Immune Biomarker Signature in the Diagnostic Workup of Fever Without Source: A Pilot Study

To the editor.

Fevers without an identified source (FWS) are frequent in all-aged patients, even in children, and subjects may be often labeled as affected with ‘fever of unknown origin' if it lasts for more than one week.¹ This definition was formally created by Petersdorf and Beeson in the early ‘60s through the evaluation of a case series of subjects displaying an unexplained rise of temperature over 38.3°C on several occasions for more than 3 weeks.² It is well-known that FWS might include infectious, non-infectious, inflammatory, tumoral diseases, but also further complex types of disorders in which fever is a predominant feature.³ Furthermore, among undiagnosed cases of FWS, the mortality rate may be relevant, varying from 6.9 to 18.6% in some reported series.⁴ In particular, investigating children with FWS may be frustrating because of multiple potential causes of fever and autoinflammatory disorders.⁵ Infectious diseases are still a substantial etiology of FWS, while non-infectious conditions may elude many common diagnostic approaches, requiring imaging studies and more specific immunological or genetic tests.⁶

Introduction

In recent years, many breakthroughs have characterized the clinical assessment of subjects with FWS; however, the final diagnosis in many cases may remain unknown. The MeMed BV assay (an automated host-immune chemiluminescence test) has been developed to differentiate bacterial and viral infections by measuring and computationally integrating 3 immune parameters in serum: C-reactive protein (CRP), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and interferon gamma-induced protein 10 (IP-10).⁷

We conducted this pilot study using the MeMed test to provide further information on FWS diagnosis, extrapolating results for CRP, TRAIL, and IP-10 from the MeMed BV assay. The rationale for this study was to determine whether each host marker could be useful for confirming pathophysiological processes related to the final diagnosis of FWS.

The study included 22 subjects with FWS who were hospitalized at the Policlinico A. Gemelli IRCCS, Rome, Italy, during the period 2019–2023; their FWS was defined according to current guidelines or active medical literature definitions.⁸ Specifically, they had a median age/IQR of 52.0/26.7 years (the M/F ratio was 17/5).

Their sera were processed through the MeMed BV on a Liaison XL platform (DiaSorin, Saluggia, Italy), as previously reported.⁹ The MeMed BV cartridges are single-use multiwell containers that work with 100 μL of subjects’ serum sample, contain all reagents with internal controls and disposables necessary to conduct the immunoassay. Upon insertion of the test cartridge into the analyzer, 3 independent assessments can be simultaneously performed to measure the cited biomarkers (CRP, TRAIL, and IP-10). The test also provides a likelihood score (the “BV score”), ranging from 0 to 100: viral infections are associated with a MeMed BV score ≤35, while bacterial infections are associated with a MeMed BV score >65–100. We individually considered serum levels of the subanalytes CRP, TRAIL, and IP-10 rather than the BV scores. The considered cut-offs were 1,9 mg/l for CRP, 74,5 pg/ml for TRAIL, and 113,6 pg/ml for IP-10.¹⁰

Subjects recruited in this retrospective investigation were stratified according to the final definitive diagnosis into 5 different categories: (a) autoinflammatory diseases (36.4%), (b) autoimmune diseases (13.6%), (c) infectious diseases (18.2%), (d) neoplastic diseases (13.6%), and (e) miscellaneous disorders (18.2%, see Figure 1); these diagnoses were established according to the routinely used serologic tests, autoantibody panels, histopathological results, imaging studies, and finally by the reported success of applied therapies. Frozen serum samples collected at the time of hospitalization were analyzed for CRP, TRAIL, and IP-10, and these data were compared with subjects’ diagnoses at the time of this study.

Figure 1. Subgroups of subjects with fever without source (FWS) who underwent MeMed BV assay.

A total of 22 subjects with FWS were analyzed in this pilot study using the CRP, TRAIL, and IP-10 subanalytes (Liaison® MeMed BV®). The figure shows the different subject subsets (expressed as percentages) according to their final diagnosis. *One subject with an autoinflammatory disease was evaluated in both active and remission phase.

Table 1.

MeMed BV markers and BV score in the cohort of patients evaluated for fever without source in the period autoinflammatory disease category in active (upper table) and remission (lower table) phases. Values over the normal range are marked in bold character. Subjects in which the biomarker was coherent with the final diagnosis are marked with ^*. One subject (n. 6) was evaluated in both presence and absence of fever.

Subjects	Diagnosis	TRAIL (pg/ml)	IP-10 (pg/ml)	CRP (mg/l)	MeMed BV score
1*	AOSD	<15	221	44.2	98
2*	AOSD, IgA deficiency, bacteriuria	<15	182	31	98
3	AOSD, UTI	<15	<100	44.1	98
4*,	AOSD	66.3	142	63.2	1
5*	MAS, loxoscelism	63.1	728	86.4	66
6*	PFAPA s.	67.3	400	20.5	33
6	PFAPA s. (nonfebrile phase)	37.1	138	8.73	63
7	PFAPA s. (nonfebrile phase)	26.9	<100	<1	46
8	AOSD (nonfebrile phase)	33.4	<100	4.09	54
9*	SLE	43.5	987	51	74
10*	IBD	100	256	16.4	10
11	IBD	65.1	100	2.17	9
12	Fever relapse in COPD	15	100	4.73	91
13*	Post-COVID 2019 pneumonia	66.3	142	63.2	57
14*	Post-surgery peritonitis	33.3	151	94.6	95
15*	UTI and Herpes zoster	17.1	679	34	96
16	Peripheral T cell lymphoma	28.4	103	52.3	93
17*	Renal cancer in Behçet’s disease	27.6	126	80.7	96
18*	BC-SLR	31.6	803	16	77
19*	IgG4-related disease	26.9	127	95.4	97
20*	Sarcoidosis	16.7	114	2.36	84
21	Sarcoidosis	16.5	<100	1.66	80
22	Fever associated with polyallergic manifestations	39.7	<100	1	21

Table legend. AOSD; adult-onset Still's disease, BC; breast cancer, COPD; chronic obstructive pulmonary disease, COVID; coronavirus disease 2019, MAS; macrophage activation syndrome, MFM; mycophenolate mofetil, NSAID; nonsteroidal anti-inflammatory drug, PFAPA; periodic fever/aphthosis/pharyngitis/adenitis, SLE; systemic lupus erythematosus, SLR; sarcoid-like reaction, IBD; inflammatory bowel disease, UTI; urinary tract infection

Among 8 subjects with autoinflammatory diseases (36.4% of the cohort), 5 had adult-onset Still’s disease (AOSD), 1 macrophage activation syndrome (MAS) after a spider bite of the genus Loxosceles, and 2 periodic fever/aphthosis/pharyngitis/adenitis (PFAPA) syndrome. One PFAPA patient was tested during both active and non-active disease phases. In these patients, both CRP and IP-10 were elevated during the active disease phase but low during the inactive phase; it was also possible to demonstrate that IP-10 elevation reflected interferon pathway activation in the subject with MAS. Three subjects had autoimmune disease: one with systemic lupus erythematosus (SLE) and 2 other ones with inflammatory bowel disease; these subjects showed consistently increased IP-10, variably increased CRP, and consistently low TRAIL. Infectious diseases were diagnosed in 4 subjects (18.2% of the cohort): fever relapse associated with chronic obstructive pulmonary disease (COPD), post-coronavirus disease 2019 (COVID-19) pneumonia, post-surgery peritonitis, and urinary tract infection (UTI) combined with Herpes zoster infection. In these cases, CRP values were high, TRAIL values were low or normal, and IP-10 values were almost normal or slightly higher than normal, except in the subject with UTI and Herpes zoster infection. Three/22 subjects (13.6% of the cohort) had neoplastic diseases: peripheral T cell lymphoma (a highly pyrogenic non-Hodgkin lymphoma), papillary renal cell cancer in combination with Behçet’s disease, and breast cancer (BC)-associated sarcoid-like reaction (SLR). CRP levels were high in the subject with lymphoma, revealing a chronic state of inflammation, whereas IP-10 was high in the other two. Four patients over a total of 22 were included in the miscellaneous category (18.2% of the cohort) with the following diagnoses: IgG4-related disease in 1, sarcoidosis in 2, and recurrent fevers related to severe polyallergy in the last 1; this group revealed the highest IP-10 result, which was also associated with the highest prednisone dose needed.

Discussion

The use of inflammatory markers may help in the differential diagnosis of FWS in combination with other first-level tests. In particular, CRP has been used as a simple marker of inflammation, common to both infectious and non-infectious inflammatory diseases. The biological role of CRP is linked to the host’s defense as part of the innate immune system, as it is a highly conserved plasma protein produced in response to various inflammatory triggers.¹¹ Indeed, high CRP levels in SLE might suggest an underlying infection,¹² but they might also disclose systemic innate immunity-mediated inflammation as in Kawasaki disease, parallel to the risk of non-responsiveness to i.v. immunoglobulin levels and risk of developing coronary artery abnormalities.^13,14 TRAIL is another marker of the TNF superfamily, capable of activating a pro-apoptotic pathway, suggesting the potential for more targeted therapies in patients with cancer.¹⁵ Beyond oncology, TRAIL also exhibits a pro-apoptotic effect on immune cells, contributing to the regulation of many immunologic processes and anti-inflammatory activities.¹⁶ Although TRAIL enhances neutrophil apoptosis and reduces inflammation, it can promote cell survival by inducing polarization of human macrophages toward a pro-inflammatory M1 phenotype via DR4 and DR5 death receptors.¹⁷ A further marker acting as a ligand of chemokine (C-X-C motif) receptor 3 (CXCR3) is IP-10 (also known as CXCL10), which usually recruits immune cells to the site of inflammation.¹⁸ Higher levels of IP-10 in the peripheral fluids can testify a T helper 1-oriented immune response, as occurring in different infections, but IP-10 and its receptor are also involved in the pathogenesis of many autoimmune diseases, either organ-specific (such as type 1 diabetes and Graves’ disease) or systemic (SLE, mixed cryoglobulinemia, Sjögren’s syndrome, and systemic sclerosis).^19,20

In this pilot study all categories of FWS of the most relevant studies have been included: autoinflammatory diseases (8 subjects, with 1 diagnosed with PFAPA syndrome considered twice as undergoing assessment during fever and not), autoimmune diseases (3 subjects), infectious diseases (4 subjects), neoplastic diseases (3 subjects), and a miscellaneous group of disorders (4 subjects). The 3 immune biomarkers in the MeMed BV test have been used to characterize the inflammatory profile of subjects with FWS and to improve the diagnostic process toward the final diagnosis that emerged during patients’ hospitalizations.

Indeed, the results of MeMed BV subanalytes in 14/22 subjects (63.6% of the whole cohort) were consistent with the final diagnosis of FWS, suggesting that routine use of this test could help address the challenging process of identifying the cause of FWS in real-life clinical practice. More specifically, in autoinflammatory diseases, CRP and IP-10 were elevated during the active phase, with IP-10 levels suggesting a marked interferon-based activation in the subject with MAS. In patients with autoimmune diseases, we observed a consistent increase in IP-10, while CRP gave variable results, and TRAIL was consistently low. The subgroup with infectious diseases had reduced TRAIL levels, despite elevated CRP and variable IP-10 levels, which were significantly increased in the case of Herpes zoster infection, as expected for a virus-induced interferon-mediated response. Two patients with a final diagnosis of malignancy had consistently elevated levels of CRP and IP-10. In the miscellaneous disease subgroup, 2/4 subjects had higher IP-10 levels, which may also correlate with the higher corticosteroid doses these patients required.

Limitations

The low number of FWS patients recruited substantially limits the significance of these preliminary results, referred to as our pilot study, reducing its statistical power, hindering generalizability, and increasing the risk of misinterpreting the true diagnostic contribution of CRP, TRAIL, and IP-10 in FWS.

Conclusions

This pilot study highlights that the MeMed BV technology provides data potentially useful for describing the inflammation pattern underlying the pathology in subjects with FWS, through the individual evaluation of CRP, TRAIL, and IP-10, revealing correlations with clinical histories and mechanisms of specific diseases. These results support larger randomized studies with similar case-subgroup distributions to better understand and define the potential discriminative role of these markers, with the aim of disclosing the final diagnosis of FWS as quickly as possible.

List of Abbreviations

AOSD

Adult-onset Still's disease

CXCL10 or IP-10

Interferon-γ-inducible protein 10

COVID

Coronavirus disease

COPD

Chronic obstructive pulmonary disease

DR

Death receptor

FWS

Fever without source

MAS

Macrophage activation syndrome

MFM

Mycophenolate mofetil

NSAID

Nonsteroidal anti-inflammatory drug

PFAPA

Periodic fever/aphthosis/pharyngitis/adenitis

SLE

Systemic lupus erythematosus

SLR

Sarcoid-like reaction

TNF

Tumor necrosis factor

TRAIL

Tumor necrosis factor-related apoptosisinducing ligand

UTIm

Urinary tract infection