Dengue Virus (DENV) is the most common Aarbovirus found in India. Around the world, it primarily affects tropical and subtropical areas. It is predominantly endemic in urban and semi-urban regions [1]

There are four dengue virus serotypes (DENV1 through DENV4) that are known to infect people. In 2013, the fifth serotype (DENV-5) was found in Bangkok, although it hasn't yet been reported in India. [2]

In patients infected with Ddengue Ffever (DF) symptom varies from asymptomatic or a mild undifferentiated fever to a more severe form like Ddengue Hhaemorrhagic Ffever (DHF) and/or Ddengue Sshock Ssyndrome (DSS). Infection with one serotype confers immunity only to that infecting serotype. Subsequent infection with one of the remaining serotypes may result in immune enhancement and can be associated with complications as in later stages of the disease spectrum. [2]

From more than 100 million cases of dengue fever, 250,000 cases result in dengue haemorrhagic fever, responsible for 25,000 deaths annually worldwide. In India, the number of dengue cases has been steadily rising for the past ten years. Over one lakh dengue cases and about 200 fatalities every year were reported over the past ten years. More than 1.37 lakh cases and a maximum of 130 fatalities were reported in 2019 in Karnataka and Gujarat. [2]

From India, all four dengue serotypes have been isolated. DENV2 is by far the most prominent, followed by DENV3.

There are obvious seasonal variations found in the occurrence of dengue, as it is a vector-borne disease. The primary vector Aedes aegypti is a nervous feeder that lives in domestic areas and bites during the day. The post-monsoon season, when fresh water has gathered in and around the dwellings, is the breeding time of the vector. During the monsoon and post-monsoon seasons, the extrinsic incubation period of the virus is significantly decreased. [3] Consequently, there has been a marked rise in Dengue and other vector-borne illnesses including Chikungunya and Malaria.

The present study was conducted in RUHS Hospital of Medical Sciences, Jaipur.  The purpose of this study was to find out the seroprevalence of Dengue in the area with ELISA to diagnose dengue serologically and to evaluate the serotype diversity in this region of India. We first performed an NS1Ag ELISA for Dengue followed by Dengue virus RT PCR to confirm the presence of Dengue RNA in all NS1Ag positive samples. For Dengue serotyping, only PCR-positive Dengue RNA samples were processed.

From September to November 2021, the peak months of Dengue, a prospective study was carried out to find out its prevalence. About 500 clinically suspected, nonconsecutive cases of Dengue/ Dengue Hemorrhagic Fever (DHF)/ Dengue Shock Syndrome (DSS) as per WHO Criteria, who either reported directly or referred to our institute for treatment from the areas in and around Jaipur, were included in the study. There was no sampling bias or any attempt to specially recruit patients for the study.

Approximately 5 ml of Blood sample was taken by venipuncture in Clot activator vacutainer (BD inc.) for separation of serum. The acute phase serum samples were collected from all the 500 individuals.

SEROLOGICAL ASSAYS

All the 500 serum samples were separated and tested on the same day for Dengue NS1 Ag by Dengue NS1Ag capture ELISA (Precision Biomed Pvt. Ltd) following standard protocol as mentioned in the kit insert. Serology was performed at RUHS HMS Jaipur and the results were uploaded on the same day.

MOLECULAR ASSAYS

Positive serum samples of Dengue NS1 Ag ELISA were further subjected to PCR (Ag Path kit of Applied Biosystems) for Dengue virus genes at DHR-VRDL lab of Department of Microbiology, SMS Medical College Jaipur. PCR positive Dengue sample were then tested for serotyping of Dengue virus into 4 Dengue serotypes (DENV1, DENV2, DENV3 and DENV4) by Multiplex RT-PCR kit (CDC DENV 1 – 4 Real-Time RT-PCR Assay for Detection and Serotype Identification of Dengue Virus).

Extraction of viral RNA

Viral RNA was extracted from the infected serum samples using the QIA amp viral RNA mini kit (Qiagen, Germany), according to the manufacturer’s protocol. RNA elute (70 µL) was collected in collection tube and stored at – 80 °C until use.

CDC dengue multiplex real time assay

For the detection and serotype identification of the dengue virus, Taqman based multiplex real time RT-PCR assay was performed in ABI 7500 Fast Dx Real time PCR instrument using the dengue specific primer and probes provided by CDC, USA (Catalog No KK0128, Package insert available at https://www.cdc.gov/dengue). The assay includes a set of oligonucleotide primers and dual labeled 50 fluorescent (Taqman) probe for detection of DENV 1 – 4. Targeted gene for DENV-1 was NS5 gene; for DENV-2, it was E gene; for DENV-3 and DENV-4, it was prM gene. Appropriate positive and negative controls were taken in each reaction during sample investigation. These positive and negative controls were provided along with CDC kit and reaction was performed according to the manufacturer’s instruction. 5 µL of extracted RNA samples, including HSC were added in wells. Plate was sealed with optical tap film and placed in Applied Biosystems 7500 Fast DX Real-time PCR system where fluorescent signal intensity was monitored during each PCR cycle. Assay was run in multiplex format (the four dengue serotypes are in same reaction) with each serotype specific probe labeled with different probes: DENV 1 – FAM; DENV 2 – HEX; DENV 3 – TEXAS RED; DENV 4 – CY5; RNase P-FAM. Thermal cycling conditions were RT 50 °C for 30 min, RT inactivation at 95 °C for 2 min, fluorescence detection at 95 °C 15 s, 60 °C 1 min for 45 cycles. Target amplification is recorded as increase accumulation of fluorescence over time in contrast to background signal.

Patients hospitalized with suspected dengue were included in the study. Clinical diagnosis was based on the WHO guidelines. Of the 500 samples tested for Dengue during the peak months i.e. September 2021 to November 2021, 289 (57.8%) samples were found positive for Dengue NS1 Ag by ELISA (Table 1), also showing gender distribution among positive patients. Table 2 shows the age distribution of samples tested positive for Dengue virus. Number of Dengue positive samples detected by PCR for Dengue virus among Positive samples of Dengue by NS1 ELISA is shown in Table 3. One hundred forty (48.4%) samples out of total positive dengue NS1Ag samples were found positive for Dengue virus gene. In rest 149 (51.5%) samples although positive for Dengue NS1 Ag by ELISA, Dengue RNA was not detected. Table 4 shows number of Dengue serotypes among Dengue positive samples as detected by Multiplex PCR. Out of 140 Dengue RT PCR positive samples 90(64.3%) were DENV2, 26(18.6%) were DENV1, 4(2.8%) were DENV3 and none were DENV4. Twenty samples (14.3%) were Dengue RNA positive but no serotype was detected.

Table 5 shows age distribution among different serotypes. Young adults between 21 – 40 years of age were found to be more affected with DENV1 (57.69%) while younger population between 0 – 20 years of age was affected more with DENV2 (53.33%) followed by adult population between 21 – 40 years with (40.44%). No DENV was detected in older population of age more than 60 yrs.

Dengue fever has is being emerged as one of the most devastating health problems. Dengue cases have been increased over the years in many parts of the world, affecting a large number of populations. As it is now endemic in India, there are occasional small outbreaks, especially during the post-monsoon season, with the spread of multiple serotypes in different parts of the country found. [4]

Moreover, complicated dengue cases like DHF and DSS have been found, which require immediate medical attention. [5] Therefore, in the absence of any licensed antiviral medication, early diagnosis is essential for proper management of this life-threatening illness.

Enhanced virus transmission to other geographical regions is probably caused by environmental changes (high humidity, high temperature, and heavy rainfall) and an inefficient water drainage system, which encourages mosquito population explosion. Additionally, a heavy human population density, rapid urbanization, and increasing globalization all contribute to dengue fever epidemics occurring often in different locations.

IgM and IgG antibodies, which appear after 7 – 8 days after clinical onset, may be employed to make the diagnosis. [6] However, it is less useful because IgG antibodies cross-react with other closely related flaviviruses, whereas IgM antibodies remain in blood for more than 90 days.

NS1 is detectable during the acute phase of dengue virus infections. NS1 tests can be as sensitive as molecular tests during the first 0-7 days of symptoms. After day 7, NS1 tests are not recommended. A positive NS1 test result is indicative of a dengue infection but does not provide serotype information. Knowing the serotype of the infecting virus is not necessary for patient care; however, if serotype information is needed for surveillance purposes, the sample should be tested by molecular test.

NS1 can be found in whole blood or plasma both, but most NS1 tests have been developed and evaluated in serum samples. While combined testing with a NS1 and IgM antibody test can usually provide a diagnostic result during the first 1-7 days of illness, a second, convalescent phase specimen should be obtained and tested for IgM when both antigen and antibody tests are negative.

Dengue virus RNA can be detected with molecular tests. For symptomatic patients during the first 1-7 days of illness, performing both molecular and IgM antibody (or NS1 and IgM antibody) tests can detect more cases than performing just one test during this time period, and usually allows diagnosis with a single sample.

It is crucial to identify the dengue serotype as secondary infection with a heterologous serotype often results in fatal DHF and DSS. [7] Numerous molecular techniques for the early serotyping of dengue viruses have been reported. [8 – 12]

Dengue infections have been observed in India since the late 1950s, but since the mid-1990s, there has been a noticeable increase in its activity. In 1964, first isolation of the Dengue virus serotypes 2 was reported from India. [13]

For the first time, DENV-1 genotype I circulation has been observed in Himachal Pradesh. Despite being present in Southern Indian states since 2012, DENV-1 genotype I has only been identified so far in the states of Maharashtra, Gujarat, and Himachal Pradesh. In 2012–2013, an outbreak was seen in Tamil Nadu and Kerala, and in 2017 in Maharashtra. [14 – 15]

In Western and Northern India, DENV-3 was a more prevalent virus. In Maharashtra, DENV-3 became the predominant serotype in 2017, and this trend continued in 2018. [15] The least prevalent serotype, DENV-4, was found in the Maharashtra region around Nagpur as well as the Andaman and Nicobar Islands. DENV-4 was one of the predominant serotypes prevalent in Tamil Nadu in 2017, according to previous studies. [16 – 18]

According to Alagarsu K et al., DEN2 was the most prevalent serotype in numerous states in 2018. Additionally, thrombocytopenia, malaise, and petechiae were more frequently associated with this serotype. [19]

According to a study by Gupta A et al [20] and an original article by Kumaria R et al [21] DENV2 was the most prevalent serotype and accounts for the highest percentage of severe dengue patients.

India experienced the largest dengue outbreak in 1996, which was caused by DENV-2. [22] This was followed by outbreak that occurred from October to December 2003 by DENV3 serotype. [23]

When dengue fever spread out in Southern India in 2007, DENV-4 and DENV-3 suddenly dominated. [24] DENV-2 was the dominating serotype during the 2014 outbreak in Pune, followed by DENV-1.[25] During the 2016 epidemic, DENV-3 was the sole serotype in New Delhi. [26]

DENV-2 was revealed to be the most prevalent serotype in many states, according to a multicentric retrospective study that involved the Virus Research Diagnostic Laboratory (VRDL Network), ICMR-NIV Pune, and New Delhi. According to 2019 data, DENV-2 was the most common serotype in Chennai, followed by DENV-3. [19]

At a tertiary care hospital in North-Western India during the 2019 outbreak, DENV-2 was the most prevalent serotype. [20] In the study by Balasubramanian S et al., DENV-2 was the predominate serotype in severe cases [27].

A male preponderance was also observed in our study, and young adults were shown to be more affected than older individuals as males spend more time outside their houses and thus are more likely to be exposed when compared to females. Ahmed et al [28] also reported male predominance. Also, risk for clinical disease after primary dengue infection is relatively low throughout childhood and then increases rapidly through adolescence and early adulthood. [29]

Dengue Multiplex real-time PCR is a rapid and accurate diagnostic test for children hospitalized patients with severe dengue as compared to Dengue NS1Ag ELISA. Inpatient with acute phase serum samples, the multiplex RT-PCR assay is the cost-effective and rapid method to concurrently identify and serotype the dengue virus, especially in endemic areas [30].

Positivity for Dengue was high – and DEN-2 was the most prevalent serotype in our area. It is important to keep an eye  on prevalent Dengue serotypes  by surveillance  to rapidly detect  emergence of newer serotypes of Dengue viruses in hitherto unknown territory.  Rapid diagnosis can help in  patient management in a timely manner due to the lack of an effective therapy and vaccine.

The genetic characterization of the Indian DENV-2 and its correlation with the clinical outcome provided important information for the development of future molecular diagnostic investigations. It is important to keep an eye out by surveillance involving every area for the emergence of newer serotypes of Dengue viruses in hitherto unknown territory.

Molecular surveillance can help in forecasting large-scale outbreaks of the disease if regional If regional variations in the serotypes and genotypes of that are most commonly circulating strains are found during the early stages of the dengue season, molecular surveillance may have implications for forecasting large-scale outbreaks of the disease, in addition to vector surveillance. Only a rapid diagnosis can benefit with patient management in a timely manner due to the lack of an effective therapy and vaccine.

CONFLICTS OF INTEREST

None

ACKNOWLEDGMENT

We thank the scientists at VRDL, Department of Microbiology, SMS Medical College, Jaipur, Rajasthan for carrying out the molecular detection and serotyping of dengue.

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