Publications by authors named "Marien I de Jonge"

123 Publications

C-Reactive Protein to rule out complicated pneumococcal disease manifestations; a retrospective cohort study in adults with pneumococcal bacteraemia.

Int J Infect Dis 2021 Aug 9. Epub 2021 Aug 9.

Department of Clinical Microbiology and Immunology, Rijnstate, Wagnerlaan 55, 6815 AD Arnhem, the Netherlands; Department of Clinical Microbiology, Radboud Centre for Infectious Diseases, Radboudumc, Geert Grooteplein Zuid 10, 6525 GA, Nijmegen, the Netherlands.

Objectives: To explore the negative predictive value (NPV) of CRP at admission to exclude complicated disease manifestations.

Methods: A Dutch multicentre retrospective cohort study was conducted between 01-01-2012 and 30-06-2020. Adults with positive blood cultures for S. pneumoniae, whose CRP was measured at admission, and whose infection focus was known, were included. Electronic medical and microbiological records were reviewed.

Results: Of the 832 bacteremic patients enrolled, 30% had complicated manifestations of pneumococcal disease. Most frequent were pleural effusion (8.9%), pleural empyema (5.4%), and meningitis (7.5%). Compared to solitary pneumonia, patients with pleural effusion and empyema presented with higher CRP levels. Although low CRP did not exclude complicated disease in general, a CRP level < 114 mg/L at admission could reliably exclude empyema among adult pneumonia patients with an NPV of 93% and a specificity of 26%. However, in cases where pleural fluid was present, CRP levels were mostly above 114 mg/L, such that suspicion of empyema could only be ruled out in a minority of cases (10%).

Conclusions: Complicated manifestations are prevalent in adult pneumococcal bacteraemia. Low blood CRP levels can reliably exclude the development of pulmonary empyema. Practical value may be largest in settings without thoracic imaging at hand.
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http://dx.doi.org/10.1016/j.ijid.2021.08.011DOI Listing
August 2021

Neisseria meningitidis Serogroup Z Meningitis in a Child With Complement C8 Deficiency and Potential Cross Protection of the MenB-4C Vaccine.

Pediatr Infect Dis J 2021 Jul 19. Epub 2021 Jul 19.

From the Pediatric Infectious Diseases and Immunology, Amalia Children's Hospital, Nijmegen, the Netherlands Section Pediatric Infectious Diseases, Laboratory of Medical Immunology, Radboud Institute for Molecular Life Sciences, Radboudumc, Nijmegen, the Netherlands Radboud Center for Infectious Diseases, Radboudumc, Nijmegen, the Netherlands Department of Medical Microbiology, Radboudumc, Nijmegen, the Netherlands Expertise Center for Immunodeficiency and Autoinflammation (REIA), Radboudumc, Nijmegen, the Netherlands Pediatric Infectious Diseases and Immunology, Wilhelmina Children's Hospital, University Medical Center, Utrecht, the Netherlands.

Complement deficient patients are susceptible to rare meningococcal serogroups. A 6-year-old girl presented with serogroup Z meningitis. This led to identification of a C8 deficiency. The MenB-4C vaccine induced cross-reactive antibodies to serogroup Z and increased in vitro opsonophagocytic killing and may thus protect complement deficient patients.
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http://dx.doi.org/10.1097/INF.0000000000003259DOI Listing
July 2021

Common haplotypes at the CFH locus and low-frequency variants in CFHR2 and CFHR5 associate with systemic FHR concentrations and age-related macular degeneration.

Am J Hum Genet 2021 08 13;108(8):1367-1384. Epub 2021 Jul 13.

Department of Ophthalmology, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, 6525EX, the Netherlands; Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Centre, 6525GA, the Netherlands. Electronic address:

Age-related macular degeneration (AMD) is the principal cause of blindness in the elderly population. A strong effect on AMD risk has been reported for genetic variants at the CFH locus, encompassing complement factor H (CFH) and the complement-factor-H-related (CFHR) genes, but the underlying mechanisms are not fully understood. We aimed to dissect the role of factor H (FH) and FH-related (FHR) proteins in AMD in a cohort of 202 controls and 216 individuals with AMD. We detected elevated systemic levels of FHR-1 (p = 1.84 × 10), FHR-2 (p = 1.47 × 10), FHR-3 (p = 1.05 × 10) and FHR-4A (p = 1.22 × 10) in AMD, whereas FH concentrations remained unchanged. Common AMD genetic variants and haplotypes at the CFH locus strongly associated with FHR protein concentrations (e.g., FH p.Tyr402His and FHR-2 concentrations, p = 3.68 × 10), whereas the association with FH concentrations was limited. Furthermore, in an International AMD Genomics Consortium cohort of 17,596 controls and 15,894 individuals with AMD, we found that low-frequency and rare protein-altering CFHR2 and CFHR5 variants associated with AMD independently of all previously reported genome-wide association study (GWAS) signals (p = 5.03 × 10 and p = 2.81 × 10, respectively). Low-frequency variants in CFHR2 and CFHR5 led to reduced or absent FHR-2 and FHR-5 concentrations (e.g., p.Cys72Tyr in CFHR2 and FHR-2, p = 2.46 × 10). Finally, we showed localization of FHR-2 and FHR-5 in the choriocapillaris and in drusen. Our study identifies FHR proteins as key proteins in the AMD disease mechanism. Consequently, therapies that modulate FHR proteins might be effective for treating or preventing progression of AMD. Such therapies could target specific individuals with AMD on the basis of their genotypes at the CFH locus.
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http://dx.doi.org/10.1016/j.ajhg.2021.06.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8387287PMC
August 2021

Kawasaki Disease Patient Stratification and Pathway Analysis Based on Host Transcriptomic and Proteomic Profiles.

Int J Mol Sci 2021 May 26;22(11). Epub 2021 May 26.

Faculty of Medicine, Imperial College London, London SW7 2AZ, UK.

The aetiology of Kawasaki disease (KD), an acute inflammatory disorder of childhood, remains unknown despite various triggers of KD having been proposed. Host 'omic profiles offer insights into the host response to infection and inflammation, with the interrogation of multiple 'omic levels in parallel providing a more comprehensive picture. We used differential abundance analysis, pathway analysis, clustering, and classification techniques to explore whether the host response in KD is more similar to the response to bacterial or viral infections at the transcriptomic and proteomic levels through comparison of 'omic profiles from children with KD to those with bacterial and viral infections. Pathways activated in patients with KD included those involved in anti-viral and anti-bacterial responses. Unsupervised clustering showed that the majority of KD patients clustered with bacterial patients on both 'omic levels, whilst application of diagnostic signatures specific for bacterial and viral infections revealed that many transcriptomic KD samples had low probabilities of having bacterial or viral infections, suggesting that KD may be triggered by a different process not typical of either common bacterial or viral infections. Clustering based on the transcriptomic and proteomic responses during KD revealed three clusters of KD patients on both 'omic levels, suggesting heterogeneity within the inflammatory response during KD. The observed heterogeneity may reflect differences in the host response to a common trigger, or variation dependent on different triggers of the condition.
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http://dx.doi.org/10.3390/ijms22115655DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8198135PMC
May 2021

Structure-Activity Relationship of Fluorinated Sialic Acid Inhibitors for Bacterial Sialylation.

Bioconjug Chem 2021 06 27;32(6):1047-1051. Epub 2021 May 27.

Cluster of Molecular Chemistry, Institute for Molecules and Materials, Radboud University Nijmegen, Nijmegen 6525 AJ, The Netherlands.

Bacterial pathogens such as Nontypeable (NTHi) can evade the immune system by taking up and presenting host-derived sialic acids. Herein, we report a detailed structure-activity relationship of sialic acid-based inhibitors that prevent the transfer of host sialic acids to NTHi. We report the synthesis and biological evaluation of C-5, C-8, and C-9 derivatives of the parent compound 3-fluorosialic acid (SiaNFAc). Small modifications are tolerated at the C-5 and C-9 positions, while the C-8 position does not allow for modification. These structure-activity relationships define the chemical space available to develop selective bacterial sialylation inhibitors.
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http://dx.doi.org/10.1021/acs.bioconjchem.1c00194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8382218PMC
June 2021

Lack of Cell Cycle Inhibitor p21 and Low CD4 T Cell Suppression in Newborns After Exposure to IFN-β.

Front Immunol 2021 12;12:652965. Epub 2021 Apr 12.

Laboratory of Medical Immunology, Section Pediatric Infectious Diseases, Department of Laboratory Medicine, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, Netherlands.

Type I IFNs, such as interferon alpha and interferon beta, are key regulators of the adaptive immune response during infectious diseases. Type I IFNs are induced upon infection, bind interferon α/β receptors on T-cells and activate intracellular pathways. The activating and inhibitory consequences of type I IFN-signaling are determined by cell type and cellular environment. The neonatal immune system is associated with increased vulnerability to infectious diseases which could partly be explained by an immature CD4 T-cell compartment. Here, we show low IFN-β-mediated inhibition of CD4 T-cell proliferation, phosphorylation of retinoblastoma protein and cytokine production in human newborns compared to adults. In addition, both naïve and total newborn CD4 T-cells are unable to induce the cell-cycle inhibitor p21 upon exposure to IFN-β in contrast to adults. The distinct IFN-β-signaling in newborns provides novel insights into T cell functionality and regulation of T cell-dependent inflammation during early life immune responses.
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http://dx.doi.org/10.3389/fimmu.2021.652965DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8071872PMC
April 2021

Complement factor D haplodeficiency is associated with a reduced complement activation speed and diminished bacterial killing.

Clin Transl Immunology 2021 3;10(4):e1256. Epub 2021 Apr 3.

Pediatric Infectious Diseases and Immunology Amalia Children's Hospital Nijmegen The Netherlands.

Objectives: Complete deficiency of alternative pathway (AP) complement factors, explained by homozygous mutations, is a well-known risk factor for invasive bacterial infections; however, this is less obvious for heterozygous mutations. We describe two siblings with a heterozygous NM_001928.3(CFD):c.125C>A p.(Ser42*) mutation in the complement factor D (fD) gene having a history of recurrent bacterial infections. We determined the effect of heterozygous fD deficiency on AP complement activity.

Methods: We determined the effect of fD levels on complement activation as measured by AP activity, complement C3 binding to the bacterial surface of (Nm), (Sp) and non-typeable (NTHi), and complement-mediated killing of Nm and NTHi. In addition, we measured the effect of vaccination of complement C3 binding to the bacterial surface and killing of Nm.

Results: Reconstitution of fD-deficient serum with fD increased AP activity in a dose- and time-dependent way. Reconstitution of patient serum with fD to normal levels increased complement C3 binding to Sp, Nm and NTHi, as well as complement-mediated killing of Nm and NTHi. Vaccination increased complement C3 binding and resulted in complete killing of Nm without fD reconstitution.

Conclusion: We conclude that low fD serum levels (< 0.5 μg mL) lead to a reduced speed of complement activation, which results in diminished bacterial killing, consistent with recurrent bacterial infections observed in our index patients. Specific antibodies induced by vaccination are able to overcome the diminished bacterial killing capacity in patients with low fD levels.
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http://dx.doi.org/10.1002/cti2.1256DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8019133PMC
April 2021

Production of inactivated gram-positive and gram-negative species with preserved cellular morphology and integrity.

J Microbiol Methods 2021 05 22;184:106208. Epub 2021 Mar 22.

Department of Pathology, Radboud University Medical Center, Nijmegen, the Netherlands. Electronic address:

There are many approaches available to produce inactive bacteria by termination of growth, each with a different efficacy, impact on cell integrity, and potential for application in standardized inactivation protocols. The aim of this study was to compare these approaches and develop a standardized protocol for generation of inactivated Gram-positive and Gram-negative bacteria, yielding cells that are metabolically dead with retained cellular integrity i.e., preserving the surface and limited leakage of intracellular proteins and DNA. These inactivated bacteria are required for various applications, for instance, when investigating receptor-triggered signaling or bacterial contact-dependent analysis of cell lines requiring long incubation times. We inactivated eight different bacterial strains of different species by treatment with beta-propiolactone, ethanol, formalin, sodium hydroxide, and pasteurization. Inactivation efficacy was determined by culturing, and cell wall integrity assessed by quantifying released DNA, bacterial membrane and intracellular DNA staining, and visualization by scanning electron microscopy. Based on these results, we discuss the bacterial inactivation methods, and their advantages and disadvantages to study host-microbe interactions with inactivated bacteria.
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http://dx.doi.org/10.1016/j.mimet.2021.106208DOI Listing
May 2021

Berberine and Obatoclax Inhibit SARS-Cov-2 Replication in Primary Human Nasal Epithelial Cells In Vitro.

Viruses 2021 02 11;13(2). Epub 2021 Feb 11.

Department of Medical Microbiology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, 6500 HB Nijmegen, The Netherlands.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged as a new human pathogen in late 2019 and it has infected over 100 million people in less than a year. There is a clear need for effective antiviral drugs to complement current preventive measures, including vaccines. In this study, we demonstrate that berberine and obatoclax, two broad-spectrum antiviral compounds, are effective against multiple isolates of SARS-CoV-2. Berberine, a plant-derived alkaloid, inhibited SARS-CoV-2 at low micromolar concentrations and obatoclax, which was originally developed as an anti-apoptotic protein antagonist, was effective at sub-micromolar concentrations. Time-of-addition studies indicated that berberine acts on the late stage of the viral life cycle. In agreement, berberine mildly affected viral RNA synthesis, but it strongly reduced infectious viral titers, leading to an increase in the particle-to-pfu ratio. In contrast, obatoclax acted at the early stage of the infection, which is in line with its activity to neutralize the acidic environment in endosomes. We assessed infection of primary human nasal epithelial cells that were cultured on an air-liquid interface and found that SARS-CoV-2 infection induced and repressed expression of specific sets of cytokines and chemokines. Moreover, both obatoclax and berberine inhibited SARS-CoV-2 replication in these primary target cells. We propose berberine and obatoclax as potential antiviral drugs against SARS-CoV-2 that could be considered for further efficacy testing.
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http://dx.doi.org/10.3390/v13020282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7918080PMC
February 2021

Infection Manager System (IMS) as a new hemocytometry-based bacteremia detection tool: A diagnostic accuracy study in a malaria-endemic area of Burkina Faso.

PLoS Negl Trop Dis 2021 03 1;15(3):e0009187. Epub 2021 Mar 1.

Nijmegen Institute of International Health, Radboudumc, Nijmegen, the Netherlands.

Background: New hemocytometric parameters can be used to differentiate causes of acute febrile illness (AFI). We evaluated a software algorithm-Infection Manager System (IMS)-which uses hemocytometric data generated by Sysmex hematology analyzers, for its accuracy to detect bacteremia in AFI patients with and without malaria in Burkina Faso. Secondary aims included comparing the accuracy of IMS with C-reactive protein (CRP) and procalcitonin (PCT).

Methods: In a prospective observational study, patients of ≥ three-month-old (range 3 months- 90 years) presenting with AFI were enrolled. IMS, blood culture and malaria diagnostics were done upon inclusion and additional diagnostics on clinical indication. CRP, PCT, viral multiplex PCR on nasopharyngeal swabs and bacterial- and malaria PCR were batch-tested retrospectively. Diagnostic classification was done retrospectively using all available data except IMS, CRP and PCT results.

Findings: A diagnosis was affirmed in 549/914 (60.1%) patients and included malaria (n = 191) bacteremia (n = 69), viral infections (n = 145), and malaria-bacteremia co-infections (n = 47). The overall sensitivity, specificity, and negative predictive value (NPV) of IMS for detection of bacteremia in patients of ≥ 5 years were 97.0% (95% CI: 89.8-99.6), 68.2% (95% CI: 55.6-79.1) and 95.7% (95% CI: 85.5-99.5) respectively, compared to 93.9% (95% CI: 85.2-98.3), 39.4% (95% CI: 27.6-52.2), and 86.7% (95% CI: 69.3-96.2) for CRP at ≥20mg/L. The sensitivity, specificity and NPV of PCT at 0.5 ng/ml were lower at respectively 72.7% (95% CI: 60.4-83.0), 50.0% (95% CI: 37.4-62.6) and 64.7% (95% CI: 50.1-77.6) The diagnostic accuracy of IMS was lower among malaria cases and patients <5 years but remained equal to- or higher than the accuracy of CRP.

Interpretation: IMS is a new diagnostic tool to differentiate causes of AFI. Its high NPV for bacteremia has the potential to improve antibiotic dispensing practices in healthcare facilities with hematology analyzers. Future studies are needed to evaluate whether IMS, combined with malaria diagnostics, may be used to rationalize antimicrobial prescription in malaria endemic areas.

Trial Registration: ClinicalTrials.gov (NCT02669823) https://clinicaltrials.gov/ct2/show/NCT02669823.
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http://dx.doi.org/10.1371/journal.pntd.0009187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7951874PMC
March 2021

Broad range detection of viral and bacterial pathogens in bronchoalveolar lavage fluid of children to identify the cause of lower respiratory tract infections.

BMC Infect Dis 2021 Feb 5;21(1):152. Epub 2021 Feb 5.

Shenzhen Children's Hospital, No. 7019 Yitian Road, Futian District, Shenzhen, 518038, Guangdong, China.

Background: Knowledge on the etiology of LRTIs is essential for improvement of the clinical diagnosis and accurate treatment. Molecular detection methods were applied to identify a broad range of bacterial and viral pathogens in a large set of bronchial alveolar lavage (BAL) fluid samples. The patterns of detected pathogens were correlated to the clinical symptoms.

Methods: BAL fluid samples and clinical data were collected from 573 hospitalized children between 1 month and 14 years of age with LRTIs, enrolled from January to December 2018. Pathogens were detected using standardized clinical diagnostics, with a sensitive, high-throughput GeXP-based multiplex PCR and with multiplex qPCR. Data were analyzed to describe the correlation between the severity of respiratory tract disease and the pathogens identified.

Results: The pathogen detection rate with GeXP-based PCR and multiplex qPCR was significantly higher than by clinical routine diagnostics (76.09% VS 36.13%,χ = 8.191, P = 0.004). The most frequently detected pathogens in the BAL fluid were human adenovirus (HADV)(21.82%), Mycoplasma pneumoniae (20.24%), human rhinovirus (13.96%), Streptococcus pneumoniae (8.90%) and Haemophilus influenzae (8.90%). In 16.4% of the cases co-detection with two or three different pathogens was found. Viral detection rates declined with age, while atypical pathogen detection rates increased with age. Oxygen supply in the HADV and Influenza H1N1 infected patients was more frequent (49.43%) than in patients infected with other pathogens.

Conclusion: Broad range detection of viral and bacterial pathogens using molecular methods is a promising and implementable approach to improve clinical diagnosis and accurate treatment of LRTI in children.
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http://dx.doi.org/10.1186/s12879-021-05834-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7864134PMC
February 2021

Respiratory Tract Infection Management and Antibiotic Prescription in Children: A Unique Study Comparing Three Levels of Healthcare in The Netherlands.

Pediatr Infect Dis J 2021 03;40(3):e100-e105

Section Pediatric Infectious Diseases, Laboratory of Medical Immunology, Department of Laboratory Medicine, Radboud Institute for Molecular Life Sciences.

Background: Respiratory tract infections (RTIs) are common in children with febrile illness visiting the general practitioner (GP) or emergency department. We studied the management of children with fever and RTI at 3 different levels of healthcare in The Netherlands, focusing on antibiotic prescription.

Methods: This prospective observational study is part of the Management and Outcome of Febrile children in Europe study. Data were used from face-to-face patient contacts of children with febrile illness in three healthcare settings in Nijmegen, The Netherlands during 2017. These settings were primary (GP), secondary (general hospital) and tertiary care (university hospital).

Results: Of 892 cases with RTI without complex comorbidities, overall antibiotic prescription rates were 29% with no differences between the 3 levels of healthcare, leading to an absolute number of 5031 prescriptions per 100,000 children per year in primary care compared with 146 in secondary and tertiary care combined. The prescription rate in otitis media was similar in all levels: 60%. In cases with lower RTI who received nebulizations prescription rates varied between 19% and 55%.

Conclusions: Antibiotic prescription rates for RTIs in children were comparable between the 3 levels of healthcare, thus leading to a majority of antibiotics being prescribed in primary care. Relatively high prescription rates for all foci of RTIs were found, which was not in agreement with the national guidelines. Antibiotic stewardship needs improvement at all 3 levels of healthcare. Guidelines to prescribe small spectrum antibiotics for RTIs need to be better implemented in hospital care settings.
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http://dx.doi.org/10.1097/INF.0000000000003019DOI Listing
March 2021

Quantitative multiplex profiling of the complement system to diagnose complement-mediated diseases.

Clin Transl Immunology 2020 9;9(12):e1225. Epub 2020 Dec 9.

Laboratory of Medical Immunology Department of Laboratory Medicine Radboud Institute for Molecular Life Sciences Radboud University Medical Center Nijmegen The Netherlands.

Objectives: Complement deficiencies are difficult to diagnose because of the variability of symptoms and the complexity of the diagnostic process. Here, we applied a novel 'complementomics' approach to study the impact of various complement deficiencies on circulating complement levels.

Methods: Using a quantitative multiplex mass spectrometry assay, we analysed 44 peptides to profile 34 complement proteins simultaneously in 40 healthy controls and 83 individuals with a diagnosed deficiency or a potential pathogenic variant in 14 different complement proteins.

Results: Apart from confirming near or total absence of the respective protein in plasma of complement-deficient patients, this mass spectrometry-based profiling method led to the identification of additional deficiencies. In many cases, partial depletion of the pathway up- and/or downstream of the absent protein was measured. This was especially found in patients deficient for complement inhibitors, such as angioedema patients with a C1-inhibitor deficiency. The added value of complementomics was shown in three patients with poorly defined complement deficiencies.

Conclusion: Our study shows the potential clinical utility of profiling circulating complement proteins as a comprehensive read-out of various complement deficiencies. Particularly, our approach provides insight into the intricate interplay between complement proteins due to functional coupling, which contributes to the better understanding of the various disease phenotypes and improvement of care for patients with complement-mediated diseases.
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http://dx.doi.org/10.1002/cti2.1225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7724921PMC
December 2020

Understanding host immune responses to pneumococcal proteins in the upper respiratory tract to develop serotype-independent pneumococcal vaccines.

Expert Rev Vaccines 2020 10 8;19(10):959-972. Epub 2020 Nov 8.

First Department of Paediatrics, "Aghia Sophia" Children's Hospital, Immunobiology Research Laboratory and Infectious Diseases Department "MAKKA", Athens Medical School , Athens, Greece.

: Nasopharyngeal colonization is a precondition for mucosal and invasive pneumococcal disease. Prevention of colonization may reduce pneumococcal transmission and disease incidence. Therefore, several protein-based pneumococcal vaccines are currently under investigation. : We aimed to better understand the host immune responses to pneumococcal proteins in the upper respiratory tract (URT) that could facilitate the development of serotype-independent pneumococcal vaccines. English peer-reviewed papers reporting immunological mechanisms involved in host immune response to pneumococcal proteins in the URT were retrieved through a PubMed search using the terms 'pneumococcal proteins,' 'nasopharyngeal colonization' and/or 'cellular/humoral host immune response.' : Although pneumococcal protein antigens induce humoral immune responses, as well as IL-17A-mediated immunity, none of them, when used as single antigen, is sufficient to control and broadly protect against pneumococcal colonization. Novel vaccines should contain multiple conserved protein antigens to activate both arms of the immune system and evoke protection against the whole spectrum of pneumococcal variants by reducing, rather than eradicating, pneumococcal carriage. The highest efficacy would likely be achieved when the vaccine is intranasally applied, inducing mucosal immunity and enhancing the first line of defense by restricting pneumococcal density in the URT, which in turn will lead to reduced transmission and protection against disease.
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http://dx.doi.org/10.1080/14760584.2020.1843433DOI Listing
October 2020

Exploring metal availability in the natural niche of to discover potential vaccine antigens.

Virulence 2020 12;11(1):1310-1328

Section Pediatric Infectious Diseases, Laboratory of Medical Immunology, Radboud Institute for Molecular Life Sciences , Nijmegen, The Netherlands.

Nasopharyngeal colonization by is a prerequisite for pneumococcal transmission and disease. Current vaccines protect only against disease and colonization caused by a limited number of serotypes, consequently allowing serotype replacement and transmission. Therefore, the development of a broadly protective vaccine against colonization, transmission and disease is desired but requires a better understanding of pneumococcal adaptation to its natural niche. Hence, we measured the levels of free and protein-bound transition metals in human nasal fluid, to determine the effect of metal concentrations on the growth and proteome of . Pneumococci cultured in medium containing metal levels comparable to nasal fluid showed a highly distinct proteomic profile compared to standard culture conditions, including the increased abundance of nine conserved, putative surface-exposed proteins. AliA, an oligopeptide binding protein, was identified as the strongest protective antigen, demonstrated by the significantly reduced bacterial load in a murine colonization and a lethal mouse pneumonia model, highlighting its potential as vaccine antigen.
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http://dx.doi.org/10.1080/21505594.2020.1825908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7550026PMC
December 2020

How the COVID-19 pandemic highlights the necessity of animal research.

Curr Biol 2020 09 10;30(18):R1014-R1018. Epub 2020 Aug 10.

Maastricht University, 6211 LK Maastricht, The Netherlands.

Recently, a petition was offered to the European Commission calling for an immediate ban on animal testing. Although a Europe-wide moratorium on the use of animals in science is not yet possible, there has been a push by the non-scientific community and politicians for a rapid transition to animal-free innovations. Although there are benefits for both animal welfare and researchers, advances on alternative methods have not progressed enough to be able to replace animal research in the foreseeable future. This trend has led first and foremost to a substantial increase in the administrative burden and hurdles required to make timely advances in research and treatments for human and animal diseases. The current COVID-19 pandemic clearly highlights how much we actually rely on animal research. COVID-19 affects several organs and systems, and the various animal-free alternatives currently available do not come close to this complexity. In this Essay, we therefore argue that the use of animals is essential for the advancement of human and veterinary health.
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http://dx.doi.org/10.1016/j.cub.2020.08.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7416712PMC
September 2020

Growth rate alterations of human colorectal cancer cells by 157 gut bacteria.

Gut Microbes 2020 11;12(1):1-20

Department of Pathology, Radboud Institute for Molecular Life Sciences (RIMLS), Radboud University Medical Center , Nijmegen, The Netherlands.

Several bacteria in the human gut microbiome have been associated with colorectal cancer (CRC) by high-throughput screens. In some cases, molecular mechanisms have been elucidated that drive tumorigenesis, including bacterial membrane proteins or secreted molecules that interact with the human cancer cells. For most gut bacteria, however, it remains unknown if they enhance or inhibit cancer cell growth. Here, we screened bacteria-free supernatants (secretomes) and inactivated cells of over 150 cultured bacterial strains for their effects on cell growth. We observed family-level and strain-level effects that often differed between bacterial cells and secretomes, suggesting that different molecular mechanisms are at play. Secretomes of , and bacteria enhanced cell growth, while most cells and secretomes inhibited growth, contrasting prior findings. In some bacteria, the presence of specific functional genes was associated with cell growth rates, including the virulence genes TcdA, TcdB in and FadA in , which both inhibited growth. cells that enhanced growth were enriched for genes of the cobalamin synthesis pathway, while cells that inhibit growth were enriched for genes of the ethanolamine utilization pathway. Together, our results reveal how different gut bacteria have wide-ranging effects on cell growth, contribute a better understanding of the effects of the gut microbiome on host cells, and provide a valuable resource for identifying candidate target genes for potential microbiome-based diagnostics and treatment strategies.
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http://dx.doi.org/10.1080/19490976.2020.1799733DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7524400PMC
November 2020

Effect of FHA and Prn on Bordetella pertussis colonization of mice is dependent on vaccine type and anatomical site.

PLoS One 2020 21;15(8):e0237394. Epub 2020 Aug 21.

Section Pediatric Infectious Diseases, Laboratory of Medical Immunology, Radboud Institute for Molecular Life Sciences, Radboudumc, Nijmegen, The Netherlands.

Bordetella pertussis vaccine escape mutants that lack expression of the pertussis antigen pertactin (Prn) have emerged in vaccinated populations in the last 10-20 years. Additionally, clinical isolates lacking another acellular pertussis (aP) vaccine component, filamentous hemagglutinin (FHA), have been found sporadically. Here, we show that both whole-cell pertussis (wP) and aP vaccines induced protection in the lungs of mice, but that the wP vaccine was more effective in nasal clearance. Importantly, bacterial populations isolated from the lungs shifted to an FHA-negative phenotype due to frameshift mutations in the fhaB gene. Loss of FHA expression was strongly selected for in Prn-deficient strains in the lungs following aP but not wP vaccination. The combined loss of Prn and FHA led to complete abrogation of bacterial surface binding by aP-induced serum antibodies. This study demonstrates vaccine- and anatomical site-dependent adaptation of B. pertussis and has major implications for the design of improved pertussis vaccines.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0237394PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7446907PMC
October 2020

Eculizumab impairs Neisseria meningitidis serogroup B killing in whole blood despite 4CMenB vaccination of PNH patients.

Blood Adv 2020 08;4(15):3615-3620

Department of Hematology, Radboudumc, Nijmegen, The Netherlands.

Complement C5 inhibitor eculizumab has a great impact on the treatment of patients with paroxysmal nocturnal hemoglobinuria (PNH). However, this treatment success has a major drawback: a substantially increased susceptibility for life-threatening Neisseria meningitidis infections. Therefore, N meningitidis vaccination is strongly advised before initiating complement C5-blocking therapy. In this study, we show that the multicomponent N meningitidis serogroup B (4CMenB) vaccination of PNH patients treated with eculizumab results in a significant increase in anti-N meningitidis serogroup B (MenB) plasma immunoglobulin G (IgG) levels. Anti-MenB IgG was able to bind to the bacterial surface and initiate complement activation; however, inhibition of the membrane attack complex formation completely blocked whole blood-mediated killing of MenB. This would suggest that, despite 4CMenB vaccination, PNH patients taking C5 inhibitors are not sufficiently protected against MenB infection, which is in line with the fact that vaccinated PNH patients still experience meningococcal infections.
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http://dx.doi.org/10.1182/bloodadvances.2020002497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7422103PMC
August 2020

Antibody Binding and Complement-Mediated Killing of Invasive Haemophilus influenzae Isolates from Spain, Portugal, and the Netherlands.

Infect Immun 2020 09 18;88(10). Epub 2020 Sep 18.

Section Pediatric Infectious Diseases, Laboratory of Medical Immunology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, the Netherlands

is a Gram-negative bacterium that can be classified into typeable (types a through f) and nontypeable (NTHi) groups. This opportunistic pathogen asymptomatically colonizes the mucosal epithelium of the upper respiratory tract, from where it spreads to other neighboring regions, potentially leading to disease. Infection with NTHi can cause otitis media, sinusitis, conjunctivitis, exacerbations of chronic obstructive pulmonary disease, and pneumonia, but it is increasingly causing invasive disease, including bacteremia and meningitis. Invasive NTHi strains are more resistant to complement-mediated killing. However, the mechanisms of complement resistance have never been studied in large numbers of invasive NTHi strains. In this study, we determined the relationship between binding of IgG or IgM and the bacterial survival in normal human serum for 267 invasive strains from Spain, Portugal, and the Netherlands, of which the majority (200 [75%]) were NTHi. NTHi bacteria opsonized with high levels of IgM had the lowest survival in human serum. IgM binding to the bacterial surface, but not IgG binding, was shown to be associated with complement-mediated killing of NTHi strains. We conclude that evasion of IgM binding by NTHi strains increases survival in blood, thereby potentially contributing to their ability to cause severe invasive diseases.
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http://dx.doi.org/10.1128/IAI.00454-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7504965PMC
September 2020

Lipidation of Pneumococcal Antigens Leads to Improved Immunogenicity and Protection.

Vaccines (Basel) 2020 Jun 17;8(2). Epub 2020 Jun 17.

Department of Molecular Genetics and Infection Biology, Interfaculty Institute of Genetics and Functional Genomics, Center for Functional Genomics of Microbes, University of Greifswald, 17489 Greifswald, Germany.

infections lead to high morbidity and mortality rates worldwide. Pneumococcal polysaccharide conjugate vaccines significantly reduce the burden of disease but have a limited range of protection, which encourages the development of a broadly protective protein-based alternative. We and others have shown that immunization with pneumococcal lipoproteins that lack the lipid anchor protects against colonization. Since immunity against is mediated through Toll-like receptor 2 signaling induced by lipidated proteins, we investigated the effects of a lipid modification on the induced immune responses in either intranasally or subcutaneously vaccinated mice. Here, we demonstrate that lipidation of recombinant lipoproteins DacB and PnrA strongly improves their immunogenicity. Mice immunized with lipidated proteins showed enhanced antibody concentrations and different induction kinetics. The induced humoral immune response was modulated by lipidation, indicated by increased IgG2/IgG1 subclass ratios related to Th1-type immunity. In a mouse model of colonization, immunization with lipidated antigens led to a moderate but consistent reduction of pneumococcal colonization as compared to the non-lipidated proteins, indicating that protein lipidation can improve the protective capacity of the coupled antigen. Thus, protein lipidation represents a promising approach for the development of a serotype-independent pneumococcal vaccine.
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http://dx.doi.org/10.3390/vaccines8020310DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7350230PMC
June 2020

Viral-bacterial (co-)occurrence in the upper airways and the risk of childhood pneumonia in resource-limited settings.

J Infect 2020 08 10;81(2):213-220. Epub 2020 Jun 10.

Section Pediatric Infectious Diseases, Laboratory of Medical Immunology, Radboud Institute for Molecular Life Sciences, Radboud Center for Infectious Diseases, Radboud University Medical Center, Nijmegen, the Netherlands.

Objective: To examine the association between bacterial-viral co-occurrence in the nasopharynx and the risk of community acquired pneumonia (CAP) in young children living in resource-limited settings.

Methods: A case-control study was conducted between January and December 2017 in Moshi, Tanzania. Children 2-59 months with CAP and healthy controls were enrolled. RSV and Influenza A/B were detected with a standardized polymerase chain reaction (PCR) method, and a simplified real-time quantitative PCR method, without sample pre-processing, was developed to detect bacterial pathogens in nasopharyngeal samples.

Results: A total of 109 CAP patients and 324 healthy controls were enrolled. Co-detection of H. influenzae and S. pneumoniae in nasopharyngeal swabs was linked with higher odds of CAP (aOR=3.2, 95% CI=1.1-9.5). The majority of the H. influenzae isolated in cases and controls (95.8%) were non-typeable. Of the viruses examined, respiratory syncytial virus (RSV) was most common (n = 31, 7.2%) in cases and controls. Children with RSV had 8.4 times higher odds to develop pneumonia than healthy children (aOR=8.4, 95%CI= 3.2 - 22.1).

Conclusions: Co-occurence of H. influenzae and S. pneumoniae in the nasopharynx was strongly associated with CAP. The high prevalence of non-typeable H. influenzae might be a sign of replacement as a consequence of Haemophilus influenzae type b vaccination.
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http://dx.doi.org/10.1016/j.jinf.2020.06.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7392802PMC
August 2020

Clinical diagnostic application of metagenomic next-generation sequencing in children with severe nonresponding pneumonia.

PLoS One 2020 4;15(6):e0232610. Epub 2020 Jun 4.

Department of Respiratory Diseases, Shenzhen Children's Hospital, Shenzhen, China.

Pneumonia is one of the most important causes of morbidity and mortality in children. Identification and characterization of pathogens that cause infections are crucial for accurate treatment and accelerated recovery. However, in most cases, the causative agent cannot be identified, which is partly due to the limited spectrum of pathogens covered by current diagnostics based on nucleic acid amplification. Therefore, in this study, we explored the application of metagenomic next-generation sequencing (mNGS) for the diagnosis of children with severe pneumonia. From April to July 2017, 32 hospitalized children with severe nonresponding pneumonia in Shenzhen Children's Hospital were included in this study. Blood tests were conducted immediately after hospitalization to assess cell counts and inflammatory markers, oropharyngeal swabs were collected to identify common pathogens by qPCR and culture. After bronchoscopy, bronchoalveolar lavage fluid (BALF) samples were collected for further pathogen identification using standardized diagnostic tests and mNGS. Blood tests were normal in 3 of the 32 children. In 9 oropharyngeal swabs, bacterial pathogens were detected, in 5 of these Mycoplasma pneumoniae was detected. Adenovirus was detected in 5 BALF samples, using the Direct Immunofluorescence Assay (DFA). In 15 cases, no common pathogens were found in BALF samples, using the current standard diagnostic tests, while in all 32 BALFs, pathogens were identified using mNGS, including adenovirus, Mycoplasma pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, cytomegalovirus and bocavirus. This study shows that, with mNGS, the sensitivity of detection of the causative pathogens in children with severe nonresponding pneumonia is significantly improved. In addition, mNGS gives more strain specific information, helps to identify new pathogens and could potentially help to trace and control outbreaks. In this study, we have shown that it is possible to have the results within 24 hours, making the application of mNGS feasible for clinical diagnostics.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0232610PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7272011PMC
August 2020

Identification of conditionally essential genes for infection in pigs.

Virulence 2020 12;11(1):446-464

Department of Infection Biology, Wageningen Bioveterinary Research (WBVR), Lelystad, The Netherlands.

is a Gram-positive bacterium and zoonotic pathogen that causes meningitis and sepsis in pigs and humans. The aim of this study was to identify genes required for infection. We created Tn-Seq libraries in a virulent strain 10, which was used to inoculate pigs in an intrathecal experimental infection. Comparative analysis of the relative abundance of mutants recovered from different sites of infection (blood, cerebrospinal fluid, and meninges of the brain) identified 361 conditionally essential genes, i.e. required for infection, which is about 18% of the genome. The conditionally essential genes were primarily involved in metabolic and transport processes, regulation, ribosomal structure and biogenesis, transcription, and cell wall membrane and envelope biogenesis, stress defenses, and immune evasion. Directed mutants were created in a set of 10 genes of different genetic ontologies and their role was determined in models. Mutants showed different levels of sensitivity to survival in whole blood, serum, cerebrospinal fluid, thermic shock, and stress conditions, as compared to the wild type. Additionally, the role of three selected mutants was validated in co-infection experiments in which pigs were infected with both wild type and isogenic mutant strains. The genetic determinants of infection identified in this work contribute to novel insights in pathogenesis and could serve as targets for novel vaccines or antimicrobial drugs.
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http://dx.doi.org/10.1080/21505594.2020.1764173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7239030PMC
December 2020

Growth on Carbohydrates from Carbonaceous Meteorites Alters the Immunogenicity of Environment-Derived Bacterial Pathogens.

Astrobiology 2020 11 8;20(11):1353-1362. Epub 2020 May 8.

Department of Internal Medicine, Radboud University Medical Center, Nijmegen, Netherlands.

The last decade has witnessed a renewed interest in space exploration. Public and private institutions are investing considerable effort toward the direct exploration of the Moon and Mars, as well as more distant bodies in the solar system. Both automated and human-crewed spacecraft are being considered in these efforts. As inevitable fellow travelers on the bodies of astronauts, spaceships, or equipment, terrestrial microorganisms will undoubtedly come into contact with extraterrestrial environments, despite stringent decontamination. These microorganisms could eventually adapt and grow in their new habitats, where they might potentially recolonize and lead to the infection of the human space travelers. In this article, we demonstrate that clinically relevant bacterial species found in the environment are able to grow in minimal media with sugar compounds identified in extraterrestrial carbon sources. As a surrogate model, we used carbohydrates previously isolated from carbonaceous meteorites. The bacteria underwent an adaptation process that caused structural modifications in the cell envelope that sparked changes in pathogenic potential, both and . Understanding the adaptation of microorganisms exposed to extraterrestrial environments, with subsequent changes in their immunogenicity and virulence, requires a comprehensive analysis of such scenarios to ensure the safety of major space expeditions in the decades to come.
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http://dx.doi.org/10.1089/ast.2019.2173DOI Listing
November 2020

Unraveling Haemophilus influenzae virulence mechanisms enable discovery of new targets for antimicrobials and vaccines.

Curr Opin Infect Dis 2020 06;33(3):231-237

Section Pediatric Infectious Diseases, Laboratory of Medical Immunology, Radboud Institute for Molecular Life Sciences.

Purpose Of Review: The human upper respiratory tract is colonized with a variety of bacterial microorganisms including Haemophilus influenzae. The species H. influenzae consists of typeable and nontypeable H. influenzae (NTHi) variants. Typeable H. influenzae are subdivided into types a through f, based on the polysaccharide capsule, whereas the NTHi strains do not express a polysaccharide capsule. In this review, we highlight the current advances in the field of H. influenzae, with the focus on bacterial virulence mechanisms that facilitate bacterial colonization and disease, particularly for NTHi.

Recent Findings: In the past decade, it has become apparent that NTHi has the ability to cause invasive infections. Recently, a number of adhesins have been shown to be crucial for bacterial colonization and invasion and these proteins were investigated as vaccine antigens. Although NTHi lacks a polysaccharide capsule, it expresses lipooligosaccharide that contribute to adhesion and evasion of complement-mediated killing, both contributing to bacterial virulence, which could potentially be targeted by novel antimicrobial drugs or vaccines.

Summary: The unraveling of H. influenzae virulence mechanisms resulted in the identification of promising targets for novel antimicrobials and vaccine antigens aiming to prevent or treat both typeable and nontypeable H. influenzae infections.
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http://dx.doi.org/10.1097/QCO.0000000000000645DOI Listing
June 2020

Immune recognition of putative alien microbial structures: Host-pathogen interactions in the age of space travel.

PLoS Pathog 2020 01 30;16(1):e1008153. Epub 2020 Jan 30.

Department of Laboratory Medicine and Radboud Center for Infectious Diseases, Radboud University Medical Center, Nijmegen, the Netherlands.

Human space travel is on the verge of visiting Mars and, in the future, even more distant places in the solar system. These journeys will be also made by terrestrial microorganisms (hitchhiking on the bodies of astronauts or on scientific instruments) that, upon arrival, will come into contact with new planetary environments, despite the best measures to prevent contamination. These microorganisms could potentially adapt and grow in the new environments and subsequently recolonize and infect astronauts. An even more challenging situation would be if truly alien microorganisms will be present on these solar system bodies: What will be their pathogenic potential, and how would our immune host defenses react? It will be crucial to anticipate these situations and investigate how the immune system of humans might cope with modified terrestrial or alien microbes. We propose several scenarios that may be encountered and how to respond to these challenges.
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http://dx.doi.org/10.1371/journal.ppat.1008153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6991955PMC
January 2020

Nasopharyngeal Microbiota Profiles in Rural Venezuelan Children Are Associated With Respiratory and Gastrointestinal Infections.

Clin Infect Dis 2021 01;72(2):212-221

Department of Pediatric Infectious Diseases and Immunology, Wilhelmina Children's Hospital, University Medical Center Utrecht, Utrecht, The Netherlands.

Background: Recent research suggests that the microbiota affects susceptibility to both respiratory tract infections (RTIs) and gastrointestinal infections (GIIs). In order to optimize global treatment options, it is important to characterize microbiota profiles across different niches and geographic/socioeconomic areas where RTI and GII prevalences are high.

Methods: We performed 16S sequencing of nasopharyngeal swabs from 209 Venezuelan Amerindian children aged 6 weeks-59 months who were participating in a 13-valent pneumococcal conjugate vaccine (PCV13) study. Using random forest models, differential abundance testing, and regression analysis, we determined whether specific bacteria were associated with RTIs or GIIs and variation in PCV13 response.

Results: Microbiota compositions differed between children with or without RTIs (P = .018) or GIIs (P = .001). Several species were associated with the absence of infections. Some of these health-associated bacteria are also observed in developed regions, such as Corynebacterium (log2(fold change [FC]) = 3.30 for RTIs and log2(FC) = 1.71 for GIIs), while others are not commonly observed in developed regions, such as Acinetobacter (log2(FC) = 2.82 and log2(FC) = 5.06, respectively). Klebsiella spp. presence was associated with both RTIs (log2(FC) = 5.48) and GIIs (log2(FC) = 7.20).

Conclusions: The nasopharyngeal microbiota of rural Venezuelan children included several bacteria that thrive in tropical humid climates. Interestingly, nasopharyngeal microbiota composition not only differed in children with an RTI but also in those with a GII, which suggests a reciprocal interplay between the 2 environments. Knowledge of region-specific microbiota patterns enables tailoring of preventive and therapeutic approaches.
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http://dx.doi.org/10.1093/cid/ciaa015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7840112PMC
January 2021
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