National Institute Of Hygiene And Epidemiology

BACKGROUND: We have previously estimated that respiratory syncytial virus (RSV) was associated with 22% of all episodes of (severe) acute lower respiratory infection (ALRI) resulting in 55 000 to 199 000 deaths in children younger than 5 years in 2005. In the past 5 years, major research activity on RSV has yielded substantial new data from developing countries. With a considerably expanded dataset from a large international collaboration, we aimed to estimate the global incidence, hospital admission rate, and mortality from RSV-ALRI episodes in young children in 2015. METHODS: We estimated the incidence and hospital admission rate of RSV-associated ALRI (RSV-ALRI) in children younger than 5 years stratified by age and World Bank income regions from a systematic review of studies published between Jan 1, 1995, and Dec 31, 2016, and unpublished data from 76 high quality population-based studies. We estimated the RSV-ALRI incidence for 132 developing countries using a risk factor-based model and 2015 population estimates. We estimated the in-hospital RSV-ALRI mortality by combining in-hospital case fatality ratios with hospital admission estimates from hospital-based (published and unpublished) studies. We also estimated overall RSV-ALRI mortality by identifying studies reporting monthly data for ALRI mortality in the community and RSV activity. FINDINGS: We estimated that globally in 2015, 33·1 million (uncertainty range [UR] 21·6-50·3) episodes of RSV-ALRI, resulted in about 3·2 million (2·7-3·8) hospital admissions, and 59 600 (48 000-74 500) in-hospital deaths in children younger than 5 years. In children younger than 6 months, 1·4 million (UR 1·2-1·7) hospital admissions, and 27 300 (UR 20 700-36 200) in-hospital deaths were due to RSV-ALRI. We also estimated that the overall RSV-ALRI mortality could be as high as 118 200 (UR 94 600-149 400). Incidence and mortality varied substantially from year to year in any given population. INTERPRETATION: Globally, RSV is a common cause of childhood ALRI and a major cause of hospital admissions in young children, resulting in a substantial burden on health-care services. About 45% of hospital admissions and in-hospital deaths due to RSV-ALRI occur in children younger than 6 months. An effective maternal RSV vaccine or monoclonal antibody could have a substantial effect on disease burden in this age group. FUNDING: The Bill & Melinda Gates Foundation.

BACKGROUND: The Direct Repeat locus of the Mycobacterium tuberculosis complex (MTC) is a member of the CRISPR (Clustered regularly interspaced short palindromic repeats) sequences family. Spoligotyping is the widely used PCR-based reverse-hybridization blotting technique that assays the genetic diversity of this locus and is useful both for clinical laboratory, molecular epidemiology, evolutionary and population genetics. It is easy, robust, cheap, and produces highly diverse portable numerical results, as the result of the combination of (1) Unique Events Polymorphism (UEP) (2) Insertion-Sequence-mediated genetic recombination. Genetic convergence, although rare, was also previously demonstrated. Three previous international spoligotype databases had partly revealed the global and local geographical structures of MTC bacilli populations, however, there was a need for the release of a new, more representative and extended, international spoligotyping database. RESULTS: The fourth international spoligotyping database, SpolDB4, describes 1939 shared-types (STs) representative of a total of 39,295 strains from 122 countries, which are tentatively classified into 62 clades/lineages using a mixed expert-based and bioinformatical approach. The SpolDB4 update adds 26 new potentially phylogeographically-specific MTC genotype families. It provides a clearer picture of the current MTC genomes diversity as well as on the relationships between the genetic attributes investigated (spoligotypes) and the infra-species classification and evolutionary history of the species. Indeed, an independent Naïve-Bayes mixture-model analysis has validated main of the previous supervised SpolDB3 classification results, confirming the usefulness of both supervised and unsupervised models as an approach to understand MTC population structure. Updated results on the epidemiological status of spoligotypes, as well as genetic prevalence maps on six main lineages are also shown. Our results suggests the existence of fine geographical genetic clines within MTC populations, that could mirror the passed and present Homo sapiens sapiens demographical and mycobacterial co-evolutionary history whose structure could be further reconstructed and modelled, thereby providing a large-scale conceptual framework of the global TB Epidemiologic Network. CONCLUSION: Our results broaden the knowledge of the global phylogeography of the MTC complex. SpolDB4 should be a very useful tool to better define the identity of a given MTC clinical isolate, and to better analyze the links between its current spreading and previous evolutionary history. The building and mining of extended MTC polymorphic genetic databases is in progress.

In recent years the outbreak of re-emerging and emerging infectious diseases has been a significant burden on global economies and public health. The growth of population and urbanization along with poor water supply and environmental hygiene are the main reasons for the increase in outbreak of infectious pathogens. Transmission of infectious pathogens to the community has caused outbreaks of diseases such as influenza (A/H5N1), diarrhea (Escherichia coli), cholera (Vibrio cholera), etc throughout the world. The comprehensive treatments of environments containing infectious pathogens using advanced disinfectant nanomaterials have been proposed for prevention of the outbreaks. Among these nanomaterials, silver nanoparticles (Ag-NPs) with unique properties of high antimicrobial activity have attracted much interest from scientists and technologists to develop nanosilver-based disinfectant products. This article aims to review the synthesis routes and antimicrobial effects of Ag-NPs against various pathogens including bacteria, fungi and virus. Toxicology considerations of Ag-NPs to humans and ecology are discussed in detail. Some current applications of Ag-NPs in water-, air- and surface- disinfection are described. Finally, future prospects of Ag-NPs for treatment and prevention of currently emerging infections are discussed.

BACKGROUND: Recent outbreaks of avian influenza A (H5N1) in poultry throughout Asia have had major economic and health repercussions. Human infections with this virus were identified in Vietnam in January 2004. METHODS: We report the clinical features and preliminary epidemiologic findings among 10 patients with confirmed cases of avian influenza A (H5N1) who presented to hospitals in Ho Chi Minh City and Hanoi, Vietnam, in December 2003 and January 2004. RESULTS: In all 10 cases, the diagnosis of influenza A (H5N1) was confirmed by means of viral culture or reverse transcriptase-polymerase chain reaction with primers specific for H5 and N1. None of the 10 patients (mean age, 13.7 years) had preexisting medical conditions. Nine of them had a clear history of direct contact with poultry (median time before onset of illness, three days). All patients presented with fever (temperature, 38.5 to 40.0 degrees C), respiratory symptoms, and clinically significant lymphopenia (median lymphocyte count, 700 per cubic millimeter). The median platelet count was 75,500 per cubic millimeter. Seven patients had diarrhea. In all patients, there were marked abnormalities on chest radiography. There was no definitive evidence of human-to-human transmission. Eight patients died, one patient has recovered, and one is recovering. CONCLUSIONS: Influenza A (H5N1) infection, characterized by fever, respiratory symptoms, and lymphopenia, carries a high risk of death. Although in all 10 cases the infection appears to have been acquired directly from infected poultry, the potential exists for genetic reassortment with human influenzaviruses and the evolution of human-to-human transmission. Containment of influenza A (H5N1) in poultry throughout Asia is therefore urgently required.

Abstract Body-mass index (BMI) has increased steadily in most countries in parallel with a rise in the proportion of the population who live in cities 1,2 . This has led to a widely reported view that urbanization is one of the most important drivers of the global rise in obesity 3–6 . Here we use 2,009 population-based studies, with measurements of height and weight in more than 112 million adults, to report national, regional and global trends in mean BMI segregated by place of residence (a rural or urban area) from 1985 to 2017. We show that, contrary to the dominant paradigm, more than 55% of the global rise in mean BMI from 1985 to 2017—and more than 80% in some low- and middle-income regions—was due to increases in BMI in rural areas. This large contribution stems from the fact that, with the exception of women in sub-Saharan Africa, BMI is increasing at the same rate or faster in rural areas than in cities in low- and middle-income regions. These trends have in turn resulted in a closing—and in some countries reversal—of the gap in BMI between urban and rural areas in low- and middle-income countries, especially for women. In high-income and industrialized countries, we noted a persistently higher rural BMI, especially for women. There is an urgent need for an integrated approach to rural nutrition that enhances financial and physical access to healthy foods, to avoid replacing the rural undernutrition disadvantage in poor countries with a more general malnutrition disadvantage that entails excessive consumption of low-quality calories.

Wild waterfowl, including ducks, are natural hosts of influenza A viruses. These viruses rarely caused disease in ducks until 2002, when some H5N1 strains became highly pathogenic. Here we show that these H5N1 viruses are reverting to nonpathogenicity in ducks. Ducks experimentally infected with viruses isolated between 2003 and 2004 shed virus for an extended time (up to 17 days), during which variant viruses with low pathogenicity were selected. These results suggest that the duck has become the "Trojan horse" of Asian H5N1 influenza viruses. The ducks that are unaffected by infection with these viruses continue to circulate these viruses, presenting a pandemic threat.

We introduce the antibody landscape, a method for the quantitative analysis of antibody-mediated immunity to antigenically variable pathogens, achieved by accounting for antigenic variation among pathogen strains. We generated antibody landscapes to study immune profiles covering 43 years of influenza A/H3N2 virus evolution for 69 individuals monitored for infection over 6 years and for 225 individuals pre- and postvaccination. Upon infection and vaccination, titers increased broadly, including previously encountered viruses far beyond the extent of cross-reactivity observed after a primary infection. We explored implications for vaccination and found that the use of an antigenically advanced virus had the dual benefit of inducing antibodies against both advanced and previous antigenic clusters. These results indicate that preemptive vaccine updates may improve influenza vaccine efficacy in previously exposed individuals.

BACKGROUND: Fatal human respiratory disease associated with influenza A subtype H5N1 has been documented in Hong Kong, and more recently in Vietnam, Thailand and Cambodia. We previously demonstrated that patients with H5N1 disease had unusually high serum levels of IP-10 (interferon-gamma-inducible protein-10). Furthermore, when compared with human influenza virus subtype H1N1, the H5N1 viruses in 1997 (A/Hong Kong/483/97) (H5N1/97) were more potent inducers of pro-inflammatory cytokines (e.g. tumor necrosis factor-a) and chemokines (e.g. IP-10) from primary human macrophages in vitro, which suggests that cytokines dysregulation may play a role in pathogenesis of H5N1 disease. Since respiratory epithelial cells are the primary target cell for replication of influenza viruses, it is pertinent to investigate the cytokine induction profile of H5N1 viruses in these cells. METHODS: We used quantitative RT-PCR and ELISA to compare the profile of cytokine and chemokine gene expression induced by H5N1 viruses A/HK/483/97 (H5N1/97), A/Vietnam/1194/04 and A/Vietnam/3046/04 (both H5N1/04) with that of human H1N1 virus in human primary alveolar and bronchial epithelial cells in vitro. RESULTS: We demonstrated that in comparison to human H1N1 viruses, H5N1/97 and H5N1/04 viruses were more potent inducers of IP-10, interferon beta, RANTES (regulated on activation, normal T cell expressed and secreted) and interleukin 6 (IL-6) in primary human alveolar and bronchial epithelial cells in vitro. Recent H5N1 viruses from Vietnam (H5N1/04) appeared to be even more potent at inducing IP-10 than H5N1/97 virus. CONCLUSION: The H5N1/97 and H5N1/04 subtype influenza A viruses are more potent inducers of proinflammatory cytokines and chemokines in primary human respiratory epithelial cells than subtype H1N1 virus. We suggest that this hyper-induction of cytokines may be relevant to the pathogenesis of human H5N1 disease.

OBJECTIVE: The aim of this study was to compare the efficacy of cloth masks to medical masks in hospital healthcare workers (HCWs). The null hypothesis is that there is no difference between medical masks and cloth masks. SETTING: 14 secondary-level/tertiary-level hospitals in Hanoi, Vietnam. PARTICIPANTS: 1607 hospital HCWs aged ≥18 years working full-time in selected high-risk wards. INTERVENTION: Hospital wards were randomised to: medical masks, cloth masks or a control group (usual practice, which included mask wearing). Participants used the mask on every shift for 4 consecutive weeks. MAIN OUTCOME MEASURE: Clinical respiratory illness (CRI), influenza-like illness (ILI) and laboratory-confirmed respiratory virus infection. RESULTS: The rates of all infection outcomes were highest in the cloth mask arm, with the rate of ILI statistically significantly higher in the cloth mask arm (relative risk (RR)=13.00, 95% CI 1.69 to 100.07) compared with the medical mask arm. Cloth masks also had significantly higher rates of ILI compared with the control arm. An analysis by mask use showed ILI (RR=6.64, 95% CI 1.45 to 28.65) and laboratory-confirmed virus (RR=1.72, 95% CI 1.01 to 2.94) were significantly higher in the cloth masks group compared with the medical masks group. Penetration of cloth masks by particles was almost 97% and medical masks 44%. CONCLUSIONS: This study is the first RCT of cloth masks, and the results caution against the use of cloth masks. This is an important finding to inform occupational health and safety. Moisture retention, reuse of cloth masks and poor filtration may result in increased risk of infection. Further research is needed to inform the widespread use of cloth masks globally. However, as a precautionary measure, cloth masks should not be recommended for HCWs, particularly in high-risk situations, and guidelines need to be updated. TRIAL REGISTRATION NUMBER: Australian New Zealand Clinical Trials Registry: ACTRN12610000887077.

BACKGROUND: The burden of shigellosis is greatest in resource-poor countries. Although this diarrheal disease has been thought to cause considerable morbidity and mortality in excess of 1,000,000 deaths globally per year, little recent data are available to guide intervention strategies in Asia. We conducted a prospective, population-based study in six Asian countries to gain a better understanding of the current disease burden, clinical manifestations, and microbiology of shigellosis in Asia. METHODS AND FINDINGS: Over 600,000 persons of all ages residing in Bangladesh, China, Pakistan, Indonesia, Vietnam, and Thailand were included in the surveillance. Shigella was isolated from 2,927 (5%) of 56,958 diarrhoea episodes detected between 2000 and 2004. The overall incidence of treated shigellosis was 2.1 episodes per 1,000 residents per year in all ages and 13.2/1,000/y in children under 60 months old. Shigellosis incidence increased after age 40 years. S. flexneri was the most frequently isolated Shigella species (1,976/2,927 [68%]) in all sites except in Thailand, where S. sonnei was most frequently detected (124/146 [85%]). S. flexneri serotypes were highly heterogeneous in their distribution from site to site, and even from year to year. PCR detected ipaH, the gene encoding invasion plasmid antigen H in 33% of a sample of culture-negative stool specimens. The majority of S. flexneri isolates in each site were resistant to amoxicillin and cotrimoxazole. Ciprofloxacin-resistant S. flexneri isolates were identified in China (18/305 [6%]), Pakistan (8/242 [3%]), and Vietnam (5/282 [2%]). CONCLUSIONS: Shigella appears to be more ubiquitous in Asian impoverished populations than previously thought, and antibiotic-resistant strains of different species and serotypes have emerged. Focusing on prevention of shigellosis could exert an immediate benefit first by substantially reducing the overall diarrhoea burden in the region and second by preventing the spread of panresistant Shigella strains. The heterogeneous distribution of Shigella species and serotypes suggest that multivalent or cross-protective Shigella vaccines will be needed to prevent shigellosis in Asia.

BACKGROUND: Typhoid fever is common in developing countries. The licensed typhoid vaccines confer only about 70 percent immunity, do not protect young children, and are not used for routine vaccination. A newly devised conjugate of the capsular polysaccharide of Salmonella typhi, Vi, bound to nontoxic recombinant Pseudomonas aeruginosa exotoxin A (rEPA), has enhanced immunogenicity in adults and in children 5 to 14 years old and has elicited a booster response in children 2 to 4 years old. METHODS: In a double-blind, randomized trial, we evaluated the safety, immunogenicity, and efficacy of the Vi-rEPA vaccine in children two to five years old in 16 communes in Dong Thap Province, Vietnam. Each of the 11,091 children received two injections six weeks apart of either Vi-rEPA or a saline placebo. Cases of typhoid, diagnosed by the isolation of S. typhi from blood cultures after 3 or more days of fever (a temperature of 37.5 degrees C or higher), were identified by active surveillance over a period of 27 months. We estimated efficacy by comparing the attack rate of typhoid in the vaccine group with that in the placebo group. RESULTS: S. typhi was isolated from 4 of the 5525 children who were fully vaccinated with Vi-rEPA and from 47 of the 5566 children who received both injections of placebo (efficacy, 91.5 percent; 95 percent confidence interval, 77.1 to 96.6; P<0.001). Among the 771 children who received only one injection, there was 1 case of typhoid in the vaccine group and 8 cases in the placebo group. Cases were distributed evenly among all age groups and throughout the study period. No serious adverse reactions were observed. In all 36 children studied four weeks after the second injection of the vaccine, levels of serum IgG Vi antibodies had increased by a factor of 10 or more. CONCLUSIONS: The Vi-rEPA conjugate typhoid vaccine is safe and immunogenic and has more than 90 percent efficacy in children two to five years old. The antibody responses and the efficacy suggest that this vaccine should be at least as protective in persons who are more than five years old.

The spread of highly pathogenic avian influenza H5N1 viruses across Asia in 2003 and 2004 devastated domestic poultry populations and resulted in the largest and most lethal H5N1 virus outbreak in humans to date. To better understand the potential of H5N1 viruses isolated during this epizootic event to cause disease in mammals, we used the mouse and ferret models to evaluate the relative virulence of selected 2003 and 2004 H5N1 viruses representing multiple genetic and geographical groups and compared them to earlier H5N1 strains isolated from humans. Four of five human isolates tested were highly lethal for both mice and ferrets and exhibited a substantially greater level of virulence in ferrets than other H5N1 viruses isolated from humans since 1997. One human isolate and all four avian isolates tested were found to be of low virulence in either animal. The highly virulent viruses replicated to high titers in the mouse and ferret respiratory tracts and spread to multiple organs, including the brain. Rapid disease progression and high lethality rates in ferrets distinguished the highly virulent 2004 H5N1 viruses from the 1997 H5N1 viruses. A pair of viruses isolated from the same patient differed by eight amino acids, including a Lys/Glu disparity at 627 of PB2, previously identified as an H5N1 virulence factor in mice. The virus possessing Glu at 627 of PB2 exhibited only a modest decrease in virulence in mice and was highly virulent in ferrets, indicating that for this virus pair, the K627E PB2 difference did not have a prevailing effect on virulence in mice or ferrets. Our results demonstrate the general equivalence of mouse and ferret models for assessment of the virulence of 2003 and 2004 H5N1 viruses. However, the apparent enhancement of virulence of these viruses in humans in 2004 was better reflected in the ferret.

Wild waterfowl are the natural reservoir of all influenza A viruses, and these viruses are usually nonpathogenic in these birds. However, since late 2002, H5N1 outbreaks in Asia have resulted in mortality among waterfowl in recreational parks, domestic flocks, and wild migratory birds. The evolutionary stasis between influenza virus and its natural host may have been disrupted, prompting us to ask whether waterfowl are resistant to H5N1 influenza virus disease and whether they can still act as a reservoir for these viruses. To better understand the biology of H5N1 viruses in ducks and attempt to answer this question, we inoculated juvenile mallards with 23 different H5N1 influenza viruses isolated in Asia between 2003 and 2004. All virus isolates replicated efficiently in inoculated ducks, and 22 were transmitted to susceptible contacts. Viruses replicated to higher levels in the trachea than in the cloaca of both inoculated and contact birds, suggesting that the digestive tract is not the main site of H5N1 influenza virus replication in ducks and that the fecal-oral route may no longer be the main transmission path. The virus isolates' pathogenicities varied from completely nonpathogenic to highly lethal and were positively correlated with tracheal virus titers. Nevertheless, the eight virus isolates that were nonpathogenic in ducks replicated and transmitted efficiently to naïve contacts, suggesting that highly pathogenic H5N1 viruses causing minimal signs of disease in ducks can propagate silently and efficiently among domestic and wild ducks in Asia and that they represent a serious threat to human and veterinary public health.

STUDY OBJECTIVE: As part of the APHEA project this study examined the association between airborne particles and hospital admissions for cardiac causes (ICD9 390-429) in eight European cities (Barcelona, Birmingham, London, Milan, the Netherlands, Paris, Rome, and Stockholm). All admissions were studied, as well as admissions stratified by age. The association for ischaemic heart disease (ICD9 410-413) and stroke (ICD9 430-438) was also studied, also stratified by age. DESIGN: Autoregressive Poisson models were used that controlled for long term trend, season, influenza epidemics, and meteorology to assess the short-term effects of particles in each city. The study also examined confounding by other pollutants. City specific results were pooled in a second stage regression to obtain more stable estimates and examine the sources of heterogeneity. MAIN RESULTS: The pooled percentage increases associated with a 10 micro g/m(3) increase in PM(10) and black smoke were respectively 0.5% (95% CI: 0.2 to 0.8) and 1.1% (95% CI: 0.4 to 1.8) for cardiac admissions of all ages, 0.7% (95% CI: 0.4 to 1.0) and 1.3% (95% CI: 0.4 to 2.2) for cardiac admissions over 65 years, and, 0.8% (95% CI: 0.3 to 1.2) and 1.1% (95% CI: 0.7 to 1.5) for ischaemic heart disease over 65 years. The effect of PM(10) was little changed by control for ozone or SO(2), but was substantially reduced (CO) or eliminated (NO(2)) by control for other traffic related pollutants. The effect of black smoke remained practically unchanged controlling for CO and only somewhat reduced controlling for NO(2). CONCLUSIONS: These effects of particulate air pollution on cardiac admissions suggest the primary effect is likely to be mainly attributable to diesel exhaust. Results for ischaemic heart disease below 65 years and for stroke over 65 years were inconclusive.

ABSTRACT The development and evaluation of a one-step single-tube accelerated real-time quantitative reverse transcription (RT) loop-mediated isothermal amplification (LAMP) assay is reported for rapid detection of the severe acute respiratory syndrome coronavirus (SARS-CoV) replicase gene. A total of 49 samples (15 throat washes, 13 throat swabs, and 21 combined throat and nasal swabs) collected from patients admitted to the Hanoi-French and Ninhbinh hospitals in Vietnam during the SARS epidemic were evaluated and compared to conventional RT-PCR. The RT-LAMP assay demonstrated 100-fold-greater sensitivity, with a detection limit of 0.01 PFU. The sensitivity and specificity of RT-LAMP assay for detecting viral RNA in clinical specimens with regard to RT-PCR were 100 and 87%, respectively. The specificity of the RT-LAMP assay was further validated by restriction analysis as well as nucleotide sequencing of the amplified product. The concentration of virus in most of the clinical samples was 0.1 PFU (0.1 to 10 2 PFU), as determined from the standard curve of SARS RT-LAMP and based on the time of positivity. The assay procedure is quite simple, wherein the amplification is carried out in a single tube under isothermal conditions at 63°C, and the result can be obtained in less than 1 h (as early as 11 min). Thus, the RT-LAMP assay reported here has the advantages of rapid amplification, simple operation, and easy detection and will be useful for rapid and reliable clinical diagnosis of SARS-CoV in developing countries.

BACKGROUND: The global burden of pediatric severe respiratory illness is substantial, and influenza viruses contribute to this burden. Systematic surveillance and testing for influenza among hospitalized children has expanded globally over the past decade. However, only a fraction of the data has been used to estimate influenza burden. In this analysis, we use surveillance data to provide an estimate of influenza-associated hospitalizations among children worldwide. METHODS AND FINDINGS: We aggregated data from a systematic review (n = 108) and surveillance platforms (n = 37) to calculate a pooled estimate of the proportion of samples collected from children hospitalized with respiratory illnesses and positive for influenza by age group (<6 mo, <1 y, <2 y, <5 y, 5-17 y, and <18 y). We applied this proportion to global estimates of acute lower respiratory infection hospitalizations among children aged <1 y and <5 y, to obtain the number and per capita rate of influenza-associated hospitalizations by geographic region and socio-economic status. Influenza was associated with 10% (95% CI 8%-11%) of respiratory hospitalizations in children <18 y worldwide, ranging from 5% (95% CI 3%-7%) among children <6 mo to 16% (95% CI 14%-20%) among children 5-17 y. On average, we estimated that influenza results in approximately 374,000 (95% CI 264,000 to 539,000) hospitalizations in children <1 y-of which 228,000 (95% CI 150,000 to 344,000) occur in children <6 mo-and 870,000 (95% CI 610,000 to 1,237,000) hospitalizations in children <5 y annually. Influenza-associated hospitalization rates were more than three times higher in developing countries than in industrialized countries (150/100,000 children/year versus 48/100,000). However, differences in hospitalization practices between settings are an important limitation in interpreting these findings. CONCLUSIONS: Influenza is an important contributor to respiratory hospitalizations among young children worldwide. Increasing influenza vaccination coverage among young children and pregnant women could reduce this burden and protect infants <6 mo.

Avian influenza A H5N1 viruses continue to spread globally among birds, resulting in occasional transmission of virus from infected poultry to humans. Probable human-to-human transmission has been documented rarely, but H5N1 viruses have not yet acquired the ability to transmit efficiently among humans, an essential property of a pandemic virus. The pandemics of 1957 and 1968 were caused by avian-human reassortant influenza viruses that had acquired human virus-like receptor binding properties. However, the relative contribution of human internal protein genes or other molecular changes to the efficient transmission of influenza viruses among humans remains poorly understood. Here, we report on a comparative ferret model that parallels the efficient transmission of H3N2 human viruses and the poor transmission of H5N1 avian viruses in humans. In this model, an H3N2 reassortant virus with avian virus internal protein genes exhibited efficient replication but inefficient transmission, whereas H5N1 reassortant viruses with four or six human virus internal protein genes exhibited reduced replication and no transmission. These findings indicate that the human virus H3N2 surface protein genes alone did not confer efficient transmissibility and that acquisition of human virus internal protein genes alone was insufficient for this 1997 H5N1 virus to develop pandemic capabilities, even after serial passages in a mammalian host. These results highlight the complexity of the genetic basis of influenza virus transmissibility and suggest that H5N1 viruses may require further adaptation to acquire this essential pandemic trait.

Highly pathogenic avian H5N1 influenza A viruses have spread throughout Asia, Europe, and Africa, raising serious worldwide concern about their pandemic potential. Although more than 250 people have been infected with these viruses, with a consequent high rate of mortality, the molecular mechanisms responsible for the efficient transmission of H5N1 viruses among humans remain elusive. We used a mouse model to examine the role of the amino acid at position 627 of the PB2 viral protein in efficient replication of H5N1 viruses in the mammalian respiratory tract. Viruses possessing Lys at position 627 of PB2 replicated efficiently in lungs and nasal turbinates, as well as in cells, even at the lower temperature of 33 8C. Those viruses possessing Glu at this position replicated less well in nasal turbinates than in lungs, and less well in cells at the lower temperature. These results suggest that Lys at PB2-627 confers to avian H5N1 viruses the advantage of efficient growth in the upper and lower respiratory tracts of mammals. Therefore, efficient viral growth in the upper respiratory tract may provide a platform for the adaptation of avian H5N1 influenza viruses to humans and for efficient person-to-person virus transmission, in the context of changes in other viral properties including specificity for human (sialic acid a-2,6-galactose containing) receptors.

The 2004 outbreaks of H5N1 influenza viruses in Vietnam and Thailand were highly lethal to humans and to poultry; therefore, newly emerging avian influenza A viruses pose a continued threat, not only to avian species but also to humans. We studied the pathogenicity of four human and nine avian H5N1/04 influenza viruses in ferrets (an excellent model for influenza studies). All four human isolates were fatal to intranasally inoculated ferrets. The human isolate A/Vietnam/1203/04 (H5N1) was the most pathogenic isolate; the severity of disease was associated with a broad tissue tropism and high virus titers in multiple organs, including the brain. High fever, weight loss, anorexia, extreme lethargy, and diarrhea were observed. Two avian H5N1/04 isolates were as pathogenic as the human viruses, causing lethal systemic infections in ferrets. Seven of nine H5N1/04 viruses isolated from avian species caused mild infections, with virus replication restricted to the upper respiratory tract. All chicken isolates were nonlethal to ferrets. A sequence analysis revealed polybasic amino acids in the hemagglutinin connecting peptides of all H5N1/04 viruses, indicating that multiple molecular differences in other genes are important for a high level of virulence. Interestingly, the human A/Vietnam/1203/04 isolate had a lysine substitution at position 627 of PB2 and had one to eight amino acid changes in all gene products except that of the M1 gene, unlike the A/chicken/Vietnam/C58/04 and A/quail/Vietnam/36/04 viruses. Our results indicate that viruses that are lethal to mammals are circulating among birds in Asia and suggest that pathogenicity in ferrets, and perhaps humans, reflects a complex combination of different residues rather than a single amino acid difference.

PURPOSE: This prospective, randomized, single-center study was designed to evaluate the influence of follow-up on detection and resectability of local recurrences and on survival after radical surgery for colorectal cancer. METHODS: Between 1987 and 1990, 207 consecutive patients who underwent curative resections for primary untreated large-bowel carcinoma were randomly assigned to a conventional follow-up group (Group A; n = 103) and to an intense follow-up group (Group B; n = 104). All the patients were followed up prospectively, and the outcome was known for all of them at five years. Patients in Group A were seen at six-month intervals for one year, and once a year thereafter. Patients in Group B were checked every three months during the first two years, at six-month intervals for the next three years, and once a year thereafter. RESULTS: Of the 103 patients in Group A, local recurrence was detected in 20; 9 (13 percent) of these patients had colon cancer, and 11 (29 percent) had rectal cancer. Of the 104 patients in Group B, local recurrence was detected in 26; 12 (16 percent) of these patients had colon cancer, and 14 (45 percent) had rectal cancer. Twelve cases (60 percent) of local recurrence in Group A and 24 cases (92 percent) in Group B were detected at scheduled visits (P < 0.05). Local recurrences were detected earlier in patients of Group B (10.3 +/- 2.7 vs. 20.2 +/- 6.1 months; P < 0.0003). Curative re-resection was possible in 2 patients (10 percent) in Group A, 1 with colon cancer and 1 with rectal cancer, and in 17 patients (65 percent) in Group B, 6 with colon cancer and 11 with rectal cancer (P < 0.01). Of the Group B patients who had curative re-resections of local recurrence, 8 (47 percent) were disease-free and long-term survivors as of the last follow-up, and 2 (11.7 percent) were alive, but with a new recurrence. The 2 patients in Group A who had curative re-resections died as a result of cancer. The five-year survival rate in Group A was 58.3 percent and in Group B was 73.1 percent. The difference is statistically significant (P < 0.02). CONCLUSIONS: Our data support use of an intense follow-up plan after primary resection of large-bowel cancer, at least in patients with rectal cancer.