Discussion

In this five-patient case series, plasma mcfDNA NGS returned with a median TAT of 3 days and, in our institutional workflow, preceded send-out IgM and CSF PCR testing when obtained. In two patients, the result initiated targeted anti-Toxoplasma therapy, including a donor-seropositive/recipient-seronegative heart transplant recipient with neurologic symptoms but non-diagnostic imaging and an outpatient with prolonged febrile hepatitis. In patients with classic ring-enhancing lesions, plasma mcfDNA supported continued empiric therapy while confirmatory studies were pending.

Our findings complement prior reports suggesting that plasma mcfDNA sequencing can detect both CNS and non-CNS toxoplasmosis. Roy et al described toxoplasma pneumonitis in AIDS diagnosed by plasma NGS before later recognition of CNS lesions. ^{Reference Roy, Siddique, Bathina and Ahmad8} Goren et al identified eight cases of T. gondii infection by plasma cfDNA sequencing in immunocompromised hosts, including clinically unsuspected and non-CNS presentations. ^{Reference Goren, Lehman, Drozdov and Thielen9}

Plasma mcfDNA may be most useful when conventional diagnostics are equivocal or infeasible. In advanced HIV, toxoplasmosis is often diagnosed presumptively based on compatible imaging and clinical response, with brain biopsy generally reserved for non-responders after 10–14 days. ^{Reference Vidal2} CSF PCR is highly specific but variably sensitive, with highest yield before or within the first week of therapy. ^{Reference Vidal2,Reference Cingolani, De Luca and Ammassari4} In our series, mcfDNA NGS often returned before send-out IgM or CSF PCR and provided actionable confirmation without lumbar puncture or brain biopsy.

Transplant recipients represent a distinct diagnostic context. Donor-derived toxoplasmosis is strongly associated with donor-seropositive/recipient-seronegative mismatch, particularly after heart transplantation, and disease can occur before seroconversion, limiting the utility of serology in early infection. ^{Reference Derouin and Pelloux3} The transplant case in our cohort illustrates this challenge: IgG remained negative, and IgM returned later, whereas plasma mcfDNA identified T. gondii within 2 days and prompted initiation of therapy. No patient underwent blood T. gondii PCR. In allogeneic hematopoietic cell transplantation, serial blood qPCR has been proposed for preemptive early detection, although blood PCR can remain negative in established disease. ^{Reference Aerts, Mehra and Groll10}

The open-ended nature of plasma mcfDNA testing necessitates diagnostic stewardship. Co-detection of additional taxa was common in our patients and is widely reported in clinical practice, where interpretation depends on pretest probability and syndrome compatibility. ^{Reference Hogan, Yang and Garner6,Reference Park, Chang and Ledeboer7} In our series, additional detections did not meaningfully change management because the T. gondii result was interpreted alongside host factors, imaging, and conventional testing rather than as a stand-alone diagnosis. A positive plasma mcfDNA result should therefore not be considered diagnostic in isolation, and false-positive or clinically irrelevant detections remain possible, particularly when orthogonal confirmation is unavailable or multiple organisms are reported. This contextual interpretation may be more difficult outside infectious diseases-supported or multidisciplinary care.

Plasma mcfDNA also has inherent limitations: it cannot localize infection, quantitative values (MPM) have not been validated as a marker of toxoplasmosis disease burden, and false-negative results can occur, particularly in isolated CNS disease or after therapy. A negative plasma mcfDNA result should therefore not dissuade empiric treatment when clinical suspicion is high. Our study is limited by retrospective design, small sample size, possible false-positive attribution in unconfirmed cases, selection bias toward patients in whom clinicians already pursued plasma mcfDNA testing, and TAT comparisons that reflect local laboratory workflows. Prospective multicenter studies are needed to define optimal patient selection and timing and to evaluate whether plasma mcfDNA meaningfully reduces invasive procedures, antimicrobial exposure, or time to appropriate therapy.