A 95 year old female resident of a nursing home developed acute respiratory symptoms, at the peak of the COVID-19 “first wave” in Italy, in March 2020. Upon hospitalization, infection by SARS-COV2 was confirmed by nasopharyngeal swab, followed by PCR detection of the Corona virus. Chest computed tomography revealed diffuse bilateral infiltrates. At admission, blood counts were in the normal range (including eosinophils, 70/ml), with elevated erythrocyte sedimentation rate (87 mm/h) and C-reactive protein (18.7 mg/l) as the only abnormal laboratory values. Underlying comorbidities included type 2 diabetes and hypertension (both moderate and well controlled). Upon hospitalization, she was treated (empirically) with hydroxychloroquine (400 mg/die) and enoxaparin (4,000 UI/die). Her conditions remained fair for two weeks (with no need for oxygen therapy), when she presented with a maculo-papular skin eruption with a pupuric aspect and scaling, involving more than 50% of the body surface (abdomen, back, upper and lower limbs), accompanied by severe pruritus and burning sensation. Fever (> 38.5 °C), lymph node swelling at multiple peripheral stations and elevated serum creatinine values completed the clinical picture. Withe blood cell count reached 15,160/ml, with eosinophil count at 1,540/ml. A DRESS diagnosis was established (RegiSCAR score = 7).6 Thus, hydroxychloroquine, associated with severe drug hypersensitivity, including DRESS,7-10 was discontinued. Enoxaparin dosage was doubled (8,000 IU/die), in consideration of increased D-dimer values (2.173 mg/ml). Prednisone (25 mg/die) and cetirizine (10 mg/die) were added to the therapy. However, a further deterioration of the clinical conditions occurred, leading to replacement of enoxaparin with the fully synthetic pentasaccharyde factor Xa inhibitor fondaparinux (2.5 mg/die, subcutaneously), generally well-tolerated in patients with local non-immediate cutaneous reactions to LMWHs, including enoxaparin.11,12
Management of the case according to this therapeutic schedule led to a slow resolution of maculo-papular lesions, over approximately one month, with a substantial fever decline achieved in 2 weeks and apyrexia in 3 weeks. Finally, the eosinophil counts also declined steadily and normalized by day 30 after the emergence of the DRESS eruption. Prednisone and fondaparinux were then discontinued.
A few days later, the patient was discharged, upon double successive negative nasopharyngeal swab.
Precision diagnosis. Following a 7-day corticosteroid wash-up period, a blood sample was obtained from the patient (fully recovered; at home). Peripheral blood mononuclear cells were isolated by gradient centrifugation (800 x g, 45’) on Lympholyte® (Cedarlane, EuroClone, Milan, Italy), upon plasma removal and suspension of the blood cellular moiety in Dulbecco’s phoshate buffered saline. Mononuclear cells were maintained in Dulbecco’s modified Eagle’s medium, with 10% autologous serum (v/v), with streptomycin (100 mg/ml), at 37 °C, in a 5% CO2, vapour-saturated atmosphere, in 64 cm2 glass Petri dishes, for 4 days, in order to allow clearance of the monocyte-macrophage component. Successively, micro-cultures were generated with the resulting purified lymphocytes (6 x 104 cells in 200 ml) and employed for carrying out LPT for enoxaparin and fondaparinux, respectively, under culturing conditions as above. LPT was performed essentially as described.13 Briefly, triplicate micro-cultures were incubated with the two drugs, respectively, at three different ten-fold concentrations: therapeutic concentration (TC; calculated on the drug distribution volume), TC/10 and TCx10 (defect and excess concentration, respectively). The distribution volume and the TC were 5.24 l and 15 mg/l, for enoxaparin,14 and 9 l and 0.27 mg/l for fondaparinux.15 Triplicate micro-cultures incubated with phytohemagglutinin-M (from Phaseolus vulgaris; 2.25 mg/ml) and the medium alone served as the positive and the negative control, respectively. Following 4-day incubation with the drugs, lymphocyte proliferation was assessed upon inclusion of the non-radioactive thymidine analogue 5-bromo-2’-deoxyuridine (BrdU; 100 mM), in the micro-cultures, for 2 hours. Incorporation of the nucleotide in proliferating cells was evaluated by an anti-BrdU monoclonal antibody (7.5 U/ml; from Roche Diagnostics GmbH, Mannheim, Germany).16,17 The LPT is deemed positive when the proliferation rate of any of the three concentrations tested compared to negative control (stimulation index) equals or exceeds 2.5,13
The assay revealed that not only enoxaparin (as suspected) but also fondaparinux induced significant lymphocyte proliferation (Figure).