Pathology and Monkeypox virus Localization in Tissues From Immunocompromised Patients With Severe or Fatal Mpox

Abstract

Background

Pathology and Monkeypox virus (MPXV) tissue tropism in severe and fatal human mpox is not thoroughly described but can help elucidate the disease pathogenesis and the role of coinfections in immunocompromised patients.

Methods

We analyzed biopsy and autopsy tissues from 22 patients with severe or fatal outcomes to characterize pathology and viral antigen and DNA distribution in tissues by immunohistochemistry and in situ hybridization. Tissue-based testing for coinfections was also performed.

Results

Mucocutaneous lesions showed necrotizing and proliferative epithelial changes. Deceased patients with autopsy tissues evaluated had digestive tract lesions, and half had systemic tissue necrosis with thrombotic vasculopathy in lymphoid tissues, lung, or other solid organs. Half also had bronchopneumonia, and one-third had acute lung injury. All cases had MPXV antigen and DNA detected in tissues. Coinfections were identified in 5 of 16 (31%) biopsy and 4 of 6 (67%) autopsy cases.

Conclusions

Severe mpox in immunocompromised patients is characterized by extensive viral infection of tissues and viremic dissemination that can progress despite available therapeutics. Digestive tract and lung involvement are common and associated with prominent histopathological and clinical manifestations. Coinfections may complicate mpox diagnosis and treatment. Significant viral DNA (likely correlating to infectious virus) in tissues necessitates enhanced biosafety measures in healthcare and autopsy settings.

Following the global eradication of smallpox in 1980, mpox (formerly monkeypox) is the most serious orthopoxviral disease in humans [1, 2]. Mpox, caused by Monkeypox virus (MPXV), is endemic in central and western Africa, where—since first documented in 1970—sporadic human cases and small outbreaks have been associated with hunting or bushmeat preparation [3, 4]. In 2003, the first outbreak of human mpox outside Africa was recognized in the United States (US), associated with bites and scratches from prairie dogs infected through comingling with imported wild African mammals [5]. Symptomatic prairie dog and human cases occurred with primarily cutaneous involvement; there was no human-to-human transmission and no human deaths [5–7]. During 2018–2021, 8 cases of mpox were identified in countries outside of Africa among travelers from Nigeria and included secondary spread only to 1 healthcare worker and 2 household contacts [8–10].

Since May 2022, global public health partners have been investigating a multinational outbreak of mpox first identified in the United Kingdom [11] and subsequently in other nonendemic countries, primarily affecting gay, bisexual, and other men who have sex with men (MSM) [12–14]. Most patients experience mucosal lesions and deep-seated and often localized cutaneous lesions [15], both of which can be very painful but typically improve within a few weeks; however, some patients experience more severe manifestations and disseminated disease that have been associated with death [15–19]. As of 30 November 2023, more than 91 000 cases and 167 deaths have been reported globally, including 55 US deaths [20, 21].

Mucocutaneous mpox is typically self-limiting and follows a febrile prodrome with a polyphasic, centrifugal rash that progresses through well-described maculopapular, vesiculopustular, and ulcerative and crusted phases over several weeks [4, 5, 22]. Lymphadenopathy is common and distinguishes mpox from smallpox. Systemic or life-threatening disease has been described [1, 4, 5, 9, 16, 23, 24]; deaths were associated with immunocompromise during a large clade II outbreak in Nigeria during 2017 [9] but have been historically reported more often for clade I than clade II [10, 25]. Characterization of the pathology and virus distribution in tissues from patients with systemic, severe, and fatal disease is limited [23, 26]. Detailed histopathologic evaluations are essential to better understand the clinical presentations and pathogenesis of mpox in immunocompromised patients and recognize how coinfections with other opportunistic pathogens may contribute to severe outcomes in these patients. To address this gap, we systematically describe the histopathologic findings, viral tissue tropism, and coinfections identified in tissues from a large series of persons with severe and fatal mpox.

METHODS

Case Selection

We describe the pathology and MPXV antigen and DNA detection in tissues from individuals with mpox who developed severe disease requiring hospitalization or resulting in fatal outcomes. Specimens were those for which clinicians and pathologists submitted samples to the US Centers for Disease Control and Prevention (CDC) Infectious Diseases Pathology Branch (IDPB) between 1 August 2022 and 28 April 2023, for pathology consultation and diagnostic evaluation for MPXV or other etiologies. All cases for which viral antigens or nucleic acids were identified in tissues by orthopoxviral immunohistochemistry (IHC) or MPXV DNA in situ hybridization (DNA-ISH) laboratory-developed assays were included in this series. Available medical and autopsy records submitted as part of diagnostic consultation were reviewed. Additional clinical data were collected during consultations between treating clinicians and CDC clinical officers. This activity was reviewed by CDC and conducted consistent with applicable federal law and CDC policy (see, eg, 45 Code of Federal Regulations [C.F.R.] part 46, 21 C.F.R. part 56; 42 United States Code [U.S.C.] §241(d); 5 U.S.C. §552a; 44 U.S.C. §3501 et seq).

Histopathological Evaluation and Detection of MPXV and Other Infectious Agents in Tissues

Formalin-fixed, paraffin-embedded (FFPE) autopsy and biopsy tissues were sectioned at 4 µm and stained with hematoxylin-eosin for histopathological evaluation, or by laboratory-developed IHC or DNA-ISH assays for detection of MPXV (see Supplementary Materials). Transmission electron microscopy was also performed for identification of orthopoxviral particles. To evaluate for coinfections, special histochemical stains, IHC, and tissue-based real-time polymerase chain reaction (PCR) and reverse-transcription PCR assays were performed for various infectious agents, based on histopathologic findings and common occurrence in immunocompromised patients. These detailed laboratory methods are provided as Supplementary Materials.

RESULTS

Clinical Data

Specimens were obtained from 22 patients from 9 US states, including 16 patients with biopsies (cases B1–B16), and 6 deceased patients with autopsy tissues (A1–A6) (Supplementary Tables 2 and 3). All were male; ages ranged from 24 to 61 (median, 40) years. All patients were severely immunocompromised. Twenty were patients with human immunodeficiency virus (HIV)/AIDS (PWH) with CD4 counts ≤150 cells/μL (14/20 with CD4 <50 cells/μL); 2 patients (1 biopsy and 1 autopsy) were renal transplant recipients receiving immunosuppressant drugs.

For biopsy patients, time from symptom onset to biopsy collection ranged from 6 to 175 (median, 40.5) days; 7 of 16 (44%) subsequently died but did not have autopsy tissues submitted to IDPB. The 6 deceased patients for whom autopsy tissues were submitted died 4–98 (median, 56.5) days after symptom onset; various digestive symptoms and respiratory failure requiring intubation were common prior to death. Eleven of 16 (69%) biopsy and all autopsy patients received some anti-orthopoxviral medication(s) prior to tissue collection. Among PWH, 7 of 15 (47%) biopsy patients and all 5 autopsy patients were not on antiretroviral therapy (ART) at the time of mpox diagnosis. Three PWH for whom autopsy tissues were received had other coinfections diagnosed during the course of mpox treatment.

Pathology of Severe and Fatal Mpox

Cutaneous Biopsy Specimens

Seven skin biopsies were received from 6 patients (Supplementary Figure 1A–E). One biopsy (6 days after symptom onset) showed an early, vesicular lesion with focal epidermal hyperplasia, ballooning degeneration, spongiosis, and exocytosis of few neutrophils. The dermis showed mild edema and perivascular, lymphohistiocytic infiltrates. Five skin biopsies (5–48 days postonset) showed full-thickness epidermal necrosis and variable presence of serocellular crust. The ulcer beds showed necroinflammatory debris with neutrophilic infiltrates, and variable edema, hemorrhage, and vascular necrosis with thrombi. Necrosis of hair follicles and adnexae was common. Lymphohistiocytic perivascular and interstitial infiltrates extended throughout the dermis. Epidermis at the ulcer margins was hyperplastic, with ballooning degeneration. Variable numbers of multinucleated cells, occasionally with glassy nuclei, were present; intracytoplasmic inclusions were not definitively identified. One skin biopsy (105 days postonset) showed marked acanthosis and thick crust, with dense lymphoplasmacytic infiltrates in the superficial dermis.

Digestive Tract Biopsies

Digestive tract biopsies from 4 patients included esophageal (n = 2), colonic (n = 1), rectosigmoid (n = 1), and rectal (n = 2) specimens (Supplementary Figure 1F and 1G). Esophageal specimens showed proliferative and necrotizing epithelial and stromal changes similar to those identified in the cutaneous biopsy specimens, and 3 intestinal biopsy specimens showed patchy to transmural necrosis and acute to subacute inflammation. Foci of stromal necrosis underlying intact mucosal epithelium were occasionally present. One rectal biopsy described as a mass-like lesion had associated epithelial hyperplasia with focal atypical changes. One colonic biopsy showed intact mucosa with peritonitis evidenced by a focal, thin layer of fibrin and hemorrhage with few admixed, degenerated inflammatory cells. One patient with esophageal and rectal biopsies had scattered enlarged cells with intranuclear and intracytoplasmic viral inclusions in both specimens.

Other Mucosal Biopsies

One tongue biopsy showed epithelial proliferation and ballooning degeneration with superficial necrosis and neutrophilic inflammation associated with fungal yeasts. Subepithelial stroma was necrotic with pronounced, acute inflammation. Nasal biopsies from this same patient comprised necrotic mucosa with foci of foreign material and bacterial cocci. One palpebral conjunctival biopsy showed ulceration with peripheral epithelial proliferative and ballooning changes and rare intracytoplasmic eosinophilic inclusions in keratinocytes [27]. The ulcer bed had dense, lymphoplasmacytic and eosinophilic inflammation (Supplementary Figure 1H).

Skeletal Muscle and Soft Tissue Biopsies

Skeletal muscle biopsies from 2 patients and soft tissue neck mass biopsies from 2 patients were examined. Muscle biopsies showed necrosis of myofibers and fibroconnective tissue, with hemorrhage, fibrin, necroinflammatory debris, and vascular necrosis with thrombi (Supplementary Figure 1I). One had rare cytomegalic cells, and 1 had scattered multinucleated cells with glassy nuclei. One soft tissue neck mass biopsy showed similar necrosis of fibroconnective tissues with fibrin and abundant, mixed inflammation, and focal clusters of cells with intranuclear inclusions. One of 2 serial biopsies from a soft tissue neck mass from another patient showed similar necrosis, with fungal yeasts; a latter biopsy from the same mass showed lymphohistiocytic inflammation.

Lung Biopsy

One lung needle core biopsy showed very small fragments of alveolar parenchyma with necrosis and organizing fibrosis with chronic inflammation.

Autopsy Findings

Reported gross autopsy findings included multiple, extensive vesiculopustular, necrotic, verrucous, and/or ulcerative cutaneous lesions as well as oral and regional digestive tract ulcerations in all cases. Lymphadenopathy and lesions in other tissues were variably reported (Supplementary Table 3) [16, 28]. Histopathologic findings in autopsy tissues are summarized in Table 1.

Autopsies of Mucocutaneous Tissues

Skin and mucosal tissue changes were similar to those in biopsy specimens, with varying stages of acanthotic and vesiculopustular or ulcerated lesions with peripheral epithelial proliferation, ballooning degeneration, syncytia, and vascular thrombosis (Supplementary Figure 1J–N). Few intracytoplasmic eosinophilic inclusions were seen in epithelium from skin, tonsil, and esophagus from 1 patient each (Supplementary Figure 1O). Intestinal lesions were severe in 3 of 5 (60%) cases for which samples were received, with transmural necrosis most prominently in rectal segments (Supplementary Figure 1P). Cytomegalic cells with variable presence of intranuclear inclusions were seen in esophagus or intestinal specimens in 3 of 5 cases.

Autopsies of PWH, Lung Tissue

Prominent lung pathology with MPXV detection by IHC and ISH was identified in 3 of 5 (60%) of autopsies of PWH. Two cases had discrete, often nodular, regions of parenchymal necrosis (infarcts) and mild acute inflammation associated with thrombotic vasculopathy (Figure 1A ). Occasional syncytia, sometimes with eosinophilic intracytoplasmic inclusions, were present within the periphery of the infarcted tissue. Two cases had necrotizing and suppurative bronchopneumonia (Figure 1B ), 1 of which had prominent bronchial and bronchiolar necrosis with epithelial syncytia and streptococcal coinfection, and the other of which had severe, lobar pneumonia with abundant, mixed bacteria and intra-alveolar eosinophilic foamy material that contained Pneumocystis cysts by Grocott methenamine silver stain. One case had features of early acute lung injury (diffuse capillary congestion and intra-alveolar edema) and focal aspiration without overt pneumonia, and 1 had diffuse alveolar damage and gram-negative bacterial pneumonia.

Autopsies of PWH, Lymphoid Tissues

Two autopsies of PWH had necrotizing lesions in lymphoid tissues associated with MPXV. Case A1 had severe necrotizing lymphadenitis with effacement of nodal tissue by necrotic debris and hemorrhage that extended into the adjacent connective and visceral tissues, forming contiguous mass-like lesions (Figure 1C ). Vascular necrosis and thrombosis were prominent. Case A2 had lymphoid necrosis in lymph nodes and prominently within the splenic white pulp, which showed karyorrhectic debris, hemorrhage, and fibrin replacing periarteriolar lymphoid sheaths (Figure 1D ). Both cases also had scattered foci of bone marrow necrosis.

Autopsies of PWH, Other Solid Organs

Autopsies of 3 PWH had foci of discrete, lytic necrosis that were variably associated with thrombotic vasculopathy, compatible with infarcts, in tissue from other evaluated solid organs. All 3 had liver necrosis; hepatocytes containing eosinophilic intracytoplasmic inclusions were identified in 2 of 3 (Figure 1E ). Necrotic foci were also variably present in adrenal gland, pancreas, and testis (Figure 1F ). Bridging ischemic hepatocellular necrosis or fibrosis, distinct from the MPXV-associated necrotizing lesions, were also present in 3 cases. Case A4 had ischemic brain lesions unassociated with MPXV antigen or DNA detection.

Organ Transplant Recipient Autopsy

Case A2 was the only non-PWH, organ transplant recipient autopsy. Mucocutaneous lesions were typical, but with notable absence of inflammation in these and other tissues (Figure 2A ). Lung showed focal alveolar hemorrhage and fibrin, suggestive of an early infarct but without identification of a thrombus. There was diffuse depletion of splenic lymphoid cells, and diffuse adrenal cortical and hepatocyte necrosis with abundant hepatocellular intracytoplasmic eosinophilic inclusions (Figure 2B and 2C ). MPXV antigen and DNA were identified extensively in tissues and prominently within intravascular leukocytes in multiple tissues (Figure 2D–I ).

MPXV Cellular Tropism by Immunohistochemistry, In Situ Hybridization, and Electron Microscopy

MPXV antigens were detected in biopsy and autopsy specimens by IHC, and DNA-ISH was performed on autopsy tissues for viral DNA localization (Table 1). Overall antigen and DNA distributions were similar; IHC and ISH staining were seen in the same tissues and cells with extensive localization to sites of tissue necrosis, and to endothelial cells and intravascular leukocytes in some autopsy tissues, sometimes in the absence of distinct histopathology.

In skin, ocular, oropharyngeal, and mucosal digestive tract tissues, viral antigens and DNA were abundant and distributed extensively within epithelial cells at the sites of vesicle formation and the acanthotic margin of ulcers (Supplementary Figure 2A and 2B). Intense globular cytoplasmic staining corresponded to intracytoplasmic inclusions, when present, and was also often seen in epithelial cells without overt inclusions. In skin, extensive staining of follicular and eccrine and sebaceous glandular epithelium was also observed (Supplementary Figure 2C). In ulcers, staining was diffuse within fibrinous necrotic debris and localized to mesenchymal cells and macrophages throughout the dermis or transmurally, for digestive tract lesions (Supplementary Figure 2D). Endothelial cell staining was also commonly present (Supplementary Figure 2E). In skeletal muscle and soft tissues biopsies, staining was similarly localized extensively within the necrotizing inflammatory infiltrate, while myofibers were spared (Supplementary Figure 2F).

For autopsy parenchymal tissues, MPXV detection colocalized with necrotic foci. In lungs with infarcts or bronchopneumonia, staining was extensive within these areas and present in scattered pneumocytes and macrophages throughout (Figure 1G and 1H ). Staining was only appreciated in aspirated material within the lung of case A5 and was not present in the lung parenchyma of case A4. In lymph node and spleen, viral antigen and DNA localized to regions of lymphoid necrosis or depletion, and variably within sinus macrophages and reticuloendothelial cells (Figure 1I and 1J ); staining was not present in spleens without lymphoid depletion. In liver, staining was confined to hepatocytes in areas of lytic necrosis and was not present in centrilobular ischemic areas; staining was also not seen in Kupffer cells outside foci of necrosis (Figure 1K ). Adrenal glands similarly had staining in necrotic foci (Figure 1L ). Staining also localized to foci of bone marrow necrosis, when present. The renal transplant autopsy had staining in skin lesions and diffuse staining in the liver, spleen, and adrenal gland (Figure 2D–F ). This case had the most widespread intravascular leukocyte staining, which was present in brain, heart, lung, kidney, prostate, and testis (Figure 2G–I ).

Orthopoxviral virions were seen by transmission electron microscopy in skin, lung, and liver samples evaluated from 3 representative cases (B3, A2, and A4). Intracellular accumulations of mature and immature virions were observed in the cytoplasm of epithelial cells of skin and lung, in pulmonary macrophages, and in hepatocytes (Supplementary Figure 2G and 2H). Mature oval-shaped virions were on average 250 nm long and 160 nm wide and had a characteristic dense, dumbbell-shaped core surrounded by laminated zones encircled by an outer membrane. Immature, spherical virions were found within Guarnieri-like inclusions in the cytoplasm of infected cells.

Coinfections Identified in FFPE Specimens

Cases were selectively tested for other infectious agents based on clinical and histopathologic findings. Coinfections were identified in specimens from 5 of 16 (31%) biopsy cases and in 4 of 6 (67%) autopsy cases (Supplementary Tables 2 and 4; Supplementary Figure 3). Among biopsy patients, cytomegalovirus (CMV) was detected in 1 or more specimens from 3 cases, and Candida spp were detected in 2 cases (Supplementary Figure 3A and 3B). For autopsy patients, 3 patients had CMV detected, and 2 of those also had polymicrobial pneumonia—1 with nonpneumococcal Streptococcus spp and human parainfluenza virus type 3, and 1 with Pneumocystis spp, and mixed bacteria, including Escherichia coli and Staphylococcus spp (Supplementary Figure 3C–F). Another autopsy patient had gram-negative bacterial pneumonia. CMV and Candida spp infections were identified by histological characteristics and confirmed by IHC testing. CMV immunoreactivity frequently localized within or near foci of MPXV-associated pathology, but was limited to scattered, individual cytomegalic cells and did not localize extensively throughout necroinflammatory debris in the same manner as MPXV antigen. Streptococcal antigens colocalized with MPXV in pneumonic lung of case A2, Pneumocystis and mixed bacteria were widespread in the pneumonic lung of case A6, and gram-negative bacteria were present without MPXV detection in pneumonic lung of case A4.

DISCUSSION

In this series of severe and fatal mpox cases from the current global outbreak, all patients were severely immunocompromised, and pathology of mucocutaneous lesions was typical of that for uncomplicated mpox [4, 5, 22]. Additionally, necrotizing digestive tract lesions were common, and in fatal cases, widespread tissue necrosis associated with thrombotic vasculopathy was also present in lung, lymphoid tissues, and various other solid organs. Abundant MPXV antigen and DNA were localized within epithelial and systemic necrotizing lesions and were also detected in endothelium and intravascular leukocytes. The severely immunosuppressed renal transplant recipient autopsy had a notable lack of inflammation in all tissues, despite widespread MPXV detection, including within endothelial cells and intravascular leukocytes. These overall findings support that tissue damage in severe mpox cases is characterized by uncontrolled MPXV replication with direct viral infection, viremia, and vasculopathic mechanisms of tissue necrosis and infarction.

Digestive tract lesions were common and represented the initial clinical manifestation in some cases. The frequency of upper and lower digestive tract mucosal lesions identified from the current outbreak is likely related, at least in part, to direct contact during sexual activity among MSM. However, histopathologic findings of stromal or mural necrosis underlying intact mucosal epithelium also suggest a component of viremic spread for some digestive tract lesions. Furthermore, digestive symptoms were common in patients from the 2003 outbreak after inoculation of the virus from bites or scratches [29] and were prominent in MPXV animal models after respiratory exposure [30]. Altogether, it is possible that digestive tract involvement is common in severe mpox, regardless of exposure route.

Pulmonary disease is common in fatal mpox [31]. Half of the autopsy patients in this study had severe clinical respiratory disease requiring intubation, and half had lung pathology overtly attributable to MPXV, including infarcts and bronchopneumonia. Perivascular pulmonary nodules have been seen on chest imaging studies of some mpox patients with advanced HIV [26]; these may represent nodular infarcts such as those seen in lungs in this study. Three cases with bronchopneumonia in this study had both MPXV and 1 or more other bacterial or fungal agents localized to the pneumonic lesions. A fourth case had a possible early infarct. Another had features of early acute lung injury and focal aspiration with MPXV detection in aspirated material. Overall lung findings highlight the possibility of pulmonary MPXV infection by multiple routes (viremia and/or inhalation) and the likelihood of pulmonary coinfections in immunocompromised, hospitalized mpox patients. Furthermore, they raise the possibility of MPXV-associated acute lung injury in absence of direct lung infection, possibly through inflammatory or cytokine-mediated processes (ie, acute respiratory distress syndrome).

Among patients with mpox and advanced HIV who are treated with antiviral agents against both, paradoxically worsening disease and poor outcomes have been observed, raising the question of whether MPXV-associated immune reconstitution inflammatory syndrome (IRIS) occurs and the extent to which it may contribute to observed outcomes [26]. All 5 autopsies of PWH in this series were initiated or reinitiated on ART after mpox diagnosis, and at least 1 had precipitous decline after initial improvement. While IRIS cannot be diagnosed by histopathology alone, the findings of limited lymphocytic infiltration in tissues with heavy MPXV viral antigen and DNA contrast with typical findings of other viral-associated mucocutaneous IRIS (eg, herpes simplex virus [HSV] IRIS), where limited viral antigen burden is overwhelmed by mixed inflammatory infiltrates [32]. Sometimes, however, pathogens may cause immune reconstitution–associated disease without overwhelming inflammation [33]. Further investigation of these phenomena through evaluation and monitoring of mpox patients receiving ART is warranted.

Analysis of global surveillance data found that immunosuppression, whether associated with HIV, medication, or other disease, resulted in significantly increased odds of hospitalization [34]. HIV was the most common immunocompromising condition among patients in this cohort, but 2 were immunosuppressed due to immunomodulating therapy in the setting of renal transplantation. The transplant recipient who underwent autopsy received high-dose steroids and thymoglobulin for treatment of acute cellular rejection, had a rapid clinical course despite receiving tecovirimat (ie, TPOXX), and died 4 days after onset. Tissues showed notable depletion of lymphoid tissue and absence of inflammation in any tissue, both of which were much more profound than in the autopsies of PWH. Thymoglobulin induces lysis of CD3⁺ T cells, and can also deplete B cells and natural killer cells, producing profound immune suppression. Tecovirimat is a virostatic agent that reduces viral spread, but complete viral clearance requires an effective host immune response [35] and drug resistance has been documented, particularly in immunocompromised patients [36, 37]. This patient had diffuse MPXV staining in spleen and adrenal glands and staining of endothelium and intravascular leukocytes in multiple tissues without other pathology. Altogether, these findings suggest decreased efficacy of tecovirimat and uncontrolled viremia in the face of profound immune suppression, regardless of cause, and emphasize the need for continued development of next-generation anti-orthopoxviral therapeutics, particularly with immunocompromised patients in mind. The refinement of animal models with immunologic competencies mimicking various human conditions will be invaluable to these endeavors [38, 39].

The histopathologic diagnosis of mucocutaneous mpox can be straightforward in the appropriate clinical and epidemiological context, but nevertheless the features we describe are nonspecific and other pathogens must be considered. Several such agents commonly cause opportunistic infections in immunocompromised patients and therefore warrant consideration in patients with severe mpox. Furthermore, coinfection by multiple pathogens is possible, making this task both more difficult and more imperative, given the urgency of effective, comprehensive treatment. Alpha-herpesviruses (HSV and varicella zoster virus [VZV]), coxsackie viruses, and some bacterial and fungal infections can cause vesiculopustular or ulcerative lesions, as well as systemic necrotizing lesions as seen in this study [22]. Alpha-herpesviruses can be particularly difficult to discern from MPXV histologically, as the presence of epithelial syncytia and intranuclear viral inclusions are characteristic. While intranuclear viral inclusions are not a classic feature of orthopoxviral infections, glassy or clear nuclei have been reported in MPXV infections and were commonly seen in syncytia in this study [22]. IHC for HSV and VZV was performed on select biopsy and autopsy specimens with prominent syncytia and glassy nuclei; all were negative. Conversely, eosinophilic intracytoplasmic inclusions (Guarnieri-like bodies) are characteristic of orthopoxviruses but are variably present, can be difficult to distinguish from condensed cytoplasmic material in necrotic cells, and were inconsistently seen in this study. As used herein, IHC can be an invaluable tool for identifying and localizing multiple pathogens within the same tissue.

Cytomegalovirus was the most common coinfection identified in this series, and while histologic features of cytomegaly and typical CMV inclusions are more readily distinguished from orthopoxviral lesions than those of the alpha-herpesviruses, clinical and histopathologic features, especially of enteric CMV infection, can be similar to MPXV. IHC in this study colocalized MPXV and CMV in tissues but also clearly differentiated CMV staining that was confined to scattered individual cells from widespread MPXV staining within necrotic inflammatory lesions. Polymicrobial pneumonias were identified in 2 cases with concurrent MPXV detection in lung, and gram-negative bacterial pneumonia was identified in another autopsy without MPXV in lung. The former 2 were assessed as primary MPXV with secondary bacterial pneumonias, while the latter was assessed as possible transfusion-associated acute lung injury and/or hospital-acquired pneumonia in this patient. These cases demonstrate the utility of histopathology and IHC for assessing presence and significance of coinfections, which is crucial both for understanding the pathogenesis of mpox and for devising optimal treatment regimens.

Limitations of this study include the small number of autopsies, only from the US, which may not be representative of all mpox deaths. These 6 autopsy cases represent 11% of the 55 US deaths reported to CDC in the current outbreak through 30 November 2023; 7 other cases for which we report the biopsy findings comprise an additional 13% of those deaths [21]. Additionally, the findings in cases with fatal outcomes encompass the complex interplay of multiple treatments, and often multiple infections, in immunocompromised patients and therefore do not represent the natural history of MPXV infection alone. Regardless, valuable insights into the pathology and pathogenesis of mpox disease, including those complicating factors, in patients with severe and fatal outcomes are gleaned.

Our IHC and ISH assays showed abundant viral antigen and DNA in mucocutaneous tissues and solid organs in fatal cases, irrespective of duration of illness, mpox treatment, or ART. While virus isolation was not performed to quantify viable virus in this study, the presence of abundant viral DNA suggests high levels of infectious virions in these tissues, and underscores the need for rigorous biosafety measures in hospital and autopsy settings, where cutaneous lesions may be extensive, where transport and handling of the body poses skin-to-skin exposure risk, where invasive medical procedures (eg, intubation) may be necessitated, and where extensive tissue collection and handling may occur. Appropriate personal protective equipment and enhanced cleaning and disinfection of hospital and autopsy room surfaces are essential [40, 41].

In conclusion, severe and fatal mpox occurred in immunocompromised persons and was characterized by mucocutaneous pathology similar to the self-limiting disease in otherwise immunocompetent persons. However, fatal cases may also have systemic involvement of other tissues by necrotizing inflammation and vascular thrombosis with direct viral infection of tissues. Digestive tract and lung involvement may have extensive histopathological and clinical manifestations, and systemic lesions with viral detection in endothelial cells and intravascular leukocytes supports viremic dissemination; optimizing immune function (eg, via ART) and early initiation of anti-orthopoxviral therapeutics are recommended to potentially prevent worsening [35]. CMV and other coinfections may be present in severe mpox and warrant consideration in diagnostic and treatment plans. Finally, the large amount of viral DNA in autopsy tissues necessitates appropriate biosafety measures by persons caring for severely affected mpox patients or performing autopsies.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org/). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.

Notes

Acknowledgments. For their work in identifying, providing clinical consultation and care for, and obtaining and evaluating autopsy and biopsy samples from mpox patients, we thank Rebecca Noe, Christine Casey, Kia Padgett, Elizabeth Rabold, Preetam Cholli, Jennifer Cope, Maureen Miller, Michelle Rose, Beatrice Gichuhi, Brook Belay, Colin Shepard, Raven Fraser, Jerry Clay Goodman, Ashley Holloman, Julia Gallardo, Hanna Siatecka, Georgia Huffman, John Powell, Lindsey B. Estetter, Marlene DeLeon-Carnes, Pamela S. Fair, Brooke Leitgeb, IDPB’s Histology and Operations Teams, and the state and local health departments, clinical institutions, and medical examiner and coroner offices.

Author contributions. J. M. R., R. B. M., J. B., C. D. P., and S. R.-S. conceptualized the study. J. M. R., R. B. M., J. B., C. D. P., S. R.-S., and J. V. N. designed the report. A. K. R., C. L. H., T. C., C. K. H., K. H., Y. X., C. A. B., J. P. G., M. M., J. C., J. L., S. C.-G., W. A. B., C. A. S., S. B. M., J. A., and M. G. E. collected and interpreted specimens or data. J. M. R., R. B. M., J. B., J. V. N., L. S.-F., E. L., H. A. B., C. D. P., S. R.-S., and J. N. S. performed specimen testing or analysis/interpretation. J. M. R. wrote the manuscript, with input from all authors. All authors reviewed and approved the manuscript. The data in this study are collected and managed by the CDC; however, the corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.

Disclaimer. The findings and conclusions in this article do not necessarily represent the official position of the US Centers for Disease Control and Prevention.

Supplement sponsorship. This article appears as part of the supplement “Mpox: Challenges and Opportunities Following the Global 2022 Outbreak,” sponsored by the Centers for Disease Control and Prevention (Atlanta, GA).

References

Author notes