Recent Trends in Dengue Virus Infection; An Analysis of Virological, Clinical and Laboratory Profiles of Patients with Dengue Virus Infection in the Center for Clinical Management of Dengue Fever and Dengue Hemorrhagic Fever, Sri-Lanka

Introduction

Dengue is one of the most evolving arbovirus infections in the world leading to significant health burden in tropical and subtropical countries. It is endemic in many of the WHO regions such as South East Asia, the Americas, the Western Pacific, Africa and Eastern Mediterranean regions and it is estimated that around 3.9 billion people are at risk of getting the infection with more than 70% of them living in Asian countries including Sri-Lanka [1, 2, 3].

The infection is due Dengue virus, a single stranded RNA virus belonging to the family Flaviviridae and is primarily transmitted by female Aedes mosquitoes; Aedesaegypti, which are prevalent in these tropical and subtropical areas.

The virus has four serotypes (DENV 1-4) which are genetically diverse but similar by about 60- 75% at amino acid level thus giving rise to the unique clinical and immunological picture in dengue [1]. Primary infection with one serotype confers lifelong homotypic immunity to the infecting serotype while providing short lived heterotypic protection to other serotypes. Therefore breakthrough infections by a different serotype (secondary dengue infection) can give rise to severe forms of dengue infection [4]. Moreover, several serotypes may co-circulate during an epidemic, thus increasing the health risks.

Clinical manifestations of the infection are diverse and may range from asymptomatic infection to severe forms such as dengue hemorrhagic fever, dengue shock syndrome or death. Many factors have been identified as prognostic markers predicting the severity of the disease such as low platelet count, improper fluid management, etc [5, 6, 7, 8]. Diagnosis of the infection is mainly clinical and may be supported by virological tests such as NS1 antigen testing, dengue specific antibody detection, dengue genome detection. There is no specific treatment for dengue and is predominantly based on fluid management and symptomatic therapy with analgesics and antipyretics.

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Sri-Lanka is hyper endemic for dengue virus infection and epidemics of dengue had been reported in the country for more than three decades. The first serologically confirmed case of Dengue occurred in 1962 following which the case burden rose dramatically over time [9]. All four serotypes of Dengue had been co-circulating in the country with time to time predominance of one or two serotypes. Dengue 1 and 2 had been predominant in 1960s, while DENV 3 & 4 were responsible for the outbreaks from 1970s to early 2000 followed by resurgence of DENV 1 in 2006-2010 and DENV 2 in 2017 epidemics [10, 11, 12]. The Dengue outbreak in 2017 due to DENV 2 was one of the largest outbreaks in the country and figure 1 depicts the phylogenetic analysis of the circulating viruses during the period. Various Scio-economic and cultural changes that occurred in Sri Lanka over time largely contributed to the spread of the vector and the virus throughout the country. Moreover, climatic changes also had a significant impact on dengue in Sri Lanka. Although, dengue cases are being reported year around throughout the country two main peaks are identifiable from October to December and from May to July.

A multitude of factors such as the infecting virus type, shifts in circulating serotypes, host immunity, vector, environment changes and accessibility to healthcare facilities play an important role in deciding the clinical outcome of dengue and occurrence of epidemics. Also the non-specific nature of the infection and lack of specific therapeutic measures makes it is important to have a high index of suspicion to identify clinical cases early and undertake necessary treatment. At the same time the circulating serotype has a significant impact on the severity of the disease due to the unique pathogenicity the virus has. Hence identification of changing serotypes over the time is important in predicting the severity and impact during epidemics.

Objectives

Therefore, the current study was formulated to describe the demographic, clinical and laboratory parameters of dengue infected patients during the inter-epidemic period in Sri Lanka, identify the circulating dengue serotypes and their association with clinical and laboratory parameters with the intent of predicting the future occurrence and severity of dengue in Sri Lanka.

Study Design

This laboratory based retrospective descriptive study was conducted at the National Arbovirus testing laboratory at the Medical Research Institute which is the premier laboratory for the diagnosis of dengue virus infection in the country. All blood/plasma samples sent from the Center for Clinical Management of Dengue Fever and Dengue Hemorrhagic Fever, Negambo, Sri-Lanka from December 2018 to February 2019) and tested positive by dengue serotyping real time PCR were included in the study. The Center for Clinical Management of Dengue Fever and Dengue Hemorrhagic Fever, Negambo, Sri-Lanka is one of two centers for the management of dengue virus infection of all age groups from all around the country. Clinical and laboratory details were collected using hospital records and the accompanying request form.

Samples tested positive for dengue virus by sero-typing real time PCR were also tested for the presence of secondary infection using the Panbio dengue IgG capture ELISA which only detect dengue IgG titers compatible with secondary dengue infection. Extraction of viral RNA (from 140µl of plasma) was carried out using the QIAmp® viral RNA mini kit by QIAGEN Germany and the extraction was carried out as per the manufacturer’s instructions. An additional centrifugation step was performed to remove any remaining ethanol and elutes were subjected to PCR assay without delay.

Dengue virus RNA detection and serotyping was done using the commercially validated Real Star® Dengue Type RT-PCR kit 1.0 according to manufacturer’s instructions using the Rotor-Gene Q 5plex real time PCR instrument. The kit detected all four dengue serotypes and had been in use for clinical diagnostics followed by verification within the laboratory for several years.

Commercially validated Panbio Dengue IgG capture ELISA kit by Abbott, USA, which detects dengue IgG of more than 2560 HAI titer, was used for the detection of secondary dengue infection. Secondary infection is characterized by high levels of specific IgG (> 2560 HAI titre) 1-2 days after the onset of infection. As per manufacture’s manual the serological sensitivity for secondary infection was 85.7% and serological specificity for primary infection was 93.3%. Serological specificity for seronegative samples was 100%.

Data analysis was conducted using the latest version of SPSS. Categorical variables were presented as frequencies/ percentages and chi-square or Fisher’s Exact Test was adopted to analyze the significance between categorical variables. The analyses were performed at 95% confidence with level of significance of p<0.05.

Results

A total of 129 Dengue real-time PCR positive patients were identified during the study period and their demographic, clinical and laboratory parameters are depicted in Table 1.

Discussion

The present study aimed at describing the demographic, clinical and laboratory parameters of patients presenting with Dengue virus infection during the inter epidemic season in Sri-Lanka and to identify the circulating Dengue serotypes and their association with the clinical presentation in order to predict the occurrence and severity of the upcoming dengue season.

In the current study, more than 75% of the infected patients were aged less than 45 years with majority being within the 6-15 year age group, which is in agreement with the characteristic age distribution seen during 2017 outbreak; one of the largest dengue outbreaks in Sri Lanka, as well as in several other previous Sri Lankan studies [13, 14, 15, 16]. At the same time globally also, frequently affected belonged to a similar age category, because they spend a significant time in public places due to work and education which make them more susceptible to be in contact with the principal vector [17, 18].

Both females and males were equally affected in the current study and the gender distribution of dengue in Sri Lanka was variable in previous studies conducted in the country as well. While some local and international studies reported that females were more frequently affected [15, 19] some others reported that males were the predominantly affected gender [16, 20, 21, 22]. Engagement of males and females in various outdoor activities due to various social and cultural reasons mainly affects the distribution and the relatively small sample size in the current study also may have had an impact on the generated value.

Clinical characteristics of the study subjects showed that majority of patients were belonging to non-severe dengue category but, had platelets less than 50,000/µL, leucopenia, elevated ALT and AST. Median AST level was more than ALT and increment of AST was observed more frequently than ALT which is a characteristic finding seen with hepatic involvement in dengue. All of these parameters closely agreed with local and international studies elsewhere [15, 16].

Furthermore, majority of the patients had primary dengue infection which usually is associated with less severe clinical presentation while the rate of secondary infections also followed closely. Local dengue sero-prevalence data is limited and one study demonstrated that the sero- prevalence was low in very young children and it gradually increased with age [23]. The pattern of age distribution in the current study, where a significant proportion was children may have had an impact on the primary infection rate.

When considering the circulating dengue serotype, all serotypes except DENV-4 were reported during the period with the predominant being DENV-2, coinciding with the hyper-endemic nature of the infection in Sri-Lanka. At the same time, it can be presumed that following it’s resurgence in 2017 DENV-2 is still circulating as the prominent serotype in Sri Lanka [12, 13].

On the other hand, DENV-1 and 3 were also circulating in significant percentages (13.2% and 27.1% respectively) with DENV 3 being higher in December to January and DENV-1 in February. At the same time it is evident that DENV 3 was more frequently associated with primary infection while DENV 1 and 2 were more associated with secondary infection keeping in line with the fact that DENV-3 had not been reported as a prominent serotype since 2004 [11]. However, none of the serotypes showed a significant association with either primary or secondary infection. These findings highlight concern whether DENV 3 can become predominant as time goes, thus paving way to another outbreak as a result of the shift in circulating serotypes.

Analysis of association of dengue serotypes with mean age of the study subjects revealed that while DENV 2 was infecting mainly adults (mean age 26 years), children were predominantly infected with DENV 1 and 3 (mean age 14 years), further confirming the data acquired for circulating serotypes over time and the rates obtained for infection status with each serotype.

With regard to association of serotypes with clinical and laboratory parameters, DENV 2 was associated with more frequent episodes of thrombocytopenia less than 50,000/µL and elevated AST, but other parameters such as severe dengue, leucopenia and high ALT were mainly associated with DENV 3. Parameters observed in the current study is comparable to studies elsewhere though none of the parameters in the present study showed a significant association. It is said that DENV 2 is associated with more severe infection by several studies [17, 18]. More so, DENV 3 is also associated with severe presentations of dengue according to a meta-analysis carried out in 2016 to analyze severity of dengue infection by different serotypes in primary and secondary infections reported that in south-east Asian region which stated that primary infections with DENV 3 was more severe and secondary infections with DENV-2, DENV-3 and DENV-4 was also more severe [24].

Considering the above factors, it is evident that in Sri Lanka during this inter epidemic period, the predominant circulating serotype is DENV-2 with co-circulation of DENV 3 and 1, thus can be predicted that severe forms of dengue would continue to be reported in the country during the upcoming season. On the other hand, both the DENV 3 and 1 show a probability of rising in numbers and may replace DENV-2. DENV 3 had not been reported in the country for some time leading to an immune naïve population who can contribute to increase spread of infection changing the predominant serotype, thus leading to an outbreak situation with severe cases.

Conclusion

Majority of the infected were children and young adults with cases of non-severe dengue being predominant. Majority had primary dengue infection. The predominant circulating serotype was DENV 2 with co-circulation of Dengue 3 and 1. DENV 2 was associated with more frequent episodes of secondary infection, thrombocytopenia and elevated AST while DENV 3 was mainly associated with primary infection, severe dengue, and leucopenia and elevated ALT.