Coronavirus Pandemic A case of Hemophagocytic lymphohistiocytosis induced by COVID-19, and review of all cases reported in the literature

Novel coronavirus infections 2019 (COVID-19) associated hyperinflammatory syndromes are well-defined clinical conditions and have a potential risk for severe infection. Hemophagocytic lymphohistiocytosis (HLH), a rare type of acute progressive hyperinflammatory syndrome, has been reported in a limited number of COVID-19 cases. In this article, we aimed to present a patient with HLH secondary to COVID-19 diagnosed by bone marrow biopsy, and to summarize and review HLH cases associated with COVID-19 in the literature. A 47-year-old male patient presented with complaints of fever, cough, abdominal discomfort, and nausea-vomiting. He had recovered from COVID-19 a month ago and was readmitted to the hospital due to the re-appearance of clinical symptoms after a two-week interval. The patient was diagnosed with HLH secondary to COVID-19 on sixth day of admission and fully recovered with systemic pulse steroid, intravenous immunoglobulin, and plasma exchange therapy. Analysis of literature searches revealed that 22 cases were definitely diagnosed with COVID19-associated HLH, 16 of them were male. They had been treated with different anti-cytokine drugs, of which nine had died. The increasing number of HLH cases, which have high mortality rates, shows the importance of hyperinflammatory syndromes in COVID-19 patients. Some patients may experience hemophagocytosis in the late period of COVID-19, even while in recovery. Increased awareness and early treatment for HLH triggered by COVID-19 can be a life-saving effort for reducing mortality in severe COVID-19 cases.


Introduction
Hemophagocytic lymphohistiocytosis (HLH), synonymously hemophagocytic syndrome, is a fatal hyperinflammatory syndrome caused by an uncontrollable proliferation of activated lymphocytes and macrophages and thus increased secretion of inflammatory cytokines [1]. It has two forms, primary and secondary HLH. Primary HLH occurs generally due to genetic disorders inherited with autosomal recessively [2]. Secondary HLH occurs reactively to underlying disease including infections, malignity, and autoimmune/autoinflammatory diseases [3]. Viral infections, especially Epstein-Barr virus (EBV) and cytomegalovirus (CMV), and other herpes viruses are well-known triggering factors of HLH. Novel coronavirus disease (COVID-19) has a potential for HLH as it has a wide clinical spectrum from mild disease to severe illness accompanying cytokine storm [3,4]. From the beginning of pandemic, similarities between severe COVID-19 and cytokine storm syndrome have drawn attention [5]. However, HLH, a marginal and potentially fatal form of cytokine storm syndrome, was detected in limited number of COVID-19 patients [6]. Here, we present a case of HLH secondary to COVID-19, in which we demonstrated hemphagocytosis in the bone marrow. In addition, we reviewed and summarized the clinical characteristics and outcomes of all the definitely diagnosed HLH cases induced by COVID-19.

Case Report
A 47-year-old male patient with no known disease before was admitted to the infectious disease's clinic with complaints of fever, cough, abdominal discomfort, nausea-vomiting, myalgia, and loss of appetite and weight (7 kg). He had been diagnosed with COVID-19 a month before, and his fever and other symptoms had resolved within 10 days with treatment. After a two-week symptom-free period, his complaints had reappeared about a week before and became more severe. The patient was clinically stable at the time of admission, and his blood tests (complete blood count, liver and renal function tests, coagulation tests and inflammation markers) were almost within normal limits except for hyperbilirubinemia (1.5 mg/dL), however, within three days, an unanticipated serious deterioration in the clinical condition and laboratory parameters was observed (Table 1). His fever continued to increase and reached 42 °C, and redness and painful swelling on the neck, and jaundice in sclera and skin appeared. On the fifth day of admission, neurological symptoms including somnolence, disorientation and meaningless speech appeared. In physical examination, cervical lymphadenopathy, liver and spleen enlargement and flapping tremor were observed. Multiple lymphadenopathies in cervical, axillar and femoral sites were detected in superficial ultrasonography. Liver and spleen were at the upper limit in abdominal ultrasonography performed on the first day of hospitalization. Three days later, massive hepatomegaly (22 cm) and splenomegaly (18 cm  negative. Although the patient's respiratory symptoms were improving, his general condition progressively deteriorated. Neurological symptoms and the complaints of fatigue, loss of appetite and weakness increased. Infection or another reason could not be found to explain his worsening. Elevated C-reactive protein (CRP), ferritin, and triglyceride level, the presence of cytopenia and hepatosplenomegaly led to the suspicion of COVID-19-induced HLH. Laboratory values of the patient are shown in detail in Table 1. On the sixth day of his admission, bone marrow aspiration/biopsy was performed, and it was observed that the bone marrow was infiltrated by activated macrophages in accordance with HLH ( Figure 1). The patient's H-Score was calculated at 209, predicting the probability of HLH at 93-96% and calculated HLH-2004 criteria met HLH [7,8]. Valid diagnostic HLH-2004 and H-Score criteria, patient parameters within these criteria, points assigned to the patient based on the parameters within the criteria, and patient's total points for HLH-2004 and H-Score are shown in Table 2. We did not detect any underlying disease triggering HLH except for COVID-19 in detailed analysis. The final diagnosis was accepted as COVID-19-induced HLH for the patient who experienced COVID-19 infection one month ago and had a positive COVID-19 antibody (IgM and IgG). The patient was given pulse steroid therapy (methylprednisolone 2 g/day for 3 days) and intravenous immunoglobulin (IVIG) (2 mg/kg dose, divided into 5 days) in accordance with the HLH-2004 protocol. After pulse steroid therapy, maintenance steroid treatment at a dose of methylprednisolone 1 mg/kg/day was given and ended within 6 weeks. Then plasma exchange treatment was started on the patient on the eleventh day of treatment and applied during the following 4 days. After this treatment, the clinical a: H-Score: Defined as hemoglobin 92 g/L or less, platelets 110 × 10 9 /L or less, and leukocytes 5 × 10 9 /L or less 1.0 × 10 9 /L; b: HLH-2004: Defined as hemoglobin less than 90 g/L, platelets less than 100 × 10 9 /L, and neutrophils less than 1.0 × 10 9 /L; c: An H-Score of ≥ 169 has a 93% sensitivity and 86% specificity for HLH.
condition and laboratory values of the patient significantly improved, and the patient was discharged with full recovery at the end of three weeks. The clinical course and consecutive laboratory tests of the patient are shown in Figure 2 and Figure 3.

Review of other COVID-19 associated HLH cases
We conducted a literature review and investigated precisely diagnosed adult HLH cases. Child cases (16 years old and younger), and suspected or unproven HLH cases were not included in the study. We found 21 more adult cases diagnosed with COVID-19-associated HLH in addition to ours [9][10][11][12][13][14][15][16][17][18][19]. Eighteen (86%) of the patients were over the age of 50 (range 17-84), 16 (76%) were male, and most of them had at least one underlying disease. All patients whose medical records were clearly described in the article had a history of fever. The patients were mostly treated with interleukine-1 receptor antagonist (anakinra), interleukin-6 receptor antibody (tocilizumab) and steroid treatment. Nine of them (43%) had died, and 2 were still on mechanical ventilation when the article was written. The characteristics, treatments and outcomes of the patients are shown in Table 3.

Ethical Approval
All procedures performed in studies involving human participants were in accordance with the 1964 Helsinki declaration and its later amendments.

Discussion
Secondary HLH is caused by a group of different reasons such as infections, malignancy, collagen tissue diseases, tissue damage, autoimmune diseases, drugs, metabolites and immunosuppression [20]. Viral infections, especially EBV, CMV, herpes simplex viruses, and other infections such as tuberculosis, malaria, and leishmaniosis can trigger hemophagocytosis [3]. COVID-19 cause a wide range of clinical spectrum from mild disease to severe infection resulting in cytokine storm. An increasing amount of clinical data indicate that cytokine storm plays an important role in the progression to severe infection resulting in acute respiratory distress syndrome (ARDS), and multi-organ failures including heart, kidney, and liver, and eventually death [21][22][23].
The clinical appearance of SARS-CoV-2 induced cytokine release syndrome has significant similarities with HLH in terms of laboratory and clinical findings. The findings of fever, cytopenia, ARDS, neurological involvement, and renal failure found in patients with severe COVID-19 are the findings that at the same time overlap with HLH [3]. Therefore, HLH should be kept in mind in patients with rapid clinical and laboratory progression to multisystemic severe disease.    Hyperinflammatory syndromes and its impact on mortality have gained a foothold in the literature about COVID- 19 pathogenesis. Various types of proinflammatory cytokines and chemokines are released at different levels and cause a heterogeneous clinical severity, from mild disease to severe disease reminiscent of secondary HLH. Cytokine levels are higher in severe COVID-19 patients than non-severe patients [23][24][25]. A recent study reported a cytokine profile associated with increased disease severity in COVID-19 patients that is similar to HLH cases [26]. Laboratory abnormalities (eg cytopenia, elevated CRP, interleukin-6 (IL-6), ferritin, and D-dimer) are more commonly detected in severe cases of COVID-19 [26,27]. Patients with rapid and progressive laboratory deterioration may be suffering from excessive cytokine production. Therefore, HLH criteria should be investigated in COVID-19 patients who are acutely ill or have a deteriorating clinic and deepening laboratory abnormalities. A recent review by Opoka-Winiarska et al. recommends investigating HLH criteria in all COVID-19 patients with worsening of general condition and multi-organ failure [5]. Timely diagnosis and starting appropriate treatment for HLH play an important role in prognosis.
Male gender was dominant in HLH cases associated with COVID-19 in published articles. Similar to COVID-19 cases, a slight predominance of male gender was also reported in non-COVID-19 associated HLH cases [28]. In fact, male dominance in HLH cases sounds reasonable, because male gender was reported as a poor prognostic factor for severe COVID-19 infection in multiple cohorts worldwide [29][30][31]. Most of the COVID-19 associated HLH patients were older than 50 years old. This was also compatible with the results of COVID-19 cohorts in which advanced age was reported as a risk factor for mortality [25][26][27].
Primary HLH seen in childhood, also called familial HLH, has a genetic inheritance and is caused by various mutations in the gene encoding perforin (PRF). There is a dysregulation in the immune system due to the perforin-mediated cell lysis pathway that results in over-activation of cytotoxic CD8+ T cells, natural killer cells, and macrophages of activated cells. The primary HLH emerges typically during early infancy and causes death within two months if left untreated. The diagnosis is established with a molecular test consistent with HLH [3,7]. There is no confirmatory test for the diagnosis of secondary HLH, and clinical and laboratory tests are mainly used [3]. Most patients with HLH experience acute multiorgan insufficiency including cytopenia, liver test abnormalities, coagulopathy and neurological symptoms which cannot be explained with any other reasons. HLH-2004 diagnostic guideline developed by Histiocyte Society and H-Score are criteria that are widely used to help diagnosis in children [7,8]. Diagnostic HLH-2004 contains eight criteria and the presence of five of the eight criteria is required to meet the HLH criteria. Nine variables (3 clinical, 5 laboratory and a cytological) are used for H-Score and the possibility are changed according to the total score. In fact, the detection of hemophagocytosis in bone marrow aspiration is neither necessary nor sufficient for diagnosis, it is only one of the criteria included in diagnostic scores. Hemaphagocytosis may not be detected in the early period in bone marrow due to a late period's finding of HLH [32,33]. Hemophagocytosis had reportedly been detected in bone marrow samples of all patients who were included in this study and who underwent bone marrow aspiration. Therefore, bone marrow aspiration / biopsy should be performed in patients with suspected HLH, especially in patients who are at the margin of diagnosis. Although hemophagocytosis cannot be detected in bone marrow aspiration, patients with suspected HLH should receive HLH-specific treatment without wasting time, preferably on the day of diagnosis. The effectiveness of the treatment depends on its timing; its early initiation improves the response and prognosis [5,7]. The initial therapy aims to suppress hyperinflammation by immunosuppressive treatment. HLH-2004 guideline recommends starting initial treatment immediately after ruling out lymphoma. HLH is a severe kind of hyperinflammatory syndrome, and its main treatment is obtained by the suppression of cytokine. Corticosteroid is the first preferable drug in treatment of infection associated HLH and often used in combination with IVIG [5,7,34]. It is recommended that plasma exchange therapy may be performed to improve coagulation test and eliminate cytokines [34]. We started corticosteroid and IVIG combination treatment on the day of diagnosis, and applied plasma exchange after five days of combination treatment. The patient responded very well to the treatment with early diagnosis and appropriate treatment. In COVID-19 associated HLH cases included in this study, anakinra and tocilizumab had been widely used. Anakinra is used with other immunosuppressive drugs in the treatment of hemophagocytic syndrome and associated with an improved response, and tocilizumab is reported to be effectively used in the treatment of cytokine release syndrome, especially following chimeric antigen receptor (CAR)-T cell therapy. However, there is insufficient evidence to recommend for or against the use of both cytokine-inhibiting agents, anakinra and tocilizumab, in cytokine storm during COVID-19 infection. [3,25,[35][36][37].

Conclusions
Hyperinflammatory syndromes are well-defined COVID-19-associated clinical conditions and may exhibit a wide range of diversity. HLH is a rarely seen, acute progressive hyperinflammatory syndrome and may accompany COVID-19. It may be overlooked due to a rapid clinical progression resulting in multiorgan failure. Early diagnosis of HLH and starting of appropriate treatment can be life-saving in patients with deteriorating general conditions. There is no specific confirmatory test for the diagnosis of adult HLH cases. HLH-2004 and H-Score guidelines can be used to help the diagnosis of HLH. Hemophagocytosis in bone marrow may not be detected at an early stage and is not required for precise diagnosis. Specific treatment should be initiated in patients with high clinical suspicion without wasting time.