Hyperferritinemia in Dengue Fever-Correlation between Serum Ferritin and Thrombocytopenia

Dengue fever is a common viral infection whose incidence in India has increased in the last 10 years. Case Fatality Rate (CFR) of dengue fever is 1%, with certain areas in rural India having CFR of 3-5% [1]. The clinical manifestations of dengue fever can be explained by the increased capillary permeability and endothelial damage. However, it is well known that there is significant cytokine stimulation during dengue [2]. Like other viral infections, the treatment is usually supportive management with Intravenous (IV) fluids and blood product transfusions as necessary.

However, for a small subset of patients, this infection is life threatening with severe cytopenias and significant systemic response. The presence of macrophage activation in dengue fever is well reported and could be one possible reason for mortality and morbidity in dengue fever [3]. MAS is a life-threatening haematological syndrome that is associated with cytopenias and fever. This is a complex entity that results from inappropriate activation of tissue macrophages leading to histiocytic proliferation, hypercytokinemia and T-cell immunosuppression. MAS can be rapidly fatal unless it is identified early and treated [4].

The treatment that has been advised for macrophage activation is mainly with steroids and other immunosuppressive agents like cyclosporine. IV immunoglobulins has been reported to be of benefit [4].

Of the overlapping features, what stand of interest for us is the serum ferritin levels. Ferritin is a protein that takes part in iron storage in the body. It is an acute phase reactant. Ferritin levels are important in MAS because it can be diagnostic for the disease as ferritin level >10,000 mcg/L is 90% sensitive and 96% specific for MAS [5]. Dengue being an infective condition, a modest rise in ferritin levels is expected in dengue fever as well. A South Indian study by Soundravally R et al., done on 48 patients with dengue fever shows that the mean ferritin levels at the time of admission was 1028.01±562.89 ng/mL. In this study hyperferritinemia (Ferritin >500 ng/mL) emerged a strong predictor of severe dengue and worsening thrombocytopenia [6]. However, there is not much evidence correlating ferritin levels and the platelet drop.

The use of intravenous steroids in severe dengue is a common clinical practice in many centres. However, there is no evidence for this usage and it is not known whether steroids have a role in this disease. Although intravenous steroids are broadly given in macrophage activation, their role with respect to dengue fever has not been studied. This study was thus done with the primary aim of assessing whether hyperferritinemia correlates with severity of thrombocytopenia. As secondary objective, we have compared the group of patients who were treated with steroids in addition to the standard treatment protocol against patients who were treated according to standard treatment protocols alone. The parameters that have been used to compare were the mean platelet drop and length of hospital stay of the patients.

It was a retrospective observational study conducted at a Tertiary Care Hospital in Kochi, South India over a period of 18 months from January 2015 to July 2016. The study was commenced after obtaining clearance from both the Institutional Ethics Committee and the Scientific Review Committee.

The study population consisted of adults admitted to the Department of General Medicine with a diagnosis of dengue fever. Only patients who were seropositive for Dengue Fever (Dengue NS 1 antigen or Dengue IgM ELISA-tested after 7 days of onset of fever) were recruited for the study. Patients included in the study also satisfied five out of eight of the Haemophagocytic Lymphohistiocytosis Diagnostic Criteria, 2004 [7]. The included study patients were those who had fever [8], hepatomegaly or splenomegaly, bicytopenia (Total leucocyte count <4000/dL, Haemoglobin <19 gm/dL, Platelet count <1,00,000/dL), elevated ferritin >500 ng/mL and triglyceride level >150 mg/dL [8-10]. Patients who were on antiplatelets, prior steroid therapy or other immunosuppressive therapy, known autoimmune disorders were excluded from the study.

During the study period, 26 patients admitted with dengue fever were found to satisfy both the inclusion and exclusion criteria.

For all 26 patients, the following clinical and biochemical data were collected as per our study protocol:

History of the present febrile illness, patient demographics, presence of WHO warning signs for severe dengue, associated co morbidities and length of hospital stay

The following analysis was done on the patients who were included in the study:

Demographic analysis;

Data were compiled in Excel sheets and were analysed using LOTUS IPSS 2.0 software. All numerical variables were expressed as mean and standard deviations. All categorical variables were expressed as percentage. Haematological and biochemical parameters of the patients at admission and after 48 hours were compared using paired t-test. Mann-Whitney U test was used to test for significance in sub group analysis of patients managed according to standard protocol versus those who have received steroids. Pearsons’ coefficient was calculated to analyse if serum ferritin levels were proportional to the mean platelet drop. Unpaired t-test was used to compare the ferritin levels of the two sub groups to ensure that they were comparable.

Of the 26 patients included in the study, 18 (69.2%) patients were male. The mean age of the patients studied was 45.77 years±16.286 years (Range: 21-75 years). The various comorbidities in this patient cohort are as follows [Table/Fig-1].

The mean and standard deviation of the important biochemical and haematological parameters are tabulated as follows. The standard deviation is more than the mean of most variables because of the considerable range over which the values were distributed [Table/Fig-2,3].

Pearsons’ coefficient was calculated to assess the relation between ferritin levels and the mean platelet drop and it showed a negative correlation which was however not statistically significant (r=-0.243, p=0.231).

Subgroup analysis done between patients on steroids versus those managed conservatively without steroids: Of the 26 patients in our study 15 of the patients had been managed with steroids and 11 without steroids. The steroid used was hydrocortisone given intravenously at a dose of 300 mg in three divided doses. The mean ferritin level of the steroid group was 11109.72 (SD=13212.05) and the mean ferritin level of the group managed without steroids was 11817.17 (SD=13935.73). The p-value for the difference between the means as determined by an unpaired t-test was 0.89. This was not significant.

Comparison between the change in platelet count and length of stay in both groups [Table/Fig-4]. Mann-Whitney U test was used as a test of significance and it showed that there was no significant difference in either the length of stay (p=0.443) or mean platelet drop (p=0.087) between the two groups.

Before embarking on a discussion of the results of our study it should be noted that there have been no previous studies that have looked at the characteristics of MAS triggered by dengue fever. Macrophage activation in dengue fever is an entity that has not been studied much. However given the increasing prevalence of dengue fever, it is an important entity. Serum ferritin level is the most important biomarker for this condition and our study primarily looks at whether this correlates with the degree of cytopenia [11]. Even though the mean ferritin level was more than 10,000 ng/mL (which is highly sensitive and specific for MAS it did not correlate with the change in platelet levels at 48 hours. Thus, a grossly high ferritin levels were not a predictor of the degree of platelet drop. This is clinically important as the treating physician should not be alarmed by the hyperferritinemia and should continue to follow standard protocol as advised by the WHO.

Of interesting note is the subgroup analysis between those treated with steroids and those treated without steroids. There was no statistical difference between the mean ferritin levels of both groups suggesting that the levels of cytokine activation due to macrophage activation is similar in both groups. It can be seen that there is a statistically significant larger drop in platelet counts in the group treated with steroids. Drastic fall in platelet counts is known to complicate the course of treatment by producing bleeding manifestations which is one of the criteria for severe dengue. While a Cochrane database systematic review which showed there is no evidence for steroids in dengue fever, more analysis should be done as to whether steroids have a negative impact on platelet counts [12]. Steroid use has not shown to affect the length of stay of the patient in hospital as well.

MAS secondary to viral infections is well described in literature. One of the earliest studies is by Risadall RJ et al., in 1979 and with low threshold of clinical suspicion this is being diagnosed with increasing frequency [13]. The most common and well known virus types to trigger this condition are Epstein barr virus, Herpes virus, HIV and cytomegalovirus. The treatment in these conditions is supportive and withdrawal of aggressive immunosuppression. Our study in patients with dengue fever who have associated MAS also conforms to this treatment principle.

However, a number of limitations do exist in present study especially the lack of mortality and patients with severe dengue, retrospective collection of data, lack of histological evidence of macrophage activation and relatively small sample size.

From this study, we conclude that there was no statistical correlation between the mean platelet drop and the ferritin levels in patients with dengue fever. The present study did not show any beneficial effect of steroids in dengue fever.

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