Hemophagocytic Lymphohistiocytosis in Dengue

Jeevitha Sivakumar¹, Poovarasan Prakasam², Vinod Gunasekaran³, suresh chelliah⁴, D. Senguttuvan⁵

¹Postgraduate Resident in Paediatrics, Kauvery Hospital, Cantonment, Trichy

²Postgraduate Resident in Paediatrics, Kauvery Hospital, Cantonment, Trichy

³Consultant, Paediatric Hematology Oncology & BMT, Kauvery Hospital, Cantonment, Trichy

⁴Senior Consultant Paediatrician, Kauvery Hospital, Cantonment, Trichy

⁵Chief Consultant Paediatrician Kauvery Hospital, Cantonment, Trichy

Background

Dengue fever, a viral illness transmitted by the Aedes mosquito, has become a significant public health concern in India. The impact is significant, as dengue fever can cause severe illness and in some cases, lead to life-threatening complications like Dengue hemorrhagic fever, Dengue Shock Syndrome and Hemophagocytic lymphohistiocytosis (HLH). HLH is a rare condition in children, but not uncommon in dengue. HLH has received increasing attention in recent years. A strong clinical suspicion of HLH is needed in any child with unusual course of prolonged fever with cytopenias or hepatosplenomegaly. The interaction between dengue virus-induced inflammation and dysregulated immune response in HLH creates a vicious cycle, perpetuating the hyper inflammatory state and exacerbating the severity of the disease. Early recognition and prompt management of HLH in dengue are crucial to prevent life-threatening complications and improve patient outcome. HLH in Dengue is not well reported in literature.

Pathophysiology in secondary HLH

HLH is a life-threatening syndrome of excessive immune activation that occurs as a familial or sporadic disorder, and it can be triggered by a variety of acquired events that disrupt immune homeostasis. It is characterized by a dysregulated innate immune system, specifically NK cells and CD 8+ cytotoxic T-cells.

In a healthy immune system, these cells produce two cytolytic enzymes: perforin and granzyme. These proteins are packaged into granules that are discharged into the immunologic synapse between the effector and target cell as shown in (Fig. 1) Perforin forms destabilizing pores in the target cell’s membrane allowing for the entrance of the strongly proteolytic granzyme as well as osmotic shifts, which result in target cell degradation.

In HLH, this process is disrupted through specific genetic mutations or acquired through some highly immunogenic stimulus (a viral infected or malignant cell). The ineffective interaction between NK cells, CD 8+ cytotoxic T-cells, and their targets leads to a vicious cycle of inflammation. More and more cytotoxic cells are recruited but remain unable to get rid of the pathogenic antigen leading to a massive increase in circulating cytokines. This hypercytokinemia leads to widespread activation of macrophages resulting in hemophagocytosis and multiorgan-damaging inflammation, which characterizes the disease ^[1].

In dengue-endemic countries, HLH syndrome in Dengue cases is likely to be underdiagnosed due to overlapping clinical features with dengue (fever, hepatosplenomegaly, leukopenia, and thrombocytopenia).

Fig. (1). Perforin forms destabilizing

Natural course of dengue fever

Dengue fever typically is a self-limited disease with a mortality rate of less than 1% when detected early and with access to proper medical care. When treated, severe dengue has a mortality rate of 2-5%, but, when left untreated, the mortality rate is as high as 20% ^[2].

Dengue begins abruptly after a typical incubation period of 5–7 days, and the course follows 3 phases: febrile, critical, and convalescent.

Febrile phase: Typical duration of 0–7 days, Biphasic fever possible. Monitoring for defervescence and warning signs is crucial to identify progression into the critical phase. Defervescence occurs between day 3 and day 8 of the illness.

Critical phase: Begins around the time of defervescence but it might begin as early as the third day after fever onset in patients who are still febrile. This is the period when those who develop severe disease will become critically ill with a rapid decline in platelet count, a rise in hematocrit and presence of warning signs for severe disease.

Convalescent phase: During this phase, the extracellular fluid, which was lost due to capillary leakage, returns to the circulatory system and the patient’s clinical status improves. This phase usually starts after 6–7 days of fever and lasts for 2–3 days.

HLH complicating Dengue

Mortality in dengue is due to severe forms of dengue: Dengue haemorrhagic fever and Dengue shock syndrome are well known complications. The pathogenesis of severe form is told to be due to occurrence of cytokine storm ^[3].

Secondary HLH following dengue is a potentially fatal form and has been seen in the severe form of infection. As discussed before, HLH is due to florid activation of macrophages by the activating cytokines, leading to phagocytosis of blood cells in the bone marrow. In a dengue patient, HLH should be suspected if fever persists more than 7 days with hepatosplenomegaly and persistent/ progressive cytopenias. These features resemble severe sepsis (due to a secondary bacterial infection) and hence should also be evaluated with blood and urine cultures and other tests as indicated. Simultaneously, investigations including serum ferritin, fasting triglyceride and fibrinogen levels and if deranged (elevated ferritin, hypertriglyceridemia and / or hypofibrinogenemia), a possibility of secondary HLH should be considered^[4]. Higher serum ferritin levels are associated with dengue as compared to other acute febrile illnesses^[5]. Presence of anemia without a clinically significant hemorrhage could be a marker to suspect HLH as usually hemoconcentration is a classical association with severe Dengue. Similarly, elevated transaminases is a common associated finding in HLH.

Diagnosis

A diagnosis of Dengue is usually made in patients with suggestive clinical features being tested positive for NS1 antigen or IgM antibodies.

Though HLH is receiving more attention among physicians nowadays, its clinical and laboratory features overlap with systemic inflammatory response syndrome (SIRS) and Multiorgan dysfunction syndrome (MODS) ^[6].

The diagnostic criteria of HLH is classically made using HLH-2004 criteria (Table 1). Five out of the eight criteria are required for the diagnosis of HLH [7]. However, all children with HLH may not meet 5 criteria and few of these criteria are not readily available for testing in clinical practice. These features are mainly due to high concentration of inflammatory cytokines and organ infiltration by activated lymphocytes and histiocytes as seen in (Table 2)

Hemophagocytosis in bone marrow may be absent in the initial stages of the disease. Though bone marrow aspirate excludes leukaemia and other causes prior to initiating steroids in HLH scenario, the role of bone marrow in a confirmed case of Dengue is not very clear. The need for gene testing for all cases of secondary HLH in Dengue is also not very clear.

A single value of serum ferritin > 500 ng/ml alone is not considered as HLH as it is an acute phase reactant. Serial rise of ferritin with presence of other signs and symptoms should be considered. But serum ferritin > 10,000 ng/ml is highly specific for HLH.

Table 1: Diagnostic criteria for HLH (HLH-2004)

Clinical

Fever

Splenomegaly

Laboratory

• Cytopenia = > 2 cell line
• Hb < 9 g/dl [in neonates, Hb < 12 g/l]
• Platelets < 1 lakh
• Neutrophils < 1000 (red flag feature)

• Hypertriglyceridemia and/or hypofibrinogenemia
• (Fasting triglycerides > 265 mg/dL / Fibrinogen < 1.5 g/l)

Ferritin > 500 ng/ml

sCD25 > 2400 U/ml

LOW or absent NK-cell cytotoxicity

Hemophagocytosis in bone marrow, CSF or lymphnodes

Table 2: Pathophysiology of HLH manifestations

Fever	IL-1, IL-6, TNF α
Hepatosplenomegaly	Organ infiltrations with activated lymphocytes and histiocytes/macrophages
Cytopenia in peripheral blood	Suppression of hematopoiesis by TNF-α, INF-γ, high ferritin; hemophagocytosis
Elevated triglycerides	TNF α inhibits lipoprotein lipase
Raised ferritin	Secreted by activated histiocytes and macrophages
Decreased fibrinogen levels	macrophages secrete plasminogen activator which stimulates plasmin causing hyperfibrinolysis
Soluble ILR2	Secreted by activated T lymphocytes

Management

Timely recognition and aggressive treatment with or without HLH specific immunotherapy is necessary to decrease the mortality and morbidity. Uncontrolled proliferation of activated lymphocytes and histiocytes resulting in production of numerous inflammatory cytokines may lead to death unless arrested by appropriate treatment [8, 9]
Child diagnosed with genetic HLH requires chemotherapy with immunosuppressants and are treated with Dexamethasone, Etoposide and Cyclosporine (induction followed by maintenance therapy). Haematopoietic stem cell transplantation (HSCT) is the only curative treatment [7].
In secondary HLH, first step is to treat and control the underlying cause. For dengue, supportive management with fluid therapy, close monitoring and supportive care remain the mainstay therapy. However, in cases associated with secondary HLH, an additional short course of immunosuppression is indicated.
There are no standard guidelines for treating secondary HLH in Dengue. It is essential to treat Dengue with HLH in an intensive care setting. Various centers follow different regimen of short course of steroids or intravenous immunogloblins (IVIG) as reported in the literature (discussed below).

Review of literature on management of HLH in Dengue

Pal et al (2014) from Institute of Child Health, Kolkatta reported 8 cases of HLH among 358 children with Dengue. All children received intravenous Dexamethasone 10 mg/m2/day till they became hemodynamically stable or accepting oral feeds. Subsequently, oral dexamethasone was tapered over 21 days. IVIG (1g/kg) was used in one child who did not respond to dexamethasone after 48 hours. All children recovered well ^[4].
Raju et al (2014) from Tirunelveli, Tamil Nadu reported 23 cases of HLH among 212 children with Dengue. Nineteen children received IVIG and all of them recovered. Of the remaining 4 children who did not receive IVIG, 3 died (2 had encephalopathy and one had cardiorespiratory complications ^[9].
Bhattacharya et al (2019) from PGIMER Chandigarh reported 7 cases of HLH among 22 children admitted with severe dengue in PICU. Four cases received intravenous methylprednisolone (30 mg/kg/day for 5 days) followed by tapering over next 2 weeks and all of them survived. Of the remaining three who did not receive steroids, 2 died ^[8].
In our center, we treat HLH in Dengue with intravenous dexamethasone 6 mg/m2/day for 3 to 5 days. If clinical and laboratory parameters are normal after 5 days, steroid is abruptly stopped. In other cases with partial response, dexamethasone is gradually tapered over 2 – 3 weeks. Though the results are encouraging, the data needs to be formally analyzed.

Take home message

To summarize, HLH is a life threatening complication of Dengue which needs to be identified early. Subsequent to immunogenic stimulus by viral infection, an ineffective interaction between NK cells, CD 8+ cytotoxic T-cells, and their targets leads to a vicious cycle of inflammation (hypercytokinemia and macrophage activation). Early diagnosis using the clinical and laboratory criteria should be established. A short course of steroids or IVIG is warranted in addition to Dengue directed treatment to decrease the morbidity and mortality.

Future directions

As the literature is insufficient in this important clinical condition, more studies need to be conducted and published on HLH in Dengue. A standard treatment guideline needs to be formulated for early diagnosis and evidence based management of this condition.

References

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