Comparison of MT-PCR with Quantitative PCR for Human Bocavirus in Respiratory Samples with Multiple Respiratory Viruses Detection.

Human bocavirus (HBoV) is an important respiratory pathogen, especially in children, but it is often found in co-detection with other respiratory viruses, which makes the diagnostic approach challenging. We compared multiplex PCR and quantitative PCR for HBoV with multiplex tandem PCR (MT-PCR) in 55 cases of co-detection of HBoV and other respiratory viruses. In addition, we investigated whether there is a connection between the severity of the disease, measured by the localization of the infection, and amount of virus detected in the respiratory secretions. No statistically significant difference was found, but children with large amount of HBoV and other respiratory virus had a longer stay in hospital.

Severe Protein Loss in a 6-month-old Exclusively Breastfed Infant with Atopic Dermatitis.

Protein loss is often the result of kidney or intestinal disease (protein-losing enteropathy) and can cause a number of serious, potentially life-threatening complications such as hypotension, thrombocytosis, electrolyte imbalance, and cerebellar ischemia. Recent research suggests an association between extremely severe atopic dermatitis (AD) and allergic enteropathy. An exclusively breastfed 6-month-old infant was admitted to our institution due to failure to thrive, electrolyte imbalance, and severe AD (SCORing Atopic Dermatitis; SCORAD 40). On admission, the infant was in poor general condition, dehydrated, malnourished (bodyweight 4870 g, -3.98 z-score), with exudative erythematous morphs scattered throughout the body. Initial laboratory results showed microcytic hypochromic anemia, hypoalbuminemia, hypogammaglobinemia, thrombocytosis, hyponatremia, high values of total immunoglobulin E (IgE), and eosinophilia. Polysensitization to a number of nutritional and inhalation allergens was demonstrated, and an exclusive amino acid-based formula has been introduced into the diet. During the hospital course, the patient developed superficial thrombophlebitis and methicillin-resistant Staphylococcus aureus (MRSA) bacteremia. Eosinophilia was found in a small intestine biopsy sample. Due to severe hypogammaglobulinemia, skin infections, and bacteremia, the differential diagnosis included primary immune deficiency (STAT3 deficiency, DOCK8 deficiency, PGM3 deficiency, IPEX), but all available immunological tests were unremarkable. Exclusive amino acid-based formula diet was continued in the infant, with topical corticosteroids under wet-dressing therapy and intravenous immunoglobulin replacement therapy. With the gradual improvement of the general condition, the introduction of solid foods was started according to the findings of allergy testing. At 17 months of age, the patient gained weight and his skin status has been improving, although frequent use of topical corticosteroids was necessary. There were no infections, no anemia or thrombocytosis, and albumin and immunoglobulin supplementation were no longer required. The main mechanism of protein loss in infants with extremely severe atopic dermatitis is probably due to damaged skin, and partially due to the eosinophilic inflammation of the small intestine. Immunoglobulin loss, potentiated by physiological or transient hypogammaglobulinemia in infants, poses a very high risk for severe, potentially life-threatening infections.

Prevalence and Molecular Characterization of Human Bocavirus Detected in Croatian Children with Respiratory Infection.

Human bocavirus (HBoV) 1 is considered an important respiratory pathogen, while the role of HBoV2-4 in clinical disease remains somewhat controversial. Since, they are characterized by a rapid evolution, worldwide surveillance of HBoVs' genetics is necessary. This study explored the prevalence of HBoV genotypes in pediatric patients with respiratory tract infection in Croatia and studied their phylogeny. Using multiplex PCR for 15 respiratory viruses, we investigated 957 respiratory samples of children up to 18 years of age with respiratory tract infection obtained from May 2017 to March 2021 at two different hospitals in Croatia. Amplification of HBoV near-complete genome or three overlapping fragments was performed, sequenced, and their phylogenetic inferences constructed. HBoV was detected in 7.6% children with a median age of 1.36 years. Co-infection was observed in 82.2% samples. Sequencing was successfully performed on 29 HBoV positive samples, and all belonged to HBoV1. Croatian HBoV1 sequences are closely related to strains isolated worldwide, and no phylogenetic grouping based on mono- or co-infection cases or year of isolation was observed. Calculated rates of evolution for HBoV1 were 10-4 and 10-5 substitutions per site and year. Recombination was not detected among sequences from this study.

The Emerging Role of Rhinoviruses in Lower Respiratory Tract Infections in Children - Clinical and Molecular Epidemiological Study From Croatia, 2017-2019.

Rhinoviruses (RVs) are increasingly implicated not only in mild upper respiratory tract infections, but also in more severe lower respiratory tract infections; however, little is known about species diversity and viral epidemiology of RVs among the infected children. Therefore, we investigated the rhinovirus (RV) infection prevalence over a 2-year period, compared it with prevalence patterns of other common respiratory viruses, and explored clinical and molecular epidemiology of RV infections among 590 children hospitalized with acute respiratory infection in north-western and central parts of Croatia. For respiratory virus detection, nasopharyngeal and pharyngeal flocked swabs were taken from each patient and subsequently analyzed with multiplex RT-PCR. To determine the RV species in a subset of positive children, 5'UTR in RV-positive samples has been sequenced. Nucleotide sequences of referent RV strains were retrieved by searching the database with Basic Local Alignment Tool, and used to construct alignments and phylogenetic trees using MAFFT multiple sequence alignment tool and the maximum likelihood method, respectively. In our study population RV was the most frequently detected virus, diagnosed in 197 patients (33.4%), of which 60.4% was detected as a monoinfection. Median age of RV-infected children was 2.25 years, and more than half of children infected with RV (55.8%) presented with lower respiratory tract infections. Most RV cases were detected from September to December, and all three species co-circulated during the analyzed period (2017-2019). Sequence analysis based on 5'UTR region yielded 69 distinct strains; the most prevalent was RV-C (47.4%) followed by RV-A (44.7%) and RV-B (7.9%). Most of RV-A sequences formed a distinct phylogenetic group; only strains RI/HR409-18 (along with a reference strain MF978777) clustered with RV-C strains. Strains belonging to the group C were the most diverse (41.6% identity among strains), while group B was the most conserved (71.5% identity among strains). Despite such differences in strain groups (hitherto undescribed in Croatia), clinical presentation of infected children was rather similar. Our results are consistent with newer studies that investigated the etiology of acute respiratory infections, especially those focused on children with lower respiratory tract infections, where RVs should always be considered as potentially serious pathogens.

HEREDITARY ANGIOEDEMA DUE TO C1-INHIBITOR DEFICIENCY IN PEDIATRIC PATIENTS IN CROATIA - FIRST NATIONAL STUDY, DIAGNOSTIC AND PROPHYLACTIC CHALLENGES.

Hereditary angioedema (HAE) is a rare autosomal dominant disease with deficiency (type I) or dysfunction (type II) of C1 inhibitor, caused by mutations in the C1-INH gene, characterized by recurrent submucosal or subcutaneous edemas including skin swelling, abdominal pain and life-threatening episodes of upper airway obstruction. The aim of this study was to investigate healthcare experiences in children with HAE due to C1 inhibitor deficiency (C1-INH-HAE) in Croatia in order to estimate the number of affected children and to recommend management protocols for diagnosis, short-term prophylaxis and acute treatment. Patients were recruited during a 4-year period at five hospitals in Croatia. Complement testing was performed in patients with a positive family history. This pilot study revealed nine pediatric patients positive for C1-INH- HAE type I, aged 1-16 years, four of them asymptomatic. Before the age of one year, C1-INH levels may be lower than in adults; it is advisable to confirm C1-INH-HAE after the age of one year. Plasma-derived C1-INH is recommended as acute and short-term prophylactic treatment. Recombinant C1-INH and icatibant are licensed for the acute treatment of pediatric patients. In Croatia, HAE is still underdiagnosed in pediatric population.

High Detection Rates of Human Bocavirus in Infants and Small Children with Lower Respiratory Tract Infection from Croatia.

BACKGROUND: Human bocavirus (HBoV) is known to cause lower respiratory tract infections (LRTI) in children and may result in substantial morbidity and mortality. The aim of this study was to determine HBoV prevalence among hospitalized infants and small children with acute LRTI in Zagreb, Croatia, as well as to evaluate HBoV DNA quantity in samples in relation to the patients' age and co-infection with other respiratory viruses. METHODS: During winter season 2016/2017, a total of 295 children younger than three years of age who were admitted to hospitals with LRTI were tested for the presence of HBoV, respiratory syncytial virus (RSV), adenovirus (ADV), parainfluenza virus (PIV) types 1 to 3, and human metapneumovirus (HMPV). HBoV was detected with a real-time PCR method, and the other viruses were diagnosed using monoclonal antibodies in direct fluorescence assay. RESULTS: Viral etiology was proven in 225/295 (76.3%) of patients. The most commonly diagnosed virus was RSV (59.3%), followed by HBoV (23.1%), PIVs (4.4%), ADV (3.1%), and HMPV (1.4%). HBoV-infected children were older than RSV-infected children; likewise, detection rates of HBoV infection increased with age, while RSV infection rates decreased with age. In 51% of HBoV-positive samples an additional respiratory virus was also detected. There was no difference in HBoV DNA quantity between samples with single virus detection and those with multiple virus detection (p = 0.056), although samples positive only for HBoV showed higher cycle threshold values. There was no difference in HBoV DNA quantity in samples of different age groups (p > 0.05). CONCLUSIONS: Frequent detection of HBoV in small children with LRTI, even in combination with other viruses, highlights its role in the pathogenesis of respiratory disease.

Metal allergy as a late-onset complication of the Nuss procedure in a pediatric patient.

We report the case of a 14-year-old boy with pectus excavatum who developed a metal allergy to stainless steel bar as a late-onset complication after the Nuss procedure. He did not have atopic diathesis. Treatment with oral steroids was effective and the metal bar was successfully removed 2 years later.

Etiology and Clinical Characteristics of Single and Multiple Respiratory Virus Infections Diagnosed in Croatian Children in Two Respiratory Seasons.

The aim of this study was to determine the causative agent of acute respiratory infection (ARI) in hospitalized children, as well as investigate the characteristics of ARIs with single and multiple virus detection in two respiratory seasons. In 2010 and 2015, nasopharyngeal and pharyngeal swabs from a total of 134 children, admitted to the hospital due to ARI, were tested using multiplex PCR. Viral etiology was established in 81.3% of the patients. Coinfection with two viruses was diagnosed in 27.6% of the patients, and concurrent detection of three or more viruses was diagnosed in 12.8% of the patients. The most commonly diagnosed virus in both seasons combined was respiratory syncytial virus (RSV) (28.6%), followed by parainfluenza viruses (PIVs) types 1-3 (18.4%), rhinovirus (HRV) (14.3%), human metapneumovirus (10.1%), adenovirus (AdV) (7.1%), influenza viruses types A and B (4.8%), and coronaviruses (4.2%). In 2015, additional pathogens were investigated with the following detection rate: enterovirus (13.2%), bocavirus (HBoV) (10.5%), PIV-4 (2.6%), and parechovirus (1.3%). There were no statistical differences between single and multiple virus infection regarding patients age, localization of infection, and severity of disease (P > 0.05). AdV, HRV, HBoV, and PIVs were significantly more often detected in multiple virus infections compared to the other respiratory viruses (P < 0.001).

Oral allergy syndrome in children.

Oral allergy syndrome (OAS) is an allergic reaction that occurs after consumption of fresh fruits and vegetables in patients with allergy to pollen. It is mediated by immunoglobulin E (IgE) antibodies and symptoms arise as a result of cross-reactivity between pollen and plant-derived food. OAS is rarely seen in young children, but the prevalence increases with age. The objectives of the study were to identify the prevalence of OAS and probable risk factors in children and adolescents with seasonal allergic rhinitis (AR). One-hundred and twenty patients with seasonal AR were included. Patients were diagnosed based on their clinical history, skin prick test outcome and specific IgE. In patients describing OAS, prick-by-prick tests with fresh fruit or vegetables were carried out. Thirty-two patients had OAS and it was more frequent in female patients than in male patients. OAS was more frequent in adolescents than in small children and in patients with higher total IgE. OAS was significantly more prevalent in patients with AR and asthma (P=0.0016), as was the case in patients with AR and atopic dermatitis (P=0.0004). OAS is rarely diagnosed in small children, partly because of an inadequate clinical history. Patients with OAS may have some risk factors in addition to pollen allergy, and those with more severe atopy are more likely to develop OAS.

[Oral allergy syndrome]. / Oralni alergijski sindrom.

Oral allergy syndrome (OAS) is an IgE antibody-mediated allergic reaction that occurs after consumption of fresh fruits and veg etables in patients with allergy to pollen. Symptoms arise due to cross-reactivity between pollen and plant-derived food and another term used for this syndrome is pollen-food allergy syndrome. The patient is sensitized with pollen and exhibits an allergic reaction to food antigen with structural similarity to the pollen. OAS is rarely seen in young children, but the prevalence increases with age and OAS is the most common manifestation of food allergy in adolescents and adults. Symptoms are usually localized in the oral mucosa, but abdominal symptoms and anaphylaxis may occur as well. Patients generally tolerate thermally processed food, but in those with atopic dermatitis it may lead to worsening of eczema. In the case of generalized symptoms and anaphylaxis, strict avoidance and first aid measures including a self-injectable adrenaline are advised.

Analysis of biennial outbreak pattern of respiratory syncytial virus according to subtype (A and B) in the Zagreb region.

BACKGROUND: The epidemic pattern of respiratory syncytial virus (RSV) in Croatia is biennial. In order to determine if the circulation of different RSV subtypes affects the outbreak cycle, the aim of the present study was to analyze the epidemic pattern of RSV in children in Croatia (Zagreb region) over a period of 3 consecutive years. METHODS: The study group consisted of 696 inpatients, aged 0-5 years, who were hospitalized with acute respiratory tract infections caused by RSV, in Zagreb, in the period 1 January 2006-31 December 2008. The virus was identified in nasopharyngeal secretions using direct immunofluorescence. The virus subtype was determined on real-time polymerase chain reaction. RESULTS: Of 696 RSV infections identified in children, subtype A virus caused 374 infections, and subtype B, 318. Four patients had a dual RSV infection (subtypes A and B). The period of study was characterized by four epidemic waves of RSV infections: the first, smaller, in the spring of 2006; the second, larger, in December 2006/January 2007; the third in spring 2008, followed by a fourth outbreak beginning in November of 2008. The biennial virus cycles were persistent although the predominant RSV subtype in the first two epidemic waves was subtype B, and in the second two it was subtype A. CONCLUSION: Over a 3 year period of observation, the biennial RSV cycle in Croatia cannot be explained by a difference in the predominant circulating subtype of RSV. Other unknown factors account for the biennial cycle of RSV epidemics in Croatia.

The distribution of HLA alleles among children with atopic asthma in Croatia.

Allergic asthma is a multifactorial disease involving well known environmental factors and less identified genetic components. In several studies the HLA genes have been implicated in the development of asthma and atopy, but the importance of these associations remains unclear. The aim of the present study was to analyse the distribution of specificities at HLA class I loci (-A and -B) and HLA class II locus (-DRB1) in a group of 143 Croatian children with atopic asthma, regarding total serum IgE and specific IgE against common inhalant allergens, as well as their connection with different asthmatic phenotypes and to identify HLA genotype which increases the risk for atopy or asthma or which has a protective effect. As controls we used a group of 163 healthy unrelated individuals. HLA class I antigens were determined by serology, while DRB1 specificities were detected by polymerase-chain reaction amplification and hybridisation with sequence specific oligonucleotide probes method (PCR-SSOP). We found no significant correlation between any of the HLA-A antigens and asthma, atopy or associated atopic phenotypes. At HLA-B locus, HLA-B8 antigen was significantly increased among asthmatic patients (p = 0.002), patients with high total serum IgE (p = 0.002), as well as among patients sensitizated to Dermatophagoides pteronyssinus (Der p) (p = 0.014) and among patients sensitizated to Der p + Dactylis glomerata (Dact g) or Ambrosia elatior (Amb a) (p = 0.004). Among HLA-DRB1 specificities, HLA-DRB1 *01 showed positive correlation with asthma and atopy (p = 0.034), while HLA-DRB1*03 specificity was observed with significantly higher frequency among patients with total serum IgE > or = 400 KU/L (p = 0.048). HLA-DRB1*16 specificity was observed with significantly lower frequency among patients with asthma only in comparison to healthy controls (p = 0.027) and to patients with asthma and allergic rhinitis (p = 0.005). In conclusion, our data suggest that HLA specificities play a relevant role in predisposition to asthma, as well as in different clinical forms of atopic diseases. HLA-B8, HLA-DRB1*01 and HLA-DRB1*03 genotype increases the risk for atopic asthma and high serum IgE.