Shuihua Lu

Clinical Findings in 111 Cases of Influenza A (H7N9) Virus Infection

BACKGROUND During the spring of 2013, a novel avian-origin influenza A (H7N9) virus emerged and spread among humans in China. Data were lacking on the clinical characteristics of the infections caused by this virus. METHODS Using medical charts, we collected data on 111 patients with laboratory-confirmed avian-origin influenza A (H7N9) infection through May 10, 2013. RESULTS Of the 111 patients we studied, 76.6% were admitted to an intensive care unit (ICU), and 27.0% died. The median age was 61 years, and 42.3% were 65 years of age or older; 31.5% were female. A total of 61.3% of the patients had at least one underlying medical condition. Fever and cough were the most common presenting symptoms. On admission, 108 patients (97.3%) had findings consistent with pneumonia. Bilateral ground-glass opacities and consolidation were the typical radiologic findings. Lymphocytopenia was observed in 88.3% of patients, and thrombocytopenia in 73.0%. Treatment with antiviral drugs was initiated in 108 patients (97.3%) at a median of 7 days after the onset of illness. The median times from the onset of illness and from the initiation of antiviral therapy to a negative viral test result on real-time reverse-transcriptase-polymerase-chain-reaction assay were 11 days (interquartile range, 9 to 16) and 6 days (interquartile range, 4 to 7), respectively. Multivariate analysis revealed that the presence of a coexisting medical condition was the only independent risk factor for the acute respiratory distress syndrome (ARDS) (odds ratio, 3.42; 95% confidence interval, 1.21 to 9.70; P=0.02). CONCLUSIONS During the evaluation period, the novel H7N9 virus caused severe illness, including pneumonia and ARDS, with high rates of ICU admission and death. (Funded by the National Natural Science Foundation of China and others.).

Association between adverse clinical outcome in human disease caused by novel influenza A H7N9 virus and sustained viral shedding and emergence of antiviral resistance

BACKGROUND On March 30, a novel influenza A subtype H7N9 virus (A/H7N9) was detected in patients with severe respiratory disease in eastern China. Virological factors associated with a poor clinical outcome for this virus remain unclear. We quantified the viral load and analysed antiviral resistance mutations in specimens from patients with A/H7N9. METHODS We studied 14 patients with A/H7N9 disease admitted to the Shanghai Public Health Clinical Centre (SPHCC), China, between April 4, and April 20, 2013, who were given antiviral treatment (oseltamivir or peramivir) for less than 2 days before admission. We investigated the viral load in throat, stool, serum, and urine specimens obtained sequentially from these patients. We also sequenced viral RNA from these specimens to study the mutations associated with resistance to neuraminidase inhibitors and their association with disease outcome. FINDINGS All patients developed pneumonia, seven of them required mechanical ventilation, and three of them further deteriorated to become dependent on extracorporeal membrane oxygenation (ECMO), two of whom died. Antiviral treatment was associated with a reduction of viral load in throat swab specimens in 11 surviving patients. Three patients with persistently high viral load in the throat in spite of antiviral therapy became ECMO dependent. An Arg292Lys mutation in the virus neuraminidase (NA) gene known to confer resistance to both zanamivir and oseltamivir was identified in two of these patients, both also received corticosteroid treatment. In one of them, wild-type sequence Arg292 was noted 2 days after start of antiviral treatment, and the resistant mutant Lys292 dominated 9 days after start of treatment. INTERPRETATION Reduction of viral load following antiviral treatment correlated with improved outcome. Emergence of NA Arg292Lys mutation in two patients who also received corticosteroid treatment led to treatment failure and a poor clinical outcome. The emergence of antiviral resistance in A/H7N9 viruses, especially in patients receiving corticosteroid therapy, is concerning, needs to be closely monitored, and considered in pandemic preparedness planning. FUNDING National Megaprojects of China for Infectious Diseases, Shanghai Municipal Health and Family Planning Commission, the National Key Basic Research Program of China, Ministry of Science and Technology, and National Natural Science Foundation of China.

Early hypercytokinemia is associated with interferon-induced transmembrane protein-3 dysfunction and predictive of fatal H7N9 infection

Significance A unique avian-origin H7N9 influenza virus caused 134 human infections with 44 deaths. The host factors contributing to moderate vs. severe disease are not clear. Here, we show that H7N9 severity was associated with a higher level of cytokines/chemokines. We demonstrate that the cytokines in the infected lung were 100- to 1,000-fold higher than those in the plasma. Furthermore, we found that the IFN-induced transmembrane protein-3 (IFITM3) C/C genotype was associated with severe clinical outcome, as reflected by reduced time in seeking medical aid; more rapid progression to acute respiratory distress syndrome; and higher viral load, cytokine/chemokine levels, and mortality rate. Overall, our data suggest that the IFITM3 genotype is a primary driver of the observed differences in clinical outcome after H7N9 infection. A unique avian-origin A/H7N9 influenza virus has so far caused 134 cases with 44 deaths. Probing the host factors contributing to disease severity, we found that lower levels of plasma inflammatory cytokines on hospital admission correlated with faster recovery in 18 patients with A/H7N9 influenza virus, whereas high concentrations of (in particular) IL-6, IL-8, and macrophage inflammatory protein-1β were predictive of a less favorable or fatal outcome. Analysis of bronchoalveolar lavage samples showed up to 1,000-fold greater cytokine/chemokine levels relative to plasma. Furthermore, patients with the rs12252-C/C IFN-induced transmembrane protein-3 (IFITM3) genotype had more rapid disease progression and were less likely to survive. Compared with patients with the rs12252-T/T or rs12252-T/C genotype of IFITM3, patients with the C/C genotype had a shorter time from disease onset to the time point when they sought medical aid (hospital admission or antiviral therapy) and a shorter interval to development of the acute respiratory distress syndrome stage (reflected by shorter intervals between clinical onset and methylprednisolone treatments and higher rates of mechanical ventilator use), as well as experiencing elevated/prolonged lung virus titers and cytokine production and higher mortality. The present analysis provides reported data on the H7N9 influenza-induced “cytokine storm” at the site of infection in humans and identifies the rs12252-C genotype that compromises IFITM3 function as a primary genetic correlate of severe H7N9 pneumonia. Together with rs12252 sequencing, early monitoring of plasma cytokines is thus of prognostic value for the treatment and management of severe influenza pneumonia.

Poor responses to oseltamivir treatment in a patient with influenza A (H7N9) virus infection

In March 2013, the cases of human infection with influenza A of H7N9 subtype were first reported. Preliminary data suggested that the H7N9 isolates are sensitive to neuraminidase inhibitors, such as oseltamivir, which is the recommended choice of treatment. On April 2nd, a 56-year-old male patient was presented with fever and cough to our hospital. He had previous history of close contact with another H7N9 patient. After caring for his wife (a confirmed H7N9 infection case died on April 3rd), this patient showed flu like symptoms on April 2nd. On the same day, oseltamivir (75 mg bid) treatment was started. Throat swab specimens were screened for H7N9 virus by real-time reverse transcriptase-polymerase chain reaction. The patient was hospitalized on April 4th. Initial specimens on April 4th and April 5th were negative for H7N9. But the specimen collected on April 10th was tested positive for H7N9. The result was confirmed by Shanghai Municipal Center of Disease Control and Prevention. By April 25th when we submitted this report, swab specimens of this patient were still positive for H7N9. This case calls for increased awareness of potential resistance of H7N9 to oseltamivir.

Clinical findings for early human cases of influenza A(H7N9) virus infection, Shanghai, China.

A novel strain of influenza A(H7N9) virus has emerged in China and is causing mild to severe clinical symptoms in infected humans. Some case-patients have died. To further knowledge of this virus, we report the characteristics and clinical histories of 4 early case-patients.

Molecular pathology analyses of two fatal human infections of avian influenza A(H7N9) virus

Aims To investigate the histopathological manifestations of two fatal cases of H7N9 influenza A virus infection. Methods Pulmonary and hepatic specimens from two fatal cases of H7N9 influenza virus infection were examined using H&E staining. Additionally, in situ hybridisation was performed with probes (ViewRNA) targeting H7N9 RNA and IP-10, interleukin (IL)-6 mRNA. The distribution of surfactant protein A (SP-A), surfactant protein B (SP-B), CD3, CD4, CD8, CD68 and C4d were determined with immunohistochemistry. Results Apart from the typical diffuse alveolar damage and hyaline membrane observed in severe influenza infection, we detected H7N9 RNA and massive intrapulmonary production of IP-10 and IL-6 mRNA using in situ hybridisation. Hyperplasia of type II pneumocytes was observed by H&E staining and immunohistochemistry. Proliferating macrophages and clustered neutrophils in the infected lungs were observed, whereas T lymphocytes, especially CD4T helper cells, were markedly depleted. No obvious complement deposition was found in lung tissues. Conclusions Our findings suggest that H7N9 influenza virus induced an immunological response towards overt pulmonary inflammation and systemic lymphopenia which led to intense alveolar damage and respiratory failure.

Sendai Virus Mucosal Vaccination Establishes Lung-Resident Memory CD8 T Cell Immunity and Boosts BCG-Primed Protection against TB in Mice

Accumulating evidence has shown the protective role of CD8+ T cells in vaccine-induced immunity against Mycobacterium tuberculosis (Mtb) despite controversy over their role in natural immunity. However, the current vaccine BCG is unable to induce sufficient CD8+ T cell responses, especially in the lung. Sendai virus, a respiratory RNA virus, is here engineered firstly as a novel recombinant anti-TB vaccine (SeV85AB) that encodes Mtb immuno-dominant antigens, Ag85A and Ag85B. A single mucosal vaccination elicited potent antigen-specific T cell responses and a degree of protection against Mtb challenge similar to the effect of BCG in mice. Depletion of CD8+ T cells abrogated the protective immunity afforded by SeV85AB vaccination. Interestingly, only SeV85AB vaccination induced high levels of lung-resident memory CD8+ T (TRM) cells, and this led to a rapid and strong recall of antigen-specific CD8+ T cell responses against Mtb challenge infection. Furthermore, when used in a BCG prime-SeV85AB boost strategy, SeV85AB vaccine significantly enhanced protection above that seen after BCG vaccination alone. Our findings suggest that CD8+ TRM cells that arise in lungs responding to this mucosal vaccination might help to protect against TB, and SeV85AB holds notable promise to improve BCG’s protective efficacy in a prime-boost immunization regimen.

Epidemiologic report and serologic findings for household contacts of three cases of influenza A (H7N9) virus infection

Abstract Background and objective We conducted epidemiologic investigations and serologic assays on household contacts that were extensively exposed to three influenza A (H7N9) virus infected case-patients before infection-control practices were implemented. Study design Data on the early clinical course of each patient and the exposure history for each patients household contacts were obtained by interviewing household members and by reviewing medical records. Viral RNA in patient samples was tested using real-time reverse transcriptase polymerase chain reaction assay. Antibodies against H7N9 virus in serum samples were tested using hemagglutination inhibition and pseudovirus based neutralization assays. Results All household contacts were extensively exposed to the case-patients without the use of measures to protect against infection. Viral RNA was detected in the specimens from case-patients for approximately 7–11 days after confirmation of infection. However, the results of the analyses of serum specimens taken from the household contacts 15–26 days post exposure revealed no evidence of transmission of H7N9 virus from the case-patients to the contacts. Conclusion Despite ample unprotected exposures to case-patients during the virus shedding period, household members in this report were not infected by the H7N9 virus.

Fluctuating Behavior and Influential Factors in the Performance of the QuantiFERON-TB Gold In-Tube Assay in the Diagnosis of Tuberculosis

Background The QuantiFERON-TB Gold In-Tube (QFT-GIT) is a newly developed but widely used interferon-γ release assay for diagnosing tuberculosis (TB). However, research has not determined whether age or the use of an immune suppressive or anti-TB treatment influences this assay’s ability to detect TB. We assessed the QFT-GIT diagnostic performance for active tuberculosis (ATB) in children and adults in an endemic country and explored the effects of glucocorticoids and anti-TB therapy on the diagnostic value of the QFT-GIT. Methods A total of 60 children and 212 adults with suspected ATB were evaluated with the QFT-GIT. The association between the QFT-GIT diagnostic value and pretreatment factors was qualitatively and quantitatively assessed. Results The sensitivity of the QFT-GIT was 83.9% (95% CI 66.3%-94.6%) in children, and 73.7% (95% CI 57.8%-85.2%) in adults. Glucocorticoids affected the mitogen-stimulated response in both children and adults. In subjects undergoing glucocorticoid pretreatment, 25.0% of the children presented with false-negative QFT-GIT results, 28.6% of adults presented with indeterminate results. For subjects pre-treated with anti-TB drugs, 44.4% presented with false-negative QFT-GIT results. Conclusions The QFT-GIT has higher sensitivity and specificity in children than adults. Glucocorticoid treatment negatively impacts the diagnostic value of the QFT-GIT in all age groups. Anti-TB treatment decreases the sensitivity of the QFT-GIT. Therefore, we recommend that the QFT-GIT assay be performed before TB-specific treatment is initiated and the test should not be used on people undergoing immunosuppression treatment, regardless of their age. A quantitative analysis of the QFT-GIT could be useful for assessing and monitoring TB-specific and non-specific immunity during conversion of the disease.

The Role of KLRG1 in Human CD4+ T-Cell Immunity Against Tuberculosis

Background KLRG1 is a marker of terminally differentiated CD8+ T cells in viral infection, but its role in human Mycobacterium tuberculosis infection remains elusive. Methods A set of cohorts of patients with tuberculosis was designed, and the expression profiles and functions of KLRG1+CD4+ T cells were determined with and without antibody blocking. Results KLRG1 expression on CD4+ T cells was significantly increased in patients with active tuberculosis, compared with healthy controls and patients without tuberculosis. Upon M. tuberculosis-specific stimulation, the ability to secrete interferon γ, interleukin 2, and tumor necrosis factor α was significantly greater in KLRG1-expressing CD4+ T cells than in their KLRG-negative counterparts and was accompanied by a decreased proportion of regulatory T cells and increased Akt signaling. However, KLRG1-expressing CD4+ T cells had a shorter life-span, which was associated with a higher apoptosis rate but a similar proliferative response. Blockade of KLRG1 signaling significantly enhanced interferon γ and interleukin 2 secretion without affecting either cell apoptosis or multiplication. Addition of a specific Akt inhibitor prevented this increased cytokine response, implicating the Akt signaling pathway. Conclusions Our study delineated the profile of KLRG1+CD4+ T cells in patients with tuberculosis and suggests that M. tuberculosis infection drives CD4+ T cells to acquire increased effector function in a terminally differentiated state, which is restrained by KLRG1 via KLRG1/Akt signaling pathway.