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Target capture sequencing reveals a monoclonal outbreak of respiratory syncytial virus B infections among adult hematologic patients

Target capture sequencing reveals a monoclonal outbreak of respiratory syncytial virus B... Background: Respiratory syncytial virus (RSV ) causes community‑acquired respiratory tract infections during winter. However, outbreaks in hospitals also occur repeatedly. In particular, patients with hematologic malignancies are at an increased risk for a severe and potentially fatal course of RSV infection. Here we present the investigation of an RSV outbreak in a hematology ward for adults following the ORION statement. Methods: An epidemiologic and molecular outbreak analysis was performed. We developed and employed a mini‑ mal oligonucleotide probe set in target capture probe sequencing that allows cost‑ effective RSV ‑A or ‑B capturing to reconstruct RSV genomes from clinical samples. Results: Four adult patients were involved in the outbreak caused by RSV‑B in March 2019. The enforcement of the pre‑ existing infection control measures by effective training of hospital staff contributed to a successful contain‑ ment. PCR‑based RSV screening on the ward enabled early detection of new cases and rapid isolation measures. The molecular analysis demonstrated that the outbreak sequences were highly related and distinct to other RSV‑B strains circulating at the same time. Conclusions: A multimodal infection control concept is essential for the timely detection and control of RSV out‑ breaks in patients with hematological disease. Among other measures, preventive screening for respiratory viruses is recommended. Furthermore, the integration of conventional and molecular epidemiology, such as whole‑ genome sequencing and variant calling, significantly contributes to the understanding of transmission pathways. Based on this, appropriate conclusions can be drawn for targeted prevention measures that have prepared us for the COVID‑19 pandemic beyond the RSV approach described here. Keywords: Respiratory syncytial virus, Outbreak, Hematology, Infection, Infection control, Capture probe sequencing, Molecular epidemiology Background *Correspondence: baier.claas@mh‑hannover.de; nfischer@uke.de Respiratory syncytial virus (RSV) is an enveloped Institute for Medical Microbiology and Hospital Epidemiology, Hannover RNA virus. It is responsible for infections of the Medical School (MHH), Carl‑Neuberg‑Straße 1, 30625 Hannover, Germany upper (URTI) and lower respiratory tract (LRTI) in Institute for Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg‑Eppendorf, Martinistraße 52, 20246 Hamburg, a seasonal rhythm in countries with temperate cli- Germany mates [1–4]. The pathogen is primarily transmitted by Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Baier et al. Antimicrobial Resistance & Infection Control (2022) 11:88 Page 2 of 8 droplets from the respiratory tract and less often by Case definition contact. Aerosol transmission might play a role under An outbreak case was a patient from the affected ward certain circumstances as well [5]. The median incuba- with nosocomial acquisition of RSV in March 2019. tion period is reported to be 4 to 5  days [6]. RSV out- Nosocomial acquisition was assumed when RSV was breaks in hematology and oncology units (e.g., [7–10]) found on day 5 or later of the patient’s stay on the ward. and other hospital settings [11] have been repeatedly reported in the last years. For infection control of RSV Routine infection control measures for RSV in healthcare facilities, several measures (e.g., isola- The infection control concept at our institution for tion, personal protective equipment) are usually com- hematology wards regarding RSV is summarized in bined to prevent nosocomial spread [12]. Patients with Table  1. It was primarily shaped by the management of a hematologic malignancy are at high risk for a severe a previous RSV outbreak in a pediatric setting in our RSV LRTI [13–16]. Especially, leukopenia seems to be institution [10] and included a weekly PCR prevalence a relevant risk factor for increased mortality [17]. screening for asymptomatic patients [19]. This screening Herein, we report the epidemiology, control and also addressed influenza. molecular investigation of a hospital outbreak caused by RSV-B affecting 4 adults on a hematology ward. In Diagnostic virology addition, we discuss how the existing infection control Combined nose/throat swabs or pharyngeal washes were concept for RSV in hematology helped us in the coro- taken for routine viral diagnostics. If available, mate- navirus disease 2019 (COVID-19) pandemic. rial from the lower respiratory tract was suitable as well. Samples were processed at the Institute of Virology of the Hannover Medical School using the reverse transcrip- tion polymerase chain reaction (RT-PCR) based Panther Methods Fusion System (Hologic) [20]. This system allowed a Epidemiologic analysis semi-quantitative diagnostic using PCR cycle threshold The epidemiologic analysis followed the principles of (ct) values. the ORION statement [18]. Patient charts, virology results and patient movement data were reviewed for RNA extraction and cDNA synthesis data acquisition. In addition, a timeline was generated. RNA was extracted from diagnostic samples using the Qiagen QIAamp MinElute Virus spin kit (Qiagen Table 1 Basic infection control measures regarding RSV at the hematology ward Isolation of RSV positive patients: RSV positive patients were isolated (single room or grouping of patients, if more than one RSV patient was present). Isolation was kept until at least one respiratory specimen was tested negative by PCR or had a ct value > = 35 and the patient’s medical condition improved Prophylactic isolation of patients with respiratory symptoms: Newly admitted patients with respiratory symptoms and already hospitalized patients with onset of respiratory symptoms were separated from other patients and tested for a panel of respiratory viruses RSV specific contact and droplet precautions: Visitors and HCWs wore a surgical mask, a gown and gloves whenever entering the room of a RSV positive patient. RSV infected patients were asked to stay preferably in their individual room and were instructed in hand hygiene. Outside of the room RSV positive patients wore surgical masks (e.g., during urgently necessary examinations) Quarantine: Other patients sharing a room with a patient who tested positive for RSV were put in quarantine for 8 days Preemptive droplet precautions on the ward (universal masking): All HCWs and visitors wore surgical masks at any time when on the ward during winter season (usually December to March). The same applied to all patients when they left their patient room. Visitors were intensively instructed in droplet precautions and hand hygiene Prophylactic RSV Screening: Newly admitted patients were tested for RSV/Influenza (admission screening). In addition, once weekly a RSV/Influenza screening for all patients on the ward took place (prevalence screening) [19, 20] Pre-Season Audits/Training: Training for HCWs provided by the infection control staff prior to the winter season Protective isolation: Nursing in separate rooms for selected patients (e.g., for hematology patients with an expected prolonged and severe leucopenia) Baier  et al. Antimicrobial Resistance & Infection Control (2022) 11:88 Page 3 of 8 #57704) according to the instructions of the manufac- room temperature involving wash buffers I-III. Beads turer. In brief, 125 µl of sample input was used and puri- were resolved in 20  µl nuclease-free water and post- fied RNA was eluted in 50 µl water. capture PCR enrichment was performed by using KAPA cDNA synthesis was performed according to the HiFi HotStart ReadyMix (Roche) with 2  μl NEXTFLEX Superscript IV RT cDNA first-strand synthesis protocol ChIP Primer Mix for Illumina (PerkinElmer Applied (Thermo Fisher Scientific) involving RiboLock RNase Genomics). 20  μl captured DNA input diluted in 50  μl Inhibitor and pdN6 random priming. 2nd strand synthe- total volume was incubated 45  s at 98  °C, followed by sis was achieved with NEB Ultra RNA non-directional 18 cycles of 15 s at 98 °C/30 s at 60 °C/30 s at 72 °C and Second Strand Synthesis Module (New England Biolabs). final extension for 1  min at 72  °C. The library was puri - cDNA was purified with Agencourt AMPure XP beads fied using Agencourt AMPure XP beads (Beckman Coul - (Beckman Coulter Life Sciences). ter Life Sciences). Concentrations of all samples were measured with a Qubit 2.0 Fluorometer (Thermo Fisher Capture probe design Scientific) and fragment lengths distribution of the final The initial step of the probe design pipeline is to select libraries was analyzed with the DNA High Sensitivity the representative strains of RSV that will be included in Chip on an Agilent 2100 Bioanalyzer (Agilent Technolo- the analysis (see Additional files 1 and 2). A total of 1101 gies). Sequencing was performed on the Illumina MiSeq complete genomes were downloaded from GenBank [21] platform (2 × 150-nt) paired reads and 3-5 Mio reads per and aligned using the MAFFT tool [22]. We then entered sample. the generated alignments into the BaitsTools tool [23] for bait generation. To reduce redundancy, we clustered Bioinformatic analysis the bait candidates with 80% identity using usearch [24] As a result of capture probe sequencing of 4 samples from and kept only the centroid of each cluster. Finally, we the patients (P1 – P4) involved in the outbreak and of 3 filtered out the self-complementary baits and the baits control patients with RSV infections on different wards that had at least 90% identity with other baits in a blastn (C1—C3, reference cohort), total numbers of 2,394,484, [25] search. To ensure the quality of the final baits, we 2,700,477, 2,790,882, 2,384,508, 2,200,353, 2,191,289, checked all final sequences for melting temperature and and 1,858,890 2 × 151-nucleotide (nt) paired-end reads GC content. The outline of the capture probe design is were generated from the Illumina MiSeq sequencer by illustrated in Additional file  3; sequences of the capture using the designed probes, respectively. Sequence raw probes are provided in Additional file 4. data were subjected to quality control using FastQC [26]. Adapter sequences of the reads and bases with a score Library preparation, hybridization capture and sequencing of less than Q30 were trimmed and any reads shorter First, ds-cDNA was enzymatically fragmented using the than 36 nt removed using Trimmomatic v0.36. [27]. By SureSelect XT HS and XT Low Input Enzymatic Frag- removing the sequence reads originating from the host mentation Kit (Agilent). Library preparation was per- organism, a set of non-host high-quality paired-end reads formed following the NEB protocol for using NEBNext were prepared and fed into the SPAdes assembler (ver- DNA Ultra Library Prep Kit protocol E7370 (New Eng- sion 3.7.1), resulting in complete genomes for all patients, land Biolabs) with the following modifications. For adap - respectively. For each sample, we mapped the non-host tor ligation, adaptors were diluted (1:10 or 1:25), if input reads to its newly assembled genome using Novoalign cDNA was low, and USER enzyme volume was reduced V3.07.00 (http:// www. novoc raft. com) with the param- to 2 µl. PCR cycles for library amplification were between eters ’-r Random -l 20 -g 40 -x 20 -t 100 -k’, resulting in 12 and 15 cycles depending on the amount of input the alignment file. Then we used the tools samtools [28] cDNA. and MarkDuplicate available in Picard tools (http:// broad Target capture enrichment was performed using xGen insti tute. github. io/ picard/) [29] to sort the alignments Lockdown Probes (see capture probe design) and rea- and remove duplicate sequences from the alignment. The gents (Integrated DNA Technologies; IDT) according to derived alignment was fed into V-Phaser2 [30] for intra- the manufacturer’s instructions. Briefly, DNA capture individual single nucleotide variation (iSNV) identifi - was achieved with 500  ng barcoded library, hybridiza- cation. In the variant identification, we only considered tion of the probes to the library was performed for 4  h variants supported by at least five reads on each strand, at 65  °C. Subsequently, probe/cDNA mix was bound to and the ratio of the number of reads on the two strands washed Streptavidin beads (M-270 Streptavidin beads is smaller than 10. Furthermore, the reads that mapped Thermo Scientific) for 45 min at 65 °C. Stringent Strepta - twice or more on the sequence were discarded. In the vidin beads washing steps (stringent washing buffer) end, the iSNVs whose allele frequency is smaller than were performed at 65  °C, followed by washing steps at 0.5% were filtered out from the final results. Baier et al. Antimicrobial Resistance & Infection Control (2022) 11:88 Page 4 of 8 Results patients 1, 2 or 3 prior to the onset of RSV infection. All Setting patients were mobile and able to leave their rooms prior The cluster took place in March 2019. The affected ward, to RSV positive testing. which managed adult chemotherapy patients for hema- tologic malignancies and autologous hematopoietic stem Clinical course cell transplantations, included 28 beds (four one-bed- All involved patients were asymptomatic at the time of rooms, six two-bedrooms, and three four-bedrooms). initial virus detection. Patient 1 quickly developed an The one-bed rooms were equipped with an anteroom URTI with dry coughing as the main symptom. Patient 2 and all rooms had en-suite bathrooms and high efficiency developed a LRTI with oxygen need (nasal cannula) and particulate air filtration. The ward was operated by quali - dyspnea. This patient had a pre-existing chronic obstruc - fied healthcare workers (HCWs) and housekeeping staff. tive pulmonary disease and was an active smoker. Patient 3 and 4 both developed only mild RSV-associated res- Epidemiology piratory tract infection symptoms with nasal congestion A total of 4 patients were tested RSV positive in respira- and mild coughing. No RSV-associated mortality was tory specimens and had been previously tested RSV neg- observed. Patients 2, 3, and 4 received antibiotics as a ative on admission. Patient characteristics and the time preventive measure against potential bacterial co-infec- course of the outbreak are shown in Table  2 and Fig.  1. tions/superinfections. Patients 2 and 3 showed prolonged Patient 1 was identified by the prevalence screening for viral shedding, up to 29  days in patient 3, as demon- RSV at the beginning of the second week of the hospi- strated by RT-qPCR positive respiratory samples. tal stay. Patient 2 was a roommate to patient 1 for several days prior to the positive test of patient 1 and was there- Control measures fore tested immediately after the positive test result of Outbreak management was initiated immediately after patient 1 became available. Patients 3 and 4 were detected the second positive patient was identified. The infec - in the prevalence screening for RSV. Patient 3 shared the tion control specialists enforced the pre-existing control room for 1 to 2 h with patient 2 one day before patient 2 measures (Table  1) by several ad hoc training sessions was tested positive. Patient 4 did not share a room with on the ward. HCWs, service personnel and visitors were Table 2 Patient’s characteristics Patient Nosocomial Underlying RSV infection White blood RSV Treatment Antibiotic Oxygen need RSV-related onset disease cells (per treatment outcome microliter) 1 Yes Recurrent AML URTI 2200 Immunoglobulins No No Recovered 2 Yes Multiple myeloma LRTI 0 Immunoglobulins Yes Yes Recovered 3 Yes Secondary URTI 1800 None Yes No Recovered hemophagocytosis 4 Yes Multiple myeloma URTI 0 Immunoglobulins Yes No Recovered At time (± 2 days) of virus detection Fig. 1 Timeline. Grey bars represent the patient’s stay on the ward. ‘X’ indicates a positive RSV testing. The first positive testing is indicated with a bold ‘X. ‘O’ indicates a negative testing. Ct = cycle threshold Baier  et al. Antimicrobial Resistance & Infection Control (2022) 11:88 Page 5 of 8 re-instructed to use personal protective equipment (pri- outbreak patients. Therefore, direct transmission due marily surgical masks) and to perform hygienic hand to co-hospitalization in the same patient room can be disinfection. Moreover, we monitored hand hygiene excluded. However, it is conceivable that there was a con- adherence by ad hoc direct observation (compliance rate tact of patient 4 on the hallway or in the ward lounge with of about 70%) and gave feedback talks. patient 3 before spatial isolation of patient 3. A random All RSV-positive tested patients were immediately and independent occurrence of different RSV strains was isolated. implausible to us because of the clear nosocomial occur- rence and the overlapping time periods. This was further Molecular analysis confirmed by virus sequencing, which confirmed infec - To confirm our hypothesis that all 4 patients were tion of the patients by the same viral strain (see below). infected with the identical RSV strain and that these To our surprise, the cluster occurred although sev- transmissions occurred on the ward, we performed RSV eral control measures recommended for hematology target capture sequencing using enrichment techniques wards were already in place [33, 34]. These control meas - in these 4 patients. In parallel, we examined 3 patients ures addressed potential sources (patients, healthcare with RSV identified at the same time on different wards workers, visitors) and transmission pathways (droplets, to test the hypothesis that this was not a common RSV contact) of RSV in the hospital setting, as previously genotype that was repeatedly introduced to the ward. described in other outbreaks [8, 10, 31]. These 3 control patients allowed us to assess the variabil - A core measure containing the spread by droplets is ity of RSV circulating in the same geographic area during universal masking (surgical masks or FFP2/KN95). Note- this time period. We were able to determine the whole worthy, this measure has been widely adopted in hos- genome sequence of 15,185 nucleotides from all 7 speci- pitals in the current COVID-19 pandemic and has also mens included. Detailed information on the sequencing shown a reduction of nosocomial RSV and influenza as of each genome and the coverage can be found in Addi- a side effect [35]. Starting March 2020, we used strict tional files 5 and 6. Phylogenetic classification identified universal masking (staff and patients) in the COVID-19 both patient and control genomes as RSV-B (Fig. 2). The pandemic with the hematologic ward affected by the out - RSV sequences of the 4 outbreak patients were identical break described here being a role model for other wards. to each other and had 83–87 single nucleotide polymor- Nursing in separate rooms of selected hematology phisms to the RSV-B controls (see Additional file 7). patients (e.g., patients with an expected prolonged and severe leucopenia) is another important measure and can Discussion contribute to outbreak prevention and containment. We investigated the epidemiologic and molecular char- In our case, the outbreak control management focused acteristics of a RSV-B cluster affecting adult hematologic explicitly on enforcing the already existing measures, patients. thereby successfully containing the outbreak. The estab - Transmission between patients, in particular patients lished PCR-based screening program (including influ - sharing a room, is mainly conducted by droplets in enza) [19] was helpful in rapid detection of the index RSV outbreaks [10, 31, 32]. Therefore, we assumed a patient and patients 3 and 4, both being asymptomatic direct transmission from patient 1 to patient 2 due to at the time of the PCR screening. The infection control the shared room occupancy. The initial RSV infection concept of prevalence and admission screening has also source of patient 1, however, remained unclear. Patient 1 been widely adopted during the COVID-19 pandemic received visits and had contact to the ward’s staff for sev - in healthcare facilities. Moreover, it might be useful to eral days prior to acquisition. However, visitors and staff extend a screening panel to other respiratory viruses were urged to wear surgical masks during patient con- such as human metapneumovirus, which can also cause tact. Because the time interval between the positive test (severe) morbidity and mortality in immunocompro- results of patients 1 and 2 was relatively short, a shared mised hematology patients. unknown source of infection would also be conceivable. Integrating molecular sequence analysis with temporal Interestingly, patients 2 and 3 shared for several hours and spatial information from classical epidemiology sig- one patient room before patient 2 was placed in quaran- nificantly increases the understanding of outbreak events. tine in a single room following the positive test result of Building on this, we tested and confirmed our epidemio - patient 1. Patient 3 tested positive 5 days later at a prev- logical hypothesis regarding the chain of transmission by alence screening. This temporal sequence agrees well target capture sequencing. The RSV-B isolates from the with the RSV incubation period. Thus, patient-to-patient 4 patients were identical but significantly distinct from contact could have also been the transmission route for other RSV-B isolates found during the same time period patient 3. Patient 4 did not share a room with any other in our institution. Given the molecular and epidemiologic Baier et al. Antimicrobial Resistance & Infection Control (2022) 11:88 Page 6 of 8 Fig. 2 Phylogenetic analysis based on whole genome sequences of RSV isolates, subgroup B. The evolutionary history was inferred using the Maximum Likelihood method based on the General Time Reversible model. The tree with the highest log‑likelihood is shown. The initial tree for the heuristic search was randomly generated. A discrete Gamma distribution was used to model evolutionary rate differences among sites with four categories. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. All positions containing gaps and missing data were eliminated analysis, we assume that patient-to-patient transmission did not focus on evaluating the economic impact of the among patients 1 to 3 is likely and that patient 4 is part outbreak here (for instance due to reduced bed capacity of the outbreak as well. The transmission route in the lat - because of isolation measures). Moreover, the prophylac- ter patient, however, remains unclear. Initial introduction tic PCR-based screening we used is a resource-intensive of the RSV outbreak strain by a point source (e.g., an oli- measure, and we are aware that this might not be avail- gosymptomatic infected relative or healthcare worker) able in other settings. is possible. This outbreak investigation illustrates that a molecular characterization is highly valuable to elucidate Conclusions the potential transmission pathways in RSV outbreaks. Although the importance of prevention and rapid con- We followed the ORION statement for outbreak tainment of RSV outbreaks in hematology is well known reporting [18] in many points in this report. However, we and mostly comprehensive infection control measures Baier  et al. Antimicrobial Resistance & Infection Control (2022) 11:88 Page 7 of 8 Declarations are in place, transmissions may occur. We therefore underline that existing measures must be enforced Ethics approval and consent to participate by intensive training sessions for the wards’ staff and We obtained ethical approval for this study from the ethics committee of the Hannover Medical School (Number 8456_BO_K_2019). Being a retrospective the patients’ visitors. Furthermore, prophylactic RSV study, the need of informed consent was waived by the ethics committee and screening is crucial for outbreak containment and even the data protection commissioner of Hannover Medical School. prevention. Integration of molecular analyses, such as Consent for publication high-resolution whole-genome sequencing, with classi- Not applicable (No individual details such as images or videos are included). cal epidemiological information on temporal and spa- tial events, can be instrumental in resolving outbreaks. Competing interests The authors declare that they have no competing interests. The infection control management described here is— beyond RSV—essential in the COVID-19 pandemic. Author details Institute for Medical Microbiology and Hospital Epidemiology, Hannover Medical School (MHH), Carl‑Neuberg‑Straße 1, 30625 Hannover, Germany. Abbreviations Institute for Medical Microbiology, Virology and Hygiene, University Medical RSV: Respiratory syncytial virus; URTI: Upper respiratory tract infection; LRTI: Center Hamburg‑Eppendorf, Martinistraße 52, 20246 Hamburg, Germany. Lower respiratory tract infection; COVID‑19: Coronavirus disease 2019; RT ‑PCR: Leibniz Institute for Experimental Virology, Martinistraße 52, 20251 Hamburg, Reverse transcription polymerase chain reaction; HCWs: Health care workers. Germany. Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School (MHH), Carl‑Neuberg‑Straße 1, 30625 Hannover, Germany. Institute of Virology, Hannover Medical School Supplementary Information (MHH), Carl‑Neuberg‑Str. 1, 30625 Hannover, Germany. Institute for Experi‑ The online version contains supplementary material available at https:// doi. mental Virology; Twincore‑ Centre for Experimental and Clinical Infection org/ 10. 1186/ s13756‑ 022‑ 01120‑z. Research; a joint venture of Hannover Medical School (MHH) and Helmholtz Centre for Infection Research (HZI), Feodor‑Lynen‑Straße 7, 30625 Hannover, Germany. Cluster of Excellence RESIST (EXC 2155), Hannover Medical School, Additional file 1. GenBank accession numbers of RSV ‑A genomes applied Carl‑Neuberg‑Straße 1, 30625 Hannover, Germany. German Center for Infec‑ in probe design tion Research (DZIF), Partner Site Hannover‑Braunschweig, 30625 Hannover, Additional file 2. GenBank accession numbers of RSV ‑B genomes applied Germany. in probe design Additional file 3. Workflow of probe design pipeline Received: 9 January 2022 Accepted: 24 May 2022 Additional file 4. Fasta files of all RSV capture probes Additional file 5. Summary of short read sequencing results Additional file 6. Coverage profiles of the RSV sequences obtained by References target capture probe sequencing 1. Borchers AT, Chang C, Gershwin ME, Gershwin LJ. Respiratory syncytial Additional file 7. Overview of SNPs of all RSV sequences obtained virus—a comprehensive review. Clin Rev Allergy Immunol. 2013;45:331– 79. https:// doi. org/ 10. 1007/ s12016‑ 013‑ 8368‑9. 2. Rodriguez R, Ramilo O. Respiratory syncytial virus: how, why and what to Acknowledgements do. J Infect. 2014;68(Suppl 1):S115–8. https:// doi. org/ 10. 1016/j. jinf. 2013. Not applicable. 09. 021. 3. Griffiths C, Drews SJ, Marchant DJ. Respiratory syncytial virus: Infection, Author contributions detection, and new options for prevention and treatment. Clin Microbiol All authors contributed to the manuscript according to the ICMJE (Interna‑ Rev. 2017;30:277–319. https:// doi. org/ 10. 1128/ CMR. 00010‑ 16. tional Committee of Medial Journal Editors) recommendations: All authors 4. Nam HH, Ison MG. Respiratory syncytial virus infection in adults. BMJ. were involved in data acquisition, analysis and interpretation. NF, JH, KR, DI, SH 2019;366: l5021. https:// doi. org/ 10. 1136/ bmj. l5021. and TP were involved in the molecular analysis. CB, F‑ CB, EE, CK and FT were 5. Kulkarni H, Smith CM, Lee DDH, Hirst RA, Easton AJ, O’Callaghan C. involved in the outbreak management and epidemiologic workup. AH carried Evidence of respiratory syncytial virus spread by aerosol. Time to revisit out routine RSV diagnostic. CB and NF wrote the manuscript. All authors have infection control strategies? Am J Respir Crit Care Med. 2016;194:308–16. read and approved the final manuscript. https:// doi. org/ 10. 1164/ rccm. 201509‑ 1833OC. 6. Lessler J, Reich NG, Brookmeyer R, Perl TM, Nelson KE, Cummings DA. Funding Incubation periods of acute respiratory viral infections: a systematic Open Access funding enabled and organized by Projekt DEAL. TP is funded by review. Lancet Infect Dis. 2009;9:291–300. https:// doi. org/ 10. 1016/ S1473‑ Germany’s Excellence Strategy—EXC 2155 “RESIST”—Project ID 390874280. 3099(09) 70069‑6. 7. Kelly SG, Metzger K, Bolon MK, Silkaitis C, Mielnicki M, Cullen J, et al. Availability of data and materials Respiratory syncytial virus outbreak on an adult stem cell transplant unit. The datasets generated during and/or analyzed during the current study are Am J Infect Control. 2016. https:// doi. org/ 10. 1016/j. ajic. 2016. 03. 075. available from the corresponding authors on reasonable request. Patient data 8. Inkster T, Ferguson K, Edwardson A, Gunson R, Soutar R. Consecutive used in this study is confidential according to the German data privacy act, yearly outbreaks of respiratory syncytial virus in a haemato‑ oncology the ethics committee and the data protection commissioner of the Hannover ward and efficacy of infection control measures. J Hosp Infect. Medical School. To protect patient confidentiality and participant’s privacy, 2017;96:353–9. https:// doi. org/ 10. 1016/j. jhin. 2017. 05. 002. data used for this study can be obtained in anonymous form only according 9. Nabeya D, Kinjo T, Parrott GL, Uehara A, Motooka D, Nakamura S, et al. to the data privacy act. The clinical and phylogenetic investigation for a nosocomial outbreak of respiratory syncytial virus infection in an adult hemato‑ oncology unit. J Med Virol. 2017;89:1364–72. https:// doi. org/ 10. 1002/ jmv. 24800. 10. Baier C, Haid S, Beilken A, Behnert A, Wetzke M, Brown RJP, et al. Molecular characteristics and successful management of a respiratory syncytial Baier et al. Antimicrobial Resistance & Infection Control (2022) 11:88 Page 8 of 8 virus outbreak among pediatric patients with hemato‑ oncological dis‑ 30. Yang X, Charlebois P, Macalalad A, Henn MR, Zody MC. V‑Phaser 2: variant ease. Antimicrob Resist Infect Control. 2018;7:21. https:// doi. org/ 10. 1186/ inference for viral populations. BMC Genomics. 2013;14:674. https:// doi. s13756‑ 018‑ 0316‑2.org/ 10. 1186/ 1471‑ 2164‑ 14‑ 674. 11. Ryu S, Kim BI, Chun BC. An outbreak of respiratory tract infection due to 31. Lehners N, Schnitzler P, Geis S, Puthenparambil J, Benz MA, Alber B, et al. Respiratory Syncytial Virus‑B in a postpartum center. J Infect Chemother. Risk factors and containment of respiratory syncytial virus outbreak in a 2018;24:689–94. https:// doi. org/ 10. 1016/j. jiac. 2018. 06. 010. hematology and transplant unit. Bone Marrow Transplant. 2013;48:1548– 12. French CE, McKenzie BC, Coope C, Rajanaidu S, Paranthaman K, Pebody R, 53. https:// doi. org/ 10. 1038/ bmt. 2013. 94. et al. Risk of nosocomial respiratory syncytial virus infection and effective ‑ 32. Jensen TO, Stelzer‑Braid S, Willenborg C, Cheung C, Andresen D, Raw‑ ness of control measures to prevent transmission events: a systematic linson W, et al. Outbreak of respiratory syncytial virus (RSV ) infection review. Influenza Other Respi Viruses. 2016;10:268–90. https:// doi. org/ 10. in immunocompromised adults on a hematology ward. J Med Virol. 1111/ irv. 12379. 2016;88:1827–31. https:// doi. org/ 10. 1002/ jmv. 24521. 13. Hijano DR, Maron G, Hayden RT. Respiratory viral infections in patients 33. Hirsch HH, Martino R, Ward KN, Boeckh M, Einsele H, Ljungman P. Fourth with cancer or undergoing hematopoietic cell transplant. Front Micro‑ European conference on infections in leukaemia (ECIL‑4): guidelines for biol. 2018;9:3097. https:// doi. org/ 10. 3389/ fmicb. 2018. 03097. diagnosis and treatment of human respiratory syncytial virus, parainflu‑ 14. Khawaja F, Chemaly RF. Respiratory syncytial virus in hematopoietic cell enza virus, metapneumovirus, rhinovirus, and coronavirus. Clin Infect Dis. transplant recipients and patients with hematologic malignancies. Hae‑ 2013;56:258–66. https:// doi. org/ 10. 1093/ cid/ cis844. matologica. 2019;104:1322–31. https:// doi. org/ 10. 3324/ haema tol. 2018. 34. Tomblyn M, Chiller T, Einsele H, Gress R, Sepkowitz K, Storek J, et al. Guide‑ 215152. lines for preventing infectious complications among hematopoietic 15. Vakil E, Sheshadri A, Faiz SA, Shah DP, Zhu Y, Li L, et al. Risk factors for cell transplantation recipients: a global perspective. Biol Blood Marrow mortality after respiratory syncytial virus lower respiratory tract infection Transplant. 2009;15:1143–238. https:// doi. org/ 10. 1016/j. bbmt. 2009. 06. in adults with hematologic malignancies. Transpl Infect Dis. 2018;20: 019. e12994. https:// doi. org/ 10. 1111/ tid. 12994. 35. Wong S‑ C, Lam GK‑M, AuYeung CH‑ Y, Chan VW‑M, Wong NL ‑D, So SY ‑ C, 16. Chemaly RF, Shah DP, Boeckh MJ. Management of respiratory viral et al. Absence of nosocomial influenza and respiratory syncytial virus infections in hematopoietic cell transplant recipients and patients with infection in the coronavirus disease 2019 (COVID‑19) era: implica‑ hematologic malignancies. Clin Infect Dis. 2014;59(Suppl 5):S344–51. tion of universal masking in hospitals. Infect Control Hosp Epidemiol. https:// doi. org/ 10. 1093/ cid/ ciu623. 2021;42:218–21. https:// doi. org/ 10. 1017/ ice. 2020. 425. 17. Azzi JM, Kyvernitakis A, Shah DP, El Haddad L, Mahajan SN, Ghantoji SS, et al. Leukopenia and lack of ribavirin predict poor outcomes in patients Publisher’s Note with haematological malignancies and respiratory syncytial virus infec‑ Springer Nature remains neutral with regard to jurisdictional claims in pub‑ tion. J Antimicrob Chemother. 2018;73:3162–9. https:// doi. org/ 10. 1093/ lished maps and institutional affiliations. jac/ dky311. 18. Stone SP, Cooper BS, Kibbler CC, Cookson BD, Roberts JA, Medley GF, et al. The ORION statement: guidelines for transparent reporting of outbreak reports and intervention studies of nosocomial infection. Lancet Infect Dis. 2007;7:282–8. https:// doi. org/ 10. 1016/ S1473‑ 3099(07) 70082‑8. 19. Baier C, Linderkamp C, Beilken A, Thol F, Heuser M, Ebadi E, et al. Influenza and respiratory syncytial virus screening for the detection of asympto‑ matically infected patients in hematology and oncology. GMS Hyg Infect Control. 2018;13:Doc08. https:// doi. org/ 10. 3205/ dgkh0 00314. 20. Ganzenmueller T, Kaiser R, Baier C, Wehrhane M, Hilfrich B, Witthuhn J, et al. Comparison of the performance of the Panther Fusion respiratory virus panel to R‑ Gene and laboratory developed tests for diagnostic and hygiene screening specimens from the upper and lower respiratory tract. J Med Microbiol. 2020;69:427–35. https:// doi. org/ 10. 1099/ jmm.0. 001133. 21. Benson DA, Cavanaugh M, Clark K, Karsch‑Mizrachi I, Ostell J, Pruitt KD, et al. GenBank. Nucleic Acids Res. 2018;46:D41–7. https:// doi. org/ 10. 1093/ nar/ gkx10 94. 22. Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013;30:772–80. https:// doi. org/ 10. 1093/ molbev/ mst010. 23. Campana MG. BaitsTools: software for hybridization capture bait design. Mol Ecol Resour. 2018;18:356–61. https:// doi. org/ 10. 1111/ 1755‑ 0998. 24. Edgar RC. Search and clustering orders of magnitude faster than BLAST. Bioinformatics. 2010;26:2460–1. https:// doi. org/ 10. 1093/ bioin forma tics/ btq461. 25. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, et al. Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : BLAST+: architecture and applications. BMC Bioinformatics. 2009;10:421. https:// doi. org/ 10. 1186/ 1471‑ 2105‑ 10‑ 421. fast, convenient online submission 26. Andrews S. FastQC: a quality control tool for high throughput sequence thorough peer review by experienced researchers in your field data. http:// www. bioin forma tics. babra ham. ac. uk/ proje cts/ fastqc/. Accessed 19 Dec 2021. rapid publication on acceptance 27. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illu‑ support for research data, including large and complex data types mina sequence data. Bioinformatics. 2014;30:2114–20. https:// doi. org/ 10. • gold Open Access which fosters wider collaboration and increased citations 1093/ bioin forma tics/ btu170. 28. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The maximum visibility for your research: over 100M website views per year Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009;25:2078–9. https:// doi. org/ 10. 1093/ bioin forma tics/ btp352. At BMC, research is always in progress. 29. BROAD INSTITUTE. Picard Toolkit. GitHub repository. http:// broad insti tute. Learn more biomedcentral.com/submissions github. io/ picard. 2019. Accessed 19 Dec 2021. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Antimicrobial Resistance & Infection Control Springer Journals

Target capture sequencing reveals a monoclonal outbreak of respiratory syncytial virus B infections among adult hematologic patients

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Abstract

Background: Respiratory syncytial virus (RSV ) causes community‑acquired respiratory tract infections during winter. However, outbreaks in hospitals also occur repeatedly. In particular, patients with hematologic malignancies are at an increased risk for a severe and potentially fatal course of RSV infection. Here we present the investigation of an RSV outbreak in a hematology ward for adults following the ORION statement. Methods: An epidemiologic and molecular outbreak analysis was performed. We developed and employed a mini‑ mal oligonucleotide probe set in target capture probe sequencing that allows cost‑ effective RSV ‑A or ‑B capturing to reconstruct RSV genomes from clinical samples. Results: Four adult patients were involved in the outbreak caused by RSV‑B in March 2019. The enforcement of the pre‑ existing infection control measures by effective training of hospital staff contributed to a successful contain‑ ment. PCR‑based RSV screening on the ward enabled early detection of new cases and rapid isolation measures. The molecular analysis demonstrated that the outbreak sequences were highly related and distinct to other RSV‑B strains circulating at the same time. Conclusions: A multimodal infection control concept is essential for the timely detection and control of RSV out‑ breaks in patients with hematological disease. Among other measures, preventive screening for respiratory viruses is recommended. Furthermore, the integration of conventional and molecular epidemiology, such as whole‑ genome sequencing and variant calling, significantly contributes to the understanding of transmission pathways. Based on this, appropriate conclusions can be drawn for targeted prevention measures that have prepared us for the COVID‑19 pandemic beyond the RSV approach described here. Keywords: Respiratory syncytial virus, Outbreak, Hematology, Infection, Infection control, Capture probe sequencing, Molecular epidemiology Background *Correspondence: baier.claas@mh‑hannover.de; nfischer@uke.de Respiratory syncytial virus (RSV) is an enveloped Institute for Medical Microbiology and Hospital Epidemiology, Hannover RNA virus. It is responsible for infections of the Medical School (MHH), Carl‑Neuberg‑Straße 1, 30625 Hannover, Germany upper (URTI) and lower respiratory tract (LRTI) in Institute for Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg‑Eppendorf, Martinistraße 52, 20246 Hamburg, a seasonal rhythm in countries with temperate cli- Germany mates [1–4]. The pathogen is primarily transmitted by Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Baier et al. Antimicrobial Resistance & Infection Control (2022) 11:88 Page 2 of 8 droplets from the respiratory tract and less often by Case definition contact. Aerosol transmission might play a role under An outbreak case was a patient from the affected ward certain circumstances as well [5]. The median incuba- with nosocomial acquisition of RSV in March 2019. tion period is reported to be 4 to 5  days [6]. RSV out- Nosocomial acquisition was assumed when RSV was breaks in hematology and oncology units (e.g., [7–10]) found on day 5 or later of the patient’s stay on the ward. and other hospital settings [11] have been repeatedly reported in the last years. For infection control of RSV Routine infection control measures for RSV in healthcare facilities, several measures (e.g., isola- The infection control concept at our institution for tion, personal protective equipment) are usually com- hematology wards regarding RSV is summarized in bined to prevent nosocomial spread [12]. Patients with Table  1. It was primarily shaped by the management of a hematologic malignancy are at high risk for a severe a previous RSV outbreak in a pediatric setting in our RSV LRTI [13–16]. Especially, leukopenia seems to be institution [10] and included a weekly PCR prevalence a relevant risk factor for increased mortality [17]. screening for asymptomatic patients [19]. This screening Herein, we report the epidemiology, control and also addressed influenza. molecular investigation of a hospital outbreak caused by RSV-B affecting 4 adults on a hematology ward. In Diagnostic virology addition, we discuss how the existing infection control Combined nose/throat swabs or pharyngeal washes were concept for RSV in hematology helped us in the coro- taken for routine viral diagnostics. If available, mate- navirus disease 2019 (COVID-19) pandemic. rial from the lower respiratory tract was suitable as well. Samples were processed at the Institute of Virology of the Hannover Medical School using the reverse transcrip- tion polymerase chain reaction (RT-PCR) based Panther Methods Fusion System (Hologic) [20]. This system allowed a Epidemiologic analysis semi-quantitative diagnostic using PCR cycle threshold The epidemiologic analysis followed the principles of (ct) values. the ORION statement [18]. Patient charts, virology results and patient movement data were reviewed for RNA extraction and cDNA synthesis data acquisition. In addition, a timeline was generated. RNA was extracted from diagnostic samples using the Qiagen QIAamp MinElute Virus spin kit (Qiagen Table 1 Basic infection control measures regarding RSV at the hematology ward Isolation of RSV positive patients: RSV positive patients were isolated (single room or grouping of patients, if more than one RSV patient was present). Isolation was kept until at least one respiratory specimen was tested negative by PCR or had a ct value > = 35 and the patient’s medical condition improved Prophylactic isolation of patients with respiratory symptoms: Newly admitted patients with respiratory symptoms and already hospitalized patients with onset of respiratory symptoms were separated from other patients and tested for a panel of respiratory viruses RSV specific contact and droplet precautions: Visitors and HCWs wore a surgical mask, a gown and gloves whenever entering the room of a RSV positive patient. RSV infected patients were asked to stay preferably in their individual room and were instructed in hand hygiene. Outside of the room RSV positive patients wore surgical masks (e.g., during urgently necessary examinations) Quarantine: Other patients sharing a room with a patient who tested positive for RSV were put in quarantine for 8 days Preemptive droplet precautions on the ward (universal masking): All HCWs and visitors wore surgical masks at any time when on the ward during winter season (usually December to March). The same applied to all patients when they left their patient room. Visitors were intensively instructed in droplet precautions and hand hygiene Prophylactic RSV Screening: Newly admitted patients were tested for RSV/Influenza (admission screening). In addition, once weekly a RSV/Influenza screening for all patients on the ward took place (prevalence screening) [19, 20] Pre-Season Audits/Training: Training for HCWs provided by the infection control staff prior to the winter season Protective isolation: Nursing in separate rooms for selected patients (e.g., for hematology patients with an expected prolonged and severe leucopenia) Baier  et al. Antimicrobial Resistance & Infection Control (2022) 11:88 Page 3 of 8 #57704) according to the instructions of the manufac- room temperature involving wash buffers I-III. Beads turer. In brief, 125 µl of sample input was used and puri- were resolved in 20  µl nuclease-free water and post- fied RNA was eluted in 50 µl water. capture PCR enrichment was performed by using KAPA cDNA synthesis was performed according to the HiFi HotStart ReadyMix (Roche) with 2  μl NEXTFLEX Superscript IV RT cDNA first-strand synthesis protocol ChIP Primer Mix for Illumina (PerkinElmer Applied (Thermo Fisher Scientific) involving RiboLock RNase Genomics). 20  μl captured DNA input diluted in 50  μl Inhibitor and pdN6 random priming. 2nd strand synthe- total volume was incubated 45  s at 98  °C, followed by sis was achieved with NEB Ultra RNA non-directional 18 cycles of 15 s at 98 °C/30 s at 60 °C/30 s at 72 °C and Second Strand Synthesis Module (New England Biolabs). final extension for 1  min at 72  °C. The library was puri - cDNA was purified with Agencourt AMPure XP beads fied using Agencourt AMPure XP beads (Beckman Coul - (Beckman Coulter Life Sciences). ter Life Sciences). Concentrations of all samples were measured with a Qubit 2.0 Fluorometer (Thermo Fisher Capture probe design Scientific) and fragment lengths distribution of the final The initial step of the probe design pipeline is to select libraries was analyzed with the DNA High Sensitivity the representative strains of RSV that will be included in Chip on an Agilent 2100 Bioanalyzer (Agilent Technolo- the analysis (see Additional files 1 and 2). A total of 1101 gies). Sequencing was performed on the Illumina MiSeq complete genomes were downloaded from GenBank [21] platform (2 × 150-nt) paired reads and 3-5 Mio reads per and aligned using the MAFFT tool [22]. We then entered sample. the generated alignments into the BaitsTools tool [23] for bait generation. To reduce redundancy, we clustered Bioinformatic analysis the bait candidates with 80% identity using usearch [24] As a result of capture probe sequencing of 4 samples from and kept only the centroid of each cluster. Finally, we the patients (P1 – P4) involved in the outbreak and of 3 filtered out the self-complementary baits and the baits control patients with RSV infections on different wards that had at least 90% identity with other baits in a blastn (C1—C3, reference cohort), total numbers of 2,394,484, [25] search. To ensure the quality of the final baits, we 2,700,477, 2,790,882, 2,384,508, 2,200,353, 2,191,289, checked all final sequences for melting temperature and and 1,858,890 2 × 151-nucleotide (nt) paired-end reads GC content. The outline of the capture probe design is were generated from the Illumina MiSeq sequencer by illustrated in Additional file  3; sequences of the capture using the designed probes, respectively. Sequence raw probes are provided in Additional file 4. data were subjected to quality control using FastQC [26]. Adapter sequences of the reads and bases with a score Library preparation, hybridization capture and sequencing of less than Q30 were trimmed and any reads shorter First, ds-cDNA was enzymatically fragmented using the than 36 nt removed using Trimmomatic v0.36. [27]. By SureSelect XT HS and XT Low Input Enzymatic Frag- removing the sequence reads originating from the host mentation Kit (Agilent). Library preparation was per- organism, a set of non-host high-quality paired-end reads formed following the NEB protocol for using NEBNext were prepared and fed into the SPAdes assembler (ver- DNA Ultra Library Prep Kit protocol E7370 (New Eng- sion 3.7.1), resulting in complete genomes for all patients, land Biolabs) with the following modifications. For adap - respectively. For each sample, we mapped the non-host tor ligation, adaptors were diluted (1:10 or 1:25), if input reads to its newly assembled genome using Novoalign cDNA was low, and USER enzyme volume was reduced V3.07.00 (http:// www. novoc raft. com) with the param- to 2 µl. PCR cycles for library amplification were between eters ’-r Random -l 20 -g 40 -x 20 -t 100 -k’, resulting in 12 and 15 cycles depending on the amount of input the alignment file. Then we used the tools samtools [28] cDNA. and MarkDuplicate available in Picard tools (http:// broad Target capture enrichment was performed using xGen insti tute. github. io/ picard/) [29] to sort the alignments Lockdown Probes (see capture probe design) and rea- and remove duplicate sequences from the alignment. The gents (Integrated DNA Technologies; IDT) according to derived alignment was fed into V-Phaser2 [30] for intra- the manufacturer’s instructions. Briefly, DNA capture individual single nucleotide variation (iSNV) identifi - was achieved with 500  ng barcoded library, hybridiza- cation. In the variant identification, we only considered tion of the probes to the library was performed for 4  h variants supported by at least five reads on each strand, at 65  °C. Subsequently, probe/cDNA mix was bound to and the ratio of the number of reads on the two strands washed Streptavidin beads (M-270 Streptavidin beads is smaller than 10. Furthermore, the reads that mapped Thermo Scientific) for 45 min at 65 °C. Stringent Strepta - twice or more on the sequence were discarded. In the vidin beads washing steps (stringent washing buffer) end, the iSNVs whose allele frequency is smaller than were performed at 65  °C, followed by washing steps at 0.5% were filtered out from the final results. Baier et al. Antimicrobial Resistance & Infection Control (2022) 11:88 Page 4 of 8 Results patients 1, 2 or 3 prior to the onset of RSV infection. All Setting patients were mobile and able to leave their rooms prior The cluster took place in March 2019. The affected ward, to RSV positive testing. which managed adult chemotherapy patients for hema- tologic malignancies and autologous hematopoietic stem Clinical course cell transplantations, included 28 beds (four one-bed- All involved patients were asymptomatic at the time of rooms, six two-bedrooms, and three four-bedrooms). initial virus detection. Patient 1 quickly developed an The one-bed rooms were equipped with an anteroom URTI with dry coughing as the main symptom. Patient 2 and all rooms had en-suite bathrooms and high efficiency developed a LRTI with oxygen need (nasal cannula) and particulate air filtration. The ward was operated by quali - dyspnea. This patient had a pre-existing chronic obstruc - fied healthcare workers (HCWs) and housekeeping staff. tive pulmonary disease and was an active smoker. Patient 3 and 4 both developed only mild RSV-associated res- Epidemiology piratory tract infection symptoms with nasal congestion A total of 4 patients were tested RSV positive in respira- and mild coughing. No RSV-associated mortality was tory specimens and had been previously tested RSV neg- observed. Patients 2, 3, and 4 received antibiotics as a ative on admission. Patient characteristics and the time preventive measure against potential bacterial co-infec- course of the outbreak are shown in Table  2 and Fig.  1. tions/superinfections. Patients 2 and 3 showed prolonged Patient 1 was identified by the prevalence screening for viral shedding, up to 29  days in patient 3, as demon- RSV at the beginning of the second week of the hospi- strated by RT-qPCR positive respiratory samples. tal stay. Patient 2 was a roommate to patient 1 for several days prior to the positive test of patient 1 and was there- Control measures fore tested immediately after the positive test result of Outbreak management was initiated immediately after patient 1 became available. Patients 3 and 4 were detected the second positive patient was identified. The infec - in the prevalence screening for RSV. Patient 3 shared the tion control specialists enforced the pre-existing control room for 1 to 2 h with patient 2 one day before patient 2 measures (Table  1) by several ad hoc training sessions was tested positive. Patient 4 did not share a room with on the ward. HCWs, service personnel and visitors were Table 2 Patient’s characteristics Patient Nosocomial Underlying RSV infection White blood RSV Treatment Antibiotic Oxygen need RSV-related onset disease cells (per treatment outcome microliter) 1 Yes Recurrent AML URTI 2200 Immunoglobulins No No Recovered 2 Yes Multiple myeloma LRTI 0 Immunoglobulins Yes Yes Recovered 3 Yes Secondary URTI 1800 None Yes No Recovered hemophagocytosis 4 Yes Multiple myeloma URTI 0 Immunoglobulins Yes No Recovered At time (± 2 days) of virus detection Fig. 1 Timeline. Grey bars represent the patient’s stay on the ward. ‘X’ indicates a positive RSV testing. The first positive testing is indicated with a bold ‘X. ‘O’ indicates a negative testing. Ct = cycle threshold Baier  et al. Antimicrobial Resistance & Infection Control (2022) 11:88 Page 5 of 8 re-instructed to use personal protective equipment (pri- outbreak patients. Therefore, direct transmission due marily surgical masks) and to perform hygienic hand to co-hospitalization in the same patient room can be disinfection. Moreover, we monitored hand hygiene excluded. However, it is conceivable that there was a con- adherence by ad hoc direct observation (compliance rate tact of patient 4 on the hallway or in the ward lounge with of about 70%) and gave feedback talks. patient 3 before spatial isolation of patient 3. A random All RSV-positive tested patients were immediately and independent occurrence of different RSV strains was isolated. implausible to us because of the clear nosocomial occur- rence and the overlapping time periods. This was further Molecular analysis confirmed by virus sequencing, which confirmed infec - To confirm our hypothesis that all 4 patients were tion of the patients by the same viral strain (see below). infected with the identical RSV strain and that these To our surprise, the cluster occurred although sev- transmissions occurred on the ward, we performed RSV eral control measures recommended for hematology target capture sequencing using enrichment techniques wards were already in place [33, 34]. These control meas - in these 4 patients. In parallel, we examined 3 patients ures addressed potential sources (patients, healthcare with RSV identified at the same time on different wards workers, visitors) and transmission pathways (droplets, to test the hypothesis that this was not a common RSV contact) of RSV in the hospital setting, as previously genotype that was repeatedly introduced to the ward. described in other outbreaks [8, 10, 31]. These 3 control patients allowed us to assess the variabil - A core measure containing the spread by droplets is ity of RSV circulating in the same geographic area during universal masking (surgical masks or FFP2/KN95). Note- this time period. We were able to determine the whole worthy, this measure has been widely adopted in hos- genome sequence of 15,185 nucleotides from all 7 speci- pitals in the current COVID-19 pandemic and has also mens included. Detailed information on the sequencing shown a reduction of nosocomial RSV and influenza as of each genome and the coverage can be found in Addi- a side effect [35]. Starting March 2020, we used strict tional files 5 and 6. Phylogenetic classification identified universal masking (staff and patients) in the COVID-19 both patient and control genomes as RSV-B (Fig. 2). The pandemic with the hematologic ward affected by the out - RSV sequences of the 4 outbreak patients were identical break described here being a role model for other wards. to each other and had 83–87 single nucleotide polymor- Nursing in separate rooms of selected hematology phisms to the RSV-B controls (see Additional file 7). patients (e.g., patients with an expected prolonged and severe leucopenia) is another important measure and can Discussion contribute to outbreak prevention and containment. We investigated the epidemiologic and molecular char- In our case, the outbreak control management focused acteristics of a RSV-B cluster affecting adult hematologic explicitly on enforcing the already existing measures, patients. thereby successfully containing the outbreak. The estab - Transmission between patients, in particular patients lished PCR-based screening program (including influ - sharing a room, is mainly conducted by droplets in enza) [19] was helpful in rapid detection of the index RSV outbreaks [10, 31, 32]. Therefore, we assumed a patient and patients 3 and 4, both being asymptomatic direct transmission from patient 1 to patient 2 due to at the time of the PCR screening. The infection control the shared room occupancy. The initial RSV infection concept of prevalence and admission screening has also source of patient 1, however, remained unclear. Patient 1 been widely adopted during the COVID-19 pandemic received visits and had contact to the ward’s staff for sev - in healthcare facilities. Moreover, it might be useful to eral days prior to acquisition. However, visitors and staff extend a screening panel to other respiratory viruses were urged to wear surgical masks during patient con- such as human metapneumovirus, which can also cause tact. Because the time interval between the positive test (severe) morbidity and mortality in immunocompro- results of patients 1 and 2 was relatively short, a shared mised hematology patients. unknown source of infection would also be conceivable. Integrating molecular sequence analysis with temporal Interestingly, patients 2 and 3 shared for several hours and spatial information from classical epidemiology sig- one patient room before patient 2 was placed in quaran- nificantly increases the understanding of outbreak events. tine in a single room following the positive test result of Building on this, we tested and confirmed our epidemio - patient 1. Patient 3 tested positive 5 days later at a prev- logical hypothesis regarding the chain of transmission by alence screening. This temporal sequence agrees well target capture sequencing. The RSV-B isolates from the with the RSV incubation period. Thus, patient-to-patient 4 patients were identical but significantly distinct from contact could have also been the transmission route for other RSV-B isolates found during the same time period patient 3. Patient 4 did not share a room with any other in our institution. Given the molecular and epidemiologic Baier et al. Antimicrobial Resistance & Infection Control (2022) 11:88 Page 6 of 8 Fig. 2 Phylogenetic analysis based on whole genome sequences of RSV isolates, subgroup B. The evolutionary history was inferred using the Maximum Likelihood method based on the General Time Reversible model. The tree with the highest log‑likelihood is shown. The initial tree for the heuristic search was randomly generated. A discrete Gamma distribution was used to model evolutionary rate differences among sites with four categories. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. All positions containing gaps and missing data were eliminated analysis, we assume that patient-to-patient transmission did not focus on evaluating the economic impact of the among patients 1 to 3 is likely and that patient 4 is part outbreak here (for instance due to reduced bed capacity of the outbreak as well. The transmission route in the lat - because of isolation measures). Moreover, the prophylac- ter patient, however, remains unclear. Initial introduction tic PCR-based screening we used is a resource-intensive of the RSV outbreak strain by a point source (e.g., an oli- measure, and we are aware that this might not be avail- gosymptomatic infected relative or healthcare worker) able in other settings. is possible. This outbreak investigation illustrates that a molecular characterization is highly valuable to elucidate Conclusions the potential transmission pathways in RSV outbreaks. Although the importance of prevention and rapid con- We followed the ORION statement for outbreak tainment of RSV outbreaks in hematology is well known reporting [18] in many points in this report. However, we and mostly comprehensive infection control measures Baier  et al. Antimicrobial Resistance & Infection Control (2022) 11:88 Page 7 of 8 Declarations are in place, transmissions may occur. We therefore underline that existing measures must be enforced Ethics approval and consent to participate by intensive training sessions for the wards’ staff and We obtained ethical approval for this study from the ethics committee of the Hannover Medical School (Number 8456_BO_K_2019). Being a retrospective the patients’ visitors. Furthermore, prophylactic RSV study, the need of informed consent was waived by the ethics committee and screening is crucial for outbreak containment and even the data protection commissioner of Hannover Medical School. prevention. Integration of molecular analyses, such as Consent for publication high-resolution whole-genome sequencing, with classi- Not applicable (No individual details such as images or videos are included). cal epidemiological information on temporal and spa- tial events, can be instrumental in resolving outbreaks. Competing interests The authors declare that they have no competing interests. The infection control management described here is— beyond RSV—essential in the COVID-19 pandemic. Author details Institute for Medical Microbiology and Hospital Epidemiology, Hannover Medical School (MHH), Carl‑Neuberg‑Straße 1, 30625 Hannover, Germany. Abbreviations Institute for Medical Microbiology, Virology and Hygiene, University Medical RSV: Respiratory syncytial virus; URTI: Upper respiratory tract infection; LRTI: Center Hamburg‑Eppendorf, Martinistraße 52, 20246 Hamburg, Germany. Lower respiratory tract infection; COVID‑19: Coronavirus disease 2019; RT ‑PCR: Leibniz Institute for Experimental Virology, Martinistraße 52, 20251 Hamburg, Reverse transcription polymerase chain reaction; HCWs: Health care workers. Germany. Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School (MHH), Carl‑Neuberg‑Straße 1, 30625 Hannover, Germany. Institute of Virology, Hannover Medical School Supplementary Information (MHH), Carl‑Neuberg‑Str. 1, 30625 Hannover, Germany. Institute for Experi‑ The online version contains supplementary material available at https:// doi. mental Virology; Twincore‑ Centre for Experimental and Clinical Infection org/ 10. 1186/ s13756‑ 022‑ 01120‑z. Research; a joint venture of Hannover Medical School (MHH) and Helmholtz Centre for Infection Research (HZI), Feodor‑Lynen‑Straße 7, 30625 Hannover, Germany. Cluster of Excellence RESIST (EXC 2155), Hannover Medical School, Additional file 1. GenBank accession numbers of RSV ‑A genomes applied Carl‑Neuberg‑Straße 1, 30625 Hannover, Germany. German Center for Infec‑ in probe design tion Research (DZIF), Partner Site Hannover‑Braunschweig, 30625 Hannover, Additional file 2. GenBank accession numbers of RSV ‑B genomes applied Germany. in probe design Additional file 3. Workflow of probe design pipeline Received: 9 January 2022 Accepted: 24 May 2022 Additional file 4. Fasta files of all RSV capture probes Additional file 5. Summary of short read sequencing results Additional file 6. Coverage profiles of the RSV sequences obtained by References target capture probe sequencing 1. Borchers AT, Chang C, Gershwin ME, Gershwin LJ. Respiratory syncytial Additional file 7. Overview of SNPs of all RSV sequences obtained virus—a comprehensive review. Clin Rev Allergy Immunol. 2013;45:331– 79. https:// doi. org/ 10. 1007/ s12016‑ 013‑ 8368‑9. 2. Rodriguez R, Ramilo O. Respiratory syncytial virus: how, why and what to Acknowledgements do. J Infect. 2014;68(Suppl 1):S115–8. https:// doi. org/ 10. 1016/j. jinf. 2013. Not applicable. 09. 021. 3. Griffiths C, Drews SJ, Marchant DJ. Respiratory syncytial virus: Infection, Author contributions detection, and new options for prevention and treatment. Clin Microbiol All authors contributed to the manuscript according to the ICMJE (Interna‑ Rev. 2017;30:277–319. https:// doi. org/ 10. 1128/ CMR. 00010‑ 16. tional Committee of Medial Journal Editors) recommendations: All authors 4. Nam HH, Ison MG. Respiratory syncytial virus infection in adults. BMJ. were involved in data acquisition, analysis and interpretation. NF, JH, KR, DI, SH 2019;366: l5021. https:// doi. org/ 10. 1136/ bmj. l5021. and TP were involved in the molecular analysis. CB, F‑ CB, EE, CK and FT were 5. Kulkarni H, Smith CM, Lee DDH, Hirst RA, Easton AJ, O’Callaghan C. involved in the outbreak management and epidemiologic workup. AH carried Evidence of respiratory syncytial virus spread by aerosol. Time to revisit out routine RSV diagnostic. CB and NF wrote the manuscript. All authors have infection control strategies? Am J Respir Crit Care Med. 2016;194:308–16. read and approved the final manuscript. https:// doi. org/ 10. 1164/ rccm. 201509‑ 1833OC. 6. Lessler J, Reich NG, Brookmeyer R, Perl TM, Nelson KE, Cummings DA. Funding Incubation periods of acute respiratory viral infections: a systematic Open Access funding enabled and organized by Projekt DEAL. TP is funded by review. Lancet Infect Dis. 2009;9:291–300. https:// doi. org/ 10. 1016/ S1473‑ Germany’s Excellence Strategy—EXC 2155 “RESIST”—Project ID 390874280. 3099(09) 70069‑6. 7. Kelly SG, Metzger K, Bolon MK, Silkaitis C, Mielnicki M, Cullen J, et al. Availability of data and materials Respiratory syncytial virus outbreak on an adult stem cell transplant unit. The datasets generated during and/or analyzed during the current study are Am J Infect Control. 2016. https:// doi. org/ 10. 1016/j. ajic. 2016. 03. 075. available from the corresponding authors on reasonable request. Patient data 8. Inkster T, Ferguson K, Edwardson A, Gunson R, Soutar R. Consecutive used in this study is confidential according to the German data privacy act, yearly outbreaks of respiratory syncytial virus in a haemato‑ oncology the ethics committee and the data protection commissioner of the Hannover ward and efficacy of infection control measures. J Hosp Infect. Medical School. To protect patient confidentiality and participant’s privacy, 2017;96:353–9. https:// doi. org/ 10. 1016/j. jhin. 2017. 05. 002. data used for this study can be obtained in anonymous form only according 9. Nabeya D, Kinjo T, Parrott GL, Uehara A, Motooka D, Nakamura S, et al. to the data privacy act. The clinical and phylogenetic investigation for a nosocomial outbreak of respiratory syncytial virus infection in an adult hemato‑ oncology unit. J Med Virol. 2017;89:1364–72. https:// doi. org/ 10. 1002/ jmv. 24800. 10. Baier C, Haid S, Beilken A, Behnert A, Wetzke M, Brown RJP, et al. Molecular characteristics and successful management of a respiratory syncytial Baier et al. Antimicrobial Resistance & Infection Control (2022) 11:88 Page 8 of 8 virus outbreak among pediatric patients with hemato‑ oncological dis‑ 30. Yang X, Charlebois P, Macalalad A, Henn MR, Zody MC. V‑Phaser 2: variant ease. Antimicrob Resist Infect Control. 2018;7:21. https:// doi. org/ 10. 1186/ inference for viral populations. BMC Genomics. 2013;14:674. https:// doi. s13756‑ 018‑ 0316‑2.org/ 10. 1186/ 1471‑ 2164‑ 14‑ 674. 11. Ryu S, Kim BI, Chun BC. An outbreak of respiratory tract infection due to 31. Lehners N, Schnitzler P, Geis S, Puthenparambil J, Benz MA, Alber B, et al. Respiratory Syncytial Virus‑B in a postpartum center. J Infect Chemother. Risk factors and containment of respiratory syncytial virus outbreak in a 2018;24:689–94. https:// doi. org/ 10. 1016/j. jiac. 2018. 06. 010. hematology and transplant unit. Bone Marrow Transplant. 2013;48:1548– 12. French CE, McKenzie BC, Coope C, Rajanaidu S, Paranthaman K, Pebody R, 53. https:// doi. org/ 10. 1038/ bmt. 2013. 94. et al. Risk of nosocomial respiratory syncytial virus infection and effective ‑ 32. Jensen TO, Stelzer‑Braid S, Willenborg C, Cheung C, Andresen D, Raw‑ ness of control measures to prevent transmission events: a systematic linson W, et al. Outbreak of respiratory syncytial virus (RSV ) infection review. Influenza Other Respi Viruses. 2016;10:268–90. https:// doi. org/ 10. in immunocompromised adults on a hematology ward. J Med Virol. 1111/ irv. 12379. 2016;88:1827–31. https:// doi. org/ 10. 1002/ jmv. 24521. 13. Hijano DR, Maron G, Hayden RT. Respiratory viral infections in patients 33. Hirsch HH, Martino R, Ward KN, Boeckh M, Einsele H, Ljungman P. Fourth with cancer or undergoing hematopoietic cell transplant. Front Micro‑ European conference on infections in leukaemia (ECIL‑4): guidelines for biol. 2018;9:3097. https:// doi. org/ 10. 3389/ fmicb. 2018. 03097. diagnosis and treatment of human respiratory syncytial virus, parainflu‑ 14. Khawaja F, Chemaly RF. Respiratory syncytial virus in hematopoietic cell enza virus, metapneumovirus, rhinovirus, and coronavirus. Clin Infect Dis. transplant recipients and patients with hematologic malignancies. Hae‑ 2013;56:258–66. https:// doi. org/ 10. 1093/ cid/ cis844. matologica. 2019;104:1322–31. https:// doi. org/ 10. 3324/ haema tol. 2018. 34. Tomblyn M, Chiller T, Einsele H, Gress R, Sepkowitz K, Storek J, et al. Guide‑ 215152. lines for preventing infectious complications among hematopoietic 15. Vakil E, Sheshadri A, Faiz SA, Shah DP, Zhu Y, Li L, et al. Risk factors for cell transplantation recipients: a global perspective. Biol Blood Marrow mortality after respiratory syncytial virus lower respiratory tract infection Transplant. 2009;15:1143–238. https:// doi. org/ 10. 1016/j. bbmt. 2009. 06. in adults with hematologic malignancies. Transpl Infect Dis. 2018;20: 019. e12994. https:// doi. org/ 10. 1111/ tid. 12994. 35. Wong S‑ C, Lam GK‑M, AuYeung CH‑ Y, Chan VW‑M, Wong NL ‑D, So SY ‑ C, 16. Chemaly RF, Shah DP, Boeckh MJ. Management of respiratory viral et al. Absence of nosocomial influenza and respiratory syncytial virus infections in hematopoietic cell transplant recipients and patients with infection in the coronavirus disease 2019 (COVID‑19) era: implica‑ hematologic malignancies. Clin Infect Dis. 2014;59(Suppl 5):S344–51. tion of universal masking in hospitals. Infect Control Hosp Epidemiol. https:// doi. org/ 10. 1093/ cid/ ciu623. 2021;42:218–21. https:// doi. org/ 10. 1017/ ice. 2020. 425. 17. Azzi JM, Kyvernitakis A, Shah DP, El Haddad L, Mahajan SN, Ghantoji SS, et al. Leukopenia and lack of ribavirin predict poor outcomes in patients Publisher’s Note with haematological malignancies and respiratory syncytial virus infec‑ Springer Nature remains neutral with regard to jurisdictional claims in pub‑ tion. J Antimicrob Chemother. 2018;73:3162–9. https:// doi. org/ 10. 1093/ lished maps and institutional affiliations. jac/ dky311. 18. Stone SP, Cooper BS, Kibbler CC, Cookson BD, Roberts JA, Medley GF, et al. The ORION statement: guidelines for transparent reporting of outbreak reports and intervention studies of nosocomial infection. Lancet Infect Dis. 2007;7:282–8. https:// doi. org/ 10. 1016/ S1473‑ 3099(07) 70082‑8. 19. Baier C, Linderkamp C, Beilken A, Thol F, Heuser M, Ebadi E, et al. Influenza and respiratory syncytial virus screening for the detection of asympto‑ matically infected patients in hematology and oncology. GMS Hyg Infect Control. 2018;13:Doc08. https:// doi. org/ 10. 3205/ dgkh0 00314. 20. Ganzenmueller T, Kaiser R, Baier C, Wehrhane M, Hilfrich B, Witthuhn J, et al. Comparison of the performance of the Panther Fusion respiratory virus panel to R‑ Gene and laboratory developed tests for diagnostic and hygiene screening specimens from the upper and lower respiratory tract. J Med Microbiol. 2020;69:427–35. https:// doi. org/ 10. 1099/ jmm.0. 001133. 21. Benson DA, Cavanaugh M, Clark K, Karsch‑Mizrachi I, Ostell J, Pruitt KD, et al. GenBank. Nucleic Acids Res. 2018;46:D41–7. https:// doi. org/ 10. 1093/ nar/ gkx10 94. 22. Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013;30:772–80. https:// doi. org/ 10. 1093/ molbev/ mst010. 23. Campana MG. BaitsTools: software for hybridization capture bait design. Mol Ecol Resour. 2018;18:356–61. https:// doi. org/ 10. 1111/ 1755‑ 0998. 24. Edgar RC. Search and clustering orders of magnitude faster than BLAST. Bioinformatics. 2010;26:2460–1. https:// doi. org/ 10. 1093/ bioin forma tics/ btq461. 25. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, et al. Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : BLAST+: architecture and applications. BMC Bioinformatics. 2009;10:421. https:// doi. org/ 10. 1186/ 1471‑ 2105‑ 10‑ 421. fast, convenient online submission 26. Andrews S. FastQC: a quality control tool for high throughput sequence thorough peer review by experienced researchers in your field data. http:// www. bioin forma tics. babra ham. ac. uk/ proje cts/ fastqc/. Accessed 19 Dec 2021. rapid publication on acceptance 27. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illu‑ support for research data, including large and complex data types mina sequence data. Bioinformatics. 2014;30:2114–20. https:// doi. org/ 10. • gold Open Access which fosters wider collaboration and increased citations 1093/ bioin forma tics/ btu170. 28. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The maximum visibility for your research: over 100M website views per year Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009;25:2078–9. https:// doi. org/ 10. 1093/ bioin forma tics/ btp352. At BMC, research is always in progress. 29. BROAD INSTITUTE. Picard Toolkit. GitHub repository. http:// broad insti tute. Learn more biomedcentral.com/submissions github. io/ picard. 2019. Accessed 19 Dec 2021.

Journal

Antimicrobial Resistance & Infection Control – Springer Journals

Published: Jun 21, 2022

Keywords: Respiratory syncytial virus; Outbreak; Hematology; Infection; Infection control; Capture probe sequencing; Molecular epidemiology

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