A. Vankeerberghen

First Case of Staphylococcus pseudintermedius Infection in a Human

ABSTRACT We present the first clinical report of a Staphylococcus pseudintermedius infection in a human. Biochemically, S. pseudintermedius can be easily misidentified as S. aureus. Therefore, the final microbiological identification requires the combination of phenotypic and genotypic tests.

Use of a multiplex real-time PCR to study the incidence of human metapneumovirus and human respiratory syncytial virus infections during two winter seasons in a Belgian paediatric hospital

ABSTRACT Viruses are an important cause of acute respiratory tract infection (ARTI) in children. This study aimed to develop and evaluate a rapid molecular diagnostic test (duplex real-time PCR) for human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV), and to determine the frequency of these two viruses as causative agents of ARTI in Belgium. Nasopharyngeal aspirates were collected over two winter and spring seasons (November 2003 to May 2004 and November 2004 to May 2005) from children aged <5 years with ARTI (n = 778). The duplex real-time PCR showed a linear range of 104–1010 copies/mL for both hMPV and hRSV. Analysis of the stability of the hRSV and hMPV genomes revealed that nasopharyngeal aspirates could be stored at room temperature for up to 1 month without significant loss of detection. hRSV was detected by antigen testing and by real-time PCR; hMPV was detected by real-time PCR only. The hRSV antigen test was less sensitive than PCR, and failed to detect one-third of the hRSV infections. Overall, 54 (6.9%) and 306 (39.3%) of the 778 samples were positive for hMPV and hRSV, respectively. Both viruses infected young infants, but the mean age of infants infected by hRSV was lower than that of infants infected by hMPV (12 months vs. 17 months, respectively).

Monitoring of herpes simplex virus in the lower respiratory tract of critically ill patients using real-time PCR: a prospective study

Herpes simplex virus (HSV) has increasingly been associated with pulmonary disease in critically ill patients. However, the clinical relevance of HSV is still a topic of debate. Monitoring of HSV in a quantitative way could potentially give relevant information on its role in the pathogenesis of lower respiratory tract infection. A fast and reliable quantitative real-time PCR (Q-PCR) for the quantitative detection of HSV-1 and HSV-2 DNA was developed. A prospective observational study was performed in an intensive-care unit (ICU) to monitor the HSV viral load in lower respiratory tract aspirates of long-term mechanically ventilated patients. HSV was common in the lower respiratory tract (LRT) of critically ill patients with mechanical ventilation for at least 48 h (62%, n = 65/105). Detection of HSV was significantly associated with prolonged mechanical ventilation (p <0.01), prolonged ICU stay (p <0.01), and development of ventilator-associated pneumonia (p = 0.02). Corticosteroid administration (p <0.01) in the ICU and anti-HSV IgG seropositivity (p <0.01) were risk factors for the occurrence of HSV in the LRT. The fact that no HSV-seronegative patient became positive suggests that all HSV DNA-positive patients had HSV reactivations. Monitoring the HSV viral load in the LRT of critically ill patients showed a typical homogeneous pattern of HSV kinetics. HSV emerged in tracheal and bronchial aspirates after a median of 7 days of intubation (5-11 days), and this was followed by an exponential increase (c. 1 log copies/mL/day) to reach very high HSV peaks (10(6)-10(10) copies/mL) in 78% of the HSV DNA-positive patients.

Circulation of HRSV in Belgium: From Multiple Genotype Circulation to Prolonged Circulation of Predominant Genotypes

Molecular surveillance of HRSV in Belgium for 15 consecutive seasons (1996–2011) revealed a shift from a regular 3-yearly cyclic pattern, into a yearly alternating periodicity where HRSV-B is replaced by HRSV-A. Phylogenetic analysis for HRSV-A demonstrated the stable circulation of GA2 and GA5, with GA2 being dominant over GA5 during 5 consecutive seasons (2006–2011). We also identified 2 new genotype specific amino acid mutations of the GA2 genotype (A122 and Q156) and 7 new GA5 genotype specific amino acid mutations (F102, I108, T111, I125, D161, S191 and L217). Several amino acid positions, all located in the second hypervariable region of HRSV-A were found to be under positive selection. Phylogenetic analysis of HRSV-B showed the circulation of GB12 and GB13, where GB13 represented 100% of the isolated strains in 4 out of 5 consecutive seasons (2007–2011). Amino acids under positive selection were all located in the aminoterminal hypervariable region of HRSV-B, except one amino acid located in the conserved region. The genotype distribution within the HRSV-B subgroup has evolved from a co-circulation of multiple genotypes to the circulation of a single predominant genotype. The Belgian GB13 strains circulating since 2006, all clustered under the BAIV branch and contained several branch specific amino acid substitutions. The demographic history of genotypes GA2, GA5 and GB13 demonstrated a decrease in the total GA2 and GA5 population size, coinciding with the global expansion of the GB13 population. The emergence of the GB13 genotype resulted in a newly established balance between the predominant genotypes.

Towards Multitarget Testing in Molecular Microbiology

Advantages of PCR assays over more conventional culture-based diagnostics include significantly higher sensitivities and shorter turnaround times. They are particularly useful when patient treatment has already been initiated or for specimens that may contain microorganisms that are slow-growing, difficult to culture, or for which culture methods do not exist. However, due to genome variability, single target testing might lead to false-negative results. This paper focuses on examples from our own experiences and the literature to provide insight into the limitations of single target testing in molecular biology. Lessons learned from these experiences include the careful design of diagnostic assays, preferably multitargeted, the importance of investigating the incidence and epidemiology of infection in detail, the frequent participation in appropriate quality assurance schemes, and the importance of continuous attentiveness by investigators when confronted with inconsistent results. In conclusion, multitargeted testing in microbiological molecular assays should be a rule.

Multicenter evaluation of BD Veritor System and RSV K-SeT for rapid detection of respiratory syncytial virus in a diagnostic laboratory setting

The recently introduced BD Veritor System RSV laboratory kit (Becton Dickinson, Sparks, MD, USA) with automatic reading was evaluated and compared with the RSV K-SeT (Coris BioConcept, Gembloux, Belgium) for the detection of respiratory syncytial virus (RSV) using 248 nasopharyngeal aspirates of children younger than 6 years old with respiratory tract infection. Compared to reverse transcriptase polymerase chain reaction as gold standard, both tests had an identical sensitivity of 78.1% and a specificity of 96.8% and 95.8% for the BD Veritor System and RSV K-SeT, respectively. Both antigen tests can be used to reliably confirm RSV in young children. However, a negative result does not definitively exclude the presence of RSV.

The “Snotbarometer”: Epidemiological data on respiratory infections

no: 150 Presentation at ESCV 2016: Poster 184 The “Snotbarometer”: Epidemiological data on respiratory infections A. Vankeerberghen ∗, K. Dierickx, A. Boel, K. Van Vaerenbergh, H. De Beenhouwer Laboratory of Microbiology, OLVZ Aalst, Belgium Molecular detection of respiratory viruses was initiated in the Laboratory of Microbiology of OLVZ Aalst, Belgium, in 2003 with the detection of human metapneumovirus (hMPV) and respiratory syncytial virus (RSV). Since then, a constant elaboration of the portfolio was performed resulting in 8 multiplex in house real time PCR’s that detect 22 respiratory pathogens including viruses (RSV, hMPV, adenovirus, bocavirus, para-influenzavirus (PIV) 1, 2, 3 and 4, Influenza A and B, coronaviruses, enterovirus and rhinovirus) and atypical bacteria (M. pneumoniae, C. pneumoniae, B. pertussis, parapertussis and holmesii). Samples are mainly obtained from our hospital but also from other hospitals from the Flanders region. On each respiratory sample for which molecular diagnostics for at least one of these pathogens is requested, the complete PCR panel of 22 pathogens is performed. This increases the accuracy of a specific diagnosis, and it also results in “local” epidemiological data. These data are translated into a graphic representation, called the “snotbarometer”, which is made available for the hospital staff through the intranet, and on the website of the hospital. The “snotbarometer” consists of a weekly and a monthly report. In the weekly report, the amount of positive samples for each pathogen separately is depicted in a graph and updated weekly. This presentation gives the physician an idea of the actually circulating pathogens, of the amount of samples analysed in the lab, and the percentage of samples positive for each pathogen. In the monthly report a seasonal overview is given for the pathogens with epidemiological data available for multiple years, so one can start to extract the characteristic seasonal patterns. Examples are RSV, influenza A and B, PIV1, PIV2, PIV3 and PIV4. This year, Influenza B exceptionally preceded Influenza A which prolonged the influenza season. For other pathogens like adenovirus, bocavirus and M. pneumoniae the seasonality is less clear and one can observe a more fluctuating presence. Together, this information is very useful to predict the upcoming viruses. Conclusion: Regional epidemiological data are powerful since they can give useful information to the physician, especially when a weekly follow-up is available. http://dx.doi.org/10.1016/j.jcv.2016.08.224 Abstract no: 181 Presentation at ESCV 2016: Poster 185 Molecular characterization of human parainfluenza virus type 3 (HPIV-3) among hospitalized patients from central Israel I. Jornist 1,∗, E. Mendelson 1, D. Ram 2, R. Azar 2, M. Mandelboim 1, M. Hindiyeh 1 1 Chaim Sheba Medical Center & Tel-Aviv University, Israel 2 Chaim Sheba Medical Center, Israel Human parainfluenza virus 3 (HPIV-3) is an enveloped, non-segmented, negative sense RNA virus that belongs to the Paramyxoviridae family. HPIV-3 is a common cause of bronchiolitis and pneumoniae in children less than 1 year of age and one of the leading causes of acute lower respiratory tract infections in children under five years of age. In Israel, the epidemiology of HPIV-3 infections is not well characterized. In this study, epidemiology and molecular characterization of HPIV-3 was performed on patient samples collected between January 2012 and September 2015. Nasopharyngeal swabs (N = 15,946) were collected from hospitalized patients presenting with respiratory illness. Viral nucleic acid was extracted from patient sample using NucliSENS® easyMAG® (bioMérieux, France) and tested for the common human respiratory viruses (influenza viruses A and B, hMPV, adenovirus, RSV and HPIV-3) using validated real time PCR multiplex assays. Furthermore, molecular characterization of HPIV-3 complete HN gene (1722 bases) was performed after sequencing the complete HN gene. The Bayesian Markov chain Monte Carlo (MCMC) method was applied using a relaxed molecular clock, as implemented in the BEAST program (version 1.7.5). Trees were visualized and edited with the FigTree program (version 1.4.2) included in the BEAST software package. Of the patient samples tested, 547 (3.43%) samples were positive for HPIV-3. Stratifying HPIV-3, by month revealed the virus major activity was during the winter and spring seasons. Not only that, but the majority of patients infected were children less than 1 year of age and elderly greater than 60 years of age. An increased HPIV-3 activity was seen in patients hospitalized in the oncology/transplants wards of the hospital. Of interest were patient’s co-infections with HPIV-3 and other respiratory viruses. Of the 547 patient infected with HPIV-3, 99 (18.1%) patients were co-infected with other human respiratory viruses. Of which, adenovirus (6.6%) and RSV (6.4%) were the most common. Molecular characterization of the complete HPIV-3 HN gene from 50 different patients infected throughout the study period revealed that the majority of the HPIV-3 strains circulating in Israel belonged to the C1b and C3a clades. These HPIV-3 clades were mainly seen in the America’s and Saudi Arabia. In addition, one HPIV-3 isolate from the year 2012 did not match with any of the C1 clades, suggesting the possibility of being a new sub clade. HPIV-3 HN sequence analysis also revealed that the isolates characterized from Israel did not acquire the substitutions T193I and I567V in the HN gene suggesting that in patients with severe infection and where Zanamivir treatment is warranted, this antiviral can be used to help in managing the HPIV-3 infection. This is the first comprehensive study that characterized HPIV3 infections in Israel. The high co-infection rate of HPIV-3 and other common human patients mandates careful evaluation of the clinical presentation of infected patients and their prognosis. In addition, in depth evaluation of the clinical presentation of patients infected with the different HPIV-3 clades should be entertained. http://dx.doi.org/10.1016/j.jcv.2016.08.225

Respiratory viruses in the intensive care unit: More frequent than expected

no: 146 Presentation at ESCV 2016: Poster 151 Case report: Unexpected cause of respiratory failure 3 days after heart transplantation K. Dierickx ∗, A. Vankeerberghen, A. Boel, K. Van Vaerenbergh, H. De Beenhouwer Laboratory of Microbiology, OLVZ Aalst, Belgium Respiratory syncytial virus is an RNA virus belonging to the Paramyxoviridae and it is mostly found in young children. This virus can also cause morbidity and mortality in immunocompromised adults. Respiratory virus infection (RSV) is an important complication in solid organ transplant patients but the longitudinal monitoring of these infections has not been extensively studied. Little has been described in literature regarding RSV pneumonia in adult heart transplant patients. Here we report an interesting case of a 56 year old female with a history of non-ischemic cardiomyopathy starting in 2011. On January the 3rd of 2015 she successfully underwent a heart transplantation. Although there were no signs of respiratory disease at the time of hospitalization she showed respiratory insufficiency three days post-transplantation. In the microbiology lab each respiratory sample is cultured and when indicated screened for a panel of 22 targets detected in 8 inhouse RT-PCR multiplexes. This molecular panel covers the most important pathogens of viral respiratory infections and atypical bacterial pneumonia. The first respiratory sample of this patient was a bronchial aspirate taken three days post transplantation. The bacterial culture was negative but the sample tested positive for RSV-A with a high viral load (Ct value of 23). Follow up samples 15 days and 35 days post-surgery were still RSV positive although with decreasing viral load (Ct value of 25 and 28 respectively). Culture of respiratory samples showed the presence of Staphylococcus aureus only 10 days after surgery so RSV is most probably the primary cause of the respiratory disease. RSV was still detectable 1 month after transplantation which might be explained by the immunosuppressive treatment of the patient. The heart transplantation was performed during the RSV season. Some days before the surgery the lady had taken care of her young grandchildren so there indeed was a potential risk of community-acquired transmission. Conclusion: Without testing for viral pathogens no accurate diagnosis for the respiratory failure of this patient could have been made. Since screening of adult patients for viral pathogens is not common practice at the IC-unit, this case illustrates the added value of molecular screening when signs of respiratory failure arise in adult immunocompromised patients. http://dx.doi.org/10.1016/j.jcv.2016.08.191 Abstract no: 148 Presentation at ESCV 2016: Poster 152no: 148 Presentation at ESCV 2016: Poster 152 Respiratory viruses in the intensive care unit: More frequent than expected A. Vankeerberghen ∗, K. Dierickx, A. Boel, K. Van Vaerenbergh, H. De Beenhouwer Laboratory of Microbiology, OLVZ Aalst, Belgium In the Laboratory of Microbiology of the OLV Hospital in Aalst respiratory samples (n = 3500/year), received from multiple hospitals spread all over Flanders, are analysed on a daily basis by in house multiplex real time PCR for a panel of viral and bacterial pathogens. The panel includes adenovirus, bocavirus, human metapneumovirus (hMPV), respiratory syncytial virus (RSV), parainfluenzavirus (PIV) 1, 2, 3 and 4, Influenza virus A and B, enterovirus, rhinovirus, coronaviruses, Bordetella pertussis & parapertussis, Mycoplasma pneumoniae and Chlamydia pneumoniae. Before 2014, the majority of samples originated from children. The severe influenza epidemic in the winter season 2014–2015 made clinicians aware that viral infections in adults are not that innocent at all. Moreover, in the “Influenza season”, not only Influenza circulated but also other viruses were cause of severe disease. Correct identification of the pathogen is indispensable to administer or withhold therapy. As a consequence, the request for the real time PCR respiratory panel on samples from adult hospitalized patients increased. In order to calculate the frequency of these pathogens in adult critically ill patients, a retrospective study was performed for the period September 2014 to May 2016 including patients transferred to the coronary care unit (CCU) and the intensive care unit (ICU) because of respiratory failure. Respiratory panel results of samples, obtained in the window from 3 days before to 5 days after transfer to the CCU and IC units, were included. From the 126 samples, 44 samples were positive (34.92%) with 41 samples (93.18%) positive for a viral pathogen and 3 samples (6.82%) positive for a bacterial pathogen (1 M. pneumoniae, 1 C. pneumoniae and 1 B. parapertussis). None of the samples were positive for adenovirus or parainfluenzavirus. As expected, Influenza A virus (n = 14) and Influenza B virus (n = 8) were the most frequent and 1 patient had a co-infection of both viruses. No other co-infection was found. Surprisingly, rhinovirus (n = 8) was found to be the third most frequent viral cause of infection. hMPV and RSV are known to cause severe respiratory problems in infants and RSV infections have also been observed in the immunocompromised host. In our study, not only RSV (n = 5) but also hMPV (n = 7) was found frequently and caused very severe “Influenza-like” disease. We can conclude that viral infections are a common cause of respiratory problems in the intensive care unit and screening of these patients might be an important clue in diagnosis and correct treatment. http://dx.doi.org/10.1016/j.jcv.2016.08.192 Abstract no: 161 Presentation at ESCV 2016: Poster 153no: 161 Presentation at ESCV 2016: Poster 153 Multidrug-resistant cytomegalovirus infection in a pediatric stem cell transplantation patient T. Bauters 1,∗, L. Florin 2, V. Bordon 3, R. Snoeck 4, G. Andrei 4, S. Gillemot 4, P. Fiten 4, G. Opdenakker 4, G. Laureys 3, E. Padalko 2 1 Department of Pharmacy, Ghent University Hospital, Ghent, Belgium 2 Department of Clinical Chemistry, Microbiology and Immunology, Ghent University and Hospital, Ghent, Belgium 3 Department of Pediatric Hemato-Oncology and Stem Cell Transplantation, Ghent University Hospital, Ghent, Belgium 4 Rega Institute for Medical Research, Department of Microbiology and Immunology, KU Leuven, Leuven, Belgium Background: Cytomegalovirus (CMV), a member of the Herpesviridae family, is characterized by a lifelong latency in the host. Clinical presentations of CMV infection are minimal in immuno-

Fluorescent in situ hybridization can be used as a complementary assay for the diagnosis of Tropheryma whipplei infection

BackgroundImmunohistochemistry and Periodic acid–Schiff (PAS) staining have been routinely used for the diagnosis of Whipple’s disease (WD). However, these methods present limitations. As a result, the last years, Fluorescence in situ hybridization (FISH) has been increasingly used as a complementary tool for the diagnosis of WD from various tissue samples.Case reportIn this study, we visualized, by FISH, Tropheryma whipplei within macrophages of a lymph node from a patient with WD. Moreover, we report in this study a patient with a pulmonary biopsy compatible with WD by PAS, immunostaining and FISH, although the specific molecular assays for T. whipplei were negative. Sequencing analysis of the 16S rDNA revealed a T. whipplei-related species with unknown classification.ConclusionFISH can be a valuable method for the detection of Tropheryma species in formalin-fixed paraffin-embedded tissues. FISH cannot replace the other already approved diagnostic techniques for WD, it can be used as a complementary tool and can provide supplementary information in a relatively short time.

Epidemiology of RSV and hMPV in Belgium: a 10-year follow-up

ABSTRACT Objectives: Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) are important respiratory pathogens. Both viral pathogens have similar clinical manifestations. The epidemiology of RSV is well known, that of hMPV is less clear. We reviewed the results of 10 consecutive years of molecular testing for RSV and hMPV in respiratory samples of Flemish patients. Methods: In the laboratory of the OLV hospital Aalst, Belgium, multiplex RT-PCR assays are used for the detection of RSV and hMPV. The lab receives invasive and noninvasive respiratory samples of patients from all over Flanders. Results: Between September 2006 and August 2016, 16,826 respiratory samples were analyzed for RSV and hMPV. Of these samples, 18% tested positive for RSV and 7.3% for hMPV. RSV consistently peaked in November/December each year within a very narrow time frame. The occurrence of hMPV was less predictable and spreaded more widely throughout the winter and spring. Both viruses were mainly found in samples from young children. RSV was most frequently detected in samples from infants <3 months, while hMPV peaked between 6 and 9 months. After the age of 1 year, RSV rapidly dropped. hMPV dropped a little later and slower. Both viruses slightly increased again at older age (>50 years). Conclusions: Despite their similarities, some of the epidemiologic characteristics of hMPV and RSV differ. The most striking difference is the annual distribution of RSV and hMPV infections.