza vaccine. Chil‑
(NIP), pediatricians should check with respective
health authorities or local guidelines to reach the dren with stable chronic pulmonary,
suggested beneficiarypopulation.
cardiovascular, renal or neurological

diseases should get the influenza vaccine

as early as possible [4]. Children with

primary immunodeficiency, HIV infection

and secondary immunodeficiency

(leukemia, carcinoma, receiving

immunosuppressive therapy, etc.) should

consult with medical professionals before

using live attenuated influenza vaccines.

SCHOOLS AND KINDERGARTENS
Staff should be vaccinated every year [17].
Schools and kin‑ dergartens should carry out

awareness and education cam‑ paigns on
prevention and control of infectious diseases
and on promotion of vaccination to students and
parents, so as to improve vaccination rates and
to prevent the outbreak of clustering epidemic

[17, 18]. Family members of chil‑ dren and
frequent visitors (such as caregivers,
housekeeping workers, etc.) also should be
vaccinated annually [17].

PEDIATRIC HEALTH CARE WORKERS
Pediatric medical staff and workers are in
frequent and close contact with children. They

should be vaccinated every year. This should and keep a physical distance of more than 1
also be considered for other staff groups, such m (1.8 m is required in the US) from others.
as hospital cleaners and porters. This protects 3. Parents or individuals accompanying
the pediatric health care workers, maintains children should use face masks and should
health care capacity by reducing sick leave, and follow strict hand hygiene and routine
also helps to reduce transmission from members disinfection according to instructions of
of staff to children and oth‑ ers, thereby reducing medical staff before vaccination.
the risk of hospital‑acquired infec‑ tions. It is a 4. After vaccination, parents should take the
concern that shedding of influenza virus has child to a designated area for routine
been shown shortly before clinical illness observation and then go home immediately
develops and that health care workers frequently to avoid a prolonged stay in the facility or
continue to work while unwell. other public areas.
5. Children and their parents or caregivers
VACCINATION SCHEDULE should apply hand hygiene and should
Influenza vaccine needs to be given every year. dispose of face masks prop‑ erly after
Countries and regions around the world should returning home.
prepare ample vac‑ cine supplies and should 6. After returning home, parents should
complete vaccination of the pedi‑ atric observe the over‑ all health of the child, keep
population about 2–3 months before the arrival his/her vaccination spot clean, and watch for
of the influenza season [4]. Although the GPPA possible complications of vaccina‑ tion.
is advocating vaccinations (as described above), 7. Expert advice should be available to support
public health priorities may differ across nations; vaccination staff in answering questions
therefore, pediatricians should check their about vaccine eligibility.
national guidelines for the eligible population,
available vaccine products and the number of In conclusion, under the COVID‑19 pandemic,
doses to administer based on age, vaccination getting the influenza vaccine in time offers
history, and vaccine product to make a final call. children a safer winter and a healthier future.

VACCINATION METHODS REFERENCES
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national flu immuni‑ sation programme

AFFILIATIONS London, UK

Kun‑Ling Shen1 · Leyla Namazova‑Baranova2 · 10Massachusetts General Hospital for Children,
Yong‑HongYang3· Gary Wing Kin Wong4 · Lanny
J.Rosenwasser5· LanceE.Rodewald6·AnneEng Boston, MA, USA
Neo Goh7 · Eitan Kerem8 · Chris O’Callaghan9 · T.
BernardKinane10·BasilElnazir11· RinaTriasih12 11Children’s Health Ireland and Trinity
· Rosemary Horne13 · Anne B. Chang14 · Jim
Buttery15 · Ruth A. Etzel16 · Kazunobu Ouchi17 College Dublin, Dublin, Ireland
·Hilary Hoey18 · Varinder Singh19 · Genesis C.
Rivera20 · Spencer S. Li21 · Yu Guan21 on behalf of 12Department of Paediatric, Faculty of
the Global Pediatric Pulmonology Alliance
(GPPA) Council · Ling Cao22 · Yue‑Jie Zheng3 · Medicine, Public Health and Nursing,
Lu‑Zhao Feng23 · Wu Zhong24 · Zheng‑De Xie25 ·
Bao‑Ping Xu1 · Rong‑Jun Lin26 · Gen Lu27 · Universitas Gadjah Mada/Dr. Sardjito
Qiang Qin1 · Chun‑Mei Zhu22 · Su‑Yun Qian28 ·
Gang Liu29 ·Cheng‑SongZhao29·ZhuangWei30· Hospital, Yogyakarta, Indonesia
Yu‑HongZhao31 onbehalf of theGlobal Pediatric
Pulmonology Alliance (GPPA) Expert Panel 13Monash University, Melbourne, Australia
on Infectious Diseases & COVID‑19
14Queensland Children’s Hospital, Brisbane,
1China National Clinical Research Center of
Respiratory Diseases, Department of Australia
Respiratory Medicine of Beijing Children’s
Hospital, Capital Medical University, 15Monash Children’s Hospital, Menzies
National Center for Children’s Health,
Beijing, China School of Health Research, Melbourne,

2Pediatrics and Child Health Research Australia
Institute, Central Clinical Hospital, Russian
Academy of Sciences, Moscow, Russian 16George Washington University, Washington,
Federation
DC, USA
3Department of Respiratory, Shenzhen
Children’s Hospital, Shenzhen, China 17Department of Pediatrics, Kawasaki

4Chinese University of Hong Kong, Hong Kong Medical School, Kurashiki‑City, Japan
SAR, China
18 Department of Pediatrics, University of
5UMKC School of Medicine, Kansas City, MO,
USA Dublin Trinity College, Dublin, Ireland

6National Immunization Program, Chinese 19Lady Hardinge Medical College and
Center for Disease Control and Prevention,
Beijing, China Assoc Kalawati Saran Children’s

7K Women’s and Children’s Hospital, Singapore, Hospital, New Delhi, India
Singapore
20Center for Medical and Allied Health
8Department of Pediatrics, Hadassah
Hebrew University Medical Center, Sciences, New Era University, Quezon
Jerusalem, Israel
City, the Philippines
9UCL Great Ormond Street Institute of Child
Health and Great Ormond Street Hospital 21Global Pediatric Pulmonology Alliance,
(GOSH) BRC, University College London,
Hong Kong SAR, China

22Department of Respiratory, the

Children’s Hospital Affiliated to the

Capital Institute of Pediatrics, Beijing,

China

23School of Population Medicine and

Public Health, Chinese Academy of

Medical Sciences/Peking Union Medical

College, Beijing, China

24National Engineering Research Center

for the Emergency Drug, Beijing, China

25National Clinical Research Center for

Respiratory Diseases, Laboratory of

Infection and Virology, Beijing Pediatric

Research Institute, Beijing

Children’sHospital, Capital Medical

University, National Center for

Children’s Health, Beijing, China26 The

Affiliated Hospital of Qingdao
University, Qingdao, China
27Department of Respiratory, Guangzhou
Women and Children’s Medical Center,
Guangzhou Medical University, Guangzhou,
China
28Pediatric Intensive Care Unit, Beijing
Children’s Hospital, Capital Medical
University, National Center for Children’s
Health, Beijing, China29 Department of
Infectious Diseases, Beijing Children’s
Hospital, Capital Medical University, National
Center for Children’s Health, Beijing, China
30Department of Health Care, Beijing
Children’s Hospital, Capital Medical
University, National Center for Children’s
Health, Beijing, China
31Department of Respiratory Medicine,
Beijing Children’s Hospital, Capital
Medical University, National Center
for Children’s Health, Beijing, China

MOLECULAR EPIDEMIOLOGY OF SARS-COV-2 ISOLATED FROM COVID-19
FAMILY CLUSTERS

Gunadi1*#, Hendra Wibawa2*#, Mohamad Saifudin Hakim3, Marcellus4, Ika Trisnawati5, RiatEl
Khair6, Rina Triasih7, Irene8, Afiahayati9, Kristy Iskandar10, Siswanto11,Nungki Anggorowati12, Edwin Widyanto
Daniwijaya13, Endah Supriyati14, Dwi Aris Agung Nugrahaningsih15, Eko Budiono16, Heni Retnowulan17, Yunika
Puspadewi18, Ira Puspitawati19, Osman Sianipar20, Dwiki Afandy21, Susan Simanjaya22, WilliamWiditjiarso23, Dyah

Ayu Puspitarani24, Fadil Fahri25, Untung Wirawan26, Aditya Rifqi Fauzi27,Alvin Santoso Kalim28, Nur Rahmi
Ananda29, Amalia Setyati30, Dwikisworo Setyowireni31, Ida Safitri Laksanawati32, Eggi Arguni33, Titik Nuryastuti34,

Tri Wibawa35, on behalf of the Yogyakarta-Central Java COVID-19 study group

1 Pediatric Surgery Division, Department of Surgery/Genetics Working Group, Faculty of Medicine, Public Health and
Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia; [email protected]
2 Disease Investigation Center, Wates, Yogyakarta, Ministry of Agriculture Indonesia; [email protected]
3 Department of Microbiology, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta,
Indonesia; [email protected]
4 Genetics Working Group, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta,
Indonesia; [email protected]
5 Pulmonology Division, Department of Internal Medicine, Faculty of Medicine, Public Health and Nursing, Universitas
Gadjah Mada/Dr. Sardjito Hospital, Yogyakarta, Indonesia; [email protected]
6 Department of Clinical Pathology and Laboratory Medicine, Faculty of Medicine, Public Health and Nursing, Universitas
Gadjah Mada/Dr. Sardjito Hospital, Yogyakarta, 55281, Indonesia; [email protected]
7 Department of Child Health, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada/Dr. Sardjito
Hospital, Yogyakarta, Indonesia; [email protected]
8 Balai Besar Teknik Kesehatan Lingkungan dan Pengendalian Penyakit, Yogyakarta, Yogyakarta, Indonesia;
[email protected]
9 Department of Computer Science and Electronics Faculty of Mathematics and Natural Sciences, Universitas Gadjah Mada,
Yogyakarta, Indonesia; [email protected]
10 Department of Child Health/Genetics Working Group, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah
Mada/ UGM Academic Hospital, Yogyakarta, Indonesia; [email protected]
11 Department of Physiology, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada/UGM Academic
Hospital, Yogyakarta, Indonesia; [email protected]
12 Department of Anatomical Pathology/Genetics Working Group, Faculty of Medicine, Public Health and Nursing,
Universitas Gadjah Mada, Yogyakarta, Indonesia; [email protected]
13 Department of Microbiology, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada/UGM Academic
Hospital, Yogyakarta, Indonesia; [email protected]
14 Centre of Tropical Medicine, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta,
Indonesia; [email protected]
15 Department of Pharmacology and Therapy/Genetics Working Group, Faculty of Medicine, Public Health and Nursing,
Universitas Gadjah Mada, Yogyakarta, Indonesia; [email protected]
16 Pulmonology Division, Department of Internal Medicine, Faculty of Medicine, Public Health and Nursing, Universitas
Gadjah Mada/Dr. Sardjito Hospital, Yogyakarta, Indonesia; [email protected]
17 Pulmonology Division, Department of Internal Medicine, Faculty of Medicine, Public Health and Nursing, Universitas
Gadjah Mada/Dr. Sardjito Hospital, Yogyakarta, Indonesia; [email protected]
18 Department of Clinical Pathology and Laboratory Medicine, Faculty of Medicine, Public Health and Nursing, Universitas
Gadjah Mada/Dr. Sardjito Hospital, Yogyakarta, 55281, Indonesia; [email protected]
19 Department of Clinical Pathology and Laboratory Medicine, Faculty of Medicine, Public Health and Nursing, Universitas
Gadjah Mada/Dr. Sardjito Hospital, Yogyakarta, 55281, Indonesia; [email protected]
20 Department of Clinical Pathology and Laboratory Medicine, Faculty of Medicine, Public Health and Nursing, Universitas
Gadjah Mada/Dr. Sardjito Hospital, Yogyakarta, 55281, Indonesia; [email protected]
21 Genetics Working Group, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta,
Indonesia; [email protected]
22 Genetics Working Group, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta,
Indonesia; [email protected]
23 Genetics Working Group, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta,
Indonesia; [email protected]
24 Genetics Working Group, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta,
Indonesia; [email protected]

25 Genetics Working Group, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta,
Indonesia; [email protected]
26 Genetics Working Group, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta,
Indonesia; [email protected]
27 Genetics Working Group, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta,
Indonesia; [email protected]
28 Genetics Working Group, Faculty of Medicine, Public
Health and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia; [email protected]
29 Pulmonology Division, Department of Internal Medicine, Faculty of Medicine, Public Health and Nursing, Universitas
Gadjah Mada/Dr. Sardjito Hospital, Yogyakarta, Indonesia; [email protected]
30 Department of Child Health, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada/Dr. Sardjito
Hospital, Yogyakarta, Indonesia; [email protected]
31 Department of Child Health, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada/Dr. Sardjito
Hospital, Yogyakarta, Indonesia; [email protected]
32 Department of Child Health, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada/Dr. Sardjito
Hospital, Yogyakarta, Indonesia; [email protected]
33 Department of Child Health, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada/Dr. Sardjito
Hospital, Yogyakarta, Indonesia; [email protected]
34 Department of Microbiology, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta,
Indonesia; [email protected]
35 Department of Microbiology, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta,
Indonesia; [email protected]

Running title: Molecular epidemiology of COVID-19 family clusters

ABSTRACT
Background: Transmission within families and multiple spike protein mutations have been associated
with the rapid transmission of SARS-CoV-2. We aimed to: 1) describe full genome characterization of
SARS-CoV-2 and correlate the sequences with epidemiological data within family clusters, and 2)
conduct phylogenetic analysis of all samples from Yogyakarta and Central Java, Indonesia and other
countries.
Methods: The study involved 17 patients with COVID-19, including two family clusters. We determined
the full-genome sequences of SARS-CoV-2 using the Illumina MiSeq next-generation sequencer.
Phylogenetic analysis was performed using a dataset of 142 full-genomes of SARS-CoV-2 from
different regions.
Results: Ninety-four SNPs were detected throughout the open reading frame (ORF) of SARS-CoV-2
samples with 58% (54/94) of the nucleic acid changes resulting in amino acid mutations. About 94%
(16/17) of the virus samples showed D614G on spike protein and 56% of these (9/16) showed other
various amino acid mutations on this protein, including L5F, V83Ll V213A, W258R, Q677H, and N811I.
The virus samples from family cluster-1 (n=3) belong to the same clade GH, in which two were
collected from deceased patients, and the other from the survived patient. All samples from this family
cluster revealed a combination of spike protein mutations of D614G and V213A. Virus samples from
family cluster-2 (n=3) also belonged to the clade GH and showed other spike protein mutations of L5F
alongside the D614G mutation.
Conclusions: Our study is the first comprehensive report associating the full-genome sequences of
SARS-CoV-2 with the epidemiological data within family clusters. Phylogenetic analysis revealed that
the three viruses from family cluster-1 formed a monophyletic group, whereas viruses from family
cluster-2 formed a polyphyletic group indicating there is the possibility of different sources of infection.
This study highlights how the same spike protein mutations among members of the same family might
show different disease outcomes.

Keywords: COVID-19 severity; family cluster; multiple spike protein mutations; phylogenetic analysis;
SARS-CoV-2 transmission; whole genome sequencing

INTRODUCTION have described the genomic epidemiology within
Many countries are still struggling to control the family clusters [6-8]. Moreover, multiple spike
COVID-19 pandemic, including Indonesia [1,2]. protein mutations have been associated with a
On February 9, 2021, Indonesia recorded higher transmissibility of SARS-CoV-2 [9]. In this
1,174,779 confirmed COVID-19 cases with study, we aimed to: 1) perform full genome
31,976 deaths and infection rate of characterization of SARS-CoV-2 and correlate
approximately 12,000 cases/day [3]. One of the the sequences with the epidemiological data
most important factors affecting the rapid within family clusters in Indonesia, and 2)
spreading of COVID-19 is transmission within conduct phylogenetic analysis of all samples
families [4,5]. Genomic epidemiology has been from Yogyakarta and Central Java, Indonesia,
suggested to be important to fill the gaps in involving the family clusters, and virus data from
identifying the SARS-CoV-2 infection sources other regions in Indonesia.
[6]. However, to our best knowledge, no reports
using Respiratory Virus Oligos Panel, whereas
METHODS
SARS-CoV-2 sample for full-genome sequencing, we used next
We collected all virus samples of confirmed
COVID-19 patients from Yogyakarta and Central generation sequencing (NGS) applied in the
Java provinces from June to November 2020. All
nasopharyngeal samples were collected in viral Illumina MiSeq instrument (Illumina, San Diego,
transport media (DNA/RNA ShieldTM Collection
Tube with Swab, Zymo Research, CA, United CA, United States) with Illumina MiSeq reagents
States) and transported to four COVID-19
diagnostic laboratories in Yogyakarta province: v3 150 cycles (2 x 75 cycles). We excluded two
1)Molecular Diagnostic Laboratory, Integrated
Laboratory Unit, Dr. Sardjito Hospital; samples for further bioinformatics analysis
2)Department of Microbiology and Laboratorium
Diagnostik Yayasan Tahija World Mosquito because of low coverages. Our sample
Program, Faculty of Medicine, Public Health and
Nursing, Universitas Gadjah Mada; 3) Balai genomes were assembled by mapping to the
Besar Teknik Kesehatan Lingkungan dan
Pengendalian Penyakit (BBTKLPP), Yogyakarta; reference genome from Wuhan, China (hCoV-
and 4) Disease Investigation Center, Wates,
Yogyakarta. SARS-CoV-2 was detected by 19/Wuhan/Hu-1/2019, GenBank accession
Real-Q 2019-nCoV Detection Kit (BioSewoom,
Seoul, South Korea) with LightCycler® 480 number: NC_045512.2) using Burrow-Wheeler
Instrument II (Roche Diagnostics, Mannheim,
Germany). Aligner (BWA) algorithm embedded in UGENE

Full-genome sequencing v. 1.30 [10]. Identification of single nucleotide
First, we performed RNA extraction of 19
nasopharyngeal swab samples by a QiAMP Viral polymorphisms (SNPs) was performed using the
RNA mini kit (Qiagen, Hilden, Germany),
synthesized the double-stranded cDNA by number of high confidence base calls
Maxima H Minus Double-Stranded cDNA
Synthesis (Thermo Fisher Scientific, MA, United (consensus sequence variations of the
States), and purified the cDNA using a GeneJET
PCR Purification Kit (Thermo Fisher Scientific, assembly) that disagree with the reference
MA, United States). For library preparations, we
utilized the Nextera DNA Flex for Enrichment bases for the genome position of interest, then

all SNPs were exported to a vcf. file and

visualized in MS Excel. The following accession

IDs for the 17 samples are: EPI_ISL_516800,

EPI_ISL_516806, EPI_ISL_516829,

EPI_ISL_525492, EPI_ISL_576383,

EPI_ISL_632936, EPI_ISL_610161,

EPI_ISL_610162, EPI_ISL_576145,

EPI_ISL_632937, EPI_ISL_575331,

EPI_ISL_576113, EPI_ISL_576114,

EPI_ISL_576115, EPI_ISL_576116,

EPI_ISL_576128, and EPI_ISL_576130 [11].

The first four IDs have been reported in our

previous study [12].

Phylogenetic analysis
We used the reference genome of hCoV-
19/Wuhan/Hu-1/2019 (NC_045512.2) for
annotation of our sequences. A dataset of 142

available SARS-CoV-2 genomes (89 sequences phylogenetic tree was constructed from 29.409
nt length of the open reading frame (ORF) of
from Indonesia and 53 from other countries) was 142 SARS-CoV-2 virus sequences using
Neighbor Joining statistical method with 2,000
retrieved from GISAID to conduct a phylogenetic bootstrap replications. The evolutionary
distances were computed using the Kimura 2-
analysis (Acknowledgment Table is provided in parameter method and the rate variation among
sites was modelled using a gamma distribution
Supplementary Data). We only used the full- with estimated shape parameter (α) for the
dataset. The estimation of α gamma distribution
genome sequences of several strains was calculated in DAMBE version 7 [13],
whereas all the other analyses were performed
representing SARS-CoV-2 clades from some in MEGA version 10 (MEGA X) [14].
Respiratory Distress Syndrome (ARDS), sepsis,
countries that had complete genome data and or septic shock, or other complications [12,15].
Our study was approved by the Medical and
no long stretches of ‘NNNN’ for the phylogenetic Health Research Ethics Committee of the
Faculty of Medicine, Public Health and Nursing,
analysis. The MAFFT program server was Universitas Gadjah Mada/Dr. Sardjito Hospital
(KE/FK/0563/EC/2020). All participants or
utilized for multiple nucleotide sequence guardians signed a written informed consent for
participating in this study.
alignment
10001|EPI_ISL_576113,YO-UGM-
(https://mafft.cbrc.jp/alignment/server/). A 10002|EPI_ISL_576114,andYO-UGM
10003|EPI_ISL_576115) formed a single group
COVID-19 severity classifications within clade GH, whereas viruses from family
COVID-19 severity was determined based on cluster-2 (YO-UGM-1004|EPI_ISL_576116,YO-
the WHO classifications: 1) mild, without UGM-1005|EPI_ISL_576128,andYO-UGM
evidence of hypoxia or pneumonia; 2) moderate, 1006|EPI_ISL_576130,) were separated into two
pneumonia but not severe; 3) severe, different nodes within clade GH (Figure 1, top-
pneumonia plus one of the following signs: right).
respiratory rate >30 breaths/minute (or based on
age for children), severe respiratory distress, or
SpO₂ <90% in room air; and 4) critical, Acute

RESULTS
Phylogenetic analysis
Phylogenetic analysis revealed that thirteen
virus samples were situated within clade GH
(GISAID classification), while two viruses were
grouped with other viruses which belonged to
clade GR, and one virus each that belonged to
clade O and clade L (Figure 1). Three viruses
from family cluster case-1 (YO-UGM-

Molecular analysis
Ninety-four SNPs were detected throughout the ORP of the SARS-CoV-2 virus samples with 60%
(54/94) of the nucleic acid changes resulting in amino acid substitutions (missense mutations) (Table 1,
detailed in Table S.1). The types of nucleic acid base changes were more often detected as transitions
(70%) compared to transversions (30%). Higher entropy values were observed more from nucleic acids
that carried more frequent base changes; however, nucleic acid changes that caused missense
mutation could have lower entropy values than those that resulted in synonymous mutation.

Table 1. Nucleic acid and amino acid mutations observed in seventeen SARS-CoV-2 virus 239
genomes collected from Yogyakarta and Central Java provinces between June and September 240
2020

`

* Nucleic acid numbering starting from ORF1ab start codon (ATG)
# Amino acid numbering starting from start codon of each gene

The majority of the virus samples (16/17) COVID-19’s severity and spike protein
possessed D614G substitution on spike protein mutations of COVID-19 samples
and 56% of these (9/16) showed other amino Based on the case definition of COVID-19
acid substitutions on this protein, including L5F severity developed for this study, 3 of 17 virus
V83Ll V213A, W258R, Q677H, and N811I. samples (17.6%) were collected each from
Second amino acid mutations that were asymptomatic cases (people) and critical cases,
frequently detected were P232L substitution on 5 virus samples (29.4%) from mild cases, and 6
NSP12 (RdRp) protein (15x), followed by Q57H virus samples (35.3%) from moderate cases
substitution on NS3 (14x) and P822L (Table 2). Two of the patients with critical stages
substitution on NSP3 protein (13x). Furthermore, eventually died. A range of Ct values was found
various amino acid mutations were also found in amongst different stages of severity,
the other proteins of virus samples, including on nevertheless all the virus samples with D614G
NSP2 (A205V, V247A, T256I, Q321K), NSP3 mutations, except one (YO-UGM-
(P679S, T1022I, A1179V, T1198K, F1354C, 10004/2020|EPI_ISL_576116), showed lower Ct
P1665L), NSP4 (A231V), NSP5 (K12R, M49I, values (clade GH, GR, and O, Ct range 16.9 to
P184S), NSP6 (L37F), NSP8 (A21T), NSP9 24.7) than those with no mutation in this position
(L42F), NSP12/RdRp (A97V, P227L, T248I, (clade L, Ct 27.9). Dual mutations of V213A and
A656S, H892Y, M906V), NSP13 (T127I, T153I, D614G on spike protein were detected in four
V169F, M576I, P203L), NSP15 (H337Y), NSP16 patients, and two of these eventually died after a
(Y222C), NS3 (A54V, A99S, T151I, D222Y), period of hospitalization.
NS7a (H73Y), and N(P13L, A119S, Q160R,
S193I, R195S, P199S, R203K, G204R, M234I).

Table 2. Severity and genetic data associated with SARS-CoV-2 viruses collected from seventeen
COVID-19 patients in 267 Yogyakarta and Central Java provinces, Indonesia from June-September
2020.

Virus samples collected from family cluster-1 *) and from family cluster-2 **) 270
CT, cycle threshold; Ref. sequence: hCoV-19/Wuhan/Hu-1/2019 (NC_045512.2)

Disease outcomes of COVID-19’s family clusters 273
The epidemiological and clinical data of COVID-19’s family clusters, including clinical 274 symptoms,
date of first symptoms appeared, diagnostic results, pathological findings, 275 comorbidity background
are provided by timeline and tabulation in Figure 2 and Table 3, 276 respectively.

Table 3. Characteristics of patients with COVID-19 from family cluster cases in Yogyakarta and Central
Java

CXR, chest X-ray; ARDS, acute respiratory distress syndrome; NLR, neutrophil to lymphocyte ratio;
DM, diabetes mellitus

In family cluster-1, all three patients showed patient-1.3 (type 2 diabetes mellitus, geriatric
critical COVID-19 and eventually, two died (YO- syndrome, history of infarction stroke). Patient-
UGM-10001|EPI_ISL_576113 and YO-UGM- 1.1 was uneventfully discharged from the
10003|EPI_ISL_576115) and one survived (YO- hospital on day 29 of hospitalization, but sadly,
UGM-10002|EPI_ISL_576114). The disease patient-1.2 and patient-1.3 passed away in the
began from patient-1.1, a 28-year-old male, who hospital after 7 and 12 days of hospitalization,
had a history of traveling from the local COVID- respectively.
19 transmission area. He complained of fever,
sore throat, cough and malaise on August 8th, Family cluster-2 involved three patients which
2020, and was tested for PCR three days were comprised of a son, 8-yo (patient-2.1),
afterward with the result of COVID-19 positive. father, 35-yo (patient-2.2) and mother, 33-yo,
His father (patient-1.2, 58-yo), who was living in with the following virus samples: YO-UGM-
the same house, showed fever, dyspnea and 10006 (EPI_ISL_576130), YO-UGM-10004/2020
diarrhea on August 13th, then was followed by (EPI_ISL_576114), and YO-UGM-10005/2020
his grandfather (patient-1.3, 88-yo) who showed (EPI_ISL_576128), respectively. Prior to the
fever and dyspnea on August 18th. The PCR index case, this family travelled from West Nusa
tests for both patients showed positive for Tenggara to Yogyakarta on August 2nd, 2020, in
COVID-19. All patients developed severe order to obtain medical treatment for patient-2.1
disease outcomes including bilateral pneumonia, who had an autoimmune disorder in a hospital in
cardiomegaly and ARDS. Several comorbidities Yogyakarta. Patient-2.1 had firstly exhibited
were recorded from patient-1.1 (obesity), symptoms of fever, cough, and runny nose on
patient-1.2 (diabetes mellitus, and obesity), and August 11th, 2020 and he was diagnosed
COVID-19 positive on August 18th. His parents

showed clinical signs of cold, headache and dry
cough (patient-2.2) and cough, sore throat, and
runny nose (patient-2.3) at the same day on
August 20th and the PCR results of both patients
were positive on August 21th. Patient-2.1
developed moderate severity with bilateral
paracardial infiltrate, whereas patient-2.2 and
patient-2.3 developed mild disease without any
abnormalities in their chest X-rays and other
laboratory findings, except eosinophilia (6.4%),
increased levels in pH of the blood (7.45), PaO2
(83.2), PaCO2 (39.2) with SaO2 96.8% and
PaO2/FiO2 value was 416 from the arterial blood
glass analysis of patient-2.2. All three patients
uneventfully recovered and were discharged
from the hospital on September 2nd, 2020.

Molecular characterizations of virus samples
collected from family clusters
Phylogenetic analysis revealed that the three
viruses from family cluster-1 were grouped
together from a single node. A matrix of nucleic
acid difference showed that YO-UGM-
10001|EPI_ISL_576113 and YO-UGM-
10003|EPI_ISL_576115 were identical on their
OR (nucleic acid and protein levels) and both
virus strains had differences of 2 nucleic acids
and 1 amino acid in the NSP2 protein which
correspond with V247A substitution in YO-UGM-
10001|EPI_ISL_576113 and YO-UGM-
10003|EPI_ISL_576115 and T256I substitution
in YO-UGM-10002|EPI_ISL_576114,
respectively (Table 4). Other unique mutations in
the other viral proteins were detected in these
three virus strains which were not shown in the
other study viruses, including V213A (Spike),
K12R (NSP5), T248I (NSP12/RdRp), A119S
and S193I (N).The virus samples from family
cluster-2 were separated in different nodes in
the phylogenetic tree (Figure 3). The tree and
the matrix sequence showed that YO-UGM-
10005/2020|EPI_ISL_576128 and YO-UGM-
10006|EPI_ISL_576130 were genetically
identical.Both virus strains had 15 nucleic acid
differences compared to YO-UGM-
10004|EPI_ISL_576114 which resulted in amino
acid variations detected in several viral proteins
(Table 4).

Table 4. Amino acid mutations detected in SARS-CoV-2 viruses collected from two family cluster cases
in Yogyakarta and Central Java provinces.

Family clusters are indicated in asterisks: Case-1 (*) and Case-2 (**)

DISCUSSION The epidemiological and genetic data indicated
Our study provides evidence of SARS-CoV-2 that local transmission occurred in family cluster-
transmission within families, in which the same 1 in which patient-1.1 (YO-UGM-
mutation of the spike protein in each family 10002|EPI_ISL_576114) was initially infected
cluster was identified. It is important to and then transmitted the virus to patient-1.2
understand the transmission routes of SARS- (YO-UGM-10003|EPI_ISL_576115) and patient-
CoV-2 to prevent and control its spreading [4]. 1.3 (YO-UGM-10001|EPI_ISL_576113).
Families have been reported as the most Interestingly, the virus that infected patient 2.2 in
dominant infection cluster of COVID-19 [16]. family cluster-2 was genetically different from
Family clusters have a higher risk of cross- that which infected both two counterparts:
infection because of frequent and close contact patient 2.1 (YO-UGM-10006/2020
among each family member [4]. Our study also (EPI_ISL_576130) and patient 2.3 (YO-UGM-
documented that although all family members 10005/2020 (EPI_ISL_576128). These viruses
showed the same multiple protein S mutations, formed a polyphyletic group indicating there is
however, they revealed different outcomes. the possibility of different sources of infection
Phylogenetic analysis revealed that three (two convergent descendants, but not their
viruses from family cluster-1 formed a common ancestors). Double mutations of V213A
monophyletic group. and D614G on spike protein were detected in

four patients, but three of them (75%) developed (Australia and some Central European), N439K
severe diseases causing critical conditions and (UK and European), N501Y (part of the new UK
two (50%) with fatal outcome. Another variant VUI-202012/01 and the new South Africa
interesting finding was documented from family variant 501.V2) [11]. These variants might be
cluster-1, in which all the virus samples in this associated with some potential advantages for
family cluster belong to the same clade GH, but these viruses. However, whether these variants
two patients died and one survived (Table 2). All are associated with COVID-19 clinical severity or
samples from family cluster-1 revealed another SARS-CoV-2 transmission efficiency is poorly
spike protein mutation, V213A, besides D614G. understood [17,18]. In addition, among eight
However, virus samples isolated from fatal clades in the GISAID classification, we only
disease outcomes carried V247A mutation in the detected five clades, i.e. L, G, GH, GR, and O, in
NSP2 protein, while those from the recovered the SARS-CoV-2 samples from Indonesia and
patient did not. In conjunction with D614G most of them (~60%) containe D614G. Globally,
mutation, substitution of valine (V) to alanine (A) D614G has been detected in ~93% samples in
in position 247 and 213 of NSP2 and spike 132 countries [11]. A recent study showed that
protein, respectively, were detected in the D614G mutation is significantly associated with
patients with fatal disease outcomes. Although the increase of SARS-CoV-2 infectivity,
both V and A, as well as G are in the non-polar competitive fitness, and transmission in primary
hydrophobic amino acid group, further human airway epithelial cells and hamsters [19].
investigations are necessary to determine
whether these dual mutations (V213A and Phylogenetic analysis showed that the full-
D614G in spike protein) or even triple mutations genome sequences of SARS-CoV-2 identified
(V213A and D614G in spike protein and V47A in within these family clusters are identical, which
NS2) are associated with increased risk of strongly indicates a direct transmission within
mortality in COVID-19 patients. Recently, more these families. Moreover, our study is also able
than 50% of the viral genome sequences in the to determine the virus clades of COVID-19 cases
UK were reported to have a new single with unknown contact history with a confirmed
phylogenetic cluster, i.e. VUI-202012/01 variant COVID-19 case. Our findings support a previous
(multiple spike protein mutations: deletion 69-70, suggestion regarding the importance of genomic
deletion 144, N501Y, A570D, D614G, P681H, epidemiology in filling the gaps of identifying
T716I, S982A, D1118H) [9]. These new variants SARS-CoV-2 infection sources [6]. Therefore, a
have been associated with a higher full-genome surveillance of SARS-CoV-2 in
transmissibility of SARS-CoV-2 up to 70% [9]. Indonesia is essential to prevent further
Until the submission date of January 2021 in transmission of SARS-CoV-2 and to identify any
GISAID, these variants were also detected in established or new variant that might affect the
Asia [11]. Interestingly, we detected other spike SARS-CoV-2 transmission and severity.
protein mutations in our collected virus strains, Notably, our study only included a limited
including those from the family clusters, i.e. L5F, number of family clusters from Yogyakarta and
V213A, W258R, Q677H, and K811I. Noteworthy, Central Java, Indonesia. These limitations
the V213A variant was identified in all patients should be considered for interpretations of our
from family cluster-1. V213A was detected in findings.
4/17 (23.5%) of our samples. This variant is only
found in only 0.01% of samples in four countries, CONCLUSIONS
including Indonesia [11]. Whether this variant is This is the first molecular epidemiology study
due to a founder effect needs further study. associating the full-genome sequences of
Currently, besides the D614G variant, several SARS-CoV-2 with the epidemiological and
mutations within the receptor binding domains clinical data within family clusters. Phylogenetic
(RBD) of the S protein have attracted most analysis revealed that the three viruses from
scientists’ attention due to their increased family cluster-1 formed a monophyletic group,
frequency in certain countries, including S477N where as viruses from family cluster-2 formed a

polyphyletic group indicating there is the Authors’contributions
possibility of different sources of infection. This G, HW, MSH, KI, and NA conceived the study.
study highlights how the same spike protein G drafted the manuscript, and HW, MSH, RT, A,
mutations among members of the same family KI, S, EA, and TW critically revised the
might show different disease outcomes. manuscript for important intellectual content. G,
Moreover, we also detected multiple spike MSH, M, IT, REK, RT, I, S, EWD, ES, DAAD,
protein mutations in our samples. Further EB, HR, YP, IP, OS, DA, SS, WW, DAP, FF,
studies are necessary to clarify the impact of UW, ARF, ASK, NRA, AS, DS, ISL, and TA
these multiple spike protein mutations in the collected the data; and G, M, and HW analyzed
transmission and severity of SARS-CoV-2 the data. All authors have read and approved
infection, especially in Indonesia. the manuscript and agreed to be accountable for
all aspects of the work in ensuring that questions
List of abbreviations related to the accuracy or integrity of any part of
SNPs, single nucleotide polymorphisms the work are appropriately investigated and
resolved.
Declarations
Ethics approval and consent to participate Acknowledgements
This study was approved by the Medical and We thank the Collaborator Members of the
Health Research Ethics Committee, Faculty of Yogyakarta-Central Java COVID-19 study
Medicine, Public Health and Nursing, group: Sumardi, Bambang Sigit Riyanto,
Universitas Gadjah Mada/ Dr. Sardjito Hospital, Munawar Gani, Satria Maulana, Cahya Dewi
Yogyakarta, Indonesia (KE/FK/0563/EC/2020) Satria, Titis Widowati, Elisabeth S. Herini, Umi
and written informed consent was obtained. The Solekhah Intansari, and Elizabeth Henny
research has been performed in accordance Herningtiyas (Faculty of Medicine, Public Health
with the Declaration of Helsinki. and Nursing, Universitas Gadjah Mada (FK-KMK
UGM)/RSUP Dr. Sardjito), Bagoes Poermadjaja
Consent to publish and Sintong HMT Hutasoit (Disease
All participants or guardians signed a written Investigation Center Wates, Yogyakarta),
informed consent for participating in this study. Indaryati and Havid Setyawan (Balai Besar
Teknik Kesehatan Lingkungan dan
Availability of data and material Pengendalian Penyakit, Yogyakarta), Ludhang
All data generated or analyzed during this study Pradipta Rizki, Nur Imma Fatimah Harahap,
are included in the submission. The sequence Kemala Athollah, Maria Patricia Inggriani, and
and metadata are shared through GISAID Sri Fatmawati (FK-KMK UGM), Safitriani and
(www.gisaid.org). Muhammad Taufiq Soekarno (PT. Pandu
Biosains). We gratefully acknowledge the
Competing interests authors, the originating and submitting
The authors declared no potential conflicts of Laboratories for their sequence and metadata
interest with respect to the research, authorship, shared through GISAID. All submitters of data
and/or publication of this article. may be contacted directly via www.gisaid.org.
The Acknowledgments Table for GISAID is
Funding reported as Supplementary material.
Our study was funded by the Universitas Gadjah
Mada (Program Penelitian Pemandatan 2020: References
5686/UN1.PIII/DIT-LIT/PT/2020) and Indonesian
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Figure 1. Phylogenetic analysis of SARS-CoV-2 genomes from Indonesia and different countries.
A phylogenetic tree was constructed from (ORF) of 142 SARS-CoV-2 virus sequences
29.409 nt length of the open reading frame using Neighbor Joining statistical method with

2,000 bootstrap replications. The evolutionary circles, while viruses for the study are indicated
distances were computed using the Kimura 2- in red and viruses from the family clusters are
parameter method and the rate variation among color-shaded in yellow. The tree is rooted to
sites was modelled with a gamma distribution Wuhan/Hu-1/2019 with the bootstrap percentage
(estimated α = 0.14566). SARS-CoV-2 virus values less than 70% hidden and it is drawn to
sequences from Indonesia (N=89) followed by scale with branch lengths measured in the
the date of collection are indicated in closed number of substitutions per site.

Figure 2. Timeline of COVID-19 symptoms and diagnosis from family clusters in Yogyakarta and
Central Java.

Figure 3. Phylogenetic analysis and nucleic acid differences of family clusters’ virus
sequences compared to the other SARS-CoV-2 virus sequences from Yogyakarta and Central
Java.
(a) The tree was constructed from 29.409 nt substitutions per site.
length of the open reading frame (ORF) of 142 (b) The number of base differences per
SARS-CoV-2 virus sequences using Neighbor sequence from between sequences are shown.
Joining statistical method and computed using Codon positions included were
the Kimura 2-parameter method with 2,000 1st+2nd+3rd+Noncoding. All ambiguous
bootstrap replications. The tree is rooted to positions were removed for each sequence pair.
Wuhan/Hu-1/2019 (NC_045512.2) with the There was a total of 29,409 positions in the final
bootstrap percentage values less than 70% dataset. Family clusters are indicated in
hidden and it is drawn to scale (0.0001) with asterisks: Case-1 (*) and Case-2 (**)
branch lengths measured in the number of

SURVEY DATA OF COVID-19 AWARENESS, KNOWLEDGE, PREPAREDNESS AND
RELATED BEHAVIORS AMONG BREAST CANCER PATIENTS IN INDONESIA

Ricvan Dana Nindreaa,∗, Nissa Prima Sarib, Wirsma Arif Harahapc, Samuel J. Haryonod, Hari Kusnantoe, Iwan
Dwiprahastof, Lutfan Lazuardig, Teguh Aryandonoh

aDoctoral Program, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta,
Indonesia
bDepartment of Midwifery, Faculty of Medicine, Universitas Andalas, Padang, Indonesia
cDivision of Surgical Oncology, Faculty of Medicine, Universitas Andalas, Padang, Indonesia
dDivision of Surgical Oncology, Dharmais Cancer Hospital, Jakarta, Indonesia
eDepartment of Family and Community Medicine, Faculty of Medicine, Public Health and Nursing,
Universitas Gadjah Mada, Yogyakarta, Indonesia
fDepartment of Pharmacology and Therapy, Faculty of Medicine, Public Health and Nursing, Universitas
Gadjah Mada, Yogyakarta, Indonesia
gDepartment of Health Policy and Management, Faculty of Medicine, Public Health and Nursing, Universitas
Gadjah Mada, Yogyakarta, Indonesia
hDepartment of Surgical Oncology, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah
Mada, Yogyakarta, Indonesia

ABSTRACT questionnaire. SPSS version 23.0 was used to
analyzed the data by descriptive and inferential
This dataset presents a survey data describing statistics and SmartPLS 3 to created the partial
least square path modeling. The data will help in
COVID-19 awareness, knowledge, pre- venting the transmission of COVID-19
among breast cancer patients and can support
preparedness and related behaviors among for health education and promotion
interventions.
breast cancer patients in Indonesia. The data
Keyword : COVID-19, Breast cancer, Indonesia,
were collected from breast cancer patients Awareness, Knowledge, Preparedness,
Behaviors
through a survey distributed by an online

questionnaire, assesing social demographic

characteristics (6 items), COVID-19 awareness

(5 items), knowledge (2 items), preparedness (2

items) and related behaviors (2 items), from 20th

June until 14th July 2020. The samples were

gathered 500 breast cancer patients in

Indonesia who were willing to fill an online

Value of the Data review and also meta-analysis in the
future
• These data are useful because this is the • These data could potentially make an
first survey that involved 500 of impact on society, involving other
respondents that explore COVID-19 variables that influence of breast cancer
awareness, knowledge, preparedness patients behaviors to prevent the
and related behaviors among breast transmission of COVID-19.
cancer patients in Indonesia.
1. Data Description
• All researchers in epidemiology, cancer The dataset provides an insightful
registry, and health psychology can information based on survey data on COVID-
benefit from these data because by using 19 awareness, knowledge, preparedness
this data to give the government and related behaviors among breast cancer
recommendations to help in pre- venting patients in Indonesia. The breast cancer
the spread of COVID-19 among breast patients collected through medical records
cancer patients and can support for review at Dr. M. Djamil General Hospital
health education and promotion Padang, Sardjito General Hospital
interventions in their country. Yogyakarta and Dharmais Cancer Hospital
Jakarta. The
• The data will be valuable to researchers
who want to compare with similar studies
on COVID- 19 awareness, knowledge,
preparedness and related behaviors
among breast cancer patients from other
countries or developing to systematic

survey data was conducted from 500 breast breast cancer patients in Indonesia are
cancer patients in Indonesia to assesing described in Tables 2–
COVID-19 awareness, knowledge, 5. Correlation between COVID-19
preparedness and related behaviors with awareness, knowledge, preparedness and
internet access. The data include five major related behaviors among breast cancer
group of variable: (a) social-demographic patients In Indonesia are described in Table
characteristics, including age, ed- ucational 6. Partial least square path modeling COVID-
background, working status, marital status 19 awareness, knowledge, preparedness
and nutritional status; (b) Five items for and related behaviors among breast cancer
COVID-19 awareness including information patients In Indonesia in Fig. 1.
about COVID-19, seriousness of COVID-19,
COVID-19 as a public health threats, 2. Experimental Design, Materials and
probability get sick from COVID-19 and Methods
someone arround of partic- ipants get This research was conducted using a
COVID-19; (c) two items assesed COVID-19 cross sectional survey design to
related to knowledge including correctly determine COVID-19 awareness,
identified 3 symptoms of the COVID-19 and knowledge and preparedness with related
correctly identified 3 prevention methods of behaviors among breast cancer patients
the COVID-19; (d) two items measured in Indonesia. The dataset in this survey
COVID-19 related to preparedness were 500 breast cancer patients collected
government confident to prevent of COVID- through medical records review at Dr. M.
19 and preparedness related to COVID-19 Djamil General Hospital Padang, Sardjito
outbreak; (e) two items assesed their General Hospital Yogyakarta and
COVID-19 related behaviors including Dharmais Cancer Hospital Jakarta, by the
COVID-19 changed daily routine and plans. written online informed consent. The data
The question- naire is provided as a re- sponses collected between 20th June
supplementary file. Social-demographic until 14th July 2020.
characteristics are presented in Table 1. The main researchers selected to use
The detailed measurement of responses on WhatsApp Messenger for enrolling
COVID-19 awareness, knowledge, potential participants. A questionnaire
preparedness and related behaviors among was designed and ex- ecuted and made
using google forms and link generated

was shared on Whatsapp messenger number of participants from medical
after main researchers got the contact records review and

Fig. 1. Partial least square path modeling COVID-19 awareness, knowledge, preparedness
and related behaviors among breast cancer patients in Indonesia.

permitted by doctors or team members who adapted used previous studies [3,4], knowl-
treated patients at Dr. M. Djamil General edge preparedness, behaviors related to
Hospital Padang, Sardjito General Hospital COVID-19 questionnaire items were adapted
Yogyakarta and Dharmais Cancer Hospital from pre- vious study by Wolf et al. [4]. The
Jakarta. The sam- pling technique in this questionnaire translating to Indonesian.
survey is convenience sampling [1]. The The respondent’s social-demographics
inclusion criteria were female patients with analyzed using frequency and percentage.
pathology examination showed positive The COVID- 19 awareness, knowledge,
breast cancer based on medical records preparedness and related behaviors among
review and never infected COVID-19 [2].The breast cancer patients analyzed using
survey items of COVID-19 awareness were Pearson correlation test. P value < 0.05 was

stated as statistically significant. References

Ethics Statement 1. R.D. Nindrea, T. Aryandono, L. Lazuardi,
This study passed the ethical review by the
ethics commiittee of the Faculty of Medicine, Breast cancer risk from modifiable and
Public Health and Nursing, Universitas
Gadjah Mada, Yogyakarta, Indonesia. The non-modifiable risk factors among
survey data was conducted according to the
Declaration of Helsinki. women in Southeast Asia: a meta-

Declaration of Competing Interest analysis, Asian. Pac. J. Cancer Prev. 18
The authors declare that they have no known
competing financial interests or personal (2017) 3201–3206,
rela- tionships which have, or could be
perceived to have, influenced the work doi:10.22034/APJCP.2017. 18.12.3201.
reported in this article.
2. R.D. Nindrea, W.A. Harahap, T.
Acknowledgments
The author would like to thanks to Aryandono, L. Lazuardi, Association of
participants who were willing to give a
response to the data of this survey. BRCA1 promoter methylation with breast

Supplementary Materials cancer in Asia: a meta-analysis, Asian.
Supplementary material associated with this
article can be found, in the online version, at Pac. J. Cancer Prev. 19 (2018) 885–889,
doi:10.1016/j.dib.2020.106145.
doi:10.22034/APJCP.2018.19.4.885.

3. B. Kelly, L. Squiers, C. Bann, A. Stinee,

H. Hansen, M. Lynch, Perceptions and

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2441- 7.

4. M.S. Wolf, M. Serper, L. Opsasnick, R.M.

O’Conor, L.M. Curtis, J.Y. Benavente, et

al., Awareness, attitudes, and actions

related to COVID-19 among adults with

chronic conditions at the onset of the U.S.

outbreak: a cross-sectional survey, Ann.

Intern. Med. 2020 (2020) M20–1239.

FULL-LENGTH GENOME CHARACTERIZATION AND PHYLOGENETIC ANALYSIS OF
SARS-COV-2 VIRUS STRAINS FROM YOGYAKARTA AND CENTRAL JAVA, INDONESIA

Gunadi1, Hendra Wibawa2, Marcellus1, Mohamad Saifudin Hakim3, Edwin Widyanto Daniwijaya4, Ludhang Pradipta Rizki3,
Endah Supriyati5, Dwi Aris Agung Nugrahaningsih6, Afiahayati7, Siswanto8, Kristy Iskandar9, Nungki Anggorowati10, Alvin
Santoso Kalim1, Dyah Ayu Puspitarani1, Kemala Athollah1, Eggi Arguni11, Titik Nuryastuti3 and Tri Wibawa3

1 Pediatric Surgery Division, Department of Surgery, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia
2 Disease Investigation Center Wates, Yogyakarta, Ministry of Agriculture, Indonesia
3 Department of Microbiology, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia
4 Department of Microbiology, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada/UGM Academic Hospital, Yogyakarta, Indonesia
5 Centre of Tropical Medicine, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia
6 Department of Pharmacology and Therapy, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia
7 Department of Computer Science and Electronics, Faculty of Mathematics and Natural Sciences, Universitas Gadjah Mada, Yogyakarta, Indonesia
8 Department of Physiology, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada/UGM Academic Hospital, Yogyakarta, Indonesia
9 Department of Child Health, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada/UGM Academic Hospital, Yogyakarta, Indonesia
10Department of Anatomical Pathology, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia
11Department of Child Health, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia

ABSTRACT whereas the clade L virus sample
Background: Recently, SARS-CoV-2 virus with (EPI_ISL_516806) was located amongst SARS-
the D614G mutation has become a public CoV-2 genomes from Asia. Using full-length
concern due to rapid dissemination of this sequences available in the GISAID EpiCoV
variant across many countries. Our study aims Database, 39 of 60 SARS-CoV-2 (65%) from
were (1) to report full-length genome sequences Indonesia harbor the D614G mutation.
of SARS-CoV-2 collected from four COVID-19 Conclusion: These findings indicate that SARS-
patients in the Special Region of Yogyakarta CoV-2 with the D614G mutation appears to
and Central Java provinces, Indonesia; (2) to become the major circulating virus in Indonesia,
compare the clade distribution of full-length concurrent with the COVID-19 situation
genome sequences from Indonesia (n = 60) worldwide. Subjects Bioinformatics, Genomics,
from March to September 2020 and (3) to Molecular Biology, Virology, Infectious Diseases
perform phylogenetic analysis of SARS-CoV-2 Keywords COVID-19, D614G mutation,
complete genomes from different countries, Indonesia, Phylogenetic analysis, SARS-CoV-2,
including Indonesia. Whole genome sequencing
Methods: Whole genome sequencing (WGS)
was performed using next-generation INTRODUCTION
sequencing (NGS) applied in the Illumina MiSeq In December 2019, an outbreak of Severe Acute
instrument. Full-length virus genomes were Respiratory Syndrome Coronavirus 2 (SARS-
annotated using the reference genome of hCoV- CoV-2) causing Coronavirus Disease 2019
19/Wuhan/Hu-1/ 2019 (NC_045512.2) and then (COVID-19) was detected in Wuhan, China and
visualized in UGENE v. 1.30. For phylogenetic has become a global pandemic, including
analysis, a dataset of 88 available SARS-CoV-2 Indonesia (World Health Organization, 2020a;
complete genomes from different countries, Phelan, Katz & Gostin, 2020). In Indonesia, the
including Indonesia, was retrieved from GISAID. first two COVID-19 cases were reported on 2
Results: All patients were hospitalized with March 2020. Since then, the confirmed cases
various severities of COVID-19. Phylogenetic have been continuously increasing although
analysis revealed that one and three virus several public health measures, involving
samples belong to clade L and GH. These three isolation of confirmed patients and community
clade GH virus samples (EPI_ISL_525492, wide containment in addition to strictly enforced
EPI_ISL_516800 and EPI_ISL_516829) were not personal health protocols, were conducted to
only located in a cluster with SARS-CoV-2 halt transmission events (World Health
genomes from Asia but also those from Europe, Organization, 2020b). Tragically, on 19

November 2020, Indonesia recorded 478,720 MATERIALS AND METHODS
COVID-19 infections and 15,503 deaths (World Classification of COVID-19 severity
Health Organization, 2020b). This situation We determined the COVID-19 severity
means that Indonesia has reported the second according to the WHO classifications:
most confirmed COVID-19 cases in the South (1) mild, symptomatic COVID-19 patients without
East Asia countries after the Philippines, yet has
the highest number of deaths caused by COVID- evidence of hypoxia or pneumonia;
19 among other South East Asia countries (2) moderate, clinical signs of pneumonia (i.e.,
(World Health Organization, 2020b). Just as
many other countries, the detection of SARS- fever, cough, dyspnea, fast breathing) but not
CoV-2 in suspected people is mainly based on severe pneumonia, including blood oxygen
reverse transcriptase real-time polymerase saturation levels (SpO2) ≥90% in room air;
chain reaction (RT-PCR). The supply of PCR (3) severe, clinical signs of pneumonia plus one of
reagents, trained lab personnel and the the conditions as follows: respiratory rate >30
availability of laboratories with sufficient breaths/minute, severe respiratory distress,
biocontainment levels are major challenges of or SpO2 <90% in room air and
SARS-CoV-2 detection in developing countries, (4) critical, Acute Respiratory Distress
such as Indonesia (Younes et al., 2020). Syndrome, sepsis, or septic shock, other
Therefore, it is not surprising that the tested complications such as acute pulmonary
people per week is still lower than the World embolism, acute coronary syndrome, acute
Health Organization (WHO) standard (World stroke and delirium (Beeching, Fletcher &
Health Organization, 2020b). Recently, SARS- Fowler, 2019).
CoV-2 with the D614G mutation became the
most frequently detected globally, including Virus Samples
South East Asia region (Korber et al., 2020; All virus samples were collected from
Nguyen et al., 2020). Interestingly, SARS-CoV-2 hospitalized patients with COVID-19 from June
with the G614 variant had significantly higher to August 2020 in Yogyakarta and Central Java
infectious titers than the original D614 virus, and provinces. Samples were collected from
COVID-19 patients with the G614 variant had a nasopharyngeal swabs and then directly put into
higher viral load than patients without the viral transport media (DNA/RNA ShieldTM
mutation (Korber et al., 2020). A recent study Collection Tube with Swab, Zymo Research,
showed that the SARS-CoV-2 with the G614 CA, USA). Samples were sent to the
variant revealed increased infectivity, Department of Microbiology and Laboratorium
competitive fitness, and transmission than the Diagnostik Yayasan Tahija World Mosquito
wild-type D614 virus in human airway epithelial Program, Faculty of Medicine, Public Health and
cells and hamster (Hou et al., 2020). However, Nursing, Universitas Gadjah Mada and the
this mutation was not associated with the Disease Investigation Center, Wates,
severity of COVID-19 (Korber et al., 2020; Yogyakarta for SARS-CoV-2 virus detection
Nguyen et al., 2020). Here, we aimed: using Real-Q 2019-nCoV Detection Kit
(1) to report full-length genome sequences of (BioSewoom, Seoul, South Korea) with
LightCycler® 480 Instrument II (Roche
SARS-CoV-2 collected from four COVID-19 Diagnostics, Mannheim, Germany).
patients in the Special Region of Yogyakarta
and Central Java provinces, Indonesia; Whole Genome Sequencing
(2) to compare the clade distribution of full- Total viral RNA was extracted from 15 original
length genome sequences from Indonesia samples (nasopharyngeal swabs) using a
(n = 60) from March to September 2020; QiAMP Viral RNA mini kit (Qiagen, Hilden,
and Germany), followed by double stranded cDNA
(3) to perform phylogenetic analysis of SARS- synthesis using Maxima H Minus Double-
CoV-2 complete genomes from different Stranded cDNA Synthesis (Thermo Fisher
countries, including Indonesia. Scientific, MA, USA), and then purified by a
GeneJET PCR Purification Kit (Thermo Fisher

Scientific, MA, USA). The Nextera DNA Flex for clades from some countries that have complete
Enrichment using Respiratory Virus Oligos
Panel was used for library preparations, and genome, high-coverage, and no stretches of
whole genome sequencing was performed using
next generation sequencing (NGS) applied in “NNNN” for the phylogenetic dataset. Sequence
the Illumina MiSeq instrument (Illumina, San
Diego, CA, USA) with Illumina MiSeq reagents alignment was performed using the MAFFT
v3 150 cycles (2 × 75 cycles). Among 15
samples that were analyzed by NGS, only four program server for multiple nucleotide sequence
samples showed good data for further
bioinformatics analysis. The paired reads were alignment
trimmed for quality and length and assembled by
mapping to the reference genome from Wuhan, (https://mafft.cbrc.jp/alignment/server/). A
China (hCoV-19/Wuhan/Hu-1/2019, GenBank
accession number: NC_045512.2) using phylogenetic tree was constructed using 29.400
Burrow-Wheeler Aligner (BWA) algorithm
embedded in UGENE v. 1.30 nucleotide length starting from the ORF1ab
(Unipro UGENE Online User Manual, 2020).
Single nucleotide polymorphism (SNP) was open reading frame of SARS-CoV-2 using a
identified based on the number of high
confidence base calls (consensus sequence maximum likelihood statistical method with
variations of the assembly) that disagree with
the reference bases for the genome position of 1,000 bootstrap replications and selected the
interest. These variations were then exported to
a vcf file and visualized in MS Excel. All four full- best-fitting substitution model (GTR+G+I) for the
genome sequences of SARS-CoV-2 had the
following accession IDs: EPI_ISL_516800, dataset. All the analyses were performed in
EPI_ISL_516806, EPI_ISL_516829 and
EPI_ISL_525492 (GISAID, 2020). Molecular Evolutionary Genetics Analysis

Genome Annotation And Phylogenetic version 10 (MEGA X) software (Kumar et al.,
Analysis
Full-length virus genomes were annotated using 2018). Because the purpose of this phylogenetic
the reference genome of hCoV-19/ Wuhan/Hu-
1/2019 (NC_045512.2). For phylogenetic analysis was to determine the evolutionary
analysis, a dataset of 88 available SARS-CoV-2
virus genomes from different countries, including relationships between our virus samples and the
those from Indonesia, was retrieved from
GISAID. Instead of using all available other SARS-CoV-2 viruses, the tree was rooted
sequences in GISAID, we used sequences of
several viruses representing SARS-CoV-2 to the oldest virus, hCoV- 19/Wuhan/Hu-1/2019.

Ethical Approval
The Medical and Health Research Ethics
Committee of the Faculty of Medicine, Public
Health and Nursing, Universitas Gadjah
Mada/Dr. Sardjito Hospital approved this study
(KE/FK/0563/EC/2020). Written informed
consent was obtained from all participants
before joining in this study.

RESULTS
Whole genome sequences of SARS-CoV-2
from the special region of Yogyakarta and
Central Java provinces, Indonesia

All patients were classified as moderate COVID-
19, except patient-4 as a mild case (Table 1;
Table S1). The details of case presentations are
described in the Table S1.

Notes:
* Name of SARS-CoV-2 proteins (bold) are followed by related genes (italic) and amino acid mutation indicated in bracket. CT, cycle threshold.

Ref. sequence: hCoV-19/Wuhan/Hu-1/2019 (NC_045512.2).

WGS revealed that the virus sample collected from patient-4 consisted of only one mutation in
from patient-1 (hCoV19/Indonesia/ YO-UGM- the NSP5 protein (M49I) (Table 1).
781481/2020, ID: EPI_ISL_516829), patient-2 The genome-wide SNPs and amino acid
(hCoV19/Indonesia/YO-UGM- 202449/2020, ID: variations of our samples are shown in Tables 2
EPI_ISL_516800) and patient-3 belonged to the and 3, respectively (positions referred to the
GH clade, while those from patient-4 reference sequence: NC_045512.2). Not all
(hCoV19/Indonesia/YO-UGM-200927/2020, ID: SNPs cause amino acid changes in our
EPI_ISL_516806) showed the L clade. samples.
Moreover, WGS of virus from patient-1, patient-2
and patient 3 showed nine amino acid mutations Clade Distribution Of Full-Length Genome
in six proteins, including NSP3 (P679S), NSP12 Sequences From Indonesia
(P323L, A656S), NSP13 (M576I), spike Whole genome sequencing revealed that one
(D614G), NS3 (A54V, Q57H, A99S), and NP virus (hCoV19/Indonesia/YO-202449/2020,
(Q160R); four amino acid mutations in four EPI_ISL_516800) had a complete SARS-CoV-2
proteins: NSP3 (P822L), NSP12 (P323L), Spike genome (29.903 nt). Although the other three
(D614G) and NS3 (Q57H); and five amino acid virus samples were shorter due to incomplete
mutations in five proteins: NSP3 (P822L), UTRs at either the 5′ or 3′, they possessed full-
NSP12 (P323L), Spike (D614G), NS3 (Q57H) length and complete open reading frames
and NS7a (H73Y), respectively; whereas those (ORFs) with a size of 29.409 nt

Figure 1 Clade distribution of SARS-CoV-2 genomes in Indonesia until the submission date of September 2020, showing that 65%
contained the D614G mutation. All G clades (G, GH and GR) carried the D614G mutation.Full-size DOI:10.7717/peerj.10575/fig-1

consisting of 11 genes (ORF1ab, S, ORF3a, E, the collection data, most (39/60, 65%) virus
M, ORF6, ORF7a, ORF7b, ORF8, N, ORF10). genomes contained the D614G mutation
Next, we compared the clade distribution of full- representing clade G (2), GR (7) and GH (30)
length genome sequences from Indonesia (n = (Fig. 1). From March to April 2020, clade L was
60) from March to September 2020. Based on dominant. On the other hand, there has been an

increase in the detection of clade GH since April Indonesia, India, United Arab Emirates and
2020 until now. Japan) (Fig. 2)

Phylogenetic Analysis DISCUSSION
Phylogenetic analysis of whole genome The present study reports four full genomes of
sequencing showed that three virus samples SARS-CoV-2 from patients with COVID-19 in
(EPI_ISL_525492, EPI_ISL_516800 and Yogyakarta and Central Java Provinces,
EPI_ISL_516829) clustered amongst viruses Indonesia. Phylogenetic analysis showed that
from the clade GH from multiple countries three of four samples collected in June and
across Asia, Middle East and Europe (Fig. 2). In August 2020 were clustered within SARS-CoV-2
particular to EPI_ISL_516829, this virus showing viruses belonging to GH clade. One of these
an extended branch length to the ancestral (EPI_ISL_516829) displayed a longer branch
human SARS-CoV-2 virus, hCoV-19/Wuhan/Hu- length compared to the other viruses indicating a
1/2019. On the other hand, one virus sample greater evolutionary distance to the ancestral
(EPI_ISL_516806) was situated between clade virus, hCoV-19/Wuhan/Hu-1/2019. This was
L viruses mainly from Asia (China, Malaysia, confirmed by the

Figure 2 Phylogenetic analysis of SARS-CoV-2 genomes from Indonesia and different countries. A phylogenetic tree was
constructed from 29.400 nt length of the open reading frame (ORF) of SARS-CoV-2 using the maximum likelihood statistical method with
1,000 bootstrap replications and best-fitting DNA substitution model (GTR+G+I) for the dataset. Virus samples from Yogyakarta and
Central Java are indicated in red, while those from other regions in Indonesia are indicated in blue. The tree is drawn to scale (0.0001), with
branch lengths measured in the number of substitutions per site. Full-size DOI:10.7717/peerj.10575/fig-2

Molecular characterization showing that more evolution closely related to the other SARS-
SNPs and amino acid mutations were found CoV-2 viruses belonged to the L clade, including
within this virus genome. The phylogenetic those from Indonesia which were collected in the
analysis also revealed that one virus sample early months of disease transmission during the
collected in May 2020 (EPI_ISL_516806) was in pandemic between March and April 2020.

Our finding corresponds with the situation in 2020). It has been reported that COVID-19
Indonesia, showing that during the early patients with the D614G mutation have a higher
pandemic only two clades, O and L, were viral load than patients infected by SARS-CoV-2
detected, and the latter clade was more without mutations (Korber et al., 2020). The
dominantly found from COVID-19 cases. patients with D614G had a CT value lower than
However, since the first detection of clade GH in one patient without the mutation (Table 1).
April 2020, this virus was more frequently Interestingly, patients infected with SARS-CoV-2
detected than the previous circulating clades. bearing D614G mutations showed moderate
Based on the data available in GISAID, 60 virus COVID-19, while the patient without mutations
samples representing five clades have been suffered from mild symptoms. These differences
detected from COVID-19 cases in Indonesia up might be associated with the small sample size
to September 2020 (based on full-length of our study (n = 4) compared with previous
genome and submission date): L (20), O (1), G studies (n = 999 (Korber et al., 2020), 175
(2), GR (7) and GH (30) (GISAID, 2020). (Wagner et al., 2020) and 88 (Lorenzo-Redondo
Whether this pattern correlates with the increase et al., 2020)). Moreover, the severity of COVID-
in the number of COVID-19 cases recently in 19 is affected by many factors, including age,
Indonesia has to be investigated further. sex, presence of comorbidities, and patients’
Interestingly, a similar situation was found in immune responses (Beeching, Fletcher &
some countries in North America (Mercatelli & Fowler, 2019; Zou et al., 2020). Further study
Giorgi, 2020) and Africa (Wruck & Adjaye, with a larger sample size and involving risk
2020), which also detected more SARS-CoV-2 factors for COVID-19 severity is mandatory to
virus strains belonging to clade GH than to the determine the association between the D614G
other clades. An increase in SARS-CoV-2 mutation and the severity of COVID-19,
detection conveys the D614G mutation particularly in Indonesia.
concurrent with the recent global situation of Among GH clades, they also consisted of
COVID-19 (GISAID, 2020). different mutations in addition to the variants that
The D614G mutation dominates globally determine the clade name (Table 1). It has
approximately 77,818/96,215 (~81%) full already been reported that the D614G variant is
genomes submitted at GISAID until 18 almost always accompanied by three other
September 2020 (GISAID, 2020). Three of four variants: a C–T change in the 5’UTR, a silent
(75%) SARS-CoV-2 in our case series also c.3307C > T variant, and P323L (Younes et al.,
consisted of D614G. According to phylogenetic 2020). All GH clade samples in the present
tree and sequence distribution analysis, it has study also contained P323L (Table 1).
been suggested that the dominating D614G Notably, whole genome sequencing is of
globally is caused by a positive selection (Korber practical importance to determine virus variants
et al., 2020), while the dominating D614G in and clades and is associated with particular
Europe is due to a founder effect (Dearlove et geographic disseminations to decide clinical and
al., 2020). Whether which mechanism occurs in political approaches at the regional and local
Indonesia is difficult to conclude since only levels (Mercatelli & Giorgi, 2020). Moreover,
limited full genomes were submitted to GISAID whether the differences in the case fatality rate
until the submission date of the end of and viral spread or transmission among different
September 2020 (n = 60) (GISAID, 2020). The countries/regions are affected by differences in
virus with the D614G mutation in Indonesia was the virus clade (Brufsky, 2020) needs to be
first detected in April 2020 in Surabaya, East further studied.
Java (GISAID, 2020), followed by other Our study has some limitations, including that
provinces, including Yogyakarta, Central Java, the samples sequenced are only 0.02%
West Java and Banten. Clade L was mostly (60/290,000) of all confirmed cases in Indonesia.
detected in These facts should be considered in the
Jakarta (7/20) and Surabaya, East Java (7/20), interpretations of our findings, especially about
followed by Papua (3/20) (Fig. 1) (GISAID, epidemiological patterns (e.g., increase vs.

decrease of frequency) of a particular clade in ADDITIONAL INFORMATION AND
Indonesia. Another limitation of our study is we
do not have any data from epidemiological DECLARATIONS
tracing, nor data concerning detection (how far,
how fast) to determine what is the size of the Funding
cluster where each patient belongs, or what is This study was funded by the Indonesian
the transmission pattern of the cluster. Ministry of Research and Technology/National
Agency for Research and Innovation (World
CONCLUSIONS Class Research 2020 scheme: 3850/UN1/
We report the full-genome sequence DITLIT/DIT-LIT/PT/2020). The funders had no
characterization and phylogenetic analysis of role in study design, data collection and
SARS-CoV-2 from Indonesia. SARS-CoV-2 with analysis, decision to publish, or preparation of
the D614G mutation appears to become the the manuscript.
major circulating virus in Indonesia, which is
concurrent with the COVID-19 situation Grant Disclosures
worldwide. Further study with a larger sample The following grant information was disclosed by
size is necessary to investigate whether the the authors:
dominating SARS-CoV-2 bearing the D614G Indonesian Ministry of Research and
mutation is due to a positive selection or a Technology/National Agency for Research and
founder effect or some other mechanism and to Innovation (World Class Research 2020
explore the role of the D614G mutation in the scheme): 3850/UN1/DITLIT/DIT-LIT/PT/2020.
pathogenesis and virulence of SARS-CoV-2.
Competing Interests
ACKNOWLEDGEMENTS The authors declare that they have no
We thank the Collaborator Members of the competing interests.
Yogyakarta-Central Java COVID-19 study
group: Kurniyanto (RSUP Dr. Soeradji Author Contributions
Tirtonegoro), Indah Juliana (RSUP Dr Soeradji ● Gunadi conceived and designed the experiments,
Tirtonegoro), Beby Dewi Sartika (RSUD Nyi
Ageng Serang), Ardorisye Saptaty Fornia (RS analyzed the data, prepared figures and/or
PKU Gamping), Dwiki Afandy (Faculty of tables, authored or reviewed drafts of the paper,
Medicine, Public Health and Nursing, and approved the final draft.
Universitas Gadjah Mada (FK-KMK UGM)), ● Hendra Wibawa conceived and designed the
Susan Simanjaya (FK-KMK UGM), William experiments, analyzed the data, prepared figures
Widitjiarso (FK-KMK UGM), Aditya Rifqi Fauzi and/or tables, authored or reviewed drafts of the
(FK-KMK UGM), Safitriani (PT. Pandu Biosains), paper, and approved the final draft.
Muhammad Taufiq Soekarno (PT. Pandu ● Marcellus performed the experiments, analyzed
Biosains) and Sri Fatmawati (FK-KMK UGM). the data, prepared figures and/or tables, and
Gunadi, Marcellus, Dwi Aris Agung approved the final draft.
Nugrahaningsih, Kristy Iskandar, Nungki ● Mohamad Saifudin Hakim performed the
Anggorowati, Alvin Santoso Kalim, Kemala experiments, authored or reviewed drafts of the
Athollah, Dyah Ayu Puspitarani are members of paper, and approved the final draft.
the Genetics Working Group (Pokja Genetik), ● Edwin Widyanto Daniwijaya performed the
Faculty ofMedicine, Public Health and Nursing, experiments, authored or reviewed drafts of the
Universitas Gadjah Mada. We gratefully paper, and approved the final draft.
acknowledge the authors, the originating and ● Ludhang Pradipta Rizki performed the
submitting laboratories for their sequence and experiments, authored or reviewed drafts of the
metadata shared through GISAID. paper, and approved the final draft.
● Endah Supriyati performed the experiments,
analyzed the data, authored or reviewed drafts of
the paper, and approved the final draft.
● Dwi Aris Agung Nugrahaningsih conceived and
designed the experiments, authored or reviewed
drafts of the paper, and approved the final draft.
● Afiahayati performed the experiments, analyzed

the data, prepared figures and/or tables, authored final draft.
or reviewed drafts of the paper, and approved ● Titik Nuryastuti conceived and designed the
the final draft.
● Siswanto performed the experiments, analyzed experiments, performed the experiments,
the data, authored or reviewed drafts of the authored or reviewed drafts of the paper, and
paper, and approved the final draft. approved the final draft.
● Kristy Iskandar analyzed the data, authored or ● Tri Wibawa conceived and designed the
reviewed drafts of the paper, and approved the experiments, authored or reviewed drafts of the
final draft. paper, and approved the final draft.
● Nungki Anggorowati analyzed the data, authored
or reviewed drafts of the paper, and approved the Human Ethics
final draft. The following information was supplied relating to
● Alvin Santoso Kalim performed the experiments, ethical approvals (i.e., approving body and any
authored or reviewed drafts of the paper, and reference numbers): The Medical and Health
approved the final draft. Research Ethics Committee of the Faculty of
● Dyah Ayu Puspitarani performed the Medicine, Public Health and Nursing, Universitas
experiments, authored or reviewed drafts of the Gadjah Mada/Dr. Sardjito Hospital approved this
paper, and approved the final draft. study (KE/FK/0563/EC/2020). Written informed
● Kemala Athollah performed the experiments, consent was obtained from all participants
authored or reviewed drafts of the paper, and before joining in this study.
approved the final draft.
● Eggi Arguni performed the experiments, authored
or reviewed drafts of the paper, and approved the

DNA Deposition
The following information was supplied regarding the deposition of DNA sequences:
The SARS-CoV-2 genome sequences used in this study are available via GISAID
(https://www.epicov.org/epi3/frontend#142ddf):
EPI_ISL_498515, EPI_ISL_512713, EPI_ISL_452350, EPI_ISL_444300, EPI_ISL_492566, EPI_ISL_453708,
EPI_ISL_482576, EPI_ISL_482585, EPI_ISL_482586, EPI_ISL_435109, EPI_ISL_515954,
EPI_ISL_481156, EPI_ISL_528821, EPI_ISL_528834, EPI_ISL_528869, EPI_ISL_435078,
EPI_ISL_529138, EPI_ISL_528745, EPI_ISL_528747, EPI_ISL_528746, EPI_ISL_528748,
EPI_ISL_528752, EPI_ISL_528751, EPI_ISL_528750, EPI_ISL_528759, EPI_ISL_528749,
EPI_ISL_458079, EPI_ISL_458081, EPI_ISL_437188, EPI_ISL_437189, EPI_ISL_435281,
EPI_ISL_437190, EPI_ISL_437191, EPI_ISL_435282, EPI_ISL_437192, EPI_ISL_467376,
EPI_ISL_435283, EPI_ISL_525492, EPI_ISL_467374, EPI_ISL_467375, EPI_ISL_518819,
EPI_ISL_516806, EPI_ISL_516800, EPI_ISL_516829, EPI_ISL_493328, EPI_ISL_451300,
EPI_ISL_496482, EPI_ISL_419301, EPI_ISL_479997, EPI_ISL_480033, EPI_ISL_480116,
EPI_ISL_529135, EPI_ISL_450186, EPI_ISL_501206, EPI_ISL_501207, EPI_ISL_501222,
EPI_ISL_455790, EPI_ISL_490048, EPI_ISL_528744, EPI_ISL_513196, EPI_ISL_513248,
EPI_ISL_513254, EPI_ISL_513264, EPI_ISL_483615, EPI_ISL_493419, EPI_ISL_516823,
EPI_ISL_527365, EPI_ISL_527377, EPI_ISL_506991, EPI_ISL_522501, EPI_ISL_526723,
EPI_ISL_526734, EPI_ISL_526746, EPI_ISL_455336, EPI_ISL_530091, EPI_ISL_435142,
EPI_ISL_513808, EPI_ISL_452134, EPI_ISL_421357, EPI_ISL_480387, EPI_ISL_511894,
EPI_ISL_511895, EPI_ISL_511896, EPI_ISL_511897, EPI_ISL_493167, EPI_ISL_493178,
EPI_ISL_402125, EPI_ISL_406798.
To access the sequence data GISAID requires user registration (https://www.gisaid.org/
registration/register/).