Influenza Outbreaks during World Youth Day 2008 Mass Gathering

Novel viruses were introduced and seasonal viruses were amplified.

I nfl uenza is caused by a highly infectious respiratory virus with the potential to rapidly spread in susceptible hosts. Infl uenza outbreaks have frequently been described in populations such as residents of nursing care facilities (1,2), residential schools or colleges (3,4), prisons (5), military facilities (6), and other enclosed communities (7).
Mass gatherings pose complex and unique challenges to public health and medical services. Because populations are increasingly mobile, and more able to attend large gatherings, the risk for outbreaks of infl uenza and other infectious diseases among a susceptible population has increased, and a substantial responsibility is placed on health services if outbreaks occur. Despite this situation, infl uenza outbreaks during mass gatherings have rarely been described (8)(9)(10), and reports have not included results of detailed virologic testing. Furthermore, the effects of outbreak strains on local infl uenza epidemiology have not been assessed.
During World Youth Day (WYD2008) celebrations, 223,000 predominately young pilgrims from 170 countries attended a series of mass religious gatherings from July 15 to July 20, 2008, in Sydney, New South Wales (NSW), Australia. At the end of the week, an outdoor evening vigil with >200,000 participants preceded the fi nal mass presided over by Pope Benedict XVI. This mass was attended by an estimated 400,000 persons (11). Approximately 100,000 pilgrims were given accommodation in sporting facilities, schools, and community centers, where temporary fl oor mats and blankets were provided and other facilities were shared. The largest site of accommodation, the Sydney Olympic Park site, provided lodging for as many as 12,000 pilgrims each night. Pilgrims remained housed overnight at the allocated accommodation sites and attended numerous outdoor religious gatherings during the day with other pilgrims. Many pilgrims traveled in Australia and New Zea-Infl uenza Outbreaks during World Youth Day 2008 Mass Gathering land before and after the WYD2008 celebrations, visiting major cities and rural areas.
We describe the epidemiologic and virologic features of an infl uenza outbreak predominantly among young adults during WYD2008 celebrations. These data provide insight into the complexity of infl uenza outbreaks during mass gatherings and their effects on the community at large. The insights gained should guide plans for mass events, particularly when held during periods of peak infl uenza activity.

Methods
Infl uenza was fi rst identifi ed among WYD2008 pilgrims on July 16, 2008. Emergency clinics were then quickly established to identify and isolate infected pilgrims. Symptomatic pilgrims were encouraged to visit the clinics, which were open 24 hours a day. Epidemiologic data were collected prospectively from all pilgrims who sought treatment. Respiratory tract samples (paired nose and throat swabs specimens) were obtained as previously described (12).
Infl uenza testing included the following: 1) point-ofcare tests (POCTs) performed either on site or in the laboratory (Quickvue A & B; Quidel, San Diego, CA, USA, or BinaxNOW Infl uenza A & B, Binax, Scarborough, ME, USA), 2) antigen detection using type-specifi c indirect fl uorescent antibodies (IFA) (Chemicon, Millipore, Billerica, MA, USA, or Bartels, Immunodiagnostic Supplies Inc., Bellevue, WA, USA); 3) validated in-house type-and subtype-specifi c nucleic acid testing (NAT) by using PCR that targeted the matrix, nonstructural, and hemagglutinin region of the infl uenza virus genome; and 4) virus culture using MDCK cells (13). IFA and NAT were the preferred diagnostic methods. The decision to perform POCT was made on a case-by-case basis by clinicians. Viral culture was performed on antigen-(POCT/IFA) or NAT-positive specimens. Testing was performed at 2 virology laboratories (Institute of Clinical Pathology and Medical Research, Westmead Hospital, Sydney, New South Wales, Australia; South Eastern Area Laboratory Service, Prince of Wales Hospital, Randwick, New South Wales).
Pilgrims with clinical or laboratory-confi rmed infl uenza who sought treatment within 48 hours of symptom onset were offered oseltamivir (75 mg 2×/d for 5 days). Public health authorities recommended that infected pilgrims remain in isolation for 48 hours or until 5 days after symptom onset.
All infl uenza isolates from WYD2008 were sent to the WHO Collaborating Centre for Reference and Research on Infl uenza in Melbourne, Victoria, Australia, where antigenic analysis was performed by using a hemagglutinationinhibition assay (14). Oseltamivir susceptibility was tested by using a fl uorescence-based neuraminidase (NA) inhibition assay (15). When no isolate was available, clinical samples were directly tested by rolling circle amplifi cation for the H274Y mutation (the most frequently reported mutation conferring oseltamivir resistance) (16). Hemagglutinin (HA) and NA gene sequencing was performed by using standard methods (17). Sequence alignment was performed by using ClustalW (www.ebi.ac.uk/Tools/clustalw2/index. html) in DNAstar Lasergene version 8 (www.dnastar.com), and phylogenetic trees were generated by using maximumlikelihood (DNAML) in PHYLIP (18).
Australia-wide laboratory-confi rmed infl uenza data were obtained from the National Notifi able Diseases Surveillance System (19). The infl uenza viruses isolated during WYD2008 were compared with viruses submitted to the WHO Collaborating Centre from all Australian states and territories during the 2008 infl uenza season. HA and NA sequences from WYD2008 viruses were compared with a representative sample of seasonal infl uenza viruses from around the world, including Australia, sequenced by the WHO Collaborating Centre.

Results
Respiratory tract samples were obtained from 227 WYD2008 attendees who sought treatment at established clinics. The true extent of infection is unknown because the pilgrims voluntarily visited the clinics, and respiratory tract sampling was limited at several accommodation sites after the outbreak was identifi ed. The median age of the pilgrims tested was 21 years (range 12-72 years, interquartile range 18-28 years); 62.8% were female. Twenty-nine percent of pilgrims tested lived in Australia, and the remainder were from overseas (Europe, 28.0%; Oceania, 20.2%; North America, 17.1%; South or Central America, 2.6%; Asia, 2.6%; Africa, 0.5%). Recent infl uenza vaccination was infrequent; 25 (21.6%) of 116 reported recent vaccination (Southern Hemisphere, 12.0%; Northern Hemisphere, 27.9%; p = 0.021). Demographic characteristics of pilgrims who visited established clinics were not signifi cantly different from those of the total pilgrim population (11).
Two or more infl uenza diagnostic tests were performed on all specimens (POCT, 80%; IFA, 100%; NAT, 97%; and virus culture, 43%). Laboratory confi rmation of infl uenza virus infection was obtained for 100 (44.1%) pilgrims. This included 69 patients whose test results were positive by both antigen detection (POCT or IFA) and NAT, 21 patients with positive results by NAT yet negative by antigen detection, 5 patients with positive results by both antigen detection and viral culture, and 5 patients with positive results by 2 antigen detection methods (when clinical material was not suffi cient for NAT or culture). Pilgrims had symptoms for a median of 2 days before they visited a clinic (95% confi dence interval 1.7-2.7 days). No signifi cant differences in age, sex, or country of origin were noted between the pilgrims with laboratory-confi rmed cas-es, pilgrims whose test results were negative, and pilgrims who were not tested (data not shown).
Infl uenza types A and B were identifi ed during WYD2008 (Table). Infl uenza A was most frequently isolated from patients from Australia and Germany, whereas infl uenza B was most frequently isolated from patients from the Solomon Islands, Papua New Guinea, Australia, and North America. Numerous distinct circulating infl uenza viruses were identifi ed: oseltamivir-resistant infl uenza A ( This outbreak occurred in the context of low seasonal infl uenza activity in Australia (Figure 1). An increase in infl uenza A and B activity was identifi ed in all Australian states and territories in the weeks after WYD2008. Infl uenza isolates from WYD2008 were compared with a representative sample of national seasonal infl uenza isolates (12.7% of the total Australian 2008 laboratoryconfi rmed infl uenza cases). Before WYD2008, infl uenza (H3N2) A/Brisbane/10/2007-like and B/Florida/4/2006like viruses were the predominant early season viral subtypes/strains observed Australia-wide (33.3% and 58.8%, respectively; Figure 2). Subsequent to WYD2008, B/ Malaysia/2506/2004-like and B/Florida/4/2006-like viruses were the most frequently identifi ed infl uenza strains (41.0% and 35.7%, respectively).
The genetic relatedness of WYD2008 viruses to preand post-WYD2008 viruses was examined by sequence alignment of the HA gene ( Figure 3) and NA gene (data not shown).
Before WYD2008, oseltamivir-resistant infl uenza A (H1N1) Brisbane/59/2007-like viruses were uncommon in Australia, and only 3 cases had been identifi ed ( Figure  2). Two cases were observed in South Australia 5 weeks and 3 days before WYD2008 (Figure 3, panel A, isolates 1-2). The remaining case was identifi ed in Victoria in a returned traveler 5 days before WYD2008 (Figure 3, panel A, isolate 3). No obvious epidemiologic links were identifi ed between pre-WYD2008 and WYD2008 oseltamivirresistant infl uenza A (H1N1) viruses. Furthermore, isolates 1-3 appeared distinct from WYD2008 isolates. All WYD2008 infl uenza A (H1N1) isolates clustered relatively closely ( Figure 3, panel A, isolates 4-16) and shared sequence homology with other 2008 Southern Hemisphere oseltamivir-resistant isolates. Epidemiologic data showed that most Australian and European pilgrims infected with oseltamivir-resistant A (H1N1) had traveled from Victoria to Sydney in the week before WYD2008. Despite this, oseltamivir-resistant A (H1N1) viruses were responsible for <10% of typed infl uenza virus infections. (Figure 2; Figure  3, panel A, isolates [17][18][19]. Genetic analysis of WYD2008 infl uenza A (H3N2) A/Brisbane/10/2007-like viruses demonstrated 2 distinct phylogenetic groups. The fi rst infl uenza A (H3N2) cluster was obtained from Australian and New Zealand pilgrims only and was genetically related to sporadic Australian pre-and post-WYD2008 infl uenza isolates (Figure 3, panel B: pre-WYD2008, isolates 1-6; WYD2008 Australian/NZ cluster, isolates 7-8; post-WYD2008, isolates 9-13). The second phylogenetic group included infl uenza A (H3N2) viruses isolated from pilgrims from Germany and Italy (Figure 3,  dence of similar strains was found in Australia before or after WYD2008. Before WYD2008, only 4 cases of infl uenza B/Malaysia-like virus infection were identifi ed Australia-wide ( Figure 2). Two isolates from NSW were found to be distinct from WYD2008 isolates by sequence analysis ( Figure  3, panel C, isolates 1-2). The remaining viruses ( Figure 3, panel C, isolates 3-4) were identifi ed in Queensland 2 and 5 days before WYD2008 and appeared to be closely related to WYD2008 isolates ( Figure 3, panel C, isolates 5-10). No epidemiologic links to WYD2008 cases were identifi ed. Pilgrims from the Solomon Islands sought treatment for a febrile illness at rural medical facilities 5 days before WYD2008, while they were staying close to the NSW/ Queensland border. Subsequent review suggests that these illnesses were most likely infl uenza. WYD2008 pilgrims with confi rmed infl uenza B/Malaysia-like virus infection were identifi ed among contacts of these original clinical case-patients. Sequence analysis of WYD2008 isolates ( Figure 3, panel C, isolates 5-10) clustered closely with isolates obtained throughout Australia post-WYD2008 ( Figure 3, panel C: isolates [11][12][13][14][15][16][17]. Because several pilgrims infected with infl uenza B/Malaysia-like viruses either lived or traveled through New Zealand en route to Sydney, we explored the idea that the infl uenza B/Malaysia from New Zealand was introduced. Peak infl uenza B/ Malaysia activity in New Zealand occurred 4 weeks before peak activity was detected in Australia (20). Furthermore, WYD2008 isolates clustered closely with pre-and post-WYD2008 New Zealand isolates (Figure 3, panel C). Infl uenza B/Malaysia-like viruses also could have been introduced into both New Zealand and Australia from Pacifi c Island countries.No clear trends were identifi ed with oseltamivir-sensitive infl uenza A (H1N1) and infl uenza B/ Florida/4/2006-like viruses when WYD2008 and community isolates were compared (data not shown).

Discussion
Thousands of mass gathering events are held each year, including major sporting events, festivals, demonstrations, and pilgrimages. Mass gatherings of a scale seen with WYD2008 have the potential to create health risks for those attending and the community at large. Although outbreaks of communicable diseases during mass gatherings have been described, insuffi cient epidemiologic and pathogen-related data have been described to characterize the outbreak, measure the impact on the wider community, or guide management of future mass gatherings (8)(9)(10). WYD2008 presented a unique opportunity to study the effects of infl uenza on mass gatherings by combining epidemiologic data acquired through prospective data collection, laboratory data obtained after respiratory tract sampling, and surveillance data obtained through established Australia-wide infl uenza laboratory networks.
A notable infl uenza outbreak occurred during WYD2008, likely exacerbated by crowded living conditions, the presence of multiple circulating infl uenza viruses, and low immunization rates. The true effects of infl uenza infection are unknown because not all infected pilgrims visited clinics and respiratory tract sampling was not performed on all clinic patients. The epidemiologic and virologic data gathered during WYD2008 highlight the complexity of an infl uenza outbreak within a large mass gathering. At least 6 distinct viruses circulated among pilgrims during WYD2008; 2 distinct infl uenza A (H1N1) viruses (oseltamivir-resistant and -sensitive), 2 distinct infl uenza A (H3N2) viruses (Australian/New Zealand and European clusters), and 2 distinct infl uenza B viruses (B/Malaysia-like and B/Florida-like). Different viruses were more likely to circulate in different groups within the pilgrim population, which suggests that exposure was not random but infl uenced by country of origin and travel before WYD2008.

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Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 16 Mass gatherings with attendees traveling from overseas allow for the introduction of novel infl uenza viruses as well as the amplifi cation of preexisting community strains. Among the strains identifi ed during WYD2008, infl uenza A (H3N2) viruses (Australian/New Zealand cluster) and B/Florida-like viruses appear closely related to viruses that were circulating in the community before WYD2008. Although rarely isolated before WYD2008, oseltamivirresistant infl uenza A/H1N1 and B/Malaysia-like viruses isolated from pilgrims are potentially related to viruses cultured from nonpilgrims in South Australia, Victoria, and Queensland, raising the possibility of local acquisition rather than introduction from overseas. Other viruses, such as infl uenza A (H3N2) (European cluster), were not detected in Australia before WYD2008 and thus appear to have been introduced with the pilgrims.
Exploring the infl uence of a mass gathering on community infl uenza activity is complex, especially when numerous viruses circulate during such an event. In 2008, the total number of laboratory-confi rmed infl uenza cases in Australia was 1.9× the 5-year average yet not signifi cantly different from the number of cases diagnosed in 2007 (19). WYD2008 coincided with the start of the normal infl uenza season in Australia, which is usually greatest between July and September (21,22). Thus, the rapid rise in infl uenza A and B activity in all states after WYD2008 may have occurred despite WYD2008. To add to the complexity are the differing and unpredictable consequences of each individual viral subtype/strain. This complexity is illustrated by oseltamivir-resistant A (H1N1) and the B/Malaysialike infl uenza viruses, which were rarely detected before WYD2008 but responsible for 24%-34% and 13%-35% of infections in pilgrims tested. Pilgrim groups had substantial contact with nonpilgrims through travel in Australia before and after WYD2008. Despite this contact, post-WYD2008 dissemination of infl uenza A (H1N1) was modest compared with dissemination of infl uenza B/Malaysia-like viruses (A [H1N1], 8.1% of all post-WYD2008 isolates; B/Malaysia, 41.0% of all post-WYD2008 isolates).
A potential explanation for the substantial effect of infl uenza B/Malaysia-like viruses observed post-WYD2008 is reduced community immunity to infl uenza B. In 2007, infl uenza B was an infrequent pathogen (6.9% of total isolates typed); infl uenza A (H3N2) and A (H1N1) viruses were most frequently detected (58.7% and 34.4% of total isolates typed or subtyped) (21). In the 2003-2006 infl uenza seasons, infl uenza B was responsible for a low prevalence of disease compared with infl uenza A (6%-35% of total infl uenza isolates typed) (22)(23)(24)(25)  Those who plan future mass gatherings need to consider the potential for infl uenza outbreaks. As observed, multiple viruses may circulate among those attending, thus increasing the opportunity for the emergence of novel reassortment viruses. Public health services need to be prepared to establish clinics rapidly and likely at many locations. Rapid diagnostic testing needs to be available, and laboratories need to be prepared for a rapid infl ux of specimens. Because viruses may be introduced, reliance on local rates of antiviral resistance may be misleading and resistance data from other countries may not be available. The circulation of oseltamivir-resistant seasonal infl uenza A (H1N1) and emergence of oseltamivir-sensitive infl uenza A pandemic (H1N1) 2009 virus (26) highlight the need for rapid typing of infl uenza viruses during outbreaks to guide the public health response. In infl uenza outbreaks in which circulating viruses include resistant seasonal infl uenza A (H1N1) virus, reliance on oseltamivir alone is likely to be insuffi cient. Infl uenza vaccination of all those attending should be recommended before mass gatherings, especially when held during the host countries' infl uenza season and given the likely emergence of antiviral resistance. Although pilgrims were encouraged to be vaccinated before attending WYD2008, the rate of recent vaccination was low. Because numerous viruses may circulate, vaccination may be insuffi cient to protect against all infl uenza strains.
This study has several limitations. First, clinics were established rapidly and involved numerous clinicians; thus, the prospectively collected data were not standardized. Sec-ond, only pilgrims who visited clinics with symptoms were assessed, and the proportion of the pilgrim population that had an infl uenza-like illness or asymptomatic infection is unknown. Third, the actual proportions of viruses detected and the pilgrim populations infected may differ from those described because respiratory tract sampling was restricted at several clinics after the identifi cation of an outbreak. Fourth, Australian infl uenza type/subtype data used for comparison may be infl uenced by a referral bias from statebased reference laboratories. Finally, only representative isolates sent to the WHO Collaborating Centre underwent sequence analysis, so further related or unrelated isolates may not have been analyzed.
After epidemiologic and virologic assessment of the WYD2008 outbreak, we highlight the complexity of infl uenza outbreaks that occur during mass gatherings with numerous viruses co-circulating among attendees. Mass gatherings enable introduction of novel infl uenza strains into the local population and the amplifi cation of circulating local seasonal infl uenza strains, or both, thereby increasing the opportunity for novel reassortment infl uenza viruses to emerge. This introduction or amplifi cation of viruses in contained outbreaks may alter seasonal infl uenza activity subsequent to a mass gathering. The resultant effect on seasonal infl uenza activity is infl uenced by many competing forces, including population movement and preexisting immunity, and thus remains unpredictable. The potential for a substantial infl uenza outbreak needs to be considered before all mass gathering events, particularly when hosted during the months of peak infl uenza activity (27,28). Greater fl exibility by public health and hospitals is required to appropriately manage and contain these outbreaks.