Contagious Period for Pandemic (H1N1) 2009

Most infected persons shed live virus after fever abated.


S ince April 2009, an infl uenza A virus, pandemic (H1N1)
2009, has spread to most countries of the world. Widespread susceptibility of persons <60 years of age may have facilitated rapid dissemination (1). Transmissibility of infl uenza viruses depends on duration of shedding, amount of virus shed, and other factors that may facilitate projection of virus into the environment, such as coughing or sneez-ing. Challenge studies in healthy volunteers inoculated with seasonal infl uenza viruses have shown that shedding generally coincides with symptom onset starting 1 day after inoculation, peaks on the second day, and generally ends 1 week after disease onset, on day 8 (2). Duration of shedding is greatly affected by age, and for seasonal infl uenza viruses is longer in young children than in adults (3)(4)(5). Since the emergence of pandemic (H1N1) 2009, the recommended duration of self-isolation has varied from complete resolution of symptoms to 1 day after fever has subsided (6,7). The objective of this study was to estimate the proportion of pandemic (H1N1) 2009-infected persons shedding infectious virus 1 week after illness onset.

Study Setting
The research ethics committee of the Centre Hospitalier Universitaire de Québec approved the study. The prospective study was conducted during May 27-July 10, 2009, in Quebec City, Quebec, Canada. All participants were community based. Eligible persons were members of households in which at least 1 person with pandemic (H1N1) 2009 confi rmed by reverse transcription-PCR (RT-PCR). Primary case-patients were either referred by their treating physicians or identifi ed among community contacts of persons with laboratory-confi rmed cases (Figure 1). At the initial home visit, a nurse obtained written informed consent from all household members and collected data for each household participant by using a standardized questionnaire. The questionnaire asked about basic sociodemographic characteristics (e.g., age, sex, occupation), presence of underlying medical conditions, presence of various symptoms or signs (i.e., fever, chills, cough, sore throat, rhinorrhea, arthralgia/myalgia, fatigue, (H1N1) 2009 dyspnea, headache, diarrhea, or vomiting), seasonal infl uenza vaccination history, and social and healthcare impact of their illness (i.e., missed workdays or schooldays, days spent in bed, medical consultations, emergency department visits, or hospital admissions).

Nasopharyngeal Swabs and Laboratory Procedures
Nasopharyngeal (NP) secretions were collected from all household members, both symptomatic and asymptomatic, with NP swabs (Nylon Flocked Swabs; Copan Innovation, Brescia, Italy) that were inserted into 3 mL of universal transport medium (Copan Innovation). Patients with results positive by RT-PCR for pandemic (H1N1) 2009 before day 7 since disease onset had a second NP swab repeated on day 8. Persons still positive on day 8 were retested on day 11.
Nucleic acids were extracted from 200 μL of specimen by using the QIAGEN Viral RNA Mini Kit (QIA-GEN, Mississauga, Ontario, Canada). Initially, pandemic (H1N1) 2009 was detected by using a conventional RT-PCR (pH1N1 PCR) specifi c for the hemagglutinin gene (8). RNA extracts from pH1N1 PCR-negative specimens were frozen at -80°C and, once specimen collection was completed, were retested by using a conventional RT-PCR assay for all infl uenza A viruses (matrix PCR), which has a higher sensitivity (9).
All samples with RT-PCR-positive results were cultured on MDCK cells in shell vials containing 1 mL of media to detect replicating viruses. Vials were observed daily for 7 days to detect cytopathic effects. Virus cultures were conducted on fresh specimens if they were positive by the pH1N1 PCR, whereas samples positive by the convention-al matrix PCR had undergone 1 freeze-thaw cycle. All cultures showing cytopathic effects were sent to the Québec provincial reference laboratory for RT-PCR confi rmation by using the previously described pH1N1 PCR.

Statistical Analyses
We compared proportions and distributions using the χ 2 test or Fisher exact test when appropriate. All statistical analyses were 2-tailed, and p values <0.05 were considered signifi cant.

Results
Of the 173 persons from 47 participating households, 35 with pH1N1 PCR-confi rmed persons (index case-patients) were referred by their treating physicians. Among the 138 other participants (household or community contacts), 73 had respiratory symptoms for <7 days at the time of enrolment and 32 (44%) of 73 were positive by pH1N1 PCR (Figure 1, Table 1). Cell culture was also positive for 97% (31/32) of these pH1N1 PCR-confi rmed cases. Of the 32 pH1N1 PCR-confi rmed cases, 78% (25/32) had fever at some point since illness onset; at the time of specimen collection, 94% (30/32) had cough and 34% (11/32) were still febrile. Specimens were retested with a matrix PCR. All results from the pH1N1 PCR-positive participants were also positive by matrix PCR; of those who were negative by pH1N1 PCR, 37% (15/41) were positive. Of the 15 participants positive by matrix PCR, 13% (2/15) were positive by cell culture. Of the 47 confi rmed cases (32 by pH1N1 PCR and 12 by matrix PCR), cell culture positivity varied from 69% to 87% for specimens obtained within 4 days after symptom onset and dropped to 33%-40% for specimens obtained days 5 and 6 after symptom onset ( Figure 2).
Of the 67 patients whose results were positive by pH1N1 PCR (35 case-patients initially referred by their physicians plus 32 detected among contacts), 62 were identifi ed <7 days after symptom onset and 43 had a second swab collected on day 8 ( Figure 1). Of these 43 cases (core participants), 47% were children <10 years of age, 26% were 10-17 years of age, and 28% were adults (>18 years of age) ( Table 1). On day 8, 18 (42%) were still positive by pH1N1 PCR, and 14 others were positive only by matrix PCR, for a total of 74% (32/43) positive by any PCR method on day 8. (Table 2) Virus culture was positive for 19% (8/43) of the patients: culture was positive only for pH1N1 PCR-positive cases (8/18, 44%) but for none of the cases positive only by matrix PCR. PCR and virus culture positivity rates did not differ among age groups ( Table 2). Only 5% (2/43) of case-patients were still febrile on day 8, but 91% (39/43) were still coughing. None of the 8 patients who had a positive virus culture on day 8 were febrile, but 7 (88%) were still coughing. Another swab was repeated on day 11 for 16 of the 18 case-patients who were positive by 784 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 16 Of the 73 symptomatic participants tested within 7 days after symptom onset, 15 of the 47 PCR-positive casepatients (in gray in Figure 1) were detected only by matrix PCR (2/15 cell culture positive) ( Table 1). Of specimens positive by pH1N1 PCR, 97% (31/32) were culture positive at diagnosis, 44% (8/18) on day 8, and none on day 11 of illness. Of those whose results were positive by matrix PCR, 13% (2/15) were culture positive at diagnosis, and none were positive on days 8 or 11 of illness. Because virus culture was much less frequently positive for specimens positive only by matrix PCR (2/39, 5%) than for specimens positive by pH1N1 PCR (39/54, 72%), the 19% virus culture positivity on day 8 among core participants (all pH1N1

Discussion
Human challenge studies with seasonal infl uenza have shown that virus shedding after day 7 is rare (2), but clinical studies have shown that shedding may persist beyond that period in some populations, such as elderly persons, immunocompromised patients, and children (3)(4)(5)(10)(11)(12). In a study among hospitalized persons infected with seasonal infl uenza A viruses, 54% remained positive by PCR beyond 7 days after symptom onset, and 29% were positive by cell culture (13). In another study, elderly hospitalized patients infected by infl uenza A (H3N2) viruses had higher virus loads than did outpatients, and their PCR positivity rate 1 week after disease onset was still high (57%) (10).
In this prospective study, the proportion of pandemic (H1N1) 2009-infected persons still shedding replicating virus on day 8 varied from 8% to 13%, with no difference between children and adults. None were still shedding infectious virus on day 11. With seasonal infl uenza, virus shedding may be longer in children because they have less preexisting immunity that would limit replication than in adults. However, children and adults <50 years of age appear equally susceptible to infection with pandemic (H1N1) 2009 virus, which would support our fi ndings of comparable virus replication and shedding across age groups studied (1). Our study had some limitations. First, our small sample size and study design may have limited our ability to directly measure culture positivity on day 8. Only a small number of patients had a specimen collected on day 8 and even fewer on day 11. In retrospect, a better design would have been to collect specimens from all 73 symptomatic household members on day 8, irrespective of the initial pH1N1 PCR result. That design would have enabled a more direct estimate of the proportion of patients who were culture positive on day 8, rather than the indirect approach we used. However, our extreme scenario (which assumes that all 73 symptomatic contacts were infected) provides the minimal positivity rate on day 8, and testing of all 73 on day 8 could only have found a proportion equal to or greater than our 8% estimate.
Second, our sampling methods could have infl uenced positivity rates. Although collection of NP specimens with a fl ocked swab is one of the best methods for obtaining specimens to detect infl uenza, those specimens might have been improperly collected by the nurses. Suboptimal collection of swabs would have yielded false-negative PCR or cell culture results, which in turn would have underestimated the proportion of patients shedding virus on day 8.
Third, PCR testing with the matrix PCR was conducted retrospectively on frozen specimens, and only 5% of those were positive by virus culture. A greater proportion of virus culture specimens might have been positive if those specimens had been processed immediately instead of going through a freeze-thaw cycle (14). Moreover, our study included only ambulatory patients ,whereas studies of seasonal infl uenza that include hospitalized or immunocompromised persons show prolonged shedding, contributing to the impression that our fi ndings most likely underestimate the true proportion of case-patients still shedding virus on day 8. The strengths of our study include its prospective design in a family setting and its use of various methods, including 2 PCR assays and virus culture, to de-   (17).
No defi nitive test is available for assessing the real contagiousness of a patient. The presence of replicating, and therefore infectious, infl uenza virus is an absolute prerequisite for contagiousness, but it does not necessarily imply it. Contagiousness depends on many factors, including viral load and presence of clinical characteristics contributing to spread of droplets (such as coughing, rhinorrea, or sneezing) and is affected by the number and proximity of contacts between a case-patient and a susceptible person. Nevertheless, our study raises concerns about current recommendations for self-isolation until only 24 hours after fever has subsided (6). With pandemic (H1N1) 2009, fever generally persists 1-4 days and may be absent in 6%-11% of patients (1,15). In our study, of the 32 pH1N1 PCR-positive household members who had been symptomatic for <7 days, 78% had fever at any time since onset of their illness, but only 34% were still febrile on the day they tested positive. Nonetheless, 97% of specimens obtained from these patients were positive by cell culture. Our sample size was insuffi cient to directly compare PCR or culture positivity by fever status or other symptom or severity indicator at specimen collection or as a component of the overall illness.
Before policy implications can directly follow from these fi ndings, the association of self-isolation with substantial social impact needs to be carefully weighed against the possible benefi ts of reducing community transmission. In the general population, a 1-week self-isolation period seems more likely to prevent transmission than does isolation until fever has resolved. However, given that 8%-13% of patients may still shed infectious virus on day 8, longer periods of self-isolation for persons expected to come into contact with vulnerable persons (e.g., pregnant women, newborns, or immunocompromised persons) also may be prudent.