Epstein-Barr virus induced haemophagocytic lymphohistiocytosis

Abstract

Haemophagocytic lymphohistiocytosis (HLH) is a rare condition of uncontrolled immune activation as a result of an inherited genetic defect or in response to malignancy, autoimmune disease, rheumatological disease, AIDS infection or post-transplant immunosuppression. Described here is the case of a 19-year-old Caucasian man who presented with complaints of worsening fever, new-onset jaundice and lethargy after failing treatment for suspected infectious mononucleosis. Physical examination was significant for fever and splenomegaly while laboratory results revealed transaminitis, cytopaenia, indirect hyperbilirubinaemia and elevated ferritin, raising the likelihood of both autoimmune haemolytic anaemia and HLH. He tested positive for Epstein-Barr virus (EBV), and bone marrow biopsy revealed hypercellular marrow with haemophagocytosis and no evidence of malignancy. High dose steroids were initiated with significant improvement in haemoglobin, resulting in a final diagnosis of HLH secondary to acute EBV infection. The patient was discharged on continued high-dose prednisone with planned taper and consideration of outpatient rituximab therapy for 4 weeks. High clinical suspicion and prompt evaluation were critical to early treatment and decreased morbidity.

Background

Haemophagocytic lymphohistiocytosis (HLH) is one disease process within the larger grouping of hyperinflammatory cytokine storm syndromes.¹ It is a rare and frequently fatal hematological disorder that often goes undiagnosed or is only diagnosed on autopsy.² The aetiology of HLH can be primary, with a genetic modification (PRF1, UNC13D and STXBP2) that is often provoked by infection or can be secondary with onset precipitated by infection or malignancy.³ HLH is generally considered a paediatric phenomenon but can occur across the lifespan. In adults, the cause in up to 50% of cases is malignancy, the most common of which are haematological.⁴

Inherited dysregulation of the cytotoxic activity of natural killer (NK) and T cells leads to the pathogenesis of primary HLH.⁵ In secondary HLH, a precipitating event, such as Epstein-Barr virus (EBV) infection, causes a disruption in the pathway of CD8+ T cell activation leading to a profound inflammatory response.⁶ There is a well-documented association between EBV and HLH, and EBV is the most common infection that triggers HLH.⁵ This uncontrolled immune response occurs more frequently in immunosuppressed patients or patients who have an X-linked lymphoproliferative disease.⁶ EBV is well known to affect B cells, yet its role in infecting T and NK cells leads to the overwhelming inflammatory response of HLH.⁵ There are also idiopathic causes and postulated mechanism is the downregulation of surface proteins, such as CD5, or increased activity of human leucocyte antigen that causes impaired activity of cytotoxic T cells.⁶

The diagnosis of HLH is challenging in that the initial presentation is composed of a wide variety of non-specific symptoms.⁷ Unlike the other hyperinflammatory disorders, including immunodeficiency-associated hyperinflammatory syndrome, malignancy-associated hyperinflammatory syndrome and macrophage activation syndrome (MAS), HLH has formal diagnostic criteria that were revised in 2004. It requires either a molecular diagnosis of HLH or presence of five of the eight diagnostic criteria including: fever; splenomegaly; two of three indications of cytopenia (haemoglobin less than 10 g/L, platelets <100 bil/L or neutropaenia <1.0 bil/L); elevated ferritin >500 ug/L; low or absent NK cell activity; hypertriglyceridaemia >265 mg/DL and/or hypofibrinogenaemia <1.5 g/L; elevated interleukin (IL)-2 receptor >2400 U/mL; and bone marrow biopsy showing haemophagocytosis, which together characterise HLH and are essential to establishing the diagnosis.^{1 7} In contrast to HLH, MAS is strongly associated with systemic juvenile idiopathic arthritis.⁸ Other triggers for HLH include infections, autoimmune or metabolic diseases.⁴

Not all patients with HLH will meet the criteria sufficiently early in the disease course to establish a diagnosis. Additionally, the differentiation between the hyperinflammatory syndromes can be difficult, and treatment can vary greatly, ranging from treating the underlying trigger to chemotherapy.¹ High clinical suspicion with timely diagnostic tests is essential to prevent delay in initiation of treatment. Treatment as per the HLH-94 protocol, which included etoposide and dexamethasone, and the updated HLH 2004, which adds cyclosporine to these agents, suppresses the overwhelming immune response.⁹ However, cyclosporine has been shown to offer limited benefit⁹; therefore, corticosteroids with chemotherapy such as etoposide are the mainstay of treatment.¹⁰ In this case report, we discuss how high clinical suspicion and prompt evaluation are critical to early treatment and the prevention of morbidity and mortality.

Case presentation

A previously healthy 19-year-old Caucasian man presented with 2 weeks of fevers and nausea after a summer camping trip in the Midwest and Northeast USA. He developed a rash that disappeared after completing a methylprednisolone dose pack prescribed by his primary care physician for infectious mononucleosis. He had fevers monitored at home for 1 week, with notable jaundice, weakness and disorientation. Due to worsening symptoms, he presented to the emergency department at our tertiary care facility. Physical examination was significant for temperature of 39.9°C (103.8°F), tachycardia, scleral and cutaneous jaundice, and splenomegaly.

Investigations

Peripheral blood smear revealed hypochromic cells and agglutination. A complete blood count revealed a haemoglobin of 5.4 g/dL, leucocytosis of 10.5 bil/L, haematocrit of 16.1%, mean corpuscular volumeof 87 fL, neutrophils of 3.6 bil/L and platelet count of 234 bil/L, while a comprehensive metabolic panel revealed sodium 134 mEq/L, potassium 4.4 mEq/L, blood urea nitrogen (BUN) 15 mg/dL, creatinine 0.79 mg/dL, protein 6.1 g/dL, albumin 3.3 g/dL, alkaline phosphate 112 U/L, aspartate transaminase 280 U/L, alanine transaminase 346 U/L, total bilirubin 8.6 ng/dL, indirect bilirubin 2.3 mg/dL and haptoglobin of <8 mg/dL. Further investigation of haemolysis revealed positive anti-C3d on the direct antiglobulin test, despite no prior history of blood transfusions. The cold agglutinin titre was 1:215. Ferritin was elevated to 21 634 ng/mL, and triglycerides were 198 mmol/L. Urinalysis showed cloudy urine with positive bilirubin, 3+ blood, 30 mg/dL protein, negative nitrites and leucocyte esterase, 11–24 red blood cells (RBC)/hpf; 3–10 white cell count/hpf, no casts and no bacteria. EBV was detected in peripheral blood by PCR, but a quantitative viral load was not performed. EBV serology revealed an EBV IgM of >160.0 U/mL, IgG 54.7 U/mL, anti-EBV-EA 89.1 U/mL and anti-EBNA <3 U/mL.

Differential diagnosis

The differential diagnosis of a young, previously healthy patient presenting with haemolytic anaemia was broad. There was no recent drug exposure to suggest drug side effects or interactions. HIV testing was negative. Because he had fever for 2 weeks at the time of presentation, though he had no arthritic symptoms, adult Still’s disease was considered as a diagnosis. His recent summer camping trip in an endogenous area was concerning for babesiosis, though studies were repeatedly negative. Consideration was given to cold agglutinin disease secondary to EBV or mycoplasma as he had positive antibodies. He had no prior history of blood transfusion, ruling out transfusion reactions. An acute hepatitis panel and cytomegalovirus testing were negative. There was also consideration given to acute leukaemia, but bone marrow biopsy did not show malignancy or aberrant cell lines. Bone marrow biopsy showed hypercellular marrow with haemophagocytosis which, alongside positive EBV serology, fever, splenomegaly, hyperferritinaemia and elevated IL-2 receptor activity of 2406 pg/mL confirmed the diagnosis of HLH (Figure 1).

Figure 1.

H&E-stained bone marrow aspirate of the case patient, showing hypercellular marrow with haemophagocytosis.

Treatment

The patient was transfused two units of the most compatible packed red blood cells, and urgent bone marrow biopsy was completed. He was admitted under medical intensive care unit surveillance and treated with 1 mg/kg of prednisone daily, as well as atovaquone and azithromycin to treat empirically for potential babesiosis. Haemoglobin responded appropriately to transfusion and remained stable. Indirect hyperbilirubinaemia improved, and the patient symptomatically improved. Atovaquone and azithromycin were discontinued prior to discharge as his peripheral blood smear repeated three times was negative for parasites. The decision was made to not start etoposide or cyclosporine as per the HLH-2004 protocol due to significant improvement solely on corticosteroids.

Outcome and follow-up

The patient and his family were counselled on treatment planning risks versus benefits. For example, the risk of etoposide causing secondary malignancy was discussed as he is young enough that this outcome would become clinically evident in his lifetime.⁵ Our patient was young and otherwise healthy, and his progression was more typical of a paediatric patient. Given the provocation of HLH by EBV and improvement on steroids, the decision to initiate chemotherapy was deferred as the family desired a second opinion, and he improved to clinically stable for discharge with normalisation of haemoglobin and bilirubin and complete resolution of symptoms on prednisone, which was continued for two more weeks with taper.

Though HLH is a T cell driven disease, there are anecdotal reports that the addition of rituximab to HLH-directed therapy may reduce the EBV reservoir as the virus replicates in B cells, which in turn may decrease cytokine storm.¹¹ This potential treatment was initially considered but was deferred due to clinical improvement. A discussion on genetic testing was initiated with his primary care physician, with a plan to complete it after discharge. At the time of this writing, he has had no recurrent episodes or similar symptoms and was doing well.

Discussion

This case of a young man with HLH after EBV infection prompts consideration of the pathophysiology of HLH in the paediatric versus adult patient. Primary HLH, more commonly diagnosed in childhood, is generally elicited by an infection, prompting a consideration of genetic predisposition in clinical detection. HLH without genetic mutations has been termed secondary HLH and is attributed to infections, commonly EBV, or malignancy, autoimmune disorder, AIDS or other immunosuppressed states^{5 11}; the patient discussed in this case likely had secondary HLH. Regardless of classification, HLH is frequently fatal and is characterised by an overwhelming immunological response.⁴ EBV-induced HLH affects B cells at first and leads to an inappropriate release of cytokines that eventually cause dysregulation of cytotoxic T and NK cells.⁴ This immunological response was evident in our patient in the presence of warm and cold agglutinin as well as positive DAT without a prior history of transfusions, and lastly, C3d-positive antibodies, which led to a delay in RBC matching in preparation for transfusion.

The generalised inflammatory state of HLH leads to a non-specific presentation and also to diagnostic complexity. A set of eight diagnostic criteria was established by the HLH-2004 trial, requiring that patients fulfil at least five criteria to confirm the diagnosis.⁷ The criteria are comprised of: fever >38.5°C; splenomegaly; bilinear or trilinear cytopaenia; elevated ferritin (500 µg/L); hypofibrinogenaemia (150 mg/dL) or hypertriglyceridaemia (265 mg/dL); elevated sIL2R (400 U/mL); reduced or absent NK cell activity; and haemophagocytosis.⁴ Other systemic life-threatening illness can also present with elevation in several of these factors. For example, lymphadenopathy, splenomegaly and hyperferritinaemia are features of adult Still’s disease and can suggest a diagnosis of MAS.⁸ Many patients will not meet the five diagnostic criteria at initial presentation, as in our case. Our patient on initial evaluation met only three out of eight criteria, including fever, splenomegaly and elevated ferritin; hence, HLH can be misdiagnosed or go undetected. The high mortality rate warrants prompt treatment, even if five diagnostic criteria are not yet fulfilled.¹⁰ For this reason, it is critical to have an on-going consideration of the entire clinical entity rather than any one factor as pathognomonic for HLH. Elevated serum ferritin over 10 000 is regarded as the best indicator to differentiate HLH from other illnesses, with sensitivity of 90% and specificity of 96%.⁴ However, elevated ferritin levels are non-specific and can be elevated as an acute phase reactant.¹⁰ Furthermore, the presence of haemophagocytosis on bone marrow biopsy alone does not confirm the diagnosis, as a quantitative threshold has not been set for this criteria.¹² Thus, confirmation of diagnosis requires the coexistence of five criteria rather than one non-specific criterion alone. Lastly, there are preliminary diagnostic guidelines for MAS, to be applied to patients with a known diagnosis of adult Still’s disease, which was not applicable to the patient described in this case.¹

Typical treatment for HLH as outlined in the HLH-2004 protocol includes early steroids and etoposide, with haematopoietic stem cell transplant for genetic or refractory cases.¹³ Soluble IL-2 receptor levels, complete blood count, liver function panels and serial ferritin levels would be used to monitor treatment response and suggest chemotherapy initiation in refractory HLH or progression.^{10 14} Our patient was diagnosed early, improved significantly with steroids and did not require chemotherapy.

Learning points.

• Haemophagocytosis lymphohistiocytosis (HLH) is a rare disease that can present in paediatric or adult patients, as a result of genetic mutations or an overwhelming immune response to an inflammatory state. In secondary HLH, such as in this case, patients infrequently may improve with corticosteroids alone to offset the proinflammatory state caused by the infectious trigger.

• The diagnosis of HLH is based on meeting clinical criteria, which are often non-specifically present in hospitalised patients, such as those with sepsis.

• HLH should be considered in patients who fail to improve with treatment for suspected infection and meet five of eight diagnostic criteria.

• The disease course of HLH varies between primary and secondary causes; treatment should be based on consideration of the inciting event and include shared decision making with the patient.

• Severe anaemia may occur in HLH and result in high rates of morbidity and mortality; this patient had delayed time to blood transfusion because of difficulties in obtaining matched blood, supported by his positive direct antiglobulin test result.