\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[ngerman,english]{babel}



\usepackage{float}








\begin{document}

\title{Virologic Characteristics of Cases of COVID-19 in Northern Vietnam,
January -- May, 2020}



\author[1]{Hang Nguyen}%
\author[2]{Son Nguyen}%
\author[1]{Phuong Hoang}%
\author[1]{Thanh Le}%
\author[2]{Huong Tran}%
\author[3]{Long Pham}%
\author[1]{Quang Thai Pham}%
\author[1]{Thuy Nguyen}%
\author[1]{Anh Dang}%
\author[2]{MAI LE}%
\affil[1]{National institute of Hygiene and Epidemiology}%
\affil[2]{National Institute of Hygiene and Epidemiology}%
\affil[3]{2.	Mohawk college, Hamilton, ON, Canada}%


\vspace{-1em}



  
  \date{May 08, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Background: Vietnam confirmed its first case of SARS-CoV-2 infection on
January 23, 2020 among travelers from Wuhan, China and experienced
several clusters of community transmission until September. Vietnam
implemented an aggressive testing, isolation, contact tracing, and
quarantine strategy in response to all laboratory-confirmed cases. We
report the results of SARS-CoV-2 testing during the first half of 2020
in northern Vietnam. Methods: From January through May, 2020, 15,650
upper respiratory specimens were collected from 14,470 suspected cases
and contacts in northern Vietnam. All were tested for SARS-CoV-2 by
real-time RT-PCR. Individuals with positive specimens were tested every
3 days until negative twice. Positive specimens from 81 individuals were
cultured. Results: Among 14,470 tested individuals, 158 (1.1\%) cases of
SARS-CoV-2 infection were confirmed; 89 were imported and 69 were
associated with community transmission. Most patients (122, 77\%) became
negative after two tests, with 11 and 4 still testing positive when
sampled a fourth and fifth time, respectively. Among 81 specimens with
Ct values \textless{}30, SARS-CoV-2 was isolated from 29 (36\%). Seven
patients testing positive again after testing negative had Ct values
\textgreater{}30 and negative culture. Conclusion: Early and widespread
testing for SARS-CoV-2 in northern Vietnam identified very few cases
which, when combined with other aggressive strategies, may have
dramatically contained the epidemic. We observed rapid viral clearance
and very few positive results following clearance. Large scale molecular
diagnostic testing is a critical part of early detection and containment
of COVID-19 in Vietnam and will remain necessary until a vaccine is
widely implemented.%
\end{abstract}%



\sloppy


\textbf{Virologic Characteristics of Cases of COVID-19 in Northern
Vietnam, January -- May, 2020.}

\textbf{Authors}

\textbf{Hang Khanh Le Nguyen \textsuperscript{1}, Son Vu
Nguyen\textsuperscript{1}, Phuong Mai Vu Hoang\textsuperscript{1}, Thanh
thi Le\textsuperscript{1}, Huong thi Thu Tran \textsuperscript{1}, Long
Hai Pham Nguyen\textsuperscript{2}, Thai Quang Pham\textsuperscript{1},
Thuy thanh Nguyen\textsuperscript{1}, Anh Duc Dang\textsuperscript{1},
Mai thi Quynh Le\textsuperscript{1}.}

\begin{enumerate}
\tightlist
\item
  \textbf{National Institute of Hygiene and Epidemiology, Hanoi,
  Vietnam}
\item
  \textbf{Mohawk college, Hamilton, ON, Canada}
\end{enumerate}

\textbf{Corresponding author} : Mai thi Quynh Le,

National Influenza Center -- National Institute of Hygiene and
Epidemiology, Hanoi, Vietnam.

Fax: + 84 4 3 8210853

Phone: + 84 912126283

Email: lom9@hotmail.com or lom9@nihe.org.vn.

\textbf{Background:} Vietnam confirmed its first case of SARS-CoV-2
infection on January 23, 2020 among travelers from Wuhan, China and
experienced several clusters of community transmission until September.
Vietnam implemented an aggressive testing, isolation, contact tracing,
and quarantine strategy in response to all laboratory-confirmed cases.
We report the results of SARS-CoV-2 testing during the first half of
2020 in northern Vietnam.

\textbf{Methods:} From January through May, 2020, 15,650 upper
respiratory specimens were collected from 14,470 suspected cases and
contacts in northern Vietnam. All were tested for SARS-CoV-2 by
real-time RT-PCR. Individuals with positive specimens were tested every
3 days until negative twice. Positive specimens from 81 individuals were
cultured.

\textbf{Results} : Among 14,470 tested individuals, 158 (1.1\%) cases of
SARS-CoV-2 infection were confirmed; 89 were imported and 69 were
associated with community transmission. Most patients (122, 77\%) became
negative after two tests, with 11 and 4 still testing positive when
sampled a fourth and fifth time, respectively. Among 81 specimens with
Ct values \textless{}30, SARS-CoV-2 was isolated from 29 (36\%). Seven
patients testing positive again after testing negative had Ct values
\textgreater{}30 and negative culture.

\textbf{Conclusion:} Early and widespread testing for SARS-CoV-2 in
northern Vietnam identified very few cases which, when combined with
other aggressive strategies, may have dramatically contained the
epidemic. We observed rapid viral clearance and very few positive
results following clearance. Large scale molecular diagnostic testing is
a critical part of early detection and containment of COVID-19 in
Vietnam and will remain necessary until a vaccine is widely implemented.

\textbf{Keyword:} COVID-19; SARS-CoV-2; real-time RT-PCR, viral culture;
containment.

\textbf{Introduction:}

SARS-CoV-2 is the cause of coronavirus disease 2019 (COVID-19), first
reported in Wuhan, China in late December 2019. As of September 2020,
SARS-CoV-2 was responsible for over 25 million cases and nearly one
million deaths(1)

Vietnam is a country of 97 million people which, despite its
lower-middle income status, managed to limit spread of SARS-CoV-2,
requiring eight months to reach 1,000 cases and seven months to record
its first fatality. The early days of the pandemic in Vietnam were
marked primarily by importations from China whereas the second cluster
of cases was characterized by importation from Europe (2--6).

Vietnam hosts two National Influenza Centers (NICs) including one at the
National Institute of Hygiene and Epidemiology (NIHE). NIHE coordinates
influenza surveillance in northern Vietnam and has played a critical
role in responding to the COVID-19 pandemic. In its role as a reference
laboratory throughout the country, NIHE received some of the earliest
specimens from suspected cases of COVID-19. Our objectives were to
describe the virologic characteristics of these specimens received
during January through April 2020 and to explore differences in these
characteristics among cases derived from different sources.

\textbf{Methods}

Vietnam established a National Steering Committee on Prevention and
Control of COVID-19 on 28 January 2020, six days after the first cases
of COVID-19 were identified in the country (2). Subsequent guidelines
issued by the Steering Committee on 19 February 2020 called for
collection nasopharyngeal and oropharyngeal (NP/OP) swabs from suspected
cases and close contacts (F1) of confirmed cases (F0) and these
guidelines were harmonized with those from WHO in March 2020 (7).
Confirmed cases of COVID-19 underwent repeated sampling every three days
during hospitalization until they recovered clinically and had at least
three negative results by real-time RT-PCR for SARS-CoV-2.

\emph{Realtime RT-PCR testing} : NP/OP swabs were placed into viral
transport medium (VTM) and maintained at 4 \selectlanguage{ngerman}°C during transport to the
NIC at NIHE during 24-48 hours (8). RNA was isolated from NP/OP using
the viral RNA extraction kit (Qiagen, Hilden, Germany) according to the
manufacturer's instructions in biosafety level 3 (BSL3) containment
laboratories. Realtime RT-PCR was conducted using SuperScript III One
-Step RT-PCR system with Platinum Taq High Fidelity DNA Polymerase
(Invitrogen, Carlsbad, CA-USA) with target of E, RdRp and N genes
following WHO recommendations. We defined confirmed cases as those with
cycle threshold (Ct) values less than 37 for at least two target genes
(E and RdRp or E and N) of SARS-CoV-2 (9).

\emph{Viral isolation:} Vero E6 cells were maintained in Eagle's minimal
essential medium (EMEM) containing 5\% (v/v) newborn calf serum (NCS);
100ul of real-time RT-PCR positive samples were inoculated onto Vero E6
cells and incubated at 37 °C. Viral growth was monitored by daily
observation of cytopathic effect. All experiments with SARS-CoV-2
viruses were performed in biosafety level 3 (BSL3) containment
laboratories (10).

\textbf{Ethical considerations}

The Ethics committee of the National Institution of Hygiene and
Epidemiology approved this study.

\textbf{Results:}

Characteristics of specimens received

During January 23 through May 25, the NIC received 15,650 NP/OP
specimens from 14,470 suspected cases from 28 cities and provinces in
northern Vietnam. Samples were submitted from two types of suspected
cases: those from 6,420 persons entering Vietnam from abroad (China
during the first cluster and other countries during the second cluster
of cases) and 8,050 from persons who were contacts of suspected or
confirmed cases. During the first cluster from January 23 to February
25, 2020, 1,741 specimens (11\% of all specimens) were collected from
persons travelling from Wuhan, China, their families, and their close
contacts. During the second cluster of cases from March 7 through May
25, 2020, we received an additional 13,909 (88.9\%) samples with nearly
two-thirds received from four locations (Hanoi, 5,366; Ha Giang, 1,603;
Thai Binh, 1,118; Lai Chau, 1,011). (Table 1).

Detection of SARS-CoV-2 by Real-time RT-PCR

For the entire study period, 15,650 samples from 158 (1.14\%) suspected
and confirmed cases tested positive for SARS-CoV-2 (Table 2).
Eighty-nine (56\%) of these were detected among suspected cases imported
from other countries and the remaining 69 (44\%) were detected among
community contacts of confirmed cases. Thirteen cases were confirmed
during the first cluster among persons returning from China or their
close contacts. Among the 158 confirmed cases, 143 (91\%) were
Vietnamese nationals and 96 (61\%) were female, although we observed
significant differences in the distribution of gender among imported
cases (44/89, or 49\% female) compared with cases detected among
community contacts (52/69, or 75\% female, P\textless{}0.0009 by
chi-square). The median age was 41years (IQR: 3 months -- 88 years old)
for community contacts and 33 years (IQR :10 years -74 years old) for
imported cases. Eleven (12\%) of the 89 imported cases were only
detected upon their second sampling while in quarantine.

Based on Ministry of Health guidelines, laboratory-confirmed cases
required follow-up testing until at least two subsequent tests were
negative. Most cases required three or four subsequent tests to meet
this criterion, but we also observed some cases undergoing collection of
10-15 subsequent specimens (Table 3).

Correlation between Ct Values, Date of illness / Days since first
positive sample, and Viral Culture results:

We analyzed the cycle threshold (Ct) values of 158 confirmed cases with
SARS-CoV-2 infection with serial sampling during hospitalization until
two consecutive negative results were obtained. The proportion of cases
that tested positive declined with the number of times they were
sampled. Among the 652 samples collected, 167 (26\%) had Ct values less
than 30 and 105 (63\%) of these were identified at the first sampling.
Among cases who were sampled a third and fourth time, only 12/124 (10\%)
and 6/60 (10\%) cases, respectively, had Ct values less than 30 (Table
3).

We identified 99 positive specimens with Ct values greater than 30
including seven cases who tested positive again after having tested
negative (``re-positives''). Cases with Ct values greater than 30
followed a pattern similar to those with Ct values less than 30: 84
(85\%) cases were positive in the first three samples only and an
additional 10 (11\%) cases remained positive in one of the next three
samples. One case was sampled 15 times with no positive results after
the tenth sampling.

A total of 81/158 (51\%) confirmed cases had appropriately stored
samples with sufficient volume to be inoculated on Vero-E6 cells, from
which we obtained 29 (36\%) SARS-CoV-2 isolates. Of these, 20 samples
had detectable cytophathic effects between 72 and 96 hours and an
additional nine isolates were harvested after a second blind passage. We
identified 28 samples with Ct values of less than 20 and, of these, 18
(64\%) yielded culturable virus (Table 4). An additional 20 cases had Ct
values of 20-25 and, from these, we were successful in culturing virus
from 10 (50\%). The additional nine isolates recovered during the second
passage had Ct values of 25-30, suggesting a low load of viable virus.
No viral isolates were recovered from samples with Ct values
\textgreater{}30 (n=20).

\textbf{Discussion}

During the first five months of the COVID-19 epidemic in Vietnam, we
characterized all upper respiratory specimens received by NIHE coming
from cities and provinces in the north of Vietnam. Just over one percent
of all samples received yielded positive results by real-time RT-PCR and
yet, by the end of May 2020, fewer than 400 cases had been identified in
total in Vietnam, with no deaths and with no community transmission
after 14 April 2020.

Rapid scale-up and decentralization of testing was a key component to
Vietnam's response to minimize entry and transmission of SARS-CoV-2.
Another component of Vietnam's response was its implementation of
centralized quarantine for all persons entering the country from
affected countries since early February 2020 (4). Travelers in
quarantine were required to provide upper respiratory specimens for
testing upon arrival and before the end of the 14-day quarantine period.
Among these travelers in quarantine centers, we identified 89
laboratory-confirmed cases, 78 (88\%) of which were positive on their
first sampling and 11 during quarantine. Lastly, quarantining and
testing of first- and second-degree exposed community members was
implemented to stop further transmission of virus from primary cases who
had not been detected on entry and subsequent cases. These data suggest
that centralized quarantine coupled with testing upon arrival and
quarantine based on exposure rather than symptoms is an effective
measure for preventing exposure of communities and transmission of
SARS-CoV-2 imported from other countries.

Although viral culture remains the gold standard for confirmation of
viral infection, because of shorter turn-around time and higher
sensitivity, detection of SARS-CoV-2 by real-time RT-PCR is the accepted
gold standard for detecting SARS-CoV-2 for purposes of isolation and
contact tracing. Semi-quantitation of viral nucleic acids using Ct value
can be used to select samples for virus isolation (2,10--12). We
observed a strong correlation between Ct values and cell culture
positivity rate (table 4). This suggests viral load data may be used as
a rough proxy for the infectivity of infected patients.

There is concern about prolonged viral nucleic acid detection in samples
from patients recovered from COVID-19. The large majority of these
samples, both in the literature and in our collection, have high Ct
values and so far culture attempts have not been successful. This
observation supports the hypothesis that prolonged shedding or
re-positivity of samples is not associated with continued replication,
but rather an indicator of removal of damaged lung tissue containing
intact stretches of viral RNA by coughing or ciliary transport.

Among these 158 confirmed COVID-19 cases, we also identified seven
individuals with positive real-time RT-PCR results after two consecutive
negative results within 15 days (Table 3). We were unable to culture
virus from these specimens, all of which had Ct values greater than 33,
suggesting that these cases represent viral remnants rather than
infectious virus. These findings are consistent with findings from South
Korea and China (13--15). Positive real-time RT-PCR results can be
confusing for patients and hospital staff who understandably wish to
prevent continued transmission, either among patients and healthcare
workers or among the general community. Our findings should provide
reassurance that patients with positive real-time RT-PCR results with Ct
values \textgreater{}30 more than 10 days after onset or first positive
result and after having had a negative result are at extremely low risk
of transmission. These findings also support a strategy of testing based
on signs of clinical recovery, rather than a ``test-of-cure'' strategy.

Our study had several limitations. First, the specimens we received were
collected as part of the national strategy for prevention and control of
COVID-19 without accompanying systematic clinical metadata and we were
thus unable to stratify between asymptomatic, mild and severe cases.
Second, we were not able to systematically assess the possible duration
of viral shedding because most of our cases were detected upon arrival,
through contact tracing, and by the quarantine process. Thus, sampling
times were determined by disease control staff in the field, rather than
in the context of a rigorously designed study. Third, the source of
specimens for viral isolation was upper respiratory tract specimens
only. We did not receive any sputum or tracheal aspirate fluids, which
may have different characteristics in terms of Ct values or culturable
virus.

In summary, we describe here the virologic and epidemiology
characteristics of cases of laboratory-confirmed COVID-19 in northern
Vietnam from two clusters of cases during the first months of the
pandemic. We determined that most cases that will be
laboratory-confirmed are confirmed within the first few samplings. We
also determined that most cases that are positive very late in their
clinical course are extremely unlikely to represent active infection
but, rather, remnants of viral RNA.

\textbf{Conflict of interest}

The authors declare there are no conflict of interest about this work

\textbf{Acknowledgement}

We thank Matt Moore (US CDC Hanoi) and Rogier van Doorn (Oxford
University Clinical Research Unit, Vietnam) for scientific review. We
recognized timely reagents supporting at beginning outbreak from WHO
Collaborating Center for Reference and Research on Tropical and Emerging
Infectious Diseases, Institute of Tropical Medicine, Nagasaki
University, Japan.

We gratefully acknowledge the contribution of health workers at Center
for Diseases Control and Prevention of cities and provinces in northern
Vietnam. We also thank health care practitioners of the clinics and
hospitals in Vietnam who supported the study.

\textbf{References}

1. WHO. Coronavirus Disease (COVID-19). WHO. 2020;Publish Ah(October).

2. Phan LT, Nguyen T V., Huynh LKT, Dao MH, Vo TAN, Vu NHP, et al.
Clinical features, isolation, and complete genome sequence of severe
acute respiratory syndrome coronavirus 2 from the first two patients in
Vietnam. J Med Virol. 2020;(April):1--7.

3. Le TQM, Takemura T, Moi ML, Nabeshima T, Nguyen LKH, Hoang VMP, et
al. Severe Acute Respiratory Syndrome Coronavirus 2 Shedding by
Travelers, Vietnam, 2020. Emerg Infect Dis. 2020;26(7):1624--6.

4. Hai Nguyen Thanh, Truong Nguyen Van TAN et al. Outbreak investigation
for COVID-19 in northern Vietnam. lancet diabetes Endocrinol.
2015;3(18):925--7.

5. Khanh NC, Thai PQ, Quach H-L, Thi N-AH, Dinh PC, Duong TN, et al.
Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 During
Long Flight. Emerg Infect Dis J {[}Internet{]}. 2020;26(11). Available
from: https://wwwnc.cdc.gov/eid/article/26/11/20-3299\_article

6. Pham Quang Thai, Maia A Rabaa DHL, Tan DQ, Quang TD, Ha-Linh, Quach,
Ngoc-Anh Hoang Thi PCD, Nghia ND, et al. The first 100 days of
SARS-CoV-2 control in Vietnam. Clin Infect Dis. 2020;

7. Plan R. Laboratory testing strategy recommendations for COVID-19:
interim guidance, 22 March 2020. 2020;(March).

8. Federation R, Africa S, Janeiro R De, Penh P, Kong H, Infectious V,
et al. WHO COVID-19 Reference Laboratory Network *. :1--2.

9. Hq WHO. TIB MOLBIOL real-time RT-PCR assay for detection of COVID-19
virus. 2020;

10. World Health Organization. Laboratory testing for coronavirus
disease 2019 (COVID-19) in suspected human cases. 2020;(March):1--7.

11. Huang C-G, Lee K-M, Hsiao M-J, Yang S-L, Huang P-N, Gong Y-N, et al.
Culture-Based Virus Isolation To Evaluate Potential Infectivity of
Clinical Specimens Tested for COVID-19. J Clin Microbiol. 2020;58(8).

12. La Scola B, Le Bideau M, Andreani J, Hoang VT, Grimaldier C, Colson
P, et al. Viral RNA load as determined by cell culture as a management
tool for discharge of SARS-CoV-2 patients from infectious disease wards.
Eur J Clin Microbiol Infect Dis. 2020;39(6):1059--61.

13. Lan L, Xu D, Ye G, Xia C, Wang S, Li Y, et al. Positive RT-PCR Test
Results in Patients Recovered from COVID-19. JAMA - J Am Med Assoc.
2020;323(15):1502--3.

14. Qiao XM, Xu XF, Zi H, Liu GX, Li BH, Du X, et al. Re-positive Cases
of Nucleic Acid Tests in Discharged Patients With COVID-19: A Follow-Up
Study. Front Med. 2020;7(June).

15. Ontario PH. COVID-19\selectlanguage{english} : Ongoing Viral Detection and Repeat Positives.
2020;1--9.

\selectlanguage{english}
\FloatBarrier
\end{document}

