Discussion
SARS-CoV-2 is associated with a wide range of symptoms ranging from a mild clinical phenotype with fever and cough to severe respiratory and/or multi-organ failure. SARS-CoV-2 has considerable morbidity and mortality, particularly among people with advanced age and co-morbities37. A significant factor contributing to the morbidity and mortality of this infection is the pulmonary and systemic inflammatory response38. Multiple SARS-CoV-2 proteins and viral RNAs trigger inflammation. Endosomal and cytoplasmic viral RNA binds TLR and NOD pathway receptors38; ORF3a, ORF3b, ORF7a, ORF8a, ORF9b and E envelope proteins are pro-apoptotic, release NF-κB or activate the NLRP3 inflammasome39,42. Subsequently, inflammasome caspases cleave interferon signal pathway components cGAS, MAVS, and IRF3 blocking antiviral interferon responses at the same time as the marked inflammatory reaction43.
We were struck by the minimal clinical findings in this high-risk, elderly woman with a co-existing hematopoietic malignancy. Our patient was on chronic ruxolitinib therapy for myelofibrosis. Ruxolitinib inhibits JAKs and TYK2 and thus downstream STATs and cytokine expression in T lymphocytes, neutrophils, and dendritic cells44. We speculate whether our patient’s minimal clinical symptoms throughout her infection could be linked to the immunosuppressive effect of the drug. Ruxolitinib may reduce the SARS-CoV-2 inflammatory state, improve the quality of life, and perhaps prolong survival from this devastating disease. This speculation is supported by several pilot studies. A trial by Giudice et al. demonstrated a significant improvement in respiratory symptoms and radiographic pulmonary lesions in seven SARS-CoV-2 patients with acute respiratory distress syndrome treated with a combination of ruxolitinib and eculizumab, an anti-C5a complement monoclonal antibody45. A retrospective study by La Rosee et al. showed ≥ 25% reduction in COVID-19 Inflammation Scores (CIS) after seven days of treatment with ruxolitinib in a subset of 14 patients with CIS ≥ 10 46. The CIS score measured chest x-ray abnormalities, levels of CRP, ferritin, triglycerides, IL6, fibrinogen, blood white cell count, blood lymphocyte count, d-dimer, PTT and presence or absence of fever. Moreover, Cao et al conducted a multicenter, randomized control trial evaluating the efficacy of ruxolitinib in 43 patients with severe SARS-CoV-2 infection. Ruxolitinib recipients showed a significant improvement in chest computed tomography and faster recovery from lymphopenia compared to the control group47. This trial also revealed that ruxolitinib was well tolerated with infrequent toxicities30. Theoretically, higher rates of aberrant JAK 2 activating mutations in older myeloproliferative neoplasm patients could enhance the hyperinflammatory state induced by SARS-CoV-233. Nevertheless, treatment with ruxolitinib should proceed cautiously as ruxolitinib and SARS-CoV-2 have both been associated with coagulopathy and increased frequency of thromboembolic events48.
An interesting facet of this case is the sustained positivity of the patient’s SARS-CoV-2 test. She was repeatedly tested for viral RNA clearance by nasal swab RT-PCR secondary to her immunocompromised state and because she required a negative test prior to discharge to her nursing facility. Many SARS-CoV-2 infected individuals have persistently positive RT-PCR tests for weeks to months after clinical recovery49. Based on viral culture, the percent of these individuals who remain infectious approaches zero by 10 to 15 days after the onset of symptoms49-51. However, shedding of infectious SARS-CoV-2 has been demonstrated by viral culture or inferred by the presence of subgenomic RNA in a subset of individuals, including immunosuppressed hosts, for months following infection52,53. Higher Cq values of SARS-CoV-2 RT-PCR reflects lower viral loads and multiple studies have demonstrated inability to culture infectious virus above certain Cq thresholds50. As demonstrated in Table 1, the Cq of the ten subsequent RT-PCR samples by nasopharyngeal swab ranged from 35.7 to 42.5 with a mean of 38.2. Based on the referenced literature, these values likely represent the presence of low quantities of viral RNA (vRNA) or vRNA fragments that are non-infectious, although the Cq thresholds are not directly comparable across assays. We were unable to culture infectious virus from our patient at day 98. However, it should be noted that respiratory viral culture is insensitive, and lack of viral growth in vitro does not ensure lack of infectiousness.
The persistent positivity of her SARS-CoV-2 testing may be potentially secondary to the immunosuppressive effective of the ruxolitinib34. Ruxolitinib targets components of both the innate and adaptive immune system. JAK/TYR2 proteins are downstream for both innate immune cytokines and adaptive immune interferon receptors54. Therefore, suppression of the pathway places a person susceptible to various infections22. With these defense mechanisms impaired, the drug contributes to increased risk of reactivation of silent viral, bacterial and fungal infections55,56. This viral susceptibility is due to JAK/TYR2 inhibitors suppressing cytokines, such as interferon, and NK cells22. This case addresses the issue of hampered anti-viral defense caused by ruxolitinib through the supplementation of interferon with subsequent T cell activation to fight SARS-CoV-2 infection. Our patient was able to clear the vRNA approximately 30 days after the administration of a total of four treatments of pegylated interferon-α2a while continuing treatment with ruxolitinib.
Viruses such as SARS-CoV-2 have evolved to facilitate their own infectivity and to evade host detection and immune response. SARS-CoV-2 activation of pro-inflammatory pathways described above39-41 generates intracellular caspases that degrade interferon and interferon signaling polypeptides43. Previous data on SARS-CoV and MERS-CoV outbreaks have revealed additional mechanisms of coronavirus type I interferon suppression41,48,57,58. To date, data exists showing that 12 of the 29 SARS CoV-2 proteins block IFN production early: nsp1 inhibits 40S ribosome participation in IFN translation; nsp3 blocks RIG-1 PAMP signaling; nsp10 performs 2-O-methyltransferase cap on vRNA to hide the vRNA; nsp13 binds and inhibits TBK1 PAMP signaling; nsp14 performs N7methyltransferase caps on vRNA again to disguise the virus; nsp15 remove 5’pU tracts from vRNA to avoid vRNA detection; nsp16 assists in 2-O-methyltransferase cap formation on vRNA; ORF3b binds and blocks IRF3 signaling; ORF6 inhibits karyopherin so cytoplasmic to nuclear PAMP signaling is blocked; M protein binds and blocks TRAF/TBK1 signaling; orf9b binds and blocks MAVS PAMP signaling and N protein binds and blocks RIGI PAMP signaling. SARS-CoV-2 produces a delayed first line antiviral defense followed by excessive inflammatory cytokinemia and dysfunctional T and NK cell responses33,48.
Interferons have been successfully used in the treatment of viral infections, such as hepatitis C, autoimmune diseases such as multiple sclerosis, and hematologic malignancies such as essential thrombocythemia, polycythemia vera, and myelofibrosis59-61. In SARS-CoV-2, interferon therapy in phase 2 and phase 3 randomized clinical trials have shown reduced the duration of virus infection, reduced inflammatory markers including IL6 and CRP and reduced mortality when administered early41,62-68. As a note of caution, type I interferons administered in later stages may cause progressive tissue damage leading to a deleterious hyperinflammation characterized by the excessive macrophage activation and hypercoagulation seen in patients with acute disease38. Interestingly, pharmacologic interferon treatment inhibits inflammation early by repressing the NLRP3 inflammasome via STAT1 and STAT369. We hypothesized that administration of interferon in our patient who was minimally symptomatic would strengthen anti-viral defense and potentially lead to viral RNA clearance. Our results support the hypothesis.