Cytokine Response Patterns in Severe Pandemic 2009 H1N1 and Seasonal Influenza among Hospitalized Adults

BACKGROUND: Studying cytokine/chemokine responses in severe influenza infections caused by different virus subtypes may improve understanding on pathogenesis. METHODS: Adults hospitalized for laboratory-confirmed seasonal and pandemic 2009 A/H1N1 (pH1N1) influenza were studied. Plasma concentrations of 13 cytokines/chemokines were measured at presentation and then serially, using cytometric-bead-array with flow-cytometry and ELISA. PBMCs from influenza patients were studied for cytokine/chemokine expression using ex-vivo culture (Whole Blood Assay,±PHA/LPS stimulation). Clinical variables were prospectively recorded and analyzed. RESULTS: 63 pH1N1 and 53 seasonal influenza patients were studied. pH1N1 patients were younger (mean±S.D. 42.8±19.2 vs 70.5±16.7 years), and fewer had comorbidities. Respiratory/cardiovascular complications were common in both groups (71.4% vs 81.1%), although severe pneumonia with hypoxemia (54.0% vs 28.3%) and ICU admissions (25.4% vs 1.9%) were more frequent with pH1N1. Hyperactivation of the proinflammatory cytokines IL-6, CXCL8/IL-8, CCL2/MCP-1 and sTNFR-1 was found in pH1N1 pneumonia (2-15 times normal) and in complicated seasonal influenza, but not in milder pH1N1 infections. The adaptive-immunity (Th1/Th17)-related CXCL10/IP-10, CXCL9/MIG and IL-17A however, were markedly suppressed in severe pH1N1 pneumonia (2-27 times lower than seasonal influenza; P-values<0.01). This pattern was further confirmed with serial measurements. Hypercytokinemia tended to be sustained in pH1N1 pneumonia, associated with a slower viral clearance [PCR-negativity: day 3-4, 55% vs 85%; day 6-7, 67% vs 100%]. Elevated proinflammatory cytokines, particularly IL-6, predicted ICU admission (adjusted OR 12.6, 95%CI 2.6-61.5, per log(10)unit increase; P = 0.002), and correlated with fever, tachypnoea, deoxygenation, and length-of-stay (Spearman's rho, P-values<0.01) in influenza infections. PBMCs in seasonal influenza patients were activated and expressed cytokines ex vivo (e.g. IL-6, CXCL8/IL-8, CCL2/MCP-1, CXCL10/IP-10, CXCL9/MIG); their 'responsiveness' to stimuli was shown to change dynamically during the illness course. CONCLUSIONS: A hyperactivated proinflammatory, but suppressed adaptive-immunity (Th1/Th17)-related cytokine response pattern was found in severe pH1N1 pneumonia, different from seasonal influenza. Cytokine/immune-dysregulation may be important in its pathogenesis

High Mortality in Adults Hospitalized for Active Tuberculosis in a Low HIV Prevalence Setting

<div><p>Background</p><p>This study aims to evaluate the outcomes of adults hospitalized for tuberculosis in a higher-income region with low HIV prevalence.</p><p>Methods</p><p>A retrospective cohort study was conducted on all adults hospitalized for pulmonary and/or extrapulmonary tuberculosis in an acute-care hospital in Hong Kong during a two-year period. Microscopy and solid-medium culture were routinely performed. The diagnosis of tuberculosis was made by: (1) positive culture of <i>M. tuberculosis</i>, (2) positive <i>M. tuberculosis</i> PCR result, (3) histology findings of tuberculosis infection, and/or (4) typical clinico-radiological manifestations of tuberculosis which resolved after anti-TB treatment, in the absence of alternative diagnoses. Time to treatment (‘early’, started during initial admission; ‘late’, subsequent periods), reasons for delay, and short- and long-term survival were analyzed.</p><p>Results</p><p>Altogether 349 patients were studied [median(IQR) age 62(48–77) years; non-HIV immunocompromised conditions 36.7%; HIV/AIDS 2.0%]. 57.9%, 16.3%, and 25.8% had pulmonary, extrapulmonary, and pulmonary-extrapulmonary tuberculosis respectively. 58.2% was smear-negative; 0.6% multidrug-resistant. 43.4% developed hypoxemia. Crude 90-day and 1-year all-cause mortality was 13.8% and 24.1% respectively. 57.6% and 35.8% received ‘early’ and ‘late’ treatment respectively, latter mostly culture-guided [median(IQR) intervals, 5(3–9) vs. 43(25–61) days]. Diagnosis was unknown before death in 6.6%. Smear-negativity, malignancy, chronic lung diseases, and prior exposure to fluoroquinolones (adjusted-OR 10.6, 95%CI 1.3–85.2) delayed diagnosis of tuberculosis. Failure to receive ‘early’ treatment independently predicted higher mortality (Cox-model, adjusted-HR 1.8, 95%CI 1.1–3.0).</p><p>Conclusions</p><p>Mortality of hospitalized tuberculosis patients is high. Newer approaches incorporating methods for rapid diagnosis and initiation of anti-tuberculous treatment are urgently required to improve outcomes.</p></div

Characteristics and outcomes of patients who received early diagnosis and treatment during the initial hospital admission, versus those who were diagnosed late (± treatment).

1<p>Altogether, 125 patients received ‘late’ treatment and 23 remained undiagnosed before death thus received no anti-TB treatment (see text). Comparisons between those who had received ‘early’ treatment and those who died before TB diagnosis were described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092077#pone.0092077.s003" target="_blank">Table S3</a>.</p>2<p>Basis for initiating ‘early’ anti-TB treatment (n = 201): positive-smear 93 (46.3%), positive PCR 8 (4.0%), positive culture 6 (3.0%), histological findings 35 (17.4%), typical clinico-radiological manifestations 59 (29.4%); ‘late’ treatment (n = 125): positive-smear 10 (8.0%), positive PCR 6 (4.8%), positive culture 86 (68.8%), histological findings 12 (9.6%), clinico-radiological manifestations 11 (8.8%).</p>3<p>Received fluoroquinolones prior to the diagnosis of TB. Levofloxacin was used in 15 cases, and ciprofloxacin in 2 cases.</p

Survival of patients with smear-negative tuberculosis (n = 203), according to time of initiation of anti-TB treatment.

<p>Patients who received late (thin solid line)(HR 3.65, 95%CI 1.07–12.40; p = 0.039) or no anti-TB treatment (dotted line)(HR 104.22, 95%CI 24.59–441.66; p<0.001) were shown to have significantly lower survival than those who received early treatment (thick solid line)(reference), as shown in the final Cox proportional hazards model, adjusted for demographics, immunocompromised conditions (HR 3.95, 95%CI 1.85–8.44; p<0.001) and supplemental oxygen requirement (HR 1.98, 95%CI 1.02–3.85; p = 0.043). Basis for initiating ‘early’ anti-TB treatment (n = 75): positive PCR 5 (6.7%), positive culture 5 (6.7%; liquid-medium, 3), histological findings 22 (29.3%), typical clinico-radiological manifestations 43 (57.3%).</p

Correlations between initial cytokine/chemokine concentrations and clinical parameters at presentation (temperature, respiratory rate, oxygen saturation) and the clinical outcomes (hospital length-of-stay, ICU admission).

<p>For correlations with temperature, respiratory rate, oxygen saturation, and length-of-stay, the Spearman's rank coefficients (<i>rho</i>) were shown. For risk of ICU admission, the adjusted odds ratio and the 95% confidence interval (CI) per log₁₀ unit increase in cytokine concentration were shown (adjusted for age, comorbidity and time from onset). Data on respiratory rate was incomplete in seasonal influenza cases, and there were too few ICU admissions to allow meaningful analysis.</p><p>*p<0.05,</p><p>**p<0.01.</p

Serial changes in plasma cytokine/chemokine concentrations during the course of hospitalization.

<p>There was sustained elevation of the proinflammatory cytokines (IL-6, CXCL8/IL-8, CCL2/MCP-1, sTNFR-1) in severe pH1N1 pneumonia; the adaptive-immunity related cytokines (CXCL10/IP-10, CXCL9/MIG, IL-17A) were markedly suppressed compared with seasonal influenza. All patients with pH1N1 influenza (severe pneumonia, n = 34; milder illness, n = 29) received antiviral treatment soon after hospitalization/recruitment; none had received high-dose corticosteroids or other immunosuppressants for ‘viral pneumonitis’ or ‘ARDS’<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026050#pone.0026050-Lee2" target="_blank">[8]</a>. Among seasonal influenza patients (most had complicated illnesses, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026050#pone-0026050-t001" target="_blank">Table 1</a> footnotes), 30(57%) received antiviral treatment. Median concentrations at each time point are shown for each group; the interquartile ranges (presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026050#pone-0026050-t001" target="_blank">Table 1</a>) are omitted here for clarity. Fewer mild pH1N1 and untreated seasonal influenza patients remained hospitalized at day 6–7 for study (Day 1, n = 116; Day 3–4, n = 62; Day 6–7, n = 30).</p

Plasma cytokine/chemokine concentrations in adults hospitalized for seasonal or pandemic H1N1 influenza, measured at presentation.

<p>Values are stated as median (interquartile range, IQR); pandemic influenza A/H1N1 ‘severe’: radiographic pneumonia plus hypoxemia; ‘mild’: hospitalized for significant respiratory or systemic symptoms (only 5/29 patients had mild pulmonary infiltrates on chest radiographs)<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026050#pone.0026050-Lee2" target="_blank">[8]</a>. Over 80% of seasonal influenza patients had respiratory/cardiovascular complications, and nearly half developed hypoxemia. Only 2 patients received long-term immunosuppressants in these cohorts. The normal plasma reference ranges of cytokines/chemokines were obtained from >100 healthy individuals <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026050#pone.0026050-Lee2" target="_blank">[8]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026050#pone.0026050-Lee4" target="_blank">[15]</a>.</p><p>Comparisons: (<b>1</b>) Seasonal influenza A (combined) vs influenza B; (<b>2</b>) seasonal influenza A/H3N2 vs A/H1N1; (<b>3</b>) severe pH1N1 vs all seasonal influenza cases (similar results when influenza B was excluded); (<b>4</b>) pH1N1 cases, ‘severe’ vs ‘mild’. Fewer pH1N1 infections had detectable levels of IFN-γ compared with seasonal influenza (8.8% vs 43.4%; p<0.001). Cytokine/chemokine concentrations were also compared between severe pH1N1 pneumonia and a subgroup of seasonal influenza patients with complicated infections and hypoxemia: CXCL10/IP-10, CXCL9/MIG and IL-17A concentrations were all significantly lower in severe pH1N1 infections (all p<0.01), and fewer had detectable IFN-γ level (p = 0.001). Complete data on plasma C-reactive protein (n = 34), serum amyloid A and cortisol were unavailable for pH1N1 cases. Mann-Whitney U test,</p><p>**p≤0.01,</p><p>*p<0.05;</p><p>#p<0.10.</p

PBMC activation and <i>ex vivo</i> cytokine/chemokine expression during seasonal influenza infection.

<p>PBMC actively expressed IL-6, (CXCL8/IL-8), CCL2/MCP-1, CXCL10/IP-10, and CXCL9/MIG during acute influenza infection; upon illness recovery, cytokine production decreased, and there was a corresponding increase in cellular responsiveness to stimuli. Cytokine response pre-/post-stimulation and the trend changes in cytokine expression across time points (with PHA/LPS stimulation – red bars; without stimulation – blue bars; folds increase in expression or the ‘responsiveness’ – gray bars) were compared using the <i>Mann-Whitney U</i> test (asterisks, underlined), and the <i>Jonckheere-Terpstra</i> test (blue/gray triangles and asterisks), respectively. IL-17A did not appear to be activated via the PHA/LPS stimulation pathway.</p

Rapid and Simultaneous Detection of Mycobacterium tuberculosis Complex and Beijing/W Genotype in Sputum by an Optimized DNA Extraction Protocol and a Novel Multiplex Real-Time PCR ▿

Rapid diagnosis and genotyping of Mycobacterium tuberculosis by molecular methods are often limited by the amount and purity of DNA extracted from body fluids. In this study, we evaluated 12 DNA extraction methods and developed a highly sensitive protocol for mycobacterial DNA extraction directly from sputa using surface-coated magnetic particles. We have also developed a novel multiplex real-time PCR for simultaneous identification of M. tuberculosis complex and the Beijing/W genotype (a hypervirulent sublineage of M. tuberculosis) by using multiple fluorogenic probes targeting both the M. tuberculosis IS6110 and the Rv0927c-pstS3 intergenic region. With reference strains and clinical isolates, our real-time PCR accurately identified 20 non-Beijing/W and 20 Beijing/W M. tuberculosis strains from 17 different species of nontuberculosis Mycobacterium (NTM). Further assessment of our DNA extraction protocol and real-time PCR with 335 nonduplicate sputum specimens correctly identified all 74 M. tuberculosis culture-positive specimens. In addition, 15 culture-negative specimens from patients with confirmed tuberculosis were also identified. No cross-reactivity was detected with NTM specimens (n = 31). The detection limit of the assay is 10 M. tuberculosis bacilli, as determined by endpoint dilution analysis. In conclusion, an optimized DNA extraction protocol coupled with a novel multiprobe multiplex real-time PCR for the direct detection of M. tuberculosis, including Beijing/W M. tuberculosis, was found to confer high sensitivity and specificity. The combined procedure has the potential to compensate for the drawbacks of conventional mycobacterial culture in routine clinical laboratory setting, such as the lengthy incubation period and the limitation to viable organisms