National H. influenzae control project

Based on the above information and the results highlighted in this thesis work, I would plan designing a national H. influenzae control project with the purpose to improve our knowledge on the epidemiology of invasive H. influenzae clinical isolates, to thwart the potential transmission across different healthcare institutions, and to determine targeted infection control interventions for identifying and preventing the dissemination of multidrug-resistant NTHi (and in some cases other specific H. influenzae) clones.

13.1.1 Objectives

This national H. influenzae control project has several unique features:

i. to identify populations at risk for invasive H. influenzae diseases;

ii. to improve and homogenize the antibiotic susceptibility testing methods used for H. influenzae;

iii. to assess the heterogeneous resistance of imipenem by using the population analysis method;

iv. to introduce the systematic detection of imipenem heteroresistance;

v. to use Next-Generation Sequencing (NGS) to describe in details the major mechanisms of antibiotic resistance and to assess a core genome multilocus sequence typing (cgMLST) scheme for H. influenzae strains isolated in laboratories/healthcare institutions across Switzerland;

vi. To develop a rapid antibiotic susceptibility testing method for invasive H. influenzae clinical strains by using WASPLab automation (automated inoculation, and automated incubation combined with timely-defined high-resolution digital imaging). A rapid antibiotic susceptibility testing method will allow the rapid prevention of therapeutic failures in the cases where the

13.1.2. Materials and methods

a. Clinical strains of interest

In the post-vaccine era, NTHi was identified as the principle cause of increased invasive H. influenzae infections in elderly persons and replaced Hib as the pathogen of primary concern. In addition, several studies in various post-vaccine populations have witnessed a steadily increase of antibiotic resistance in NTHi. Thus, NTHi clinical strains are a high-priority in this project. In order to facilitate progressive capacity building, I propose a stepwise approach and recommend to sequentially implement this national H. influenzae control project. For the first step, I plan to analyze only invasive H. influenzae strains by using the mandatory reporting setup by Federal Office of Public Health. For the second step, I will extend the analysis to NTHi strains isolated from upper and lower respiratory specimens (e.g. broncho-alveolar lavages and bronchial aspirations). For the last step, I will include the NTHi strains isolated from gynecological specimens.

b. Project concept

Rule-1: University hospitals and private routine clinical microbiology laboratories, which voluntarily participate in this project, will directly send H. influenzae isolates to the bacteriology laboratory at Geneva University Hospitals with basic and anonymized epidemiological data (date and place of collection, infection type, age and gender of the patient, and antibiotic susceptibility profile). Isolates should be shipped by using ESwab within no longer than 7 days after detection.

Rule-2: All the experiments will be performed at Geneva University Hospitals. Results will be communicated to the submitting institution and all NGS data will be available to all institutions participating in the project.

Rule-3: In case of a confirmed clone (i.e. match between isolates from the same institution), the institution concerned will be informed as well as the Federal Office of

Public Health. The same approach in case of clustering of strains from different institutions.

c. Antimicrobial susceptibility testing

The antibiotic susceptibility testing will be performed according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) methods. The following antibiotics will be tested: penicillin, ampicillin, amoxicillin/clavulanic acid, piperacillin/tazobactam, cefuroxime, ceftriaxone, imipenem, meropenem, ciprofloxacin, levofloxacin, and co-trimoxazole. Isolates that display an imipenem MICs

>2 mg/mL according to the E-test method will be further analyzed using the population analysis method.

d. Alterations in PBPs and acrR gene will be investigated by NGS

This project will benefit from all the relevant methods used in this thesis work. The genomic DNA will be sequenced using Illumina MiSeq (150 bp paired-end reads) technology (Illumina, San Diego CA, USA). The Illumina sequence quality will be evaluated using the Fastqc program (http://www.bioinformatics.babraham.ac.uk/

projects/fastqc/) and filtered using the Fastq-mcf programs (Eautils:

http://code.google.com/p/ea-utils/). The genome sequences will be assembled using the Edena v3 assembler. The genome annotation will be performed using the National Center for Biotechnology Information annotation pipeline.

e. Core genome multi-locus sequence typing target genes

The core genome multi-locus sequence typing (cgMLST) scheme will be defined by using Ridom SeqSphere+ software version 5 (Ridom GmbH, Germany). H. influenzae Rd KW20 will be chosen as the reference strain.

13.1.3. Epidemiological data

For all clinical isolates, some epidemiologic data such as sample type, age of the

will be used to identify the potential clustering within the same institution or across different institutions. In addition, they will be required to assess the distribution of invasive NTHi according to the age group of the patients.

13.1.4. Importance of this project

To our knowledge, no other research group is working on H. influenzae in Switzerland.

In addition, this project is innovative and can be the starting point for the creation of a reference center on H. influenzae in Switzerland as is already the case for Neisseria meningitidis or Streptococcus pneumoniae.

To date, an epidemiological surveillance for H. influenzae by genotypic analysis and accurate antibiotic susceptibility testing methods including the assessment of the heterogeneous resistance of imipenem has not been established in Switzerland and these analyses are not mandatory. Furthermore, we do have currently no idea about the frequency of treatment failure caused by imipenem heteroresistant strains.

I am convinced that this project fills an important gap in the surveillance and control of H. influenzae in Switzerland. The experience and knowledge acquired during my thesis work applied to a public health question should constitute a sound basis for this project.

13.1.5. Budget

a. Volume

We expect to analyse approximatively 150 strains within the first year, fulfilling the predefined features and permitting capacity building of this project. A rule to cap the number of strains to 150 will be elaborated if the number of collected strains would be significantly larger.

b. Costs

The expected project costs for the upcoming three years will be defined after submission of this project to Federal Office of Public Health and Geneva University Hospitals.

c. Timeline

The pilot phase of this project will last approximatively 6 months (from July to December 2020). A first evaluation of the pilot phase will be scheduled for March 2021.

On the basis of experience acquired during the pilot phase, the operating procedures of the project will be adjusted. At the end of the project an exhaustive report will be written to evaluate the real impact of this project and the potential creation of the reference centre of H. influnezae at Geneva University Hospitals.

13.2. Rapid antibiotic susceptibility testing method