








key: cord-102586-mx534xbx
authors: Javaid, W.; Ehni, J.; Gonzalez-Reiche, A. S.; Carreno, J. M.; Hirsch, E.; Tan, J.; Khan, Z.; Kriti, D.; Ly, T.; Kranitzky, B.; Barnett, B.; Cera, F.; Prespa, L.; Moss, M.; Albrecht, R. A.; Mustafa, A.; Herbison, I.; Hernandez, M. M.; Pak, T.; Alshammary, H.; Sebra, R.; Smith, M.; Krammer, F.; Gitman, M.; Sordillo, E. M.; Simon, V.; van Bakel, H.
title: Real Time Investigation of a large Nosocomial Influenza A Outbreak Informed by Genomic Epidemiology
date: 2020-05-15
journal: nan
DOI: 10.1101/2020.05.10.20096693
sha: 
doc_id: 102586
cord_uid: mx534xbx

Background: Nosocomial respiratory virus outbreaks represent serious public health challenges. Rapid and precise identification of cases and tracing of transmission chains is critical to end outbreaks and to inform prevention measures. Methods: We combined conventional surveillance with Influenza A virus (IAV) genome sequencing to identify and contain a large IAV outbreak in a metropolitan healthcare system. A total of 381 individuals, including 91 inpatients and 290 health care workers (HCWs), were included in the investigation. Results: During a 12-day period in early 2019, infection preventionists identified 89 HCWs and 18 inpatients as cases of influenza-like illness (ILI), using an amended definition, without the requirement for fever. Sequencing of IAV genomes from available nasopharyngeal (NP) specimens identified 66 individuals infected with a nearly identical strain of influenza A H1N1 (43 HCWs, 17 inpatients, and 6 with unspecified affiliation). All HCWs infected with the outbreak strain had received the seasonal influenza virus vaccination. Characterization of five representative outbreak viral isolates did not show antigenic drift. In conjunction with IAV genome sequencing, mining of electronic records pinpointed the origin of the outbreak as a single patient and a few interactions in the emergency department that occurred one day prior to the index ILI cluster. Conclusions: We used precision surveillance to identify and control a large nosocomial IAV outbreak, mapping the source of the outbreak to a single patient rather than HCWs as initially assumed based on conventional epidemiology. These findings have important ramifications for more effective prevention strategies to curb nosocomial respiratory virus outbreaks.

Nosocomial outbreaks of pathogens represent major challenges for health care providers and 63 institutions. It is critical for hospitals and health systems to not only quickly identify infected cases but 64 also determine the source of the outbreak in order to mitigate the threat to patients and health care 65 workers (HCWs). Nosocomial influenza virus outbreaks have been described worldwide (1-3); 66 children, the elderly, institutionalized and immuno-compromised patients are particularly vulnerable. In 67 some instances, nosocomial outbreaks have been caused by HCWs who work while ill (4).

Influenza virus is a single-stranded, negative sense, segmented RNA virus that causes an acute 

Here we report the integration of our conventional infection prevention measures with precision 82 surveillance, including sequencing the genome of IAV from clinical biospecimens and data mining of 83 electronic medical records (EMRs), to successfully identify and control a large nosocomial IAV 84 outbreak affecting both inpatients and HCWs.

Epidemiology of the nosocomial influenza outbreak. In early 2019, symptoms suggestive of 88 influenza like illness (ILI) were first observed in several HCWs as well as in inpatients receiving critical 89 care at Hospital A in NYC. At the direction of the Infection Prevention Department, the hospital's 90 incident command system was activated. An extensive outbreak investigation was started, which 91 included mandatory staff symptom checks and testing of all inpatients with any respiratory symptoms, 92 regardless of fever status. Enhanced cleaning of patient care areas and clinical staff workspaces was Over the course of the hospital-wide outbreak investigation, a total of 381 individuals (91 inpatients 95 and 290 HCWs) were screened by regular body temperature checks, symptom surveys and/or 96 molecular diagnostic testing for IAV, IBV and respiratory syncytial virus (RSV). A total of 18 inpatients 97 (19.8%) and 89 HCWs (29.7%) included in the epidemiological investigation tested positive for IAV 98 ( Figure 1A ).

Subtyping of IAV from the nasopharyngeal (NP) samples collected during the epidemiological 100 investigation (N=104), the routine influenza surveillance at Hospital A (N=150) and Hospital B 101 (N=231), revealed a stark increase of IAV/H1 at day 4, 5 and 6 of the investigation ( Figure 1B) . Of 102 note, all the samples from inpatients and HCWs included in the investigation that we successfully 103 subtyped harbored IAV/H1N1, suggesting a single transmission chain.

The 89 positive HCWs were distributed across 29 different work assignment categories (Figure 1C ), 105 predominantly front line care providers, including 24 resident physicians (residents, fellows, or interns), 106 16 registered nurses, 8 patient care assistants, and 6 attending physicians. Eighty-seven of these 89 107 HCWs (>90%) had been vaccinated with the quatrivalent seasonal influenza virus vaccine two to five 108 months (average: 108 days) prior to being tested positive for IAV ( Figure 1D) . Importantly, these 109 infected HCWs presented various, and mostly minor, clinical symptoms, and most individuals would 110 not have been classified as having "influenza-like illness" given the lack of fever ( Figure 1E ). Because 111 of this altered influenza disease manifestation in vaccinated HCWs, the case definition was amended 112 early in the context of our investigation.

Genomics of the nosocomial influenza virus outbreak. In order to determine whether there was 115 transmission of a single IAV strain or there were several independent introductions into the hospital 116 system, we performed next generation sequencing (NGS) of IAV from the NP specimens that were 117 banked following the initial diagnostic testing. As part of our Institution's Pathogen Surveillance

Program, we routinely sequence influenza virus from a subset of the patients seeking care at our 119 hospitals (termed "surveillance"). Thus, in addition to cases identified in the outbreak investigation, we 120 included surveillance samples obtained from the general patient population seeking care at our 121 hospitals as part of our Institution's Pathogen Surveillance Program in order to determine potential 122 community circulating strains.

Complete genomic sequences were obtained from 214 IAV isolates (Figure 2A) , including 126 from 124 Hospital A (investigation and surveillance) and 88 from Hospital B (surveillance only). Pairwise 125 comparison of these viral genomic sequences showed a large cluster of 66 viral isolates that differed 126 by no more than 3 single-nucleotide variants (SNVs), indicating that a single virus clone was 127 responsible for a large portion of the nosocomial outbreak ( Figure 2B) . Additionally, our analyses 128 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted May 15, 2020 . . https://doi.org/10.1101 /2020 indicated that other independent introductions of IAV H1N1 strains, with limited forward transmissions, 129 had caused smaller clusters of ILI at both Hospital A and Hospital B. We also noted two small 130 independent clusters due to transmission of IAV H3N2 viruses.

Correlating virus genomic sequences with the timing and the source of these isolates showed that all 132 of the virus isolates obtained on Day 0 and most of virus isolates on Day 1 of the infection prevention 133 investigation were distinct from the viral isolate that caused the large outbreak. Although two HCWs 134 were tested positive for IAV on Day 0, their viruses were different from the one that caused the 135 outbreak, and not associated with any nosocomial transmission. All HCWs infected with the outbreak 136 virus had received the seasonal influenza virus vaccine. The first isolate that clustered with the 137 outbreak virus strain was obtained from a patient identified on Day 1 of the investigation ( Figure 2C ).

Altogether, the genomic analyses of available clinical influenza isolates showed that cases identified 139 by the conventional epidemiological investigation encompassed patients and HCWs who together CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 15, 2020 . . https://doi.org/10.1101 /2020 sample matched the outbreak strain, and we used the PathoSPOT framework (https://pathospot.org) 162 to query various electronic hospital records in order to create a timeline ( Figure 4A ).

The data showed that 4 of the 9 initial nosocomial IAV cases were seen (patients p1, p2, and p3) or 164 worked (HCW1) in the emergency department (ED) on the same day (Day -1), during overlapping 165 time periods. The 3 patients were admitted from the ED to different wards and had no other shared 166 interactions with HCWs, indicating that the common exposure most likely occurred in the ED.

Similarly, because one of the patients who acquired nosocomial IAV did not have direct contact with 168 HCW1 and had already been transferred out prior to the time HCW1 was present in the ED, the 169 evidence indicates HCW1 was exposed during that work shift rather than being the primary case.

In contrast, patient p3, the putative primary case for this outbreak, was brought to the ED in the 171 morning of Day -1, several hours before p1 and p2, and interacted with HCW1. P3 was admitted to a 172 medical unit the same day ( Figure 4A , grey) with fever, shaking chills, dyspnea, and abdominal pain,

but developed systemic inflammatory response syndrome and was transferred to the ICU where the 174 patient was intubated, a procedure that can generate significant aerosols (7) Because blood cultures 175 of p3 grew Gram-negative bacteria, the diagnosis of IAV was delayed. However, the patient remained 176 febrile despite antibiotics prompting diagnostic testing for influenza virus on day 3.

The next three early cases (HCW2, p4 and p5) most likely acquired infection in the ICU from p3, 178 although p4 and p5 had further overlapping stays following transfer to the same medical/surgical 179 inpatient unit from the ICU. Our data suggests that cases p6 and p7, who were admitted through the 180 ED several days after the start of the outbreak, acquired IAV infection from ED HCWs who had been 181 exposed to p3 and became IAV-test positive in the days thereafter.

The interaction network based on available contact records ( Figure CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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Since we routinely sequence influenza virus isolates from patients receiving care at our health system 219 as part of our Pathogen Surveillance Program, we could compare the strains from the outbreak 220 investigation conducted at Hospital A to the strains found in the surveillance of Hospital A as well as 221 Hospital B (Figure 3) . These additional data allowed us to not only identify previously unrecognized 222 smaller transmission events (four inpatients and two HCWs at Hospital A and six patients at Hospital 223 B) but also ascertain that there was a large (in number) but limited (in time) outbreak of H1N1 in our 224 health system (Figures 2 and 3) . Importantly, this specific H1N1 virus outbreak strain did not spread 225 further in the community.

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 15, 2020. . https://doi.org/10. 1101 /2020 A limitation of our study is that we did not have access to biospecimens from 22 HCWs whose tests 227 were performed at laboratories outside our health system. Additionally, only partial viral genomes 228 could be retrieved from two of the available biospecimens linked to the epidemiological outbreak 229 investigation. However, we were able to obtain viral genomes for all the patients identified during the 230 first three days of the outbreak, providing a solid foundation for the reconstruction of the transmission 231 chain.

Our data suggest that the outbreak began in the ED most likely through introduction of the virus by a 233 single patient, who had received aggressive resuscitative care and was subsequently transferred to 234 the intensive care unit of Hospital A (Figure 4) . A possible solution to mitigate such risks to HCWs 235 and patients in the future is to enhance screening and isolation of patients coming into the ED with 236 any respiratory symptoms, even when an alternative diagnosis seems to be the predominant 

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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Among these 381 individuals, a total of 89 HCWs and 18 patients were found to be infected with IAV.

A high incidence of cases was found among HCWs, including interns, residents, fellows, and rotators, 267 as well as nursing and associated fields. Clusters were found in several areas inside the hospital 268 including intensive care units and several medical/surgical floors and an inpatient psychiatric unit.

Prophylaxis with oseltamivir was offered to exposed HCWs and inpatients. Treatment with oseltamivir 270 was provided to infected inpatients and HCWs, in addition to extended sick leave for HCWs who 271 remained afebrile but symptomatic. There was no mortality reported in association with the outbreak.

One patient required readmission secondary to ILI. We educated staff throughout the hospital 273 including the trainees on symptoms associate with influenza and encouraged all to report to their 274 supervisors if they had any symptoms. Mandatory symptom checks at beginning of each shift were 275 also implemented. Anyone symptomatic was encouraged to get tested. We were able to contain this 276 outbreak from detection to eradication within 10 days, with incredible collaboration between many The time frame from which surveillance and investigation samples were included covered a total of 27 284 days, starting from six days before the investigation to seven days after the 12 days long outbreak 285 investigation. Viral RNA was extracted from 280µL of NP-UTM using the QIAamp Viral RNA Minikit 286 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 15, 2020. were then used to construct a multi-genome alignment of isolates in each cluster using parsnp.

(9)Pairwise distances between genomes were then calculated as the number of single nucleotide 319 variants (SNVs) between genome alignments in each cluster for further analysis. To identify 320 transmission events we used the PathoSPOT heatmap view (Fig. 2) . We set a threshold of ≤3 SNVs 321 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 15, 2020 . . https://doi.org/10.1101 /2020 to identify potential transmissions. Next, the dendro-timeline view was used to perform phylogenetic 322 analysis of outbreak isolates, and to reconstruct the early outbreak timeline based on the full 323 admission/transfer/discharge (ADT) history for each patient obtained from the electronic medical 324 record. Finally, the ADT history was combined with patient-HCW interaction data to reconstruct a 325 network of all known contacts in Cytoscape.(10). 

In order to confirm phylogenetic grouping, we inferred a second ML analysis at the whole genome 340 level for all strains that belonged to the highest supported clade that contained the outbreak isolates. 

Hemagglutination inhibition (HI) assay. These assays were performed as described before (12, 13).

Briefly, serum samples from eight healthy study participants receiving the seasonal influenza virus 355 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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Palese for guidance and many thought provoking discussions. We are indebted to Dr. C. Cordon-

Cardo for continued support of the Pathogen Surveillance Program.

We would like to thank the staff, the Nursing leadership, including Christine Mahoney and Maria 

We gratefully acknowledge the authors, originating and submitting laboratories of sequences from 379 GISAID's EpiFlu (www.gisaid.org) that were used as background for our phylogenetic inferences. The 380 list of authors and submitting laboratories is shown in Table S1 . 

(S10OD018522 and S10OD026880) and institutional seed funds.

This manuscript was edited by Life Science Editors. 386 387 388 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 15, 2020 . . https://doi.org/10.1101 /2020 Authors contributions: CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 15, 2020. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted May 15, 2020 . . https://doi.org/10.1101 /2020 CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted May 15, 2020. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

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Nosocomial transmission of influenza: A retrospective cross-467 sectional study using next generation sequencing at a hospital in England

Hospital-acquired influenza infections detected by a surveillance system over six 472 seasons

Characterisation of 474 nosocomial and community-acquired influenza in a large university hospital during two 475 consecutive influenza seasons

Nosocomial outbreak of 478 influenza A H3N2 in an inpatient oncology unit related to health care workers presenting 479 to work while ill

Donnell 482 MR. 2019. Precision Surveillance for Viral Respiratory Pathogens: Virome Capture 483

Sequencing for the Detection and Genomic Characterization of Severe Acute Respiratory 484 Infection in Uganda

Use of Whole

Sequencing in the Investigation of a Nosocomial Influenza Virus Outbreak

Aerosol generating 490 procedures and risk of transmission of acute respiratory infections to healthcare workers: 491 a systematic review

Origins of the 2009 H1N1 influenza pandemic in swine in 495 Mexico. Elife 5

The Harvest suite for rapid core-497 genome alignment and visualization of thousands of intraspecific microbial genomes

Cytoscape: a software environment for integrated models of 501 biomolecular interaction networks

RAxML version 8: a tool for phylogenetic analysis and post-analysis 503 of large phylogenies

Antigenic sites in influenza 506 H1 hemagglutinin display species-specific immunodominance



