Are we missing respiratory viral infections in infants and children? Comparison of a hospital-based quality management system with standard of care.

OBJECTIVES: Hospital-based surveillance of influenza and acute respiratory infections relies on International Classification of Diseases (ICD) codes and hospital laboratory reports (Standard-of-Care). It is unclear how many cases are missed with either method, i.e. remain undiagnosed/coded as influenza and other respiratory virus infections. Various influenza-like illness (ILI) definitions co-exist with little guidance on how to use them. We compared the diagnostic accuracy of standard surveillance methods with a prospective quality management (QM) programme at a Berlin children's hospital with the Robert Koch Institute. METHODS: Independent from routine care, all patients fulfilling pre-defined ILI-criteria (QM-ILI) participated in the QM programme. A separate QM team conducted standardized clinical assessments and collected nasopharyngeal specimens for blinded real-time quantitative PCR for influenza A/B viruses, respiratory syncytial virus, adenovirus, rhinovirus and human metapneumovirus. RESULTS: Among 6073 individuals with ILI qualifying for the QM programme, only 8.7% (528/6073) would have undergone virus diagnostics during Standard-of-Care. Surveillance based on ICD codes would have missed 61% (359/587) of influenza diagnoses. Of baseline ICD codes, 53.2% (2811/5282) were non-specific, most commonly J06 ('acute upper respiratory infection'). Comparison of stakeholder case definitions revealed that QM-ILI and the WHO ILI case definition showed the highest overall sensitivities (84%-97% and 45%-68%, respectively) and the CDC ILI definition had the highest sensitivity for influenza infections (36%, 95% CI 31.4-40.8 for influenza A and 48%, 95% CI 40.5-54.7 for influenza B). CONCLUSIONS: Disease-burden estimates and surveillance should account for the underreporting of cases in routine care. Future studies should explore the effect of ILI screening and surveillance in various age groups and settings. Diagnostic algorithms should be based on the WHO ILI case definition combined with targeted testing.

[Diagnostically Approach to Pediatric Carpal Fractures: a Retrospective Analysis]. / Diagnostisches Vorgehen bei Handwurzelknochenfrakturen im Kindesalter: eine retrospektive Analyse.

INTRODUCTION: Carpal fractures in children are rare, but can be missed, as their clinical symptoms are unspecific and discrete. Even X-ray diagnosis is difficult. Timely diagnosis and consistent therapy are especially important for scaphoid fractures, as they can help to avoid complications such as non-union or avascular necrosis. A diagnostic approach to paediatric carpal fractures will be discussed on the basis of the following group of patients. METHODS: Retrospective analysis of children under 14 years treated in our institution between 09/2010 and 02/2012 for clinically suspected carpal fracture. In the primary evaluation, all children underwent standard X-rays of the hand and/or wrist. All patients were treated by cast immobilisation until complete clinical recovery. All patients with clinical signs of carpal fracture were treated by cast immobilization, even with normal X-rays. The clinical follow-up examination was after 10 to 14 days. In patients with persistent complaints, MRI was performed. We retrospectively evaluated the records of all patients: the fractured carpal bone, and X-ray and MRI-diagnosis were stated. We calculated the mean difference between first presentation and MRI and the mean period for total recovery, in patients with fracture or non-fracture. RESULTS: 61 children (27 boys and 34 girls, mean age 11.5 y) were included in our study. The mean delay between accident and time of first presentation to our paediatric ED was 0.6 days. In primary X-rays, a carpal fracture was demonstrated in only in 2 (3.3 %) patients, but was suspected in only 6 (9.8 %) of patients. In 53 (87.9 %) patients, there was no radiographic evidence of carpal fracture. 14 patients underwent additional scaphoid views, but scaphoid fracture was confirmed in only 1 (7 %) of these patients. In 3 (21.4 %) patients, a scaphoid fracture was suspected and in 10 patients a carpal fracture could be excluded. After a mean time of 11.8 days, all patients underwent a clinical follow-up examination. 32 (54 %) patients had persistent symptoms and MRI was done after a mean time of 17 days. Carpal fracture was then excluded in 12/32 (37 %) patients and was diagnosed in another 20/32 (63 %) children. There were 14 scaphoid fractures, including 3× bone bruise lesions, 4 capitate fractures, 3 triquetral fractures, including 1× bone bruise lesion and 1 bone bruise lesion of the trapezoid. In patients with proven carpal fracture, it took a mean time of 56 days for complete recovery, in comparison with 15 days in patients with excluded carpal fracture. Surgical therapy was unnecessary in any of the patients, and there were no complications. CONCLUSION: In children with clinical and radiographic carpal fracture, diagnosis is difficult and often unsuccessful at first. Even in discrete clinical complaints, generous cast immobilization is essential and clinical follow up is recommended not later then 14 days. In patients with persistent clinical symptoms, MRI is the imaging method of choice, as it is capable of detecting carpal fractures and even bone bruise lesions with high sensitivity, thereby avoiding unnecessary diagnostic or therapeutic stress for the patients.

Age-related prevalence of cross-reactive antibodies against influenza A(H3N2) variant virus, Germany, 2003 to 2010.

To estimate susceptibility to the swine-origin influenza A(H3N2) variant virus (A(H3N2)v) in the German population, we investigated cross-reactive antibodies against this virus and factors associated with seroprotective titre using sera from representative health examination surveys of children and adolescents (n = 815, 2003­06) and adults (n = 600, 2008­10). Antibodies were assessed by haemagglutination inhibition assay (HI); in our study an HI titre ≥ 40 was defined as seroprotective. We investigated associated factors by multivariable logistic regression. Overall, 41% (95% confidence interval (CI): 37­45) of children and adolescents and 39% (95% CI: 34­44) of adults had seroprotective titres. The proportion of people with seroprotective titre was lowest among children younger than 10 years (15%; 95% CI: 7­30) and highest among adults aged 18 to 29 years (59%; 95% CI: 49­67). Prior influenza vaccination was associated with higher odds of having seroprotective titre (odds ratio (OR) for children and adolescents: 3.4; 95% CI: 1.8­6.5; OR for adults: 2.4; 95% CI: 1.7­3.4). Young children showed the highest and young adults the lowest susceptibility to the A(H3N2)v virus. Our results suggest that initial exposure to circulating seasonal influenza viruses may predict long-term cross-reactivity that may be enhanced by seasonal influenza vaccination.

Postinjection Muscle Fibrosis from Lupron.

We describe the case of a 6.5-year-old girl with central precocious puberty (CPP), which signifies the onset of secondary sexual characteristics before the age of eight in females and the age of nine in males as a result of stimulation of the hypothalamic-pituitary-gonadal axis. Her case is likely related to her adoption, as children who are adopted internationally have much higher rates of CPP. She had left breast development at Tanner Stage 2, adult body odor, and mildly advanced bone age. In order to halt puberty and maximize adult height, she was prescribed a gonadotropin releasing hormone analog, the first line treatment for CPP. She was administered Lupron (leuprolide acetate) Depot-Ped (3 months) intramuscularly. After her second injection, she developed swelling and muscle pain at the injection site on her right thigh. She also reported an impaired ability to walk. She was diagnosed with muscle fibrosis. This is the first reported case of muscle fibrosis resulting from Lupron injection.

[The injured child--diagnostic work-up in the emergency room]. / Das verletzte Kind--diagnostisches Vorgehen im Schockraum.

The diagnosis of an injured child in the emergency room requires interdisciplinary collaboration and should be performed in a level 1 or 2 trauma center, if possible. Here, the basic trauma team could be complemented with (pediatric) surgeons. In a pediatric trauma center, specially trained pediatric surgeons or trauma surgeons, anesthetists, and radiologists who are experienced in the treatment of children should be available. The initial emergency room treatment does not differ significantly from that of adults. Ionizing radiation is the greatest hazard for children in the diagnosis of trauma patients. The CT scan is responsible for most of the radiation. To reduce the risk of developing a malignancy, the most harmful consequence of radiation, differentiated use is necessary. This can be achieved by using the presented algorithms. However, the differentiated use of the CT should not result in additional risk to the child. If the child is in a critical condition and obviously has multiple life-threatening injuries, the use of a whole-body CT is justified, due to time saving and targeted therapy of the child.

Ultrasound diagnosis of supracondylar fractures in children.

PURPOSE: The objective of our study was to evaluate the safety and accuracy of ultrasound (US) compared to standard radiographs in diagnosing supracondylar fractures (SCFs) of the humerus in children. PATIENTS AND METHODS: A total of 106 children (aged between 1 and 13 years) with clinically suspected SCF of the humerus were primarily examined by US followed by standard two-plane radiographs of the elbow. US was conducted with a linear scanner viewing the distal humerus from seven standardized sectional planes. US fracture diagnosis was established either by a cortical bulging or cortical gap, or by a positive dorsal fat pad (dFP) sign. X-ray diagnosis was stated by an independent pediatric radiologist and, afterwards, compared to our US findings. Sonographic and radiographic findings were collected in a contingency table. The sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) for US fracture diagnosis were calculated according to the radiographs. In addition, by identifying significant angulation and/or disrupture, SCFs were classified as non-operative/stable and operative/instable SCFs according to the AO Pediatric Fracture Classification System. RESULTS: By US, a SCF could be excluded in 43 patients and in 63 patients, a fracture was diagnosed. In contrast, by radiographs, an SCF could be excluded in 46 patients and in 60 patients, a fracture was diagnosed. For US fracture diagnosis in comparison to radiographs, we calculated a sensitivity of 100 %, a specificity of 93.5 %, an NPV of 100 %, and a PPV of 95.2 %. Thirty-nine SCFs were sonographically classified as stable grades 1/2 SCFs and confirmed in 37 patients by X-rays. All four operative/instable SCFs were correctly identified by US. CONCLUSION: By identifying a positive dFP sign and/or cortical lesions of the distal humerus, SCFs can be detected very sensitively by US. Even the estimation of fracture displacement seems to be possible. We suggest US as an applicable alternative method in the primary evaluation of suspected SCF in children, guiding further diagnostics, where appropriate. After minor injuries, if clinical assessment for an elbow fracture is low and US examination is negative for fracture, additional radiographs are dispensable. Thereby, the amount of X-ray burden during childhood can be reduced, without loss of diagnostic safety.

Clinical and mutation data in 12 patients with the clinical diagnosis of Nager syndrome.

Nager syndrome (MIM #154400) is the best-known preaxial acrofacial dysostosis, mainly characterized by craniofacial and preaxial limb anomalies. The craniofacial abnormalities mainly consist of downslanting palpebral fissures, malar hypoplasia, micrognathia, external ear anomalies, and cleft palate. The preaxial limb defects are characterized by radial and thumb hypoplasia or aplasia, duplication of thumbs and proximal radioulnar synostosis. Haploinsufficiency of SF3B4 (MIM *605593), which encodes SAP49, a component of the pre-mRNA spliceosomal complex, has recently been identified as the underlying cause of Nager syndrome. In our study, we performed exome sequencing in two and Sanger sequencing of SF3B4 in further ten previously unreported patients with the clinical diagnosis of Nager syndrome, including one familial case. We identified heterozygous SF3B4 mutations in seven out of twelve patients. Four of the seven mutations were shown to be de novo; in three individuals, DNA of both parents was not available. No familial mutations were discovered. Three mutations were nonsense, three were frameshift mutations and one T > C transition destroyed the translation start signal. In three of four SF3B4 negative families, EFTUD2 was analyzed, but no pathogenic variants were identified. Our results indicate that the SF3B4 gene is mutated in about half of the patients with the clinical diagnosis of Nager syndrome and further support genetic heterogeneity for this condition.

Contact investigation of a case of human novel coronavirus infection treated in a German hospital, October-November 2012.

On 24 October 2012, a patient with acute respiratory distress syndrome of unknown origin and symptom onset on 5 October was transferred from Qatar to a specialist lung clinic in Germany. Late diagnosis on 20 November of an infection with the novel Coronavirus (NCoV) resulted in potential exposure of a considerable number of healthcare workers. Using a questionnaire we asked 123 identified contacts (120 hospital and three out-of-hospital contacts) about exposure to the patient. Eighty-five contacts provided blood for a serological test using a two-stage approach with an initial immunofluorescence assay as screening test, followed by recombinant immunofluorescence assays and a NCoV-specific serum neutralisation test. Of 123 identified contacts nine had performed aerosol-generating procedures within the third or fourth week of illness, using personal protective equipment rarely or never, and two of these developed acute respiratory illness. Serology was negative for all nine. Further 76 hospital contacts also tested negative, including two sera initially reactive in the screening test. The contact investigation ruled out transmission to contacts after illness day 20. Our two-stage approach for serological testing may be used as a template for similar situations.

[Detection of local influenza outbreaks and role of virological diagnostics]. / Erkennung von Influenzaausbrüchen und Rolle der virologischen Diagnostik.

For many years, the Working Group on Influenza (AGI) has been the most important influenza surveillance system in Germany. An average sample of the population is covered by both syndromic and virological surveillance, which provides timely data regarding the onset and course of the influenza wave as well as its burden of disease. However, smaller influenza outbreaks cannot be detected by the AGI sentinel system. This is achieved by the information reported by the mandatory notification system (Protection Against Infection Act, IfSG), which serves as the second pillar of the national influenza surveillance. Approaches to recognize such outbreaks are based either on reported influenza virus detection and subsequent investigations by local health authorities or by notification of an accumulation of respiratory diseases or nosocomial infections and subsequent laboratory investigations. In this context, virological diagnostics plays an essential role. This has been true particularly for the early phase of the 2009 pandemic, but generally timely diagnostics is essential for the identification of outbreaks. Regarding potential future outbreaks, it is also important to keep an eye on animal influenza viruses that have repeatedly infected humans. This mainly concerns avian influenza viruses of the subtypes H5, H7, and H9 as well as porcine influenza viruses for which a specific PCR has been established at the National Influenza Reference Centre. An increased incidence of respiratory infections, both during and outside the season, should always encourage virological laboratory diagnostics to be performed as a prerequisite of further extensive investigations and an optimal outbreak management.

Special considerations in the interpretation of plain radiographs of the cervical spine in children. A review of the literature.

INTRODUCTION: This review provides an overview of the special considerations with regard to correct diagnosis of plain radiographs of the pediatric cervical spine. Injuries to the cervical spine are rare in children. The leading trauma mechanism is motor vehicle injury. Plain radiographs are a common tool in the search for a diagnosis. Taking the growth process into account there are many differences to be found compared to the adult c-spine. Knowledge of these differences is important when working towards the correct interpretation of plain radiographs of the pediatric c-spine. METHODS: To create this review, a literature search of the electronic databases Cochrane, PubMed/MEDLINE and Embase was conducted. RESULTS: Special considerations of plain radiographs of the pediatric c-spine are presented. Biomechanical and embryology specifics have been a focus of this review. They are explained relating on the development of the c-spine. The known auxiliary lines used in the interpreting of the pediatric c-spine are reported. A selection of these auxiliary lines is shown. CONCLUSION: Knowledge of the c-spines characteristics is of major importance for every physician involved in pediatric trauma care. This could lead to not only avoiding misdiagnosis but could also lead to avoiding the overuse of computed tomography of the pediatric c-spine.

Diagnosis of cervical spine injuries in children: a systematic review.

OBJECTIVE: The objective of this systematic review was to discuss current knowledge of the diagnostic management of cervical spine (c-spine) injuries in children. METHODS: Studies dealing with this topic were collected from the following sources: MEDLINE via PubMed, Embase, and Cochrane. Where possible, a meta-analysis was performed. Furthermore, the level of evidence for all the included publications was assigned. RESULTS: The incidence of cervical spine injury (CSI) in children is rare (1.39 %). It seems that the upper c-spine is more often injured in children younger than 8 years of age. When a CSI is expected, immobilization should be performed. The best immobilization is achieved with a combination of a half-spine board, rigid collar, and tape. The literature for thoracic elevation or an occipital recess in children younger than 8 years of age is inhomogeneous. The c-spine in children can be cleared by a combination of the National Emergency X-Radiography Utilization Study (NEXUS) low-risk criteria and the Canadian C-Spine Rule. Caution is advised for nonverbal and/or unconscious children. In these children, plain radiographs should be performed. If these images are inadequate or show hints for bony injuries, a computed tomography (CT) of the c-spine should be considered. Additional views of the c-spine offer only little information for clearing the c-spine.

Assays for laboratory confirmation of novel human coronavirus (hCoV-EMC) infections.

We present a rigorously validated and highly sensitive confirmatory real-time RT-PCR assay (1A assay) that can be used in combination with the previously reported upE assay. Two additional RT-PCR assays for sequencing are described, targeting the RdRp gene (RdRpSeq assay) and N gene (NSeq assay), where an insertion/deletion polymorphism might exist among different hCoV-EMC strains. Finally, a simplified and biologically safe protocol for detection of antibody response by immunofluorescence microscopy was developed using convalescent patient serum.

[Ultrasound as a viable alternative to standard X-rays for the diagnosis of distal forearm fractures in children]. / Die Sonografie als sichere Alternative zur konventionellen Röntgendiagnostik bei distalen Unterarmfrakturen im Kindesalter.

AIM: Distal forearm fractures are very common in childhood. Radiography of the wrist is the standard diagnostic procedure. But because of higher sensitivity of growing bones to ionising radiation the diagnostic use of X-rays should be minimised as much as possible. Recent studies have shown that distal forearm fractures in children can be safely and reliably diagnosed using only ultrasound. The aim of our study was to evaluate and confirm the safety and applicability of the ultrasound diagnostic procedure in comparison to X-ray diagnosis under routine conditions of our paediatric emergency department. PATIENTS AND METHODS: We investigated 115 patients aged 2-14 years. After clinical assessment patients with suspected forearm fractures first underwent ultrasound examination of the metaphyseal forearm followed by standard two view radiographs of the wrist. Ultrasound and radiographic findings were then compared and sensitivity and specificity for ultrasound were calculated. In 9 patients with suspected displacements, sonographic and radiographic axis measurement were done and also compared. RESULTS: Radiologically we found 62 patients with 78 distal forearm fractures. By ultrasound we also diagnosed 52 fractures. All patients with no fractures were correctly diagnosed as well. Referring to X-rays we calculated for ultrasound a sensitivity of 94.9 %, a specificity of 98 %, a negative predictive value of 97.4 % and a positive predictive value of 96.1 %. The mean difference of the deformities of the radius in the sagittal section measured sonographically and radiologically were 1.7° (SD 1.6°). CONCLUSION: We confirm ultrasound is an applicable, rapid and safe alternative to X-rays in diagnosing metaphyseal forearm fractures in children. Even sonographic axis measurement seems to be a viable method. Thereby ultrasound potentially reduces the X-ray burden in children and additionally accelerates the diagnostic procedure.

Comparison of the novel ResPlex III assay and existing techniques for the detection and subtyping of influenza virus during the influenza season 2006-2007.

Influenza virus is a major cause of disease worldwide. The accurate detection and further subtyping of influenza A viruses are important for epidemiologic surveillance, and subsequent comprehensive characterization of circulating influenza viruses is essential for the selection of an optimal vaccine composition. ResPlex III is a new multiplex reverse transcriptase polymerase chain reaction (RT-PCR)-based method for detecting, typing, and subtyping influenza virus in clinical specimens. The ResPlex III assay was compared with other methods with respect to sensitivity and accuracy, using 450 clinical specimens obtained from subjects throughout Germany during the 2006-2007 influenza season. Samples were analyzed for the presence of influenza virus in Madin-Darby canine kidney (MDCK) cells by rapid cell culture using peroxidase staining and conventional cell culture confirmed by hemagglutination inhibition assay, a rapid diagnostic assay (Directigen Flu A+B test; BD Diagnostic Systems, Heidelberg, Germany), in-house real-time RT-PCR (RRT-PCR), and ResPlex III (Qiagen, Hilden, Germany). ResPlex III had the highest sensitivity for detecting influenza virus in clinical specimens, followed by in-house RRT-PCR (96% compared with ResPlex III). Conventional cell culture in MDCK cells, rapid culture, and quick test assays were substantially less sensitive (55%, 72%, and 39%, respectively). Virus subtyping results were identical using ResPlex III and the standard virological subtyping method, hemagglutination inhibition. ResPlex III is a quick, accurate, and sensitive assay for detecting and typing influenza A and B viruses and subtyping influenza A viruses in clinical specimens, and might be considered for a supplemental role in worldwide seasonal and pandemic influenza surveillance.

A Novel Homozygous WDR72 Mutation in Two Siblings with Amelogenesis Imperfecta and Mild Short Stature.

Amelogenesis imperfecta (AI) is a clinically and genetically heterogeneous group of inherited defects of enamel formation. In isolated AI (no additional segregating features), mutations in at least 7 genes are known so far, causing dominant, recessive or X-linked AI and allowing the identification of the molecular etiology in 40-50% of affected families. We report on 2 siblings (an 11-year-old female and a 7-year-old male) born to consanguineous Turkish parents, with AI and mild, proportionate short stature. Both parents have normal teeth, but mother, maternal grandmother and great-grandfather are/were also of short stature. A spine X-ray performed in the girl excluded brachyolmia. Affymetrix GenomeWide SNP6.0 Array analysis identified no pathogenic copy number changes, but showed sharing of large homozygous regions, including chromosome band 15q21.3 containing the WDR72 gene. WDR72 sequence analysis in both siblings revealed homozygosity for a novel stop mutation in exon 10 (c.997A>T, p.Lys333X) explaining the AI phenotype. Mutations in WDR72 are a very rare cause of autosomal-recessive hypomaturation type of isolated AI. The mutation described in our patients specifies the diagnosis AI IIA3 and represents only the sixth WDR72 mutation reported so far. The WDR72 protein is critical for dental enamel formation, but its exact function is still unknown.

Vaccination against pandemic H1N1 (2009) in patients after allogeneic hematopoietic stem cell transplantation: a retrospective analysis.

PURPOSE: Limited data are available on immunologic responses to primary pandemic H1N1 (2009) vaccination in recipients of allogeneic hematopoietic stem cell transplantation (HSCT) recipients. In 2009 serologic responses to either pandemic H1N1 (2009) vaccine (n = 36) or pandemic H1N1 (2009) infection (n = 2) were studied in 38 HSCT recipients. METHODS: Responses were measured with a standard hemagglutination-inhibition assay. Fourteen patients had active chronic graft-versus-host disease (cGvHD) at the time of vaccination/infection and seven patients had cGvHD in remission; 11 patients had no immunosuppressive therapy, and 27 patients were on immunosuppressive therapy. Nineteen patients (53%) responded to pandemic H1N1 (2009) vaccination. Two patients had pandemic H1N1 (2009) infection without prior vaccination, and one patient had severe pandemic H1N1 (2009) infection with acute respiratory distress syndrome despite prior single vaccination. RESULTS: Non-responders to pandemic H1N1 (2009) vaccination more often had cGvHD (65 vs. 53%) and received second- or third-line therapy (53 vs. 11%), while responders mostly had first-line therapy for cGvHD. While vaccine responders had no or single agent immunosuppressive therapy, non-responders frequently received moderate or intense immunosuppressive therapy. All vaccine recipients previously treated with rituximab were non-responders. CONCLUSIONS: In summary, the overall response to pandemic H1N1 (2009) vaccination in HSCT recipients was modest. Patients receiving combined immunosuppressive therapy for steroid-refractory cGvHD barely responded to pandemic H1N1 (2009) vaccination.

How deadly is seasonal influenza-associated pneumonia? The German Competence Network for Community-Acquired Pneumonia.

The emergence of new influenza virus subtypes has rekindled the interest in the clinical course and outcome of patients with influenza-associated pneumonia. Based on prospective data from 5,032 patients with community-acquired pneumonia (CAP) included in the German Competence Network for Community-Acquired Pneumonia (CAPNETZ), we studied the incidence, clinical characteristics and outcome of patients with influenza-associated CAP and compared these findings with patients without influenza. Diagnosis relied on a positive PCR for influenza in throat washings. 160 patients with influenza-associated CAP were identified (3.2% of total population, 12% of those with defined aetiology). 34 (21%) patients with seasonal influenza had a concomitant pathogen (mostly Streptococcus pneumoniae). Patients with influenza-associated CAP were significantly older, had been vaccinated less often and had preceding antibacterial treatment less often. 30-day mortality was low (4.4%) and not different to that of patients with pneumonia caused by bacterial (6.2%) or viral (other than influenza) pathogens (4%). Patients with influenza plus a bacterial pathogen (mixed influenza-associated pneumonia) had a higher mortality than those with pure influenza-associated pneumonia (9% versus 3.2%). Mortality was higher in patients with mixed compared with pure influenza-associated pneumonia. However, we could not observe any excess mortality in patients with influenza-associated pneumonia.

Comparison of hemagglutination inhibition assay, an ELISA-based micro-neutralization assay and colorimetric microneutralization assay to detect antibody responses to vaccination against influenza A H1N1 2009 virus.

The hemagglutination inhibition (HI) assay has been the main method used to investigate immune responses to vaccination against influenza H1N1 (2009) virus. However microneutralization tests (MNT) have been shown to be more sensitive and more specific. In this study, the three methods of choice: (i) the HI assay, (ii) an ELISA-based conventional MNT and (iii) a colorimetric MNT in terms of their ability to detect antibody responses in serum pairs collected from 43 healthy individuals before and 21 days after vaccination were compared. The colorimetric MNT was established yielding intra- and inter-run imprecisions of 7.5% and 12.4%, respectively. Testing of antisera to seasonal influenza viruses demonstrated the assay to be specific for antibodies to influenza H1N1 (2009) virus. A good correlation between the three methods was found, being highest for the ELISA-MNT and the colorimetric MNT (r=0.714 for geometric mean titers (GMT) and r=0.695 for titer increases). Similar rates of fourfold titer increases were detected: 95.3% in the ELISA-MNT vs. 93.0% in colorimetric MNT and 95.3% in HI assay. The ELISA-based MNT demonstrated the highest titer range leading to the highest postvaccination GMT and the highest titer increase (>50-fold). The lowest GMTs were measured with the HI assay, while the colorimetric MNT detected the highest GMT in prevaccination sera. Taken together, similar seroconversion rates were obtained with the three assays. The ELISA-MNT appeared to be the best method to compare absolute pre- and postvaccination GMTs. The colorimetric MNT, being less labour-intensive than the ELISA-MNT, seems to be a suitable tool in vaccination studies.

Influence of antiretroviral therapy on immunogenicity of simultaneous vaccinations against influenza, pneumococcal disease and hepatitis A and B in human immunodeficiency virus positive individuals.

OBJECTIVES: Immune response to many vaccinations is impaired in human immunodeficiency virus (HIV) positive patients. METHODS: A total of n = 131 HIV positive patients were vaccinated against influenza, pneumococcal disease, hepatitis A and B, with n = 82 patients (62.6%) receiving 2 or more simultaneous vaccinations. Safety and immunogenicity of simultaneous vaccinations were assessed. Current antiretroviral therapy (ART) regimens were evaluated as potential predictors for antibody response. RESULTS: Immune response rates were 45% (influenza), 68% (pneumococcus), 63.6% (hepatitis A) and 62.5% (hepatitis B). Adverse reactions after vaccination were documented in 2 of 131 patients (1.5%). No statistically significant difference between pre- and post-vaccination CD4+ T-cell counts (CD4) and HIV plasma load was observed. 85% of patients received ART containing nucleotide reverse transcriptase inhibitors, non-nucleotide reverse transcriptase inhibitors and/or protease inhibitors (PI). Higher ratio of CD4 to CD8 and intake of PI were statistically significant, independent predictors for antibody response after influenza vaccination (OR 1.9 and 2.8, p = 0.01 and 0.04, respectively). CONCLUSIONS: Simultaneous vaccinations in HIV positive patients were safe and well tolerated. The positive effect of PI on antibody response after influenza vaccination should be confirmed in larger studies.