{'fraction_non_alphanumeric': 0.021659843691849645, 'fraction_numerical': 0.033569036099739484, 'mean_word_length': 5.008944543828265, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Identification of influenza urban transmission patterns by geographical, epidemiological and whole genome sequencing data: protocol for an observational study

Introduction Urban transmission patterns of influenza viruses are complex and poorly understood, and multiple factors may play a critical role in modifying transmission. Whole genome sequencing (WGS) allows the description of patient-to-patient transmissions at highest resolution. The aim of this study is to explore urban transmission patterns of influenza viruses in high detail by combining geographical, epidemiological and immunological data with WGS data. Methods and analysis The study is performed at the University Hospital Basel, University Children’s Hospital Basel and a network of paediatricians and family doctors in the Canton of Basel-City, Switzerland. The retrospective study part includes an analysis of PCR-confirmed influenza cases from 2013 to 2018. The prospective study parts include (1) a household survey regarding influenza-like illness (ILI) and vaccination against influenza during the 2015/2016 season; (2) an analysis of influenza viruses collected during the 2016/2017 season using WGS—viral genomic sequences are compared with determine genetic relatedness and transmissions; and (3) measurement of influenza-specific antibody titres against all vaccinated and circulated strains during the 2016/2017 season from healthy individuals, allowing to monitor herd immunity across urban quarters. Survey data and PCR-confirmed cases are linked to data from the Statistics Office of the Canton Basel-City and visualised using geo-information system mapping. WGS data will be analysed in the context of patient epidemiological data using phylodynamic analyses, and the obtained herd immunity for each quarter. Profound knowledge on the key geographical, epidemiological and immunological factors influencing urban influenza transmission will help to develop effective counter measurements. Ethics and dissemination The study is registered and approved by the regional ethics committee as an observational study (EKNZ project ID 2015–363 and 2016–01735). It is planned to present the results at conferences and publish the data in scientific journals. Trial registration number NCT03010007.


209
Participants and sample size. We used the probability-proportional-to-size (PPS) sampling as 210 a basis to plan the survey, as each quarter's base population varies as well as the systematic 211 equal probability of selection (EPS) sampling method within each quarter, so that each 212 household had an equal probability of being selected. Both sampling methods account for the 213 heterogeneity of urban quarters. In order to gain insights into community variation in attitudes,

214
beliefs and behaviors on influenza vaccination from a representative sample, the survey was 215 distributed in ten selected urban quarters (Figure 2) of Basel, which displayed a great variety 216 in terms of socio-demographic and -economic and building structures. Everyone not living in 217 one of the ten selected urban quarters was excluded from the survey.

219
We calculated the number of responses needed (n) to make the data representative with the We calculate the number of necessary questionnaires to be distributed per quarter by using 228 the amount of responses needed per quarter, assuming a response rate of 12% (Table 1).

229
The questionnaire is translated into the six most commonly spoken languages (German,

230
English, Italian, Serbo-Croatian, Albanian, and Turkish). Distribution is according to the three 231 most commonly spoken language per quarter based on data from the Statistics Office of the 232 Canton Basel-City ( Table 2)

242
In this project part, patients with ILI were recruited, and confirmed Influenza viruses are 243 sequenced using WGS (15). This allows us to determine the phylogeny and transmission

263
Additional samples. In addition to the nasopharyngeal swab for influenza, we also collect the 264 following materials: whole blood (EDTA) samples for analysis of host genetics and serum 265 samples for measurement of HIA titers as previously described (Kaufmann L et al. JOVE 2017) 266 against vaccine and circulating influenza strains.

268
Additional data. Patient data from influenza cases were merged with metadata from the

290
Measurement of antibody titers. Antibody titers are determined as previously described (16).

291
Briefly, antibody titers are determined using the hemagglutination inhibition (HI) assay against

423
Sensitivity analyses: The final regression models will be repeated after exclusion of outliers in 424 the dataset and with changing some of the underlying baseline assumptions (depends on the 425 variables to be included).

429
The combined analysis of the viral sequencing data, the metadata from the Statistics Office of 430 the Canton Basel-City, and the two questionnaires is done using phylodynamic methods. The 431 data collected in the questionnaires filled out by the patients is cleaned up and processed 432 analog to the data in B1.

433
In phylodynamics the transmission chain between hosts is reconstructed using the sequenced

446
The herd immunity estimates per urban quarter as percentage above a sero-protection 447 threshold is visualized using GIS mapping as previously described.

463
Prospective household survey. Given the distribution of the foreign-born or persons in the 464 urban quarter who speak other languages than German (Table 2), the Basel Cantonal

467
Nevertheless, a language bias is likely induced into the questionnaire as not every spoken

502
Ethics and dissemination

503
The study is approved by the regional ethics committee as observational study (EKNZ project

508
We plan to present the results of this research project at national and international scientific 509 meetings. We aim to publish our results in open-access journals so they are widely available 510 to interested international audiences. We aim to make our sequencing data available to the 511 research community so that distribution of viruses can be assessed on both a national and 512 international level.

Declarations
Ethics approval and consent to participate: The study is approved by the regional ethical review board (Project-ID 2015-363 and 2016-01735).

Consent for publication:
Written informed consent is provided by every patient or legal representative.

Questionnaire on Influenza in the City of Basel
Within the scope of the research project on influenza in the city of Basel, we are conducting a survey in selected quarters of Basel.
The participation in the survey takes approximately 15 minutes and is voluntary. Your anonymity will be ensured. We ask you to return this brochure with the completed questionnaire until 1 st May 2016 with the postage-paid envelope.
Participants can partake in a lottery (Apple Laptop worth CHF 2500.-and two "Pro Innerstadt"-coupons each worth CHF 500.-). See end of questionnaire. Many thanks for your participation.

121
The specific aims of the retrospective study parts are:

129
The specific aims of the prospective study parts are:

216
We calculated the number of responses needed (n) to make the data representative with the  We calculate the number of necessary questionnaires to be distributed per quarter by using 225 the amount of responses needed per quarter, assuming a response rate of 12% (Table 1).

226
The questionnaire is translated into the six most commonly spoken languages (German,

227
English, Italian, Serbo-Croatian, Albanian, and Turkish). Distribution is according to the three 228 most commonly spoken language per quarter based on data from the Statistics Office of the 229 Canton Basel-City ( Table 2)

287
Measurement of antibody titers. Antibody titers are determined as previously described (16).

372
We also include the location of a surveyed person within the urban quarter. This information 373 is obtained from surveyed persons who precisely locate their place of residence on a map or 374 by roughly indicating their location within a grid map of the city that we developed for this 375 purpose. Locations are georeferenced and added to the data set.

377
Data quality. The data cleaning and harmonization procedure yields a data set that is accurate, 378 complete and consistent, that allows us to go back to original source data (questionnaires), 379 accounts for incomplete or missing values, and that conforms with data handling and   Model checking: The Pearson and deviance goodness-of-fit tests will be performed to assess 417 the fit of the data to a Poisson distribution in the final regression models. Furthermore, the 418 distributions of the deviance residuals will be analysed.

420
Sensitivity analyses: The final regression models will be repeated after exclusion of outliers in 421 the dataset and with changing some of the underlying baseline assumptions (depends on the 422 variables to be included).

426
The combined analysis of the viral sequencing data, the metadata from the Statistics Office of 427 the Canton Basel-City, and the two questionnaires is done using phylodynamic methods. The 428 data collected in the questionnaires filled out by the patients is cleaned up and processed 429 analog to the data in B1.

430
In phylodynamics the transmission chain between hosts is reconstructed using the sequenced

442
B3. Determination of herd immunity in health individuals.

443
The herd immunity estimates per urban quarter as percentage above a sero-protection 444 threshold is visualized using GIS mapping as previously described. The distribution of herd 445 immunities in urban quarters will be correlated with incidence rates of influenza and Influenza viruses. Therefore, we would expect around 3500 to 4300 PCR-confirmed influenza 458 cases. To capture every single case of influenza will not be possible.

460
Prospective household survey. Given the distribution of the foreign-born or persons in the 461 urban quarter who speak other languages than German (Table 2), the Basel Cantonal households that should receive questionnaires in the major foreign languages (Table 3).

464
Nevertheless, a language bias is likely induced into the questionnaire as not every spoken 465 language could be captured with the questionnaire. Data entry and data handling errors can 466 almost be excluded. In population (census) surveys performed by the Federal Office of 467 Statistics of Switzerland, a 6% error margin is usually expected.

468
Finally, for logistic and cost reasons, we cannot distribute the household survey to all 19 official 469 urban quarters but focus our questionnaire on 10 selected quarters. Although this might induce 470 a certain selection bias, we feel that the included quarters reflect the diversity similar in a 471 representative number. Also, the questionnaires distributed and received per quarters allows 472 us to perform a statistical representative analysis.

474
Prospective study of influenza transmission. The prospective trial aims to include as many are more likely to respond. In addition, during patient recruitment not all influenza cases will 494 be captured by the study. In the prospective study part, we include patients with influenza-like 495 illness and use in a second step a PCR to confirm an influenza infection. However, as with the 496 retrospective study, not all patients with influenza infection can be captured within a city as 497 only symptomatic and patients presenting at the family doctor will be included.

498
We have recently finished the recruitment for the study and started with data analysis and 499 manuscript writing. We expect that results will be available by end of 2019.

501
Ethics and dissemination

502
The study is approved by the regional ethics committee as observational study (EKNZ project

507
We plan to present the results of this research project at national and international scientific 508 meetings. We aim to publish our results in open-access journals so they are widely available 509 to interested international audiences. We aim to make our sequencing data available to the

Declarations
Ethics approval and consent to participate: The study is approved by the regional ethical review board (Project-ID 2015-363 and 2016-01735).

Consent for publication:
Written informed consent is provided by every patient or legal representative.
Availability of data and material: Data analysis is ongoing, however, we will publish all WGS data of the patient in public repositories for WGS data. Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.

Questionnaire on Influenza in the City of Basel
Within the scope of the research project on influenza in the city of Basel, we are conducting a survey in selected quarters of Basel.
The participation in the survey takes approximately 15 minutes and is voluntary. Your anonymity will be ensured. We ask you to return this brochure with the completed questionnaire until 1 st May 2016 with the postage-paid envelope.

Map for question D5
Please look at the map of your quarter and mark the approximate location of your residence. Please state the coordinates (a combination of a letter and a number on the horizontal respectively vertical axis).

Example (right)
You live at the corner of Bläsiring / Müllheimerstrasse. Answer: N12 Thank you for your participation.