Panspecies molecular assays detect viral pathogens missed by real-time PCR/reverse-transcriptase PCR among pneumonia patients, Sarawak, Malaysia.

BACKGROUND: In a year-long pneumonia etiology study conducted June 2017 to May 2018 in Sarawak, Malaysia, 599 patients' nasopharyngeal swab specimens were studied with real-time polymerase chain reaction (rPCR)/ reverse-transcription (rRT-PCR) assays for respiratory pathogens known to contribute to the high burden of lower respiratory tract infections. The study team sought to compare real-time assay results with panspecies conventional molecular diagnostics to compare sensitivities and learn if novel viruses had been missed. METHODS: Specimens were studied for evidence of adenovirus (AdV), enterovirus (EV) and coronavirus (CoV) with panspecies gel-based nested PCR/RT-PCR assays. Gene sequences of specimens positive by panspecies assays were sequenced and studied with the NCBI Basic Local Alignment Search Tool software. RESULTS: There was considerable discordance between real-time and conventional molecular methods. The real-time AdV assay found a positivity of 10.4%; however, the AdV panspecies assay detected a positivity of 12.4% and the conventional AdV-Hexon assay detected a positivity of 19.6%. The CoV and EV panspecies assays similarly detected more positive specimens than the real-time assays, with a positivity of 7.8% by the CoV panspecies assay versus 4.2% by rRT-PCR, and 8.0% by the EV panspecies assay versus 1.0% by rRT-PCR. We were not able to ascertain virus viability in this setting. While most discordance was likely due to assay sensitivity for previously described human viruses, two novel, possible zoonotic AdV were detected. CONCLUSIONS: The observed differences in the two modes of amplification suggest that where a problem with sensitivity is suspected, real-time assay results might be supplemented with panspecies conventional PCR/RT-PCR assays.

Frequency of influenza H3N2 intra-subtype reassortment: attributes and implications of reassortant spread.

BACKGROUND: Increasing evidence suggests that influenza reassortment not only contributes to the emergence of new human pandemics but also plays an important role in seasonal influenza epidemics, disease severity, evolution, and vaccine efficacy. We studied this process within 2091 H3N2 full genomes utilizing a combination of the latest reassortment detection tools and more conventional phylogenetic analyses. RESULTS: We found that the amount of H3N2 intra-subtype reassortment depended on the number of sampled genomes, occurred with a steady frequency of 3.35%, and was not affected by the geographical origins, evolutionary patterns, or previous reassortment history of the virus. We identified both single reassortant genomes and reassortant clades, each clade representing one reassortment event followed by successful spread of the reassorted variant in the human population. It was this spread that was mainly responsible for the observed high presence of H3N2 intra-subtype reassortant genomes. The successfully spread variants were generally sampled within one year of their formation, highlighting the risk of their rapid spread but also presenting an opportunity for their rapid detection. Simultaneous spread of several different reassortant lineages was observed, and despite their limited average lifetime, second and third generation reassortment was detected, as well as reassortment between viruses belonging to different vaccine-associated clades, likely displaying differing antigenic properties. Some of the spreading reassortants remained confined to certain geographical regions, while others, sharing common properties in amino acid positions of the HA, NA, and PB2 segments, were found throughout the world. CONCLUSIONS: Detailed surveillance of seasonal influenza reassortment patterns and variant properties may provide unique information needed for prediction of spread and construction of future influenza vaccines.

The Epidemiology, Virology and Clinical Findings of Dengue Virus Infections in a Cohort of Indonesian Adults in Western Java.

BACKGROUND: Dengue has emerged as one of the most important infectious diseases in the last five decades. Evidence indicates the expansion of dengue virus endemic areas and consequently the exponential increase of dengue virus infections across the subtropics. The clinical manifestations of dengue virus infection include sudden fever, rash, headache, myalgia and in more serious cases, spontaneous bleeding. These manifestations occur in children as well as in adults. Defining the epidemiology of dengue in a given area is critical to understanding the disease and devising effective public health strategies. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report the results from a prospective cohort study of 4380 adults in West Java, Indonesia, from 2000-2004 and 2006-2009. A total of 2167 febrile episodes were documented and dengue virus infections were confirmed by RT-PCR or serology in 268 cases (12.4%). The proportion ranged from 7.6 to 41.8% each year. The overall incidence rate of symptomatic dengue virus infections was 17.3 cases/1,000 person years and between September 2006 and April 2008 asymptomatic infections were 2.6 times more frequent than symptomatic infections. According to the 1997 WHO classification guidelines, there were 210 dengue fever cases, 53 dengue hemorrhagic fever cases (including one dengue shock syndrome case) and five unclassified cases. Evidence for sequential dengue virus infections was seen in six subjects. All four dengue virus serotypes circulated most years. Inapparent dengue virus infections were predominantly associated with DENV-4 infections. CONCLUSIONS/SIGNIFICANCE: Dengue virus was responsible for a significant percentage of febrile illnesses in an adult population in West Java, Indonesia, and this percentage varied from year to year. The observed incidence rate during the study period was 43 times higher than the reported national or provincial rates during the same time period. A wide range of clinical severity was observed with most infections resulting in asymptomatic disease. The circulation of all four serotypes of dengue virus was observed in most years of the study.

Geographical Assessment of Rickettsioses in Indonesia.

To expand the documentation of rickettsioses in Indonesia, we conducted an ectoparasite and small mammal investigation involving four major islands: Java, Sumatra, Sulawesi, and Kalimantan. Coastal and highland regions on each island surveyed were chosen to represent different ecologies in Indonesia. Indication of the presence of Rickettsia spp. was evident in all areas sampled. Typhus group rickettsiae-specific antibodies had significantly higher prevalence among small mammals captured in Java compared to the other islands surveyed (78% in coastal and 50% in highland regions) and the prevalence of spotted fever group rickettsiae-specific antibodies was significantly higher in Kalimantan than the other islands investigated. Hosts and vectors were restricted by Rickettsia spp. but not by coastal or highland regions. Our findings expand the range in which rickettsial pathogens have been documented within the Indonesian archipelago and point to a significant risk to human health.

Diversity of coronavirus in bats from Eastern Thailand.

BACKGROUND: Bats are reservoirs for a diverse range of coronaviruses (CoVs), including those closely related to human pathogens such as Severe Acute Respiratory Syndrome (SARS) CoV and Middle East Respiratory Syndrome CoV. There are approximately 139 bat species reported to date in Thailand, of which two are endemic species. Due to the zoonotic potential of CoVs, standardized surveillance efforts to characterize viral diversity in wildlife are imperative. FINDINGS: A total of 626 bats from 19 different bat species were individually sampled from 5 provinces in Eastern Thailand between 2008 and 2013 (84 fecal and 542 rectal swabs). Samples collected (either fresh feces or rectal swabs) were placed directly into RNA stabilization reagent, transported on ice within 24 hours and preserved at -80°C until further analysis. CoV RNA was detected in 47 specimens (7.6%), from 13 different bat species, using broadly reactive consensus PCR primers targeting the RNA-Dependent RNA Polymerase gene designed to detect all CoVs. Thirty seven alphacoronaviruses, nine lineage D betacoronaviruses, and one lineage B betacoronavirus (SARS-CoV related) were identified. Six new bat CoV reservoirs were identified in our study, namely Cynopterus sphinx, Taphozous melanopogon, Hipposideros lekaguli, Rhinolophus shameli, Scotophilus heathii and Megaderma lyra. CONCLUSIONS: CoVs from the same genetic lineage were found in different bat species roosting in similar or different locations. These data suggest that bat CoV lineages are not strictly concordant with their hosts. Our phylogenetic data indicates high diversity and a complex ecology of CoVs in bats sampled from specific areas in eastern regions of Thailand. Further characterization of additional CoV genes may be useful to better describe the CoV divergence.

Epidemiologic, clinical, and virologic characteristics of human rhinovirus infection among otherwise healthy children and adults: rhinovirus among adults and children.

BACKGROUND: human rhinovirus (HRV) is a major cause of influenza-like illness (ILI) in adults and children. Differences in disease severity by HRV species have been described among hospitalized patients with underlying illness. Less is known about the clinical and virologic characteristics of HRV infection among otherwise healthy populations, particularly adults. OBJECTIVES: to characterize molecular epidemiology of HRV and association between HRV species and clinical presentation and viral shedding. STUDY DESIGN: observational, prospective, facility-based study of ILI was conducted from February 2010 to April 2012. Collection of nasopharyngeal specimens, patient symptoms, and clinical information occurred on days 0, 3, 7, and 28. Patients recorded symptom severity daily for the first 7 days of illness in a symptom diary. HRV was identified by RT-PCR and genotyped for species determination. Cases who were co-infected with other viral respiratory pathogens were excluded from the analysis. We evaluated the associations between HRV species, clinical severity, and patterns of viral shedding. RESULTS: eighty-four HRV cases were identified and their isolates genotyped. Of these, 62 (74%) were >18 years. Fifty-four were HRV-A, 11HRV-B, and 19HRV-C. HRV-C infection was more common among children than adults (59% vs. 10%, P<0.001). Among adults, HRV-A was associated with higher severity of upper respiratory symptoms compared to HRV-B (P=0.02), but no such association was found in children. In addition, adults shed HRV-A significantly longer than HRV-C (P trend=0.01). CONCLUSIONS: among otherwise healthy adults with HRV infection, we observed species-specific differences in respiratory symptom severity and duration of viral shedding.

High rate of A(H1N1)pdm09 infections among rural Thai villagers, 2009-2010.

BACKGROUND: Pandemic influenza A(H1N1)pdm09 emerged in Thailand in 2009. A prospective longitudinal adult cohort and household transmission study of influenza-like illness (ILI) was ongoing in rural Thailand at the time of emergence. Symptomatic and subclinical A(H1N1)pdm09 infection rates in the cohort and among household members were evaluated. METHODS: A cohort of 800 Thai adults underwent active community-based surveillance for ILI from 2008-2010. Acute respiratory samples from ILI episodes were tested for A(H1N1)pdm09 by qRT-PCR; acute and 60-day convalescent blood samples were tested by A(H1N1)pdm09 hemagglutination inhibition assay (HI). Enrollment, 12-month and 24-month follow-up blood samples were tested for A(H1N1)pdm09 seroconversion by HI. Household members of influenza A-infected cohort subjects with ILI were enrolled in household transmission investigations in which day 0 and 60 blood samples and acute respiratory samples were tested by either qRT-PCR or HI for A(H1N1)pdm09. Seroconversion between annual blood samples without A(H1N1)pdm09-positive ILI was considered as subclinical infection. RESULTS: The 2-yr cumulative incidence of A(H1N1)pdm09 infection in the cohort in 2009/2010 was 10.8% (84/781) with an annual incidence of 1.2% in 2009 and 9.7% in 2010; 83.3% of infections were subclinical (50% in 2009 and 85.9% in 2010). The 2-yr cumulative incidence was lowest (5%) in adults born ≤ 1957. The A(H1N1)pdm09 secondary attack rate among household contacts was 47.2% (17/36); 47.1% of these infections were subclinical. The highest A(H1N1)pdm09 secondary attack rate among household contacts (70.6%, 12/17) occurred among children born between 1990 and 2003. CONCLUSION: Subclinical A(H1N1)pdm09 infections in Thai adults occurred frequently and accounted for a greater proportion of all A(H1N1)pdm09 infections than previously estimated. The role of subclinical infections in A(H1N1)pdm09 transmission has important implications in formulating strategies to predict and prevent the spread of A(H1N1)pdm09 and other influenza virus strains.

Dramatic decline of respiratory illness among US military recruits after the renewed use of adenovirus vaccines.

BACKGROUND: In late 2011, after a 12-year hiatus, oral vaccines against adenovirus types 4 (Ad4) and 7 (Ad7) were again produced and administered to US military recruits. This study examined the impact of the new adenovirus vaccines on febrile respiratory illness (FRI) and adenovirus rates and investigated if new serotypes emerged. FRI rates and their associated hospitalizations had markedly risen since vaccine production ceased in 1999. METHODS: From 1996 to 2013, the Naval Health Research Center conducted FRI surveillance at 8 military recruit training centers in the United States. During this period, 58 103 FRI pharyngeal swab specimens were studied, yielding 37 048 adenovirus-positive cases, among which 64% were typed. RESULTS: During the 2 years after reintroduction of the vaccines, military trainees experienced a 100-fold decline in adenovirus disease burden (from 5.8 to 0.02 cases per 1000 person-weeks, P < .0001), without evidence that vaccine pressure had increased the impact of adenovirus types other than Ad4 and Ad7. Although the percentage of type 14 increased following reintroduction of the vaccination, the actual number of cases decreased. We estimate that the vaccines prevent approximately 1 death, 1100-2700 hospitalizations, and 13 000 febrile adenovirus cases each year among the trainees. CONCLUSIONS: These data strongly support the continued production and use of Ad4 and Ad7 vaccines in controlling FRI among US military trainees. Continued surveillance for emerging adenovirus subtypes is warranted.

Health-related behaviors and effectiveness of trivalent inactivated versus live attenuated influenza vaccine in preventing influenza-like illness among young adults.

BACKGROUND: Vaccination is the preferred preventive strategy against influenza. Though health behaviors are known to affect immunity and vaccine delivery modes utilize different immune processes, data regarding the preferred influenza vaccine type among adults endorsing specific health-related behaviors (alcohol use, tobacco use, and exercise level) are limited. METHODS: The relative effectiveness of two currently available influenza vaccines were compared for prevention of influenza-like illness during 2 well-matched influenza seasons (2006/2007, 2008/2009) among US military personnel aged 18-49 years. Relative vaccine effectiveness was compared between those self-reporting and not reporting recent smoking history and potential alcohol problem, and by exercise level using Cox proportional hazard modeling adjusted for sociodemographic and military factors, geographic area, and other health behaviors. RESULTS: 28,929 vaccination events and 3936 influenza-like illness events over both influenza seasons were studied. Of subjects, 27.5% were smokers, 7.7% had a potential alcohol-related problem, 10.5% reported minimal exercise, and 4.4% reported high exercise levels. Overall, the risk of influenza-like illness did not significantly differ between live attenuated and trivalent inactivated influenza vaccine recipients (hazard ratio, 0.98; 95% confidence interval, 0.90-1.06). In the final adjusted model, the relative effectiveness of the 2 vaccine types did not differ by smoking status (p = 0.10), alcohol status (p = 0.21), or activity level (p = 0.11). CONCLUSIONS: Live attenuated and trivalent inactivated influenza vaccines were similarly effective in preventing influenza-like illness among young adults and did not differ by health-related behavior status. Influenza vaccine efforts should continue to focus simply on delivering vaccine.

Little evidence of subclinical avian influenza virus infections among rural villagers in Cambodia.

In 2008, 800 adults living within rural Kampong Cham Province, Cambodia were enrolled in a prospective cohort study of zoonotic influenza transmission. After enrollment, participants were contacted weekly for 24 months to identify acute influenza-like illnesses (ILI). Follow-up sera were collected at 12 and 24 months. A transmission substudy was also conducted among the family contacts of cohort members reporting ILI who were influenza A positive. Samples were assessed using serological or molecular techniques looking for evidence of infection with human and avian influenza viruses. Over 24 months, 438 ILI investigations among 284 cohort members were conducted. One cohort member was hospitalized with a H5N1 highly pathogenic avian influenza (HPAI) virus infection and withdrew from the study. Ninety-seven ILI cases (22.1%) were identified as influenza A virus infections by real-time RT-PCR; none yielded evidence for AIV. During the 2 years of follow-up, 21 participants (3.0%) had detectable antibody titers (≥ 1:10) against the studied AIVs: 1 against an avian-like A/Migratory duck/Hong Kong/MPS180/2003(H4N6), 3 against an avian-like A/Teal/Hong Kong/w312/97(H6N1), 9 (3 of which had detectible antibody titers at both 12- and 24-month follow-up) against an avian-like A/Hong Kong/1073/1999(H9N2), 6 (1 detected at both 12- and 24-month follow-up) against an avian-like A/Duck/Memphis/546/74(H11N9), and 2 against an avian-like A/Duck/Alberta/60/76(H12N5). With the exception of the one hospitalized cohort member with H5N1 infection, no other symptomatic avian influenza infections were detected among the cohort. Serological evidence for subclinical infections was sparse with only one subject showing a 4-fold rise in microneutralization titer over time against AvH12N5. In summary, despite conducting this closely monitored cohort study in a region enzootic for H5N1 HPAI, we were unable to detect subclinical avian influenza infections, suggesting either that these infections are rare or that our assays are insensitive at detecting them.

Serum antibody response to matrix protein 2 following natural infection with 2009 pandemic influenza A(H1N1) virus in humans.

Natural infection-induced humoral immunity to matrix protein 2 (M2) of influenza A viruses in humans is not fully understood. Evidence suggests that anti-M2 antibody responses following influenza A virus infection are weak and/or transient. We show that the seroprevalence of anti-M2 antibodies increased with age in 317 serum samples from healthy individuals in the United States in 2007-2008. Infection with 2009 pandemic H1N1 influenza A virus (A[H1N1]pdm09) elicited a recall serum antibody response to M2 protein of A(H1N1)pdm09 in 47% of the affected 118 individuals tested. Anti-M2 antibody responses were more robust among individuals with preexisting antibodies to M2 protein. Moreover, the antibodies induced as a result of infection with A(H1N1)pdm09 were cross-reactive with M2 protein of seasonal influenza A viruses. These results emphasize the need to further investigate the possible roles of anti-M2 antibodies in human influenza A virus infection.

The diagnostic and prognostic value of dengue non-structural 1 antigen detection in a hyper-endemic region in Indonesia.

As dengue fever is undifferentiated from other febrile illnesses in the tropics and the clinical course is unpredictable, early diagnosis is important. Several commercial assays to detect dengue NS1 antigen have been developed; however, their performances vary and data is lacking from hyper-endemic areas where all four serotypes of dengue are equally represented. To assess the sensitivity of the Bio-Rad platelia Dengue NS1 antigen assay according to virus serotype, immune status, gender, and parameters of severe disease, acute sera from 220 individuals with confirmed dengue and 55 individuals with a non-dengue febrile illness were tested using the Bio-Rad platelia Dengue NS1 antigen assay. The overall sensitivity of the NS1 ELISA was 46.8% and the specificity was 100%. The sensitivity in primary infections was significantly higher than in secondary infections (100% vs. 35.7%). In secondary infections, the sensitivity of NS1 detection was highest in DENV-3 (47.1%), followed by DENV-1 (40.9%), DENV-2 (30%) and DENV-4 (27%) infections. NS1 was less frequently detected in sera with high titers of HI antibodies or in acute samples from patients whose pre-illness sera showed neutralizing antibodies to more than one serotype. The detection of NS1 was higher in females, severe cases, and individuals with lower platelet counts (<100,000/mm(3)). While the overall sensitivity of this NS1 ELISA is poor, our data suggest that in secondary infections, detection may be predictive of a more severe illness.

Prospective study of avian influenza virus infections among rural Thai villagers.

BACKGROUND: In 2008, 800 rural Thai adults living within Kamphaeng Phet Province were enrolled in a prospective cohort study of zoonotic influenza transmission. Serological analyses of enrollment sera suggested this cohort had experienced subclinical avian influenza virus (AIV) infections with H9N2 and H5N1 viruses. METHODS: After enrollment, participants were contacted weekly for 24 mos for acute influenza-like illnesses (ILI). Cohort members confirmed to have influenza A infections were enrolled with their household contacts in a family transmission study involving paired sera and respiratory swab collections. Cohort members also provided sera at 12 and 24 months after enrollment. Serologic and real-time RT-PCR assays were performed against avian, swine, and human influenza viruses. RESULTS: Over the 2 yrs of follow-up, 81 ILI investigations in the cohort were conducted; 31 (38%) were identified as influenza A infections by qRT-PCR. Eighty-three household contacts were enrolled; 12 (14%) reported ILIs, and 11 (92%) of those were identified as influenza infections. A number of subjects were found to have slightly elevated antibodies against avian-like A/Hong Kong/1073/1999(H9N2) virus: 21 subjects (2.7%) at 12-months and 40 subjects (5.1%) at 24-months. Among these, two largely asymptomatic acute infections with H9N2 virus were detected by >4-fold increases in annual serologic titers (final titers 1:80). While controlling for age and influenza vaccine receipt, moderate poultry exposure was significantly associated with elevated H9N2 titers (adjusted OR = 2.3; 95% CI, 1.04-5.2) at the 24-month encounter. One subject had an elevated titer (1:20) against H5N1 during follow-up. CONCLUSIONS: From 2008-10, evidence for AIV infections was sparse among this rural population. Subclinical H9N2 AIV infections likely occurred, but serological results were confounded by antibody cross-reactions. There is a critical need for improved serological diagnostics to more accurately detect subclinical AIV infections in humans.

Sequential waves of gene expression in patients with clinically defined dengue illnesses reveal subtle disease phases and predict disease severity.

BACKGROUND: Dengue virus (DENV) infection can range in severity from mild dengue fever (DF) to severe dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). Changes in host gene expression, temporally through the progression of DENV infection, especially during the early days, remains poorly characterized. Early diagnostic markers for DHF are also lacking. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we investigated host gene expression in a cohort of DENV-infected subjects clinically diagnosed as DF (n = 51) and DHF (n = 13) from Maracay, Venezuela. Blood specimens were collected daily from these subjects from enrollment to early defervescence and at one convalescent time-point. Using convalescent expression levels as baseline, two distinct groups of genes were identified: the "early" group, which included genes associated with innate immunity, type I interferon, cytokine-mediated signaling, chemotaxis, and complement activity peaked at day 0-1 and declined on day 3-4; the second "late" group, comprised of genes associated with cell cycle, emerged from day 4 and peaked at day 5-6. The up-regulation of innate immune response genes coincided with the down-regulation of genes associated with viral replication during day 0-3. Furthermore, DHF patients had lower expression of genes associated with antigen processing and presentation, MHC class II receptor, NK and T cell activities, compared to that of DF patients. These results suggested that the innate and adaptive immunity during the early days of the disease are vital in suppressing DENV replication and in affecting outcome of disease severity. Gene signatures of DHF were identified as early as day 1. CONCLUSIONS/SIGNIFICANCE: Our study reveals a broad and dynamic picture of host responses in DENV infected subjects. Host response to DENV infection can now be understood as two distinct phases with unique transcriptional markers. The DHF signatures identified during day 1-3 may have applications in developing early molecular diagnostics for DHF.

Contributions of the Global Emerging Infections Surveillance and Response System Network to global health security in 2011.

In its 15th year, the Global Emerging Infections Surveillance and Response System (GEIS) continued to make significant contributions to global public health and emerging infectious disease surveillance worldwide. As a division of the US Department of Defense's Armed Forces Health Surveillance Center since 2008, GEIS coordinated a network of surveillance and response activities through collaborations with 33 partners in 76 countries. The GEIS was involved in 73 outbreak responses in fiscal year 2011. Significant laboratory capacity-building initiatives were undertaken with 53 foreign health, agriculture and/or defense ministries, as well as with other US government entities and international institutions, including support for numerous national influenza centers. Equally important, a variety of epidemiologic training endeavors reached over 4,500 individuals in 96 countries. Collectively, these activities enhanced the ability of partner countries and the US military to make decisions about biological threats and design programs to protect global public health as well as global health security.

Identification of a novel human papillomavirus by metagenomic analysis of samples from patients with febrile respiratory illness.

As part of a virus discovery investigation using a metagenomic approach, a highly divergent novel Human papillomavirus type was identified in pooled convenience nasal/oropharyngeal swab samples collected from patients with febrile respiratory illness. Phylogenetic analysis of the whole genome and the L1 gene reveals that the new HPV identified in this study clusters with previously described gamma papillomaviruses, sharing only 61.1% (whole genome) and 63.1% (L1) sequence identity with its closest relative in the Papillomavirus episteme (PAVE) database. This new virus was named HPV_SD2 pending official classification. The complete genome of HPV-SD2 is 7,299 bp long (36.3% G/C) and contains 7 open reading frames (L2, L1, E6, E7, E1, E2 and E4) and a non-coding long control region (LCR) between L1 and E6. The metagenomic procedures, coupled with the bioinformatic methods described herein are well suited to detect small circular genomes such as those of human papillomaviruses.

Seroepidemiologic investigation of an outbreak of pandemic influenza A H1N1 2009 aboard a US Navy vessel--San Diego, 2009.

BACKGROUND: During summer 2009, a US Navy ship experienced an influenza-like illness outbreak with 126 laboratory-confirmed cases of pandemic influenza A (H1N1) 2009 virus among the approximately 2000-person crew. METHODS: During September 24-October 9, 2009, a retrospective seroepidemiologic investigation was conducted to characterize the outbreak. We administered questionnaires, reviewed medical records, and collected post-outbreak sera from systematically sampled crewmembers. We used real-time reverse transcription-PCR or microneutralization assays to detect evidence of H1N1 virus infection. RESULTS: Retrospective serologic data demonstrated that the overall H1N1 virus infection attack rate was 32%. Weighted H1N1 virus attack rates were higher among marines (37%), junior-ranking personnel (34%), and persons aged 19-24 years (36%). In multivariable analysis, a higher risk of illness was found for women versus men (odds ratio [OR] = 2.2; 95% confidence interval [CI]: 1.1-4.4), marines versus navy personnel (OR = 1.7; 95% CI, 1.0-2.9), and those aged 19-24 versus ≥ 35 years (OR = 3.9; 95% CI, 1.2-12.8). Fifty-three percent of infected persons did not recall respiratory illness symptoms. Among infected persons, only 35% met criteria for acute respiratory illness and 11% for influenza-like illness. CONCLUSIONS: Approximately half of H1N1 infections were asymptomatic, and thus, the attack rate was higher than estimated by clinical illness alone. Enhanced infection control measures including pre-embarkation illness screening, improved self-reporting of illness, isolation of ill and quarantine of exposed contacts, and prompt antiviral chemoprophylaxis and treatment might be useful in controlling shipboard influenza outbreaks.

Evidence for avian H9N2 influenza virus infections among rural villagers in Cambodia.

BACKGROUND: Southeast Asia remains a critical region for the emergence of novel and/or zoonotic influenza, underscoring the importance of extensive sampling in rural areas where early transmission is most likely to occur. METHODS: In 2008, 800 adult participants from eight sites were enrolled in a prospective population-based study of avian influenza (AI) virus transmission where highly pathogenic avian influenza (HPAI) H5N1 virus had been reported in humans and poultry from 2006 to 2008. From their enrollment sera and questionnaires, we report risk factor findings for serologic evidence of previous infection with 18 AI virus strains. RESULTS: Serologic assays revealed no evidence of previous infection with 13 different low-pathogenic AI viruses or with HPAI avian-like A/Cambodia/R0404050/2007(H5N1). However, 21 participants had elevated antibodies against avian-like A/Hong Kong/1073/1999(H9N2), validated with a monoclonal antibody blocking ELISA assay specific for avian H9. CONCLUSIONS: Although cross-reaction from antibodies against human influenza viruses cannot be completely excluded, the study data suggest that a number of participants were previously infected with the avian-like A/Hong Kong/1073/1999(H9N2) virus, likely due to as yet unidentified environmental exposures. Prospective data from this cohort will help us better understand the serology of zoonotic influenza infection in a rural cohort in SE Asia.

Surveillance of influenza in Indonesia, 2003­2007.

BACKGROUND: Longitudinal data are limited about the circulating strains of influenza viruses and their public health impact in Indonesia. We conducted influenza surveillance among outpatients and hospitalized patients with influenza-like illness (ILI) across the Indonesian archipelago from 2003 through 2007. METHODOLOGY: Demographic, clinical data, and respiratory specimens were collected for 4236 ILI patients tested for influenza virus infection by RT-PCR and viral culture. PRINCIPAL FINDINGS: Influenza A and B viruses co-circulated year-round with seasonal peaks in influenza A virus activity during the rainy season (December­January). During 2003­2007, influenza viruses were identified in 20·1% (4236 / 21 030) of ILI patients, including 20·1% (4015 / 20 012) of outpatients, and 21·7% (221 / 1018) of inpatients. One H5N1 case was identified retrospectively in an outpatient with ILI. Antigenic drift in circulating influenza A and B virus strains was detected during the surveillance period in Indonesia. In a few instances, antigenically drifted viruses similar to the World Health Organization (WHO) vaccine strains were detected earlier than the date of their designation by WHO. CONCLUSIONS: Influenza A and B virus infections are an important cause of influenza-like illness among outpatients and hospitalized patients in Indonesia. While year-round circulation of influenza viruses occurs, prevention and control strategies should be focused upon the seasonal peak during rainy season months. Ongoing virologic surveillance and influenza disease burden studies in Indonesia are important priorities to better understand the public health impact of influenza in South-East Asia and the implications of influenza viral evolution and global spread.