Morbidity and Mortality Associated With Nosocomial Transmission of Oseltamivir- Resistant Influenza A(H1N1) Virus FREE

A global emergence and rapid spread of oseltamivir-resistant influenza A(H1N1) viruses carrying a neuraminidase (NA) gene with an H274Y (N2 numbering; H275Y in N1 numbering) amino acid substitution has been observed since January 2008.^1- 3 Viruses carrying this mutation are presumed to exhibit attenuated pathogenicity,⁴ compromised transmission,⁵ and reduced lethality.⁶ However, current widespread circulation of oseltamivir-resistant influenza A(H1N1) viruses associated with typical influenza illnesses and viral pneumonia suggest that these viruses retain significant transmissibility and pathogenicity.^2- 3,7- 8 While these resistant variants may cause significant mortality and retain efficient transmission, these properties have not yet been firmly established.

In February 2008, an outbreak of influenza A(H1N1) virus occurred in a medical ward at a Dutch university hospital. Clinical specimens from symptomatic contact patients of the presumed index patient were tested by influenza polymerase chain reaction (PCR) and sequences were further analyzed. Medical records of contact patients with related influenza virus infection were reviewed for underlying disease, clinical findings, and outcome. Screening specimens were obtained from contacts of the last outbreak patient (patient 4) to rule out further spread of the virus.

Influenza virus detection was performed on clinical respiratory specimens using rapid antigen testing and PCR (as described previously⁹), along with viral culture. Antigenic characterization (hemagglutination inhibition testing) and phenotypic oseltamivir and zanamivir susceptibility (IC₅₀, concentration of drug needed to inhibit enzyme activity by 50%) were determined as described.^10- 11 Viral RNA extracted from clinical specimens was further transcribed and amplified. Hemagglutinin chain 1 (HA₁) and neuraminidase gene sequences from patients with confirmed influenza virus infection were analyzed and phylogenetically related to other 2007-2008 seasonal influenza viruses obtained at the hospital or sentinel surveillance isolates collected nationwide using Bionumerics version 5.1 (Applied Maths, Sint-Martens-Latem, Belgium).

The investigation of this outbreak did not involve any planned activity that could have been reviewed prospectively by an institutional review board or ethics committee. Nevertheless, all necessary precautions were taken to prevent identification of the patients and health care workers involved. The physicians involved all gave signed permission to use clinical data and were informed on the outcome of the investigation. All of them agreed with the intention to publication. Details including the age and role of the health care workers were omitted or described in a nonspecific way, while we also took care to preserve all clinically meaningful details. The chairman of the ethics committee at the Leiden University Medical Center was consulted retrospectively and agreed to the approach as described for reporting the clinical information obtained during the investigation and included herein.

The clinical characteristics and timeline of the outbreak of influenza A(H1N1) virus are shown in the Table and in Figure 1, respectively. Community-acquired oseltamivir-resistant influenza A(H1N1) virus with NA gene H274Y mutation, isolated from the presumed index case, was detected in 3 additional patients (mean oseltamivir IC₅₀, 484 nM; mean zanamivir IC₅₀,1.1 nM). The presumed index case (patient 1), who was vaccinated for 2007-2008 seasonal influenza and received high-dose (cumulative) prednisolone therapy for systemic lupus erythematosus, was admitted to the hospital on January 29, 2008, with fever, cough, dyspnea, and lymphocytopenia. Mechanical ventilation and broad-spectrum empirical antibacterial treatment were initiated for acute respiratory failure and apparent pulmonary consolidations by chest radiography. Oseltamivir administration was initiated following influenza A virus detection using rapid antigen testing and PCR along with contact and droplet isolation. No other viral and bacterial respiratory pathogens were detected and blood cultures remained negative. The patient was transferred to a medical ward following clinical improvement on February 1 and isolation precautions were continued for the duration of symptoms until February 3. Viral clearance was confirmed by PCR upon lymphocyte reconstitution in an outpatient setting on March 4, 2008.

Two hematopoietic stem cell transplant recipients (patient 2 and patient 4) and an 89-year-old elderly patient (patient 3) developed hospital-acquired influenza A virus infection (Table). These patients were present at the same time as the index patient at the medical ward without isolation procedures (Figure 1), but the 4 patients never shared rooms. Patient 2 developed mild influenza symptoms with rapid viral clearance, whereas both patients 3 and 4 developed pulmonary consolidations and fatal respiratory failure with viral excretion under broad-spectrum antibacterial therapy. The influenza vaccination status of these patients is not known. The attribution of mortality to influenza was supported by detection of influenza A(H1N1) viral RNA from postmortem pulmonary tissue and histopathological pulmonary findings consistent with viral pneumonia in patient 4, with the exclusion of other pathogens. Further nosocomial spread to other contacts within the wards was excluded by PCR.

HA₁ gene and NA gene sequence analysis of viruses from the 4 outbreak patients revealed indistinguishable nucleotide sequences and phylogenetic clustering (Figure 2). In addition to NA gene H274Y substitution, a rare T284A substitution was sequenced (NA gene sequence GenBank accession numbers AB476754, AB476755, AB476756, AB462370) and could therefore be recognized as a marker for transmission. An unlinked national surveillance isolate (A/NL/088/08) with identical HA₁ gene nucleotide sequence revealed no NA gene phylogenetic clustering and lacked the specific T284A substitution. The NA gene T284A mutation was not observed in other 2007-2008 seasonal influenza A(H1N1) viruses or in worldwide sequences submitted to public databases, reinforcing the unique genetic relatedness of this influenza A(H1N1) virus patient cluster.

Five health care workers developed influenza-like illness (onset February 4-6, 2008) during admission of the presumed index patient. One health care worker, vaccinated for the 2007-2008 seasonal influenza, developed influenza-like illness following established contact with the index patient and continued working. Four other health care workers took sick leave within 24 to 48 hours of symptom onset. However, no samples for influenza testing were obtained from any of these 5 health care workers. Thus, their role in possibly contributing to this apparent nosocomial spread of influenza could not be confirmed.

Viruses cultured from the patient cluster revealed a poor antigenic match with the 2007-2008 vaccine reference strain A/Solomon Islands/3/06 (approximately 16-fold difference by duplicate hemagglutination inhibition testing). This may in part explain the sustained susceptibility and infectivity of the vaccinated index patient and suspected health care worker resulting in the hospital outbreak.

This outbreak provided evidence that circulating oseltamivir-resistant influenza A(H1N1) viruses with NA gene H274Y mutation are transmitted between humans. Limitations of this observational study include the small number of patients, therefore the findings require careful interpretation and do not allow conclusions on the frequency of this complication in hospital settings. The vaccination status of secondarily infected cases (patients 2, 3, and 4) remained unclarified. Information on the mechanism of spread was limited by the circumstances in this study. Data obtained from clinical specimens suggest different routes of transmission; however, this could not be further explored because the sampling of symptomatic health care workers and testing of fomites are not routinely performed. However, analysis of data obtained from clinical specimens provided some insight to different routes of transmission and suggested a limited viral spread.

Early identification and prolonged isolation precautions appear prudent in the care for infected immunocompromised patients to prevent nosocomial influenza virus outbreaks. This study confirmed that circulating H274Y-mutated A(H1N1) viruses can retain significant pathogenicity and lethality, as shown in these elderly or immunocompromised patients with lymphocytopenia, underlining the urgency for the introduction of new effective antiviral agents and therapeutic strategies.¹²