Volume 14, Issue 12 , Pages 1348-1355, December 2008
Molecular Characterization of Strains of Respiratory Syncytial Virus Identified in a Hematopoietic Stem Cell Transplant Outpatient Unit Over 2 Years: Community or Nosocomial Infection?
Article Outline
Abstract
Respiratory syncytial virus (RSV) is recognized as the leading cause of nosocomial respiratory infection among hematopoietic stem cell transplant (HSCT) recipients, causing considerable morbidity and mortality. RSV is easily transmitted by contact with contaminated surfaces, and in HSCT units, more than 50% of RSV infections have been characterized as of nosocomial origin. From April 2001 to October 2002, RSV was identified by direct immunofluorescent assay in 42 symptomatic HSCT recipients. Seven RSV strains from 2001 and 12 RSV strains from 2002 were sequenced. RNA extraction, cDNA synthesis, and seminested polymerase chain reaction (PCR) with primers complementary to RSV genes G and F were performed. PCR products were analyzed by nucleotide sequencing of the C-terminal region of gene G for typing (in group A or B). Of the 7 strains analyzed in 2001, only 2 belonged to group B; the other 5 belonged to group A. Of these 7 strains, 3 were identical and were from recipients receiving outpatient care. In 2002, of the 12 strains analyzed, 3 belonged to group A and the other 9 belonged to group B. Of these 9 strains, 7 were genetically identical and were also from recipients receiving outpatient care. Therefore, multiple strains of RSV cocirculated in the hematopoietic stem cell transplant units (ward and outpatient units) between 2001 and 2002. Nosocomial transmission was more likely to occur at the HSCT outpatient unit than in the HSCT ward. Infection control practices should also be implemented in the outpatient setting.
Key Words: Respiratory synctial virus, Hematopoietic stem cell transplantation, Molecular epidemiology, Nosocomial transmission, Outpatient care
Introduction
Respiratory syncytial virus (RSV) infection can cause significant morbidity and mortality in hematopoietic stem cell transplant (HSCT) recipients. RSV circulates throughout the year, but increased activity is observed in late fall and winter in countries with a subtropical climate.
RSV is classified in the genus Pneumovirus, subfamily Pneumovirinae, family Paramyxoviridae. Two antigenic groups of RSV (A and B) have been described on the basis of reactions with monoclonal antibodies (mAbs) specific for the G protein, the most variable gene product among RSV isolates. Nucleotide sequencing has shown that, at the amino acid level, there is only 53% identity between the G proteins of the prototype strains of groups A and B, and up to 20% sequence variation has been observed among the G proteins of RSV isolates of the same antigenic group [1]. Sequencing of phylogenetically informative regions of the G gene has been useful in identifying lineages (or genotypes) of RSV within the 2 major groups and in refining our ability to differentiate between RSV isolates. Therefore, phylogenetic analysis has been recommended for epidemiologic investigation of RSV outbreaks [2].
Respiratory viruses (RV) are easily transmitted by contact with contaminated surfaces. In HSCT units, more than 50% of RSV infections have been characterized as of nosocomial origin, based on the interval between patient admission and the diagnosis of RSV infection 3, 4. More recent studies have used molecular epidemiology to better characterize RSV transmission in hospital outbreaks 5, 6. American and European guidelines recommend strict infection-control measures to limit RSV transmission in HSCT units [7]. However, the implementation of such policies is limited in the outpatient setting, and, therefore, transmission is likely to occur.
Although previous studies have highlighted the possibility of nosocomial transmission, molecular characterization of RSV strains during the RV season and the mechanisms involved in RSV transmission have been poorly addressed, especially in the outpatient setting.
Patients and Methods
Study Population
Nasal wash (NW) samples from HSCT recipients with symptoms of upper respiratory tract infection were collected in 2001 and 2002. NWs were also taken from 37 symptomatic health care workers (HCW) who assisted the HSCT recipients during the study period. The samples were collected according to Englund et al. [8] and processed within 2 hours of sampling by direct immunofluorescent assay (DFA) (Imagen® Dako, Cambridgeshire, UK), which allows rapid diagnosis of the most relevant RV infections. The remaining material was stored at −80°C.
RSV infection was diagnosed by DFA in 21 of the 141 patients tested in 2001, 21 of the 181 patients tested in 2002, and in 13 of the 172 patients tested in 2003 [9]. These patients were either hospitalized in the HSCT unit at the Hospital das Clínicas, Faculty of Medical Sciences, University of São Paulo (HC-FMUSP), or attending the outpatient unit at the same hospital.
RV infection was detected in 6 HCWs (16.2%): RSV was diagnosed in 1 HCW (2.7%), influenza A in 3 (8.1%), and influenza B in 2 (5.4%).
Facilities and Infection Control Policies
The inpatient unit of the Bone Marrow Transplant Division has rooms with filtered air and positive pressure. HSCT recipients are hospitalized in single rooms either prior to the transplant or in the event of posttransplant complications. The control of RV transmission in the HSCT unit includes the following measures: (1) influenza vaccination is recommended for all HSCT recipients after the sixth month of transplantation for family members and HCWs, (2) NW samples are taken from all symptomatic patients to test for respiratory virus using the DFA, (3) symptomatic patients are kept under contact precautions until a specific diagnosis is made, (4) patients who test positive for respiratory viruses are transferred from rooms with positive pressure to rooms without positive pressure, (5) HCWs with respiratory symptoms are tested for respiratory virus infection, (6) visits by children under 12 years of age are forbidden during the respiratory virus season.
The outpatient unit operates from 7am to 7pm hours and attends patients at various stages after transplant for medical care or laboratory tests. Patients share a small waiting room, as well as the bathrooms and treatment room, where there are 6 reclining chairs. NW samples were collected in the treatment room by the nurses wearing a mask, gown, and gloves. Patients were seen by a physician in a separate room (consulting room), which was also used to “cohort” patients with respiratory virus infections who needed to stay in the outpatient unit. The control of RV infections in the outpatient unit included the following measures: (1) NWs were collected from all symptomatic patients to test for respiratory viruses using DFA, (2) face masks were used in the unit when patients had symptoms of respiratory infection and/or a respiratory virus was detected, (3) disposable sheets were used on the reclining chairs, and (4) when available, individual consulting rooms were used for patients with RV infections who needed to stay in the outpatient unit or, failing this, patients were cohorted according to the virus detected.
Definition of RSV Nosocomial Infection
A patient was considered to have nosocomial RSV infection in the presence of signs and symptoms of respiratory disease in the upper and/or lower respiratory tract together with the following criteria: (1) no signs or symptoms of respiratory disease on admission, (2) NW sample tested negative for RSV by DFA on admission, and (3) positive DFA test at least 5 days after admission, based on the average incubation period for RSV infection, according to Garcia et al. [10].
RNA Extraction, Reverse Transcription Polymerase Chain Reaction (PCR), and DNA Sequencing
The RNA of the samples that tested positive for RSV by DFA was extracted by the silica gel method (NucliSens®). cDNA was synthesized using random primers, and a seminested PCR was performed with primers complementary to the fusion protein (F) and attachment protein (G). The following 3 primers were used: GAB (YCA YTT TGA AGT GTT CAA CTT) [11], FV (GTT ATG ACA CTG GTA TAC CAA CC) [12], and F1AB (CAA CTC CAT TGT TAT TTG CC) [13].
The PCR products were purified with a Microcon Purification kit (Millipore, Bedford, MA). GAB and F1AB primers and the Big Dye v2.0 kit (Applied Biosystems, Bedford, MA) were used for the sequencing reaction performed on an ABI PRISM 377 DNA sequencer (Applied Biosystems Inc.).
Nucleotide Sequence Analysis
Electropherograms were analyzed using the SeqMan PC program (DNASTAR Inc., Madison, WI) to obtain a 270-nucleotide sequence corresponding to the second variable region of the glycoprotein G gene (G2), including nucleotides 649-918 and 652-921 for RSV A and B, respectively [9]. The contigs corresponding to the G2 region were aligned in 2 groups (A and B) using the ClustalX program and converted into NEXUS format.
To determine whether the RSV strains circulating in both HSCT units were similar to community strains, sequences of community-acquired RSV obtained from children seen at the emergency room of the University Hospital, University of São Paulo, from 2001 to 2002 were included in the analysis. Two sequences of RSV isolated from HSCT recipients in 2003 were also included in the analysis to establish the persistence of strains in consecutive years.
Phylogenetic Analysis
Sequence alignment for groups A and B was performed using the multiple sequence alignment method implemented in ClustalX. Maximum likelihood (ML) analysis was performed using PAUP∗4.0 b10 (Sinauer Associates Inc., Sunderland, MA) for Power Macintosh and Unix [14].
Results
Samples Studied
Of the 21 samples that were positive for respiratory syncytial virus by DFA in 2001, only 7 could be analyzed for the following reasons: (1) some samples were not stored because of insufficient material after DFA processing, (2) in some samples the RNA was lost, possibly because of a delay in processing and/or inadequate storage, or (3) amplified product was inadequate for sequencing. Similarly, of the 20 positive samples in 2002, only 12 could be analyzed. Of the 19 virus strains analyzed, 11 were from females and 8 from males; the underlying diseases were chronic myelogenous leukemia(CML) in 9 patients, acute myelogenous leukemia(AML) in 6, multiple myeloma(MM) in 3 and non-Hodgkin's lymphoma(NHL) in 1 patient. Eighteen patients were receiving medical care at the outpatient unit.
Analysis of Virus Strains in 2001 and 2002
In 2001, samples from 7 patients were analyzed, 6 of whom were receiving outpatient care when RSV infection was diagnosed. Of these 7 strains, 5 belonged to group A (71.4%) and 2 to group B. The predominant genotype for that year in our patient population was GA2, followed by GA5 and SAB1 (Table 1). The predominant strains circulating in the community in the same year belonged to group B (E.L. Durigon, personal communication, data not published).
Table 1. RSV Group and Genotype of Patients Infected in 2001 and 2002
| Year | Patient No. | RSV Group | Genotype | Location |
|---|---|---|---|---|
| 2001 | 687 | B | SAB1 | Outpatient unit |
| 2001 | 997 | A | GA2 | Outpatient unit |
| 2001 | 445 | B | SAB1 | Outpatient unit |
| 2001 | 1034 | A | GA2 | Outpatient unit |
| 2001 | 1032 | A | GA2 | Outpatient unit |
| 2001 | 1023 | A | GA5 | Hospital HSCT ward |
| 2001 | 1152 | A | GA5 | Outpatient unit |
| 2002 | 147 | A | GA2 | Outpatient unit |
| 2002 | 1322 | B | SAB3 | Outpatient unit |
| 2002 | 1329 | B | SAB3 | Outpatient unit |
| 2002 | 1370 | B | SAB3 | Outpatient unit |
| 2002 | 1321 | B | SAB3 | Outpatient unit |
| 2002 | 1149 | B | SAB3 | Outpatient unit |
| 2002 | 817 | A | GA2 | Outpatient unit |
| 2002 | 1220 | A | GA5 | Outpatient unit |
| 2002 | 1328 | B | SAB3 | Outpatient unit |
| 2002 | 858 | B | SAB1 | Outpatient unit |
| 2002 | 1248 | B | SAB1 | Outpatient unit |
| 2002 | 1308 | B | SAB3 | Outpatient unit |
Three of the 5 strains from group A (60%), identified in 2001, were considered to be very closely related phylogenetically (TMO9971, TMO10321, and TMO10341; Figure 1). All 3 patients were receiving outpatient care.
Figure 1
Phylogenetic tree constructed using the maximum-likelihood method for samples from RSV group A.
In 2002, samples from 12 patients were analyzed, all of whom were receiving outpatient care when the infection was diagnosed. Whereas the predominant RSV strains circulating in the community in 2002 were from group A (E.L. Durigon, personal communication, data not published), 9 of the 12 strains (66.6%) from HSCT recipients receiving outpatient care belonged to group B (Table 1).
Seven of the 9 strains from group B in 2002 (77.7%) were considered phylogenetically identical (TMO13222, TMO13292, TMO13702, TMO13212, TMO11492, TMO13282, and TMO13082; Figure 2). The predominant genotype for that year was SAB3, followed by SAB1 and GA2 (Table 1).
Figure 2
Phylogenetic tree constructed using the maximum-likelihood method for samples from RSV group B.
Further data on RSV infection in 2001 and 2002, according to month of diagnosis and day of outpatient visits are shown in Figure 3.
Figure 3
Light gray squares indicate duration of RSV shedding; squares with black borders indicate the samples that were sequenced; N = day of nasal wash sampling; • = period of hospital admission; V = visit to the outpatient unit without NW sampling; S = sequenced; NS = not sequenced.
Discussion
São Paulo is a city with a subtropical climate in the southeast region of Brazil. In general, respiratory viruses circulate year round with similar activity except for respiratory syncytial virus and influenza A and B, which peak in fall and winter, respectively. The start of the RSV season in São Paulo was in April 2001 and in March 2003. In 2001, the peak occurred in May, and cases were registered until the end of the winter that year, in agreement with data from other regions in Brazil with the same climate [15]. In 2002, the RSV season had a peak at the end of fall/beginning of winter and lasted until the beginning of spring, in October.
To prevent nosocomial transmission, it is recommended that for patients with established RSV: (1) separate rooms should be used; (2) individual equipment, such as pulse oximeters, electronic thermometers, stethoscope, etc., should be used; (3) a contact precaution sign should be placed on the patients' door; and (4) contact isolation precautions should be used, such as cohort nursing, wearing of gown and gloves, and strict attention to good hand washing practices in any contact with the infected patient [16].
Such measures are difficult to implement in the outpatient setting. In this study, most of the virus strains analyzed in 2001 and 2002 were from patients who had been attending the outpatient department of the HSCT unit. Two clusters of closely related strains were found in the ML analysis: 1 in 2001 with group A strains of RSV (TMO9971, TMO10321, and TMO10341), and the other in 2002, with group B strains (TMO13702, TMO13222, TMO13292, TMO13212, TMO11492, TMO13282, and TMO13082).
Some of the patients infected in 2002 attended the outpatient unit on the same day or within a few days of each other. Interestingly, the strains identified in these patients were from group B, different from those circulating in the community that year (group A). These observations suggest that transmission may have taken place within the outpatient unit as a result of the small area shared by patients and the difficulty in implementing RSV control measures in this area. Nichols et al. [17] observed evidence of crosstransmission of the parainfluenza-3 virus, which also belongs to the Paramyxoviridae family, in the outpatient unit at the Fred Hutchinson Cancer Research Center in Seattle during a parainfluenza outbreak at the institution. The authors observed a cluster of parainfluenza-3 genotype in outpatients, whereas this genotype was not found in hospitalized patients receiving medical care during the same period.
The main mechanism of RSV transmission (namely, contact with surfaces that are contaminated with the virus) favors transmission in hospitals [18]. During the study period, no hospital-acquired RSV infection could be demonstrated in the inpatient ward at the HSCT Unit. RSV infection was diagnosed in patient TMO10231 2 days after admission to the ward and was therefore considered to be community acquired. However, other authors have shown that the virus can be transmitted within inpatient wards 2, 3, 6, 10, 19, 20.
There are 2 major limitations in this study that prevented a definitive conclusion about crosstransmission of RSV in the outpatient unit. First, we were unable to characterize molecularly all the virus strains from patients with RSV infection. Moreover, more cases of RSV would probably be identified if PCR was also performed on the NW samples. Second, clinical samples from patients' family members and from employees at the HSCT unit were lacking. Consequently, the whole transmission chain could not be demonstrated, particularly in samples from 2001. Patients can contract community-acquired infection through contact with family members, especially those family members under 12 years of age, and may already be infected when they arrive at the outpatient unit 21, 22. Similarly, asymptomatic or oligosymptomatic health staff can transmit the infection within the work environment [21]. Although NWs were collected from employees with symptoms of respiratory infection, these samples were not stored for later analysis. One case of RSV was detected in a HCW , and crosstransmission may therefore have taken place, although this could not be demonstrated.
HSCT recipients were infected with multiple strains in 2001 and 2002, corroborating the findings of Mazzulli et al. [2] and Englund et al. [19], who also reported that multiple strains can be introduced and spread in hospitals. As most of the patients were not hospitalized, they were in contact with viruses circulating in the community, which could explain the variety of strains and genotypes in the HSCT outpatient unit. As reviewed by Cane, in 2001 [23], the A and B groups of RSV can circulate together in a community, although group A strains predominate over group B strains. Surprisingly, the strains analyzed from 2001 were mainly from group A, with GA2 as the predominant genotype followed by GA5; the predominant strains circulating in the community in São Paulo that year were from group B. In 2002, the strains isolated from HSCT recipients were mainly from group B, and the predominant genotype was SAB3 followed by SAB1; the predominant strains circulating in the community were from group A (E.L. Durigon, data not published). These data reinforce the hypothesis of crosstransmission of RSV in the outpatient unit in the present study. Another possible explanation is the selective advantage of the virus, ensuring that genotypes against which the community is probably immune can circulate and persist in immunologically compromised individuals 1, 24.
In the present study, we observed that multiple strains cocirculated in the hematopoietic stem cell transplant unit (outpatient unit and inpatient ward) at the Hospital das Clínicas, São Paulo, in 2001 and 2002. The detection of phylogenetically related RSV strains in the day hospital that were different from the predominant community strains in circulation in the respective years suggests the occurrence of RSV transmission in the outpatient setting. Thus, strict control measures should also be implemented as much as possible in the outpatient setting, to avoid transmission of RSV and other respiratory viruses among HSCT recipients.
Acknowledgments
Financial disclosure: This study was supported by Fapesp Grant 2001/11087-2 and 2002 08465-8. Adriana Freire Machado received a grant from CAPES, Brazilian Ministry of Education. The authors thank Dr Frederico L. Dulley and the clinicians and nurses assisting the HSCT recipients for excellent health care.
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Financial disclosure: See Acknowledgments on page 1354.
PII: S1083-8791(08)00403-5
doi:10.1016/j.bbmt.2008.09.012
© 2008 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.
Volume 14, Issue 12 , Pages 1348-1355, December 2008
