Detection of herpes simplex and varicella-zoster virus from skin lesions: comparison of RT-PCR and isothermal amplification for rapid identification.

We compared 2 molecular tests for detection of herpes simplex viruses 1 and 2 (HSV-1, HSV-2) and varicella-zoster virus (VZV): real-time polymerase chain reaction (RT-PCR) (Argene, BioMerieux, France) performed on an LC480 platform (Roche Applied Science, Mannheim, Germany) and isothermal amplification using a Solana HSV1 + 2/VZV assay (Quidel Corporation Worldwide Headquarters, San Diego, CA) with helicase-dependent amplification performed by a Solana® instrument. With both methods, HSV-1 was detected in 68/291 (23.4%), HSV-2 in 23/291 (7.9%), and VZV in 48/291 (16.5%) skin lesions. Both methods agreed completely only in detection of HSV-2 (kappa = 1). Concordance between Solana HSV1 + 2/VZV and RT-PCR was 98.3% (kappa = 0.95) for HSV-1 and 99.3% (kappa = 0.98) for VZV. Rapid detection of HSV-1, HSV-2, and VZV using the Solana platform is a useful method for routine diagnostics and for urgent swab samples requiring a short turnaround time.

Respiratory and Enteric Virus Detection in Children.

The majority of children with febrile seizures have viral infections and viruses were detected in 22% to 63% of children in published studies. Using molecular methods, viruses were also detected in asymptomatic persons. A prospective study was conducted to detect respiratory and enteric viruses in 192 children with febrile seizures and compare the detection rates to those found in 156 healthy age-matched controls. A respiratory or enteric virus was detected in 72.9% of children with febrile seizures and in 51.4% of healthy controls. The viruses most strongly associated with febrile seizures were influenza, respiratory syncytial virus, parainfluenza, human coronavirus, and rotavirus. Compared to healthy controls, the age-adjusted odds ratios for nasopharynx virus positivity in febrile seizure patients were 79.4, 2.8, 7.2, and 4.9 for influenza virus, parainfluenza virus, respiratory syncytial virus, and human coronavirus, respectively, and 22.0 for rotavirus in stool. The detected virus did not influence clinical features of febrile seizure.

Narrowing of the Diagnostic Gap of Acute Gastroenteritis in Children 0-6 Years of Age Using a Combination of Classical and Molecular Techniques, Delivers Challenges in Syndromic Approach Diagnostics.

BACKGROUND: Twenty-five percent to 50% of acute gastroenteritis (AGE) cases remain etiologically undiagnosed. Our main aim was to determine the most appropriate list of enteric pathogens to be included in the daily diagnostics scheme of AGE, ensuring the lowest possible diagnostic gap. METHODS: Two hundred ninety seven children ≤6 years of age, admitted to hospital in Slovenia, October 2011 to October 2012, with AGE, and 88 ≤6 years old healthy children were included in the study. A broad spectrum of enteric pathogens was targeted with molecular methods, including 8 viruses, 6 bacteria and 2 parasites. RESULTS: At least one enteric pathogen was detected in 91.2% of cases with AGE and 27.3% of controls. Viruses were the most prevalent (82.5% and 15.9%), followed by bacteria (27.3% and 10.2%) and parasites (3.0% and 1.1%) in cases and controls, respectively. A high proportion (41.8%) of mixed infections was observed in the cases. For cases with undetermined etiology (8.8%), stool samples were analyzed with next generation sequencing, and a potential viral pathogen was detected in 17 additional samples (5.8%). CONCLUSIONS: Our study suggests that tests for rotaviruses, noroviruses genogroup II, adenoviruses 40/41, astroviruses, Campylobacter spp. and Salmonella sp. should be included in the initial diagnostic algorithm, which revealed the etiology in 83.5% of children tested. The use of molecular methods in diagnostics of gastroenteritis is preferable because of their high sensitivity, specificity, fast performance and the possibility of establishing the concentration of the target. The latter may be valuable for assessing the clinical significance of the detected enteric, particularly viral pathogens.

The Role of Human Coronaviruses in Children Hospitalized for Acute Bronchiolitis, Acute Gastroenteritis, and Febrile Seizures: A 2-Year Prospective Study.

UNLABELLED: Human coronaviruses (HCoVs) are associated with a variety of clinical presentations in children, but their role in disease remains uncertain. The objective of our prospective study was to investigate HCoVs associations with various clinical presentations in hospitalized children up to 6 years of age. Children hospitalized with acute bronchiolitis (AB), acute gastroenteritis (AGE), or febrile seizures (FS), and children admitted for elective surgical procedures (healthy controls) were included in the study. In patients with AB, AGE, and FS, a nasopharyngeal (NP) swab and blood sample were obtained upon admission and the follow-up visit 14 days later, whereas in children with AGE a stool sample was also acquired upon admission; in healthy controls a NP swab and stool sample were taken upon admission. Amplification of polymerase 1b gene was used to detect HCoVs in the specimens. HCoVs-positive specimens were also examined for the presence of several other viruses. HCoVs were most often detected in children with FS (19/192, 9.9%, 95% CI: 6-15%), followed by children with AGE (19/218, 8.7%, 95% CI: 5.3-13.3%) and AB (20/308, 6.5%, 95% CI: 4.0-9.8%). The presence of other viruses was a common finding, most frequent in the group of children with AB (19/20, 95%, 95% CI: 75.1-99.8%), followed by FS (10/19, 52.6%, 95% CI: 28.9-75.6%) and AGE (7/19, 36.8%, 95% CI: 16.3-61.6%). In healthy control children HCoVs were detected in 3/156 (1.9%, 95% CI: 0.4-5.5%) NP swabs and 1/150 (0.7%, 95% CI: 0.02-3.3%) stool samples. It seems that an etiological role of HCoVs is most likely in children with FS, considering that they had a higher proportion of positive HCoVs results than patients with AB and those with AGE, and had the highest viral load; however, the co-detection of other viruses was 52.6%. TRIAL REGISTRATION: ClinicalTrials.gov NCT00987519.

Detection of human coronaviruses in simultaneously collected stool samples and nasopharyngeal swabs from hospitalized children with acute gastroenteritis.

BACKGROUND: Human coronaviruses (HCoVs) are a well-known cause of respiratory infections but their role in gastrointestinal infections is unclear. The objective of our study was to assess the significance of HCoVs in the etiology of acute gastroenteritis (AGE) in children <6 years of age. METHODS: Stool samples and nasopharyngeal (NP) swabs collected from 260 children hospitalized for AGE (160 also had respiratory symptoms) and 157 otherwise healthy control children admitted for elective surgery were tested for the presence of four HCoVs using real time RT-PCR. Registered at ClinicalTrials.gov (reg. NCT00987519). RESULTS: HCoVs were more frequent in patients with AGE than in controls (23/260, 8.8% versus 4/151, 2.6%; odds ratio, OR 3.3; 95% confidence interval, CI 1.3-10.0; P = 0.01). Three of four HCoV-positive members in the control group, asymptomatic when sampled, recalled gastrointestinal or respiratory symptoms within the previous 14 days. In patients with AGE, HCoVs were present in NP samples more often than in stools (22/256, 8.6%, versus 6/260, 2.3%; P = 0.0004). In 5/6 children with HCoVs detected in stools, the viruses were also detected in NP swabs. Patients had a significantly higher probability of HCoV detection in stool (OR 4; 95% CI 1.4-15.3; P = 0.006) and also in stool and/or NP (OR 3.3, 95% CI 1.3-10.0; P = 0.01) than healthy controls. All four HCoVs species were detected in stool and NP samples. CONCLUSIONS: Although HCoVs were more frequently detected in patients with AGE than in the control group, high prevalence of HCoVs in NP swabs compounded by their low occurrence in stool samples and detection of other viruses in stool samples, indicate that HCoVs probably play only a minor role in causing gastrointestinal illness in children <6 years old.

Coronavirus infections in hospitalized pediatric patients with acute respiratory tract disease.

BACKGROUND: Acute viral respiratory infections are an important cause of morbidity and mortality in humans worldwide. The etiological backgrounds of these infections remain unconfirmed in most clinical cases. The aim of this study was to estimate the prevalence of human coronavirus infections in a series of children hospitalized with symptoms of acute respiratory tract disease in a one-year period in Slovenia. METHODS: The 664 specimens from 592 children under six years of age hospitalized at the University Children's Hospital in Ljubljana were sent for the routine laboratory detection of respiratory viruses. Respiratory viruses were detected with a direct immunofluorescence assay and human coronaviruses were detected with a modified real-time RT-PCR. RESULTS: HCoV RNA was detected in 40 (6%, 95% CI: 4.3%-8.1%) of 664 samples. Of these specimens, 21/40 (52.5%) were identified as species HKU1, 7/40 (17.5%) as OC43, 6/40 (15%) as 229E, and 6/40 (15%) as NL63. Infection with HCoV occurred as a coinfection with one or more other viruses in most samples (70%). Of the HCoV-positive children, 70.3% had lower respiratory tract infections. CONCLUSION: The results of our study show that HCoV are frequently detected human pathogens, often associated with other respiratory viruses and acute respiratory tract infections in hospitalized children. An association between age and the viral load was found. The highest viral load was detected in children approximately 10 months of age.

Human bocavirus and other respiratory viral infections in a 2-year cohort of hospitalized children.

Human bocavirus (HBoV) infection is reported worldwide and may cause severe respiratory tract infections. The aim of the present study was to assess the prevalence of HBoV, and other respiratory viral pathogens, in a 2-year retrospective study of children admitted to hospital, and to investigate whether viral loads of HBoV DNA were associated with severity of infection. Between April 2007 and March 2009, 891 respiratory samples from 760 children admitted to hospital with acute respiratory tract infection were tested for the presence of respiratory viruses by real-time PCR or direct immunofluorescence testing. HBoV DNA was detected by using internally controlled real-time quantitative PCR assay and 25 samples selected at random were sequenced. The virus detected most frequently was rhinovirus, followed by respiratory syncytial virus, HBoV, and human metapneumovirus. HBoV DNA was detected in 18.4% of children admitted to hospital. HBoV was the only viral pathogen detected in 66/164 (40.2%) of HBoV DNA-positive children and in 7.4% of all 891 samples. Ninety-seven percent (64/66) of children with an HBoV single infection were diagnosed as having lower respiratory tract infection. Median HBoV DNA viral load was significantly higher in children when HBoV was detected as a single pathogen. Higher HBoV DNA viral loads were associated with prematurity and age. HBoV seems to be an important and frequent pathogen in respiratory tract infections in children, and it is likely that the severity of illness is comparable to the severity of RSV illness.