Duration of Influenza A Virus Shedding in Hospitalized Patients and Implications for Infection Control
Objective. To assess the duration of shedding of influenza A virus detected by polymerase chain reaction (PCR) and cell culture among patients hospitalized with influenza A virus infection.
Setting. Mayo Clinic (Rochester, Minnesota) hospitals that cater to both the community and referral populations.
Methods. Patients 18 years old and older who were hospitalized between December 1, 2004, and March 15, 2005, with a laboratory‐confirmed (ie, PCR‐based) diagnosis of influenza A virus infection were consecutively enrolled. Additional throat swab specimens were collected at 2, 3, 5, and 7 days after the initial specimen (if the patient was still hospitalized). All specimens were tested by PCR and culture (both conventional tube culture and shell vial assay). Information on demographic characteristics, date of symptom onset, comorbidities, immunosuppression, influenza vaccination status, and receipt of antiviral treatment was obtained by interview and medical record review. Patients were excluded if informed consent could not be obtained or if the date of symptom onset could not be ascertained.
Results. Of 149 patients hospitalized with influenza A virus infection, 50 patients were enrolled in the study. Most patients were older (median age, 76 years), and almost all (96%) had underlying chronic medical conditions. Of 41 patients included in the final analysis, influenza A virus was detected in 22 (54%) by PCR and in 12 (29%) by culture methods at or beyond 7 days after symptom onset. All 12 patients identified by culture also had PCR results positive for influenza A virus.
Conclusion. Hospitalized patients with influenza A virus infection can shed detectable virus beyond the 5‐ to 7‐day period traditionally considered the duration of infectivity. Additional research is needed to assess whether prolonging the duration of patient isolation is warranted to prevent nosocomial outbreaks during the influenza season.
Received December 18, 2006; accepted April 30, 2007; electronically published July 6, 2007.
Influenza is a highly infectious, acute respiratory illness caused by the influenza A and influenza B viruses. Influenza A infections generally occur in the form of epidemics or the influenza season that typically peaks during the period from November through February in temperate climates. In the United States, influenza A virus infection is responsible for more than 110,000 hospitalizations and 36,000 deaths annually, with most cases occurring in people with well‐defined risk factors such as age older than 65 years or younger than 2 years; chronic cardiovascular, pulmonary, or renal disease; diabetes mellitus; or immunosuppression.1 Moreover, the burden of influenza has been found to be steadily increasing in older patients despite increases in vaccination coverage. Because most people in this population with influenza A require care in the hospital, special measures are required to prevent spread of infectious influenza virus in the healthcare setting.
The influenza virus is readily transmitted by respiratory droplets that are produced when an infected person coughs or sneezes. Adults are generally considered infectious from the day before onset of symptoms until approximately 5 days after onset.1 Among healthy adult volunteers aged 18‐49 years who received trivalent intranasal live attenuated influenza vaccination, only 1 of 18 volunteers was found to be shedding influenza virus 7 days after vaccine administration.2 More prolonged periods of viral shedding have been documented in children and immunocompromised patients.3‐8
In accordance with the estimated period of infectivity in adults, current Centers for Disease Control and Prevention infection control guidelines for the prevention and control of influenza in acute care facilities advocate that patients hospitalized with suspected or confirmed influenza be placed under standard isolation precautions and droplet isolation precautions for 5 days after symptom onset.9,10 The Centers for Disease Control and Prevention also recommend that healthcare workers with influenza virus infection should not provide patient care for 5 days after symptom onset and that sick individuals should not visit hospitalized patients for 5 days after symptom onset.10 We conducted a study to assess the duration of influenza A virus shedding by use of culture and real‐time polymerase chain reaction (PCR) assay of serial specimens obtained from patients hospitalized with influenza A virus infection at our institution during the 2004‐2005 influenza season.
Methods
Study Setting, Patients, and Data Collection
This study was conducted among all patients 18 years and older who were hospitalized and had received a laboratory‐confirmed diagnosis of influenza A infection from December 1, 2004, through March 15, 2005. Information on demographic characteristics, duration of symptoms (including date of onset), receipt of antiviral treatment, comorbidities, immunosuppression, and influenza vaccination status, was obtained through direct patient interviews and a review of electronic medical records. Initial testing of respiratory tract specimens (ie, throat swab specimens, nasal swab specimens, nasopharyngeal swab specimens, bronchoalveolar lavage fluid specimens, and sputum specimens) obtained from patients with symptoms of influenza‐like illness was performed by PCR for influenza A. If the initial PCR assay was positive for influenza, additional throat swab samples were obtained at 2, 3, 5, and/or 7 days from the time the first specimen was obtained, if the patient remained in the hospital. (The initial protocol included obtaining throat swab specimens at 2, 3, and 7 days after the first specimen was obtained; however, because of short hospital stays, the protocol was modified during the study to include additional samples at 5 days after the first specimen was obtained.) Samples were collected at these predefined times regardless of the result from the preceding sample (ie, regardless of whether it tested positive or negative for influenza A). Informed consent was obtained for all testing subsequent to obtaining the initial specimen. Patients were excluded from the study if informed consent could not be obtained or if the date of symptom onset could not be ascertained.
Microbiologic Methods
All specimens were tested using real‐time PCR, shell vial assay, and tube culture.
Nucleic acid extraction for PCR. Throat swab specimens rinsed in M5 medium were extracted with a nucleic acid purification system (MagNA Pure; Roche Applied Science), reverse transcribed, and amplified with PCR by use of primers and probes specific for the matrix gene of the influenza virus. The primers were as follows: primer 1, 5′‐TAA CCG AGG TCG AAA CGT ATG TTC T; primer 2, 5′‐GGC ATT TTG GAC AAA GCG TCT A; probe (fluorescien labeled), 5′‐CGA AAT CGC GCA GAG ACT TGA AGA TGT; and probe (red), 5′‐TTG CTG GGA AAA ACA CAG ATC TTG AGG C.
Cell cultures. Throat swab specimens rinsed in M5 medium were inoculated into shell vial cell cultures that contained a mixed monolayer of human adenocarcinoma cells (strain A549) and mink lung cells (Mv1Lu) (R‐Mix; Diagnostic Hybrids). After 48 hours of incubation at 35°C‐37°C, monoclonal antibodies to influenza type A virus (Chemicon International) were added and viral‐specific foci were detected microscopically (×100 magnification) by use of direct immunofluorescence after staining with influenza A antisera. Each specimen in M5 medium was also inoculated into primary rhesus monkey kidney cell tube culture (Diagnostic Hybrids and Viromed Laboratories) and incubated at 35°C‐37°C for up to 14 days. The presence of influenza A virus in conventional tube cultures was initially detected by hemadsorption with guinea pig erythrocytes and specifically identified with the NOW Influenza A/B kit (Binax).
Data Entry and Analysis
All demographic, clinical, and laboratory data were entered into a database constructed with Microsoft Access (Microsoft). Using the date of symptom onset as day 1, the time that influenza virus was detected by each method was calculated. For the calculation of the final duration of viral shedding, patients were considered to have stopped shedding virus if the last sample obtained before discharge from the hospital was negative for influenza A by all methods. The χ2 test was used to evaluate the association of prolonged viral shedding with patient characteristics, and the Student t test was used to evaluate differences between mean duration of viral shedding. Analyses were performed with SAS software (SAS Institute). Findings were considered statistically significant at a P value of .05.
Results
Patient Characteristics
A total of 149 patients were hospitalized with a diagnosis of influenza A virus infection (confirmed by PCR) at our institute between December 1, 2004, and March 15, 2005; of these, 50 patients were enrolled in our study. The main reason patients were excluded from the study was a short hospital stay (ie, less than 48 hours) after the first diagnostic sample was found to be positive for influenza A virus. A few patients were excluded because we were unable to obtain informed consent or because we were unable to determine the time of symptom onset.
Study patients had a mean age of 72 years and a median age of 76 years (range, 21‐91 years). There was a slight predominance of males (62%). Almost all patients—48 (96%) of 50—had 1 or more of the following underlying diseases: chronic heart or lung disease, diabetes, renal failure, or immunocompromising illness. Influenza vaccination status could be determined for 47 (94%) of the patients, and 36 (77%) of these 47 had been vaccinated more than 2 weeks before receiving the diagnosis of influenza. Twenty‐seven patients (54%) received antiviral therapy. The median length of hospital stay was 5 days (range, 1‐16 days). Clinical outcomes for patients enrolled in our study are summarized in Table 1.
Duration of Influenza A Virus Shedding
The respiratory tract samples obtained at initial diagnosis were as follows: throat swab specimens, 22; nasal swab specimens, 19; both throat and nasal swab specimens (both samples positive for influenza A), 1; nasopharyngeal swab specimens, 6; sputum specimens, 1; and bronchoalveolar lavage fluid specimens, 1. Additional throat swab specimens were obtained from 49 of the 50 patients who remained in the study on day 2 after the initial diagnostic (ie, PCR‐positive) sample was obtained, from 38 of the 50 patients who remained in the study on day 3 after the sample was obtained, and from 15 of the 50 patients who remained in the study on day 5 and/or day 7 after the sample was obtained. The number of samples that tested positive for influenza A virus by each method was found to decrease with increasing time from symptom onset (Table 2).
In this study, we defined prolonged viral shedding as the detection of influenza A virus RNA by PCR at or beyond 7 days after symptom onset. Throat swab specimens were obtained from 35 patients at or beyond 7 days after symptom onset. In addition, 6 patients provided samples that returned negative results earlier than 7 days after symptom onset. These 41 patients were included in the denominator for further analyses. For the remaining 9 patients, it could not be determined whether viral shedding persisted at or beyond 7 days after symptom onset, and they were excluded from analysis. Influenza A virus target RNA was amplified by PCR in specimens from 22 (54%) of 41 patients, and influenza A virus was detected by culture (both tube culture and shell vial assay) in specimens from 12 (29%) of 41 patients at or beyond 7 days of onset of symptoms. All 12 patients identified by culture also had PCR results positive for influenza A virus. When we restricted the analysis to only those patients who had throat culture samples used for initial diagnosis (
), 20 patients were included in the denominator, and 9 (45%) tested positive for influenza A virus by PCR at or beyond 7 days after symptom onset. The longest duration of influenza A virus shedding identified by any method in this study was 14 days after symptom onset.
The characteristics of the subset of 22 patients found to have prolonged viral shedding were similar to those of the overall study population: the median age was 76 years (range, 21‐90 years), 14 (67%) were male, 20 (91%) had chronic illnesses, and 4 had overt causes of immunosuppression. The median length of hospital stay was 5 days.
We also evaluated the association of prolonged viral shedding with influenza vaccination status and antiviral treatment. Of 41 patients, 28 (68%) had received the influenza vaccine and 10 (24%) had not; vaccination status was unknown for 3 (7%) of the patients (Table 3). A total of 23 (56%) of these 41 patients were treated with antiviral drugs (Table 4). Neither prior vaccination nor antiviral therapy was found to be significantly associated with prolonged viral shedding.
A total of 32 patients were followed up to the end of viral shedding, which was defined by the last sample obtained before discharge from the hospital (ie, before a specimen with test results negative for influenza A virus). Fourteen (44%) of the patients had 1 sample with negative results before discharge from the hospital, and 18 (56%) had 2 or more samples with negative results. Of these 32 patients, 17 (53%) had received antiviral treatment and 15 (47%) had not. For those treated with antiviral therapy, the mean duration of viral shedding was 5.7 days, and the median durations was 6 days, compared with a mean of 7.5 days and a median of 7 days for those not treated with antiviral therapy. These differences were not statistically significant. The longest duration of viral shedding observed after starting antiviral treatment was 4 days (range, 0‐4 days; median, 1 day after starting antiviral treatment).
Discussion
In this study, hospitalized patients were found to shed influenza A virus detectable by both PCR (22 [54%] of 41 patients) and culture methods (12 [29%] of 41 patients) at or beyond 7 days after symptom onset. This finding raises the concern that hospitalized patients who are older and/or have chronic illnesses could shed influenza A virus beyond the 5‐ to 7‐day period that has traditionally been considered to be the time of infectivity. A period of droplet isolation precautions limited to 5 days after symptom onset, as is currently recommended by the Centers for Disease Control and Prevention to control influenza in acute care settings,10 could be insufficient for such patients, and prolonging isolation measures for the entire duration of the hospital stay might be more prudent to prevent outbreaks in hospitals during the influenza season.
Prolonged shedding of influenza viruses has been described in immunocompromised adults3‐6 and immunocompetent children.7,8 Immunity to influenza viruses is believed to result from some combination of innate immunity, specific serum immunoglobulin G antibody, mucosal immunoglobulin A antibody, and cell‐mediated immunity.11,12 It is not surprising to observe prolonged replication and shedding of influenza virus in immunocompromised hosts. Young children, who are experiencing only their first or second influenza infection and who—in the absence of vaccination—have no preexisting humoral or cellular immunity, may have similarly protracted illnesses and periods of viral shedding.
Immunity against influenza is also known to decline with advancing age, a phenomenon that is particularly well documented in association with influenza vaccination. In a recent review of 32 vaccine antibody response studies, Goodwin et al.13 reported that people over 65 years old have significantly reduced antibody response to influenza vaccination, compared with younger adults. This may be due to poor stimulation of both T cell–dependent antibody responses and cellular immune responses,14 and it is consistent with the prolonged viral shedding among older patients that we observed in our study. In addition, this same patient population is also well known to be at high risk for complications and death from influenza. Despite increases in vaccination, annual rates of hospitalization among older patients with influenza virus infection have continued to increase15 and, in conjunction with our results, this increase emphasizes the need for measures to prevent nosocomial transmission of influenza.
Almost all of the patients enrolled in our study had experienced the onset of symptoms that met the criteria for influenza‐like illness during the 2‐week period prior to hospital admission. Hospital‐acquired influenza A was suspected in 2 study patients but was not definitely traced to other patients or healthcare personnel. Although nosocomial outbreaks of influenza A did not occur at our institution during the 2004‐2005 influenza season, the results of our study also support recommendations in national guidelines for universal influenza vaccination for healthcare workers1,9 to reduce the risk of transmission of influenza virus among hospitalized patients.
In our study, the mean duration of viral shedding in those treated with antiviral therapy was 5.7 days and the median duration was 6 days. When compared with previous reports, this duration is intermediate between that demonstrated in experimental infection in healthy adults (for whom the median duration of viral shedding was 58 hours among patients treated with oseltamivir16) and that in immunocompromised hosts (for whom the mean duration of viral shedding was 9.7 days among those treated with M2 inhibitors and 7.5 days among those treated with neuraminidase inhibitors6). In addition, antiviral therapy was shown to significantly decrease the duration of influenza virus shedding in both of these patient populations but this decrease did not appear in another study of children treated with neuraminidase inhibitors.17 In the present study, the duration of shedding appeared to be shorter for those who received antiviral therapy, but this difference did not reach statistical significance.
Prolonged viral shedding has previously been found to be associated with drug‐resistant virus; patients are also more likely to shed drug‐resistant virus later in treatment.4,18 In light of the observed increases in amantadine‐resistant influenza, it has been recommended that contact be minimized between persons taking antiviral drugs and those who are not.18 Although the issue of drug‐resistant viruses was not specifically addressed in our study, the finding of prolonged viral shedding among hospitalized patients (including those treated with antiviral therapy) emphasizes the importance of this recommendation and the need to continue isolation precautions when patients receive antiviral therapy.
Some limitations of our study need to be recognized. First, our patient population was clearly a selected group of mostly older patients with chronic medical conditions. Sicker patients with prolonged hospital stays were more likely to be recruited into the study, compared with those who were dismissed sooner because of milder disease or fewer complications. However, we believe that the results of our study are applicable to similar patients hospitalized with influenza A. It is unclear if these results are generalizable to all adults with influenza, particularly younger, otherwise healthy adults treated in the outpatient setting. Second, regardless of the source of the initial diagnostic specimen, all follow‐up specimens were throat swab samples. It has been suggested that the sensitivity of throat swab samples for influenza diagnosis might be low,19 and if some throat swab specimens gave false‐negative results, we could have underestimated the duration of viral shedding in those cases. Third, we were able to detect viral shedding for longer durations by PCR, compared with the durations we could detect by culture. It is possible that PCR could amplify inactive viral RNA,20 but it is also possible that culture results could be false‐negative because of a lower level of virus excretion21 or virus inactivation during transportation.22 Because we did not obtain specimens from other patients and healthcare personnel to monitor for transmission of infectious viruses, it is unclear whether the fact that influenza A could be detected by PCR meant that the patient was infective. Finally, our determination of the total duration of viral shedding and our comparisons of hospitalized patients with and without prolonged viral shedding are subject to bias. Patients were not followed up after discharge from the hospital, and the final duration of viral shedding was unknown for several patients; in many cases, a single negative sample was used to identify the end of viral shedding, which could lead to underestimation of duration if those samples had false‐negative results. Small numbers of subjects also precluded adjustment for potential confounders, such as age or comorbid conditions. Future studies should attempt to follow up all patients until the end of viral shedding to minimize bias. Surveillance of contacts should also be attempted to help assess whether prolonged shedding is associated with continued infectivity.
In conclusion, the results of our study suggest that hospitalized patients who are older and/or have chronic illnesses could shed influenza A virus for several days beyond the period these individuals are traditionally thought to be infective. A recommendation for prolonged isolation precautions might be necessary to prevent influenza outbreaks in acute care facilities.
Acknowledgments
Potential conflicts of interest. All authors report no conflicts relevant to this article.
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