1.Definition and classification of fever of unknown origin (FUO) is a clinical concept proposed by foreign scholars in 1961, which is defined as a fever lasting for more than 3 weeks, with a temperature greater than 38.3℃ several times, and undiagnosed after at least 1 week of intensive examination. This strict definition excludes common fever and self-limiting fever. Some scholars have suggested that the definition of fever of unknown origin should be more comprehensive to include diseases that have changed with medical advances, such as human immunodeficiency virus (HIV) infection and increasing neutropenia; others have argued that changing the definition would be detrimental to the diagnosis and treatment of FUO. Subsequently, FUO was classified into 4 categories based on suspected etiology: classic (including cases meeting previous criteria for FUO, with emphasis on dynamic evaluation of these previously healthy patients), medical-derived, immunodeficiency, and HIV-associated types. The characteristics of each type of FUO differ, as do the differential diagnosis and diagnostic procedures. In China, at the 1998 National Symposium on Febrile Diseases, FUO was defined as a person with fever lasting more than 2-3 weeks, with a body temperature ≥ 38.5 °C, who cannot be clearly diagnosed despite a detailed history, physical examination, and routine laboratory tests. Some authors also suggest that those with body temperature >37.5 ℃ and fever lasting for more than 2 weeks should be categorized as FUO. At present, most scholars prefer the classical foreign definition of FUO, as it is more applicable in clinical practice. Once the diagnosis of FUO is established, the cause of FUO should be actively searched for, and there are many causes of FUO, up to more than 200. According to the etiology FUO can be divided into 4 categories. However, because the clinical manifestations of FUO often lack characteristics, and routine laboratory tests may not have specific results, the diagnosis of FUO has been a rather difficult problem in clinical work. A complete and comprehensive medical history and careful physical examination are particularly important in the diagnosis. (1) Medical history The medical history should be comprehensive, with detailed questions about the type and degree of fever and accompanying symptoms, such as cough, sputum, chills, chills, nausea, vomiting, abdominal pain, joint and muscle pain, and rash. In addition, we should ask about the general condition such as diet, urine and stool, weight change, previous diagnosis and treatment and its efficacy, and whether there is a history of tuberculosis and tumor in the past. The personal history should pay attention to the history of pet keeping, the history of living in infected areas, etc. The history of family history of hereditary febrile diseases should also be noted. The history should be taken in order to provide potential diagnostic clues (PDCs). Many of the misdiagnosed cases are caused by the lack of detailed history taking and systematic physical examination. (2) Physical examination should emphasize a careful and thorough physical examination, and any suspicious signs in any part of the body should be examined in detail. Positive findings, such as enlarged lymph nodes, enlarged thyroid, temporal artery tenderness, and heart murmur, are helpful in diagnosing lymphoma, lymph node tuberculosis, thyroiditis, temporal arteritis, and infective endocarditis. Physical examination sometimes requires repeated examinations to detect positive signs, with particular attention to the skin, mucous membranes, and lymphatic system. In abdominal palpation, special attention should be paid to the presence of enlargement of the liver and spleen and other organs or the presence of masses. more than half of the abnormal signs found in patients with FUO can provide PDCs to make a preliminary diagnosis. (3) Adjunctive examinations In the clinical diagnosis of FUO, PDCs can often be found through comprehensive and meticulous history taking and physical examination, combined with the results of appropriate adjuvant examinations, and then targeted adjuvant examinations can be performed accordingly, and for most patients with FUO, a clear diagnosis can be made. For some patients with fever as the main symptom, but lacking symptoms and signs that clearly reflect organ damage, ancillary examinations are more important for diagnosis and differential diagnosis. The initial diagnostic procedures of FUO include blood routine, urine routine, stool routine, liver function, blood sedimentation, and bacterial culture, from which clues are sought to classify the etiology of FUO. Further tests are often invasive or expensive and should not be performed blindly. The PPD test is simple and inexpensive, and is a very economical screening method. It should be done in all patients with FUO who are not known to have a positive reaction, but a positive test result does not prove the presence of active TB disease. Chest imaging is useful for screening for infection, collagen vascular disease, and malignancy. If the cause of fever is not clear from the initial examination, special tests such as serology, ultrasound, CT, MRI and nuclear medicine should be used. It is recommended that ultrasound or CT examination of the abdomen and pelvis can be performed early in the diagnosis of FUO based on the results of the preliminary examination, which is valuable to exclude common causes of FUO such as abdominal or pelvic abscesses and malignant tumors. If the diagnosis is still not confirmed, MRI or radionuclide imaging may be an option. A nuclide scan may detect inflammatory and neoplastic lesions that are not defined by CT. Endoscopy can diagnose inflammatory bowel disease and sarcoidosis. Invasive tests such as lumbar puncture, bone aspiration, liver biopsy, and lymph node biopsy are considered only when clinical symptoms are highly suggestive or when all other tests are ineffective. Approximately 8-20% of patients with FUO who cannot be found the cause of the disease even after active refinement of various ancillary tests can be temporarily observed and diagnostic treatment can be considered if necessary. Diagnostic treatment should be carried out selectively according to the more likely etiology without compromising further identification of the cause, and therapeutic drugs should be selected with high specificity, exact efficacy and safety, and in sufficient quantity, throughout the course, and should not be changed at will without special reasons. ①Laboratory tests Routine blood tests: can reflect the body’s reaction status to pathogenic factors, especially infection. Leukocytosis generally refers to neutrophilia and can be seen in acute septic infections, certain viral infections (infectious mononucleosis), malignancies, etc. Extreme leukocytosis is seen in leukemia and leukemia-like reactions. Most viral infections do not have leukocytosis. Leukopenia is seen in certain infectious diseases (typhoid, paratyphoid, malaria, viral hepatitis, Salmonella spp. infections, etc.), non-leukemic or subleukemic leukemias, granulocyte deficiency, malignant histiocytosis, etc. Eosinophilia is seen in parasitic infections, allergic diseases, and certain blood disorders; eosinophilia is seen in typhoid and paratyphoid fever, and if eosinophils are absent is a strong point of support for the diagnosis of typhoid fever. Lymphocytosis is seen in infectious mononucleosis (with large numbers of heterogeneous lymphocytes), lymphocytic leukemia, and certain chronic infections. Monocytosis is seen in subacute infective endocarditis, typhus, malaria, active tuberculosis, etc. A significant increase in monocytes and the presence of naïve, primitive monocytes is seen in monocytic leukemia. Blood sedimentation: The main causes of accelerated blood sedimentation are increased plasma fibrinogen, globulin and decreased albumin. Blood sedimentation as one of the acute phase indicators is most often seen in inflammation, connective tissue disease, malignancy, severe liver disease, anemia, etc. After 30 hours of acute infection, the hematocrit may begin to increase and often remains normal until the recovery phase of the disease. In patients with chronic hypothermia, if all other tests are normal but the blood sedimentation is increased, it often indicates that the cause is non-functional and that there is probably some hidden disease, especially tuberculosis and malignant tumors. Blood sedimentation is a good reference for determining the activity of tuberculosis and rheumatism. C-reactive protein: It is a special glycoprotein that appears in the blood during certain disease processes. Positive C-reactive protein is seen in various tissues with purulent inflammation, tissue necrosis, malignant tumors, connective tissue disease and active rheumatic fever. In acute viral infections, C-reactive protein is often negative, which helps to differentiate bacterial inflammation from viral infections. In conclusion, it is important to distinguish infectious from non-infectious fever, determine the degree of tissue damage and identify the activity of inflammatory diseases. Serum enzymatic tests: γ-glutamyl transferase and alkaline phosphatase are significantly increased in obstructive jaundice and hepatocellular carcinoma. Creatine phosphokinase and aldolase are significantly elevated in polymyositis and dermatomyositis, while rheumatoid arthritis, scleroderma, systemic lupus erythematosus and other connective tissue diseases do not have high or only slightly elevated enzyme activity, so the detection of these two enzymes can help differentiate polymyositis and dermatomyositis from other connective tissue diseases. Blood and urine cultures: Patients with unexplained fever without other local symptoms should have blood and urine cultures routinely performed to help in the diagnosis of sepsis and urinary tract infectious diseases. Specimen collection should strive to be performed before antibiotics are administered; in fact, because almost all patients with FUO have received antimicrobial therapy prior to admission, the positive rate of bacterial cultures decreases, causing diagnostic difficulties. In infective endocarditis, a positive blood culture has a decisive diagnostic value and can guide the choice of antibiotics. Urine culture must be done in strict accordance with the middle urine collection method to collect specimens so as not to contaminate the specimen and affect the culture results. Rheumatoid immunological examination: immunoglobulin, serum complement, rheumatoid factor, antinuclear antibody profile, anti-neutrophil cytoplasmic antibody (ANCA) and anti-basement membrane antibody should be performed when autoimmune disease is suspected. An elevated anti-streptococcal hemolysin “0” titer can indicate recent hemolytic streptococcal infection and can be used as an indicator of rheumatic fever. Infection-related pathogenesis and serum antibody tests: anti-HIV antibody test for suspected acquired immunodeficiency syndrome (AIDS). Viral antibodies, especially cytomegalovirus and EBV antibody tests are helpful in diagnosing viral infections. Antibody tests for atypical pathogens such as mycoplasma, chlamydia and Legionella are useful for diagnosis. In antibody testing, changes in antibody titers during the early onset and recovery period are more diagnostic. Thyroid function and thyroid autoantibody test: This test should be done when hyperthyroidism or thyroiditis is suspected. Tumor markers: Patients with fever when malignancy is suspected can be tested for relevant tumor markers. Other: Patients with high fever may have abnormal liver function, such as elevated alanine aminotransferase, which usually returns to normal after the fever subsides. In viral hepatitis fever, liver function may be significantly abnormal and alanine aminotransferase significantly elevated. Fester reaction can be used for the diagnosis of typhoid fever and paratyphoid fever. ②Instrumental examination X-ray examination: frontal and lateral chest films are helpful for the diagnosis of pneumonia, tuberculosis, lung abscess and malignant tumor of the lung. Skeletal radiographs can rule out diseases such as septic osteomyelitis. Enhanced CT of the abdomen or pelvis: can detect abscesses and tumors in the abdomen or pelvis. Echocardiography: In patients with heart valve disease, congenital cardiovascular malformation or prosthetic valve replacement, with unexplained fever for more than 1 week, often irregular, the possibility of bacterial endocarditis should be alerted. The presence of anemia, peripheral embolism, and a heart murmur should be considered for the diagnosis of this disease. Blood cultures and transthoracic or esophageal echocardiography should be performed immediately. The finding of bacterial redundancy on the heart valves may clarify the diagnosis. Transesophageal cardiac ultrasound is more diagnostic because it avoids interference from the chest wall and lungs and can detect smaller flora (<1 mm). Vascular Doppler examination of both lower extremities: It can detect whether there is thrombosis in the deep veins of both lower extremities. MRI: Useful for the diagnosis of FUO due to systemic vasculitis. Lesions in aortitis are most often seen in the aortic arch and its branches, followed by the descending aorta, abdominal aorta and renal arteries. MRI can show lesions and edema in the walls of the involved arteries and can assist in determining whether the disease is active. Radionuclide imaging: Radionuclide imaging is able to detect local pathologic tissue changes early in the disease, before morphologic changes are present. It can show the location and number of lesions and is diagnostic for detecting lesions in areas not clinically suspected. Commonly used methods include 67Ga scan, indium-labeled leukocyte and 99mTc scan, which can show the site of infection and inflammation in vivo and help in the diagnosis of FUO. In recent years, the role of radioactive 18-fluorine-labeled fluorodeoxyglucose positron emission tomography (18F-FDG PET) and positron emission tomography-computed tomography (PET/CT) in the diagnosis of FUO is gaining attention. 18F-FDG is taken up by tissues with high glucose consumption and is able to accumulate in malignant tissues as well as in infectious, inflammatory, autoimmune and granulomatous lesion sites. Compared to labeled blood cells, 18F-FDG PET has a broader spectrum of diagnostic disease and is more sensitive compared to 67Ga scans. 18F-FDG PET appears to have a high negative predictive value for excluding fever caused by inflammation. The limited number of studies available suggest that 18F-FDG PET imaging is useful for the final diagnosis in 25-69% of patients with FUO, and presumably it is expected to occupy a central position in the second-line means of FUO diagnosis. In non-infectious inflammatory diseases, 18F-FDG PET is useful in the diagnosis of vasculitis of large vessels, inflammatory bowel disease, nodular disease, and painless subacute thyroiditis. In patients with FUO due to tumors, 18F-FDG PET can assist in the diagnosis of Hodgkin's disease, progressive non-Hodgkin's lymphoma, colorectal cancer, and sarcoma. 18F-FDG PET has the potential to replace other imaging tests in the future in the diagnosis of patients with FUO.