Offizielles Organ der SULM Schweizerische Union für Labormedizin | Organe officiel de l’USML Union Suisse de Médecine de Laboratoire | www.sulm.ch | N R . 3 | J U N I 2 0 2 2
S W I S S
Microbiology and the pandemic Professor Gilbert Greub
A century of antibiotic resistance Surveillance of pathogens in Switzerland The reference centre for tick-borne infections The Human Microbiota Network Le réchauffement climatique et les microbes
L A B O R A T O R Y
M E D I C I N E
2
CD OU NC TA ET NITOSN E
Inhalt · Sommaire 3 EDITORIAL 4 NEWS
Microbiology and the pandemic. 6 NEWS
A century of antibiotic resistance: targets, mechanisms and susceptibility testing. 9 NEWS
Molecular surveillance of pathogens in Switzerland – Focus: SARS-CoV-2 and its variants. 12 NEWS
The Swiss national reference centre for tick-borne infections. 17 NEWS
Le réchauffement climatique: possibles -impacts sur les microbes, leurs réservoirs, les infections et les épidémies. 22 NEWS
The Human Microbiota Network of the Swiss National Centre of Competences in Research (NCCR) on microbiomes: objectives and main approaches 25 MARKETPLACE 26 NEWS
En mémoire de Prof. Claude Bachmann.
P I P E T T E – S W I S S L A B O R AT O R Y M E D I C I N E | WWW. S U L M . C H
NR. 3 | JUNI 2022
IMPRESSUM «pipette», offizielles Publikationsorgan der SULM / Organe officiel de l’USML 18. Jahrgang, Nr 3/2022, erscheint 2022 5-mal, ISSN 1661-09 Herausgeber | Editeur Schweizerische Union für Labormedizin (SULM) Prof. Dr. Dr. Michael Nagler, Inselspital Universitätsspital, Universitätsinstitut für Klinische Chemie, Z entrum für Labormedizin 3010 Bern michael.nagler@insel.ch www.sulm.ch Redaktionskomitee | Comité de rédaction Prof. Dr. Dr. Michael Nagler Dr. Roman Fried Prof. Dr. Gilbert Greub Prof. Dr. Alexander Leichtle Marianne Schenk Dr. Véronique Viette Dr. med. et Dr. phil. II Elsbeth Probst-Müller Redaktion | Rédaction Peter Neyer pipette@sulm.ch Redaktionsadresse | Adresse de la rédaction pipette@sulm.ch Cover © Prof. Gilbert Greub
Richtlinien für Autoren | Instructions pour les auteurs www.sulm.ch/pipette Verlag | Editeur Stämpfli AG Wölflistrasse 1 Postfach 3001 Bern Telefon: 031 300 66 66 Herstellung | Production Stämpfli AG Wölflistrasse 1 Postfach 3001 Bern Telefon: 031 300 66 66 Inserate | Annonces Stämpfli AG Ruzica Dragicevic, Anzeigenleiterin Wölflistrasse 1, Postfach 3001 Bern Telefon: 031 300 63 87 E-Mail Ruzica.Dragicevic@staempfli.com Abonnemente | Abonnements www.sulm.ch/pipette/abonnement info@sulm.ch Einzelpreis CHF 20.– Jahresabo CHF 80.– Auflage | Tirage 8000 Exemplare Nächste Ausgabe | Prochain numéro «At the bedside» Erscheinungsdatum | Date de parution: 10.8.2022
A G E N D A
www.sulm.ch/aktuell/agenda – Termine zu Kongressen, Tagungen und Versammlungen – Dates des congrès, conférences et réunions P I P E T T E O N L I N E W W W . S U L M . C H / P I P E T T E
– Lesen Sie die «pipette» online als E-Paper, im Browser oder auf dem Tablet. Alle Artikel können im «pipette»-Archiv als PDF heruntergeladen werden. – Lire la «pipette» en ligne comme e-paper, dans le navigateur ou sur la tablette. Tous les articles de la «pipette» peuvent être téléchargés en format PDF.
P I P E T T E – S W I S S L A B O R AT O R Y M E D I C I N E | W W W. S U L M . C H
EDITORIAL
NR. 3 | JUNI 2022
Microbiology and the pandemic
Microbiologie et pandémie
This special issue on microbes is going in press just when a large European outbreak of Monkeypox virus is on the frontpage of most newspapers. Indeed, this large poxviridae DNA virus has spread and has infected a huge number of persons, mainly male having sex with male, likely due to some superspreading events such as the pride held in Maspalomas (Gran Canaria). Monkeypox is disscussed in an article on climate change, since global warming and reduced size of tropical rainforests partially explain the progressive increased number of human cases in endemic countries in sub-Saharian Africa. Such outbreaks needs rapid detection of cases by PCR and molecular epidemiology as done earlier for SARSCoV-2. To be effective, molecular epidemiology needs (i) a network of laboratories providing excellent quality sequences and (ii) a common repository with comprehensive metadata. This is discussed – using SARS-CoV-2 as an example – in the article by Neves et al. SARSCoV-2 is also discussed in the interview done by Michael Nagler, our new editor-in-Chief. Global warming also explain the increased surfaces suitable for ticks (16 to 25 % in ten years in Switzerland) and the increased incidence of tick-borne infections, which make the work of the National Center for tick-borne infections (CNRTNRZK) increasingly important. The tasks of this national center and the health issues associated with Borrelia & Coxiella, as well as the TBE virus are discussed in another article. Finally, given the increased threat of antibiotics resistance, Caruana et al presents current knowledge on antibiotics. Good lecture about microbes.
Ce numéro spécial sur les microbes part sous presse alors même que l’Europe se trouve confrontée à une vaste épidémie du virus de la variole du singe qui fait la une de la plupart des journaux. En effet, ce gros virus à ADN de la famille des Poxviridae se répand et a contaminé un nombre conséquent de personnes, principalement des hommes ayant des relations sexuelles avec d’autres hommes, probablement en raison d’événements de super-propagation comme la pride de Maspalomas (Grande Canarie). Il est question de la variole du singe dans un article sur le changement climatique, car le réchauffement mondial et la réduction des forêts tropicales expliquent en partie le nombre progressivement croissant de cas humains dans les pays d’endémie de l’Afrique subsaharienne. Ce type d’épidémies requiert une détection rapide des cas par PCR et le recours à l’épidémiologie moléculaire, comme précédemment dans le cas du SARS-CoV-2. Pour être efficace, l’épidémiologie moléculaire nécessite (i) un réseau de laboratoires fournissant des séquences d’excellente qualité et (ii) un référentiel commun de métadonnées exhaustives. Ce sujet est abordé dans l’article de Neves et coll. en prenant en exemple le SARS-CoV-2. Il est également question du SARSCoV-2 dans l’entretien avec notre nouveau rédacteur en chef, Michael Nagler. Le réchauffement climatique explique aussi l’élargissement du territoire adapté aux tiques (de 16 % à 25 % en dix ans en Suisse) et l’incidence croissante des infections transmises par les tiques, raisons pour lesquelles le travail du Centre national de référence pour les maladies à tiques (CNRT-NRZK) prend de plus en plus d’importance. Un autre article aborde d’ailleurs les tâches auxquelles s’attelle ce Centre national, et d’autres questions de santé associées aux bactéries Borrelia et Coxiella, ainsi qu’au virus de l’encéphalite à tiques. Enfin, étant donné la menace accrue d’antibiorésistance, Giorgia Caruana et coll. présentent les dernières connaissances en matière d’antibiotiques. Nous vous souhaitons une bonne lecture en compagnie des microbes.
Gilbert Greub
Gilbert Greub
SULM – Schweizerische Union für Labormedizin | USML – Union Suisse de Médecine de Laboratoire Angeschlossene Fachgesellschaften BAG CSCQ FAMH FMH H+ KHM labmed MQ pharmaSuisse SGED
Bundesamt für Gesundheit – Abteilung KU Schweizerisches Zentrum für Qualitätskontrolle Die medizinischen Laboratorien der Schweiz Verbindung der Schweizer Ärztinnen und Ärzte Die Spitäler der Schweiz Kollegium für Hausarztmedizin Schweizerischer Berufsverband der biomedizinischen Analytikerinnen und Analytiker Verein für medizinische Qualitätskontrolle Schweizerischer Apothekerverband Schweizerische Gesellschaft für Endokrinologie und Diabetologie
SGKC/SSCC SGM SGMG SGRM SSAI/SGAI SGH/SSH SVA SVDI
Schweizerische Gesellschaft für Klinische Chemie Schweizerische Gesellschaft für Mikrobiologie Schweizerische Gesellschaft für Medizinische Genetik Schweizerische Gesellschaft für Rechtsmedizin Schweizerische Gesellschaft für Allergologie und Immunologie Schweizerische Gesellschaft für Hämatologie Schweizerischer Verband Medizinischer Praxis-Fachpersonen Schweizerischer Verband der Diagnosticaund Diagnostica-Geräte-Industrie
3
Prof. Gilbert Greub, MD-PhD, Chef de Service et Directeur de l’Institut de Microbiologie. Médecin chef des laboratoires de microbiologie diagnostique Institut de microbiologie de l’Université de Lausanne Département des laboratoires
4
ED N EW UC SA T I O N
P I P E T T E – S W I S S L A B O R AT O R Y M E D I C I N E | WWW. S U L M . C H
NR. 3 | JUNI 2022
Microbiology and the pandemic The COVID-19 pandemic posed major challenges for medical laboratories. The Pipette discusses the difficulties, opportunities and lessons learned with Professor Gilbert Greub. He is Director of the Institute of Microbiology, Lausanne University Hospital (CHUV), and Professor at the University of Lausanne. Professor Greub, you are interested in the discovery of new pathogens of pneumonia. Was the COVID-19 pandemic a stroke of luck for you? This was not a stroke of luck for anyone. It has affected the whole world and more than 6 million people have died from it. However, it was an opportunity to test our knowledge and approach towards new pathogens. When I first learned about COVID-19 in the news, I knew we had to develop new diagnostic tools. Right after the Christmas 2019 vacations, we started looking for primers. We were happy to be able to use an established R&D approach that we had used with various previous emerging pathogens like Parachlamydia, since this expertise in R & D of our team reduced much the time to get an established reliable diagnostic SARS-CoV-2 PCR.
Your laboratory has been busy with something that has affected everybody. Did you feel the public’s attention, and did that lead to increased pressure on you or your laboratory? Yes, the lab was under a lot of pressure because we had to be able to deliver the test with a high accuracy and a short turn-around time. We were also exposed to the risk of a shortage of reagents and a shortage of manpower. This was the first time we did such a huge number of analyses per day. But it was also possible to interact with the lay public and improve their perception of microbes in general. In the past, we developed a game about microbes to help people understand the risks and how to get protected. I also tried to continue lay communication and we developed a second game including SARS-CoV-2. With this, we wanted to moderate the debate.
You pointed out the importance of lay communication in a recent publication. Are there lessons that we need to learn from the pandemic?
In terms of communication, we encountered several problems. Different people have communicated contradictory messages. In the beginning, we did not have the knowledge about the disease and about the best testing strategy. One expert had one opinion and another expert had a different opinion. We did not discuss this in scientific meetings, but openly in the newspaper. And that’s not good, because it leads to a loss of confidence in science and medicine in the population.
As it was the case with the antigen tests? Regarding the antigen tests, that was really weird. We have known for years that antigen tests are not good enough for respiratory viruses like influenza. We already knew in April 2020 that the mean viral load of SARS-CoV-2 was comparable to that of influenza and other respiratory viruses. There was no clue that it would work any better for SARS-CoV-2. Of course, this could be a solution when reagents are not available or for very remote regions without access to PCR. Moreover, it is only possible to use antigen tests at the time of highest infectivity (day 1 to 4 of infection) and not in asymptomatic patients. This should have been better communicated so that people do not completely trust antigen tests. If I, as a dutiful layman, want to protect my grandmother from infection and perform an antigen test before visiting her, I will possibly transmit the disease. We have to do a better job here in the future.
But how can we organize a better communication in the future and who should do this job? One problem is that experts in Switzerland are organized in national societies, for example the Swiss Society of Microbiology. These would be the ideal people to contact, but they have not been asked at all regarding antigen testing. The decision was made at the political level and everyone was surprised. The epidemiologists were driving this decision without asking the microbiologists.
Indeed, I had the impression that some specialists were not asked at all – such as microbiologists and laboratory physicians – and others were asked again and again. Do you think that the composition of the Swiss National COVID-19 Science Task Force was balanced in this regard? Yes, I think that microbiologists have not been involved enough in the diagnostic group. In the Swiss Society of Microbiology, we have a coordinated clinical commission for many years and none of the members were part of the diagnostic task force. But that was partly due to the origins of the task force. Various researchers from EPFL and ETH wanted to be active here. In contrast, we were too busy organizing the daily routine in the medical laboratory. Until April 2022, we didn’t have time to worry enough about political issues. As an example, some researchers asked us why did we initially test only fragile, symptomatic people? But in the March 2020, we simply had too few tests available, there were no commercial tests available. In this situation, the focus of testing had to be diagnosis (at-risk subjects), not epidemiology (all the population, including asymptomatic subjects without history of close contact).
The situation in developing the tests and setting up the test structures was difficult; there were no commercial reagents, few personnel, and a competitive situation regarding new analytical equipment. How did you deal with this situation? In our lab, we had an automated PCR solution with pipetting robots and extraction machines already before COVID-19. Fortunately, our solution allowed for large analysis numbers. The analyzers of the other large laboratories in French-speaking Switzerland all required reagents from one company, which then could no longer deliver. By chance, this was not the case in our laboratory. But we also had many reagents
P I P E T T E – S W I S S L A B O R AT O R Y M E D I C I N E | W W W. S U L M . C H
NR. 3 | JUNI 2022
E D U C ANTEI W ON S
As a final question, what were the three most important things you learned during the pandemic that laboratory medicine in Switzerland should do better next time?
was simply too long, and this led to the unpleasant story with the antigen tests. Thirdly, communication with the stakeholders is extraordinarily important – also with industry partners, authorities, The most important point is that we and politicians. And if I may make a should define clearly when and how to fourth point – this is good communicado a test. Because it is nonsense to do a tion with lay people. test when it is not needed. As an exam- Question were asked by Michael Nagler, So, the key of success was an ex- ple, in the spring of 2020, we did a huge Editor-in-Chief, «pipette» on 29 March isting automation system before number of tests without any being posi- 2022. the pandemic? tive. This has led to large costs for the so- Correspondence Yes, with this strategy we were ready be- ciety. Secondly, we must prioritize a michael.nagler@insel.ch fore the outbreak in Switzerland of this short time period to test result. In various laboratories, the time to test result world pandemic. available because we analyze 110 different pathogens on our system and most of the reagents are interchangeable. Since February 14, 2020, we could provide the tests. As of March 24, we had the Roche Cobas Platform in addition and could use both at the same time, thus positively impacting the access to PCR reagents.
Anzeige
A CLINISYS GROUP COMPANY
GLIMS Genetics Software designed with you in mind
Diagnostics intelligence - software for life GLIMS Genetics allows you to create integrated, digital workflows for the entire genetic spectrum. lt combines the strengths of a mature Laboratory Information System (LIS) with specialist genetic requirements, delivering unique advantages: • High-level automation to minimise risk of errors • Complete genetics workflow • Family management with a comprehensive drawing pedigree solution for clear analysis • Automated decision making and flexible configuration To discover more, visit us on the ESHG Conference in Vienna, 11 - 14 June 2022. You find us on Booth X5 - 134.
www.clinisysgroup.com
MIPS_Advertentie 168x133 EN_100522.indd 1
CliniSys | MIPS – A trusted partner to hospitals and private healthcare organisations across Europe, for more than 30 years.
12/05/2022 09:33
5
6
ED N EW UC SA T I O N
P I P E T T E – S W I S S L A B O R AT O R Y M E D I C I N E | WWW. S U L M . C H
NR. 3 | JUNI 2022
Giorgia Caruana 1
A century of antibiotic resistance: targets, mechanisms and susceptibility testing Stephen Hawking famously said, «Intelligence is the ability to adapt to change». Indeed, bacteria proved to be smart, having been in constant evolution, skillfully responding to the (selective) pressure imposed by antibiotics with useful modifications that earned some of them the precious title of multi-drug resistant (MDR). Almost a century after the discovery of penicillin, as foreseen by its far-sighted discoverer, antibiotic resistance constitutes a growing global threat with potentially major consequences, both on the ability to treat patients and on our capacity of infection prevention and control. The increased consumption of antibiotics both in humans and in animals (food industry), together with inappropriate prescribing, contributed to the growing of this problem. Consequently, antibiotics passed from representing the «wonder-drug» with healing powers to the most feared of evils. This was worsened by the small pipeline of new molecules developed in the last 30 years, which contributed to the need of implementation of antibiotic stewardship programs to control the use of antibiotics. With infectious diseases specialists on one side, busy limiting prescriptions and teaching how to better adapt the antibiotic spectrum, microbiologists have been deploying increasingly advanced technologies in order to rapidly detect resistance and allow clinicians a faster fine-tuning of antibiotic treatment.
the nucleic acid synthesis (Table 1). Each class share the same target, but they can differ by i) their ability to reach their target inside the bacteria, ii) their capacity to resist hydrolysis by bacterial enzymes, iii) their action spectrum (narrow versus large) and iv) their pharmacological properties. Some of them deploy a bactericidal effect, being capable of killing bacteria because acting on a defense or replication mechanism essential for germ survival. Most of the molecules inhibiting the synthesis of peptidoglycan, an essential component of the bacterial wall, are bactericidal. Among these, we find beta-lactamines (such as penicillin, cephalosporins or carbapenems), glycopeptides (such as vancomycine) and fosfomycin. Another bactericidal effect is to block some specific enzymes (such as topoisomerase, RNA polymerase) deputed to the deoxyribonucleic acid (DNA) synthesis: examples of molecules in this class are quinolones (such as ciprofloxacine) and rifampicine. A third bactericidal action is the alteration of the permeability of the cytoplasmic membrane, effect deployed by antibiotics such as polymixines, an «old» class Antibiotic molecules of molecules that has been revisited in The past two years aside, where CO- the past years as rescue molecule when VID19 claimed the headlines as «new dealing with MDRs. Finally, some other kid on the block», the enter on the antibiotics can induce bacterial death market of new antimicrobial mole- by inhibiting the protein synthesis, like cules has often been the hit of the day aminoglycosides. on newspapers, as they have been com- Not all molecules have the capacity of ing with promising novel actions directly killing the bacteria: several against MDR «super bugs». classes are defined bacteriostatic for Antibiotics can be divided into four their ability to inhibit bacterial replicamain classes, according to their site of tion. This can function both as support action: i) the cell wall, ii) the cell mem- for the immune system, which can efbrane, iii) the protein synthesis or i) fectively complete the job by killing the pre-hit bacteria, or as a booster in combination with other bactericidal molecules. Here we find molecules like tetracyclines (such as doxycycline) and 1 Institute of Microbiology, Lausanne macrolides (azythromicine, clarythroUniversity Hospital, University of Lausanne, 1011 Lausanne, Switzerland. micine), which (like aminoglycosides)
are capable of entering the cell wall of the bacteria using energy-dependent transport mechanisms in ribosomal sites, which subsequently leads to the inhibition of the protein synthesis. Another intra-cellular target is the synthesis of folates (vitamins essential for the synthesis of DNA), mechanisms used by molecules such as sulphonamides and trrimethoprimes, which work as metabolite analogues. Researchers keep looking for new mechanisms in order to develop molecules that might hit these or other targets and spare the antibiotics we (and bacteria) already know. An example is a novel mechanism of action, recently discovered in Switzerland (University of Zurich), targeting an outer membrane protein, essential for Gram-negative bacteria, thus destroying the integrity of the bacterial membranes: the target is promising, but no molecules have been released to the market yet.
Mechanisms of resistance We can distinguish four main mechanisms of bacterial resistance (Table 2), which can be intrinsic (always expressed) or induced (expressed under stress such as antibiotic exposure) and can occur mainly because of natural resistance, gene transfer or mutations. The first mechanism is the reduction of permeability of the cell membrane. This is the most common intrinsic resistance mechanisms, but can also be induced by antibiotic use, with the effect of preventing the entry of the antibiotic into the bacterial cell. This mechanism can take place through different mechanisms: by modifying the number of porins (water-filled channels) in bacterial membrane, by inducing structural, chemical or polar modification of the wall, as well as collaborating with the bacterial «community» and creating a biofilm, which entangle the contact with antibiotics.
P I P E T T E – S W I S S L A B O R AT O R Y M E D I C I N E | W W W. S U L M . C H
Mechanism of action
Target Wall penicillin-binding protein (PBP)
Cell wall synthesis inhibition
Wall peptidoglycan
Disruption of cell membrane
Lipopolysaccharides (external membrane of Gram-negative bacteria)
Protein synthesis inhibition Nucleic acid synthesis inibition/ disruption
30s ribosomial subunit (intra-cellular)
50s ribosomial subunit (intra-cellular)
Nucleic acid Enzymes for acid folic synthesis
E D U C ANTEI W ON S
NR. 3 | JUNI 2022
Antibiotic class ß-lactams: penicillins, cephalosporines, carbapenems Glycopetides Aminogycosides
Effect on bacteria Bactericidal
Example of molecules Penicillin G, amoxicillin, cephalosporin, meropenem
Bactericidal Bactericidal
Tetracyclines Polymixins
Bacteriostatic Bactericidal
Vancomycin Streptomycin, amikacin, gentamicin Tetracycline, doxycycline Colistine, polymyxin B
Macrolides Oxazolidinones Rifamycin Fluoroquinolones Nitroimidazoles Sulfonamides and trimethoprim
Bacteriostatic Bacteriostatic Bactericidal Bactericidal Bactericidal Bacteriostatic
Erythromycin, azithromycin Linezolid Rifampin Levofloxacin, ciprofloxacin Metronidazole Sulfamethoxazole, trimethoprim
Table 1. Classes of antibiotics and action mechanisms.
The second mechanism is the push of antibiotics outside the cell by means of efflux pumps, which are transporters whose genes are usually chromosomally encoded and then either naturally expressed or expressed under stressful circumstances. While these first two mechanisms are often part of natural resistance, other mechanisms can be acquired by different means: most commonly by horizontal genes transfer, as well as by random mutations on their DNA. Horizontal gene transmission is often plasmid-mediated via conjugation but can also take place by transfer of free DNA (transformation) or, less frequently, by means of bacteriophages (microscopic viruses capable of infecting bacteria) via transduction. As consequence of these gene acquisition, we can find the production of enzymes capable of inactivating the antibiotic molecule: a typical example are the enzymes called beta-lactamases, inactivating penicillin and its derivates. Finally, bacteria can resist antibiotics by modifying the antibiotic target (penicillin binding proteins, cell wall, ribosomes or enzymes for acid nucleic synthesis), making impossible for the molecules to bind at the surface or in the inside of the bacterial cell. No matter what the resistance method used, some clones of resistant bacteria selected by the pressure of antibiotics spread in the environment and to other people, and this is how clusters (or epidemics) of MDR infections are generated.
General resistance mechanisms Principal classes involved ß-lactams Glycopetides Cell permeability reduction Aminoglycosides Aminoglycosides ß-lactams Aminoglycosides Tetracyclines Macrolides Efflux pumps Oxazolidinones Fluoroquinolones Sulfonamides and trimethoprim ß-lactams Molecule inactivation
Aminoglycosides Tetracyclines Fluoroquinolones ß-lactams
Antibiotic target modification
Glycopetides Aminoglycosides Tetracyclines Macrolides Oxazolidinones Fluoroquinolones Sulfonamides and trimethoprim
Example of mechanism detail Reduction in wall p orins Thickening of cell wall Changes in wall p olarity Changes in wall p olarity
Pumping antibiotics out of b acterial cells.
Production of ß-lactamases enzymes Production of aminoglycosides modifying enzymes Antibiotic structure m odification Antibiotic chemical modifications Modification in penicillin binding proteins (PBP) Modification in wall p eptidoglycan Mutations in ribosomes Protection of ribosomes Mutations in ribosomes Ribosomial chemical modifications Modification in enzymes for acid nucleic synthesis Overproduction/reduction in enzymes for acid folic synthesis
Table 2. Summary of principal bacteria resistance mechanisms Readapted from Reygaert WC. AIMS Microbiol. 2018.
Susceptibility testing and resistance detection
in helping clinicians to preserve the currently available antibiotics. The In lack of a wide pipeline of new mol- main AST and AMR detection methods ecules, innovations and advances in are summarized in Table 3. antimicrobial susceptibility testing AST employs a phenotypic approach, (AST) and antimicrobial resistance which mostly relies on pure bacterial (AMR) detection play an essential role cultures in contact with different con-
7
8
ED N EW UC SA T I O N
centrations of antibiotics: when the bacteria are resistance to a certain concentration of antibiotic, the growth will stop and the arrest will be visible. Molecules can be tested through different methods: agar (solid media) or broth (liquid media) dilution, disk diffusion (measuring the diameter of inhibition zone around the disk with a pre-determined concentration of antibiotic) or gradient tests (decreasing concentrations of antibiotics contained in a single strip with different marked levels). Based on bacterial growth, the major limitation for most of phenotypic tests is the speed. For this reason, rapid AST methods have been investigated, and several promising technologies are under study. Rapid phenotypic tests are already on the market. They are capable to detect the presence or absence of a specific enzyme link to resistance through chromogenic media, changing colors according to the susceptibility. Nanomotion technologies instead, are based on fluctuations caused by the nanomotion due to metabolically active bacteria: through cantilevers (micro-electromechanical sensors), they are capable of detecting the changes in nanomotion caused by the exposure to a drug, without the need to wait for bacterial growth on cultures. Other examples of new phenotypic methods, which rely on the combination of imaging and artificial intelligence, are the use of nuclear magnetic resonance or mass spectroscopy to detect susceptibility based on a specific image pattern. Flow cytometry is also a technique that has been readapted for the study of antibiotic susceptibility and resistance detection. Aside from phenotypic AST, there is also the possibility of directly detecting some specific genes associated with resistance, as well as mutations or variations in genes’ expression. These are known as molecular methods for AMR detection and are based on nucleid-acid amplification (genetics), sequence databases and bioinformatics (genomics). They represent the future of AMR detection, but nowadays they are still limited by prohibitive costs and long turnaround-times, making them a luxury that only research centers can afford.
P I P E T T E – S W I S S L A B O R AT O R Y M E D I C I N E | WWW. S U L M . C H
Testing
Type of method
Classical phenotypic Antibiotic susceptibility testing Innovative phenotypic
Antimicrobial resistance detection
Genetic Genomic
NR. 3 | JUNI 2022
Method details Solid media dilution Liquid media dilution Disk diffusion Gradient strip tests Chromogenic media Nanomotion technologies Nuclear magnetic resonance Mass spectrometry Flow cytometry Nucleic-acid amplification Next-generation sequencing
Table 3. Principal available methods for antibiotic susceptibility and antimicrobial resistance testing.
Conclusion The several promising laboratory technologies being developed and researched are a precious help to compensate the lack of novel molecules in the antimicrobial pipeline. Nevertheless, the reduction of turnaround time for detection of AMR can have a very different impact according to the epidemiology of resistance present in the country and it cannot supply one hundred percent the need on new «weapons». Indeed, we are still left with the question: where did these resistant bacteria come from? Why they are endemic is some countries and others are able, instead, to keep their rate under control? The possibility of non-humans (animals, environment) reservoirs has been widely debated, but large studies have disproved this hypothesis and evidence seems in favor of human-human transmission and infection control habits as main responsible for the increase of antimicrobial resistance. A lot is still to investigate, but now it is our turn to show intelligence, to adapt to change, to fight back antibiotic resistance, to develop revolutionary methods for AST, to update the pipeline of new antimicrobials and to learn how to use antibiotics appropriately. Humans and bacteria will continue to evolve adapting to each other, that’s the beauty of nature, but the fight against «super-bugs» is not over yet: who do you think is going to win? Correspondence giorgia.caruana@chuv.ch
Referenzen 1. Reygaert WC. An overview of the antimicrobial resistance mechanisms of bacteria. AIMS Microbiology 2018;4(3):482-501. 2. Luther A, Urfer M, Zahn M et al. Chimeric peptidomimetic antibiotics against Gramnegative bacteria. Nature 2019 Dec;576(7787):452-458. 3. Uddin TM, Chakraborty AJ, Khusro A et al. Antibiotic resistance in microbes: History, mechanisms, therapeutic strategies and future prospects. Journal of Infection and Public Health 2021;14(12):1750-1766. 4. van Belkum A, Burnham CAD, Rossen JW, Mallard F, Rochas, Dunne WM. Innovative and rapid antimicrobial susceptibility testing systems. Nature Reviews Microbiology 2020;18(5):299-311. 5. Lautenbach E, Patel JB, Bilker WB, Edelstein PH, Fishman NO. Extended-spectrum ß-lactamase-producing Escherichia coli and Klebsiella pneumoniae: risk factors for infection and impact of resistance on outcomes. Clinical infectious diseases 2001;32(8):1162-1171.
P I P E T T E – S W I S S L A B O R AT O R Y M E D I C I N E | W W W. S U L M . C H
E D U C ANTEI W ON S
NR. 3 | JUNI 2022
Aitana Neves 1 , Dominique Blanc 2 , Gilbert Greub 2 , Hans H. Hirsch 3 , Michael Huber 4 , Laurent Kaiser 5 , Stephen L. Leib 6 , Vincent Perreten 6 , J acques Schrenzel 5 , Roger Stephan 4 , Reinhard Zbinden 4 , Adrian Egli 3
Molecular surveillance of pathogens in Switzerland – Focus: SARS-CoV-2 and its variants Currently, the BA.2 variant of SARS-CoV-2 dominates in Switzerland as it does in places around the globe. In recent months, circulating virus lineages have been widely monitored by sequencing the genomes of thousands of SARS-CoV-2 isolates. These efforts were coordinated by the National Reference Centre for Emerging Viral Infections (CRIVE) in Geneva. A total of 15 laboratories from diagnostics and research supported this and thus contributed significantly to the surveillance and risk assessment of viral evolution in Switzerland. The large quantities of sequencing data and associated metadata were centralized at the «Swiss Pathogen Surveillance Platform» (SPSP), where quality was checked, the sequences annotated, and finally transmitted to other databases. Data was visualized for example in the dashboard of the Federal Office of Public Health (FOPH) or using international comparisons via GISAID on platforms such as nextstrain.org. SPSP was developed together with the SIB Swiss Institute of Bioinformatics as a «One Health» focused platform to share genomes and associated metadata between institutions for surveillance and research. The project was initially funded by the National Research Programme (NRP72) of the Swiss National Science Foundation and has since been further developed. Today, SPSP represents a key platform for the molecular monitoring of human, animal, and environmental pathogens (viruses, bacteria, fungi) in Switzerland and clearly passed the maturity test during the COVID19 pandemic. A key challenge now is to secure sustainable funding for SPSP and to motivate diagnostic and research laboratories to share sequence data via SPSP as an essential step in comprehensive «One Health» surveillance and outbreak monitoring of infectious diseases.
Monitoring of SARS-CoV-2 variants at national level Yesterday Alpha and Delta, today Omicron, and tomorrow maybe Pi: SARSCoV-2 rapidly evolves and adapts to selection pressures. Within the past two years, the surveillance of SARS-CoV-2 using whole genome sequencing of viral isolates has resulted in an unprecedented global program to tightly monitor the evolutionary steps. Also Swiss
1 SIB Swiss Institute of Bioinformatics, Geneva, Switzerland 2 University of Lausanne and University Hospital Lausanne, Lausanne, Switzerland 3 University of Basel and University Hospital Basel, Basel, Switzerland 4 University of Zurich, Zurich, Switzerland 5 University of Geneva and University Hospital Geneva, Geneva, Switzerland 6 University of Bern, Bern, Switzerland
laboratories are on a daily lookout for new SARS-CoV-2 variants and their transmission chains. Sequencing of the viral genome makes it possible to identify the variant in question and its complete genetic profile with detection of single nucleotide polymorphisms at high spatiotemporal resolution. Indeed, early detection of new viral variants that are more contagious or lead to vaccine failure (immune escape) are critical for public health decisions and information of the general public. In addition, outbreaks and transmission patterns can also be studied in detail in order to adapt protective measures and to guide decisions regarding vaccine adaptation. Knowledge about variants also matters for the timely and targeted treatment of individual infected patients when using monoclonal antibodies or newly developed antivirals for therapy. In 2021, more than 100’000 SARS-CoV-2 viruses were sequenced and analyzed by 15 academic or private laboratories throughout Switzerland (Figure 1). However, sequencing data is only useful if it is linked and shared as quickly as possible – hence the essential need for coordination and standardization of sequencing programs. The sequencing effort in Switzerland was coordinated by the National Reference Centre for Emerging Viral Infections (CRIVE, Geneva) and funding
was provided by the Federal Office of Public Health (FOPH). By April 2022, more than 135,000 genomes have been examined. Due to federal cost restrictions, the sequencing efforts were centralized in April 2022 over two scientific core facilities (H2030 in Geneva and ETH Zurich in Basel). Nevertheless, «diagnostic» sequencing, as an alternative to «surveillance» sequencing, may still be applied in certain cases by diagnostic laboratories to keep research expertise in place. For harmonized, standardized analysis and rapid sharing of sequencing data, the «Swiss Pathogen Surveillance Platform» (www.spsp.ch) was established. This collaborative and secure infrastructure was originally developed by the SIB Swiss Institute of Bioinformatics together with the University Hospitals Basel, Geneva and Lausanne (CHUV) and the Universities of Basel, Bern and Zurich as a "One Health"-related platform between 2018 and 2020. The platform core development was funded by the National Research Programme 72 with a focus on antibioticresistant bacteria with methicillin-resistant Staphylococcus aureus (MRSA) as a proof of concept. However, during the SARS-CoV-2 pandemic, we rapidly adapted and optimized the data- and workflows of the platform for viral genomes including specific bioinformatic
9
10
ED N EW UC SA T I O N
P I P E T T E – S W I S S L A B O R AT O R Y M E D I C I N E | WWW. S U L M . C H
Figure 1: SARS-CoV-2 sequencing centers (until March 2022). Names in blue are members of the SPSP Executive Board.
tools. The SARS-CoV-2 genome sequences and the associated metadata such as laboratory and sample identifiers, date of PCR test, type of sample collection, reason for sequencing, sequencing machine used, sex and age of the patient are loaded onto SPSP in a secured and centralised way, harmonised, annotated and shared with the FOPH.
lance programme. Since the end of July 2021, all Swiss laboratories participating in the surveillance programme have been obliged to submit their SARSCoV-2 sequences to SPSP (Figure 2). Thus, the data collected on SPSP today allow a comprehensive and representative overview of the circulating variants in Switzerland. Three times a week, the platform sends its genomic surveillance report to the FOPH. This notably Accelerated surveillance of the epi- allows the FOPH to have a precise picdemic in Switzerland ture of the variants circulating in SwitSPSP was adapted for SARS-CoV-2 sur- zerland. The FOPH then integrates veillance at the beginning of 2021 and these data into statistics (https://www. has been sending uniform reports on covid19.admin.ch/en/overview) . The SARS-CoV-2 variants circulating in sequencing data can also be used by Switzerland to the FOPH since May FOPH to match sequence data with 2021 as part of the national surveil- available patient data on hospitalisa-
NR. 3 | JUNI 2022
tions, vaccinations, symptoms at the time of the test, etc. In this way, FOPH could determine whether a variant or specific mutation is associated with higher pathogenicity or vaccine resistance among all observed mutations. Thanks to this monitoring platform, the FOPH can access a centralized and standardized database via a single access point instead of receiving reports in different formats from each laboratory. This results in significant time and cost savings, as well as greater granularity in the analysis of sequencing data. We hope that the value and important advantages of SPSP is recognized and will be applied to other pathogens in the future, thereby better understanding the complex transmission routes of pathogens in humans, animals, the environment and the foodchain.
Boost international surveillance and research by facilitating Open Data SPSP enables the central storage of virus sequences in Switzerland with rich and structured metadata that is very useful for research. As this is potentially sensitive data, SPSP is hosted on a highly secure infrastructure of a BioMedIT node, making this data available for research in an ethically and legally controlled framework. In addition, the SPSP platform also transfers anonymised viral data to the open data platform European COVID-19 portal (https://www.covid19dataportal.org/) and to GISAID to support global surveillance and research. Thanks to these
Figure 2: Data on SPSP in 2021. A. Available sequences. Blue, data depending on date of sampling; Orange, data depending on date of submission to SPSP (start-up in March 2021 for SARS-CoV-2). B. Turn-around times from acceptance to publication of sequence data.
P I P E T T E – S W I S S L A B O R AT O R Y M E D I C I N E | W W W. S U L M . C H
efforts, Switzerland currently ranks fourth after the UK, the USA and Germany in terms of the absolute number of openly shared SARS-CoV-2 sequences (Table 1 and Figure 3). These public databases are essential for studying and understanding the role of observed variations on the pathogenicity of the virus, its interaction with host cells at the time of infection, or even for the development of vaccines and treatments.
E D U C ANTEI W ON S
NR. 3 | JUNI 2022
Country
Sequences submitted
USA
2,077,366
Raw sequences submitted 1,396,278
United Kingdom
1,811,616
2,462,155
Germany
304,074
7,510
Switzerland
129,060
38,107
Denmark
41,834
753
Australia
26,589
15,889
Table 3. Statistics from the EU Covid-19 Data Portal – ranking by country. R eproduced from https://www.covid19dataportal.org/statistics
A secure platform and effective governance for future surveillance. SPSP is a collaborative platform that follows the «One Health» approach, i.e. is multidisciplinary and aims, among other things, to explore and optimize human and animal health outcomes, but still meets the data security standards of the Swiss Personalized Health Network (SPHN). The Executive Executive Board of SPSP includes representatives from five universities and university hospitals as well as from SIB, and decides on important strategic decisions. There is also an Advisory and a Scientific Board with various stakeholders, including regulatory and scientific representatives, which allows rapid integration of new tools into the platform e.g. an automated cluster detector. SPSP can count on an efficient governance and clearly defined ethical and legal framework to quickly integrate new laboratories wishing to contribute to SPSP. Contracts for data exchange and sharing as open data were concluded with all centers. The FAIR principles of data including findability, accessibility, interoperability, and reusability are central values for SPSP. After successfully testing the platform during the SARS-CoV-2 pandemic, we now look to expand surveillance with new pathogens and hope for sustained funding from federal agencies to optimize infectious disease surveillance between humans, animals,the environment and the foodchain and generate reliable data with regularity. More recent outbreaks like the Hepatitis E outbreak in 2021, the Salmonella outbreak of chocolate eggs before Eastern, or Listeria monocytogenes outbreaks are covered in the media and have an important impact on food safety and reputation of the food industry. Similar
Figure 3. statistics from the EU Covid-19 Data Portal - ranking by institution. https://www.covid19dataportal.org/statistics
monitoring of Legionella pneumophila can be very important to link an environmental source with a severe pneumonia of a patient. Often sequencing data is lacking or not exchanged, which hinders clear conclusions. SPSP can close this important gap between different stakeholders involved in an outbreak investigation. Our vision is that SPSP can be used by different specialists with highly customized analytical and visualization tools to quickly detect the emergence and spread of pathogens and take early action to contain transmission by tracking them in near real time. The data will also be made available to researchers to monitor the dynamics of diseases and measures such as vaccinations and antibiotics. The possibility of using SPSP in the long term to link genomic data of bacteria or viruses emerging in Switzerland with epidemiological data is promising for ensuring Switzerland’s exemplary public health response capacity and providing patients with the most appropriate treatment.
Acknowledgements We thank our colleagues in the laboratories for reliably sequencing SARSCoV-2 and sharing the data via the Swiss Pathogen Surveillance Platform. In particular, we also thank: Prof. Richard Neher (Biozentrum, University of Basel), Prof. Tanja Stadler (ETH Zurich), Prof. Lorenz Risch (Risch), Adrian Härri (Biolytix), PD Dr. Oliver Nolte (Centre for Laboratory Medicine St. Gallen), Prof. Alexander Imhof (SRO), Dr. Arnaud Tanguy (Genesupport), Dr. Alexis Dumoulin (HVS), Dr Gladys Martinelli (EOC), Dr. Cinzia Zehnder and Dr. Etleva Lleshi (Synlab). We would like to thank SERI, swissuniversities, Bangerter Rhyner Foundation and the NRP72 programme of the Swiss National Science Foundation for their financial support.
Correspondence Dr. Aitana Neves, aitana.lebrand@sib.swiss Prof. Dr. Dr. Adrian Egli, adrian.egli@usb.ch
11
12
ED N EW UC SA T I O N
P I P E T T E – S W I S S L A B O R AT O R Y M E D I C I N E | WWW. S U L M . C H
NR. 3 | JUNI 2022
Greub Gilbert 1 , Ackermann Rahel 2 , Cagno Valeria 3 , Coste Alix 4 , Croxatto Antony 5 , Opota Onya 6 , Lienhard Reto 7
The Swiss national reference centre for tick-borne infections Due to climate changes, the geographical surfaces suitable for ticks has increased in ten years from 16% to 25%, as shown by Rochat et al [1]. Moreover, due to societal changes (partially boostered by the SARS-CoV-2 pandemics), the Swiss population appears to be increasingly exposed to ticks due to more diversified and more regular outdoors activities. Thus, in 2021, according to a recent SUVA press communicate, as many as 14’000 human tick bites have been recorded in Switzerland as compared to about 10’000 human tick bites per year a few years earlier. This trend may have a significant impact on public health given the different tick-borne pathogens observed in Switzerland, such as Borrelia spp. and the tick-borne encephalitis virus (TBE virus). Due to globalisation and increased travel rates in exotic areas (slowed down by the SARS-CoV-2 pandemics but rapidly regrowing in 2022), we also have to deal in Switzerland with a number of patients returning home suffering from spotted fever due to various Rickettsia species [2]. In addition, there are a number of tick-borne infections that remain likely underdiagnosed (infections with Anaplasma phagocytophilum and Candidatus Neoehrlichia mikurensis, for example). Moreover, several novel agents related to Chlamydia have been documented in ticks [3], emerging as new pathogens or having yet an unknown pathogenic role [4, 5]. This leaves to the Swiss national reference centre for tick-borne diseases (CNRT) a number of challenges that will be described in the present article. We will also summarize key aspects about some of these microbes studied at the CNRT.
tute of Microbiology of the University of Lausanne (V. Cagno, A. Coste, and O. Opota), he is actively collaborating with ADMED Microbiologie at La Chaux-de-Fonds (R. Ackermann, A Croxatto and R. Lienhard). G. Greub, R Lienhard and R. Ackermann were already actively driving the CNRT since more than a decade [6]. The Federal Office of Public Health (FOPH) defined Q fever and Lyme disease as the two main priorities of the CNRT, despite the fact that Coxiella burnetii is a zoonotic disease mainly transmitted by exposure to goats and sheep, and which was undetected in Swiss ticks in a vast survey [7]. However, the tasks and perimeter of the CNRT also includes a number of other infections and pathogens, as summarized in Figure 1 & Table 1.
Lyme disease Lyme Disease is endemic in Switzerland. The first clinical feature de-
scribed in Europe was already in 1883 as «acrodermatitis chronica atrophicans». Erythema migrans was described medically in 1909 by the Swedish dermatologist A. Afzelius. However, the agent named Borrelia burgdorferi was only described in 1982. Often considered the new great imitator, this bacterium can be responsible for skin, articular, neurological, cardiac and optical manifestations [8]. Its main clinical sign is the pathognomonic «erythema migrans», a localized acute stadium usually recognized by physician and some patients, even when it reaches as much as 1 meter in diameter. Less frequently, this skin manifestation can disseminate in multi-erythema all over the body. As disseminated acute disease, Lyme neuroborreliosis is the second most frequent clinical feature. Reported as «tick paralysis» in 1922, Garin and Boujadoux described the poly-meningo-radiculoneuritis after a
The Swiss national reference centre for tick-borne diseases (CNRT) Since 1st January 2022, the Swiss National Reference Center for tick-borne infection is directed by Professor Gilbert Greub. With his team at the Insti1 Prof. Gilbert Greub, Institute of Microbiology, University Hospital Center of Lausanne, Lausanne, Switzerland 2 Dr. Rahel Ackermann, ADMed Microbiologie, La Chaux-de-Fonds, Switzerland 3 Dr. Valeria Cagno, Institute of Microbiology, University Hospital Center of Lausanne, Lausanne, Switzerland 4 Dr. Alix Coste, Institute of Microbiology, University Hospital Center of Lausanne, Lausanne, Switzerland 5 Dr. Antony Croxatto, ADMed Microbiologie, La Chaux-de-Fonds, Switzerland 6 Dr. Onya Opota, Institute of Microbiology, University Hospital Center of Lausanne, Lausanne, Switzerland 7 Dr. Reto Lienhard, ADMed Microbiologie, La Chaux-de-Fonds, Switzerland
Figure 1. General organization of the Swiss National Reference Center for Tick-Borne Infections (CNRT). This chart highlights the diversity of pathogens that are part of the tasks and perimeter of the CNRT, as revised in 2021 by the FOPH. It also shows that the CNRT is active at the epidemiological level but also represents a reference resource in diagnostic microbiology and infectious diseases.
P I P E T T E – S W I S S L A B O R AT O R Y M E D I C I N E | W W W. S U L M . C H
NR. 3 | JUNI 2022
Provision of expertise (control) of positive C. burnetii serologies for all positive cases detected in diagnostic laboratories in Switzerland (including individual cases) using complementary serologies available in the CNRT laboratories. Evaluation and verification of commercial tests for PCRs and serologies for Lyme borreliosis and Q fever, as well as some other tick-borne agents. Publicly disseminate evaluation reports. Creating and maintaining a homepage. Evaluate a novel test for serology for Lyme borreliosis Implement PCRs for Lyme borreliosis, Q fever and tick-borne encephalitis in the two CNRT laboratories taking into account the different target genes so that two independent analyses can be offered for confirmatory diagnosis and to ensure method redundancy Mapping of laboratories performing diagnostic methods for Lyme borreliosis, Q fever and tick-borne encephalitis. Publication of recommendations in the diagnosis of Lyme borreliosis and Q fever for physicians (RMS -SMW) and laboratories (SSM CCCM). Designing immunofluorescence tests for C. burnetii serology (home-made) Creation of an EQA for the control of C. burnetii serologies in collaboration with the CSCQ (provision of samples, technical expertise, analyses, reporting). Table 1. Some of the projects that will be tackled by the CNRT during the next 3 years. These tasks are largely focused on Q fever, Lyme disease and tick-borne encephalitis, since so far these three infectious diseases represent the main identified public health zoonotic threats in Switzerland.
tick bite. This aseptic meningitis is typical enough to be recognized as Lyme disease when occurring during the tick season, especially when following erythema migrans, which occurs weeks before. However it becomes more difficult out of this context and when more aspecific neurological manifestations appear. Concerning the last stage of the disease, late disseminated borreliosis is mainly featured by the arthritis described first at Lyme (Connecticut, USA) as an emergent disease in 1975, and Acrodermatitis Chronica Atrophicans (ACA) associated sometime to peripheral neurological disorder. Since Borrelia burgdorferi was identified as the etiologic agent of Lyme disease, diagnostic tools have been rapidly implemented. By the end of the 80’s, the «Diagnostic Parasitaire Laboratory» (DPL) from the Zoology Institute of the University of Neuchâtel, set up the first test in Switzerland. It was a home-brew indirect immunofluorescence assay (IF) using as antigen a culture of the B31 Borrelia burgdorferi sensu stricto strain. This first tool has then rapidly been complemented by an ELISA with a whole antigenic extract of the B31 bacterial strain. This 96 wells plate Enzyme Immunoassay (EIA) enabled to handle more analyses at once, as the diagnostic requests extended and epidemiological studies
became more frequent. The serology sat as the main diagnostic tool. However, the use of native antigens rapidly questioned the specificity of the assays, with cross-reactions with syphilis markers (TPHA) and with rhumatoid factors (RF). In the early 90’s, a more specific capture test was validated to eliminate RF negative impact for IgM diagnostics. This new assay uses whole purified flagella antigen as described in Hansen and Lebech [4]. This was especially meant to enhance and to facilitate neuroborreliosis diagnostic. Specificity problems were partially resolved by using «lab-made» westernblot as confirmatory test [10]. These western-blot assays included two Swiss B. burgdorferi sensu lato isolates from ticks. The use of other strains was then motivated by the identification of the new etiological species present in Europe. Thus, a US B. burgdorferi strain (B31), a B. garinii strain (NE84) and a B. afzelii strain (NE17) were added to the blot panel used to confirm Lyme borreliosis. This confirmation procedure was shown to be useful, allowing a specificity of 95%. However, the high sero-prevalence in the Swiss population remain a challenge for the diagnostic, particularly in high risk groups such as orienteer runner, old rural population or hunters since positive serology may only reflect a past infection without any
E D U C ANTEI W ON S
causal relationship with the current symptoms that triggered the serology. Borrelia culture and PCR were then introduced as complementary tools, since these assays are highly specific. However, their systematic routine diagnostic use is not possible as (i) it needs more invasive sampling such as biopsies or lumbar punctures and (ii) they both lack sensitivity. Beginning of this century, the use of recombinant antigens in EIA for Lyme borreliosis diagnostic launched a new era [9]. Proteins as outer surface protein C (OspC) or Variable Lipoprotein Surface-Exposed protein (VlsE) showed their importance and utility [reviewed in 10]. Moreover, with such recombinant proteins, we can achieve a higher quality and an improved stability in the production of diagnostic assays. These two antigens are now used in instruments-based EIA (such as chemiluminescence immunoassays or enzyme-linked fluorescence assays) and in immunoblot assays on different matrices. They enhance both sensitivity and specificity of serological assays, but are still not able to differentiate passed contact or infection with active disease. Today, serology remains the main tool to diagnose Lyme borreliosis. In Switzerland as in many country in Europe, it is recommended to use an EIA as first test and if reactive, to confirm by an immunoblot. The CNRT has the mission to lead the microbiological diagnostic of borreliosis. By evaluation, validation and verification of new commercial methods, we are able to help the companies to introduce the appropriate testing methods. This is only possible with an established and qualified collection of sera for the different disease stages. We will continue to bridge the communication between the actors of diagnostics in Europe grouped in ESGBOR, test companies and diagnostic laboratories to help introduce new methods that are of interest. The goal of the CNRT is to help the population, the physician and the authorities to find answers and solutions to problems. As a service, we aim to rapidly (=same day results) diagnose acute disseminated stage such as neuroborreliosis. For this purpose, we set up rapid ELISA tests to exclude Borrelia as the aetiolog-
13
14
ED N EW UC SA T I O N
ical agent (if negative) and we confirm diagnosis within 48h (if ELISA was positive). Parameters such as CSF IgG and IgM antibody levels, specific intrathecal production and the new CXCL13 marker detection have been now tested routinely for more than 5 years for our and other hospitals’ patients. Culture and PCR are systematically tested when enough CSF is available, in order to complete diagnosis with very high positive predictive value and to also study the epidemiological profile of Lyme Borreliosis in Switzerland. This is a needed step in the prospective vision to develop a Lyme vaccine. The role of the CNRT extends of course beyond diagnostics and epidemiological surveys and we are highly active by providing advices on patients care and in lay communication, providing for instance in collaboration with the «tick league» a comprehensive set of answers to the most frequently asked questions (https://www.bag.admin.ch/dam/bag/ de/dokumente/mt/infektionskrankheiten/zecken/FAQ-Zecken-Zeckenstiche.pdf.download.pdf/FAQ%20 Zecken%20DE.pdf).
Q fever Q fever is a disease caused by a bacterium called Coxiella burnetii. The natural reservoir of this bacterium is domestic animals, pets, some wild animals and more rarely ticks. Infection in humans usually occurs through inhalation of dust containing the infectious agent excreted by carrier animals, also by ingestion of contaminated food, and more rarely by tick bites. Noteworthy, the main ticks associated with Coxiella transmission seems to be Rhipicephalus evertsi and Amblyomma variegatum, according to a recent meta-analysis, which are tick species uncommonly seen in Switzerland contrarily to Ixodes ricinus. Symptoms in the acute phase are generally a prolonged fever (> 10 days) with normal white blood cell count, thrombocytopenia and elevated liver enzymes [12]. Subjects with predisposing factors, such as heart valve disease, may develop chronic disease. Endocarditis and infections of aneurysms or vascular prostheses are the most common forms of chronic Q fe-
P I P E T T E – S W I S S L A B O R AT O R Y M E D I C I N E | WWW. S U L M . C H
ver [13] and are usually fatal if left untreated. Underdiagnosis may especially occur in subjects with an initially pauci-symptomatic endocarditis that may then get superinfected by S. aureus or other Gram positive cocci [14] and Coxiella should thus be considered systematically. Chronic Coxiella hepatitis are also commonly seen [12]. While Q fever is rarely reported in children, the chronic picture is very different from adults with osteomyelitis being the most common. In Switzerland, Q fever has only been notifiable since November 2012, following an epidemic in the Lavaux region linked to the presence of infected sheep flocks that caused a dozen human cases [15]. The introduction of this mandatory notification was done despite the small outbreak in humans since the epidemic potential of C. burnetti is much higher, as demonstrated by the epidemics in Bagnes in 1982 and in the Netherlands in 2009, where more than 1000 human cases have been documented [16, 17]. Today in Switzerland, each year, between 40 to 60 cases are reported. In addition to sporadic cases, outbreaks occurs also occasionally, but is generally limited thank to active preventive measures. Diagnosis is based on PCR or serology. PCR can be performed on whole blood or serum during acute phase [18], and helps diagnose acute Q fever in the first 2 weeks of infection. Pcr may also then be done on infected tissue (heart valve samples, liver biopsy, bone biopsy, …). In Lausanne, we are using a Coxiella burnetii specific TaqMan PCR [18], that exhibits x% sensitivity on valve samples and which was also proven sensitive enough for the diagnosis of hepatitis, uveitis, aortic prosthetic infection and spondylodiscitis. Serology, on the other hand, can be done by using a highly sensitive screening test followed by an immunofluorescence test for confirmation. A quadrupling of the phase II IgG antibody titre by immunofluorescence between matched acute and convalescent specimens is the diagnostic gold standard for confirming the diagnosis of acute Q fever. However, a negative serology in the acute phase does not exclude Q fever. Indeed, the immunofluorescence is negative during the early stages of acute illness, when PCR is gen-
NR. 3 | JUNI 2022
erally still positive. Most patients seroconvert by the third week of illness. A single high ’convalescent’ serum sampled after the acute phase of the disease is sufficient for the diagnosis (positive if > 1/256); however, a fourfold increase between acute and convalescent samples exhibits a much higher sensitivity (may be considered positive with titers > 1/64) and specificity than a single high convalescent titre. The diagnosis of chronic Q fever requires demonstration of a high titer of phase I IgG antibody (≥ 1:800) and an identifiable focal infection (e.g. endocarditis, hepatitis or spondylodiscitis). PCR, immunohistochemistry, or culture of affected tissues can provide definitive confirmation of C. burnetii infection. However, those approaches have a low sensitivity and are somehow difficult to implement, since samples are not systematically available. Thus, the diagnosis of Q fever relies heavily on serological monitoring of the patient. It should be noted that immunofluorescence requires considerable expertise. The implementation of specific and relatively sensitive PCR is also a challenge and few laboratories offer it.
Tick-borne encephalitis (TBE) Tick-borne encephalitis (TBE), the most important tick-borne viral disease of humans in Eurasia, is caused by tick-borne encephalitis virus (TBEV), a member of the genus Flavivirus of the family Flaviviridae. TBEV is mainly transmitted to humans via tick bites. The distribution of TBEV correlates with the occurrence of its vector ticks and ranges from Western Europe to Russia, Siberia, and FarEastern countries. Three viral subtypes, i.e. European, Siberian, and Far Eastern, have been described; in addition, two new subtypes (Himalayan and Baikalian) have been proposed. When transmitted to humans, TBEV may cause disease of variable severity, ranging from subclinical infections to severe courses with neurological involvement and potentially fatal outcome. While the infection is asymptomatic in 70–95 % of cases, symptomatic disease may occur as meningeal, encephalitic, poliomyelitic, and myelo-radiculitic forms. [19]. In
P I P E T T E – S W I S S L A B O R AT O R Y M E D I C I N E | W W W. S U L M . C H
Switzerland, neurological disease most frequently manifests as meningoencephalitis (55%), followed by meningitis (22%) and encephalomyelitis (3%) [20]. Overall, the FOPH recorded 454 cases in 2020 and 285 in 2021, with an incidence of 5.11 and 3.27 per 100,000 inhabitants, respectively [21]. The case fatality rate during the acute phase is 0.9% and 45% of symptomatic patients present neurological sequelae [22]. Currently, no specific treatment against TBEV is available. Besides preventing tick bites, active immunization is the most important protective measure against infections with TBEV. Six licensed vaccines exist, all of which use inactivated whole virus strains [23] (8). In Europe, two vaccines based on European subtype isolates are available, which can be used interchangeably: FSME-IMMUN® (Pfizer) and ENCEPUR® (Bavarian Nordic). The vaccination schedules comprise three doses for primary immunization. Thereafter, vaccine manufacturers prescribe booster vaccinations for maintaining protection (first booster dose three years after primary immunization, subsequent booster doses every five years [<50 years] or every three years [≥50 years]). Deviating from this schedule, Switzerland has extended the booster intervals to ten years [24]. Virus-neutralizing antibody titers are believed to be responsible for longterm immunity after natural infection and vaccination [25]. While after a 3-dose primary series seropositivity persist for more than 10 years in >90% of younger subjects, it drops to 37.5% in those 60 years or older. However, a systematic review found field effectiveness to remain high in irregularly vaccinated subjects and thus not to correlate well with the percentage of subjects achieving an arbitrarily defined threshold of persisting antibodies [26]. Since the decision for the prolongation of vaccination intervals by the FOPH Switzerland in 2006, no indication was found that extended booster intervals resulted in an increased rate of breakthrough infection. On the other hand, there was a marked public health benefit with respect to increased acceptability of TBE immunization in the general population [27]. Various information campaign have been done to
NR. 3 | JUNI 2022
E D U C ANTEI W ON S
fever. The latter, due to various rickettsial species, are not yet recognized as significant public health problems in Switzerland, likely due to underdiagnosis of Rickettsia slovacae and Rickettsia helveticae infections. The same is true for Anaplasma phagocytophilum, which has been largely documented in Ixodes ticks collected in various Swiss region [7], but remain often undiagnosed, when causing leucopenia or pancytopenia considered of unexplained etiology. Presence of various chlamydia-related bacteria have also been documented in Swiss ticks [3, 33], with especially several documentation in Ixodes ricinus ticks of very high bacterial load of Rhabdochlamydia bacteria, that allowed direct full genome sequencing [34]. Thus, in addition to tackle the problems raised by the circulation in Switzerland of Borrelia, Coxiella and to the tick-borne encephalitis virus, by providing (i) prevention advices, (ii) up-to-date diagnosis and (iii) expert opinions on clinical aspects, the national center for tick-borne infections is also in charge of improving knowledge about novel emerging pathogens such as Rhabdochlamydia and Parachlamydia [35]. Moreover, since a number of tick-borne infections are acquired during travel abroad, surveillance of imported tickborne infections and advices to travelers are also important, especially for (i) African-tick bite fever, that represents the more common spotted fever observed in Swiss travelers, (ii) Mediterranean spotted fever, which exhibits the highest mortality of European tick-borne rickettsial infections, and (iii) Rocky Mountain spotted fever, which exhibits a very high mortality of up to 30%. Finally, the CNRT has also the task to better understand ticks biology and to determine the regions where current climatic conditions are favorable for ticks & tick-borne pathogens and we recently demonstrated an increased proConclusions portion of surfaces suitable for ticks in The ticks are able to transmit a num- Switzerland, [1], leading to changes in ber of significant pathogens and in the FOPH recommendations regarding addition to Borrelia, Coxiella and to the vaccine against the tick-borne enthe tick-borne encephalitis virus cephalitis virus [24]. (TBEV), there are a number of additional major human pathogens trans- Correspondence gilbert.greub@chuv.ch mitted by ticks such as Anaplasma, References ➤ Ehrlichia and all the agents of spotted increase awareness about TBEV, vaccine and tick-bite prevention, including the development of a game called Krobs, which preventive messages are provided on a corresponding dedicated website (www.krobs.ch). The diagnosis of TBE must be established in the laboratory because of the non-specific clinical presentation. The detection of specific nucleic acid (RNA) in the blood by RT-PCR is only successful during the first viremic phase of the disease before seroconversion. The method of choice is thus the demonstration of specific IgM- and IgG-serum antibodies, since these antibodies are detectable in practically every case at time of hospitalization. Most often Enzyme-linked Immunosorbent Assays (ELISA) are used. However, in cases of contacts with other flavivirus (e.g. vaccinations against yellow fever or Japanese encephalitis; dengue virus infections), a neutralization assay should be performed to assess immunity due to the interference of flavivirus cross-reactive antibodies in ELISA test [29]. Seroneutralization assays can be performed in biosafety level (BSL)-3 conditions with the wildtype virus. Alternatively pseudovirions and recombinant viral particles exposing structural proteins of TBEV (and other flaviviruses) can be produced [30, 31]. These systems allow to assess the presence of specific antibodies for TBEV in a BSL-2 condition and in parallel for different flaviviridae. For the differentiation of vaccinationinduced antibodies from those induced by natural infection, assays detecting antibodies against the non-structural protein 1 (NS1) of the virus have been introduced. As available vaccines are highly purified and inactive, without substantial amounts of NS1, there is no TBEV replication and therefore no formation of NS1 protein and/or NS1specific antibodies [32].
15
16
ED N EW UC SA T I O N
P I P E T T E – S W I S S L A B O R AT O R Y M E D I C I N E | WWW. S U L M . C H
NR. 3 | JUNI 2022
References 1. Rochat E, Vuilleumier S, Aeby S, Greub G, Joost S. Nested Species Distribution Models of Chlamydiales in Ixodes ricinus (Tick) Hosts in Switzerland. Appl Environ Microbiol. 2020 Dec 17;87(1):e01237-20. 2. Bellini C, Monti M, Potin M, Dalle Ave A, Bille J, Greub G. Cardiac involvement in a patient with clinical and serological evidence of African tick-bite fever. BMC Infect Dis. 2005 Oct 20;5:90. 3. Pilloux L, Baumgartner A, Jaton K, Lienhard R, Ackermann-Gäumann R, Beuret C, Greub G. Prevalence of Anaplasma phagocytophilum and Coxiella burnetii in Ixodes ricinus ticks in Switzerland: an underestimated epidemiologic risk. New Microbes New Infect. 2018 Sep 6;27:22-26. 4. Greub G. Parachlamydia acanthamoebae, an emerging agent of pneumonia. Clin Microbiol Infect. 2009 Jan;15(1):18-28. 5. Lamoth F, Greub G. Amoebal pathogens as emerging causal agents of pneumonia. FEMS Microbiol Rev. 2010 May;34(3):260-80. 6. Greub G, Lienhard R & Ackermann R. Le Centre national de référence pour les maladies transmises par les tiques (CNRT/NRZK). Pipette 2017 ; 5: 8-9. 7. Pilloux L, Baumgartner A, Jaton K, Lienhard R, Ackermann-Gäumann R, Beuret C, Greub G. Prevalence of Anaplasma phagocytophilum and Coxiella burnetii in Ixodes ricinus ticks in Switzerland: an underestimated epidemiologic risk. New Microbes New Infect. 2018 Sep 6;27:22-26. 8. Radolf JD, Strle K, Lemieux JE, Strle F. Lyme Disease in Humans. Curr Issues Mol Biol. 2021;42:333-384. 9. Hansen K, Lebech AM. Lyme neuroborreliosis: a new sensitive diagnostic assay for intrathecal synthesis of Borrelia burgdorferi--specific immunoglobulin G, A, and M. Ann Neurol. 1991 Aug;30(2):197-205. 10. Lienhard R. Quelle est l’utilité de la sérologie de Lyme. Rev Med Suisse. 2015 Oct 7;11(489):1830-4. 11. Yessinou RE, Katja MS, Heinrich N, Farougou S. Prevalence of Coxiella-infections in ticks - review and meta-analysis. Ticks Tick Borne Dis. 2022 May;13(3):101926.Xx 12. Delaloye J, Greub G. Fievre Q: une zoonose souvent méconnue. Rev Med Suisse. 2013 Apr 24;9(383):879-84 13. Melenotte C, Protopopescu C, Million M, Edouard S, Carrieri MP, Eldin C, Angelakis E, Djossou F, Bardin N, Fournier PE, Mège JL, Raoult D. Clinical Features and Complications of Coxiella burnetii Infections From the French National Reference Center for Q Fever. JAMA Netw Open. 2018 Aug 3;1(4):e181580. 14. Kaech C, Raoult D, Greub G. Incidental live-saving polymerase chain reaction in a case of prosthetic valve dual-pathogen endocarditis. Clin Infect Dis. 2008 Jul 1;47(1):144. 15. Bellini C, Magouras I, Chapuis-Taillard C, Clerc O, Masserey E, Peduto G, Péter O, Schaerrer S, Schuepbach G, Greub G. Q fever outbreak in the terraced vineyards of Lavaux, Switzerland. New Microbes New Infect. 2014 Jul;2(4):939. 16. Dupuis G, Péter O, Pedroni D, Petite J. Aspects cliniques observés lors d’une épidémie de 415 cas de fièvre Q [Clinical aspects observed during an epidemic of 415 cases of Q fever]. Schweiz Med Wochenschr. 1985 Jun 15;115(24):814-8. French. PMID: 3892664. 17. Hackert VH, van der Hoek W, Dukers-Muijrers N, de Bruin A, Al Dahouk S, Neubauer H, Bruggeman CA, Hoebe CJ. Q fever: single-point source outbreak with high attack rates and massive numbers of undetected infections across an entire region. Clin Infect Dis. 2012 Dec;55(12):1591-9. 18. Jaton K, Peter O, Raoult D, Tissot JD, Greub G. Development of a high throughput PCR to detect Coxiella burnetii and its application in a diagnostic
laboratory over a 7-year period. New Microbes New Infect. 2013 Oct;1(1):612. 19. Lindquist L. 2014. Tick-borne encephalitis, p 531-559. In Tselis ACB, J. (ed), Handbook of Clinical Neurology, vol 123. 20. Federal Office for Public Health Switzerland. 2016. Bulletin 41/16: 622-626. 21. Federal Office for Public Health Switzerland. Data on Infectious diseases. https://www.bag.admin.ch/bag/de/home/zahlen-und-statistiken/zahlen-zu-infektionskrankheiten.exturl.html/aHR0cHM6Ly9tZWxkZXN5c3RlbWUuYmFnYXBwcy5jaC9pbmZyZX/BvcnRpbmcvZGF0ZW5kZXRhaWxzL2QvZnNtZS5odG1sP3dlYmdy/YWI9aWdub3Jl.html 22. Robert Steffen, MD, Heinz-Josef Schmitt, MD, PhD, Dace Zavadska, MD, Tick-borne encephalitis vaccine—a wave of news, Journal of Travel Medicine, Volume 29, Issue 2, March 2022. 23. Kollaritsch H, Paulke-Korinek M, Holzmann H, Hombach J, Bjorvatn B, Barrett A. 2012. Vaccines and vaccination against tick-borne encephalitis. Expert Rev Vaccines 11:1103-19. 24. Federal Office for Public Health Switzerland. 2006. Recommendations for TBE vaccination. Bulletin 6:12-14. 25. Pierson TC, Fremont DH, Kuhn RJ, Diamond MS. 2008. Structural insights into the mechanisms of antibody-mediated neutralization of flavivirus infection: implications for vaccine development. Cell Host Microbe 4:229-38. 26. Steffen R, Erber W, Schmitt HJ. Can the booster interval for the tick-borne encephalitis (TBE) vaccine ’FSME-IMMUN’ be prolonged? - A systematic review. Ticks Tick Borne Dis. 2021 Sep;12(5):101779. 27. Schmidt AJ, Altpeter E, Graf S, Steffen R. Tick-borne encephalitis (TBE) in Switzerland: does the prolongation of vaccine booster intervals result in an increased risk of breakthroughs? J Travel Med. 2022 Mar 21;29(2):taab158. 28. Greub G, Kebbi C. KROBS: un jeu innovant sur les microbes. Pipettes 2018; 6:22-23. 29. Holzmann H. Diagnosis of tick-borne encephalitis. Vaccine. 2003 Apr 1;21 Suppl 1:S36-40. 30. Agudelo M, Palus M, Keeffe JR, Bianchini F, Svoboda P, Salát J, Peace A, Gazumyan A, Cipolla M, Kapoor T, Guidetti F, Yao KH, Elsterová J, Teislerová D, Chrdle A, Hönig V, Oliveira T, West AP, Lee YE, Rice CM, MacDonald MR, Bjorkman PJ, Růžek D, Robbiani DF, Nussenzweig MC. Broad and potent neutralizing human antibodies to tick-borne flaviviruses protect mice from disease. J Exp Med. 2021 May 3;218(5):e20210236. 31. Hu HP, Hsieh SC, King CC, Wang WK. Characterization of retrovirus-based reporter viruses pseudotyped with the precursor membrane and envelope glycoproteins of four serotypes of dengue viruses. Virology. 2007 Nov 25;368(2):376-87. 32. Girl P, Bestehorn-Willmann M, Zange S, Borde JP, Dobler G, von Buttlar H. Tick-Borne Encephalitis Virus Nonstructural Protein 1 IgG Enzyme-Linked Immunosorbent Assay for Differentiating Infection versus Vaccination Antibody Responses. J Clin Microbiol. 2020 Mar 25;58(4):e01783-19. 33. Croxatto A, Rieille N, Kernif T, Bitam I, Aeby S, Péter O, Greub G. Presence of Chlamydiales DNA in ticks and fleas suggests that ticks are carriers of Chlamydiae. Ticks Tick Borne Dis. 2014 Jun;5(4):359-65. X 34. Pillonel T, Bertelli C, Aeby S, de Barsy M, Jacquier N, Kebbi-Beghdadi C, Mueller L, Vouga M, Greub G. Sequencing the Obligate Intracellular Rhabdochlamydia helvetica within Its Tick Host Ixodes ricinus to Investigate Their Symbiotic Relationship. Genome Biol Evol. 2019 Apr 1;11(4):1334-1344. 35. Ackermann R, Lienhard R, Greub G. Waldmikroben: von Borrelia burgdorferi zum Zeckenenzephalitisvirus. Pipettes 2019; 4; 11-13.
Anzeige
PRÄZISES LIQUID-HANDLING Präzision, Reproduzierbarkeit und perfekte Ergonomie für Ihre Liquid Handling-Anwendungen. Sie wählen aus einem kompletten Angebot manueller und elektronischer Mikropipetten, Dispensern, Repetierpipetten, Pipettierhilfen und Spritzen aus. Socorex Isba SA CH-1024 Ecublens • Tel. +41 (0)21 651 6000 socorex@socorex.com • www.socorex.com
Service Center
P I P E T T E – S W I S S L A B O R AT O R Y M E D I C I N E | W W W. S U L M . C H
NR. 3 | JUNI 2022
MAR CN E EK DE UTCPALTAI O
Greub Gilbert 1
Le réchauffement climatique: possibles impacts sur les microbes, leurs réservoirs, les infections et les épidémies. Le réchauffement climatique est une nouvelle réalité. Ses conséquences sont multiples avec par exemple la survenue (i) en mai 2022 de tornades en Allemagne, (ii) davantage de «vents du Sahara» qui ont couverts l’hiver dernier d’une robe beige nos montagnes enneigés, (iii) de périodes de sécheresse et de vagues de chaleur prolongées avec des incendies d’une ampleur inhabituelle et (iv) la survenue de pluies diluviennes rappelant les moussons, mais observés en Europe continentale, avec des inondations majeures telles que celles observées en été 2021 en Belgique. Globalement, l’augmentation des températures moyennes de 1.5 degrés a des impacts au niveau mondial sur l’accès à l’eau potable et à la nourriture, avec un impact majeur en terme de santé pour certaines populations (africaine sub-saharienne par exemple). Le réchauffement climatique impacte aussi l’épidémiologie des maladies infectieuses et il est essentiel de comprendre l'impact de ces changements climatiques sur les microbes et les infections. Ainsi dans cet article, nous allons discuter de l'impact du réchauffement climatique sur les microbes, notamment: • la documentation de cyanobactéries toxinogènes dans le lac de Neuchâtel, • le changement de la répartition géographique des tiques et du moustique tigre, vecteurs de diverses maladies dont la borréliose de Lyme et l’encéphalite à tiques • le changement de la répartition géographique d’espèces animales réservoirs, qui ont probablement contribué à la large épidémie de peste à Madagascar en 2017, et à la recrudescence de cas de Monkeypox au Nigeria, en république centrafricaine et en république démocratique du Congo • la possibilité que le dégel du permafrost puisse libérer divers agents pathogènes dont le virus de la variole • Par contre, nous n’aborderons pas les risques sanitaires liées aux inondations (leptospirose, gastroentérites virales, tourista, choléra, …), malgré l’importance de cette problématique.
Figure 1: Phormidium, une cyanobactérie également appelée Microcoleus. Notez la nature filamenteuse des cas bactéries capables de photosynthèse (Photographie du Dr Peter Sausal avec Tycoiver, Visuals unlimited, Science photo Library)
le lac de Neuchâtel des suites d'une intoxication liée à la présence de cyanobactéries observées dans les mêmes eaux [1]. Cette prolifération de cyanobactéries est clairement le reflet du réchauffement climatique. Ce fait divers nous permet de rappeler ici que le rôle pathogène des microbes n'est pas toujours lié à sa prolifération au sein de l'organisme et que parfois une simple toxine peut avoir un effet majeur. Ainsi les cyanobactéries peuvent proLes cyanobactéries du lac de duire différentes toxines qui affectent Neuchâtel le foie (hépato-toxine), la peau (derDurant la canicule de fin juillet 2020, mato-toxine) ou le système nerveux plusieurs chiens sont décédés à proxi- central (neuro-toxine) [2, 3]. Les neumité de l'embouchure de l’Areuse sur rotoxines des cyanobactéries peuvent induire une paralysie rapide comme 1 Prof. Gilbert Greub, Institute of Microbiology, celle constatée chez les chiens qui University Hospital Center of Lausanne, Lausanne, Switzerland sont allés se désaltérer et ont bu de
l'eau dans une région où proliféraient des cyanobactéries toxiques. Au total 8 chiens sont décédés en l'espace de 20 à 30 minutes sur une période de 2 jours. Le résultat des analyses effectuées dans le contenu des estomacs de 2 chiens morts le 30 juillet 2020 a confirmé la présence de 2 cyanobactéries (Tychonema et Microcoleus) toutes deux connues pour produire des neurotoxines [1]. Ces cyanobactéries d’allure filamenteuse (Figure 1) ont longtemps été considérées comme des algues (algues bleues). Il s'agit en fait de bactéries dont l'apparition il y a 3 milliards d'années a constitué le tournant le plus important de l'évolution du vivant puisque ces bactéries sont capables de photosynthèse et ont permis une diversification des biotopes.
17
18
ED N EW UC SA T I O N
Il est important de noter que ce n’est pas directement les hautes chaleurs qui furent la cause de cet évènement, mais davantage la baisse prolongée de la pluviométrie et le fait d’eaux stagnantes sur les derniers kilomètres de l’Areuse suite au moindre débit d’eau dans cette rivière. Ces eaux stagnantes (plus chaudes que la température usuelle de la rivière furent favorables à la multiplication des cyanobactéries productrices de neurotoxines puis suite à un orage, ces cyanobactéries et leurs toxines se sont retrouvées dans le lac de Neuchâtel, proche de l’embouchure de l’Areuse, où les chiens sont venus se désaltérer. Notez que ces cyanobactéries sont bien connues dans le Nord de l’Europe où en été certains lacs ou étang (eaux stagnantes) sont interdits à la baignade par période en raison de prolifération de cyanobactéries.
Arthropodes vecteurs et réchauffement climatique Les tiques Les tiques sont des vecteurs de nombreux pathogènes différents et le réchauffement climatique a vu la répartition des tiques se modifier avec davantage de tiques du genre Ixodes se localiser en altitude au-delà de 1500 m, comme nous l’avons bien démontré [4]. Ainsi, la surface favorable aux tiques s’est élevée en 10 ans de 16% à 25% du territoire Suisse [4]. Sur la base de l’analyse des zones où des tiques ont pu être documentés, nous avons observés que les tiques se retrouvaient davantage proche de points d’eau (ruisseaux, étangs, …) et que leur présence dépendait clairement des extrêmes de température, à la fois en été et en hiver [4]. L'arrivée de nouvelles espèces de tiques dont Rhipicephalus peut aussi être le reflet du réchauffement climatique. Cette dernière est le vecteur de la fièvre boutonneuse méditerranéenne généralement présente autour de la Méditerranée, mais documentée pour la première fois en 2020 en Suisse de manière autochtone, sans notion de voyage hors de Suisse, ce qui contraste avec les cas observées par le passé, clairement lié à des voyages au bord de la Méditerranée [5].
P I P E T T E – S W I S S L A B O R AT O R Y M E D I C I N E | WWW. S U L M . C H
Cet accroissement des zones favorables aux tiques n’est pas le seul déterminant du nombre accru d’infections liées aux tiques observées ces dernières années. En effet, le nombre accru de personnes allant se baladées dans les forêts et dans les près explique aussi le nombre plus élevé de maladies de Lyme documentées (14'000 par année en 2021) et d’encéphalite à tiques (plus de 300 ces dernières années) [6]. Ces maladies sont transmises principalement par la tique Ixodes ricinus, appelée dans le langage vernaculaire «tique du mouton». Comme démontré près de New York par des collègues américains, l’accroissement des maladies transmises par les tiques est multifactoriel, incluant non seulement l’extension des zones géographiques favorables aux tiques, mais également l’accroissement de la proportion de tiques infectées avec dans leur région (i) 57% des tiques Ixodes scapularis documentées porteurs de Borelia burgdorferi et (ii) de nombreuses co-infections [7]. Cette notion de co-infection est importante puisque l’impact du climat non-seulement peut s’exercer sur l’étendue des zones favorables aux tiques, mais également sur la présence ou l’absence de co-infections par plus d’un pathogène ainsi que sur la présence d’endosymbiontes. Ainsi, dans une étude européenne, les symbiontes du genre Spiroplasma se retrouvent plus souvent en association avec un autre symbionte de tique (Lariksella et/ou Ricketsiella) et moins souvent (compétition) avec les pathogènes tels que Borrelia valaisiana et diverses rickettsies [8]. Ainsi, le nombre accru de cas d’infections humaines par des pathogènes transmis par des tiques en Suisse est probablement multifactoriel et le reflet: • de l’extension des zones favorables aux tiques liée au changement climatique • d’une diversité accrue de tiques dans nos régions liée au changement climatique • d’une exposition accrue aux tiques par des changements de mode de vie, partiellement lié au changement climatique avec davantage de journées d’ensoleillement propices aux ballades
NR. 3 | JUNI 2022
• d’un changement des taux de tiques infectées, partiellement lié au changement climatique • d’une amélioration des méthodes de détection des pathogènes et de diagnostic des maladies liées aux tiques • d’une meilleure surveillance • par une bonne communication au grand public et une sensibilisation, qui conduit davantage de personnes chez le médecin. Le moustique Tigre De la même manière que la répartition des tiques s'est modifiée avec le réchauffement climatique, la répartition géographique du moustique tigre (Aedes albopictus) s’étend progressivement. Ainsi, dans une récente étude, Ravasi et al. ont démontrés que les régions favorables au moustique tigre s’étend au delà des Alpes, notamment au plateau suisse, à la région bâloise et à la vallée du Rhône [9]. Ceci est inquiétant vu que ce moustique est le vecteur de plusieurs virus dont le virus Chikungunya, le Zikavirus et l’agent de la dengue. C’est surtout le Chikungunya qui est craint dans nos régions puisque cette maladie qui était principalement documenté dans la région de Madagascar et dans l'île de la Réunion, est aujourd’hui documenté dans la plaine du Pô (Italie) dès 2019. L’intervention «n’invitons pas le moustique tigre pour l’apéro» qui vise à éviter de laisser des coupelles d’eau par exemple sous les bacs de fleurs ou laisser de l’eau stagner dans des pneus laissés à l’abandon comme le suggère une étude comparant la même région frontalière, du côté italien et suisse et démontrant une réduction de l’ordre de 4x du nombre d’oeufs d’Aedes albopictus documentés au tessin par rapport à la région italienne où aucune mesure n’est prise [10]. Ainsi, il est évident que le réchauffement climatique modifie les maladies infectieuses auxquelles nous sommes exposés en Suisse, ce qui doit nous conduire à davantage de vigilance. Cependant, des interventions telles que la campagne actuelle pourrait limiter la dispersion du moustique tigre. Des modifications de la répartition des moustiques vecteurs similaires sont également observées dans
P I P E T T E – S W I S S L A B O R AT O R Y M E D I C I N E | W W W. S U L M . C H
NR. 3 | JUNI 2022
les autres pays du monde, où le ré- • virus plus contagieux chauffement climatique modifie les • circulation accrue parmi la populazones d’endémies de diverses malation d’hommes ayant des relations dies transmises par les moustiques, y sexuelles avec d’autres, avec dispercompris le paludisme (parfois favorasion géographique dans un monde blement, parfois défavorablement). globalisé La 2ème hypothèse est privilégiée Réchauffement climatique et puisque les orthopoxvirus sont des viréservoirs de pathogènes rus ADN peu enclins à des mutations multiples qui modifieraient leur Les rats, les puces et la peste contagiosité. L'épidémie de peste à Madagascar a Heureusement, les premières séété médiatisée, en raison du nombre quences disponibles pour les cas porde cas significatifs qui ont été docu- tugais suggèrent que l’épidémie est mentés durant les mois d'août à oc- due à la souche provenant de l’Afrique tobre 2017 [11]. Notons que cette zoo- de l’Ouest [15], un virus causant une nose due à Yersinia pestis est maladie dont la mortalité est inféclassiquement transmise par les rieure à 1% dans le contexte médical puces de rats causant des adénopa- local. Même si la prudence est de thies nécrotiques (peste bubonique). mise, la mise en place rapide de diaLa peste était endémique dans la ré- gnostic par PCR et l’effort de séquengion rurale de Madagascar, mais suite çage des souches circulantes devrait au réchauffement climatique et à un permettre de bien comprendre cette été particulièrement chaud en 2017, nouvelle épidémie multifocale docules rats infestés dans des régions ru- mentée dans de nombreux pays d’Eurales sont remontés plus en altitude, rope, dont la Suisse et de proposer les là où se trouve la ville principale de mesures de mitigation appropriées. l'île de Madagascar, Antananarivo, Cette épidémie multifocale eurocausant soudainement une épidémie péenne n’est clairement pas liée au majeure avec des transmissions se- réchauffement climatique. Par contre condaires de personnes à personnes les cas fréquents documentés au Nipar aérosol (peste pulmonaire) [12]. geria dès 2017 sont probablement le reflet du déplacement des réservoirs Les singes et le virus monkeypox (rongeurs et singes) suite au changeEn 2 semaines, entre le 6 et le 20 mai ment climatique. De même il est pos2022, 20 cas de monkeypox ont été sible que la très importante augmendocumentés au Royaume-Uni [13]. tation du nombre de cas documentés Ceci est tout à fait inhabituel puisque en Afrique centrafricaine ces 10 derla variole du singe était jusque-là can- nières années [16] soit lié indirectetonnée en Afrique centrale (répu- ment au déplacement des réservoirs blique centrafricaine, république dé- animaux. Ces réservoirs se retrouvent mocratique du Congo) et en Afrique alors plus proche des populations hude l’Ouest (Nigéria). Le monkeypox maines et une prévalence accrue en été découvert initialement en 1958 zone d’endémie fournit davantage chez un singe (d’où le nom du virus) d’opportunité pour le virus monkeymais il peut aussi infecté divers ron- pox de se retrouver à infecter un voyageurs (qui peuvent servir de réser- geur, qui: voirs et vecteurs) et l’être humain. De- (i) présente une infection peu sévère puis le 1er cas humain documenté en (passant inaperçue) 1970, le monkeypox était jusqu’à ce (ii) est contagieux durant plus de 14 printemps 2022 qu’occasionnellement jours par gouttelettes de salive, documenté hors des pays d’Afrique et/ou par contacts avec des musubsaharienne où la maladie est enqueuses et/ou peaux infectées (uldémique et de surcroit les cas eurocérations, pustules) péens étaient jusqu’alors liés à des (iii) retourne en Europe où il transvoyages dans ces zones [13, 14]. Si les mettra à d’autres, débutant poraisons de l’épidémie multifocale actentiellement un début d’épidétuelle restent inconnues, plusieurs mie hypothèses sont considérées: Dans ces pays endémiques, des ac-
E D U C ANTEI W ON S
tions doivent être également être entreprises afin de limiter la morbidité et la mortalité lié à ce virus, car avec l’arrêt de la vaccination anti-varioloique, la protection croisée conférée par le vaccin rend progressivement l’ensemble de la population mondiale non-immune aux différents orthopoxvirus dont le smallpox virus et le monkeypox virus [17].
Le réchauffement climatique et la variole Aujourd'hui la variole est une maladie éradiquée. Mais il se pourrait que la maladie puisse réapparaître par exemple suite à une dissémination accidentelle ou volontaire à partir d’un des stocks connus de variole ou d’un stock non répertorié [18]. De plus, il se pourrait que le réchauffement climatique ramène le corps pétrifié par la glace, d'une personne infectée il y a plusieurs siècles que ce soit suite à la fonte des glaciers ou du permafrost [19]. L'hypothèse est étayée par la découverte d'ADN de la variole à partir du corps d'une femme sibérienne datant d'environ 300 ans [20] et par le fait que l'équipe de Jean-Michel Claverie a pu cultiver un ancien virus datant de plus de 30 000 ans à partir du permafrost [21]. Ce virus appelé Pithovirus sibericum est apparemment non pathogène pour l'être humain, se multipliant uniquement au sein des amibes libres du genre Acanthamoeba et pas en culture de cellules mammifères [21]. Cependant, il représente une preuve de la longévité des virus ADN présents dans le permafrost. Ainsi outre une réapparition de la variole, on peut aussi craindre l'apparition de virus inconnus qui ont peutêtre été extrêmement pathogènes dans un passé lointain. En cas de réapparition de variole, il faudrait alors craindre une pandémie d'une maladie transmissible par voie respiratoire (comme le Coronavirus) mais grevé d'une mortalité bien supérieure que celle du SARS-CoV-2 (de l'ordre de 30% environ). De plus, compte tenu du délai probable entre les éventuels premiers cas et l'alerte, le relatif peu de doses de vaccins disponibles (vu que cette maladie est éradiquée) et l'absence d'immunité chez près de 70% de la population ac-
19
20 OCTOBER 2022 SWISS SYMPOSIUM IN
H
POINT-OF-CARE DIAGNOSTICS FHNW Campus Muttenz, Region Basel
SCAN & REGISTER
SCAN & REGISTER Exciting keynotes by: •
Gyorgy Abel, Lahey Hospital & Medical Center; Harvard
•
Antonella Santuccione, Womens’ Brain Project; Alzheimer’s Disease Biogen
Pre-event workshop on regulatory requirements hosted by Effectum Medical Exhibition by leading companies Networking with academic and industrial investigators Scientific poster competition
Seegene STARlet-AIOS
All-in-One full automation solution for molecular testing (sample in – result out) Space-efficient Optimized for small to mid-sized hospitals and C-labs
Simple-to-use Easy installation, operation and maintenance
Test expandability
All Seegene’s syndromic assays are available using one system
Affordable
Adoption of a Full automation system with a low budget
BÜHLMANN Laboratories AG CH - 4124 Schönenbuch/Basel
|
Phone: +41 61 487 12 12
|
Fax: +41 61 487 12 34
|
E-mail: info@buhlmannlabs.ch
www.buhlmannlabs.ch
P I P E T T E – S W I S S L A B O R AT O R Y M E D I C I N E | W W W. S U L M . C H
tuelle, une pandémie majeure serait à craindre [18]. Heureusement que ce scénario reste très peu probable et que la population mondiale, qui vient de faire face à la pandémie de Coronavirus est de facto de mieux en mieux préparée à faire face à des virus transmissibles par gouttelettes ou aérosols. Rappelons ici que le virus de la variole au passé dévastateur, probablement apparu en Afrique il y a plus de 4000 ans, se serait disséminé en Inde et en Chine puis vers le 6ème siècle après J.-C. en Europe [19]. Totalement inconnu dans le Nouveau Monde la variole a été introduite par les conquistadors espagnols et portugais et a presque entièrement décimé la population locale jouant de ce fait
E D U C ANTEI W ON S
NR. 3 | JUNI 2022
un rôle majeur dans la chute de l'empire Aztèque. La présence de variole aux Amériques a aussi été la conséquence de la traite des esclaves provenant d'Afrique où la maladie était alors endémique [19]. Il est important de souligner que non seulement la variole a été associée à une mortalité majeure avec plus de 400 000 décès annuels en Europe au XVIIIe siècle mais a causé également une morbidité significative puisqu'un tiers des survivants devenaient aveugles [22]. De plus les survivants présentaient des cicatrices majeures sur le corps qui défiguraient les visages [22]. En raison de sa grande mortalité, des efforts considérables ont été fait pour éradiquer cette mala-
die et la variole représente aujourd'hui le seul exemple de maladie éradiquée grâce à un vaccin [19].
Conclusion Ainsi, le changement climatique actuel présente un impact majeur sur diverses maladies infectieuses et il est important que le corps médical ainsi que les acteurs de la médecine de laboratoire soient conscients des changements épidémiologiques liés au climat et aux changements de la répartition des vecteurs et des réservoirs de maladies infectieuses. Correspondence gilbert.greub@chuv.ch
Références 1. Aragno M, Greub G. Cyanobactéries toxiques. Swiss Laboratory Medicine Journal (Pipette) 2020: 5: 21-22. 2. C. Svrcek, D. W. Smith, Cyanobacteria toxins and the current state of knowledge on water treatment options: a review, J. Environ. Eng. Sci. 3: 155–184, 2004. 3 3. Centers for Disease Control and Prevention (CDC). Facts about cyanobacteria and cyanobacterial harmful algal blooms (www.cdc.gov/hab/cyanobacteria/facts.htm). 4. Rochat E, Vuilleumier S, Aeby S, Greub G, Joost S. Nested Species Distribution Models of Chlamydiales in Ixodes ricinus (Tick) Hosts in Switzerland. Appl Environ Microbiol. 2020 Dec 17;87(1):e01237-20. 5. Chamot E, Chatelanat P, Humair L, Aeschlimann A, Bowessidjaou J. Cinq cas de fièvre boutonneuse méditerranéenne en Suisse [5 cases of Mediterranean boutonneuse fever in Switzerland]. Ann Parasitol Hum Comp. 1987;62(5):371-9. French. 6. Greub G, Ackermann R, Cagno V, Coste A, Croxatto A, Opota O, and Lienhard R. The Swiss national reference centre for tick-borne infections. Swiss Laboratory Medicine Journal (Pipette) 2022: this issue. 7. Sanchez-Vicente S, Tagliafierro T, Coleman JL, Benach JL, Tokarz R. Polymicrobial Nature of Tick-Borne Diseases. mBio. 2019 Sep 10;10(5):e02055-19. 8. Aivelo T, Norberg A, Tschirren B. 2019. Bacterial microbiota composition of Ixodes ricinus ticks: the role of environmental variation, tick characteristics and microbial interactions. PeerJ 7:e8217 https://doi.org/10.7717/ peerj.8217 9. Ravasi D, Mangili F, Huber D, Azzimonti L, Engeler L, Vermes N, Del Rio G, Guidi V, Tonolla M, Flacio E. Risk-Based Mapping Tools for Surveillance and Control of the Invasive Mosquito Aedes albopictus in Switzerland. Int J Environ Res Public Health. 2022 Mar 9;19(6):3220. 10. Ravasi D, Parrondo Monton D, Tanadini M, Flacio E. Effectiveness of integrated Aedes albopictus management in southern Switzerland. Parasit Vectors. 2021 Aug 16;14(1):405. 11. Nguyen VK, Parra-Rojas C, Hernandez-Vargas EA. The 2017 plague outbreak in Madagascar: Data descriptions and epidemic modelling. Epidem-
ics. 2018 Dec;25:20-25. 12. Burki T. Plague in Madagascar. Lancet Infect Dis. 2017 Dec;17(12):1241. 13. Beeching R, de Valdoleiros SR, and Greub G for EITaF. Monkeypox – reemerging in unexpected places and risk groups. ESCMID Emerging Infections Task Force, 20 may 2022. 14. Kozlov M. Monkeypox goes global: why scientists are on alert. Nature. 2022 May 20. 15. Isidro J et al. First draft genome sequence of Monkeypox virus associated with the suspected multi-country outbreak, May 2022 (confirmed case in Portugal). Published online on 20 May 2022 at https://virological.org/t/firstdraft-genome-sequence-of-monkeypox-virus-associated-with-the-suspected-multi-country-outbreak-may-2022-confirmed-case-in-portugal/799 16. Bunge EM, Hoet B, Chen L, Lienert F, Weidenthaler H, Baer LR, Steffen R. The changing epidemiology of human monkeypox-A potential threat? A systematic review. PLoS Negl Trop Dis. 2022 Feb 11;16(2):e0010141. 17. Smithson C, Imbery J, Upton C. ReAssembly and Analysis of an Ancient Variola. Virus Genome. Viruses. 2017 Sep 8;9(9):253. 18. Barras V, Greub G. History of biological warfare and bioterrorism. Clin Microbiol Infect. 2014;20(6):497–502. 19. Greub G, Barras V. La variole: un passé dévastateur, une vaccination salvatrice et de possibles risques futurs. Swiss Laboratory Medicine Journal (Pipette) 2020: 5: 12-14. 20. Biagini P, Thèves C, Balaresque P, Géraut A, Cannet C, Keyser C, Nikolaeva D, Gérard P, Duchesne S, Orlando L, Willerslev E, Alekseev AN, de Micco P, Ludes B, Crubézy E. Variola virus in a 300-year old Siberian mummy. N Engl J Med 2012; 367:2056–2058. 21. Legendre M, Bartoli J, Shmakova L, Jeudy S, Labadie K, Adrait A, Lescot M, Poirot O, Bertaux L, Bruley C, Couté Y, Rivkina E, Abergel C, Claverie JM. Thirty-thousand-yearold distant relative of giant icosahedral DNA viruses with a pandoravirus morphology. Proc Natl Acad Sci U S A. 2014;111(11):4274–9 22. Ellner PD. Smallpox: gone but not forgotten. Infection. 1998 SepOct;26(5):263-9.
21
22
ED N EW UC SA T I O N
P I P E T T E – S W I S S L A B O R AT O R Y M E D I C I N E | WWW. S U L M . C H
NR. 3 | JUNI 2022
Gilbert Greub 1 , Claire Bertelli 2 , Benoit Guery 3 , Grégory Resch 4 , Pascale Vonaesch 5
The Human Microbiota Network of the Swiss National Centre of Competences in Research (NCCR) on microbiomes: objectives and main approaches Studying the human microbiota is pivotal to improve our understanding of a number of infectious and non-infectious diseases. In Lausanne, microbiota related research started about 15 years ago when new generation technologies became available. However, it is only in 2018 that amplicon-based metagenomics was accredited in the diagnostic laboratory of the Institute of Microbiology at CHUV and was used for patients’ care. Still, the indications remain limited and require investigations on the perimeter and limitations of this novel technology for targeted applications in the clinics.
To fill that gap, the critical mass of research on microbiota needed to be increased, which has been made possible with the new National Center of Competences in Research (NCCR) on microbiomes that started its activity on July 1st 2020. This NCCR co-directed by Jan van der Meer (UNIL) and Julia Vorholt (ETHZ) was initially composed of 18 different research groups, organized in 6 work packages (WPs), located in Lausanne (UNIL, CHUV and EPFL), Zurich (UNIZH and ETHZ) and Bern (UNIBE). WP1, led by Gilbert Greub, is focusing Figure 1. Methods that are developed or improved by the NCCR human microbiota network. on human microbiota, mainly in the gut, and currently exhibit nine research axis (see Table 1), i.e. four on specific diseases (D1 to D4) and five on method improvement or tool development (M1 to M5) to study the microbiota. The objectives of the six work packages is listed in Table 2.
1 Prof. Gilbert Greub, Institute of Microbiology, University Hospital Center of Lausanne, Lausanne, Switzerland 2 Dr. Claire Bertelli, Head of bioinformatics, Genomics and Metagenomics Laboratory, University Hospital Center of Lausanne, Lausanne, Switzerland 3 Prof. Benoit Guery, Médecin chef, Service des maladies infectieuses, University Hospital Center of Lausanne, Lausanne, Switzerland 4 Dr. Grégory Resch, Head of bacteriophage research, University Hospital Center of Lausanne, Lausanne, Switzerland 5 Dr. Pascale Vonaesch, Assistant Professor, Department of Fundamental Microbiology, University of Lausanne
Figure 2. Main translational projects done by the NCCR human microbiota network.
Methods developed, implemented or improved by the human microbiota network of the NCCR The methods developed in WP1 to study the human microbiota are summarized in Figure 1 and Table 1. Ded-
icated bioinformatics tools are being developed for diagnostic and research applications of metagenomics and other type of microbiota analyses. Fast (and relatively cheap) methods, such as flow cytometry, will be used
P I P E T T E – S W I S S L A B O R AT O R Y M E D I C I N E | W W W. S U L M . C H
Project number1
NR. 3 | JUNI 2022
E D U C ANTEI W ON S
Main objective
Possible deliverables
M1
Improve the metagenomics tools, including both wet lab protocols and bioinformatics pipelines; this project is initially focused on 16S rRNA amplicon-based bacterial profiling2 but also aims to expand to shotgun metagenomics3.
Provide efficient and robust diagnostic tools enabling the routine use of both amplicon-based2 and direct metagenomics3 in accredited laboratories
M2
Develop a culturomics4 pipeline by combining automated inoculation, semi-automated colonies detection and MALDITOF identification. Define the robustness of culturomics as compared to amplicon-based microbiota profiling2.
Provide a new modern culture-based approach to study the microbial composition, complementary to metagenomics and enabling isolation of key strains, that may be further studied.
M3
Implement phage production as a medical product for patient treatment. Develop phage-related tools to define the phages present in complex samples, such as the gut microbiota, and study the impact of phages on the diseases listed below (D1 to D4).
Improve knowledge and know-how on phages, phage production, and phage therapy.
M4
Implement flow cytometry wetlab protocols and bioinformatic pipelines to perform microbiota analyses and benchmark this new approach with PCR-based metagenomics. Identify flow cytometry signatures associated with gut dysbiosis.
Provide reliable and robust algorithm to Use flow cytometry as a routine tool in microbiological diagnostic arsenal.
M5
Develop new bioinformatics tools and adapt existing ones to improve the identification of virulence and antibiotic resistance encoding genes, as well as metabolic pathways.
Provide a comprehensive, robust and easy-to-use annotation tool for a flexible analysis both for research and diagnostic purposes with short time to results.
D1
Better understand the pathogenesis of Salmonella infection and whether the presence (n+1) or absence (n-1) of some gut bacteria may modify the natural course of the disease, including severity of the disease and duration of the bacterial shedding in absence of antibiotic treatment.
Define bacterial species associated with better or worse outcome and identify some protective bacteria that might be used as probiotics to improve patient care
D2
Better understand the pathogenesis of Clostridioides difficile and whether the presence (n+1) or absence (n-1) of some gut bacteria may modify the natural course of the disease, including severity of the disease, recurrent infections and treatment success.
Define bacterial species associated with better or worse outcome and identify some protective bacteria that might be used as probiotics to improve patient care
D3
Investigate the impact of SARS-CoV-2 infection on the gut microbiota and the impact of exposure to antibiotics and ICU stays on the antibiotic resistance profile of COVID-19 hospitalized patients
For subjects with severe SARS-CoV-2 infection, define strategies that might decrease the colonization with resistant pathogens.
D4
To investigate the pathophysiology underlying undernutrition and cognitive development in young children living in sub-Saharan Africa and describing the impact of the presence (n+1) and absence (n-1) of specific members of the microbiota on stunting/ cognitive delay
Define bacterial species and bacterial functions associated with better or worse outcome and identify potential protective bacteria that might be used as next generation probiotics to improve patient care
D = diseases; M = methods; 2 «amplicon-based metagenomics» often use the V3-V4 region of the 16S rRNA-encoding gene and thus only allows «bacterial profiling»; 3 “shotgun metagenomics» and «direct metagenomics» are synonyms that both refer to «metagenomics sensu-stricto», i.e. providing sequences of all genes present in a given microbiota; 4 culturomics corresponds to high-throughput culture. 1
( h t t p s : / / n c c r- m i c r o b i o m e s . c h / r e search/work-package-1/), two translaWP1 Human microbiomes (translational microbiome research) tional research projects are focusing WP2 Microbiomes in animal systems respectively on Salmonella & ClostridWP3 Plant microbiomes ioides difficile. These pathogens have WP4 Environmental microbiomes been selected since they correspond to WP5 Synthetic and engineered microbiomes two very different clinical situations WP6 Data analysis and modelling and both exhibit a yet poorly underTable 2. The different work packages of the NCCR microbiomes, as outlined on the NCCR stood pathogenesis. However, we inwebsite (https://nccr-microbiomes.ch/research/overview/) tend to further expand this «N+1 and N-1 approach» (see Table 3) applied to bacterial agents implicated in diaron different samples obtained through metagenomics) will only be performed rheal diseases by also studying the imthe various clinical projects and on a subset of samples selected from pact of additional pathogens such as benchmarked to amplicon-based subjects of the main clinical cohorts. Campylobacter jejuni and Trophmetagenomics metagenomics, which eryma whipplei on the gut microbiota is already accredited and used in the Translational projects of the of infected and colonized individuals. diagnostic microbiology laboratory at human microbiota network of We also consider enlarging the curCHUV. Conversely, more comprehen- the NCCR rent research by (i) better defining sive analyses (metabolomics, shotgun As outlined on the NCCR website what is a normal gut microbiota, (ii) Worpackage Thematics
23
24
ED N EW UC SA T I O N
P I P E T T E – S W I S S L A B O R AT O R Y M E D I C I N E | WWW. S U L M . C H
Terms
Definitions
Microbiome
Literally, all genomes present in a given sample. Generally corresponds to all sequences obtained by direct sequencing of microbial DNA present in a sample, allowing not only a taxonomic profiling but also providing insight on virulence-related genes and genes encoding for various metabolic activities.
Microbiota
Diversity of microbes present in a given sample. Currently, the microbiota analysis is commonly done by sequencing the V3-V4 regions of the 16S rRNA encoding gene, directly providing information on the taxonomic profile of microbes, but not on their metabolic competencies or virulence-associated genes. Microbiota may also be performed using non-sequences based approaches such as microscopy (see dysbiosis below), flow cytometry, or culturomics.
N+1
Analytic approach where the effect of the presence of an additional microbe on the others microbes is investigated. Typically, the additional microbes may have a positive impact in an additive or synergistic way on the effect on a studied phenotype of the other microbes. Alternatively, this microbe may have a negative impact, which is than better studied by using the N-1 approach.
Healthy microbiota
Normal microbiota, without any pathogen and also without specific bacterial composition that might be detrimental. Please note that two commensals non-pathogenic bacteria could be detrimental and led to «dysbiosis» if upon their association they might, for example, complete a given metabolic pathway that could led to the production of a toxic compound. Antimicrobial compounds produced by some specific bacterial strains represents another example on how the presence of a single new strain may significantly impact the microbiota.
Dysbiosis
Situation where the microbial composition is detrimental to health. Definition of dysbiosis in directly related to the definition of healthy microbiota, since due to various causes the healthy microbiota may be modified and be dysbiotic. One of the best defined dysbiosis is the vagina microbiota, which is defined by the disappearance of the usual predominance of lactobacilli; lactobacilli are involved in the local acidification of the vaginal mucosa and are producers of antimicrobial product, preventing overgrowth of pathogens associated with vaginitis, vaginosis or other local dysbiotic condition.
Metagenomics
High throughput sequencing. This approach entered all microbiota research lab given the high quality of sequences obtained using approaches such as Illumina and the relative low cost of the instruments and the reagents.
Amplicon-based metagenomics
Relatively efficient and cheap approach to perform an analysis of the taxonomic composition of a given sample; based on a PCR amplification step, this approach is more sensitive than shotgun metagenomics; this approach generally use the V3-V4 region of the 16S rRNA-encoding gene and thus only allows «bacterial profiling» and does not provide any information on the presence of virus or on the presence of eucaryotes such as yeasts, filamentous fungi, protozoa or helminths in the sample.
Shotgun metagenomics
Analysis of all the genomes present in a given sample with a preliminary amplification step. Also called «direct metagenomics». The main limitations of this approach is the high cost and the low sensitivity (needs > 100’000 bacteria/ml)
Amplicon sequence variants
Sequences obtained by high throughput sequencing are compared and each unique sequence corresponds to an ampliconsequence variant (ASV). Several ASVs may correspond to a defined taxonomic group (species, genus,family, order or class). ASVs ae used to perform bacterial profiling; however, to do a precise taxonomic profiling, the use of additional genes or at least the full 16S rRNA sequences is preferred. Such precision may be important since V3-V4 for example does not allow to discriminate the different streptococci species and Streptococcus gallolyticus was strongly associated with colon cancer – thus, such precise taxonomic affiliation may be of importance. The same is true with Lactobacillus, which includes some species associated with weight gain and others with weight loss.
Culturomics
High-throughput culture. This approach is now developed in Lausanne thank to the availability of both automated high-throughput culture system (BD-Kiestra) and MALDI-TOF (Bruker).
Table 3. Glossary on some microbiota-related concepts (non-exhaustive list)
NR. 3 | JUNI 2022
studying more in depth the gut-brain axis and (iii) extending investigations towards non-infectious diseases, such as Crohn, ulcero-hemorrhagic rectocolitis, irritable bowel disease and gluten deficiency, as well as nutritionassociated diseases (undernutrition, obesity).
Conclusions. The NCCR allowed hiring of two new Professors at UNIL (Claire Bertelli, Pascale Vonaesch) and one MER (Grégory Resch) in the human microbiota work package. Thus, initially composed of two group leaders (Gilbert Greub & Benoit Guery), the human microbiota network (i.e WP1 of the NCCR) includes now five group leaders who collectively sign this short article. Today, the five P.Is, all from UNIL, interact tightly together during the weekly work-in-progress meeting and several projects specific meetings. Noteworthy, the WP1 also interacts closely with several group leaders of other work packages (WP 2 to 6) on fruitful collaborations, and both (i) proposes the new tools developed by WP1 to others, but also (ii) beneficiates from tools and know-how of the other NCCR researchers. Human microbiota research is still at its infancy and many clinical applications will likely emerge from current research performed within the NCCR with future benefit for patients’ care.
P I P E T T E – S W I S S L A B O R AT O R Y M E D I C I N E | W W W. S U L M . C H
E D U C ANTEI W ON S
NR. 3 | JUNI 2022
Neu: STARlet-AIOS - Atellica® Integrated vollautomatisiertes Automation modulares MDxSiemens Healthineers investiert in innoWeiterentwicklungen für Atellica System von Seegene vative Solution. Automatisieren Sie manuelle ®
Mit dem STARlet-AIOS bringt Seegene das erste vollautomatisierte MDx-System auf den Markt. Diese einzigartige All-in-One-Lösung für molekulare Tests mit ihrem modularen Design bietet einen vollständig automatisierten Arbeitsablauf und ist mit allen Seegene Assays kompatibel. Das STARlet-AIOS System besteht aus einem STARlet IVD Gerät zur Probenaufreinigung und PCR-Setup sowie zwei CFX96TM Real-Time PCR Systemen zur Messung und Auswertung der Proben. Mit diesem vollautomatisierten
Arbeitsschritte und entlasten Sie Ihr Personal bei repetitiven Arbeiten. Integrieren Sie die Deckelentfernung der Patientenröhrchen in den analytischen Arbeitsablauf von Atellica® Solution mithilfe des Atellica® Decapper Moduls. Automatisieren Sie die Qualitätskontrolle: Abarbeitung, Auswertung und lagern Sie bis zu 60 Kontrollen im dafür vorgesehen gekühlten Fach, integriert im Atellica® Sample Handler. Darüber hinaus verfügt der Atellica® Sample Handler über integrierte automatische, spezifische Probensortierung in Kombination mit Abruf-
Sophisticated, yet simple, built-in software seamlessly connects to your existing LIS to make it easy to run laboratory operations Built-in revolutionary sample management technology
System (Sample In- Result Out), welches einen Durchsatz von bis zu 234 Proben in 8 Stunden ermöglicht, wird die Laborroutine nochmal deutlich schneller und effizienter. Durch das modulare Design bietet der STARlet-AIOS die einzigartige Möglichkeit, dass bereits im Labor bestehende Seegene Geräte (STARlet IVD und CFX96) in das STARlet-AIOS System integriert werden können, was nebst einer grossen Kostenersparnis auch mehr Nachhaltigkeit bedeutet. Ein weiterer grosser Vorteil ist, dass das System äusserst kompakt ist im Vergleich zu anderen MDx-Vollautomaten mit ähnlichem Durchsatz. Weitere Informationen:
BÜHLMANN Laboratories AG Baselstrasse 55, 4124 Schönenbuch, Switzerland Tel. 061 487 12 12, Fax 061 487 12 34 info@buhlmannlabs.ch www.buhlmannlabs.ch
Flexibility to add decapping with little or no additional footprint Integrated sample sorting, retrieval, and archiving Integrated sample sorting, functions throughout all retrieval, and archiving functions phases of all testing throughout phases of testing
Sorting Feature NOT Enabled
Sorting Feature Enabled
und Archivierungsfunktionen und ermöglicht eine Verwaltung von Proben in allen Phasen (präanalytisch, analytisch und postanalytisch). Der bidirektionale magnetische Probentransport – Atellica Magline® Transport – mit intelligenter Zeitplanung und variablen Geschwindigkeiten gestattet eine vorhersehbare Priorisierung – aller über 40 verschiedenen Probentypen – der Durchlaufzeiten und Notfallproben. Mit der benutzerfreundlichen Software Atellica Asa™ – eine kachelbasierte Tablet-Anwendung – welche die Aufgaben des Bedieners organisiert, priorisiert und rationalisiert, bekommt Ihr Laborpersonal noch mehr Freiheiten und kann sich auf das Wesentliche konzentrieren.
Siemens Healthineers www.siemens-healthineers.com
25
26
ED N EW UC SA T I O N
P I P E T T E – S W I S S L A B O R AT O R Y M E D I C I N E | WWW. S U L M . C H
NR. 3 | JUNI 2022
En mémoire de Prof. Claude Bachmann
CLAUDE BACHMANN (1941 – 2022) Médecin chef de service du Laboratoire de chimie clinique du CHUV et Professeur ordinaire à la Faculté de biologie et médecine de l’Université de Lausanne 1988 – 2006; Membre honoraire de la Société Suisse de Chimie Clinique (SSCC) et de la Society for the Study of Inborn Errors of Metabolism (SSIEM).
Pédiatre de formation (titre FMH, 1973), Claude Bachmann a toujours démontré un vif intérêt tant pour la recherche scientifique que pour les développements analytiques, déjà lors de ses études de médecine (Université de Bâle), et lors de sa formation d’interne en pédiatrie (Kinderspital, Bâle; Dept of Pediatrics, UCSD, La Jolla, California [Prof WL Nyhan]).
ment des laboratoires au CHUV (qu’il dirigera de 1993 à 1997).
domaine dans lequel il développera, en collaboration avec JP Colombo, le dosage des enzymes et où il engagera une recherche soutenue par le FNRS. Son expertise dans ce domaine, reconnue au niveau international (SSIEM), aura contribué à une meilleure compréhension et au traitement de ces maladies rares.
Durant toutes ses années d’activité, avec enthousiasme et passion, sans jamais perdre de vue l’intérêt du patient, Claude Bachmann s’attachera à développer des méthodes analytiques en vue de soutenir la détection des EIM (acides aminés, acides organiques, acide orotique, acylcar- Son excellence en recherche pédiaC’est à l’occasion de la prise en charge nitines). Il se spécialisera dans le do- trique et celle de chimiste clinicien d’un patient atteint d’une erreur innée maine des EIM du cycle de l’urée, ont été honorées en suisse respectivedu métabolisme (hyperglycinémie non-cétosique) que Claude Bachmann a donné à sa carrière médicale une orientation qui le conduira sur le chemin de la pratique de la médecine de laboratoire en chimie clinique (diplôme de chimie clinique de la SSCC, 1977; spécialiste FAMH en chimie clinique, 1991). Son attachement au domaine des erreurs innées du métabolisme (EIM) donnera sa spécificité à l’ensemble de sa carrière de chimiste clinicien. Vice-directeur du laboratoire central de chimie clinique à l’Inselspital (Berne) il en dirige la section des analyses spéciales de 1975 à 1988 sous la direction du Prof. JP Colombo. En 1988 il prend la direction du service du laboratoire de chimie clinique du CHUV où il achèvera sa carrière en 2006, date de son départ à la retraite. En visionnaire, il y aura fortement œuvré à l’automatisation et à l’informatisation de la chimie clinique ainsi qu’à la mise en place d’un départe-
Au sein de la SSCC il assurera toutes les fonctions, participant à de nombreux groupes de travail (médicaments ; valeurs de références ; paramètres significatifs ; indication et validation d’analyses de chimie clinique pour le diagnostic médical). Il est élu à la présidence de la Commission Scientifique de la SSCC en 1978. Il est élu président de la SSCC en 1991. image source: www.annahartmann.net
P I P E T T E – S W I S S L A B O R AT O R Y M E D I C I N E | W W W. S U L M . C H
NR. 3 | JUNI 2022
E D U C ANTEI W ON S
27
Prof. Claude Bachmann (1941 – 2022) Bis zu seiner Pensionierung im Jahr 2006 war Claude Bachmann ein wichtiger Akteur der klinischen Chemie in der Schweiz. Sein Humanismus, seine umfassende Kultur und seine Fähigkeit, Verbindungen zu knüpfen standen immer im Dienst der Forschung und der Patienten. Er war vielen ein treuer Freund und vielen ein anspruchsvoller Mentor. Die Werte, die Claude Bachmann im Laufe seiner medizinischen und wissenschaftlichen Karriere verkörperte, bilden sein Vermächtnis. Dafür ist ihm die Schweizerische Gesellschaft für Klinische Chemie zutiefst dankbar.
ment par le Prix commémoratif Guido la recherche et de la formation. Acteur Fanconi (1989) et la médaille Richte- incontournable de la chimie clinique rich de l’USLM (2008). helvétique jusqu’à sa retraite en 2006, son leadership aura contribué à Attaché à la qualité de la formation l’émergence d’une culture en médedes professionnels de laboratoire il cine de laboratoire fondée sur le dias’engagera fortement pour la mise en logue entre le savoir scientifique et œuvre d’une formation post graduée médical, toujours à la recherche de la des universitaires de ce domaine. Il mise en évidence des aspects biochiprésidera, à l’Académie Suisse des miques et analytiques d’importance Sciences Médicales (ASSM), la Com- clinique. mission « formation post graduée pour chefs de laboratoire médical » Doté d’une très grande curiosité inteldont les travaux ont conduit à la mise lectuelle, et d’une capacité à penser auen œuvre de l’actuelle formation delà du problème à résoudre, son esprit FAMH des spécialistes en médecine de acéré et critique a souvent défié ses inlaboratoire. Il soutiendra activement terlocuteurs scientifiques, les incitant à le développement de la formation des formuler de nouvelles hypothèses. TABs en œuvrant activement dans le conseil d’école de l’actuelle ESSANTé. Il aura été l’ami fidèle de plusieurs, le Son humanisme, sa vaste culture et mentor exigeant de bien d’autres. Il son aptitude à créer des liens sauront aura su susciter le meilleur de cerajouter à la valorisation de ses rela- tains, soit en médecine, soit en retions engagées au service de la re- cherche soit encore dans la transmischerche et des patients. En témoignent sion du savoir. Ceux-ci exercent encore son vaste réseau qui le conduira à aujourd’hui, reconnaissants de l’héricontribuer à plus de 300 publications tage reçu et dont les valeurs incarnées ou chapitres de livres et à participer à par Claude Bachmann tout au long de plusieurs comités de rédaction (dont sa carrière médicale et scientifique Journal of Inherited Metabolic Di- perdurent encore à ce jour dans la sease ; Pediatric Research ; Clinica pratique d’une médecine de laboraChimica Acta ; Enzyme and Protein, ; toire d’excellence, en perpétuelle évoLabormedizin ; Annales de biologie lution. clinique ; European Journal of LaboPour la SSCC ratory Medicine ;…). Dr Olivier Boulat, Médecin chef de service, Service de chimie clinique, CHUV
Tout au long de sa carrière en chimie clinique, Claude Bachmann se sera Prof Olivier Braissant, Responsable de secteur de recherche, Service de chimie clinique, CHUV engagé, tant sur le plan de la médecine de laboratoire, que sur le plan de Dr André Deom, Membre Honoraire SSCC
Warum Wochen warten ...
Dermatomykose-Erreger sicher und schnell nachweisen Einzigartiger EUROIMMUN-PCR-Chip in der Pilzdiagnostik ... in weniger als 5 Stunden zum Laborbefund
Klinische Bedeutung Dermatomykosen sind Infektionen der Haut, Haare und Nägel, die in den meisten Fällen durch Dermatophyten, in selteneren Fällen durch Hefen und Schimmelpilze verursacht werden. Hautpilzinfektionen zählen mit 20 bis 25 % Betroffenen weltweit zu den häufigsten Infektionskrankheiten mit hoher Rezidivrate, Tendenz steigend.
EUROArray HPV Nachweis und vollständige Subtypisierung von 18 Hochrisiko-HPVTypen und 1 Niedrigrisiko-HPV-Typ. Bestimmung von persistierenden oder multiplen Hochrisiko-Infektionen DNA-basierter Test zum Nachweis der Onkogene E6 und E7
Weltweit umfangreichster Test (> 50 Subtypen) für die Haut-, Haar- und Nagelpilzdiagnostik
EUROArray STI 11 Zuverlässig auch bei asymptomatischen oder multiplen Infektionen
Spezifische Identifizierung von Mischinfektionen auch mit Hefen und Schimmelpilzen
Nachweis von 11 Krankheitserregern
Gesteigerter Behandlungserfolg dank frühzeitiger, erregerspezifischer Therapie Hohe Sensitivität selbst für schwer kultivierbare Dermatophyten, auch nach Therapiebeginn Probenmaterial Nagelspäne, Hautschuppen, Haarwurzeln, Kulturmaterial, Histologisches Material/Gewebeschnitte dermatophyten-pcr.de
Patient ID : Result from :
0 05.07.2021
Lot :
Test : Protocol :
I180217JG 29.06.2021 15:28
Scan date :
:50
Print date : Patient name
Page :
Partial result
Result
Cross contaminat
ion control
valid
Internal Control DNA positive contro
Dermatophyte
(universal) Trichophyton equin um
Nannizzia gypse a Nannizzia incurv ata Nannizzia persic olor
Microsporum canis Microsporum ferrug
ineum
Microsporum audou inii M. canis/audou inii
Candida parap silosis Candida guillie rmondii Candida albica ns
Fusarium solan i Fusarium oxysp orum Scopulariopsis brevicaulis Test result
Dermatophyte Yeast/Mould
Signature :
Ihr Partner für innovative und moderne medizinische Diagnostik
1 c3h6w0 e9i z0 A 0 0G S c h w e i z A GS c h w e i z AT Ge l + 4 1 4 S o iemd m M e d i z i n i s c hM e e d i z i n i s cm h ea i l@e u r M i zu i nni.scchh e u ina. g ch L a b o r d i a g n oLsatbi koar d i a g nwows twi k. ea u r Lo ai m b omr d nostika
Chip 1
valid DETECTED
Trichophyton tonsu rans Trichophyton interd igitale Trichophyton menta grophytes T. interdigitale/ment agrophytes Trichophyton quinc keanum Trichophyton schoe nleinii Trichophyton simii T. quinckeanum/sch oenleinii/simii Trichophyton benha miae(white/afr.) Trichophyton benha miae (yellow) T. bullosum/be nhamiae (afr.) T. concentricum/erin acei Trichophyton erinac ei
T. verrucosum /eriotrephon Trichophyton rubrum
Slide 1 Field A
DETECTED
icity control
Trichophyton violac eum Epidermophyton floccosum Nannizzia fulva
1 Automatic evalu ation with the EURO ArrayScan softw are
valid
l
Hybridisation specif
:
Dermatomycosi s Demo EUROArray Dermatomycosi s 2_0 22.07.2021 10:10 :27 0
not detected not detected not detected not detected not detected not detected not detected not detected not detected not detected not detected not detected not detected not detected not detected DETECTED not detected not detected not detected not detected
not detected not detected not detected not detected not detected not detected not detected not detected DETECTED not detected not detected not detected Result Trichophyton
rubrum Candida albica ns
Slide 1 Field A
Chip 2