Obstacles in Dengue Diagnosis and Severity Prediction

Abstract

Background:

Dengue emerges as a perennial public health menace. Identification of patients at risk of developing dengue hemorrhagic fever (DHF)/dengue shock syndrome is still a huge challenge.

Objective:

In this background, this study was done to predict the severity of dengue by measuring the interleukin (IL) 6 and IL8 by enzyme-linked immunosorbent assay (ELISA).

Materials and Methods:

A total of 88 samples were collected from patients with clinical signs suggestive of dengue were tested for NS1 and immunoglobulin (Ig) M (NIV Pune), and proinflammatory markers IL-6 and IL-8 (Diaclone SAS Pvt Ltd., France) by ELISA.

Results:

All the 88 patients were positive for dengue IgM. Dengue NS1 was positive in 36 patients. IL-6 and IL-8 were raised in 56 cases and 46 cases, respectively. Out of 32 severe dengue cases, 24 had raised IL-6 and IL-8.

Conclusion:

The dynamic nature of dengue requires close monitoring and frequent clinical and laboratory evaluations. Early prediction requires prompt testing of all the markers (IgM, IgG, and NS1) irrespective of the day of illness. Although cytokine estimation aids in assessing the severity, other cost-effective alternatives (hematocrit and platelet count) are copacetic.

INTRODUCTION

The World Health Organization (WHO) estimates that dengue is endemic in almost 129 countries, emerges as a public health menace throughout the year, and often peaks during and after rainy season. It becomes hyperendemic in most of the countries due to the cocirculation of all the four serotypes. Epidemiological data reported that 100 million cases of dengue fever (DF) and half a million cases of dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS) occur worldwide every year, with a mortality rate of <1%–20% depending on the awareness and quality of treatment.^[1-3] The clinical manifestations may vary from self-resolving DF to the more devastating DHF/DSS. Immunity to one serotype does not offer protection against other serotypes. However, it exhibits extensive cross reactivity in serological tests because they are closely related antigenically.^[2,4] DHF occurs mostly in persons with heterotypic secondary infection. It has been well recognized that the inflammatory response and deregulated cytokine production play a crucial role in DHF/DSS. Identification of patients at risk of developing DHS/DSS is still a huge challenge. In this background, this study was done to predict the severity of dengue by measuring the interleukin (IL) 6 and IL8 by enzyme-linked immunosorbent assay (ELISA).

MATERIALS AND METHODS

A total of 88 samples were collected from patients admitted in a tertiary care teaching hospital with clinical signs suggestive of dengue. All the samples were tested for complete blood count including platelet count, immunoglobulin M (Ig) M (NIV Pune), NS1, and proinflammatory markers IL-6 and IL-8 (Diaclone SAS Pvt Ltd., France) by ELISA.

The ELISA kit used is highly sensitive to detect IL-6 and IL-8 with minimum value of 12.3 pg/mL and 2 pg/mL, respectively. The specificity of both the kits was 100% as mentioned by the manufacturer.

The Institutional Ethics Committee clearance was obtained before starting the study (TIREC-Ref 1701/MICRO/2020 date January 07, 2020). Statistical analysis was done using IBM SPSS Statistics for Windows, Version 27.0. (Armonk, NY: IBM Corp).

Inclusion criteria

• Patients of any age with clinical signs suggestive of dengue.

Exclusion criteria

• Fever treated as outpatient

• Those with other known infectious diseases.

RESULTS

Of the 88 patients, 45 were children (<14 years) and 43 were adults (>14 years). Of these 45 children, 29 were male and 16 were female. Among the 43 adults, 23 were male and 20 were female. According to the WHO clinical scoring criteria, 56 patients had dengue without complications (score 1 and 2) and 32 had severe dengue (score 3 and 4).

Platelet count <100,000 cells/mm³ was observed among 42 and 29 children and adults, respectively. All the 88 patients were positive for dengue IgM. Dengue NS1 was positive in 36 patients [Table 1].

Table 1

Distribution of serological markers of dengue

IL-6 and IL-8 were raised (>100 pg/mL) in 53 cases and 52 cases, respectively. Out of 32 severe dengue cases (WHO clinical score 3 and 4), 24 had raised (>100 pg/mL) IL-6 and 20 had (>100 pg/mL) IL-8. With the WHO clinical score 1, 21 cases had (>100 pg/mL) IL-6 and 24 cases had (>100 pg/mL) IL-8 [Table 2]. There was no significant association of WHO clinical score with IL6 (P = 0.894) and IL8 (P = 0.810) among the four groups.

Table 2

Distribution of interleukin 6 and interleukin 8

DISCUSSION

Dengue usually presents with nonspecific symptoms such as fever, headache, malaise, and skin rash which are seen in malaria, chikungunya, typhoid, Brucella, Leptospira, and scrub typhus. Laboratory investigations play a crucial role in the diagnosis, owing to the cocirculation of other febrile illness in the community.

Dengue IgM antibody titers are significantly higher in primary infection than in secondary infection and may persist for more than 90 days; it falls to undetectable level mostly in 60 days. Hence, a single acute phase serum for IgM MAC-ELISA is less sensitive in the diagnosis. IgM is more specific and appears only after 3–5 days of illness in primary infection, and in the secondary infection, it is not always positive. False negative can occur, if the sample is taken before detectable IgM.^[5]

Dengue IgG can be useful to differentiate primary from secondary infection; four-fold rise in titer implies recent infection.^[6] False positive can occur with both Ig M and IgG in other Flavivirus infection.^[7]

NS1 is a highly conserved glycoprotein necessary for the replication of virus which can be detected from 1 to 9 days after the onset of infection and may persist up to 18 days. It is detected only for a short period in secondary infection, because IgG may mask or rapid clearance due to immune complex formation.^[8,9]

Hence, simultaneous detection of all these three markers is essential in the diagnosis and differentiates primary infection from secondary infection.

Reverse transcription polymerase chain reaction and virus isolation in the first 4 days of infection are the recommended methods for the confirmation of dengue.^[10] Dengue virus (DEN) replicates in mononuclear cells including skin dendritic cells, tissue macrophages, blood monocytes, and hepatocytes.^[11] At present, the host cell receptors involved in viral entry are particularly not known.

Studies reported that the patients with secondary infection are at significant risk of DHF/DSS, but only 5% of patients with primary infection can develop severe form of dengue. It is common in children <1 year, born to dengue immune mothers.^[12] Severity of dengue depends on the age and immune status of the infected person, DEN serotype/genotype, and genetic makeup of the population.^[13] Cocirculation of multiple serotypes has been consistently reported and might be responsible for the escalation of DHF cases.^[14] DEN-1 is the predominant serotype followed by DEN-2 and DEN 3 in severe dengue.^[15]

Immunity is type specific, and secondary infection with other serotypes is due to antibody dependent enhancement. Studies explained that the avidity or titer of preexisting antibodies is not sufficient to neutralize the second serotype and promotes higher systemic viral load. This in turn leads to excessive immune activation with a cytokine storm, profound plasma leakage due to defect in vascular permeability. At the defervescence stage, fever subsides and viral load falls suddenly, but severe symptoms such as vascular leakage, hemoconcentration, hemorrhage, and shock appear. This suggests that host immune response also plays a role in the severity. T cell mediated tissue damage also contributes to vascular defect by damaging infected endothelial cells, platelets, and coagulation factors.^[16,17]

In this study, out of 32 severe dengue cases (WHO clinical score 3 and 4), IL-6 and IL-8(>100 pg/mL) were noted in 24 and 20 cases, respectively. With the WHO clinical score 1, 21 cases had IL-6 and 24 cases had (>100 pg/mL) IL-8. Hence, the importance of circulating levels of cytokines as inflammatory mediators is debatable and difficult to interpret. Some authors reported correlation of cytokine level with severity, and some noted low level in DHF/DSS. The levels are increased at the initial stage of disease followed by a decreasing tendency until shock occurs, then it rises again.^[2]

Hence, the measured levels mainly depend on the day of sample collection, require sequential monitoring, and involve huge cost. Although cytokines play a key a role in the pathology of dengue, cytokine storm can occur with other viral infections too. Instead, cost effective, acceptable markers used to assess the severity of dengue are continuous monitoring of hematocrit and platelet count.

The management of dengue is predominantly supportive and symptomatic. Until now, there is no effective tetravalent vaccine to offer long term protection against homotypic and hetertypic dengue serotypes. Another challenge is lack of animal model result in uncertainity about its safety and efficacacy. The development and spread of resistance to insecticides is a burgeon issue in the vector control.

Limitation

1. In the present study, IL-6 and IL8 were not performed in accordance with the duration of febrile illness.

2. The duration of illness with respect to the thrombocytopenia was not recorded.

CONCLUSION

As the number of severe dengue cases and case-fatality are increasing, which are due to late health-seeking behaviour and access to health care facility. The dynamic nature of dengue requires close monitoring, frequent clinical and laboratory evaluations. As there is no specific treatment, early prediction requires prompt testing of all the markers (IgM, IgG, and NS1). Though cytokine estimation aids in assessing the severity, other cost effective alternatives (hematocrit and platelet count) are sufficient. Symptomatic management of severe form of dengue is imperative to reduce the mortality. Further, case definition, diagnosis and classification of dengue, based on the WHO DF/DHF/DSS criteria, vary between countries and regions. Changes in the epidemiology of dengue lead to difficulties in adapting existing WHO criteria. Variations in the defined warning signs are observed in different regions, thus require local adaptation of the guidelines in the future.