Fever of unknown origin (FUO) is a clinical syndrome first described over 60 years ago. In 1961, Robert G. Petersdorf and Paul B. Beeson defined it as an illness lasting more than three weeks, with a fever exceeding 38.3°C on several occasions, and a diagnosis that remains undetermined after one week of inpatient diagnostic evaluation.^[1] Thirty years later, David T. Durack and Alan C. Street proposed two significant modifications: first, distinguishing classical FUO from three other forms — nosocomial, neutropenic, and HIV-associated FUO; and second, enabling outpatient management of patients.^[2] In 1997, De Kleijn and colleagues refined the FUO definition by incorporating standardized investigations to improve diagnostic accuracy and comparability across studies.^[3] They further emphasized the importance of identifying potentially diagnostic clues (PDCs) in guiding the diagnostic evaluation of FUO patients.^[3] The endemic diseases present in a given country or region strongly influence the etiologic spectrum of FUO. Depending on the setting, infections such as tuberculosis, malaria, b diagnosis.^[4]

Visceral leishmaniasis (VL) is a neglected vector-borne parasitic disease caused by parasites of the Leishmania donovani complex, which are transmitted by the bite of infected female sandflies.^[5] It is estimated that 50,000 to 90,000 cases of VL occur annually worldwide, though the true burden is likely higher. Approximately 90% of the global VL burden is concentrated in Eastern Africa, South Asia, and Brazil.^[6]VL is also endemic in parts of the Mediterranean basin, including North Macedonia, where the estimated incidence ranges from 0.42 to 0.53 cases per 100,000 population.^[7] The clinical spectrum of VL ranges from asymptomatic infection to a severe, life-threatening disease if left untreated.^[8] The incubation period is usually two to eight months but can range from a few weeks to several years. Symptoms typically develop subacutely with gradual onset of malaise, weight loss, hepatosplenomegaly, and prolonged fever—often intermittent or remittent in pattern.^[8]

Despite its recognition as part of the differential diagnosis of FUO^[9], the role of VL remains underrecognized and poorly characterized in the literature.

The goal of this study was to evaluate the clinical and laboratory characteristics of patients with VL presenting as FUO and to compare them with those of patients with other FUO etiologies.

The study was approved by the Ethics Committee for Research Involving Humans at the Medical Faculty in Skopje (approval no. 03-2031/18), and written informed consent was obtained from all participants. The research was conducted in accordance with the principles of the Declaration of Helsinki.^[10]

The study included 94 patients with FUO, of whom 62 (66%) were male and 32 (34%) were female. The median age was 49 years (IQR 36–65 years). The median maximum body temperature during hospitalization was 39.0°C (IQR 38.5–40.0°C), and the median duration of fever before diagnosis was 29 days (IQR 21–60 days).

Infectious diseases were the most common cause of FUO, identified in 49 patients (52.1%), followed by noninfectious inflammatory disorders in 19 (20.2%), miscellaneous conditions in 16 (17%), and malignancies in 10 (10.6%). The most frequent single diagnosis was visceral leishmaniasis (VL), identified in 17% of cases, followed by infective endocarditis (14.9%) and adult-onset Still’s disease (9.6%). Subacute thyroiditis and habitual hyperthermia were present in 6.4% of cases. The full etiological distribution is presented in Table 1.

Demographics and clinical manifestations of patients with VL and other FUO etiologies are summarized in Table 2. Compared to patients with other FUO causes, those with VL were significantly more likely to present with weight loss, diaphoresis, splenomegaly (all p<0.001), and hepatomegaly (p=0.002). They also had a higher febrile peak (p=0.026), and male sex was more common in this group (p=0.046). The frequency of other symptoms, including malaise, rigors, and anorexia, did not differ significantly between the groups.

The laboratory parameters of patients with VL and other FUO etiologies are shown in Table 3. Compared to other FUO patients, those with VL had more pronounced cytopenias, with lower hemoglobin and erythrocyte levels (both p=0.006), reduced hematocrit (p=0.004), as well as significantly lower leukocyte and platelet counts (both p<0.001). Neutrophil percentages were significantly lower (p<0.001), whereas lymphocyte percentages were elevated (p<0.001) in the VL group. Among biochemical parameters, VL patients had higher AST (p=0.005), while ALT, ALP, and GGT did not differ significantly. Bilirubin and creatinine were slightly higher in VL patients (p=0.008 and p=0.011, respectively), although still within normal ranges. VL patients also had lower albumin (p=0.029) and higher globulin levels (p=0.001). CRP levels were elevated in both groups, but the difference was not statistically significant (p=0.115). The remaining laboratory parameters demonstrated comparable values between the groups.

In this study, visceral leishmaniasis emerged as a prominent cause of FUO in immunocompetent adults, accounting for 17% of cases in our cohort. This finding aligns with previous data from North Macedonia, where infections, and particularly VL, were also identified as leading FUO etiologies.^[11] To our knowledge, this is the first published study from the region to specifically examine VL in the context of FUO and to describe its clinical and laboratory profile relative to other causes.

Across Europe, VL is rarely reported as a cause of FUO, usually in the form of isolated case reports or small series. However, two studies from Turkey documented VL as a recognized FUO etiology: a nine-year series of 87 FUO patients identified VL in 5.9% of cases^[12], and a separate report described 20 cases of VL presenting as FUO.^[13] In Greece, VL was reported in 3.6% and 3.0% of FUO patients during two consecutive time periods (1992–2000 and 2001–2007, respectively).^[14] A large multicenter study conducted through the ID-IRI platform across 21 countries—including several European nations, as well as countries from Asia, Africa, and the Middle East—found VL in less than 1% of 407 infectious FUO diagnoses.^[15] Reports from India have shown greater variation, with VL accounting for 1.5% to 9.4% of FUO cases, depending on the population and clinical setting.^{[16, 17]} Beyond immunocompetent adults, VL has also been described as a cause of FUO in pediatric patients and in those with HIV-associated immunosuppression, where it often presents atypically and complicates the diagnostic workup.^{[18, 19]}

These findings demonstrate that, while rare, VL is still a clinically relevant FUO etiology in endemic areas. In our cohort, the proportion of VL among FUO cases was notably higher than that reported in most European series, even though human leishmaniasis has been documented in at least 14 European countries between 2005 and 2020. ^[7] Nevertheless, VL presenting specifically as FUO remains uncommon in European literature. Possible reasons for its higher prevalence in our study include delayed recognition due to its nonspecific clinical presentation and broad differential diagnosis, limited familiarity of healthcare providers with the disease, and restricted access to diagnostic tools in peripheral hospitals.

Patients with VL in our cohort displayed a distinctive profile compared to those with other FUO etiologies. Male sex was significantly more common among patients diagnosed with visceral leishmaniasis, consistent with patterns reported in endemic regions. For example, data from Eastern Africa show that approximately two-thirds of VL cases occur in males, suggesting a clear male predominance in disease burden.^[20] Several factors may contribute to this disparity, including biological influences, such as sex hormone-modulated immune responses, and sociocultural or occupational factors that result in greater exposure of men to sandfly vectors.^[21]

VL patients more frequently presented with weight loss, diaphoresis, hepatomegaly, and splenomegaly—findings consistent with earlier reports.^[22] The higher febrile peaks may reflect systemic inflammation driven by sustained parasite proliferation and elevated cytokines such as IL-6 and TNF-α, which contribute to fever and other systemic symptoms in active VL.^[23] Hematologic abnormalities were another hallmark, with previous studies reporting anemia in 85%–92%, leukopenia in 83%–85%, and thrombocytopenia in over 70% of patients.^{[24, 25]} In line with these observations, VL patients in our cohort exhibited more pronounced cytopenias compared to other FUO cases. Hypergammaglobulinemia and hypoalbuminemia were also evident in our VL group, as described in other studies.^{[22, 26]}

Our findings confirm that visceral leishmaniasis can be an important and often underrecognized cause of FUO in endemic regions. It may present with subtle and nonspecific findings, as seen in our cohort, where six patients were diagnosed through bone marrow biopsy for suspected hematologic malignancy (unpublished data). The remaining cases were treated with empirical antibiotics by general practitioners or other specialists for suspected bacterial infections; as the disease progressed and initial therapies failed, they were referred to our clinic and were later diagnosed using indirect immunofluorescence serology (unpublished data). These findings highlight the need for greater clinical awareness and a high index of suspicion to avoid diagnostic delays and inappropriate treatment, especially in regions where VL is endemic but frequently overlooked. Given the relatively high prevalence of VL among FUO cases in the Republic of North Macedonia, early consideration and prompt testing for VL should be incorporated into the initial FUO workup, even in the absence of typical clinical signs.

A key strength of our study is that it addresses an underexplored area: VL as a cause of FUO in immunocompetent adults, a topic for which reports remain scarce. The prospective design and systematic evaluation of patients using standardized criteria allowed us to reliably describe the clinical and laboratory profile of VL compared to other FUO causes in an endemic setting.

Limitations include the fact that it was a single-center study, so the findings might not fully apply to other places or populations. Additionally, the relatively small number of VL cases, although reflecting its frequency in the population, reduced the statistical power for more detailed analyses and subgroup comparisons.

Kostadin Poposki: conceptualization, methodology, formal analysis, investigation, data curation, writing – original draft, writing – review and editing, visualization, project administration; Dejan Jakimovski: methodology, validation, investigation, resources, data curation, software; Zaklina Sopova: methodology, validation, investigation, resources, data curation, supervision; Arlinda Osmani Loga: methodology, investigation, resources, data curation, software; Irena Trajkova: methodology, investigation, resources, data curation; Mile Bosilkovski: conceptualization, validation, methodology, investigation, writing – review and editing, validation, visualization, supervision, project administration.

The authors have no funding to report.

The authors have declared that no competing interests exist.