Surveillance of the respiratory syncytial virus outside infancy: impact of testing methods, a retrospective observational study.

Background: The European Medicines Agency has approved several vaccines to protect the elderly against respiratory syncytial virus (RSV) infections. However, differences in performance between antigen and PCR tests, especially in adults, can make monitoring RSV difficult. This study aims to assess the impact of the chosen diagnostic methods on the surveillance of RSV. Methods: RSV and influenza test results obtained from July 2022 to June 2023 in a consolidated clinical laboratory in Brussels, Belgium, were collected. These results included antigen tests, quadruplex PCR tests and viral cultures on respiratory samples. Epidemiological trends related to the age of patients and the diagnostic methods were analysed. Results: Among 14 761 RSV tests, the overall number of positive tests for infants until 1 year of age peaked on 5 November 2022 (67 per 7 days) whereas it peaked on 22 December 2022 for adults (33 per 7 days). Positive antigen tests peaked on 7 November 2022 (56 per 7 days) whereas positive PCRs peaked on 19 December 2022 (36 per 7 days). Nevertheless, the positivity rate of RSV PCRs had peaked 1 month previously. Infants were mainly diagnosed through antigen testing, contrary to older patients. The influenza epidemic was probably the cause of the increased use of a quadruplex PCR, leading to a delayed increase in the absolute number of PCRs positive for RSV. Conclusion: This study shows that the use of different diagnostic methods could lead to an erroneous representation of RSV epidemiology in adults due to the lack of sensitivity of antigen detection. RSV surveillance in the elderly should rely rather on molecular methods.

Multiple interspecies recombination events documented by whole-genome sequencing in multidrug-resistant Haemophilus influenzae clinical isolates.

Introduction: Haemophilus influenzae (Hi) was long known as an easy-to-treat bacterium, but increasing resistance against beta-lactams and other critically important antibiotics is now a growing concern. We describe here the whole-genome sequencing (WGS) analysis of three non-typeable Hi isolates received in 2018-2019 by the Belgian National Reference Centre (NRC) for Haemophilus influenzae, as they presented an unusual multi-resistant profile. Methods: All three isolates were sequenced by WGS and mapped to the reference isolate Hi Rd KW20. Shorten uptake signal sequences (USSs) known to be associated with homologous recombination were sought in ftsI, murE and murF genes, and inner partial sequences were compared against the blast nucleotide database to look for similarity with other Haemophilus species. Their antimicrobial resistance (AMR) genotype was studied. Core-genome multilocus sequence typing (MLST) was performed on the NTHi database pubMLST to place our isolates in the actual worldwide epidemiology. Results: The isolates also harboured interspecies recombination patterns in the murF-murE-ftsI region involved in cell wall synthesis. The three isolates were multidrug resistant and two of them were also resistant to amoxicillin-clavulanic acid and showed a reduced susceptibility to meropenem. All three isolates belonged to the MLST clonal complex (CC) 422, and WGS revealed that the three were very similar. They harboured mobile genetic elements (carrying blaTEM-1B, mefA and msrD genes associated with resistance), mutations in gyrA and parC linked to fluoroquinolone resistance as well as remodelling events in ompP2 that might be related to lower carbapenem susceptibility. Conclusion: The Hi evolution towards antimicrobial multiresistance (AMR) is a complex and poorly understood phenomenon, although probably linked to a large degree to the presence of USSs and exchange within the family Pasteurellaceae. To better understand the respective roles of clonal expansion, horizontal gene transfers, spontaneous mutations and interspecies genetic rearrangements in shaping Hi AMR, both analysis of Hi communities over time within individuals and worldwide monitoring of non-typeable Hi causing infections should be conducted.

Evaluation of two automated real-time PCR-based quantification methods for whole blood Epstein-Barr viral load.

Quantification of EBV DNA is important in transplantation settings for the diagnosis of post-transplantation. We evaluated the performance of the AltoStar® EBV PCR Kit 1.5 on whole blood specimens: limit of detection, linearity, accuracy, and precision were determined using the WHO NIBSC 09/260 international standard. Results of 69 clinical samples were compared between the AltoStar® EBV PCR Kit 1.5 (altona Diagnostics) and the RealTime EBV assay (Abbott). The LoD of the AltoStar® Kit was 148 IU/mL and linearity was between 375 and 500000. A high concordance was found between nominal value of the NIBSC dilutions and the AltoStar EBV result. The total variation ranged from 2.2% to 9.6%. Out of 69 clinical samples tested, there was a high concordance between the 22 paired results within the overlapping linear ranges of both tests. The AltoStar® EBV assay is reliable and accurate for EBV viral load determination on whole blood samples.

Haemophilus influenzae carriage and antibiotic resistance profile in Belgian infants over a three-year period (2016-2018).

Background: Non-typeable Haemophilus influenzae has become increasingly important as a causative agent of invasive diseases following vaccination against H. influenzae type b. The emergence of antibiotic resistance underscores the necessity to investigate typeable non-b carriage and non-typeable H. influenzae (NTHi) in children. Methods: Nasopharyngeal swab samples were taken over a three-year period (2016-2018) from 336 children (6-30 months of age) attending daycare centers (DCCs) in Belgium, and from 218 children with acute otitis media (AOM). Biotype, serotype, and antibiotic resistance of H. influenzae strains were determined phenotypically. Mutations in the ftsI gene were explored in 129 strains that were resistant or had reduced susceptibility to beta-lactam antibiotics. Results were compared with data obtained during overlapping time periods from 94 children experiencing invasive disease. Results: Overall, NTHi was most frequently present in both carriage (DCC, AOM) and invasive group. This was followed by serotype "f" (2.2%) and "e" (1.4%) in carriage, and "b" (16.0%), "f" (11.7%), and "a" (4.3%) in invasive strains. Biotype II was most prevalent in all studied groups, followed by biotype III in carriage and I in invasive strains. Strains from both groups showed highest resistance to ampicillin (26.7% in carriage vs. 18.1% in invasive group). A higher frequency of ftsI mutations were found in the AOM group than the DCC group (21.6 vs. 14.9% - p = 0.056). Even more so, the proportion of biotype III strains that carried a ftsI mutation was higher in AOM compared to DCC (50.0 vs. 26.3% - p < 0.01) and invasive group. Conclusion: In both groups, NTHi was most frequently circulating, while specific encapsulated serotypes for carriage and invasive group were found. Biotypes I, II and III were more frequently present in the carriage and invasive group. The carriage group had a higher resistance-frequency to the analyzed antibiotics than the invasive group. Interestingly, a higher degree of ftsI mutations was found in children with AOM compared to DCC and invasive group. This data helps understanding the H. influenzae carriage in Belgian children, as such information is scarce.

Moderate forms of COVID-19 among healthcare workers in a Belgian hospital and their immune response.

INTRODUCTION: Healthcare professionals have been treating patients with COVID-19 since the pandemic started in early 2020 while also trying to limit disease spread among their coworkers and communities. This study aimed to identify and follow potentially infected healthcare workers in one hospital in order to develop an epidemiological baseline for COVID-19 infection and spread rates in this population. MATERIALS AND METHODS: This prospective study was conducted between 1 April and 30 June 2020 at a single Belgian hospital. Healthcare workers with symptoms consistent with COVID-19 were included. Participants underwent testing for SARS-CoV-2 infection by nasopharyngeal (NP) swab and analysis of blood samples for antibody response at different timepoints (day 0, 7, 14 and day 30 or 60). Patient exposures, symptoms, and disease progression were collected. RESULTS: Of a total of 150 healthcare workers with symptoms compatible with SARS-CoV-2 infection, 31% (47) tested positive for the virus by NP swab. Of the 47 participants with positive NP swabs, 66% also had positive IgG serology. Of the 99 participants with negative NP swabs who underwent blood sample collection, 5% had positive IgG serology results. Of the 29 participants who presented with at least 3 major symptoms, 15 (52%) had positive NP swabs and 14 (48%) had positive serology. CONCLUSIONS: This study demonstrates that evidence of symptoms, even when major, is a poor predictor for SARS-CoV-2 positivity in health care workers and raises the question of the best way to efficiently screen this population especially during the upcoming flu period.

Leveraging of SARS-CoV-2 PCR Cycle Thresholds Values to Forecast COVID-19 Trends.

Introduction: We assessed the usefulness of SARS-CoV-2 RT-PCR cycle thresholds (Ct) values trends produced by the LHUB-ULB (a consolidated microbiology laboratory located in Brussels, Belgium) for monitoring the epidemic's dynamics at local and national levels and for improving forecasting models. Methods: SARS-CoV-2 RT-PCR Ct values produced from April 1, 2020, to May 15, 2021, were compared with national COVID-19 confirmed cases notifications according to their geographical and time distribution. These Ct values were evaluated against both a phase diagram predicting the number of COVID-19 patients requiring intensive care and an age-structured model estimating COVID-19 prevalence in Belgium. Results: Over 155,811 RT-PCR performed, 12,799 were positive and 7,910 Ct values were available for analysis. The 14-day median Ct values were negatively correlated with the 14-day mean daily positive tests with a lag of 17 days. In addition, the 14-day mean daily positive tests in LHUB-ULB were strongly correlated with the 14-day mean confirmed cases in the Brussels-Capital and in Belgium with coinciding start, peak, and end of the different waves of the epidemic. Ct values decreased concurrently with the forecasted phase-shifts of the diagram. Similarly, the evolution of 14-day median Ct values was negatively correlated with daily estimated prevalence for all age-classes. Conclusion: We provide preliminary evidence that trends of Ct values can help to both follow and predict the epidemic's trajectory at local and national levels, underlining that consolidated microbiology laboratories can act as epidemic sensors as they gather data that are representative of the geographical area they serve.

SARS-CoV-2 Diagnostic Tests: Algorithm and Field Evaluation From the Near Patient Testing to the Automated Diagnostic Platform.

Introduction: Since the first wave of COVID-19 in Europe, new diagnostic tools using antigen detection and rapid molecular techniques have been developed. Our objective was to elaborate a diagnostic algorithm combining antigen rapid diagnostic tests, automated antigen dosing and rapid molecular tests and to assess its performance under routine conditions. Methods: An analytical performance evaluation of four antigen rapid tests, one automated antigen dosing and one molecular point-of-care test was performed on samples sent to our laboratory for a SARS-CoV-2 reverse transcription PCR. We then established a diagnostic algorithm by approaching median viral loads in target populations and evaluated the limit of detection of each test using the PCR cycle threshold values. A field performance evaluation including a clinical validation and a user-friendliness assessment was then conducted on the antigen rapid tests in point-of-care settings (general practitioners and emergency rooms) for outpatients who were symptomatic for <7 days. Automated antigen dosing was trialed for the screening of asymptomatic inpatients. Results: Our diagnostic algorithm proposed to test recently symptomatic patients using rapid antigen tests, asymptomatic patients using automated tests, and patients requiring immediate admission using molecular point-of-care tests. Accordingly, the conventional reverse transcription PCR was kept as a second line tool. In this setting, antigen rapid tests yielded an overall sensitivity of 83.3% (not significantly different between the four assays) while the use of automated antigen dosing would have spared 93.5% of asymptomatic inpatient screening PCRs. Conclusion: Using tests not considered the "gold standard" for COVID-19 diagnosis on well-defined target populations allowed for the optimization of their intrinsic performances, widening the scale of our testing arsenal while sparing molecular resources for more seriously ill patients.

Development and Potential Usefulness of the COVID-19 Ag Respi-Strip Diagnostic Assay in a Pandemic Context.

Introduction: COVID-19 Ag Respi-Strip, an immunochromatographic (ICT) assay for the rapid detection of SARS-CoV-2 antigen on nasopharyngeal specimen, has been developed to identify positive COVID-19 patients allowing prompt clinical and quarantine decisions. In this original research article, we describe the conception, the analytical and clinical performances as well as the risk management of implementing the COVID-19 Ag Respi-Strip in a diagnostic decision algorithm. Materials and Methods: Development of the COVID-19 Ag Respi-Strip resulted in a ready-to-use ICT assay based on a membrane technology with colloidal gold nanoparticles using monoclonal antibodies directed against the SARS-CoV and SARS-CoV-2 highly conserved nucleoprotein antigen. Four hundred observations were recorded for the analytical performance study and thirty tests were analyzed for the cross-reactivity study. The clinical performance study was performed in a retrospective multi-centric evaluation on aliquots of 328 nasopharyngeal samples. COVID-19 Ag Respi-Strip results were compared with qRT-PCR as golden standard for COVID-19 diagnostics. Results: In the analytical performance study, the reproducibility showed a between-observer disagreement of 1.7%, a robustness of 98%, an overall satisfying user friendliness and no cross-reactivity with other virus-infected nasopharyngeal samples. In the clinical performance study performed in three different clinical laboratories during the ascendant phase of the epidemiological curve, we found an overall sensitivity and specificity of 57.6 and 99.5%, respectively with an accuracy of 82.6%. The cut-off of the ICT was found at CT <22. User-friendliness analysis and risk management assessment through Ishikawa diagram demonstrate that COVID-19 Ag Respi-Strip may be implemented in clinical laboratories according to biosafety recommendations. Conclusion: The COVID-19 Ag Respi-Strip represents a promising rapid SARS-CoV-2 antigen assay for the first-line diagnosis of COVID-19 in 15 min at the peak of the pandemic. Its role in the proposed diagnostic algorithm is complementary to the currently-used molecular techniques.

Bigger and Better? Representativeness of the Influenza A Surveillance Using One Consolidated Clinical Microbiology Laboratory Data Set as Compared to the Belgian Sentinel Network of Laboratories.

Infectious diseases remain a serious public health concern globally, while the need for reliable and representative surveillance systems remains as acute as ever. The public health surveillance of infectious diseases uses reported positive results from sentinel clinical laboratories or laboratory networks, to survey the presence of specific microbial agents known to constitute a threat to public health in a given population. This monitoring activity is commonly based on a representative fraction of the microbiology laboratories nationally reporting to a single central reference point. However, in recent years a number of clinical microbiology laboratories (CML) have undergone a process of consolidation involving a shift toward laboratory amalgamation and closer real-time informational linkage. This report aims to investigate whether such merging activities might have a potential impact on infectious diseases surveillance. Influenza data was used from Belgian public health surveillance 2014-2017, to evaluate whether national infection trends could be estimated equally as effectively from only just one centralized CML serving the wider Brussels area (LHUB-ULB). The overall comparison reveals that there is a close correlation and representativeness of the LHUB-ULB data to the national and international data for the same time periods, both on epidemiological and molecular grounds. Notably, the effectiveness of the LHUB-ULB surveillance remains partially subject to local regional variations. A subset of the Influenza samples had their whole genome sequenced so that the observed epidemiological trends could be correlated to molecular observations from the same period, as an added-value proposition. These results illustrate that the real-time integration of high-throughput whole genome sequencing platforms available in consolidated CMLs into the public health surveillance system is not only credible but also advantageous to use for future surveillance and prediction purposes. This can be most effective when implemented for automatic detection systems that might include multiple layers of information and timely implementation of control strategies.

Epidemiology and genotype 3 subtype dynamics of hepatitis E virus in Belgium, 2010 to 2017.

BackgroundHepatitis E virus (HEV) is an emerging public health concern in high-income countries and can cause acute and chronic hepatitis. Reported numbers of indigenously acquired HEV infection have increased in the past decade in many European countries. Since 2010, the National Reference Centre (NRC) for Hepatitis Viruses has been testing samples of suspected hepatitis E cases in Belgium.AimIn this surveillance report, we present the epidemiological trends of symptomatic HEV infections in Belgium, from the distribution by age, sex and geography to the molecular characterisation of the viral strains.MethodSerum samples of suspected cases sent to the NRC between 2010 and 2017 were analysed for the presence of HEV-specific IgM and RNA. Virus was sequenced for genotyping and phylogenetic analysis in all samples containing sufficient viral RNA.ResultsThe NRC reported an increase in the number of samples from suspected cases (from 309 to 2,663 per year) and in the number of laboratory-confirmed hepatitis E cases (from 25 to 117 per year). Among 217 sequenced samples, 92.6% were genotype 3 (HEV-3), followed by 6.5% of genotype 1 and 0.9% of genotype 4. HEV-3 subtype viruses were mainly 3f, 3c and 3e. HEV-3f was the most common subtype until 2015, while HEV-3c became the most common subtype in 2016 and 2017.ConclusionThe increasing trend of HEV diagnoses in Belgium may be largely explained by increased awareness and testing.

Detection of beta-lactamase-negative ampicillin resistance in Haemophilus influenzae in Belgium.

Haemophilus influenzae, a frequent colonizer of the respiratory tract, is the causative agent of several clinically important infections. In cases that require therapeutic intervention, laboratory susceptibility testing can detect beta-lactam antibiotic resistance and guide the best treatment course. In the absence of a beta-lactamase, beta-lactam resistance may be due to an altered form of the PBP3 protein, encoded by the ftsI gene. While these so-called beta-lactamase-negative ampicillin-resistant (BLNAR) strains are of serious clinical interest, identification in the clinical laboratory is not always straightforward. In the current study, the ftsI genes of a set of phenotypic BLNAR H. influenzae isolates taken from samples collected in the UZ Brussel hospital in Belgium were sequenced and re-tested at the National Reference Laboratory (NRC). Non-silent mutations in the ftsI gene were found in 100% of the isolates. Although 30% of the isolates were classified by the NRC as beta-lactamase-negative ampicillin-sensitive (BLNAS) strains based on the EUCAST guidelines on ampicillin minimal inhibitory concentration (MIC), all isolates showed MIC values ≥1 mg/L. These relatively high MIC values indicate a decreased susceptibility to ampicillin, and suggest that sequencing of the ftsI gene should be used as part of an antibiotic susceptibility testing (AST) algorithm in the clinical laboratory. This would allow clinicians to make better informed decisions regarding patient treatment.

Campylobacter Concisus and Acute Gastroenteritis in Children: Lack of Association.

The role of Campylobacter concisus as a cause of acute gastroenteritis remains to be demonstrated. This prospective study includes 184 cases and 176 controls. It shows no evidence that C. concisus plays a role in acute gastroenteritis. Considering the very low prevalence in cases and controls, if there is an etiologic link, it would be moderate and difficult to demonstrate.

Clinical burden of hepatitis E virus infection in a tertiary care center in Flanders, Belgium.

BACKGROUND: Hepatitis E virus (HEV) infection is increasingly recognized as a cause of hepatitis in developed countries. A high HEV IgG seroprevalence in humans and pigs is reported as well as sporadic clinical cases of autochtonous HEV but there are currently no data available on the clinical burden of HEV in Belgium. OBJECTIVES: The objective of the current study was to evaluate the actual clinical burden of HEV infections in our tertiary care center in Flanders, Belgium. STUDY DESIGN: In the setting of Ghent University Hospital, patients were assessed for the presence of HEV IgG and IgM as well as HEV RNA if no other cause was found for one of the following clinical presentations: a) elevation of liver enzymes in post-liver transplant; b) suspicion of acute or toxic hepatitis; c) unexplainable elevation of liver enzymes; d) cirrhosis with acute-on-chronic exacerbation. RESULTS: In a period of 39 months (January 2011-April 2014) 71 patients were enrolled. HEV IgG was found positive in 13 (18,3%) patients; HEV IgM in 6 patients (8,5%) and HEV RNA in 4 (5,6%) patients. All HEV IgM/RNA positive patients were male, aged 41-63, and classified in the clinical groups a), b) or d). HEV IgG seroprevalence was slightly higher but not significantly different from the seroprevalence in the general population in this region in Belgium previously reported to be 14% (p-value 0.41) by our group. CONCLUSIONS: HEV should be considered as a cause of liver pathology especially in middle-aged men with elevation of liver enzymes.

Campylobacter coli meningitis in a 57-year-old patient.

Campylobacter coli is a major pathogen rarely responsible for extra-intestinal infections. We report a case of Campylobacter coli bacteremia and meningitis in a 57-year-old immunocompetent Belgian man. The strain, isolated from cerebrospinal fluid, belonged to the unusual sequence type 8418 (ST8418). The patient fully recovered after meropenem treatment.

Hepatitis E virus serology and PCR: does the methodology matter?

Hepatitis E virus (HEV) genotype 3 is an emerging pathogen in the developed world. As the clinical manifestations and routine laboratory parameters are often nonspecific, accurate diagnostic tests are crucial. In the current study, the performance of six serological assays and three PCR assays for the detection of HEV was evaluated. In the setting of the Ghent University Hospital, patients with clinically suspected HEV infection were tested for the presence of HEV IgM and IgG as well as HEV RNA. Serology was performed using six commercial HEV ELISA assays: Biorex, Wantai and Mikrogen IgM and IgG. HEV RNA was detected using one commercial assay (Altona RealStar®), and two optimized in-house real-time RT-PCR assays (according to Jothikumar et al., 2006 and Gyarmati et al., 2007). In addition, all three PCR assays were performed on 16 external quality control (EQC) samples. In a period of 39 months (January 2011-April 2014), 70 patients were enrolled. Using different ELISA assays, the prevalence of antibodies varied from 5.7% to 14.3% for HEV IgM and from 15.7% to 20.0% for IgG. All but two of the results of the PCR assays performed on clinical samples agreed. However, 10 out of 16 EQC samples results showed major discrepancies. We observed important differences in the performance of various serological and PCR assays. For this reason, results of both serological and molecular tests for HEV should be interpreted with caution.

Prevalence, antimicrobial resistance and genetic diversity of Campylobacter coli and Campylobacter jejuni in Ecuadorian broilers at slaughter age.

Thermotolerant Campylobacter spp. are a major cause of foodborne gastrointestinal infections worldwide. The linkage of human campylobacteriosis and poultry has been widely described. In this study we aimed to investigate the prevalence, antimicrobial resistance and genetic diversity of C. coli and C. jejuni in broilers from Ecuador. Caecal content from 379 randomly selected broiler batches originating from 115 farms were collected from 6 slaughterhouses located in the province of Pichincha during 1 year. Microbiological isolation was performed by direct plating on mCCDA agar. Identification of Campylobacter species was done by PCR. Minimum inhibitory concentration (MIC) values for gentamicin, ciprofloxacin, nalidixic acid, tetracycline, streptomycin, and erythromycin were obtained. Genetic variation was assessed by RFLP-flaA typing and Multilocus Sequence Typing (MLST) of selected isolates. Prevalence at batch level was 64.1%. Of the positive batches 68.7% were positive for C. coli, 18.9% for C. jejuni, and 12.4% for C. coli and C. jejuni. Resistance rates above 67% were shown for tetracycline, ciprofloxacin, and nalidixic acid. The resistance pattern tetracycline, ciprofloxin, and nalidixic acid was the dominant one in both Campylobacter species. RFLP-flaA typing analysis showed that C. coli and C. jejuni strains belonged to 38 and 26 profiles respectively. On the other hand MLST typing revealed that C. coli except one strain belonged to CC-828, while C. jejuni except 2 strains belonged to 12 assigned clonal complexes (CCs). Furthermore 4 new sequence types (STs) for both species were described, whereby 2 new STs for C. coli were based on new allele sequences. Further research is necessary to estimate the impact of the slaughter of Campylobacter positive broiler batches on the contamination level of carcasses in slaughterhouses and at retail in Ecuador.

Estimation of hepatitis E virus (HEV) pig seroprevalence using ELISA and Western blot and comparison between human and pig HEV sequences in Belgium.

Zoonotic transmission of hepatitis E virus (HEV) is of special concern, particularly in high income countries were waterborne infections are less frequent than in developing countries. High HEV seroprevalences can be found in European pig populations. The aims of this study were to obtain prevalence data on HEV infection in swine in Belgium and to phylogenetically compare Belgian human HEV sequences with those obtained from swine. An ELISA screening prevalence of 73% (95% CI 68.8-77.5) was determined in Belgian pigs and a part of the results were re-evaluated by Western blot (WB). A receiver operating characteristic curve analysis was performed and scenarios varying the ELISA specificity relative to WB were analysed. The seroprevalences estimated by the different scenarios ranged between 69 and 81% and are in agreement with the high exposure of the European pig population to HEV. Pig HEV sequences were genetically compared to those detected in humans in Belgium and a predominance of genotype 3 subtype f was shown in both swine and humans. The high HEV seroprevalence in swine and the close phylogenetic relationships between pig and human HEV sequences further support the risk for zoonotic transmission of HEV between humans and pigs.