Alterations in faecal microbiome and resistome in Chinese international travellers: a metagenomic analysis.

BACKGROUND: International travel increases the risk of acquisition of antibiotic-resistant bacteria and antibiotic resistance genes (ARGs). Previous studies have characterized the changes in the gut microbiome and resistome of Western travellers; however, information on non-Western populations and the effects of travel-related risk factors on the gut microbiome and resistome remains limited. METHODS: We conducted a prospective observational study on a cohort of 90 healthy Chinese adult residents of Hong Kong. We characterized the microbiome and resistome in stools collected from the subjects before and after travelling to diverse international locations using shotgun metagenomic sequencing and examined their associations with travel-related variables. RESULTS: Our results showed that travel neither significantly changed the taxonomic composition of the faecal microbiota nor altered the alpha (Shannon) or beta diversity of the faecal microbiome or resistome. However, travel significantly increased the number of ARGs. Ten ARGs, including aadA, TEM, mgrB, mphA, qnrS9 and tetR, were significantly enriched in relative abundance after travel, eight of which were detected in metagenomic bins belonging to Escherichia/Shigella flexneri in the post-trip samples. In sum, 30 ARGs significantly increased in prevalence after travel, with the largest changes observed in tetD and a few qnrS variants (qnrS9, qnrS and qnrS8). We found that travel to low- or middle-income countries, or Africa or Southeast Asia, increased the number of ARG subtypes, whereas travel to low- or middle-income countries and the use of alcohol-based hand sanitizer (ABHS) or doxycycline as antimalarial prophylaxis during travel resulted in increased changes in the beta diversity of the faecal resistome. CONCLUSIONS: Our study highlights travel to low- or middle-income countries, Africa or Southeast Asia, a long travel duration, or the use of ABHS or doxycycline as antimalarial prophylaxis as important risk factors for the acquisition/enrichment of ARGs during international travel.

Three-Dose Primary Series of Inactivated COVID-19 Vaccine for Persons Living with HIV, Hong Kong.

In a cohort of persons living with HIV in Hong Kong, surrogate virus neutralization testing for COVID-19 yielded a median level of 89% after the third dose of an inactivated COVID-19 vaccine, compared with 37% after the second dose. These results support using a 3-dose primary series for enhanced immune protection.

Surrogate neutralization responses following severe acute respiratory syndrome coronavirus 2 vaccination in people with HIV: comparison between inactivated and mRNA vaccine.

OBJECTIVE: People with HIV (PWH) co-infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are at higher odds of severe diseases. Whereas the immunogenicity of mRNA vaccine and adenovirus-vectored vaccine was similar between PWH in stable condition and healthy adults, the effects of inactivated vaccines are not known. DESIGN: Prospective longitudinal observational study in real-world setting. METHODS: Adult PWH in care and planning to receive either inactivated (day 0 and day 28) or mRNA-based (day 0 and day 21) vaccine against SARS-CoV-2 were recruited, with blood samples collected over 6 months for surrogate virus neutralization test (sVNT). Demographic and clinical data including age, sex, CD4 + cell count, and suppressed viral load (SVL) status were transcribed for analyses, by simple and multivariable linear regression models, and multivariable linear generalized estimating equations (GEE). RESULTS: A total of 611 HIV patients, 91% male patients, were recruited, of whom 423 and 184 have received mRNA-based and inactivated vaccine, respectively. The seroconversion rate was 99% for mRNA-based vs, 86% for inactivated vaccine [odds ratio (OR) = 21.56, P  = 0.004]. At 6 months, mRNA-based vaccine continued to give a higher response (94 vs. 57%, P  < 0.001). The temporal pattern varied between the two vaccines. By GEE, mRNA-based vaccine ( B  = 40.59, P  < 0.001) and latest SVL status ( B  = 10.76, P  = 0.01) were positively associated with sVNT level, but not latest CD4 + cell count. CONCLUSION: In HIV patients, inactivated vaccine gave a lower peak and shorter duration of sVNT responses compared with mRNA vaccine. The results suggested that different strategies may be needed in boosting the immunity in anticipation of the emergence of variants in the community.

Molecular epidemiology of acute HCV infection in HIV-positive patients from Hong Kong, Taipei, Tokyo.

BACKGROUND: Acute hepatitis C virus (HCV) infections have been increasingly reported among human immunodeficiency virus (HIV)-positive men who have sex with men (MSM) in the Asia-Pacific region. It remains unknown whether international network of HCV transmission has occurred in this region. METHODS: HIV-positive patients with acute HCV infection, defined as HCV seroconversion within a year or documented acute hepatitis with seroconversion, diagnosed in Hong Kong, Taipei and Tokyo during 2010-2016 were included in this molecular epidemiology study. The NS5B region of the HCV genome (365 bp) was amplified using nested polymerase chain reaction and sequenced. RESULTS: Of 234 HIV-positive patients with acute HCV infection, all were male with 94% being MSM. At the diagnosis of acute HCV infection, 73.5% had concurrent sexually transmitted diseases and 88.0% were receiving combination antiretroviral therapy. The most prevalent HCV genotype was 3a, 2a and 1b in Hong Kong, Taipei and Tokyo respectively. Nine independent clusters belonging to five genotypes (1b, 2a, 2c, 3a and 6a) were identified, each of which occurred in one city without overlapping except for one 3a sequence from Taipei that was closely related genetically to the Hong Kong cluster. CONCLUSIONS: No international network of HCV transmission was identified among HIV-positive patients in the three Asia-Pacific cities. The transmission dynamics of sexually acquired HCV differed by city, but the risk of intercity clustering should not be ignored.

Role of human Toll-like receptors in naturally occurring influenza A infections.

BACKGROUND: We investigated the roles of Toll-like receptors (TLRs) in naturally occurring influenza. METHODS: A prospective, case - control study was conducted. Adults hospitalized with virologically confirmed influenza A infections (onset <48 hours, before treatment) were compared with age-/gender-matched controls. TLRs (2, 3, 4, 7, 8, 9) expression in monocytes and dendritic cells (DCs - total, myeloid, plasmacytoid) was quantitated using flow cytometry. Gene expression of RLRs (RIG-1, MDA-5) was evaluated using real-time PCR. Concomitant signaling molecules expression, plasma cytokine/chemokine concentrations, and respiratory tract viral loads were measured. PBMCs were cultured and stimulated ex vivo with TLR-specific ligands for cytokine responses. RESULTS: Forty two patients with influenza (24 A/H3N2, 18 A/H1N1pdm09) and 20 controls were studied. Patients' mean age was 68 ± 16 years; 81% had respiratory/cardiovascular complications. There were increased cellular expressions of TLR9, TLR8, TLR3, and TLR7 during influenza; TLR2 and TLR4 were suppressed. Results were similar for both virus strains. Higher TLR expression levels at presentation significantly correlated with lower viral loads (Spearman's rho: -0.46 to -0.69 for TLR9, TLR8, and TLR3; P-values <0.05). Multivariate regression models (adjusted for age, comorbidity, disease severity, time from onset) confirmed their independent associations. Increased signaling molecules (phospho-MAPKs, IκB) and inflammatory cytokines (IL-6, sTNFR-1, CCL2/MCP-1; CXCL10/IP-10, IFN-γ) correlated with increased TLR expression. RLRs were upregulated simultaneously. PBMCs of patients with influenza showed significant, dynamic changes in their cytokine responses upon TLR stimulation, compared with controls. CONCLUSIONS: Our results suggest that TLRs play an important role in early, innate viral inhibition in naturally occurring influenza. Inflammatory cytokine responses are concomitantly induced. These findings support investigation of TLR targeting as a novel intervention approach for prophylaxis against influenza.

A "pre-seasonal" hospital outbreak of influenza pneumonia caused by the drift variant A/Victoria/361/2011-like H3N2 viruses, Hong Kong, 2011.

BACKGROUND: Beginning from late 2011 and early 2012, increasing circulation of antigenically drifted influenza A/Victoria/361/2011-like H3N2 viruses within genotype 3 of the A/Victoria/208/2009 clade have been reported in multiple European countries and elsewhere. Whether these emerging viruses are associated with increased disease severity is unclear. OBJECTIVES: To report the clinical and virological findings of a moderately severe hospital outbreak of A/Victoria/361/2011-like viruses that occurred in November 2011 in Hong Kong. STUDY DESIGN: Clinical and virological hospital outbreak investigation. RESULTS: The outbreak occurred in an adult psychiatric ward in November 2011, a time well before the usual local seasonal influenza winter peak. Altogether, 7 patients and 1 healthcare-worker were affected (mean age, 47 [range, 34-61] years). The attack rates among patients and healthcare-workers were 33% (7/21) and 7% (1/15), respectively. Pneumonia developed in 38% (3/8) of cases; none had underlying immunocompromised conditions. High nasopharyngeal viral loads were detected. All cases responded to antiviral treatment. Multiple amino acid mutations with reference to earlier A(H3N2) vaccine strains were mapped to key antigenic sites on hemagglutinin; however, no critical mutations on receptor binding sites were detected. Viral sequence variations jeopardized the performance of molecular diagnostic assays. CONCLUSIONS: Severe disease and pneumonia occurred in a substantial proportion of non-immunocompromised adults in a hospital outbreak attributed to the emerging antigenically drifted A/Victoria/361/2011-like H3N2 viruses. Close monitoring of the transmission of this drift variant is required. Further studies are also necessary to determine virus virulence.

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.

Complications and outcomes of pandemic 2009 Influenza A (H1N1) virus infection in hospitalized adults: how do they differ from those in seasonal influenza?

BACKGROUND: It is unclear whether pandemic 2009 influenza A (pH1N1) infection caused more significant disease among hospitalized adults than seasonal influenza. METHODS: A prospective, observational study was conducted in adults hospitalized with polymerase chain reaction-confirmed pH1N1 infection in 2 acute-care general hospitals from June 2009 to May 2010 (n = 382). Complications and outcomes were described and compared with those in a seasonal influenza cohort (2007-2008, same hospitals; n = 754). RESULTS: Hospitalized patients with pH1N1 influenza were younger than those with seasonal influenza (mean age ± standard deviation, 47 ± 20 vs 70 ± 19 years) and fewer had comorbid conditions (48% vs 64%). The rate of positive immunofluorescence assay results was low (54% vs 84%), and antiviral use was frequent (96% vs 52%). Most patients in both cohorts developed complicated illnesses (67.8% vs 77.1%), but patients with pH1N1 influenza had higher rates of extrapulmonary complications (23% vs 16%; P = .004) and intensive care unit admission and/or death (patient age <35 years, 2.3% vs 0%; 35-65 years, 12.4% vs 3.2%; >65 years, 13.5% vs 8.5%; adjusted odds ratio [OR] 2.13; 95% confidence interval [CI], 1.25-3.62; P = .005). Patients who received antiviral treatment within 96 h after onset had better survival (log-rank test, P < .001). However, without timely treatment, the mortality risk was higher with pH1N1 infection (9.0% vs 5.8% for seasonal influenza; adjusted OR, 6.85; 95% CI, 1.64-28.65; P = .008]. Bacterial superinfection worsened outcomes. CONCLUSIONS: Adults hospitalized for pH1N1 influenza had significant complications and mortality despite being younger than patients with seasonal influenza. Antiviral treatment within 96 h may improve survival.

Viral clearance and inflammatory response patterns in adults hospitalized for pandemic 2009 influenza A(H1N1) virus pneumonia.

BACKGROUND: Little is known about the virological and inflammatory responses of severe pandemic 2009 influenza A(H1N1) virus pneumonia during antiviral treatment. METHODS: In a prospective observational study, we recruited consecutive adults hospitalized with confirmed pandemic 2009 H1N1 infection during a 16-week period. Nasopharyngeal aspirate and non-respiratory samples (blood, stool and urine) were collected at presentation, and serial nasopharyngeal flocked swabs (NPFS) and tracheal aspirates (TA) were collected after initiating oseltamivir treatment for quantitative viral RNA assay, using real-time reverse transcriptase-PCR. Serial plasma samples were collected for cytokine/chemokine assay using cytometric bead array. Patients with severe pneumonia (lung infiltrates and hypoxaemia) were compared to those with milder illnesses. RESULTS: A total of 66 patients were studied (mean age 43 ±20 years); 28 (42%) developed severe pneumonia, of whom 10 (15%) required intubation. Severe pneumonia was associated with older age, dyspnoea, delayed presentation >2 days from onset, extrapulmonary virus detection (13-28%) and higher viral concentration despite late-presentation (multiple linear regression, ß=0.94, 95% confidence interval 0.15-1.74; P=0.02). Patients with severe pneumonia exhibited slow viral clearance with oseltamivir treatment, particularly in the lower respiratory tract (median [interquartile range] durations of RNA positivity after antiviral initiation were NPFS 6.0 days [3.0-8.0], TA 11.0 days [7.8-14.3] versus milder illness group NPFS of 2.0 days [1.0-3.0] days; P<0.01). High viral load in lower respiratory tract despite upper-tract RNA negativity and viral rebound after stopping treatment were noted in some patients. H275Y mutation was absent. High plasma levels of interleukin (IL)-6, CXCL-8 (IL-8), CCL2 (monocyte chemoattractant protein-1) and soluble tumour necrosis factor receptor-1 were observed, which correlated with the extent and progression of pneumonia in hospital. CONCLUSIONS: In severe 2009 H1N1 pneumonia, viral clearance is slow with treatment, particularly in the lower respiratory tract. A more sustained antiviral regime appears warranted.

Possible role of aerosol transmission in a hospital outbreak of influenza.

BACKGROUND: We examined the role of aerosol transmission of influenza in an acute ward setting. METHODS: We investigated a seasonal influenza A outbreak that occurred in our general medical ward (with open bay ward layout) in 2008. Clinical and epidemiological information was collected in real time during the outbreak. Spatiotemporal analysis was performed to estimate the infection risk among patients. Airflow measurements were conducted, and concentrations of hypothetical virus-laden aerosols at different ward locations were estimated using computational fluid dynamics modeling. RESULTS: Nine inpatients were infected with an identical strain of influenza A/H3N2 virus. With reference to the index patient's location, the attack rate was 20.0% and 22.2% in the "same" and "adjacent" bays, respectively, but 0% in the "distant" bay (P = .04). Temporally, the risk of being infected was highest on the day when noninvasive ventilation was used in the index patient; multivariate logistic regression revealed an odds ratio of 14.9 (95% confidence interval, 1.7-131.3; P = .015). A simultaneous, directional indoor airflow blown from the "same" bay toward the "adjacent" bay was found; it was inadvertently created by an unopposed air jet from a separate air purifier placed next to the index patient's bed. Computational fluid dynamics modeling revealed that the dispersal pattern of aerosols originated from the index patient coincided with the bed locations of affected patients. CONCLUSIONS: Our findings suggest a possible role of aerosol transmission of influenza in an acute ward setting. Source and engineering controls, such as avoiding aerosol generation and improving ventilation design, may warrant consideration to prevent nosocomial outbreaks.

Viral loads and duration of viral shedding in adult patients hospitalized with influenza.

BACKGROUND: The goal of this study was to characterize viral loads and factors affecting viral clearance in persons with severe influenza. METHODS: This was a 1-year prospective, observational study involving consecutive adults hospitalized with influenza. Nasal and throat swabs were collected at presentation, then daily until 1 week after symptom onset. Real-time reverse-transcriptase polymerase chain reaction to determine viral RNA concentration and virus isolation were performed. Viral RNA concentration was analyzed using multiple linear or logistic regressions or mixed-effect models. RESULTS: One hundred forty-seven inpatients with influenza A (H3N2) infection were studied (mean age+/-standard deviation, 72+/-16 years). Viral RNA concentration at presentation positively correlated with symptom scores and was significantly higher than that among time-matched outpatients (control subjects). Patients with major comorbidities had high viral RNA concentration even when presenting>2 days after symptom onset (mean+/-standard deviation, 5.06+/-1.85 vs 3.62+/-2.13 log10 copies/mL; P=.005; beta, +0.86 [95% confidence interval, +0.03 to +1.68]). Viral RNA concentration demonstrated a nonlinear decrease with time; 26% of oseltamivir-treated and 57% of untreated patients had RNA detected at 1 week after symptom onset. Oseltamivir started on or before symptom day 4 was independently associated with an accelerated decrease in viral RNA concentration (mean beta [standard error], -1.19 [0.43] and -0.68 [0.33] log10 copies/mL for patients treated on day 1 and days 2-3, respectively; P<.05) and viral RNA clearance at 1 week (odds ratio, 0.10 [95% confidence interval, 0.03-0.35] and 0.30 [0.10-0.90] for patients treated on day 1-2 and day 3-4, respectively). Conversely, major comorbidities and systemic corticosteroid use for asthma or chronic obstructive pulmonary disease exacerbations were associated with slower viral clearance. Viral RNA clearance was associated with a shorter hospital stay (7.0 vs 13.5 days; P=.001). CONCLUSION: Patients hospitalized with severe influenza have more active and prolonged viral replication. Weakened host defenses slow viral clearance, whereas antivirals started within the first 4 days of illness enhance viral clearance.

Cytokine responses in a severe case of glandular fever treated successfully with foscarnet combined with prednisolone and intravenous immunoglobulin.

Viral loads and cytokine responses Epstein-Barr virus (EBV) were measured in an 18-year-old boy with severe glandular fever complicated by a mild anaemia, severe thrombocytopaenia and neutropaenia. Hepatosplenomegaly was detected by abdominal ultrasound in the presence of significant hepatitis. Cytokine testing demonstrated elevated cell-mediated Th1 (IFN-gamma, IL-12, sTNFR1, CXCL10, CXCL9 and CCL3) and humoral Th2 (IL-4) immune responses. Serum antibodies to EBV virus capsid antigen (VCA) IgM and IgG antibodies were detected, together with a raised EBV DNA level (up to about 70,000 DNA copies/mL) in the acute phase of the illness. This EBV DNA load decreased rapidly in response to treatment with a combination of foscarnet, intravenous immunoglobulin and prednisolone, and the boy's symptoms settled eventually after approximately 50 days of illness, following this combined antiviral and immune-modulating therapy. Detailed immunological, virological, haematological and biochemical laboratory parameters are presented to document this patient's severe EBV disease and eventual recovery.

Failure to confirm HIV infection in two end-stage HIV/AIDS patients using a popular commercial line immunoassay.

Immunoassays using either viral lysate (Western blot) or recombinant/synthetic antigen (immunoblot) for anti-HIV capture are still the preferred method to confirm HIV infection. Two cases of HIV-1-infected patients presented with acquired immunodeficiency syndrome (AIDS)-defining illnesses. Laboratory tests were performed using multiple commercial HIV test kits on multiple sera from both patients over several weeks. Both patients were strongly positive on the anti-HIV/p24 antigen combined screening assay. Yet, HIV-1 infection could not be confirmed using a popular commercial immunoassay. Eventually, HIV infection was confirmed using an alternative commercial Western blot assay as well as an HIV quantitative PCR test. In laboratories without nucleic acid testing (NAT) for HIV, indeterminate results may delay confirmation of HIV infection, if commercial line immunoassays alone are available. Some end-stage HIV/AIDS patients may not produce antibodies to specific HIV antigens and may therefore give indeterminant or negative results on some immunoassays, depending on the type of antigen used. This report highlights the utility of having NAT available when diagnosing difficult cases of HIV infection, especially in light of the recent Centers for Disease Control and Prevention move towards more universal, routine, HIV testing.