Patient no. 2 was a 2-year-old boy, present on the inpatient pediatric hematology-oncology floor for over a month, receiving inpatient care for hepatoblastoma and associated complications. This patient was likely exposed to the 2009 influenza A (H1N1) virus by HCW no. 1, on day 4 of the outbreak, and became febrile on day 6. Staff and family noted increased fussiness and a runny nose prior to development of fever, but otherwise no new symptoms. Nasopharyngeal swabs were obtained within 24 hours of the onset of fever, and oseltamivir therapy and isolation precautions were initiated. The patient initially did well, was without fever after 48 hours, and isolation precautions were discontinued after 7 days (on day 13 of the outbreak). On day 13 of the outbreak, Patient no. 2 began a new cycle of chemotherapy and developed new fevers beginning on day 20 of the outbreak with no new symptoms noted other than nasal congestion on exam. Testing for respiratory viruses, including H1N1, was performed that same day, isolation and treatment with oseltamivir were reinitiated, and the patient gradually improved with resolution of fever after 4 days.
In response to the outbreak, emphasis was placed on enhanced respiratory precautions. The nursing staff were cohorted and vaccinated with inactivated pandemic H1N1 vaccine. Other exposed patients, many of whom were immunocompromised, received oseltamivir prophylaxis. The prophylactic treatment was well tolerated, and no additional patients developed symptoms.
Initially, the outbreak appeared to be successfully contained and Patient no. 2, who had recovered well, was taken out of isolation precautions after 7 days. Approximately 1 week after the discontinuation of isolation precautions (14 days following the initial positive test), Patient no. 2 underwent chemotherapy, developed fever, and a second nasopharyngeal swab tested positive for Influenza A (the 2009 pandemic H1N1 virus) by RT-PCR. Testing for other respiratory viruses was negative. A chest radiograph was normal. The patient remained hospitalized throughout this course of events. While it is possible that the second episode represented new nosocomial acquisition, given lack of evidence of ongoing transmission, reactivation is a more likely explanation of the patient's recurrent infection. A healthcare worker (HCW no. 4) caring for Patient no. 2 between episodes, while no isolation precautions were in place, subsequently developed H1N1 infection as well. Observations to determine HCW compliance with isolation precautions were undertaken during the period of the outbreak and increased over the course of a few days from 80% to 98%. However, observations were not undertaken routinely prior to the outbreak.
Three of the four healthcare workers tested positive for H1N1 at onset of symptoms - one was treated presumptively and did not undergo testing. All four healthcare workers were treated with oseltamivir for 5 days and responded to treatment. They returned to work 1 week after treatment was initiated. A follow-up nasopharyngeal swab was not required or obtained, but all were free of symptoms at the time of returning to work.
To determine whether these epidemiologically linked cases of influenza were initiated by a single source patient, we performed genetic analyses on viruses isolated from nasopharyngeal swabs taken from both patients and from the two healthcare workers who were tested. We first isolated influenza vRNA from each sample and determined its concentration using a quantitative reverse-transcription (QRT-) PCR assay. The concentration of viral vRNA ranged from 70 000 to 51 million copies/ml.
Because of the relatively low concentration of vRNA in Patient no. 2's samples, we chose to focus our genetic analysis on two of the eight segments of the influenza virus RNA genome. The highly variable HA gene encodes the viral attachment protein and is the major target of virus-specific antibodies, which can drive diversification of virus sequences. 7  We reasoned that if the nosocomial outbreak was initiated by more than one virus, then sequence differences distinguishing the strains would be most apparent in HA.
We also analyzed the basic polymerase subunit 2 (PB2) gene, which is more tightly conserved than HA, but encodes well-characterized pathogenicity determinants. 8  We were able to sequence full-length HA and PB2 genes directly from all available samples except for Patient no. 2's second sample; sequence data from this sample was, therefore, derived from an in vitro-expanded virus stock. The HA and PB2 segments of viruses isolated from all patients were completely identical to each other (Figure S1). blast database searches revealed no previously identified influenza viruses that matched these sequences exactly at the nucleotide level, but both the HA and PB2 genes in viruses isolated from these patients showed 99% identity to contemporaneous viruses isolated in Southeastern Wisconsin (http://www.ncbi.nlm.nih.gov/nuccore). To estimate the genetic distance of viruses in this outbreak from the contemporaneous virus population, we performed a phylogenetic analysis of HA and PB2 genes using the vaccine strain A/California/07/2009 (CA07; H1N1), isolated early in the pandemic, as an outgroup (Figure S2). Although the topologies of the HA and PB2 trees differed slightly, in both trees, the UW Hospital isolates clustered together, as expected for identical sequences. Indeed, the CA07 sequences, isolated in April 2009, clustered closely together with Southeastern Wisconsin isolates from September to December 2009, suggesting that all these viruses are closely related. Our results are consistent with the conclusion that Patient no. 1 had a community-acquired infection with 2009 influenza A (H1N1) virus and was the sole source of this nosocomial outbreak.