Herpesvirus-Associated Acute Urticaria: An Age Matched Case-Control Study

Background Acute and recurrent acute urticaria are often associated with multiple factors including infections and recent data suggest a role for herpesviruses. Objective To test the null hypothesis, that is, there is no association of herpesvirus infections with urticaria. Methods Thirty-seven patients between one month and 15 years of age were age matched to 37 controls who were healthy or had mild acute respiratory infections but without urticaria. Patients and controls were followed for 1 to 6 years. Diagnostic studies included DNA detection by real-time PCR for herpes simplex virus (HSV) types 1 and 2, Epstein-Barr virus (EBV), cytomegalovirus (CMV) and human herpesvirus-6 (HHV-6). Tests for other infections included adenovirus, parvovirus B 19, respiratory syncytial virus, influenza A, Group A streptococci, rotavirus, and parasites. Results Specific infections were diagnosed in 26 of 37 cases and among 9 of 37 control children (P=0.0002). Single or concomitant herpesvirus infections occurred in 24 cases and in 4 controls (65% vs 11 %, p=0.0003). Cases had 10 HHV-6 infections, 8 CMV infections, 5 EBV infections, and 4 HSV-1 infections. Conclusion Herpesvirus infections are associated with acute or recurrent acute urticaria.


Introduction
Urticaria is a common skin reaction characterized by itchy short-lived elevated erythematous wheals anywhere on the skin. Acute urticaria is associated with symptoms for less than 6 weeks, and is considered to be recurrent acute if the duration of symptom-free periods between urticaria episodes is longer than 6 weeks [1]. Although a specific cause is not frequently identified, the onset of acute urticaria has been associated with allergic reactions to foods and drugs, contact with chemicals, physical stimuli, or infections.
Nearly all of the numerous studies reporting evidence for infectious agents triggering acute or recurrent acute urticaria are retrospective observational studies without appropriate controls or are case reports. Different studies reported a rate ranging from 37% to 58% of multiple infections among 54 or 79 patients with acute urticaria, respectively [2,3]. Another study highlighted the role of Mycoplasma pneumoniae infection, which occurred in 32% of 65 children with acute urticaria [4]. Other reports focused on an association between acute urticaria and streptococcal infection, hepatitis A and B viruses, parvovirus B19, cytomegalovirus (CMV), coxsackie A9 virus, enterovirus, influenza A and parainfluenza viruses [5][6][7][8][9][10]. Acute urticaria has also been observed after influenza vaccination [11]. Upper respiratory or digestive symptoms are common with acute urticaria associated with infections [10,12,13]. These studies do not provide a compelling case for causal link between a specific infection and the development of urticaria. Reports of herpesvirus infections are frequent but reports of associated urticaria are few. We thus investigated the association of herpes simplex virus (HSV) types 1 and 2, Epstein-Barr virus (EBV), CMV and human herpesvirus-6 (HHV-6) with acute or recurrent acute urticaria.

Ethical statement
The study was approved by the local ethical committee named Comitato Etico del Dipartimento di Medicina Sperimentale e Sanità Pubblica, Università dell'Aquila, L'Aquila, Italy. Written consent was obtained from each subject or their parent or guardian. The local ethical committee approved the written consent process. Consent was documented in writing.

Subjects
A prospective study was performed from November 1, 2005 to October 30, 2010, and included 37 patients, 26 of whom were outpatients and 11 were hospitalized due to a first attack of acute urticaria. All patients had a physical examination and history that included time of onset of disease, frequency duration size and distribution of wheals, diurnal variation, shape, associated angioedema, family history of urticaria and atopy, previous or current allergies, infections, gastric/intestinal problems, use of drugs, and correlation to food. The diagnosis of urticaria was made by the appearance of circumscribed, slightly elevated, erythematous, edematous papules or wheals. Lesions varied from moment to moment and disappeared within minutes or hours. As controls 37 children who were healthy or had mild acute respiratory infections, who were predominantly siblings or playmates of the patients, were agematched to within one year of the cases and had the same diagnostic studies. Both cases and controls were followed every 3 to 6 months for 1 to 6 years (mean: 4.2 years and 3.8 respectively).
In both cases and controls at enrollment EBV-DNA, CMV-DNA and HHV-6-DNA were detected in the serum and saliva with the use of Real-Time polymerase-chain-reaction. IgG and IgM antibodies against HSV, EBV, CMV, and HHV-6 were examined in serum samples, using commercial enzyme-linked immunosorbent assay from Radim (Pomezia, Italy) and Sorin (Saluggia, Italy). A viral infection was considered as active if high levels of virus-specific IgM antibodies were initially detected with an increase in concentration of viral specific IgG antibodies or HSV-1/2, EBV, CMV or HHV-6 DNA was detected in at least one sample. Other enrollment studies for cases and controls included IgG and IgM anti-adenovirus, respiratory syncythial virus, influenza virus and parvovirus B19 as well as rotavirus detection in the stools. Group A streptococcal infection was diagnosed if a throat swab was positive and/or a significant ASO increase (≥ double of previous one) occurred. Specific IgG and IgM antibodies towards Mycoplasma and Chlamydia pneumoniae were performed in children with upper respiratory infections. Helicobacter pylori and specific IgG antibodies were tested for in children with recurrent abdominal pain.
Allergic studies included skin prick and total and specific IgE. Skin test was determined by using undiluted commercial allergens (cow's milk, whole eggs, chocolate, tomato, grasses, olive, Parietaria, Artemisia, Alternaria, Ambrosia, Betulla, Dermatophagoides pteronyssinus, Dermatophagoides farinae, Penicillium, dog, cat). A positive histamine control skin test was performed. A positive skin test was defined as inducing a reaction with a wheal size measuring 3 mm or more after subtraction of the control value. Specific IgE antibodies to bovine milk proteins (α-lactalbumin, β-lactoglobulin, and casein) was determined by radioallergosorbent assay (RAST).

Statistical analysis
Statistical analyses were done in part with JMP8.0 (SAS Institute Inc.). McNemar's chi square tests were used for paired comparisons. Variables with a univariate P value of <0.2 were included in a logistic regression model. Multinomial logistic regression to analyze a 1-1 matched case-control study was performed using IBM SPSS Statistics Version 19 (2010, SPSS Inc).

Clinical manifestations of urticaria
Tables 1 and 2 describe the characteristics of cases and controls. The mean age of the cases and controls was 3.8 years (Table 3) with a range from 1 month to 15 years. Twenty of 37 (54%) cases had a single episode of acute urticaria, and the remaining 17 (46%) had recurrent acute urticaria. While acute urticaria prevailed in children younger than 2 or older than 6 years of age, recurrent urticaria occurred more frequently in children between 2 and 6 years (p <0.05). Urticaria was associated with other clinical manifestations in 27 cases (73%).

Risk factors
Ten cases (37%) had past allergic manifestations (Table 1). Six cases had increased levels of specific IgE antibodies towards cow's milk, egg, Dermatophagoides pteronissimus/ farinae. One case had an increased level of total IgE antibodies. Only two cases had positive skin prick tests to Graminaceae. Ibuprofen was the possible cause of acute urticaria in only one case.

Virologic findings
Specific infections were diagnosed in 26 of 37 cases and among 9 of 37 control children (70% vs 24%; P=0.0002). Of infected cases, 25 had viral infections, six had viral-bacterial co-infections, six were infected with two viruses, and one had M. pneumoniae infection. Two or more infections occurred in 7 of 17 cases (41%) who had recurrent urticaria compared with The only infections significantly associated with urticaria were infections with one of the four herpesviruses, CMV, EBV, HSV, or HHV-6 ( Table 3). In a multinomial regression analysis infections by HHV-6, HSV, and EBV were each independently predictive of acute or recurrent urticaria and CMV approached statistical significance (P = 0.1). None of the other infections whether viral or bacterial approached statistical significance (Table 3). Among 24 cases with a herpesvirus infection, 13 (54%) had recurrent acute urticaria compared with recurrent acute urticaria in 4 of 13 (31%) cases without a herpesvirus infection (p = 0.3). Atopy or allergies were not statistically (p =0.3) more frequent among cases with herpesvirus infections (13/17 = 76%) than cases without (11/20 =55%).

Discussion
Acute urticaria occurs in 15% to 25% of people at a some point in their life, being associated with transudation of fluid from skin blood vessels and release of mediators from cutaneous mast cells and basophils [14] The release of mast cell-derived mediators may be caused by both immune and non immune mechanisms [15]. The association of acute urticaria with atopy is common, being found up to 50% of the cases and occasionally associated with an increase in total IgE level, which may represent a nonspecific marker of the immune mechanisms involved in urticaria rather than a sign of underlying atopy [12].
In children, acute urticaria is often considered as a food or drug allergy, leading to frequently unnecessary diet restrictions or elimination of drugs and administration of antihistaminic or corticosteroids. Identification of the true casual factors for urticaria would be very helpful in the management and prognosis. Possible causes, however, are numerous having been reported in between 21% to 83% of cases [2,16]. This high variability is due to the various criteria used in evaluating the causes and the enrollment criteria. The various criteria used have included acute or chronic infections, non allergic hypersensitivity reactions to foods and drugs, and autoimmunity mediated by functional autoantibodies directed against the IgE receptors [13,[17][18][19]. Between 37% and 58%, of children with acute urticaria also have infections, usually virus-induced upper respiratory infections [2,3]. Viral infections have also been associated to the onset of atopic manifestations and an increase in systemic IgE levels [20]. Infections, however, frequently also occur in patients with drug allergies making it difficult to distinguish between the two possible causal factors [16].
Ours is the first prospective age-matched case-control study to evaluate the association between acute or recurrent acute urticaria and a broad range of infectious causes in children. One previous study reported a 32% rate of M. pneumoniae infections among 65 children but a so called control group of 49 children with respiratory infections was not tested for M. pneumoniae. We found no evidence that acute urticaria was associated with Mycoplasma or streptococcal or respiratory viral infections. We did however observed that the onset of acute urticaria was strongly associated with an herpesvirus infection, including children who developed recurrent acute urticaria. Herpesviruses can persist long life alternating latent and reactivation phases, thus primary or reactivated herpesvirus infections may be associated with acute or recurrent acute urticaria. Cellular immunity controls herpesvirus infections and both primary and recurrent infections are associated with altered cell-mediated responsiveness and production of a number of cytokines [21]. As occurs in virusinduced asthma exacerbations after viral induction of mast cell activation, the activation of mast cells may be involved in herpesvirus-induced diseases, including the urticaria in genetically predisposed children [15,22].
HHV-6, CMV and EBV infections are common in children. In particular the seroprevalence rate of HHV-6 is between 80% and 100% by the age of 3 years [23]. HHV-6 usually causes exanthema subitum but can affect the immunosurveillance  production of interferon-α and tumor necrosis factor-α, and upregulates NK cell cytotoxicity via interleukin-15 [24,25]. CMV infection is common both pre and postnatally with an overall prevalence of between 15% and 25% among children, and also has many effects on the immune system mostly via cellmediated immunity [21]. Eight children had persistent CMV DNA detection for more than 3 months after acute urticaria: six of these children developed recurrent acute urticaria. EBV and HSV-1 infections also stimulate cellular and antibody immune responses [21,26]. Caubet   patients with a positive OCT (50%) had findings suggestive of an acute or recent EBV infection [27].
Herpesviruses are a component of the human "microbiome" since they are adapted to lifelong infection of their hosts. Recent evidence suggest a possible role for herpesviruses in allergy and atopy [9]. Our results with urticaria and those with allergic and atopic reactions suggest physicians caring for allergic and atopic populations should be familiar with the manifestations of herpes-related disease [27,28].
Because herpesvirus infections are common among children, possible limitations of our study include the small sample size and the practical limitation of only being able to obtain, in most cases, single specimens for diagnostic testing. Perhaps more subjects and repetitive testing would skew the results. Nevertheless with a significantly higher rate than controls, we observed a herpesvirus infection in 65% of our patients which suggests herpesviruses may be component of the process involved in development of acute or recurrent acute urticaria.