A 20-years-old man presented with a new rash to his primary care provider in June 2020. He had been previously diagnosed with SCID-X1 due to hypomorphic interleukin-2 receptor subunit gamma-chain (IL2RG) mutation in 2017, approximately 3 years prior to the onset of his rash. His immunologic evaluation was undertaken due to a history of severe eczema and frequent infections as well as a family history of immunodeficiency. Immunologic work-up was initially notable for hypogammaglobulinemia and he was placed on standard monthly intravenous immunoglobulin (IVIG) infusions. Genetic testing then returned positive for the c.460C>T (p.P154S) mutation in the IL2RG gene. At the time of presentation, the patient reported a six-month history of hyperpigmented, violaceous macules and plaques involving the extremities and torso but sparing the palms, soles, and mucous membranes ( Figure 1 ). His skin lesions began on both feet and spread to the legs, torso, arms, and face over the course of a few months. At the time of initial evaluation, Tinea corporis was confirmed by potassium hydroxide preparation of a skin scraping, and flesh-colored papules with central umbilication clinically resembling those of molluscum contagiosum were also noted. Review of systems was negative for fevers, chills, arthralgias, respiratory, gastrointestinal, or genitourinary symptoms. Laboratory tests obtained eight months after appearance of the skin lesions were notable for a normal serum immunoglobulin G of 798 mg/dL and low CD4+ and CD8+ T-cell counts at 199 and <45 cells/microL, respectively. Very unusual for a SCID-X1 patient, he had above upper normal range numbers of CD3/CD16+56+ natural killer cells (1909 cells/microL, normal range 126-729 cells/microL). Prior to SCID-X1 diagnosis, he received routine childhood immunizations, including two doses of MMR vaccine at 1 and 4 years of age and a varicella vaccine. The patient reported two previous episodes of herpes zoster, persistent molluscum contagiosum, bacterial skin and soft-tissue infections, Tinea corporis, onychomycosis, and several episodes of viral and bacterial pneumonia. Prior to the onset of the skin lesions, he had no known exposure to tuberculosis or animal exposures. At the time of presentation, he was enrolled as a university student and lived with two roommates. During university breaks, he lived with his parents and a younger sibling, hereinafter referred to as close contacts.
The patient underwent biopsy of a violaceous macular lesion on his left flank. Dermatopathology revealed superficial and deep dermal infiltrates, predominantly histocytes, aggregating into poorly formed granulomas centered around vessels and adnexal structures. The Gram, Periodic Acid-Schiff, fluorescent rhodamine acid-fast, Fite, and Grocott methenamine silver stains were negative, as were fungal and mycobacterial cultures. Double fluorescent immunohistochemical (IHC) staining of formalin-fixed paraffin-embedded (FFPE) tissue sections of the skin biopsy with mouse monoclonal antibody against RuV capsid and rabbit polyclonal antibody against CD206 revealed the presence of RuV in M2 macrophages in the cores of granulomas located in the upper ( Figures 2A, C ) and deep dermis ( Figures 2B, D ). No staining for RuV capsid was detected in a few myeloperoxidase (MPO)-positive neutrophils near the granulomas (not shown). IHC staining for measles virus and varicella zoster virus (VZV) antigens were negative (not shown).
Following consultation with an infectious disease specialist, an NP swab and fresh-frozen skin biopsy sample were collected for the detection of RuV RNA by RT-qPCR in December 2020. RuV RNA was not detected in the skin biopsy sample. The failure to detect RuV RNA by RT-PCR in the fresh-frozen skin biopsy despite detecting RuV capsid by IHC in FFPE tissue sections is most likely due to the absence of RuV-positive cells in the frozen biopsy, as RuV is known to be unevenly distributed in lesions. However, RuV RNA was detected in the NP swab, and replication-competent RuV was isolated in Vero cell culture from the NP swab ( Figure 3 ). Because the patient had no known recent exposures to persons with suspected or confirmed rubella, there was concern that the RuV had been acquired earlier, potentially through MMR vaccination. The complete genomic sequence of the isolated virus was determined using the Sanger method and designated RVi/Oregon.USA/49.20 (GenBank accession number ON861827). Phylogenetic analysis showed that the isolated virus was vaccine-derived ( Figure S1 ) with 97.3% nucleotide identity to the genomic sequence of the RA27/3 strain used in an MMR vaccine. A 36-nucleotide deletion in RVi/Oregon.USA/49.20 resulted in the N-terminal deletion of 12 amino acids of the E2 glycoprotein. Despite this deletion, the virus replicated efficiently in Vero cells, producing titers around 106 focus forming units (ffu) per mL of the culture medium, which is 1-2 logs higher than those produced by other previously described VDRV isolates. Following these results, it was recommended that the patient adhere to droplet precautions until further information regarding potential VDRV shedding could be ascertained.
 Treatment. No specific treatment is currently indicated for rubella virus infection. The progression of the rubella virus infection and waning immunity, as indicated by a decline in CD4+ and CD8+ T-cell numbers in the absence of available anti-viral therapy for rubella, provided the rationale for the patient to undergo ex vivo lentiviral gene therapy at the National Institutes of Health (NCT01306019) with busulfan (6 mg/kg total) myeloid conditioning in April 2021. In September 2021, approximately 5 months after gene therapy, the patient was admitted for facial rash and meningitis. A swab collected from a facial skin lesion was positive for VZV by PCR. Cerebrospinal fluid (CSF) was positive for VZV as well as for VDRV by RT-qPCR. VDRV viral load was 4.3x102 RNA copies/mL CSF; live VDRV was not recovered from CSF ( Figure 3 ). He received IV acyclovir 10 mg/kg every 8 hours for 10 days and IV hydrocortisone 50 mg daily for 3 days for VZV treatment during his hospital admission. He was discharged home on oral valacyclovir 1 g every 8 hours for 11 days to complete a total 21-day course of antiviral therapy, then transitioned to acyclovir prophylaxis.
At time of report, patient was 9 months post gene therapy and pancytopenic with absolute neutrophil counts in the range of approximately 500-1000 cells/microL. Reconstitution of T-cells in older X-SCID patients takes years and at this early time point, his T-cell counts remained low with a CD4 count of 65 cells/microL (21%) and CD8 count of <45 cells/microL (4.1%). However, his violaceous skin lesions had mostly resolved by 9 months following gene therapy.
A bone marrow biopsy performed in July 2022 to evaluate ongoing pancytopenia (WBC 1.34 K/microL, hemoglobin 9.6 g/dL, and platelet count 20 K/microL) was notable for the presence of multiple foci of granulomatous inflammation. Grocott methenamine silver and acid-fast bacilli stains did not detect fungal and mycobacterial organisms in the bone marrow biopsy. There was no evidence of Epstein-Barr virus on in-situ hybridization. Qualitative PCR for cytomegalovirus, Epstein-Barr virus, human herpesvirus 6, and adenovirus was negative. To evaluate the possibility of VRDV as a possible etiology of the patient's pancytopenia, the sequential tissue sections of bone marrow core biopsy were double immunostained with RuV capsid antibody and either CD206, MPO, or CD3 antibody ( Figure 4 ). The bone marrow was strongly positive for rubella capsid with MPO+ neutrophils being the main infected cells and almost all neutrophils were rubella-positive. There was heavy infiltration of CD206+ macrophages, many of them forming granulomas. Numerous CD3+ T cells were predominately localized inside the granulomas. The areas occupied by granulomas were free from rubella-positive neutrophils, but some macrophages inside the granulomas contained rubella antigen. In addition, macrophages with rubella-positive neutrophils or globules of rubella antigen inside the cells could be seen throughout the sample outside of granulomas. The lack of IHC staining of neutrophils and macrophages with VZV monoclonal antibody cocktail confirmed the specificity of rubella antibody staining of these cells (not shown).
To monitor virus shedding by this patient, 11 sequential respiratory samples (eight NP swabs, one nasal swab, two oropharyngeal swabs) and two urine samples were collected over 15 months beginning in December 2020. The samples were analyzed by quantitative RT-qPCR and viral cell culture. VDRV RNA was detected in all eight NP swabs; replication-competent viruses were recovered from six of these ( Figure 3 ). The amount of virus in the NP swabs varied between 4.3x101 to 4.3x102 RNA copies/mL of swab media with two spikes of RuV RNA concentration. One spike of 1.4x104 RNA copies/mL was observed three months after gene therapy in July 2021, and the second spike of 9.9x103 RNA copies/mL occurred in March 2022, four months after treatment with steroids. IVIG had been administered monthly during this period. Two oropharyngeal swabs, a nasal swab, and urine collected in July 2021, contained replication-competent VDRV as well. Only one urine sample and two NP swab samples were culture-negative, and each was collected at a different time point. Viral RNA loads in the culture-negative samples were compatible to those in the culture-positive samples.
Though VDRVs have been detected in some individuals with inborn errors of immunity by culture in skin lesions and by RT-PCR in NP swabs, and RuV vaccine has been hypothesized to be an antigenic trigger for development of cutaneous granulomas, transmissibility of VDRVs remains unknown. To assess potential transmission of VDRV from the patient to his close contacts, NP swabs and serum samples were collected from three close contacts in the family, including an immunocompetent adolescent sibling. NP swabs were tested by rubella RT-qPCR. Serologic investigation included measuring immunoglobulin M (IgM) and IgG to rubella virus, and 50% neutralization titer (NT50) against the RA27/3 rubella virus strain as previously described. The patient's rubella serologic status was also assessed. The NP swabs of these close contacts were negative by RT-qPCR. None of the serum samples contained rubella IgM. IgG titers of the close contacts ranged from 25-60 IU/mL, and the NT50 was 80 ( Supplementary Table 1 ); both results were similar to those of typical immunocompetent vaccinees. In contrast, the serum neutralization titer of the patient was substantially higher (NT50 = 640) than that of his close contacts or other immunocompetent vaccinees. Laboratory testing did not provide evidence of VDRV transmission to close contacts, including a younger sibling who could be potentially exposed prior to receipt of MMR vaccination, suggesting that VDRV is unlikely to have been transmitted to vaccinated contacts with competent immune systems. In light of these results, the infectious disease physician and public health officials advised the patient that the droplet precautions suggested earlier for infection control could be discontinued.