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\def\RunningAuthor{Tram Van Ta et al.}
\firstPage{2578}
\articleType{Original Article}
\receivedDate{17.04.2019}
\acceptedDate{07.07.2019}
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\title{The correlation of clinical and subclinical presentations with dengue serotypes and plasma viral load: the case of children with dengue hemorrhagic fever in Vietnam}
\author{Tram~Van~Ta\textsuperscript{*}\textsuperscript{1},  
            Hai~Thanh~Tran\textsuperscript{2},  
            Quyen~Nguyen~Than~Ha\textsuperscript{3},  
            Xuong~Tuyet~Nguyen\textsuperscript{4},  
            Vu~Kien~Tran\textsuperscript{5},  
            Hien~The~Pham\textsuperscript{6}, 
            Cameron~Simmons\textsuperscript{3}~\\[5pt]{\textsuperscript{1}Tien Giang General Hospital, Tien Giang province, Vietnam}~\\{\textsuperscript{2}Tien Giang Medical College, Tien Giang province, Vietnam}~\\{\textsuperscript{3}Oxford University Clinical Research Unit, Ho Chi Minh City, Vietnam}~\\{\textsuperscript{4}Vietnam National Hospital of Pediatrics, Vietnam}~\\{\textsuperscript{5}Tra Vinh General Hospital, Tra Vinh province, Vietnam}~\\{\textsuperscript{6}Tra Vinh University, Tra Vinh province, Vietnam}}

\begin{abstract}
Numerous studies have been carried out on patients afflicted with dengue hemorrhagic fever (DHF), but various issues related to the disease, including the characteristics of the dengue virus (DENV), remain unclear. To address this deficiency, the current research was conducted to determine the correlation of clinical and subclinical presentations with dengue serotypes and plasma viral load. This prospective cohort study, which was performed at Tien Giang General Hospital from 2009 to 2014, involved 481 children who were under 15 years of age and had DHF for less than 72 hours. Results showed that among the patients, the highest proportion were composed of those suffering from DENV-1 infection (44.7\%). The progression of the disease to dengue shock syndrome (DSS) owing to infection with DENV-2 and DENV-1 was significantly higher than that caused by infection with DENV-3 and DENV-4. No statistically significant differences in DENV viremia were found between the non-shock DHF and DSS groups. Finally, no correlation was found between dengue plasma viral load and clinical and subclinical presentations. The findings led to the conclusion that dengue serotypes can be used as a basis in ascertaining the prognosis of DSS and DHF.
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Correlation,\newline Clinical,\newline Dengue serotypes,\newline Dengue hemorrhagic fever,\newline Subclinical presentations,\newline Vietnam
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Tram~Van~Ta~\\ Phone:\ (+84) 913771779~\\  Email:\ tavantram@gmail.com
        \par}\vspace*{-11pt}\hrulefill\par{\fontsize{12}{14}\selectfont ISSN: 0975-7538}\par% 
        \textsc{DOI:}\ \href{https://doi.org/10.26452/\@journalDoi}{\textcolor{blue}{\underline{\smash{https://doi.org/10.26452/\@journalDoi}}}}\par%
        \vspace*{-11pt}\hrulefill\\{\fontsize{9.12}{10.12}\selectfont Production and Hosted by}\par{IJRPS |~\href{https://ijrps.com}{https://ijrps.com}}\par%
        \vspace*{-2pt}{\fontsize{9.12}{10.12}\selectfont\textcopyright\ \@copyrightYear\ $|$ All rights reserved.}\par%
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\section{Introduction}
Dengue fever (DF) is an acute infection caused by the dengue virus (DENV) and transmitted to humans by \textit{Aedes} mosquitoes, mainly \textit{Aedes aegypti}\unskip~\citep{584802:13551244}. These mosquitoes cause about 390 million dengue cases in the world, of which 96 million (67-136 million) are severe cases; out of these extreme instances, 90\% occur in the age group under 15 years \unskip~\citep{584802:13551228}. In Vietnam, DF incidence is equally critical and accompanied with huge treatment costs given that it is a tropical monsoon country characterized by ongoing urbanization and a population with limited knowledge of the disease \unskip~\citep{584802:13551236,584802:13551231}. As estimated in 2017, the cost of treating a child with dengue averages at about 151 USD - a situation that seriously affects the developing economy of Vietnam \unskip~\citep{584802:13551237}. Clinical and subclinical symptoms are very important aspects in the diagnosis of DF and its complications, especially severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Symptoms such as plasma loss and coagulopathy, for example, render DHF patients prone to hypovolemic shock, which eventually results in severe bleeding and death. Many patients afflicted with DHF have died despite the release of \unskip~\cite{584802:13551244} guidelines on the diagnosis and treatment of DF, thus motivating a number of studies on factors related to dengue and dengue-induced death. Some of these determinants are virulence, infection and reinfection, local factors (age, nutritional status, gender, etc.), pre-existing severe illnesses (gastrointestinal hemorrhage, liver failure, metabolic acidosis, etc.), and management issues (late detection and treatment, inappropriate management, uncontrolled monitoring) \unskip~\citep{584802:13551231,584802:13551232,584802:13551234}. Understanding these factors plays a critical role in managing and improving the treatment and prognosis of DHF. 

Prospective studies in Latin America and Southeast Asia concluded that most cases of DHF shock are associated with secondary immune responses. Factors such as originating virus and patient location are likewise critical contributors to severe illness. The amount of virus in the blood may also be an essential aspect because viral concentrations in DSS cases are often high. In some countries, different levels of virulence are considered a culprit in various consequences \unskip~\citep{584802:13551240}. These findings underscore the necessity of looking into the association between a patient's body and DENV as well as immunological factors because such explorations are expected to enhance the prognosis of dengue patients. Correspondingly, the current work determined the relationship of dengue serotypes and DENV infection levels with clinical and subclinical DHF symptoms in children. This association is an important new issue with respect to diagnosing and managing potentially fatal DF.
    
\section{Materials and Methods}
\textbf{Study design and setting}

This prospective cohort research was carried out at the Pediatrics Department of Tien Giang General Hospital from 2009 to 2014. Children with DF were selected on the basis of the following criteria: (1) the presence of one or more clinical symptom markers, such as abdominal pain, persistent vomiting, clinical fluid accumulation, mucosal bleeding, lethargy, restlessness, and liver enlargement ({\textgreater}2 cm) corresponding with criteria in the \unskip~\citet{584802:13551243} guidelines for DHF; (2) ages below 15 years; (3) DF lasting less than 72 hours; and (4) consent from the children's families. The exclusion criteria were the existence of diseases such as liver failure, kidney failure, nephrotic syndrome, heart failure, and congenital heart disease and the refusal of families to participate in the research.

\textbf{Sample size}

To ensure the validity of results, the minimum sample size was calculated using the formula recommended by the WHO \unskip~\citep{584802:13551235}
\begin{eqnarray*}N=\frac{Z_{\alpha/2}\times P(1-P)}{d^{2}} \end{eqnarray*}
This study assumed a 95\% confidence interval (Z\ensuremath{_{1-\ensuremath{\alpha\ }/2}} = 1.96) with a 5\% margin of error (\textit{d}). The prevalence of DHF and DSS in children was calculated on the basis of Ha's (2003) study on isolated DENV strains, for which the author determined the ratio of DHF and DSS incidence to population size as follows: 16/84 = 0.19.\ensuremath{^{6}} Therefore, the minimum sample size appropriate for the present study was 236 children. For accurate results, we endeavored to recruit twice this number of patients, but a total of 481 patients were found eligible for inclusion.

\textbf{Data collection and analysis}

Data were collected through face-to-face interviews with the patients' guardians and a review of the patients' medical records. The interviewer first explained the meaning of the study as well as the projected benefits and risks encountered over the course of the research. Information was then collected on the following patient characteristics: age and gender; clinical symptoms, such as vomiting, abdominal pain, mucosal bleeding, bleeding under the skin, temperature, liver enlargement, and shock; and subclinical symptoms, namely, white blood cell count, aspartate aminotransferase (ASP) and alanine aminotransferase (ALT) concentrations, platelet count, hematocrit (HCT) level, and DENV serotype. These data were uploaded onto Microsoft Excel (v. 2007) for storage and statistical calculations. The relationship among the patients' clinical characteristics, DENV serotype, and DENV concentration in the blood was determined via a chi-square test to run on the Statistical Package for the Social Sciences (v. 16.0).

\textbf{Ethical considerations}

This study was approved by the ethics committee of the Tien Giang General Hospital. The patients were informed that participation in this study is completely voluntary and that their identities will be kept anonymous. The guardians were asked to sign a statement confirming that they willingly took part in the research.
    
\section{Results and Discussion}
\textbf{Clinical characteristics }

Table~\ref{tw-33f5c14b20d4}  presents the observed clinical symptoms of the 481 patients, of which 40.1\% exhibited vomiting; most of these patients (22.4\%) started vomiting from day 3 of disease onset. Abdominal pain was experienced by 29.5\% of the patients, out of whom the highest number (12.3\%) suffered from this symptom on the third day from disease occurrence. A total of 38.5\% of the patients presented with subcutaneous hemorrhaging, with most (19\%) presenting this symptom on day three of the disease. The same timing was observed as to mucosal hemorrhaging, which manifested in 19.4\% out of 38.7\% of the patients. Hepatomegaly was a commonly occurring symptom, manifesting in 20.4\% of the patients and occurring most often on the fourth day from disease onset.


\begin{table*}[!htbp]
\caption{\boldmath {Distribution of Patients According to Clinical Manifestations} }
\label{tw-33f5c14b20d4}
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\tbltoprule \rowcolor{kwdboxcolor}\multicolumn{2}{p{\dimexpr(.6584\linewidth-2\tabcolsep)}}{\cAlignHack Clinical manifestations} &   N & \%\\
\tblmidrule 
\multicolumn{1}{p{\dimexpr(.3079\linewidth-2\tabcolsep)}}{\multirow{5}{\linewidth}{Vomiting }} &
  No  &
  288 &
  59.9\\
 &
  Yes &
  193 &
  40.1\\
 &
  Day 1\ensuremath{^{st}} &
  21 &
  4.4\\
 &
  Day 2\ensuremath{^{nd }} &
  64 &
  13.3\\
 &
  Day 3\ensuremath{^{rd }} &
  108 &
  22.4\\
\multicolumn{1}{p{\dimexpr(.3079\linewidth-2\tabcolsep)}}{\multirow{6}{\linewidth}{Abdominal pains }} &
  No &
  339 &
  70.5\\
 &
  Yes  &
  142 &
  29.5\\
 &
  Day 2\ensuremath{^{nd }} &
  40 &
  8.3\\
 &
  Day 3\ensuremath{^{rd}} &
  59 &
  12.3\\
 &
  Day 4\ensuremath{^{th }} &
  26 &
  5.4\\
 &
  Day 5\ensuremath{^{th}} &
  17 &
  3.5\\
\multicolumn{1}{p{\dimexpr(.3079\linewidth-2\tabcolsep)}}{\multirow{6}{\linewidth}{Bleeding under the skin }} &
  No &
  296 &
  61.5\\
 &
  Yes  &
  185 &
  38.5\\
 &
  Day 2\ensuremath{^{nd}} &
  31 &
  6.4\\
 &
  Day 3\ensuremath{^{rd}} &
  91 &
  19.0\\
 &
  Day 4\ensuremath{^{th }} &
  41 &
  8.5\\
 &
  Day 5\ensuremath{^{th}} &
  22 &
  4.6\\
\multicolumn{1}{p{\dimexpr(.3079\linewidth-2\tabcolsep)}}{\multirow{6}{\linewidth}{Mucosal bleeding }} &
  No &
  295 &
  61.3\\
 &
  Yes  &
  186 &
  38.7\\
 &
  Day 2\ensuremath{^{nd }} &
  39 &
  8.1\\
 &
  Day 3\ensuremath{^{rd}} &
  93 &
  19.4\\
 &
  Day 4\ensuremath{^{th }} &
  31 &
  6.4\\
 &
  Day 5\ensuremath{^{th}} &
  23 &
  4.8\\
\multicolumn{1}{p{\dimexpr(.3079\linewidth-2\tabcolsep)}}{\multirow{7}{\linewidth}{Highest day temperature }} &
  Mean \ensuremath{\pm} SD &
  \multicolumn{2}{p{\dimexpr(.34160000000000004\linewidth-2\tabcolsep)}}{38.62 \ensuremath{\pm} 0.66}\\
 &
  Day 1\ensuremath{^{st}} &
  22 &
  4.6\\
 &
  Day 2\ensuremath{^{nd }} &
  166 &
  34.5\\
 &
  Day 3\ensuremath{^{rd}} &
  217 &
  45.1\\
 &
  Day 4\ensuremath{^{th }} &
  41 &
  8.5\\
 &
  Day 5\ensuremath{^{th}} &
  14 &
  2.9\\
 &
  Day 6\ensuremath{^{th }} &
  21 &
  4.4\\
\multicolumn{1}{p{\dimexpr(.3079\linewidth-2\tabcolsep)}}{\multirow{6}{\linewidth}{Liver enlargement }} &
  No &
  383 &
  79.6\\
 &
  Yes  &
  98 &
  20.4\\
 &
  Day 3\ensuremath{^{rd}} &
  16 &
  3.3\\
 &
  Day 4\ensuremath{^{th }} &
  38 &
  7.9\\
 &
  Day 5\ensuremath{^{th}} &
  31 &
  6.5\\
 &
  Day 6\ensuremath{^{th }} &
  13 &
  2.7\\
\multicolumn{1}{p{\dimexpr(.3079\linewidth-2\tabcolsep)}}{\multirow{6}{\linewidth}{DSS }} &
  No &
  457 &
  95\\
 &
  Yes  &
  24 &
  5\\
 &
  Day 3\ensuremath{^{rd}} &
  2 &
  0.4\\
 &
  Day 4\ensuremath{^{th }} &
  11 &
  2.3\\
 &
  Day 5\ensuremath{^{th}} &
  10 &
  2.1\\
 &
  Day 6\ensuremath{^{th }} &
  1 &
  0.2\\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}
\textbf{Subclinical characteristics}

Table~\ref{tw-79a64ff3316d} shows the lowest average white blood cell count was 4.72\ensuremath{\pm}1.96/mm \unskip~\citep{584802:13551236}, which occurred among most of the patients on days 4 and 5 of the disease. The highest AST concentration was 68.58\ensuremath{\pm}9.00 U/L, which manifested in the majority of the patients (93.6\%) also on the fourth and fifth days. The second and third highest proportions of patients developed this symptom on the third and sixth days, respectively. The highest average ALT concentration was 40.61\ensuremath{\pm}7.17 U/L, found in 93.6\% of the sample and occurring on the fourth and fifth days from disease occurrence. The lowest platelet count was 80.19\ensuremath{\pm}33.91/mm \unskip~\citep{584802:13551236}, manifesting in nearly all the children on days 4 to 6 of the disease. The highest average HCT concentration was 42.86\ensuremath{\pm}4.30\%. 


\begin{table*}[!htbp]
\caption{\boldmath {Distribution of Patients According to Subclinical Symptoms (N = 481)} }
\label{tw-79a64ff3316d}
\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.4135\linewidth-2\tabcolsep}p{\dimexpr.2401\linewidth-2\tabcolsep}p{\dimexpr.18529999999999998\linewidth-2\tabcolsep}p{\dimexpr.1611\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\multicolumn{2}{p{\dimexpr(.6536\linewidth-2\tabcolsep)}}{\cAlignHack Characteristic} & N & \%\\
\tblmidrule 
\multicolumn{1}{p{\dimexpr(.4135\linewidth-2\tabcolsep)}}{\multirow{4}{\linewidth}{DENV serotype}} &
  DENV-1  &
  215  &
  44.7 \\
 &
  DENV- 2  &
  92  &
  19.1 \\
 &
  DENV-3  &
  61  &
  12.7 \\
 &
  DENV-4  &
  113  &
  23.5 \\
Characteristic &
  Time &
   &
  \\
\multicolumn{1}{p{\dimexpr(.4135\linewidth-2\tabcolsep)}}{\multirow{7}{\linewidth}{White cell counts (highest)}} &
  Mean \ensuremath{\pm} SD  &
  \multicolumn{2}{p{\dimexpr(.3464\linewidth-2\tabcolsep)}}{4.72 \ensuremath{\pm} 1.96/mm\ensuremath{^{3}}}\\
 &
  Day 2\ensuremath{^{nd}} &
  60  &
  12.5 \\
 &
  Day 3\ensuremath{^{rd }} &
  76  &
  15.8 \\
 &
  Day 4\ensuremath{^{th }} &
  105  &
  21.8 \\
 &
  Day 5\ensuremath{^{th }} &
  102  &
  21.2 \\
 &
  Day 6\ensuremath{^{th }} &
  84  &
  17.5 \\
 &
  Day 7\ensuremath{^{th }} &
  54 &
  11.2\\
\multicolumn{1}{p{\dimexpr(.4135\linewidth-2\tabcolsep)}}{\multirow{4}{\linewidth}{AST (highest)}} &
  Mean \ensuremath{\pm} SD  &
  \multicolumn{2}{p{\dimexpr(.3464\linewidth-2\tabcolsep)}}{68.58 \ensuremath{\pm} 9.0 U/L}\\
 &
  Day 3\ensuremath{^{rd }} &
  31 &
  6.4\\
 &
  Day 4\ensuremath{^{th }} &
  198  &
  41.2 \\
 &
  Day 5\ensuremath{^{th }} &
  252  &
  52.4 \\
\multicolumn{1}{p{\dimexpr(.4135\linewidth-2\tabcolsep)}}{\multirow{4}{\linewidth}{ALT (highest)}} &
  Mean \ensuremath{\pm} SD  &
  \multicolumn{2}{p{\dimexpr(.3464\linewidth-2\tabcolsep)}}{40.61 \ensuremath{\pm} 7.17 U/L}\\
 &
  Day 3\ensuremath{^{rd }} &
  31 &
  6.4\\
 &
  Day 4\ensuremath{^{th }} &
  198  &
  41.2 \\
 &
  Day 5\ensuremath{^{th }} &
  252  &
  52.4 \\
\multicolumn{1}{p{\dimexpr(.4135\linewidth-2\tabcolsep)}}{\multirow{7}{\linewidth}{Platelet (lowest)}} &
  Mean \ensuremath{\pm} SD  &
  \multicolumn{2}{p{\dimexpr(.3464\linewidth-2\tabcolsep)}}{80.19 \ensuremath{\pm} 33.91/mm\ensuremath{^{3}}}\\
 &
  Day 2\ensuremath{^{nd}} &
  11  &
  2.3 \\
 &
  Day 3\ensuremath{^{rd }} &
  12  &
  2.5 \\
 &
  Day 4\ensuremath{^{th }} &
  84  &
  17.5 \\
 &
  Day 5\ensuremath{^{th }} &
  143  &
  29.7 \\
 &
  Day 6\ensuremath{^{th }} &
  181  &
  37.6 \\
 &
  Day 7\ensuremath{^{th }} &
  50  &
  10.4 \\
\multicolumn{1}{p{\dimexpr(.4135\linewidth-2\tabcolsep)}}{\multirow{7}{\linewidth}{Hct (highest)}} &
  Mean \ensuremath{\pm} SD &
  \multicolumn{2}{p{\dimexpr(.3464\linewidth-2\tabcolsep)}}{42.86 \ensuremath{\pm} 4.30 \%}\\
 &
  Day 2\ensuremath{^{nd}} &
  13 &
  2.7\\
 &
  Day 3\ensuremath{^{rd }} &
  83 &
  17.3\\
 &
  Day 4\ensuremath{^{th }} &
  131  &
  27.2 \\
 &
  Day 5\ensuremath{^{th }} &
  160  &
  33.3 \\
 &
  Day 6\ensuremath{^{th }} &
  83  &
  17.3 \\
 &
  Day 7\ensuremath{^{th }} &
  11  &
  2.3 \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}
\textbf{DENV concentrations}

Table~\ref{tw-3d0f7bac212c} shows the average daily viral dengue concentration in the patients was 3.5\ensuremath{\times}10\ensuremath{^{8}} (copies/ml). The comparison of virus types showed no significant differences in DENV concentration (p = 0.645), but the highest occurring dengue variant was DENV-3 infection, whereas the lowest was DENV-4 infection. The average DENV concentrations differed in terms of the time of DENV infection, with the highest concentration occurring on day 1 of the fever. Viral concentration in the blood decreased on days 2 and 3, with the difference between these days being statistically significant (p = 0.01). Average DENV levels in cases of DENV reinfection (3.7\ensuremath{\times}10\ensuremath{^{8}} copies/ml) were higher than cases of initial infection (3.2\ensuremath{\times}10\ensuremath{^{8}} copies/ml), but the difference was non-significant (p = 0.513).


\begin{table*}[!htbp]
\caption{\boldmath {DENV Concentrations} }
\label{tw-3d0f7bac212c}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.27939999999999998\linewidth-2\tabcolsep}p{\dimexpr.3057\linewidth-2\tabcolsep}p{\dimexpr.2375\linewidth-2\tabcolsep}p{\dimexpr.17740000000000002\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}\multicolumn{2}{p{\dimexpr(.5851\linewidth-2\tabcolsep)}}{DENV concentrations (copies/ml)} & Mean & {\multirow{2}{*}{P-value*}}\\
\rowcolor{kwdboxcolor}\multicolumn{2}{p{\dimexpr(.5851\linewidth-2\tabcolsep)}}{\cAlignHack Mean} & 3.5 x 10\ensuremath{^{8}} & \\
\tblmidrule 
\multicolumn{1}{p{\dimexpr(.27939999999999998\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{Day has fever }} &
  Day 1\ensuremath{^{st}} &
  9.4 x 10\ensuremath{^{8}} &
  \multicolumn{1}{p{\dimexpr(.17740000000000002\linewidth-2\tabcolsep)}}{\multirow{3}{\linewidth}{0.010}}\\
 &
  Day 2\ensuremath{^{nd }} &
  4.5 x 10\ensuremath{^{8}} &
  \\
 &
  Day 3\ensuremath{^{rd}} &
  1.5 x 10\ensuremath{^{8}} &
  \\
\multicolumn{1}{p{\dimexpr(.27939999999999998\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{History of DF or DHF}} &
  No &
  3.2 x 10\ensuremath{^{8}} &
  \multicolumn{1}{p{\dimexpr(.17740000000000002\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{0.513}}\\
 &
  Yes &
  3.7 x 10\ensuremath{^{8 }} &
  \\
\multicolumn{1}{p{\dimexpr(.27939999999999998\linewidth-2\tabcolsep)}}{\multirow{4}{\linewidth}{DENV serotype}} &
  DENV-1 &
  4.6 x 10\ensuremath{^{8}} &
  \multicolumn{1}{p{\dimexpr(.17740000000000002\linewidth-2\tabcolsep)}}{\multirow{4}{\linewidth}{0.645}}\\
 &
  DENV-2 &
  1.4 x 10\ensuremath{^{8}} &
  \\
 &
  DENV-3 &
  8.4 x 10\ensuremath{^{8}} &
  \\
 &
  DENV-4 &
  0.5 x 10\ensuremath{^{8}} &
  \\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{(*) Chi-square test }
\end{tablenotes}
\end{threeparttable}

\end{table*}
\textbf{Correlation of clinical and subclinical characteristics with DENV type and concentration}

Table~\ref{tw-ac9f65d555b1} presents the results on the relationship between clinical characteristics and DENV serotypes. No correlation was found between DHF and vomiting, abdominal pain, and hepatomegaly in the children (p {\textgreater} 0.05). Subcutaneous hemorrhaging and mucosal hemorrhaging most frequently occurred in patients with DENV-1 infection, followed by those infected with DENV-3 and DENV-2. The lowest manifestations were found in the children infected with DENV-4. The difference among these incidences was statistically significant (p = 0.01). Table~\ref{tw-5d6f4d22111d} shows that no correlation was found between DHF and the average DENV concentration in the patients exhibiting clinical symptoms (p {\textgreater} 0.05).


\begin{table*}[!htbp]
\caption{\boldmath {Relationship between Clinical Characteristics and DENV Serotypes (N = 481)} }
\label{tw-ac9f65d555b1}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.2029\linewidth-2\tabcolsep}p{\dimexpr.11129999999999999\linewidth-2\tabcolsep}p{\dimexpr.10720000000000002\linewidth-2\tabcolsep}p{\dimexpr.1188\linewidth-2\tabcolsep}p{\dimexpr.1299\linewidth-2\tabcolsep}p{\dimexpr.1239\linewidth-2\tabcolsep}p{\dimexpr.09349999999999998\linewidth-2\tabcolsep}p{\dimexpr.11249999999999998\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Clinical manifestations & DENV \mbox{}\protect\newline serotype & DENV-1 & DENV-2 & DENV-3 & DENV-4 & Total & P-value*\\
\tblmidrule 
\multicolumn{1}{p{\dimexpr(.2029\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{Vomiting}} &
  Yes &
  88 (45.6) &
  33 (17.1) &
  23 (11.9) &
  49 (25.4) &
  193 &
  \multicolumn{1}{p{\dimexpr(.11249999999999998\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{0.700}}\\
 &
  No &
  127 (44.1) &
  59 (20.5) &
  38 (13.2) &
  64 (22.2) &
  288 &
  \\
\multicolumn{1}{p{\dimexpr(.2029\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{Abdominal pains}} &
  Yes &
  65 (45.8) &
  28 (19.7) &
  21 (14.8) &
  28 (19.7) &
  142 &
  \multicolumn{1}{p{\dimexpr(.11249999999999998\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{0.560}}\\
 &
  No &
  150 (44.2) &
  64 (18.9) &
  40 (11.8) &
  85 (25.1) &
  339 &
  \\
\multicolumn{1}{p{\dimexpr(.2029\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{Mucosal bleeding}} &
  Yes &
  110 (59.1) &
  31 (16.7) &
  43 (23.1) &
  2 (1.1) &
  186 &
  \multicolumn{1}{p{\dimexpr(.11249999999999998\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{0.010}}\\
 &
  No &
  105 (35.6) &
  61 (20.7) &
  18 (6.1) &
  111 (37.6) &
  295 &
  \\
\multicolumn{1}{p{\dimexpr(.2029\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{Bleeding under the skin}} &
  Yes &
  114 (61.6) &
  30 (16.2) &
  40 (21.6) &
  1 (0.5) &
  185 &
  \multicolumn{1}{p{\dimexpr(.11249999999999998\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{0.010}}\\
 &
  No &
  101 (34.1) &
  62 (20.9) &
  21 (7.1) &
  112 (37.8) &
  296 &
  \\
\multicolumn{1}{p{\dimexpr(.2029\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{Liver enlargement}} &
  Yes &
  45 (45.9) &
  18 (18.4) &
  8 (8.2) &
  27 (27.6) &
  98 &
  \multicolumn{1}{p{\dimexpr(.11249999999999998\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{0.400}}\\
 &
  No &
  170 (44.4) &
  74 (19.3) &
  53 (13.8) &
  86 (22.5) &
  383 &
  \\
\multicolumn{1}{p{\dimexpr(.2029\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{DSS}} &
  Yes &
  14 (58.3) &
  8 (33.3) &
  1 (4.2) &
  1 (4.2) &
  24 &
  \multicolumn{1}{p{\dimexpr(.11249999999999998\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{0.027}}\\
 &
  No &
  201 (44.0) &
  84 (18.4) &
  60 (13.1) &
  112 (24.5) &
  457 &
  \\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{(*) Chi-square test}
\end{tablenotes}
\end{threeparttable}

\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Relationship between Clinical Characteristics and DENV Concentrations} }
\label{tw-5d6f4d22111d}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.241\linewidth-2\tabcolsep}p{\dimexpr.087\linewidth-2\tabcolsep}p{\dimexpr.1058\linewidth-2\tabcolsep}p{\dimexpr.39480000000000004\linewidth-2\tabcolsep}p{\dimexpr.1714\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Clinical manifestations  & \multicolumn{2}{p{\dimexpr(.1928\linewidth-2\tabcolsep)}}{\cAlignHack n} & Mean DENV concentration (copies/ml)  & P-value* \\
\tblmidrule 
\multicolumn{1}{p{\dimexpr(.241\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{Vomiting }} &
  Yes  &
  193  &
  2.80x10\ensuremath{^{8 }} &
  \multicolumn{1}{p{\dimexpr(.1714\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{0.18 }}\\
 &
  No  &
  288  &
  4.19x10\ensuremath{^{8}} &
  \\
\multicolumn{1}{p{\dimexpr(.241\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{Abdominal pains }} &
  Yes  &
  142  &
  2.65x10\ensuremath{^{8}} &
  \multicolumn{1}{p{\dimexpr(.1714\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{0.52 }}\\
 &
  No  &
  339  &
  4.05x10\ensuremath{^{8}} &
  \\
\multicolumn{1}{p{\dimexpr(.241\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{Mucosal bleeding }} &
  Yes  &
  186  &
  5.27x10\ensuremath{^{8}} &
  \multicolumn{1}{p{\dimexpr(.1714\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{0.19 }}\\
 &
  No  &
  295  &
  2.60x10\ensuremath{^{8 }} &
  \\
\multicolumn{1}{p{\dimexpr(.241\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{Bleeding under the skin }} &
  Yes  &
  185  &
  4.72x10\ensuremath{^{8 }} &
  \multicolumn{1}{p{\dimexpr(.1714\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{0.38 }}\\
 &
  No  &
  296  &
  2.95x10\ensuremath{^{8 }} &
  \\
\multicolumn{1}{p{\dimexpr(.241\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{Liver enlargement }} &
  Yes  &
  98  &
  6.77x10\ensuremath{^{8 }} &
  \multicolumn{1}{p{\dimexpr(.1714\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{0.34 }}\\
 &
  No  &
  383  &
  2.83x10\ensuremath{^{8 }} &
  \\
\multicolumn{1}{p{\dimexpr(.241\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{DSS }} &
  Yes  &
  24  &
  3.44x10\ensuremath{^{8 }} &
  \multicolumn{1}{p{\dimexpr(.1714\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{0.96 }}\\
 &
  No  &
  457  &
  3.64x10\ensuremath{^{8}} &
  \\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{(*) Chi-Square test}
\end{tablenotes}
\end{threeparttable}

\end{table*}
\textbf{The relationship between subclinical characteristics and DENV type and concentration }

The average number of leukocytes and platelets is lowest in the patients infected with DENV-2, followed by the group with DENV-1 infection. The difference between these groups was statistically significant (p = 0.01). The highest average HCT was observed in the DENV-2 group, followed by the DENV-1 group, also with the difference being statistically significant (p = 0.01). No correlation was found between the highest concentrations of AST and ALT and different types of DENV (Table~\ref{tw-7f476fff8cee}).


\begin{table*}[!htbp]
\caption{\boldmath {Relationship between Subclinical Characteristics and DENV Serotypes} }
\label{tw-7f476fff8cee}
\centering 
\begin{threeparttable}

\def\arraystretch{1.1}
\ignorespaces 
\centering 
\begin{tabulary}{\linewidth}{p{\dimexpr.2597\linewidth-2\tabcolsep}p{\dimexpr.19400000000000002\linewidth-2\tabcolsep}p{\dimexpr.1196\linewidth-2\tabcolsep}p{\dimexpr.14879999999999999\linewidth-2\tabcolsep}p{\dimexpr.14850000000000003\linewidth-2\tabcolsep}p{\dimexpr.1294\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Subclinical & DENV serotype-  &   n & Mean  & SD  & P-value* \\
\tblmidrule 
\multicolumn{1}{p{\dimexpr(.2597\linewidth-2\tabcolsep)}}{\multirow{4}{\linewidth}{White cell count (/mm\ensuremath{^{3}}) }} &
  1 &
  215  &
  4.53 &
  2.49  &
  \multicolumn{1}{p{\dimexpr(.1294\linewidth-2\tabcolsep)}}{\multirow{4}{\linewidth}{0.01}}\\
 &
  2  &
  92  &
  4.32  &
  1.76  &
  \\
 &
  3 &
  61  &
  5.19 &
  2.45  &
  \\
 &
  4  &
  113  &
  5.15  &
  2.03  &
  \\
\multicolumn{1}{p{\dimexpr(.2597\linewidth-2\tabcolsep)}}{\multirow{4}{\linewidth}{AST concentrations (U/L) }} &
  1 &
  215  &
  65.85 &
  64.98  &
  \multicolumn{1}{p{\dimexpr(.1294\linewidth-2\tabcolsep)}}{\multirow{4}{\linewidth}{0.38}}\\
 &
  2  &
  92  &
  75.19  &
  70.36  &
  \\
 &
  3 &
  61  &
  80.49 &
  58.96  &
  \\
 &
  4  &
  113  &
  61.98  &
  33.49  &
  \\
\multicolumn{1}{p{\dimexpr(.2597\linewidth-2\tabcolsep)}}{\multirow{4}{\linewidth}{ALT (U/L) }} &
  1 &
  215  &
  40.33 &
  53.34  &
  \multicolumn{1}{p{\dimexpr(.1294\linewidth-2\tabcolsep)}}{\multirow{4}{\linewidth}{0.50}}\\
 &
  2  &
  92  &
  41.79  &
  65.44  &
  \\
 &
  3 &
  61  &
  50.29 &
  11.97  &
  \\
 &
  4  &
  113  &
  34.95  &
  37.87  &
  \\
\multicolumn{1}{p{\dimexpr(.2597\linewidth-2\tabcolsep)}}{\multirow{4}{\linewidth}{Platelet (/mm\ensuremath{^{3}}) }} &
  1 &
  215  &
  77.89 &
  36.75  &
  \multicolumn{1}{p{\dimexpr(.1294\linewidth-2\tabcolsep)}}{\multirow{4}{\linewidth}{0.01}}\\
 &
  2  &
  92  &
  73.61  &
  30.95  &
  \\
 &
  3 &
  61  &
  83.26 &
  31.31  &
  \\
 &
  4  &
  113  &
  88.27  &
  30.43  &
  \\
\multicolumn{1}{p{\dimexpr(.2597\linewidth-2\tabcolsep)}}{\multirow{4}{\linewidth}{Hct (\%) }} &
  1 &
  215  &
  43.27 &
  4.41  &
  \multicolumn{1}{p{\dimexpr(.1294\linewidth-2\tabcolsep)}}{\multirow{4}{\linewidth}{0.01}}\\
 &
  2  &
  92  &
  43.96  &
  4.30  &
  \\
 &
  3 &
  61  &
  42.33 &
  3.79  &
  \\
 &
  4  &
  113  &
  41.50  &
  4.01  &
  \\
\tblbottomrule 
\end{tabulary}\par 
\begin{tablenotes}\footnotesize 
    
\item{(*) Chi-square test }
\end{tablenotes}
\end{threeparttable}

\end{table*}
The patients participating in this study were identified as potentially afflicted with DHF, with the proportion of patients who were likely to develop DSS being approximately 5\%. Out of these patients, 3\% and 2.1\% suffered from shock on days 4 and 5 of the disease, respectively. The rate of DSS found in the current study is much lower than that observed by Hung (2004), whose investigation involved 62 children under 12 months of age with DHF, including no-shock DHF (grade II, 69.3\%) and DHF accompanied by shock (grade III, 15 cases; grade IV, four cases). Among the patients, 30.7\% showed improved management of DF diagnosis and treatment. The findings of the present study are also lower than those derived in research in Brazil (i.e., 6.5\%) \unskip~\citep{584802:13551242}. The distribution of patients, according to DENV serotype, was as follows. The most prevalent was DENV-1 (44.7\%), followed by DENV-4 (23.5\%), DENV-2 (19.1\%), and DENV-3 (12.7\%). \unskip~\citet{584802:13551241} found that the occurrence rates of DENV-1, DENV-2, DENV-3, and DENV-4 in Vietnam are 62.8\%, 27.4\%, 8.8\%, and 0.9\%, respectively. As can be seen, DENV-1 is the predominant disease-causing serotype, as was similarly found by \unskip~\citet{584802:13551239} and \unskip~\cite{584802:13551242} (77.3\%). In Thailand, \unskip~\cite{584802:13551230} found that DENV-4 still has a relatively low prevalence (36\%) in the Southern region, which is a finding supported by research in other parts of the world \unskip~\citep{584802:13551232,584802:13551242,584802:13551238}. \textit{\textbf{\space }}

A statistically significant difference in DENV serotype prevalence was found between DHF and DSS (p = 0.027, p{\textless}0.05), which differs from the survey results derived in Thailand (Fried et al. 2010), where no difference in serotypes between dengue groups was discovered. The current results indicated that patients with DENV-1 and, particularly, DENV-2 had higher shock rates than the rest of the patients. This finding is similar to that derived by \unskip~\cite{584802:13551241}, who recorded a higher and more severe case of shock in the DENV-2 infected group than in the DENV-1 infected group \unskip~\cite{584802:13551241}. The present work is also consistent with studies conducted in Taiwan and Thailand \unskip~\citep{584802:13551230,584802:13551229}. The limitations of this study are worth noting. First, the subjects were selected on the basis of DHF classification, but no clear distinction exists among DF, DHF, and DSS cases; thus, it is possible to confuse DF and DHF \unskip~\citep{584802:13551233} given that patients afflicted with these conditions display the same symptoms as those observed in other infections. Second, the study was conducted in only one hospital, thus preventing generalizability to the entire Vietnam or other parts of the world. Finally, because this work found no correlation among plasma concentrations of DENV, this precludes approaches to evaluating severe cases of DHF for enhanced management. The strengths of the research include a five-year data collection period, complete with virus isolation and serotyping. In addition, the study monitored patients over the course of several days, thus acquiring complete data on the timing of clinical symptoms and a more general view of DHF and DSS cases.
    
\section{Conclusion}
The susceptibility of patients infected with DENV-2 and DENV-1 was significantly higher than those infected with the other virus types. Subcutaneous hemorrhaging and mucosal hemorrhaging occurred most frequently in patients with DENV-1 infection, followed by those infected with DENV-3 and DENV-2. These symptoms manifested themselves to the lowest extent in the DENV-4 cases. The difference among the subjects was statistically significant (p = 0.01). The average number of leukocytes and platelets was lowest in the DENV-2 patients, followed by the DENV-1 group, with the difference between them being statistically significant (p = 0.01). The highest average HCT was observed in the group infected with DENV-2, followed by the patients infected with DENV-1. The difference between these groups was also statistically significant (p = 0.01). No correlation was found between DENV serotype and vomiting, abdominal pain, hepatomegaly, and AST and ALT levels in the children with DHF (p {\textgreater} 0.05). DENV concentrations were unassociated with the clinical and subclinical symptoms examined.
\section*{Acknowledgement}The authors would like to acknowledge all the staff of Tien Giang General Hospital for data collection.
    

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