European Journal of Cardiovascular Medicine

A severe hazard to global public health in tropical and sub-tropical regions of the world, particularly in urban and semi-urban areas, dengue is an arboviral illness spread by mosquitoes [1]. According to estimates from the World Health Organization (WHO), between 50 and 100 million people contract dengue each year, and over 2.5 billion people are at risk of contracting the disease in more than 100 countries. Rapid unplanned urbanization, population movement from rural to urban areas, a lack of vector management, shifting climate patterns, and inadequate sanitary facilities have all led to the creation of favorable breeding grounds for Aedes aegypti, the dengue vector [2].

The dengue virus has four distinct serotypes, which are referred to as DENV 1, DENV 2, DENV 3, and DENV 4. An infection can result from any one or more of the four serotypes. Anyone with dengue serotype infection confers homotypic immunity to that specific serotype for life. It is well-documented that subsequent infections with different dengue virus serotypes increase the risk of developing severe dengue [3].

Non-structural protein-1 (NS1) antigen capture assays, which can detect the virus up to nine days after symptoms in primary infection, are commonly used to diagnose dengue. However, because of an amnestic reaction, NS-1 is detectable for a significantly shorter time in individuals with secondary infections. The immunoglobulin G or immunoglobulin M antibody capture assay, which is used for serological diagnosis, exhibits cross-reactivity with different influenza viruses. In the acute phase of the disease (the phase with viremia), molecular techniques like reverse transcription-polymerase chain reaction (RT-PCR) allow for the same-day diagnosis of DENV and can identify the serotype (even in individuals with secondary infection). Compared to virus isolation approaches, PCR-based procedures are more affordable, quicker, more precise, sensitive, and less difficult [4].

The clinical and epidemiological characteristics of dengue illness have been examined in several earlier investigations [3,5–9]. We conducted a cross-sectional study at tertiary care facility with the goal of examining the various dengue serotypes, their clinical severity, and the biochemical test profiles of dengue patients.

Objectives and goals

The current study's goals are to: (1) ascertain the prevalence of dengue serotypes; (2) calculate co-infection rates; (3) establish a correlation between the serotypes and the degree of thrombocytopenia, total and differential leucocyte count, and disturbance of hepatic and renal function.

The study was conducted on One hundred (100) patients admitted (in-patient) in the department with dengue fever were selected for the study from the period of May 2023 to April 2024 and informed consent was obtained from all the participants.

Patient Participation

This cross-sectional study included patients over 12 years old who tested positive for dengue by reverse transcription-polymerase chain reaction (RT-PCR). At the time of admission, every patient had a comprehensive physical examination along with a comprehensive medical history. Patients with known malignancies, haematological disorders, or those taking any medication that could cause thrombocytopenia were excluded, as were those with co-morbid conditions such as diabetes mellitus, hypertension, hypothyroidism, liver disease, and chronic renal disease.

Analysis in the laboratory is essential. 

Serum albumin, blood urea nitrogen (BUN), serum creatinine, aspartate aminotransferase (ALT), alanine aminotransferase (AST), platelet count, total leucocyte count (TLC), differential leucocyte count (DLC), and platelet count were measured at admission. By using ribonucleic acid (RNA) amplification by ABI 7500 Fast Dx (Applied Biosystems, Foster City, California) RT-PCR, the dengue serotype was verified. According to the WHO categorization, patients were divided into three categories: non severe dengue without warning signals, non-severe dengue with warning indications, and severe dengue.

STATISTICAL ANALYSIS:

The data thus generated was analysed Statistically using the student ‘t’ test to compare the mean of two groups. ANOVA for comparison of mean in more than two groups. Pearson’s coefficient of correlation was used to calculate the correlation between different parameters. P <0.05 was considered statistically significant.

Table 1 reveals that 21 patients had dengue infection with serotype-1, 34 patients had serotype-2, 26 patients had serotype-3, and 15 patients had serotype-4. Additionally, three patients and one patient each had co-infections with serotypes 1 and 3. Between the various infection serotypes, there was no statistically significant variation in the distribution of severity (p-value 0.491).

TABLE 1: Patient distribution based on severity and serotype

*NS without W represents non-severe dengue without warning signs

**NS with W represents non-severe dengue with warning indications.

DENV 1 is the serotype 1 of the dengue virus; DENV 2 is the serotype 2 of the virus; DENV 3 is the serotype 3 of the virus; and DENV 4 is the serotype 4 of the virus.

TABLE 2: Based on serotypes, the mean total leucocyte counts and the percentages of neutrophils and lymphocytes

Table no.2 indicates a statistically significant difference in the mean neutrophil and lymphocyte percentages in the groups (p-value <0.001 and 0.004, respectively), but no significant difference in the mean leucocyte count in the groups (p-value 0.575). A post hoc analysis revealed that serotype-4 had a substantially larger percentage of neutrophils than serotypes-1 and 2 (p-values 0.001 and 0.016, respectively), while serotype-1 had a significantly lower proportion of lymphocytes than serotypes-3 and 4 (p-values 0.043 and 0.02, respectively).

TABLE 3: Mean platelets count according to serotypes among the groups

Table 3 shows no significant difference in the mean platelet count among the groups (p-value 0.190).

TABLE 4: Based on serotypes, the groups' mean serum ALT, AST, and albumin levels

The mean ALT, mean AST and serum albumin levels differ statistically significantly (one-way ANOVA, p-values <0.001, <0.001, and <0.001, respectively), as shown in Table 4. When comparing serotypes 2, 3, and 4 to serotype-1, post hoc analysis using the Bonferroni technique revealed significantly higher mean ALT and AST values (p-value <0.001, 0.002, 0.006 and <0.001, <0.001, <0.001, respectively). Comparing serotypes 3 and 4 to serotypes 1 and 2, the mean serum albumin levels were considerably lower (p-values 0.002, 0.002, and <0.001, <0.001, respectively).

Table 5: Mean values of serum creatinine and BUN according to serotypes in the groups

The mean serum creatinine and blood urea nitrogen (BUN) for each group are shown in Table 5 and are within the normal range.

TABLE 6: Comparison of the mean laboratory values between individuals with and without co-infection

The comparison of mean laboratory values between patients with and without co-infection is presented in Table 6. Compared to the no co-infection group, both co-infected groups had lower lymphocyte percentages and albumin levels and higher mean neutrophil percentages, platelet counts, AST, and ALT. BUN and creatinine were greater in the co-infection group with serotypes 1 and 3 than in the no co-infection group.

One of the prominent arboviral illnesses that has undergone a significant epidemiological shift in the last 20 years is dengue, which is a serious global public health issue, particularly in tropical and subtropical regions.

We looked at 100 patients who had dengue that was verified by RT-PCR. Sixty percent of those one hundred cases had dengue serotype-2 (DENV 2) or serotype-3 (DENV 3) infection (Table 1). There were the highest number of cases (34%) of dengue serotype 2, followed by 26% of DENV 3 cases, 21% of DENV 1 cases, and 15% of DENV 4 cases. This is consistent with earlier research from Mumbai, Delhi, Uttar Pradesh, and other locations [5-7]. Four individuals in our study were simultaneously infected with two different dengue virus serotypes. Serotypes 1 and 3 co-infected three patients, while serotypes 2 and 3 co-infected one patient. The first instances of concurrent infections by distinct dengue serotypes were documented in a Delhi investigation earlier this year [5].

According to Table 1's serotype link with severity, DENV 2 (20.6%) and DENV 4 (20%) had the highest percentage of severe dengue cases. Nevertheless, the p-value of 0.491 indicates that this distributional difference was not statistically significant. In their 2018 investigation, Racherla et al. [8] discovered that DENV 2 was the most common serotype, with increased infection severity with DENV 3 and DENV 4 With the exception of co-infection, the mean TLC (Table 2) for all infection serotypes was within normal bounds. However, there were notable differences in the groups' mean lymphocyte and neutrophil percentages (Table 2) (p-value 0.004 and <0.001, respectively). These findings are consistent with a 2017 study by Wardhani et al. [9]. Leukopenia’s lack may be a predictor of severe dengue, according to a prior Thai study [10]. Although it is typical for mean lymphocyte counts to rise after many viral infections, they discovered that they were within the normal range. Serotype-4 was one of the serotypes with the highest percentage of severe dengue (20%), although post hoc analysis in our study showed that patients infected with serotype-4 had a considerably lower lymphocyte percentage than serotype-1 (p-value = 0.02). Our findings contradict those of earlier research, which showed that normal lymphocyte counts remained virtually unchanged in dengue illness but that the number of atypical lymphocytes increased significantly [11]. The death or suppression of myeloid progenitor cells, which are observed to exhibit mild hypocellularity during the first seven days of fever and normal cellularity during the convalescent phase, is thought to be the cause of leukopenia in dengue [12]. Remarkably, in comparison to previous cohorts, the patients in our study who co-infected with serotypes 2 and 3 had a much higher TLC, a higher neutrophil percentage, and a lower lymphocyte percentage.

Thrombocytopenia was observed in all dengue infection serotypes in our investigation; DENV 1 and DENV 3 had the lowest value (13x10^3 /mm3). The difference in platelet counts between the various infection serotypes were statistically not significant (p-value = 0.190). Most of the patients in Chaloemwong et al.'s [13] study showed thrombocytopenia, although their bleeding diathesis was only 5.8% because most of the patients had a non-severe form of dengue illness. Azin et al.'s discovery of thrombocytopenia [14] occurring in haemorrhagic and severe variants of dengue from the moment symptoms appeared and remaining constant as the illness progressed. In our study, the co-infection groups' mean platelet counts were greater than the means of the other groups.

There may be more hepatotropic potential for serotypes-2, -3, and -4 due to the higher mean ALT and AST levels observed in co-infection with dengue viruses compared to infection with a single serotype (Table 6), and in serotypes-2, -3, and 4 compared to serotype-1 (Table 4).

In our investigation, the mean AST was greater than the mean ALT for every serotype. According to Ferede et al. [15], there was an increase in AST and ALT in 45.1% and 17.6% of the patients, respectively, with AST showing a higher relative elevation in more cases than ALT. This is consistent with a different study by Mariappan T [16], 68.5% and 39.2% of the cases, respectively, had increased AST and ALT values. Another study [17] found similar results, with increased AST in 72.7% of cases and ALT in 27.3% of cases. The observed pattern may be explained by the excess release of AST from injured muscle cells (a non-hepatic source) during infection, which results in more increase of AST than ALT. This is because, despite being hepatotropic, the dengue virus also destroys other organs. Hepatocytes are mostly linked to ALT, with minimal involvement from cardiac and skeletal muscle. Erythrocytes, kidney, brain, heart, and skeletal muscle have significantly greater concentrations of AST; this is typically due to damage to these tissues as well as a reaction to hepatic injury [17].

Overall, all infection serotypes had reduced albumin values according to our research (Table 4); however, hypoproteinaemia was more noticeable in co-infection cases (Table 6). Serotypes 3 and 4 had a mean blood albumin level that was noticeably lower than serotypes 1 and 2. Hypoproteinaemia may result from plasma leakage caused by the intricate relationship between the virus, host immune response, and endothelial cells that compromise the integrity of the endothelium cells' barrier and their functions. Similar findings were reported by Ferede et al. [15]. In 21.6% of the instances, hypoproteinaemia was noted, per their investigation.

According to our analysis, the most common dengue serotypes in the study conducted, were DENV 2 and DENV 3. There were also four cases of concurrent infection with two different dengue virus serotypes. Although not statistically significant, the highest percentage of cases of severe dengue was found in DENV 2 and DENV 4 infections. The four serotypes did not significantly differ in TLC. In comparison to DENV 1, DENV 4 had a lower mean lymphocyte percentage and a greater percentage of cases with severe dengue (20% vs. 14.3%, non-significant). Every serotype exhibited thrombocytopenia, and there was no discernible variation in the severity of the condition between the serotypes. In DENV 2, 3, and 4, significantly higher mean AST and ALT levels were observed, especially in co-infected cases. For all serotypes, AST levels were greater than ALT levels, and DENV 3 and 4 were found to have considerably lower mean serum albumin levels. For every infection serotype, the mean BUN and creatinine were within the normal range. It is advisable to test for dengue infection serotype at the time of diagnosis, as this can assist the physician in being on the lookout for patients who have co-infection or who are infected with dengue serotypes that have higher illness severity (DENV 2 and DENV 4). In larger investigations, the potential increased hepatotropic nature of DENV 2, 3, and 4 has to be assessed. One possible method for the assessment and treatment of dengue could be the use of RT-PCR to confirm the presence the dengue virus and the infecting serotype. Though there were some intriguing results in the co-infection patients, the limited sample size makes it impossible to determine how these findings will affect the severity and duration of the illness.

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