Clara P. Y. Li

Broad-spectrum antivirals for the emerging Middle East respiratory syndrome coronavirus.

Summary Objectives Middle East respiratory syndrome coronavirus (MERS-CoV) has emerged to cause fatal infections in patients in the Middle East and traveler-associated secondary cases in Europe and Africa. Person-to-person transmission is evident in outbreaks involving household and hospital contacts. Effective antivirals are urgently needed. Methods We used small compound-based forward chemical genetics to screen a chemical library of 1280 known drugs against influenza A virus in Biosafety Level-2 laboratory. We then assessed the anti-MERS-CoV activities of the identified compounds and of interferons, nelfinavir, and lopinavir because of their reported anti-coronavirus activities in terms of cytopathic effect inhibition, viral yield reduction, and plaque reduction assays in Biosafety Level-3 laboratory. Results Ten compounds were identified as primary hits in high-throughput screening. Only mycophenolic acid exhibited low EC50 and high selectivity index. Additionally, ribavirin and interferons also exhibited in-vitro anti-MERS-CoV activity. The serum concentrations achievable at therapeutic doses of mycophenolic acid and interferon-β1b were 60–300 and 3–4 times higher than the concentrations at which in-vitro anti-MERS-CoV activities were demonstrated, whereas that of ribavirin was ∼2 times lower. Combination of mycophenolic acid and interferon-β1b lowered the EC50 of each drug by 1–3 times. Conclusions Interferon-β1b with mycophenolic acid should be considered in treatment trials of MERS.

Delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel Middle East respiratory syndrome coronavirus: implications for pathogenesis and treatment

The high mortality associated with the novel Middle East respiratory syndrome coronavirus (MERS-CoV) has raised questions about the possible role of a cytokine storm in its pathogenesis. Although recent studies showed that MERS-CoV infection is associated with an attenuated IFN response, no induction of inflammatory cytokines was demonstrated during the early phase of infection. To study both early and late cytokine responses associated with MERS-CoV infection, we measured the mRNA levels of eight cytokine genes [TNF-α, IL-1β, IL-6, IL-8, IFN-β, monocyte chemotactic protein-1, transforming growth factor-β and IFN-γ-induced protein (IP)-10] in cell lysates of polarized airway epithelial Calu-3 cells infected with MERS-CoV or severe acute respiratory syndrome (SARS)-CoV up to 30 h post-infection. Among the eight cytokine genes, IL-1β, IL-6 and IL-8 induced by MERS-CoV were markedly higher than those induced by SARS-CoV at 30 h, whilst TNF-α, IFN-β and IP-10 induced by SARS-CoV were markedly higher than those induced by MERS-CoV at 24 and 30 h in infected Calu-3 cells. The activation of IL-8 and attenuated IFN-β response by MERS-CoV were also confirmed by protein measurements in the culture supernatant when compared with SARS-CoV and Sendai virus. To further confirm the attenuated antiviral response, cytokine response was compared with human HCoV-229E in embryonal lung fibroblast HFL cells, which also revealed higher IFN-β and IP-10 levels induced by HCoV-229E than MERS-CoV at 24 and 30 h. Whilst our data supported recent findings that MERS-CoV elicits attenuated innate immunity, this represents the first report to demonstrate delayed proinflammatory cytokine induction by MERS-CoV. Our results provide insights into the pathogenesis and treatment of MERS-CoV infections.

Dose sparing intradermal trivalent influenza (2010/2011) vaccination overcomes reduced immunogenicity of the 2009 H1N1 strain

BACKGROUND We hypothesized that low dose intradermal vaccination of the trivalent influenza vaccine (TIV) delivered by the MicronJet600™ (NanoPass Technologies, Israel) would be non-inferior to the full dose intramuscular and mid dose Intanza(®) vaccination in the elderly and the chronically ill adults. METHODS We performed a prospective randomized trial on elderly and chronically ill adults. Subjects were randomly assigned into 4 groups. Groups ID3 and ID9 received reduced dose ID TIV (3 μg and 9 μg of hemagglutinin (HA) per strain respectively) delivered by MicronJet600™ (NanoPass Technologies, Israel). Group INT9 received reduced dose ID TIV (9 μg) delivered by Becton Dickinsons Soluvia™ device (Intanza(®)9, Sanofi-Pasteur, France). Control group IM15 received a full dose IM TIV (15 μg). We measured antibody titers by hemagglutination inhibition (HAI) and microneutralization (MN) assays at baseline and day 21. RESULTS Baseline characteristics for all groups were similar (group and sample sizes: ID3=63; ID9=68; INT9=65; and IM15=66). At day 21 post vaccination, the GMT ratio and the seroconversion rates difference for all three strains of the ID vaccine groups were non-inferior to the IM vaccine group. The seroconversion rate, seroprotection rate, and the GMT of the H1N1 strains by HAI and MN assays were significantly higher in the ID groups compared with the full dose IM vaccine group. The seroconversion rates of the H3N2 strain by HAI assay were also significantly higher in the ID groups when compared with the full dose IM group. Direct comparison among the three ID groups showed no significant differences. No serious adverse events related to vaccination were reported. CONCLUSION Dose-sparing ID TIV can overcome reduced immunogenicity of the H1N1 strain, and according to some measures, for the H3N2 strain. At risk subjects indicated for the TIV should be considered for intradermal immunization to compensate for reduced immunogenicity.

Immunogenicity of Intradermal Trivalent Influenza Vaccine With Topical Imiquimod: A Double Blind Randomized Controlled Trial

BACKGROUND Imiquimod, a synthetic Toll-like receptor 7 agonist enhanced immunogenicity of influenza vaccine in a mouse model. We hypothesized that topical imiquimod before intradermal influenza vaccination (TIV) would produce similar effect in human. METHODS We performed a prospective 1-year follow-up, double-blind, randomized, controlled trial with adults with comorbidities. Participants were randomized to 1 of the following 3 vaccinations: topical 5% 250 mg imiquimod ointment followed by intradermal TIV, topical aqueous-cream followed by intradermal TIV, or topical aqueous-cream followed by intramuscular TIV. Patients and investigators were blinded to the type of topical treatment applied. Hemagglutination inhibition (HI) and microneutralization antibody titers were measured. The primary outcome was the day 7 seroconversion rate. RESULTS Ninety-one recruited participants completed the study. The median age was 73 years. On day 7, 27/30 (90%) patients who received imiquimod and intradermal TIV achieved seroconversion against the H1N1 strain by HI, compared with 4/30 (13.3%) who received aqueous-cream and intramuscular TIV (P < .001), and 12/31 (38.7%) who received aqueous-cream and intradermal TIV (P < .001). The seroconversion, seroprotection, and geometric mean titer-fold increase were met in all 3 strains in the imiquimod and intradermal TIV group 2 weeks earlier, and the better seroconversion rate was sustained from day 7 to year 1 (P ≤ .001). The better immunogenicity was associated with fewer hospitalizations for influenza or pneumonia (P < .05). All adverse reactions were self-limited. CONCLUSIONS Pretreatment with topical imiquimod significantly expedited, augmented, and prolonged the immunogenicity of influenza vaccination. This strategy for influenza immunization should be considered for the elderly population.

Analytical Sensitivity of Seven Point-of-Care Influenza Virus Detection Tests and Two Molecular Tests for Detection of Avian Origin H7N9 and Swine Origin H3N2 Variant Influenza A Viruses

Rapid virological diagnosis is important for early case identification, initiation of specific antiviral therapy, and implementation of infection control measures for patients suffering from influenza. Most point-of-care (POC) test kits commonly used at the outpatient setting which provide results

Comparison of Pyrosequencing, Sanger Sequencing, and Melting Curve Analysis for Detection of Low-Frequency Macrolide-Resistant Mycoplasma pneumoniae Quasispecies in Respiratory Specimens

ABSTRACT Macrolide-resistant Mycoplasma pneumoniae (MRMP) is emerging worldwide and has been associated with treatment failure. In this study, we used pyrosequencing to detect low-frequency MRMP quasispecies in respiratory specimens, and we compared the findings with those obtained by Sanger sequencing and SimpleProbe PCR coupled with a melting curve analysis (SimpleProbe PCR). Sanger sequencing, SimpleProbe PCR, and pyrosequencing were successfully performed for 96.7% (88/91), 96.7% (88/91), and 93.4% (85/91) of the M. pneumoniae-positive specimens, respectively. The A-to-G transition at position 2063 was the only mutation identified. Pyrosequencing identified A2063G MRMP quasispecies populations in 78.8% (67/88) of the specimens. Only 38.8% (26/67) of these specimens with the A2063G quasispecies detected by pyrosequencing were found to be A2063G quasispecies by Sanger sequencing or SimpleProbe PCR. The specimens that could be detected by SimpleProbe PCR and Sanger sequencing had higher frequencies of MRMP quasispecies (51% to 100%) than those that could not be detected by those two methods (1% to 44%). SimpleProbe PCR correctly categorized all specimens that were identified as wild type or mutant by Sanger sequencing. The clinical characteristics of the patients were not significantly different when they were grouped by the presence or absence of MRMP quasispecies, while patients with MRMP identified by Sanger sequencing more often required a switch from macrolides to an alternative M. pneumoniae-targeted therapy. The clinical significance of mutant quasispecies should be investigated further with larger patient populations and with specimens obtained before and after macrolide therapy.

Clinical significance of Pneumocystis jiroveci in patients with active tuberculosis

Pneumocystis colonization has been associated with airway inflammation and obstruction. We conducted a retrospective cohort study to investigate the clinical significance of Pneumocystis in the airway of patients with active tuberculosis. Of the 108 respiratory specimens tested positive for M. tuberculosis by polymerase chain reaction (PCR), 11 (10.2%) were also positive for Pneumocystis by PCR. Compared with patients tested negative for Pneumocystis, those with Pneumocystis had a higher serum alanine transaminase level, a greater likelihood of requiring oxygen supplementation, and a worse 30-day mortality. The proportion of patients with chronic obstructive pulmonary disease was not significantly different between the 2 groups, but lung malignancy was more prevalent among patients with Pneumocystis. Multivariate analysis showed that Pneumocystis was independently associated with oxygen supplementation. Our study has shown an association between the detection of Pneumocystis in lower respiratory tract specimens and greater impairment of pulmonary function among patients with active tuberculosis.

Immunogenicity and safety of intradermal trivalent influenza vaccination in nursing home older adults: a randomized controlled trial.

OBJECTIVE To compare the immunogenicity and safety between full-dose (15 μg) intramuscular (i.m.) and full-dose (15 μg) intradermal (i.d.) immunization of the trivalent influenza vaccine in nursing home older adults. DESIGN A single-center, randomized, controlled, open-label, parallel group trial from October 2013 to April 2014. SETTING Nine nursing homes in Hong Kong. PARTICIPANTS Hundred nursing home older adults (mean age: 82.9 ± 7.4 years). INTERVENTION Fifty received i.d. (Intanza) and 50 received i.m. (Vaxigrip) vaccination. MEASUREMENTS Baseline measurements included demographics, comorbidity, frailty and nutritional status. Day 21 and day 180 immunogenicity (seroconversion rate, seroprotection rate, geometric mean titer [GMT] fold increase in antibody titer) using hemagglutination-inhibition and adverse events were measured. Noninferiority and superiority of i.d. compared with i.m. vaccination in immunogenicity were analyzed. The study was registered on ClinicalTrials.gov; identifier: NCT 01967368. RESULTS At day 21, noninferiority in immunogenicity of the i.d. vaccination was demonstrated. The seroconversion rate of the H1N1 strain was significantly higher in the i.d. group. At day 180, immunogenicity of both groups fell but the GMT of all strains in i.d. group was higher and the difference was significant for H3N2 strain. The seroconversion rate and GMT fold increase of H3N2 strain was significantly higher in the i.d. group. Local adverse events was significantly more in i.d. group, but they were mild and resolved in 72 hours. CONCLUSIONS I.d. vaccination is noninferior, and even superior in some parts of immunogenicity assessment, to i.m. vaccination without compromising safety in nursing home older adults. I.d. vaccination is a good alternative to i.m. vaccination in this population.

Assessment of antigen and molecular tests with serial specimens from a patient with influenza A(H7N9) infection.

Kwok-Hung Chan, Kelvin K. W. To, Jasper F. W. Chan, Clara P. Y. Li, Kit-Man Chan, Honglin Chen, Pak-Leung Ho, Kwok-Yung Yuen Department of Microbiology, The University of Hong Kong, Hong Kong Special Administrative Region, China; State Key Laboratory for Emerging Infectious Diseases, Carol Yu Centre for Infection, Research Centre of Infection and Immunology, The University of Hong Kong, Hong Kong Special Administrative Region, China

Response to Comments on “Immunogenicity and Safety of Intradermal Trivalent Influenza Vaccination in Nursing Home Older Adults: A Randomized Controlled Trial”

To the Editor: The recent report on “Intradermal Trivalent Influenza Vaccination in Nursing Home Older Adults” is very interesting.1 Chan et al1 concluded that “ID vaccination is noninferior, and even superior in some parts of immunogenicity assessment, to IM vaccination without compromising safety in nursing home older adults.” In fact, there aremany considerations on the present study. First, it must be noted that “the immune response appears to partially depend on the delivery device and route of injection.”2 The technique and injection device must be well selected to get the most appropriate immune response.2,3 The basic pitfall in medical practice is the incorrect intradermal administration technique. In addition, to get the best immunogenicity, a specific device for intradermal administration is required for successful intradermal delivery.2 Second, although the intradermal influenza vaccination is considered safe, a higher rate of local adverse effects (such as pain and erythema) is reported in some settings.4,5