Diagnosis Of Small Vessel Vasculitis Research Articles

E018 Prophylactic co-trimoxazole prescribing practice in patients who received rituximab therapy: comparison across indications and specialties

Abstract Background/Aims Rituximab is a chimeric anti-CD20 monoclonal antibody targeting B-cells used as part of treatment algorithms for malignancy and autoimmune diseases. As is the case of other immunosuppressive agents, rituximab increases the risk of infections. Primary prophylaxis with co-trimoxazole may be considered in high-risk cohort of patients to prevent opportunistic infection, particuarly Pneumocystis jerovecii pneumonia (PJP). To investigate the prescribing practice for prophylactic co-trimoxazole in patients who received rituximab infusion in a large tertiary referral centre. To compare practices between rheumatology and nephrology department. To compare the incidence of hospitalisation for infection (particularly PJP) between those prescribed prophylactic co-trimoxazole and those who did not. Methods This was a single centre retrospective observational study over a 3 year period assessing prophylactic co-trimoxazole prescribing practice in 185 patients attending rheumatology or renal services in Cork University Hospital (CUH) and receiving rituximab. Co-trimoxazole prophylactic treatment was defined as prophylaxis dose of co-trimoxazole (either 480mg daily or 960mg three times per week) received during rituximab treatment and at least 6 months post completion of treatment. Results 185 patients’ medical charts were reviewed. Only 28 patients received prophylaxis with co-trimoxazole. The majority of patients (n = 24, 85%) who received prophylaxis with co-trimoxazole had an underlying diagnosis of small vessel vasculitis (SVV). Co-trimoxazole was more commonly prescribed by nephrologists (n = 17, 71% primarily for SVV) as compared to rheumatologist (n = 7, 29% primarily for SVV). 156 patients were not prescribed prophylactic co-trimoxazole. The primary indication for rituximab in these patients was inflammatory arthropathy (IA) (n = 72,46%), SVV (n = 26, 16%), systemic lupus erythematosus (n = 19, 12%), with the remainder accounted for by scleroderma, undifferentiated connective tissue disease, membranous nephropathy (MN), myositis, focal segmental glomerulosclerosis and amyloid renal disease. No patient was admitted over the course of the study period with PJP. 59 patients required hospital admissions due to other infections, most commonly patients with SVV. The most common infections were respiratory tract and urinary tract infections. Overall infections requiring hospitalization were more common in the group receiving prophylaxis, perhaps highlighting the vulnerable nature and greater prednisolone prescribing in the SVV cohort. No difference was seen between groups in the prevalence of UTIs. Conclusion In this cohort co-trimoxazole prophylaxis was more commonly prescribed for patient with small vessel vasculitis and differences in prescribing patterns were seen between nephrologists and rheumatologists. With greater awareness of the heightened risk of PJP in patents receiving rituximab consideration should be given to use of antibiotic prophylaxis for other indications. Disclosure S. Tio: None. J. Morrow: None. S. Cowley: None. M. Clarkson: None. G. Murphy: None.

PULMONARY LIMITED SERONEGATIVE GRANULOMATOSIS WITH POLYANGIITIS VASCULITIS

PULMONARY LIMITED SERONEGATIVE GRANULOMATOSIS WITH POLYANGIITIS VASCULITIS

A RARE PRESENTATION OF GRANULOMATOSIS WITH POLYANGITIS

SESSION TITLE: Fellows Pulmonary Manifestations of Systemic Disease Posters SESSION TYPE: Fellow Case Report Posters PRESENTED ON: October 18-21, 2020 INTRODUCTION: Granulomatosis with polyangiitis (GPA) is a small to medium vessel vasculitis, usually ANCA positive. Clinically this disease is difficult to diagnose, as presentations vary. Patients can have ear, nose, and sinus disease as well as rapidly progressive glomerulonephritis.1 Renal complications occur in up to 75% of patients.1 Rare pulmonary complications include alveolar hemorrhage, nodules, and cavitary lesions. Seldom these patients have mediastinal lymphadenopathy and obstructive lung disease.2 Here we present a case of a woman who developed pulmonary nodules, lymphadenopathy, and obstructive lung disease due to GPA. CASE PRESENTATION: Our patient is a 75yo female nonsmoker with a history of polymyalgia rheumatica in remission that endorsed one year of dyspnea on exertion, hoarseness, and 20 lb weight loss. PFTs noted obstructive ventilatory defect meeting criteria for severe COPD. CT chest revealed multiple pulmonary nodules, mediastinal lymphadenopathy, and emphysematous change throughout of unknown duration as she did not have any previous imaging. CT-guided biopsy pathology showed chronic inflammatory changes, but no malignant cells. PET scan had hypermetabolic nodules bilaterally worrisome for metastasis. Bronchoscopy noted subglottic edema, but we were unable to pass ETT, so EBUS and ENB were aborted. She was noted to have mucosal inflammation throughout the tracheobronchial tree. Repeat CT-chest showed bilateral spiculated nodules increasing in size and number compared to prior scan. Second CT-guided biopsy pathology showed chronic inflammation and fibrosis with small noncaseating granulomas. Based on in-house and outside pathology consult, it was decided she likely had GPA. After evaluation by rheumatology she started rituximab infusions, but pulmonary nodules remained stable in size. P and c-ANCA were negative, along with negative c-ANCA by immunofluorescence. RF, CCP, ESR, CRP and renal function were within normal limits. CT maxillofacial was negative. DISCUSSION: When a vasculitis is suspected, a negative ANCA does not exclude the diagnosis of GPA, so further investigations, such as biopsy, should be considered. The lung is the most common site biopsied. Our case presented a diagnostic challenge given her presentation with a COPD-like syndrome and significant weight loss, as well as p-ANCA negativity. By some estimates <10% of cases of GPA present with isolated disease, such as in our patient.3 CONCLUSIONS: Prompt diagnosis for GPA is needed as the mortality is high without treatment. Treatment includes immunosuppression with glucocorticoids, methotrexate, azathioprine or cyclophosphamide.1 Our case highlights the importance of the pathologist in the diagnosis of small vessel vasculitis to lead the care team to the correct and rapid diagnosis. Reference #1: Seo P, Stone JH. The Antineutrophil Cytoplasmic Antibody-Associated Vasculitides. Am J Med. 2004; 117: 39-50. Reference #2: George TM, Cash JM, Farver C, et al. Mediastinal mass and hilar adenopathy: rare thoracic manifestations of Wegener's granulomatosis. Arthritis Rheum. 1997;40:1992–1997. Reference #3: Ananthakrishan L, Sharma N, Kanne JP. Wegner's Granulomatosis in the Chest: High-Resolution CT Findings. AJR 2009: 192; 676-682. DISCLOSURES: My spouse/partner as a Consultant relationship with Abbvie Please note: $1-$1000 Added 05/27/2020 by Ebtesam Islam, source=Web Response, value=Consulting fee No relevant relationships by Ximena Solis, source=Web Response

Utility of Direct Immunofluorescence in the Diagnosis of Small Vessel Vasculitis of the Skin: A Cross-Sectional Study.

Utility of Direct Immunofluorescence in the Diagnosis of Small Vessel Vasculitis of the Skin: A Cross-Sectional Study.

Urinary Soluble CD163 in Active Renal Vasculitis

A specific biomarker that can separate active renal vasculitis from other causes of renal dysfunction is lacking, with a kidney biopsy often being required. Soluble CD163 (sCD163), shed by monocytes and macrophages, has been reported as a potential biomarker in diseases associated with excessive macrophage activation. Thus, we hypothesized that urinary sCD163 shed by crescent macrophages correlates with active glomerular inflammation. We detected sCD163 in rat urine early in the disease course of experimental vasculitis. Moreover, microdissected glomeruli from patients with small vessel vasculitis (SVV) had markedly higher levels of CD163 mRNA than did those from patients with lupus nephritis, diabetic nephropathy, or nephrotic syndrome. Both glomeruli and interstitium of patients with SVV strongly expressed CD163 protein. In 479 individuals, including patients with SVV, disease controls, and healthy controls, serum levels of sCD163 did not differ between the groups. However, in an inception cohort, including 177 patients with SVV, patients with active renal vasculitis had markedly higher urinary sCD163 levels than did patients in remission, disease controls, or healthy controls. Analyses in both internal and external validation cohorts confirmed these results. Setting a derived optimum cutoff for urinary sCD163 of 0.3ng/mmol creatinine for detection of active renal vasculitis resulted in a sensitivity of 83%, specificity of 96%, and a positive likelihood ratio of 20.8. These data indicate that urinary sCD163 level associates very tightly with active renal vasculitis, and assessing this level may be a noninvasive method for diagnosing renal flare in the setting of a known diagnosis of SVV.

Outcomes of patients admitted to intensive care units for acute manifestation of small-vessel vasculitis: a multicenter, retrospective study

BackgroundThe outcomes of patients admitted to the intensive care unit (ICU) for acute manifestation of small-vessel vasculitis are poorly reported. The aim of the present study was to determine the mortality rate and prognostic factors of patients admitted to the ICU for acute small-vessel vasculitis.MethodsThis retrospective, multicenter study was conducted from January 2001 to December 2014 in 20 ICUs in France. Patients were identified from computerized registers of each hospital using the International Classification of Diseases, Ninth Revision (ICD-9). Inclusion criteria were (1) known or highly suspected granulomatosis with polyangiitis, eosinophilic granulomatosis with polyangiitis, microscopic polyangiitis (respectively, ICD-9 codes M31.3, M30.1, and M31.7), or anti–glomerular basement membrane antibody disease (ICD-9 codes N08.5X-005 or M31.0+); (2) admission to the ICU for the management of an acute manifestation of vasculitis; and (3) administration of a cyclophosphamide pulse in the ICU or within 48 h before admission to the ICU. The primary endpoint was assessment of mortality rate 90 days after admission to the ICU.ResultsEighty-two patients at 20 centers were included, 94 % of whom had a recent (<6 months) diagnosis of small-vessel vasculitis. Forty-four patients (54 %) had granulomatosis with polyangiitis. The main reasons for admission were respiratory failure (34 %) and pulmonary-renal syndrome (33 %). Mechanical ventilation was required in 51 % of patients, catecholamines in 31 %, and renal replacement therapy in 71 %. Overall mortality at 90 days was 18 % and the mortality in ICU was 16 %. The main causes of death in the ICU were disease flare in 69 % and infection in 31 %. In univariable analysis, relevant factors associated with death in nonsurvivors compared with survivors were Simplified Acute Physiology Score II (median [interquartile range] 51 [38–82] vs. 36 [27–42], p = 0.005), age (67 years [62–74] vs. 58 years [40–68], p < 0.003), Sequential Organ Failure Assessment score on the day of cyclophosphamide administration (11 [6–12] vs. 6 [3–7], p = 0.0004), and delayed administration of cyclophosphamide (5 days [3–14] vs. 2 days [1–5], p = 0.0053).ConclusionsPatients admitted to the ICU for management of acute small-vessel vasculitis benefit from early, aggressive intensive care treatment, associated with an 18 % death rate at 90 days.Electronic supplementary materialThe online version of this article (doi:10.1186/s13054-016-1189-5) contains supplementary material, which is available to authorized users.

The use of muscle biopsy in the diagnosis of systemic vasculitis affecting small to medium-sized vessels: a prospective evaluation in Japan

Objectives: The aim of this study was to assess the use of muscle biopsy for histopathological confirmation of small vessel vasculitis (SVV) or medium vessel vasculitis (MVV).Method: Muscle biopsies were performed for all consecutive cases of suspected SVV or MVV seen at Tokyo Metropolitan Tama Medical Centre between February 2012 and May 2014 except those for which a skin or renal biopsy was indicated.Results: Forty-nine patients underwent muscle biopsies. All patients were followed for a minimum of 6 months. Diagnosis of SVV or MVV was made in 35 patients. An unrelated condition was diagnosed in 11 patients and no diagnoses were made in three patients. Of the 35 patients in whom SVV or MVV was diagnosed, positive muscle biopsies were obtained in 20 patients [15 microscopic polyangiitis (MPA), three polyarteritis nodosa (PAN), and two eosinophilic granulomatosis with polyangiitis (EGPA)], while other findings led to the same diagnosis in 15 (seven MPA, four GPA, three PAN, and one rheumatoid vasculitis). The sensitivity of the muscle biopsy was 57% [20/35; 95% confidence interval (CI) 50–57]. Of 13 patients presenting with peripheral neuropathy, the muscle biopsy demonstrated vasculitis in nine patients, with 75% sensitivity (9/12; 95% CI 69–75). There were no complications in the procedure apart from delayed wound healing in one patient.Conclusions: Muscle biopsy is a safe method that offers a high diagnostic yield for SVV or MVV, especially in patients with vasculitic neuropathy.

Clinically-Irrelevant Positivity for Serum Proteinase 3-ANCA in HIV Disease

Diagnosis of small-vessel vasculitides (granulomatosis with polyangiitis, microscopic polyangiitis, Churg-Strauss syndrome, and their localized forms) is aided by the detection of serum antineutrophil cytoplasmic antibodies (ANCA). However, serum ANCA positivity does not always mean vasculitis. Here, we report on a 61-year-old female patient with very high serum levels of proteinase 3 ANCA, marked hypergammaglobulinemia, low complement levels, and a 16-mm lung nodule on chest CT scan, who was referred to our Institution with a provisional diagnosis of possible granulomatosis with polyangiitis. On admission, history-taking disclosed two recent episodes of viral reactivation (namely, cytomegalovirus and Varicella-Zoster virus), while physical examination revealed lingual and nail involvement suggestive of Candida infection. An immunodeficiency disorder was eventually suspected. Search for antibodies against human immunodeficiency virus (HIV) 1 and 2 turned positive. Western blot analysis confirmed HIV infection. Thus, although ANCA detection may be helpful in diagnosing small-vessel vasculitis in the appropriate clinical scenario, screening for HIV infection should sometimes be considered to discriminate clinically irrelevant serum ANCA positivity.

AB0770 Systematic review of the value of positron emission tomography imaging in the diagnosis & management of large vessel vasculitis

BackgroundA systematic literature review was conducted to evaluate the value of Positron Emission Tomography (PET) scan in the diagnosis and management of large vessel vasculitis. Vasculitis is classified by American...

AB0766 The role of color-doppler-sonography in the diagnosis of giant cell arteritis characterized by adventitial inflammation of the temporal arteries

BackgroundThe classic histological appearance of inflamed temporal arteries (TA) in giant cell arteritis (GCA) is transmural cell infiltration. However, periadventitial small vessel vasculitis (SVV) surrounding uninflamed TA and/or isolated vasculitis...

Clinical features and diagnosis of small-vessel vasculitis

Vasculitis is inflammation of the blood vessel. Granulomatosis with polyangiitis (GPA), microscopic polyangiitis, and eosinophilic GPA are three small-vessel vasculitic diseases that share certain features, but also have important differences. Distinguishing these entities may influence the diagnostic approach, treatment decisions, and outcomes. Circulating antineutrophil cytoplasmic antibodies (ANCA) characterize all three diseases, although their immunofluorescence patterns and target antigen specificities differ. While the presence of ANCA can suggest these diagnoses, the diseases are best viewed as separate entities, each defined by specific clinical and histologic characteristics.

Antineutrophil cytoplasmic antibodies

Antineutrophil cytoplasmic antibodies (ANCA) are predominantly IgG autoantibodies directed against constituents of primary granules of neutrophils and monocytes lysosomes. Although several antigenic targets have been identified, those ANCA directed to proteinase 3 or myeloperoxidase are clinically relevant, whereas the importance of other ANCA remains unknown. Both are strongly associated with small vessel vasculitides, the ANCA-associated vasculitides, which include Wegener's granulomatosis, microscopic polyangiitis, and Churg-Strauss syndrome, and the localised forms of these diseases (eg, pauci-immune necrotising and crescentic glomerulonephritis). ANCA is a useful serological test to assist in diagnosis of small-vessel vasculitides. 85-95% of patients with Wegener's granulomatosis, microscopic polyangiitis, and pauci-immune necrotising and crescentic glomerulonephritis have serum ANCA. ANCA directed to either proteinase 3 or myeloperoxidase are clinically relevant, yet the relevance of other ANCA remains unknown. Besides their diagnostic potential, ANCA might be valuable in disease monitoring. In addition, data seem to confirm the long-disputed pathogenic role of these antibodies. There is increasing evidence that myeloperoxidase-ANCA are directly involved in the pathogenesis of necrotizing vasculitis. This is less clear for proteinase 3-ANCA, markers for Wegener's granulomatosis. With respect to proteinase 3-ANCA, complementary proteinase 3, a peptide translated from the antisense DNA strand of proteinase 3 and homologous to several microbial peptides, may be involved in induction of proteinase 3-antineutrophil cytoplasmic autoantibodies.

Direct immunofluorescence of skin biopsy: Perspective of an immunopathologist

By direct immunofluorescence (DIF), presence of immune complexes in the skin biopsy at various locations such as the dermo-epidermal junction, dermal blood vessels, etc. help to arrive at a diagnosis. (1) To study the role of DIF in confirmation or exclusion of diseases involving skin vis-à-vis histopathology and clinical diagnosis, (2) to describe the annual spectrum of dermatologic conditions that present to a tertiary referral center and require DIF examination of skin biopsy for confirmation of diagnosis. A total of 267 biopsies received over a period of 16 months in the Department of Immunopathology were analyzed along with clinical and histopathological details and the correlation between them was studied. DIF was positive in 204 skin biopsies. Of these, 127 biopsies showed good clinico-immuno-histopathological correlation. In 10 cases, only DIF could clinch the diagnosis. In another nine cases, immune deposits were noted, which were unexpected in light of clinical and histopathological diagnosis. The most common skin involvement was seen in vasculitides. DIF was, however, non-contributory in lesions like erythema multiformè, post Kala-azar dermal leishmaniasis, sarcoidosis, lupus vulgaris, pyoderma gangrenosum and prurigo nodularis. The DIF of skin in conjunction with histopathology gives the best diagnostic yield. It is invaluable in confirming the diagnosis of small vessel vasculitides and bullous lesions of skin and can be used as an additional tool to pinpoint the diagnosis of systemic and localized autoimmune diseases involving the skin.

Use of Likelihood Ratios Can Improve the Clinical Usefulness of Enzyme Immunoassays for the Diagnosis of Small-Vessel Vasculitis

Antineutrophil cytoplasmic antibodies (ANCAs)1 are associated with small-vessel vasculitis (SVV), such as Wegener granulomatosis, microscopic polyangiitis, and Churg–Strauss syndrome (1). Indirect immunofluorescence (IIF) analyses can distinguish 2 major patterns: cytoplasmic and perinuclear. The cytoplasmic ANCA pattern is typically associated with antibodies to proteinase 3 (PR3), whereas the perinuclear ANCA pattern is typically associated with antibodies to myeloperoxidase (MPO). PR3 ANCAs are predominantly seen in patients with Wegener granulomatosis, whereas MPO ANCAs are predominantly seen in patients with microscopic polyangiitis(1)(2)(3). Studies that have addressed the clinical usefulness of specific enzyme immunoassays (EIAs) for PR3 and MPO for the diagnosis of SVV have used a single cutoff value. In this letter, we illustrate how the likelihood ratio (LR) for SVV depends on the titer of anti-PR3 or anti-MPO antibodies. Our calculations are based on a clinically well-defined group of 37 consecutive patients with newly diagnosed SVV (data collected over a 10-year period) and 285 consecutive control individuals with disease (data collected over 21 months) that a physician suspected to be a …

Small vessel vasculitis

Background Vasculitis is a primary inflammatory process of large, medium-sized, and small blood vessels. There are multiple entities particularly among small vessel vasculitides. Some are life threatening and require treatment with toxic agents. Diagnostic challenges are posed by low prevalence, controversial classification, inconsistency of clinical and pathological features, and the existence of clinical and pathological “look-alikes”. Also, patients may present to a variety of medical specialists, and the diagnosis is often unexpected. The cause often is unknown. Review Classification is by vessel size and then on an immunopathologic basis. Generalised and “organ-limited” forms occur. The principal clinical and pathological features are outlined for each of antibasement membrane disease, immune complex disorders including Henoch–Schönlein purpura and cryoglobulinemic vasculitis and the pauci-immune group, which is often associated with antineutrophil cytoplasmic antibody (ANCA), comprising microscopic polyangiitis, Wegener's granulomatosis, and Churg–Strauss syndrome. A brief account is given of “look-alikes” including microthrombotic conditions, which can confound the diagnosis of small vessel vasculitis. Conclusion Requirements for diagnosis include full disclosure of the past and present medical history with review of laboratory results, especially diagnostic immunology. Histology should be of targeted biopsies of recent active lesions in preference to blind biopsies. Sampling should be extensive using high-quality thin sections. Systematic microscopic evaluation of architectural features and cellular detail is necessary. Tissue immunofluorescence is a useful adjunct. The final opinion must take all available information into account but may ultimately depend on a critical judgement by the pathologist.

Les titres d’ANCA répétés peuvent-ils prédire la rechute des vascularites ANCA positives ?

Antineutrophil cytoplasm autoantibodies (ANCA) are essential for the diagnosis of small-vessel vasculitis. Information for and against their usefulness to predict relapse exists. Different approaches, using various methods and definitions, have been used to answer the role of ANCA in predicting disease relapse. Multinational, multicentric prospective studies are needed to address this question using uniform definitions and preferably the best latest tools for ANCA detection.

C3 and C4 allotypes in anti-neutrophil cytoplasmic autoantibody (ANCA)-positive vasculitis.

In ANCA-associated small vessel vasculitis few genetic factors have proven to be of importance for disease susceptibility, an exception being deficiency of alpha1-anti-trypsin, the main inhibitor of proteinase 3 (PR3). Alerted by our finding that myeloperoxidase has affinity for C3, and the finding of an increased frequency of the C3F allele in systemic vasculitis in a British cohort, we examined polymorphism of C3 and C4 in patients with ANCA+ small vessel vasculitis. After identification of all patients at our department with a positive ANCA test during the period 1991-95 and a diagnosis of small vessel vasculitis, blood samples were collected after informed consent. The 67 included patients were grouped according to ANCA serology and disease phenotype using the Chapel Hill nomenclature. The gene frequency of C3F was found to be increased (0. 32) compared with controls (0.20; P < 0.05) in the PR3-ANCA+ subgroup. The frequency of C4A3 was increased in the group as a whole, but no increase of C4 null alleles was seen. The findings imply a role for the complement system in the pathogenesis of ANCA-associated small vessel vasculitis.

Cutaneous vasculitis and collapse.

Accepted 23 April 1998 A 20-year-old man was admitted with a 1-week history of a vasculitic rash over both legs and aching in both calves. He felt otherwise well although...

Patients hospitalized because of small vessel vasculitides with renal involvement in the period 1975-95: organ involvement, anti-neutrophil cytoplasmic antibodies patterns, seasonal attack rates and fluctuation of annual frequencies.

To study organ involvement, anti-neutrophil cytoplasmic antibodies (ANCA) patterns, trends in yearly frequencies and seasonal variations of symptom onset in patients hospitalized because of small vessel vasculitides during a 21 year period (1975-95). A retrospective investigation was conducted of 138 patients hospitalized with a diagnosis of small vessel vasculitides, as defined by the Chapel Hill Consensus Conference, within the County of Orebro, a mixed urban and rural area of central Sweden. Orebro Medical Center Hospital, Orebro, Sweden and two district hospitals within the County of Orebro, Sweden. During the studied period there were 19 patients with a diagnosis of Wegener's granulomatosis (WG), 70 patients with microscopic polyangiitis (MPA), 36 patients with renal limited vasculitis (RLV), two with Churge-Strauss vasculitis (C-S), seven with Henoch-Schönleins purpura (HSP) and four with essential cryoglobulinemic vasculitis (ECV). Renal involvement was present in 123 patients (89.1%). A positive c- and/or pANCA was found in nearly 90% of the 111 patients where sera were available. Calculations of frequency data, restricted to the primary catchment area for patients with ANCA associated vasculitis and renal involvement (WG, MPA, RLV) during a 21-year period (1975-95) gave a mean annual frequency of 1.6 per 100,000 adults (95% CI: 1.2-3.1); for this group of patients with the inclusion of those with C-S, HSP and ECV during the last 10 year period (1986-95) gave a mean annual frequency of 2.5 per 100,000 adults (95% CI: 1.7-3.4), for male adults 3 per 100,000 (95% CI: 1.6-4.4), and female adults 1.9 (95% CI: 0.9-2.8). A frequency peak of 6.3 per 100,000 was seen for men aged 55-64. A periodic fluctuation of the frequencies with peaks every 3-4 years was noted for patients with ANCA related vasculitis (WG, MPA, RLV) during the 21-year period 1975-95. Onset of symptoms was predominantly noticed during the winter months (December-February) for patients with a positive cANCA. The observed frequencies in our study of patients with small vessel vasculitides were higher than those previously documented. We also showed a periodic fluctuation of the annual frequencies and a seasonal variation of symptom onset.