Molecular characterization of a respiratory syncytial virus outbreak in a hematology unit in Heidelberg, Germany

Abstract

In 2011 and 2012, a large outbreak of respiratory syncytial virus (RSV) infections affecting 57 laboratory-confirmed patients occurred in an adult hematology unit in Heidelberg, Germany. During the outbreak investigation, we performed molecular genotyping of RSV strains to differentiate between single versus multiple introductions of the virus into the unit. Furthermore, we assessed the time of viral shedding of consecutive samples from the patients in order to better understand the possible impact of prolonged shedding for outbreak control management. We used subtype-specific reverse transcription-PCR on nasopharyngeal and bronchoalveolar specimens for routine diagnostics and for measuring the viral shedding period. Samples of 47 RSV-infected patients involved in the outbreak were genotyped by sequence analysis and compared to samples from RSV-infected hospitalized children representing the timing of the annual RSV epidemic in the community. Molecular investigation of the virus strains from clinical samples revealed a unique cluster with identical nucleotide sequences of RSV type A (RSV A outbreak strain) for 41 patients, while 3 patients were infected with different RSV A (nonoutbreak) strains and three other patients with RSV type B. Outbreak strains were identified in samples from November 2011 until January 2012, while nonoutbreak strains were from samples coinciding with the community epidemic in February and March 2012. Median duration of viral shedding time was 24.5 days (range, 1 to 168 days) with no difference between outbreak and nonoutbreak strains (P = 0.45). Our investigation suggests a single introduction of the RSV A outbreak strain into the unit that spread among the immunocompromised patients. Prolonged viral shedding may have contributed to nosocomial transmission and should be taken into account in the infection control management of RSV outbreaks in settings with heavily immunosuppressed patients.

Fig 1

Cases of RSV in the hematology unit (columns) and in non-outbreak-related pediatric patients (line) by week of laboratory confirmation.

Fig 2

Sequence alignment of the second variable region of the RSV G gene (nucleotide positions 634 to 895 of GA2; GenBank accession number JF950053) constructed by a Clustal W algorithm. RSV G gene nucleotide sequences were deposited in GenBank under accession numbers JX967562 (outbreak), JX967571 (P40), JX967563 (P41), JX967564 (P45), JX967572 (P47), JX967565 (C5), JX967566 (C11), JX967567 (C13), JX967568 (C15), JX967569 (C19), JX967570 (C21), JX967573 (C22), JX967574 (C27), and JX967575 (C29). Sequences are grouped by RSV A and RSV B sequences. RSV A outbreak represents 41 identical sequences from patients of the hematology unit. Dots indicate nucleotide identities, and dashes are used for adjustment of nucleotide insertions. P, patient from hematology unit; C, outbreak-unrelated pediatric patient. Reference sequences for RSV A and RSV B genotypes selected from GenBank are indicated by their accession numbers.

Fig 3

Phylogenetic tree of RSV A and RSV B sequences from the second variable region of the G gene constructed with MEGA version 5.05 using the maximum likelihood method. RSV A outbreak represents identical sequences from patients of the hematology unit. P, patients of the hematology unit; C, outbreak-unrelated pediatric patient. Reference sequences for RSV A and RSV B genotypes selected from GenBank are indicated by their accession numbers. The bar indicates 0.1 nucleotide substitutions per site. Bootstrap values greater than 60 are displayed on branch nodes.

Fig 4

Representation of the shedding periods and temporal distribution of RSV infection for long-term RSV-infected hematology patients. The sampling dates of RSV-positive specimens are indicated with triangles. Black triangle, RSV A outbreak strain; gray triangle, RSV A nonoutbreak strain; white triangle, RSV B strain. Circles denote RSV-negative samples indicating clearance of infection. Thirty-five cases with two or more sequential RSV-positive specimens and a positivity period of 7 days or longer were identified. Patients P12, P23, and P40 were lost to follow-up, and patients P4, P5, P9, P10, P38, and P41 died (†) while still shedding RSV.