Metagenomic Diagnosis of Unknown Fever: Advances, Clinical Applications, and Future Perspectives

Abstract

Fever of unknown origin (FUO) remains a diagnostic challenge in clinical practice, often leading to extensive investigations with inconclusive results. The advent of metagenomic next-generation sequencing (mNGS) has revolutionized the diagnostic landscape, enabling unbiased pathogen detection from clinical samples. This review explores the scientific basis, clinical applicability, and practical advantages of metagenomic approaches in diagnosing unknown fevers. It critically examines the epidemiology, pathophysiology, diagnostic workflow, and latest advances, with particular attention to guideline recommendations and real-world implementation for healthcare professionals.

Introduction

Fever of unknown origin (FUO) is a recurring clinical syndrome defined by prolonged fever without an established diagnosis despite comprehensive evaluation. Traditionally, FUO workup requires a battery of microbiological, serological, and imaging tests, yet many cases remain unexplained. Recent developments in metagenomic next-generation sequencing (mNGS) have provided a powerful alternative for pathogen identification, offering the promise of unbiased, comprehensive detection within a single test. This article reviews the evidence for metagenomic diagnosis in FUO, focusing on scientific mechanisms, clinical impact, and integration into modern clinical workflows.

Epidemiology / Disease Burden

FUO accounts for a significant proportion of hospital admissions worldwide, with reported incidence rates ranging from 2% to 10% among inpatients with prolonged fever. The etiological landscape varies by geography, patient population, and healthcare setting, with infectious, inflammatory, neoplastic, and miscellaneous causes. Despite advances in diagnostic modalities, up to 30% of FUO cases remain undiagnosed, resulting in prolonged hospitalizations, increased healthcare costs, and patient morbidity. The burden is especially pronounced in immunocompromised populations, where atypical and rare pathogens are more prevalent.

Pathophysiology

The mechanisms underlying FUO are intrinsically linked to the host-pathogen interaction and immune response. Infectious causes may be due to fastidious or rare organisms, atypical presentations, or low microbial loads not detected by conventional diagnostics. Autoimmune and neoplastic etiologies may produce fever through cytokine-mediated pathways, further complicating the diagnostic algorithm. Metagenomic sequencing overcomes these barriers by enabling comprehensive analysis of genetic material in clinical specimens, thus circumventing the need for prior knowledge of the suspected pathogen.

Risk Factors

Risk factors predisposing patients to FUO include immunosuppression (e.g., HIV/AIDS, organ transplantation, chemotherapy), travel to endemic regions, exposure to zoonotic agents, and underlying chronic diseases. Additionally, elderly patients, children, and those with recent invasive procedures may be at higher risk for atypical or occult infections. Recognizing these risk factors is crucial for optimizing specimen selection and interpreting metagenomic findings within the clinical context.

Clinical Features

Patients with FUO present with persistent or recurrent fever, typically defined as temperature ≥38.3°C for more than three weeks with no diagnosis after one week of inpatient investigation. Associated symptoms are often non-specific, including malaise, weight loss, night sweats, and localized signs depending on underlying etiology. The absence of clear clinical, laboratory, or imaging clues underscores the need for advanced diagnostic approaches such as metagenomics.

Diagnosis

Traditional FUO diagnostic workup involves a stepwise approach: thorough clinical evaluation, broad laboratory testing (CBC, ESR, CRP, liver/renal panels), blood cultures, serology, and imaging (CT, MRI, PET-CT). Despite this, many cases remain unresolved. Metagenomic next-generation sequencing (mNGS) enables hypothesis-free detection of bacterial, viral, fungal, and parasitic DNA or RNA directly from blood, cerebrospinal fluid, or tissue samples. The workflow includes unbiased nucleic acid extraction, library preparation, high-throughput sequencing, and bioinformatics analysis to match sequences against large pathogen databases. Numerous studies have demonstrated superior sensitivity and breadth of mNGS over conventional diagnostics, particularly in immunocompromised hosts and atypical infections.

Treatment & Management

Management of FUO is guided by the underlying cause when identified; however, empirical treatment is often initiated in critically ill or immunocompromised patients. Metagenomic diagnosis enables pathogen-directed therapy, reducing unnecessary broad-spectrum antimicrobial use and associated adverse effects. In non-infectious causes, mNGS can exclude infectious etiologies, facilitating early consideration of autoimmune or neoplastic processes and appropriate consultation with specialists. The timely and accurate identification of causative agents enhances outcomes and reduces length of hospital stay.

Recent Advances / Emerging Therapies

Recent advances in mNGS have focused on improved sensitivity, faster turnaround times, and streamlined bioinformatics pipelines. Single-molecule sequencing, improved sample processing, and machine-learning algorithms for data interpretation are rapidly expanding the clinical utility of metagenomics. Integration of host transcriptomic and proteomic data further refines diagnostic accuracy and may provide prognostic information. Emerging point-of-care metagenomic platforms hold promise for rapid bedside diagnosis in resource-limited settings, although regulatory, cost, and infrastructure barriers remain.

Guideline Recommendations

Several professional societies, including the Infectious Diseases Society of America (IDSA), now recognize the utility of mNGS in the FUO workup, especially for immunocompromised hosts or when conventional testing fails. Guidelines emphasize the importance of multidisciplinary collaboration, pre-analytic specimen optimization, and careful interpretation of mNGS results within clinical context to avoid misdiagnosis or overdiagnosis due to contamination or commensal detection. Ongoing updates to guidelines reflect the rapidly evolving evidence base and technological advances.

Conclusion

Metagenomic next-generation sequencing represents a paradigm shift in the diagnosis of fever of unknown origin, enabling rapid, comprehensive pathogen detection that transcends the limitations of traditional methods. For clinicians, mNGS offers enhanced diagnostic yield, informs targeted therapy, and supports evidence-based management in complex cases. Continued research, technological innovation, and guideline development are essential for optimizing clinical integration, cost-effectiveness, and patient outcomes in the era of precision infectious disease diagnostics.