LettersAugust 2023

Tecovirimat Resistance in an Immunocompromised Patient With Mpox and Prolonged Viral Shedding

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    Background: Tecovirimat, an inhibitor of the viral VP37 envelope-wrapping protein used to treat severe monkeypox virus (MPXV) infection, has a low barrier to resistance (1, 2). A tecovirimat-resistant MPXV variant was recently identified at the autopsy of an immunocompromised patient with mpox after prolonged tecovirimat treatment (3).

    Objective: To describe the rapid selection of a tecovirimat-resistant MPXV variant during treatment of a severely immunocompromised patient with prolonged MPXV infection.

    Case Report: A 53-year-old man who had been vaccinated for smallpox in childhood presented in November 2022 with weight loss; a longstanding large, painful anal ulcer; and proctitis, but without skin lesions. Work-up revealed HIV-1 infection with pronounced immunosuppression (plasma viral load, 523 000 copies/mL; CD4+ T-lymphocyte count, 0.02 × 109 cells/L), chronic hepatitis B virus infection, latent syphilis, Cryptococcus neoformans antigenemia, anal Chlamydia trachomatis infection, and cytomegalovirus infection.

    The initiation of antiretroviral therapy (ART) was deferred due to travel. Six weeks after the HIV diagnosis, the patient was admitted for worsening anal pain. Antiretroviral therapy (bictegravir, 50 mg; emtricitabine, 200 mg; and tenofovir alafenamide, 25 mg) and intravenous ganciclovir for suspected cytomegalovirus colitis were started. Perianal disease deteriorated, and necrotizing vesicles appeared on the neck and buttocks on days 15 and 20 of ART, respectively (in addition to cerebral toxoplasmosis). Mpox immune reconstitution inflammatory syndrome was suspected, and polymerase chain reaction testing of the different lesions, stored plasma samples, and anal biopsy specimens was performed. All tested positive for MPXV (Figure 1).

    Figure 1. Clinical and virologic evolution over time relative to the initiation of the 2 consecutive courses of tecovirimat.

    The 2 courses of tecovirimat are indicated as “1” and “2”. Sequencing results (method as described in reference 5, using an R10.4.1 flow cell [Oxford Nanopore Technologies]) are represented as pie charts above the graph: The external ring of each pie represents the proportions of wild-type (N267 [WT]; green) and resistant (N267D [R]; orange) MPXV variant populations in anal lesion samples. Numbers above each pie indicate the percentage of the N267D (R) allele frequency (orange) and the total (N267D [R] and N267 [WT]) allele depth (black) (only considering bases with a minimum quality score [Phred score] of Q30). The evolution of Ct values of anal lesions (black circles) and blood samples (red circles), HIV VL (triangles), and CD4+ cell count (squares) are presented on the graph. Crosses indicate positive MPXV culture samples. The timing of the appearance and resolution of the anal ulcer and skin lesions is indicated by the red bars underneath the graph. To convert CD4+ cell counts to ×109/L, multiply by 0.001. ART = antiretroviral therapy; Ct = PCR cycle threshold; LOD = limit of detection; MPXV = monkeypox virus; PCR = polymerase chain reaction; R = resistant; VL = viral load; WT = wild-type. Consensus sequence National Center for Biotechnology Information GenBank accession numbers: d-87 02 Nov_2022_anal: OQ672570; d-9 19_Jan_2023_anal: OQ672571; d-2 26_Jan_2023_anal: OQ672572; d-2 26_Jan_2023_neck: OQ672573; d5 02_Feb_2023_anal: OQ672574; d5 02_Feb_2023_gluteal: OQ672575; d11 08_Feb_2023_anal: OQ672576; d16 13_Feb_2023_anal: OQ672577; d25 23_Feb_2023_anal: OQ672578; d31 28_Feb_2023_anal: OQ672579.

    Oral tecovirimat (600 mg twice daily) was started 1 day after the confirmation of MPXV infection (ART day 39). During the initial 2-week course, all lesions improved but anal lesion resolution was incomplete and viral shedding persisted. Tecovirimat treatment was extended for 2 weeks, after which the clinical evolution continued to be favorable. Daily intake was reliably reported throughout treatment. After 25 days of tecovirimat treatment, MPXV DNA became undetectable in blood. In contrast, except for a temporary decrease on day 11 of treatment (cycle threshold [Ct], 28.39), the anorectal viral load remained high up to day 48 (Ct, 21.58) and detectable up to the end of follow-up (Ct, 33.94 at day 88).

    Retrospective MPXV sequencing of the anorectal samples revealed a dominant variant population (at 94.11% allele frequency) carrying an F13L gene mutation (encoding an N267D variant of VP37) as early as 11 days after initiation of tecovirimat treatment (Figure 1). This variant was already detectable as a minor population before tecovirimat initiation in anorectal (3.41% at day −87 and 0.64% at day −2), neck (0.23% at day −2), and gluteal (0.05% at day −11) lesion samples. In vitro, this N267D mutation was associated with a 350-fold increase in the half maximal effective concentration of tecovirimat compared with wild-type (WT) virus (2103 nM for N267D vs. 5.9 nM for WT MPXV) (Figure 2) and with reduced viral outgrowth in a direct competition assay.

    Figure 2. In vitro drug susceptibility testing.

    MPXV was isolated from anal swab samples on days −2 and 31 by spinoculation on vero cells, and virus stock was prepared by double passaging on vero cells (for 3 and 4 days, respectively). Viral stocks were confirmed for the presence of WT and N267D mutant virus by whole-genome sequencing (i.e., 100% WT/0% R and 36% WT/64% R in viral stocks derived from day −2 and 31 samples; indicative of a comparative outgrowth disadvantage for the N267D mutant virus present at 98.34% in the patient sample at day 31). Serial dilutions of tecovirimat were incubated with 10 plaque-forming units (0.0005 multiplicity of infection) of dominant WT (green circles) and dominant N267D mutant virus (orange circles) containing viral stocks, on confluent vero cells for 4 days in 96 well plates in a plaque reduction assay. EC50 values were calculated using a 4-variable logistic model in GraphPad Prism v.9. Cmin = reported minimal concentration of tecovirimat (after 600-mg oral dose; from reference 1); EC50 = half maximal effective concentration; MPXV = monkeypox virus; R = resistant; WT = wild-type.

    * The proportion of viral growth (expressed in amount of plaques) compared with the untreated condition.

    Discussion: Our report confirms the potential rapid selection of resistant mutant virus during tecovirimat monotherapy, and we believe this report is the first to study this phenomenon longitudinally. A variant (VP37 N267D) with substantiated tecovirimat resistance was selected within the standard 2-week treatment.

    Although MPXV infections are mostly self-limiting, prolonged disease has been described in immunocompromised patients (4). Our patient had protracted viral shedding of at least 87 days before initiation of tecovirimat treatment, when a minor fraction of the resistant variant was already detectable. Indeed, intrahost viral evolution enhanced by prolonged replication may influence the viral population’s diversity and subsequent ability to escape antiviral pressure. However, in the absence of pharmacokinetic and pharmacodynamic monitoring, we cannot rule out subtherapeutic tecovirimat levels due to factors such as insufficient high-fat food intake.

    Despite the emergence of tecovirimat resistance in our patient, the clinical evolution was favorable, possibly due to further immune reconstitution. Similar to other VP37 amino acid substitutions that confer resistance to tecovirimat in orthopox viruses (2), decreased viral fitness may play a role, as suggested by the in vitro outgrowth assay. Whether in vivo fitness is affected in the N267D variant described here remains to be determined.

    The rapid selection of resistance in our patient highlights the risks of tecovirimat monotherapy, especially in the context of prolonged disease and immunosuppression. In such cases, we advocate for surveillance for resistant variants, emphasis on immune reconstitution, monitoring of viral clearance, and strict adherence to infection prevention measures. Additional research is needed on strategies that increase the barrier to resistance, including the use of antiviral combination treatments.