Two Years of Genomic Surveillance in Belgium during the SARS-CoV-2 Pandemic to Attain Country-Wide Coverage and Monitor the Introduction and Spread of Emerging Variants.

An adequate SARS-CoV-2 genomic surveillance strategy has proven to be essential for countries to obtain a thorough understanding of the variants and lineages being imported and successfully established within their borders. During 2020, genomic surveillance in Belgium was not structurally implemented but performed by individual research laboratories that had to acquire the necessary funds themselves to perform this important task. At the start of 2021, a nationwide genomic surveillance consortium was established in Belgium to markedly increase the country's genomic sequencing efforts (both in terms of intensity and representativeness), to perform quality control among participating laboratories, and to enable coordination and collaboration of research projects and publications. We here discuss the genomic surveillance efforts in Belgium before and after the establishment of its genomic sequencing consortium, provide an overview of the specifics of the consortium, and explore more details regarding the scientific studies that have been published as a result of the increased number of Belgian SARS-CoV-2 genomes that have become available.

Two Separate Clusters of SARS-CoV-2 Delta Variant Infections in a Group of 41 Students Travelling from India: An Illustration of the Need for Rigorous Testing and Quarantine.

We report two clusters of SARS-CoV-2 B.1.617.2 (Delta variant) infections in a group of 41 Indian nursing students who travelled from New Delhi, India, to Belgium via Paris, France. All students tested negative before departure and had a second negative antigen test upon arrival in Paris. Upon arrival in Belgium, the students were quarantined in eight different houses. Four houses remained COVID-free during the 24 days of follow-up, while all 27 residents of the other four houses developed an infection during quarantine, including the four residents who were fully vaccinated and the two residents who were partially vaccinated. Genome sequencing revealed two distinct clusters affecting one and three houses, respectively. In this group of students, vaccination status did not seem to prevent infection nor decrease the viral load. No severe symptoms were reported. Extensive contact tracing and 3 months of nationwide genomic surveillance confirmed that these outbreaks were successfully contained and did not contribute to secondary community transmission in Belgium. These clusters highlight the importance of repeated testing and quarantine measures among travelers coming from countries experiencing a surge of infections, as all infections were detected 6 days or more after arrival.

Sneathia amnii bacteraemia and chorioamnionitis leading to second trimester abortion: a case report.

BACKGROUND: Sneathia amnii (formerly designated as Leptotrichia amnionii ) was first described in 2002 in the USA. Members of the genus Sneathia can be part of the normal flora of the genitourinary tract, but have been implicated in invasive (mostly gynaecological) infections. CASE PRESENTATION: To the best of our knowledge, here we present the first case of S. amnii infection in Belgium, in a young woman presenting with fever leading to second trimester septic abortion. CONCLUSION: Despite its pathogenicity, S. amnii remains an underrated cause of infections due to inherent difficulties with conventional laboratory methods. By extracting the bacterial DNA directly from the blood culture broth and performing a 16S ribosomal RNA gene sequence analysis we succeeded in identifying S. amnii as the most probable cause of the septic abortion in our patient.

Bacteremia and complicated parapneumonic effusion caused by Bordetella holmesii in an elderly patient.

We describe a case of bacteremia and a complicated parapneumonic effusion caused by Bordetella holmesii, in an elderly patient with underlying chronic hepatitis C infection.

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

BACKGROUND: Immunohistochemistry 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 REPORT: In 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. CONCLUSION: FISH 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.

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.

Multicenter evaluation of the revised RIDA® QUICK test (N1402) for rapid detection of norovirus in a diagnostic laboratory setting.

The updated RIDA® QUICK (N1402) immunochromatographic assay (R-Biopharm) for detection of norovirus was evaluated during a prospective, multicenter study using 771 stool samples from patients with gastroenteritis. Compared to real-time reverse transcriptase polymerase chain reaction (RT-rtPCR) as gold standard, the RIDA® QUICK had an overall sensitivity of 72.8% (91/125) and a specificity of 99.5% (640/643). Genotype analysis of the polymerase (ORF1) and capsid (ORF2) region of the genome indicated that the RIDA® QUICK assay could detect a broad range of genotypes including new variants (15 of 125 positive samples) which were detected by an in-house SYBR®Green RT-rtPCR, but not by the RIDA® GENE PCR PG1415 (R-Biopharm) and mostly not by the RIDA® GENE PCR PG1405 and the Xpert® Norovirus assay (Cepheid). The RIDA® QUICK can be used to reliably confirm norovirus in stool samples, but a negative result does not definitively exclude the presence of norovirus.

How is the Xpert MRSA Gen 3 assay (Cepheid) performing on pooled eSwab medium?

The performance of the Xpert MRSA Gen 3 was compared to the Xpert MRSA on pooled eSwab media from nose, throat, and perineum using broth enriched cultured as gold standard. A lower specificity was found for the Xpert MRSA Gen 3 compared to the Xpert MRSA (91.8% versus 97.9%; P<0.05).

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.

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.

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.

Implementation of real-time RT-PCR for detection of human metapneumovirus and its comparison with enzyme immunoassay.

Human metapneumovirus (hMPV) is responsible for outbreaks of bronchiolitis in winter and early spring in young children. Due to the relatively recent discovery of hMPV, the diagnostic opportunities are limited, while differential diagnosis with respiratory syncytial virus (RSV) remains important. We validated the RT-PCR by comparing various methods of RNA extraction, one-step RT-PCR kits and primer-probe combinations. The optimized RT-PCR was evaluated using 47 nasopharyngeal aspirates (NPAs) collected from children younger than 5 years, with clinically suspected RSV infection. The evaluated RT-PCRs were also compared to a commercially available hMPV enzyme immunoassay (EIA). We found 8.5% hMPV positivity with both RT-PCRs, in agreement with published literature. hMPV EIA showed positive and indeterminate results in 17% and 8.5%, respectively, of the tested NPAs. Positive RT-PCR samples were positive or indeterminate by hMPV EIA. Samples that were positive for RSV and influenza A virus interfered with the hMPV EIA. In conclusion, although RT-PCR is already a valuable tool for diagnosing hMPV infections, further optimization of the RT-PCR method is recommended. The hMPV EIA kit shows poor specificity and therefore needs further improvement.

First case of Staphylococcus pseudintermedius infection in a human.

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.

Differential induction of human beta-defensin expression by periodontal commensals and pathogens in periodontal pocket epithelial cells.

BACKGROUND: To investigate the possible role of beta-defensins in gingival health and periodontal disease, we examined the effect of several stimuli on the expression of interleukin-8 (IL-8), human beta-defensin-1, -2, -3, and -4 (hBD) in primary human diseased gingival epithelial (HGE) cell cultures from periodontitis patients by quantitative TaqMan reverse transcription polymerase chain reaction (RT-PCR). METHODS: Several strains of the periodontopathogens Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis were added to the cells, as well as the oral commensal bacteria Fusobacterium nucleatum and Escherichia coli. The induction by the proinflammatory stimuli phorbol 12-myristate 13-acetate (PMA) and tumor necrosis factor-alpha (TNF-alpha) was also tested. RESULTS: In addition to the published observations (PMA induces hBD-2 and -4; TNF-alpha induces hBD-2 and -3), it was found that PMA can upregulate hBD-1 and hBD-3, whereas TNF-alpha can induce hBD-4. The commensal bacteria were significant inducers of hBD-2, hBD-3, and IL-8. The pathogen P. gingivalis induced hBD-1 and hBD-3 at different time points than the commensals, but no induction of IL-8 and hBD-2 could be observed. These data fit with the chemokine paralysis theory. A correlation was found between the pathogenicity of different serotypes of A. actinomycetemcomitans and the induction profiles of defensins and IL-8. CONCLUSION: The results suggest that a correlation can be found in diseased oral epithelium between the defensin profiles that are induced and the pathogenicity of the oral bacterial strains.

Distribution of human beta-defensin polymorphisms in various control and cystic fibrosis populations.

Human beta defensins contribute to the first line of defense against infection of the lung. Polymorphisms in these genes are therefore potential modifiers of the severity of lung disease in cystic fibrosis. Polymorphisms were sought in the human beta-defensin genes DEFB1, DEFB4, DEFB103A, and DEFB104 in healthy individuals and cystic fibrosis (CF) patients living in various European countries. DEFB1, DEFB4, and DEFB104 were very polymorphic, but DEFB103A was not. Within Europe, differences between control populations were found for some of the frequent polymorphisms in DEFB1, with significant differences between South-Italian and Czech populations. Moreover, frequent polymorphisms located in DEFB4 and DEFB104 were not in Hardy Weinberg equilibrium in all populations studied, while those in DEFB1 were in Hardy Weinberg equilibrium. Sequencing of a monochromosomal chromosome 8 mouse-human hybrid cell line revealed signals for multiple alleles for some loci in DEFB4 and DEFB104, but not for DEFB1. This indicated that more than one DEFB4 and DEFB104 gene was present on this chromosome 8, in agreement with recent findings that DEFB4 and DEFB104 are part of a repeat region. Individual DEFB4 and DEFB104 PCR amplification products of various samples were cloned and sequenced. The results showed that one DNA sample could contain more than two haplotypes, indicating that the various repeats on one chromosome were not identical. Given the higher complexity found in the genomic organization of the DEFB4 and DEFB104 genes, association studies with CF lung disease severity were performed only for frequent polymorphisms located in DEFB1. No association with the age of first infection by Pseudomonas aeruginosa or with the FEV1 percentage at the age of 11-13 years could be found.

The cystic fibrosis transmembrane conductance regulator: an intriguing protein with pleiotropic functions.

Cystic fibrosis is a frequent autosomal recessive disorder that is caused by the malfunctioning of a small chloride channel, the cystic fibrosis transmembrane conductance regulator. The protein is found in the apical membrane of epithelial cells lining exocrine glands. Absence of this channel results in imbalance of ion concentrations across the cell membrane. As a result, fluids secreted through these glands become more viscous and, in the end, ducts become plugged and atrophic. Little is known about the pathways that link the malfunctioning of the CFTR protein with the observed clinical phenotype. Moreover, there is no strict correlation between specific CFTR mutations and the CF phenotype. This might be explained by the fact that environmental and additional genetic factors may influence the phenotype. The CFTR protein itself is regulated at the maturational level by chaperones and SNARE proteins and at the functional level by several protein kinases. Moreover, CFTR functions also as a regulator of other ion channels and of intracellular membrane transport processes. In order to be able to function as a protein with pleiotropic actions, CFTR seems to be linked with other proteins and with the cytoskeleton through interaction with PDZ-domain-containing proteins at the apical pole of the cell. Progress in cystic fibrosis research is substantial, but still leaves many questions unanswered.