Menno D. de Jong

Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia.

Avian influenza A (H5N1) viruses cause severe disease in humans, but the basis for their virulence remains unclear. In vitro and animal studies indicate that high and disseminated viral replication is important for disease pathogenesis. Laboratory experiments suggest that virus-induced cytokine dysregulation may contribute to disease severity. To assess the relevance of these findings for human disease, we performed virological and immunological studies in 18 individuals with H5N1 and 8 individuals infected with human influenza virus subtypes. Influenza H5N1 infection in humans is characterized by high pharyngeal virus loads and frequent detection of viral RNA in rectum and blood. Viral RNA in blood was present only in fatal H5N1 cases and was associated with higher pharyngeal viral loads. We observed low peripheral blood T-lymphocyte counts and high chemokine and cytokine levels in H5N1-infected individuals, particularly in those who died, and these correlated with pharyngeal viral loads. Genetic characterization of H5N1 viruses revealed mutations in the viral polymerase complex associated with mammalian adaptation and virulence. Our observations indicate that high viral load, and the resulting intense inflammatory responses, are central to influenza H5N1 pathogenesis. The focus of clinical management should be on preventing this intense cytokine response, by early diagnosis and effective antiviral treatment.

Avian Influenza Virus (H5N1): a Threat to Human Health

SUMMARY Pandemic influenza virus has its origins in avian influenza viruses. The highly pathogenic avian influenza virus subtype H5N1 is already panzootic in poultry, with attendant economic consequences. It continues to cross species barriers to infect humans and other mammals, often with fatal outcomes. Therefore, H5N1 virus has rightly received attention as a potential pandemic threat. However, it is noted that the pandemics of 1957 and 1968 did not arise from highly pathogenic influenza viruses, and the next pandemic may well arise from a low-pathogenicity virus. The rationale for particular concern about an H5N1 pandemic is not its inevitability but its potential severity. An H5N1 pandemic is an event of low probability but one of high human health impact and poses a predicament for public health. Here, we review the ecology and evolution of highly pathogenic avian influenza H5N1 viruses, assess the pandemic risk, and address aspects of human H5N1 disease in relation to its epidemiology, clinical presentation, pathogenesis, diagnosis, and management.

Memory T cells established by seasonal human influenza A infection cross-react with avian influenza A (H5N1) in healthy individuals

The threat of avian influenza A (H5N1) infection in humans remains a global health concern. Current influenza vaccines stimulate antibody responses against the surface glycoproteins but are ineffective against strains that have undergone significant antigenic variation. An alternative approach is to stimulate pre-existing memory T cells established by seasonal human influenza A infection that could cross-react with H5N1 by targeting highly conserved internal proteins. To determine how common cross-reactive T cells are, we performed a comprehensive ex vivo analysis of cross-reactive CD4+ and CD8+ memory T cell responses to overlapping peptides spanning the full proteome of influenza A/Viet Nam/CL26/2005 (H5N1) and influenza A/New York/232/2004 (H3N2) in healthy individuals from the United Kingdom and Viet Nam. Memory CD4+ and CD8+ T cells isolated from the majority of participants exhibited human influenza-specific responses and showed cross-recognition of at least one H5N1 internal protein. Participant CD4+ and CD8+ T cells recognized multiple synthesized influenza peptides, including peptides from the H5N1 strain. Matrix protein 1 (M1) and nucleoprotein (NP) were the immunodominant targets of cross-recognition. In addition, cross-reactive CD4+ and CD8+ T cells recognized target cells infected with recombinant vaccinia viruses expressing either H5N1 M1 or NP. Thus, vaccine formulas inducing heterosubtypic T cell-mediated immunity may confer broad protection against avian and human influenza A viruses.

Avian influenza A (H5N1)

Abstract Since their reemergence in 2003, highly pathogenic avian influenza A (H5N1) viruses have reached endemic levels among poultry in several southeast Asian countries and have caused a still increasing number of more than 100 reported human infections with high mortality. These developments have ignited global fears of an imminent influenza pandemic. The current knowledge of the virology, clinical spectrum, diagnosis and treatment of human influenza H5N1 virus infections is reviewed herein.

Streptococcus suis Meningitis in Adults in Vietnam

BACKGROUND Streptococcus suis infection is an emerging zoonosis in Asia. We determined the detailed epidemiological, clinical, and microbiological characteristics of S. suis meningitis in adults. METHODS We prospectively studied 450 patients with suspected bacterial meningitis. Four hundred thirty-five (96.7%) of the patients participated in a trial to determine the effect of adjunctive dexamethasone treatment. For patients with S. suis infection, bacterial DNA load at hospital admission and during treatment was analyzed in cerebrospinal fluid specimens using quantitative real-time polymerase chain reaction. S. suis strains were characterized using pulsed-field gel electrophoresis and multilocus sequence typing. Putative virulence factors, including extracellular protein factor, suilysin, and muramidase released protein, were detected using polymerase chain reaction and Western blot assay. Predictors of outcome were identified using logistic regression analysis. RESULTS S. suis was the most common pathogen and was detected in 151 (33.6%) of the patients. Fifty (33.1%) of these 151 patients reported exposure to pigs or pork. Mortality was low (2.6%; 4 of 151 patients died), but mild to severe hearing loss occurred in 93 (66.4%) of 140 patients. Severe deafness at hospital discharge was associated with age >50 years (odds ratio, 3.65; 95% confidence interval, 1.15-11.6), a strain carrying the epf gene (odds ratio, 3.42; 95% confidence interval, 1.02-11.4), and dexamethasone therapy (odds ratio, 0.23; 95% confidence interval, 0.06-0.78) but was not associated with cerebrospinal fluid bacterial DNA load. Bacterial DNA was still detectable in 58 (63%) of 92 cerebrospinal fluid samples after 6-10 days of antimicrobial treatment. Ninety-one of 92 S. suis strains had serotype 2. Thirty-three (36%) of these epidemiologically unrelated strains belonged to 1 pulsed-field gel electrophoresis cluster of multilocus sequence type 1, indicating clonal spread. CONCLUSION S. suis serotype 2 is the most frequent cause of bacterial meningitis in adults in southern Vietnam and is associated with substantial morbidity attributable to hearing loss.

Prophylactic and therapeutic efficacy of human monoclonal antibodies against H5N1 influenza.

Background New prophylactic and therapeutic strategies to combat human infections with highly pathogenic avian influenza (HPAI) H5N1 viruses are needed. We generated neutralizing anti-H5N1 human monoclonal antibodies (mAbs) and tested their efficacy for prophylaxis and therapy in a murine model of infection. Methods and Findings Using Epstein-Barr virus we immortalized memory B cells from Vietnamese adults who had recovered from infections with HPAI H5N1 viruses. Supernatants from B cell lines were screened in a virus neutralization assay. B cell lines secreting neutralizing antibodies were cloned and the mAbs purified. The cross-reactivity of these antibodies for different strains of H5N1 was tested in vitro by neutralization assays, and their prophylactic and therapeutic efficacy in vivo was tested in mice. In vitro, mAbs FLA3.14 and FLD20.19 neutralized both Clade I and Clade II H5N1 viruses, whilst FLA5.10 and FLD21.140 neutralized Clade I viruses only. In vivo, FLA3.14 and FLA5.10 conferred protection from lethality in mice challenged with A/Vietnam/1203/04 (H5N1) in a dose-dependent manner. mAb prophylaxis provided a statistically significant reduction in pulmonary virus titer, reduced associated inflammation in the lungs, and restricted extrapulmonary dissemination of the virus. Therapeutic doses of FLA3.14, FLA5.10, FLD20.19, and FLD21.140 provided robust protection from lethality at least up to 72 h postinfection with A/Vietnam/1203/04 (H5N1). mAbs FLA3.14, FLD21.140 and FLD20.19, but not FLA5.10, were also therapeutically active in vivo against the Clade II virus A/Indonesia/5/2005 (H5N1). Conclusions These studies provide proof of concept that fully human mAbs with neutralizing activity can be rapidly generated from the peripheral blood of convalescent patients and that these mAbs are effective for the prevention and treatment of H5N1 infection in a mouse model. A panel of neutralizing, cross-reactive mAbs might be useful for prophylaxis or adjunctive treatment of human cases of H5N1 influenza.

Frequent Detection of Respiratory Viruses without Symptoms: Toward Defining Clinically Relevant Cutoff Values

ABSTRACT Highly sensitive techniques, such as PCR, have greatly improved the detection of respiratory viruses. However, the sensitivity of PCR tests also complicates clinical interpretation, as the presence of small amounts of viral targets may not necessarily have clinical relevance. We performed a prospective case-control study in asymptomatic and symptomatic young children. PCR detection of 14 respiratory viruses was performed in nasal washes, and results were quantified in copies per milliliter. A total of 141 cases and 157 controls were included. In 72% of the cases and 28% of the controls, at least one virus was identified. When stratified for age, at least one virus was identified in 47% of the controls younger than 1 year old. Rhinovirus (RV) was frequently detected in both symptomatic and asymptomatic individuals. Receiver operating characteristic analysis for quantitative rhinovirus detection showed that cutoff values for clinical relevance are feasible for RV. In contrast to rhinovirus, respiratory syncytial virus (RSV) was rarely detected in controls, suggesting that a positive RSV test result is almost always of clinical relevance, independent of viral quantity. In conclusion, our study shows that asymptomatic carriage of a respiratory virus occurs frequently in young children. However, significant differences in the amount of virus present were observed between cases and controls. This suggests that defining cutoff levels should be feasible and represents the next necessary step for diagnosing viral respiratory infections using molecular tests.

Triple Combination of Amantadine, Ribavirin, and Oseltamivir Is Highly Active and Synergistic against Drug Resistant Influenza Virus Strains In Vitro

The rapid emergence and subsequent spread of the novel 2009 Influenza A/H1N1 virus (2009 H1N1) has prompted the World Health Organization to declare the first pandemic of the 21st century, highlighting the threat of influenza to public health and healthcare systems. Widespread resistance to both classes of influenza antivirals (adamantanes and neuraminidase inhibitors) occurs in both pandemic and seasonal viruses, rendering these drugs to be of marginal utility in the treatment modality. Worldwide, virtually all 2009 H1N1 and seasonal H3N2 strains are resistant to the adamantanes (rimantadine and amantadine), and the majority of seasonal H1N1 strains are resistant to oseltamivir, the most widely prescribed neuraminidase inhibitor (NAI). To address the need for more effective therapy, we evaluated the in vitro activity of a triple combination antiviral drug (TCAD) regimen composed of drugs with different mechanisms of action against drug-resistant seasonal and 2009 H1N1 influenza viruses. Amantadine, ribavirin, and oseltamivir, alone and in combination, were tested against amantadine- and oseltamivir-resistant influenza A viruses using an in vitro infection model in MDCK cells. Our data show that the triple combination was highly synergistic against drug-resistant viruses, and the synergy of the triple combination was significantly greater than the synergy of any double combination tested (P<0.05), including the combination of two NAIs. Surprisingly, amantadine and oseltamivir contributed to the antiviral activity of the TCAD regimen against amantadine- and oseltamivir-resistant viruses, respectively, at concentrations where they had no activity as single agents, and at concentrations that were clinically achievable. Our data demonstrate that the TCAD regimen composed of amantadine, ribavirin, and oseltamivir is highly synergistic against resistant viruses, including 2009 H1N1. The TCAD regimen overcomes baseline drug resistance to both classes of approved influenza antivirals, and thus may represent a highly active antiviral therapy for seasonal and pandemic influenza.

Validation of Clinical Application of Cytomegalovirus Plasma DNA Load Measurement and Definition of Treatment Criteria by Analysis of Correlation to Antigen Detection

ABSTRACT Successful preemptive cytomegalovirus (CMV) therapy in transplant patients depends on the availability of sensitive, specific, and timely diagnostic tests for CMV infections. The pp65 antigenemia assay has been used for this purpose with considerable success. Quantification of CMV DNA is currently regarded to be an alternative diagnostic approach. The precise relationship between these two methods has still to be defined, but is essential to compare diagnostic results. This study compared the results of both assays with a large series of transplant recipients in different categories. An internally controlled quantitative real-time CMV DNA PCR was used to test 409 plasma samples from solid organ transplant (SOT) and stem cell transplant (SCT) patients. Levels of CMV DNA in plasma correlated well with classified outcomes of the pp65 antigenemia test. Despite this correlation, the quantitative CMV PCR values in a class of antigen test results were within a wide range, and the definition of an optimal cutoff value for initiating treatment required further analysis by a receiver-operating characteristic curve analysis. This is essential for reactivating infections in particular. For the SCT patients the optimal cutoff value of CMV DNA load defining relevant viral reactivation (in this assay, 10,000 copies/ml) was slightly higher than that for the SOT patients (6,300 copies/ml). Based on a comparison with the established pp65 antigenemia assay, quantification of CMV DNA in plasma appeared to be capable of guiding the clinical management of transplant recipients. This approach may have important advantages, which include a superior reproducibility and sensitivity, allowing the inclusion of kinetic criteria in clinical guidelines.

Kinetics of neutralizing antibodies in patients naturally infected by H5N1 virus

BACKGROUND Little is known about the kinetics of anti-H5 neutralizing antibodies in naturally H5N1-infected patients with severe clinical illness or asymptomatic infection. METHODS Using H5N1 microneutralisation (MN) and H5-pseudotype particle-based microneutralisation assays (H5pp) we analyzed sera sequentially obtained from 11 severely ill patients diagnosed by RT-PCR (follow-up range 1-139 weeks of disease onset) and 31 asymptomatically infected individuals detected in a sero-epidemiological study after exposure to H5N1 virus (follow-up range: 1-2 month-11 months after exposure). RESULTS Of 44 sera from 11 patients with H5N1 disease, 70% tested positive by MN (antibody titre > or = 80) after 2 weeks and 100% were positive by 3 weeks after disease onset. The geometric mean MN titers in severely ill patients were 540 at 1-2 months and 173 at 10-12 months and thus were higher than the titers from asymptomatic individuals (149 at 1-2 months, 62.2 at 10-12 months). Fractional polynomial regression analysis demonstrated that in all severely ill patients, positive titers persisted beyond 2 years of disease onset, while 10 of 23 sera collected 10-11 months after exposure in asymptomatically infected individuals tested negative. CONCLUSIONS Our results indicate that people with asymptomatic H5N1 infection have lower H5N1 antibody titres compared to those with severe illness and that in many asymptomatically infected patients the antibody titer decreased to levels below the threshold of positivity within one year. These data are essential for the design and interpretation of sero-epidemiological studies.