Early plasma cell-free DNA diagnosis of fatal Acanthamoeba granulomatous amebic encephalitis in a liver transplant recipient

Abstract

Background

Acanthamoeba granulomatous amebic encephalitis (GAE) is a rare, highly lethal opportunistic infection in immunocompromised hosts. Diagnosis is difficult because clinical and radiographic findings are nonspecific and conventional cerebrospinal fluid (CSF) studies may be nondiagnostic.

Case

We report a 68-year-old woman who developed fever, seizures, and progressive encephalopathy 13 days after orthotopic liver transplantation. Brain MRI demonstrated multifocal supratentorial and infratentorial enhancing lesions with vasogenic edema and peripheral diffusion restriction. Initial CSF bacterial, viral, and fungal studies were negative. Plasma microbial cell-free DNA (mcfDNA) sequencing identified Acanthamoeba species 5 days after admission, prompting expedited Centers for Disease Control and Prevention (CDC) consultation and initiation of multidrug anti-amoebic therapy. CDC CSF real-time PCR subsequently confirmed Acanthamoeba infection. Despite antimicrobial escalation and reduction of immunosuppression, cerebral abscesses, progressive edema and uncal herniation developed and the patient died.

Conclusion

Plasma mcfDNA provided rapid, noninvasive organism identification of an unsuspected free-living amoebic CNS infection and directed confirmatory testing and therapy. It should be considered an adjunct diagnostic tool rather than a replacement for CSF or tissue testing. Results require clinical correlation and confirmation with site-specific testing when feasible.

Highlights

• •Plasma mcfDNA detected Acanthamoeba prior to confirmatory CSF PCR.
• •The blood-based test identified an unsuspected pathogen five days after hospitalization.
• •mcfDNA directed targeted CDC testing and rapid initiation of anti-amebic therapy.
• •Plasma mcfDNA is noninvasive and agnostic but requires clinical and microbiologic correlation.

Introduction

Acanthamoeba species are free-living amoebae commonly present in the environment, yet they rarely cause human disease. Exposure has been documented through contact with soil, natural and treated water sources, seawater, swimming pools, sewage, sediments, air-conditioning units, domestic tap water, bottled water, dental treatment units, hospitals and dialysis units, eyewash stations, and contact lenses and lens cases [1,2]. Infection may manifest as granulomatous amebic encephalitis (GAE) or disseminated disease. GAE typically follows a subacute to chronic course and presents with nonspecific symptoms such as headache, confusion, nausea, vomiting, fever, lethargy, neck stiffness, focal neurologic deficits, or signs of increased intracranial pressure. Neuroimaging frequently demonstrates moderate to severe cerebral edema with multifocal lesions involving the midbrain, brainstem, corpus callosum, and cerebellum [3,4].

Non-keratitis Acanthamoeba infections affect an estimated 3–12 persons annually in the United States, with 173 cases reported between 1956 and 2020 and approximately 85% resulting in death [5]. In a comprehensive review, 18% of infections occurred in solid organ transplant recipients, underscoring the vulnerability of this population [5]. Among reported cases, 71% presented as GAE, with over half lacking preceding cutaneous manifestations, which may contribute to delayed recognition [5]. Opportunistic infections in transplant recipients vary, and transplant-associated Acanthamoeba infections have been described within weeks to years post-transplant. Published cases in orthotopic liver transplant recipients remain rare and are frequently associated with delayed diagnosis, disseminated disease, and poor outcomes. Despite advances in diagnostic modalities and antimicrobial therapy, Acanthamoeba CNS infection remains associated with exceptionally high mortality. We describe a post–liver transplant case in which plasma microbial cell-free DNA (mcfDNA) identified Acanthamoeba prior to confirmatory cerebrospinal fluid (CSF) PCR, enabling early Centers for Disease Control and Prevention (CDC)-guided therapy, yet rapid progression to multifocal abscesses and fatal herniation physiology—highlighting diagnostic utility amid persistent therapeutic limitations.

Case presentation

A 68-year-old woman with a history of eczema and resolved herpes zoster infection presented with nausea, anorexia, fatigue, and elevated aminotransferases in 09/2025. Despite corticosteroid therapy, she developed rapidly progressive liver failure secondary to autoimmune hepatitis and suspected drug-induced liver injury, requiring deceased-donor orthotopic liver transplantation on 10/25/2025. Pre-transplant infectious evaluation was negative. She denied recent freshwater exposure or recent travel, although later reported intermittent nasal irrigation use. Induction immunosuppression consisted of corticosteroids followed by tacrolimus and mycophenolate mofetil. She was discharged on postoperative day 7 on valganciclovir and trimethoprim-sulfamethoxazole prophylaxis; antifungal prophylaxis was not administered. Her immediate postoperative course was otherwise uncomplicated.

She was readmitted on 11/7/2025 with fever, headache, tremors, dry cough, and new-onset focal seizures. Evaluation demonstrated right upper lobe pneumonia and Escherichia coli urinary tract infection. Head CT without contrast showed patchy vasogenic edema without a discrete intra-axial mass lesion. Brain MRI with and without contrast on 11/8/2025 (Fig. 1) demonstrated multifocal supratentorial and infratentorial lesions involving the bilateral frontal lobes, right temporal operculum, left precentral gyrus, and bilateral cerebellar hemispheres. Lesions demonstrated gyriform and rounded mass-like enhancement with associated vasogenic edema and focal peripheral diffusion restriction, most prominent within the right temporal operculum and left precentral gyrus. Faint parenchymal susceptibility was present without significant blooming. Given recent transplantation and predominantly peripheral diffusion restriction, differential considerations included atypical posterior reversible encephalopathy syndrome, infectious encephalitis, and post-transplant lymphoproliferative disorder. Cerebrospinal fluid analysis demonstrated neutrophilic pleocytosis and elevated protein. CSF Gram stain and bacterial, anaerobic, acid-fast bacillus, and fungal cultures; cryptococcal antigen; VDRL; meningitis/encephalitis panel; and PCR testing for HSV, VZV, EBV, CMV, and Toxoplasma were obtained as part of the infectious evaluation.

Fig. 1.

MRI progression from November 8 to November 12.

Empiric antimicrobial therapy was initiated with vancomycin and cefepime, and subsequently broadened to meropenem and acyclovir. Acyclovir was discontinued following negative cerebrospinal fluid meningitis/encephalitis panel and HSV PCR testing, while meropenem was continued for treatment of Escherichia coli urinary tract infection. Given concern for calcineurin inhibitor neurotoxicity, tacrolimus was held on 11/8/2025. Maintenance immunosuppression transitioned to cyclosporine while diagnostic evaluation continued.

Despite broad-spectrum antimicrobial therapy and adjustment of immunosuppression, the patient experienced progressive encephalopathy necessitating transfer to the intensive care unit on 11/10/2025. Additional infectious testing included blood cultures, serum cryptococcal antigen, CMV and EBV PCR, West Nile virus PCR and serologies, Histoplasma, Blastomyces, Coccidioides, and Aspergillus antibodies, Brucella antibodies, dengue serologies, and Ehrlichia and Anaplasma PCR. These studies were unrevealing. Because of worsening encephalopathy and multifocal CNS lesions without an identified cause, plasma mcfDNA sequencing (Karius test) was obtained on 11/9/2025 and resulted on 11/12/2025, identifying Acanthamoeba species at 1030 molecules/100 nanoliters and Escherichia coli at 2385 molecules/100 nanoliters with CTX-M resistance marker detection. The Acanthamoeba result prompted expedited CDC consultation and targeted testing; subsequent CDC CSF real-time PCR was positive for Acanthamoeba, confirming GAE.

In consultation with the CDC, multidrug anti-amoebic therapy was initiated including isavuconazole, flucytosine, sulfadiazine, pentamidine, miltefosine, and nitroxoline. Cyclosporine was discontinued, while corticosteroids were continued at reduced dosing given recent liver transplantation and concern for graft rejection. Despite rapid escalation of therapy, repeat brain MRI with and without contrast on 11/12/2025 (Fig. 1) demonstrated progression to more discrete multifocal cerebral abscesses with central diffusion restriction and ring-like enhancement, and multiple lesions demonstrated susceptibility changes concerning for angioinvasive infection. Vasogenic edema worsened, most pronounced in the right frontal and right temporal lobes, with progressive local mass effect resulting in right lateral ventricular compression, a 2 mm right-to-left midline shift, and features of early right uncal herniation. CT imaging of the chest, abdomen, and pelvis was obtained during hospitalization, and no cutaneous lesions were documented. No dedicated PET/CT evaluation for disseminated infection was performed.

The patient experienced rapidly progressive neurologic deterioration. Hypertonic saline was initiated for cerebral edema; however, neurosurgical intervention was deemed infeasible because of extensive multifocal disease. Following multidisciplinary discussions regarding prognosis, the family elected comfort-focused care, and the patient died on 11/16/2025.

Discussion

Acanthamoeba GAE remains difficult to diagnose because its clinical presentation and imaging findings overlap with other infectious, inflammatory, neoplastic, and treatment-related conditions. Symptoms such as headache, fever, nausea, seizures, altered mental status, and progressive loss of consciousness are nonspecific and may resemble other CNS infections. Given its rarity, diagnosis depends on clinical suspicion for amoebic infection, particularly when evaluation for bacterial, viral, and fungal causes is unrevealing. A recent review found that only 34% of reported Acanthamoeba GAE cases in the United States were diagnosed antemortem [2]. Among reported solid organ transplant cases, diagnoses have also been made only after advanced neurologic disease or at autopsy [6]. These challenges are particularly important in transplant recipients, where the differential diagnosis for multifocal CNS lesions is broad and neurologic decline may occur rapidly.

Once suspected, diagnosis of Acanthamoeba infection may rely on identification of trophozoites or cysts by microscopy, culture, histopathology, or molecular testing. Microscopic diagnosis can be challenging because trophozoites may be mistaken for host cells such as monocytes and macrophages, while cysts are usually not observed in the CSF [7]. Molecular testing with PCR can provide more sensitive detection than conventional morphologic methods [8]. However, targeted PCR generally requires Acanthamoeba to already be considered in the differential diagnosis. In patients with unexplained encephalitis and negative routine bacterial, viral, and fungal studies, this dependence on clinical suspicion may delay appropriate testing and diagnosis.

In this case, plasma mcfDNA sequencing was obtained during the evaluation of unexplained encephalitis and identified Acanthamoeba before confirmatory CSF PCR. The sample was obtained on hospital day 3 and resulted three days later, five days after hospitalization. Because Acanthamoeba was not initially suspected, the agnostic nature of mcfDNA testing allowed identification of a pathogen that may otherwise have required additional targeted testing or tissue biopsy. The result prompted CDC consultation, targeted CSF PCR, and initiation of multidrug therapy. The value of plasma mcfDNA in this case was not that it replaced CSF testing, but that it identified an unexpected pathogen early enough to direct subsequent testing and treatment. Plasma mcfDNA testing is also noninvasive and can be obtained without repeat lumbar puncture or tissue biopsy.

CSF metagenomic next-generation sequencing (mNGS) was not performed in this case. CSF mNGS and plasma mcfDNA provide different advantages during the evaluation of unexplained CNS infection. CSF mNGS evaluates the involved compartment directly and has been used to diagnose otherwise unexplained meningoencephalitis, including infections caused by free-living amoebae [[9], [10], [11]]. Plasma mcfDNA is less invasive and may identify pathogens from deep-seated infections when CSF testing is negative or additional lumbar puncture or tissue biopsy is not feasible. However, plasma testing is not specific to the CNS, and its sensitivity for free-living amoebic CNS infection is not well defined. In one reported case of Balamuthia encephalitis, plasma mNGS was positive despite negative CSF mNGS and CSF PCR, with the diagnosis later confirmed in brain tissue [12]. These tests may therefore be complementary, and plasma mcfDNA results should be interpreted in the clinical context and confirmed with CSF or tissue testing when feasible.

Published experience with blood-based cell-free DNA testing for free-living amoebae remains limited but is growing. A CDC series described nine U.S. free-living amoebic infections initially identified by blood cell-free DNA testing, including four Acanthamoeba, four Balamuthia, and one Naegleria infection [13]. Free-living amoebae had not been suspected before the result in eight of the nine cases, and confirmatory CSF or tissue testing was positive in all seven patients for whom confirmatory specimens were available [13]. Plasma-based sequencing has also been reported in Balamuthia GAE [14], and a recent report described plasma mcfDNA detection of Acanthamoeba in a transplant recipient after initial CSF studies were nondiagnostic [15]. These reports suggest that plasma mcfDNA may be a useful adjunct when severe encephalitis remains unexplained, particularly when an unusual pathogen is not initially suspected.

Despite earlier organism identification, the patient continued to deteriorate after initiation of multidrug therapy. By the time Acanthamoeba was identified, MRI already demonstrated extensive multifocal CNS disease, with repeat imaging showing rapid progression to cerebral abscesses, worsening edema, and early uncal herniation. Treatment of Acanthamoeba GAE remains based largely on case reports, in vitro susceptibility data, and expert guidance, with no standardized or reliably effective regimen [2,6]. Several factors likely contributed to the poor outcome in this patient, including extensive multifocal disease, uncertain CNS penetration of available therapies, inability to surgically debulk the lesions, and the need to balance reduction of immunosuppression with the risk of graft rejection. This case highlights that earlier diagnosis may shorten the time to targeted treatment, but advanced Acanthamoeba GAE remains difficult to treat once extensive CNS disease is present.

The source of infection in this case could not be established. The patient reported intermittent nasal irrigation with tap water, which represented a possible exposure because Acanthamoeba infection has previously been associated with nasal rinsing [1]. However, a causal relationship could not be established, and the route of acquisition remains unknown. Donor-derived infection was also considered and appropriate notifications were made; however, no donor-derived Acanthamoeba infection was identified at the time of this report.

Conclusion

Acanthamoeba GAE is a rare but highly lethal cause of encephalitis in solid organ transplant recipients. In this case, plasma mcfDNA identified Acanthamoeba prior to confirmatory CSF PCR and facilitated early CDC-guided therapy, yet extensive multifocal disease progressed rapidly to abscess formation, cerebral edema, and fatal herniation physiology. Plasma mcfDNA may be a useful adjunct to accelerate organism identification in unexplained encephalitis, but results require clinical correlation and confirmation.

Ethics declaration

Written informed consent to take part in the study and to publish the article has been obtained from all participants or their legal representatives. The privacy rights of participants have been observed.

This study was performed in compliance with relevant laws, regulatory frameworks and guidelines where the research took place. The Annette C. and Harold C. Simmons Transplant Institute granted an exemption. The authors provided the following explanation: Since this is a single case report form it is not considered research.

This research follows the CARE guidelines and the CARE checklist.

CRediT authorship contribution statement

Julie Hill-Mak: Writing – original draft. J. Sebastian Danobeitia: Writing – review & editing. Alejandro Perez: Writing – review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper