OTUB1 contributes to the stability and function of Influenza A virus NS2.

The influenza A virus (IAV) consists of 8 single-stranded, negative-sense viral RNA (vRNA) segments. After infection, vRNA is transcribed, replicated, and wrapped by viral nucleoprotein (NP) to form viral ribonucleoprotein (vRNP). The transcription, replication, and nuclear export of the viral genome are regulated by the IAV protein, NS2, which is translated from spliced mRNA transcribed from viral NS vRNA. This splicing is inefficient, explaining why NS2 is present in low abundance after IAV infection. The levels of NS2 and its subsequent accumulation are thought to influence viral RNA replication and vRNP nuclear export. Here we show that NS2 is ubiquitinated at the K64 and K88 residues by K48-linked and K63-linked polyubiquitin (polyUb) chains, leading to the degradation of NS2 by the proteasome. Additionally, we show that a host deubiquitinase, OTUB1, can remove polyUb chains conjugated to NS2, thereby stabilizing NS2. Accordingly, knock down of OTUB1 by siRNA reduces the nuclear export of vRNP, and reduces the overall production of IAV. These results collectively demonstrate that the levels of NS2 in IAV-infected cells are regulated by a ubiquitination-deubiquitination system involving OTUB1 that is necessary for optimal IAV replication.

Antifungal susceptibility testing with YeastONE™ is not predictive of clinical outcomes of Cryptococcus neoformans var. grubii fungemia.

Mortality rates due to Cryptococcus neoformans var. grubii fungemia remain significant despite treatment with antifungal drugs. The predictive function of antifungal susceptibility and its correlation with treatment outcome remains controversial. A retrospective study was conducted from January 1, 2009, to December 31, 2016, on 85 patients with C. neoformans var. grubii fungemia confirmed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Antifungal drug susceptibility was determined using the YeastONE™ colorimetric broth microdilution method coupled with Vizion™ System following the Clinical and Laboratory Standards Institute guidelines. Six antifungal agents-amphotericin B, fluconazole, flucytosine, itraconazole, posaconazole, and voriconazole-were tested. The patients' demographic data and clinical information were abstracted for further analyses. Antifungal regimens consisting of amphotericin B with or without fluconazole or flucytosine were administered for induction treatment of these patients, followed with intravenous or oral fluconazole for maintenance therapy. Clinical outcomes were defined by 14- and 30-day mortality rates. Risk factors associated with outcomes were fitted in a logistic regression model by univariate or multivariate method. Eighty-five patients with C. neoformans var. grubii fungemia were enrolled in the study. The Sequential Organ Failure Assessment Score, Glasgow Coma Scale, Charlson comorbidity score, and adequate duration of therapy for amphotericin B were predictors for mortality in univariate analysis. Antifungal susceptibility testing with YeastONE™ does not predict clinical outcomes of C. neoformans var. grubii fungemia. Greater disease severity, high comorbidities, poor consciousness level, and inappropriate treatment were associated with increased mortality in cryptococcemia cases.

Cryptococcus neoformans is an encapsulated yeast living in both plants and animals that is composed of three main serotypes: C. neoformans var. grubii, C. neoformans var. gattii, and C. neoformans var. neoformans. C. neoformans var. grubii is the most common disease-causing Cryptococcus species worldwide. C. neoformans var. gattii is more prevalent than C. neoformans var. neoformans in both tropical and subtropical regions of Asia. C. neoformans causes severe, even fatal, diseases such as pulmonary infection, bloodstream infection, skin and soft tissue infection, bone and joint infection, central nervous system infection, and disseminated infection, regardless of host immunocompetence. We conducted a retrospective study on 85 patients who contracted cryptococcemia from January 1, 2009, to December 31, 2016. This work conducted both microbiological and clinical studies involving in vitro susceptibility testing, demographic data, comorbidities, treatment modalities, and treatment outcomes. We utilized a modern medical technique-based instrument, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS; Biotyper, Bruker Daltonics, Inc.), which determines the unique proteomic fingerprint of an organism, to identify the C. neoformans serotype. We utilized Thermo Fisher Scientific™ Sensititre™ YeastONE™ colorimetric broth microdilution plates coupled with a Vizion™ Digital MIC Viewing System (a computer-assisted optical reading machine) to determine the in vitro susceptibility of amphotericin B, flucytosine, fluconazole, itraconazole, posaconazole, and voriconazole against 85 C. neoformans var. grubii blood isolates. In conclusion, the susceptibility patterns of these antifungal agents did not correlate significantly with treatment outcomes. However, a lower disease severity score, a lower Glasgow Coma Scale score, fewer comorbidities, and adequate amphotericin B treatment duration were predictors for treatment success in univariate analysis.

Diagnostic performance of the Sofia® influenza A+B fluorescent immunoassay in adult outpatients in Northern Taiwan.

To evaluate the diagnostic performance of the Sofia influenza A+B fluorescent immunoassay (Sofia FIA), we performed a prospective study at the Chang Gung Memorial Hospital in Taiwan from January 2012 to December 2013. Patients who presented at out-patient clinics or the emergency department with influenza-like illness were included. Upper respiratory tract specimens were collected from oropharynx or nasopharynx. Performance of the Sofia FIA was compared to that of the Formosa One Sure Flu A/B Rapid Test. A Real-time reverse transcriptase-polymerase chain reaction assay (RT-PCR) and/or virus culture were used as reference standards. Of the 109 enrolled patients, the sensitivity, specificity, positive, and negative predictive values of the Sofia FIA to detect influenza A virus were 82%, 89%, 77%, and 89%, respectively. These parameters were 100% when the samples were from nasopharynx. The positive predictive value for influenza B virus detection was 29%. The sensitivity of the Sofia FIA for detection of influenza A virus was 93% between days 2 and 4 after onset of symptoms. For specimens with low viral loads (RT-PCR cycle threshold between 30 and 34.9), the sensitivity of The Sofia FIA was 83% (10/12). The Sofia FIA performed effectively in detecting influenza A virus infection. With nasopharyngeal samples, the performance was comparable to RT-PCR. Although influenza viral load typically decreases with time, the Sofia FIA was sensitive enough to identify influenza infecting patients presenting after several days of illness. However, a high false positive rate limits the assay's usefulness to identify influenza B virus infection.

Antimicrobial resistance in Mycobacterium abscessus complex isolated from patients with skin and soft tissue infections at a tertiary teaching hospital in Taiwan.

Background: Mycobacterium abscessus complex (MABC) is the most common non-tuberculous mycobacterium that causes complicated skin and soft tissue infections (cSSTIs). The selection of antimycobacterial agents for successful treatment of such infections is a critical issue. Objectives: To investigate the antimicrobial susceptibility patterns of MABC isolates from skin and soft tissue to a variety of antimycobacterial agents. Methods: Sixty-seven MABC isolates were collected and partial gene sequencing of secA1, rpoB and hsp65 was used to classify them into three subspecies: M. abscessus subsp. abscessus (MAB), M. abscessus subsp. massiliense (MMA) and M. abscessus subsp. bolletii (MBO). The MICs of 11 antimycobacterial agents for these 67 isolates were determined using a broth microdilution method and commercial Sensititre RAPMYCOI MIC plates, as recommended by CLSI. Results: In total, 28 MAB, 38 MMA and 1 MBO were isolated from patients with cSSTIs at our hospital. Most MABC strains were resistant to ciprofloxacin, doxycycline, imipenem, linezolid, minocycline, moxifloxacin and trimethoprim/sulfamethoxazole. In addition, most MABC strains were intermediately susceptible or resistant to cefoxitin. Eighteen of the 28 MABs and 1 MBO isolate harboured the T28 polymorphism in the erm(41) gene. Two of the 38 MMA isolates had an rrl A2059G point mutation. Most of the MABC strains were susceptible to amikacin and tigecycline. Conclusions: In Taiwan, amikacin, clarithromycin and tigecycline have good activity against MMA and MAB erm(41) C28 sequevar isolates, whereas amikacin and tigecycline, rather than clarithromycin, have good activity against both MBO and MAB erm(41) T28 sequevar isolates. Clinical trials are warranted to correlate these data with clinical outcomes.

Lack of Association between YEASTONE Antifungal Susceptibility Tests and Clinical Outcomes of Cryptococcus Meningitis.

The relation between antifungal susceptibility and treatment outcomes is not well-characterized. There is paucity of surveillance data for cerebrospinal fluid (CSF) isolates of cryptococcus investigated with YEASTONE colorimetric broth microdilution susceptibility testing. A retrospective study of laboratory-confirmed cryptococcus meningitis (CM) patients was conducted. The antifungal susceptibility of CSF isolates was determined using YEASTONE colorimetric broth microdilution. Clinical parameters, CSF laboratory indices, and antifungal susceptibility results were analyzed to identify risk factors for mortality. High rates of resistance to fluconazole and flucytosine were observed in this cohort. Voriconazole had the lowest MIC (0.06 µg/mL) and lowest rate of resistance (3.8%). In a univariate analysis, hematological malignancy, concurrent cryptococcemia, high Sequential Organ Failure Assessment (SOFA) score, low Glasgow coma scale (GCS) score, low CSF glucose level, high CSF cryptococcal antigen titer, and high serum cryptococcal antigen burden were associated with mortality. In a multivariate analysis, meningitis with concurrent cryptococcemia, GCS score, and high CSF cryptococcus burden, were independent predictors of poor prognosis. Both early and late mortality rates were not significantly different between CM wild type and non-wild type species.

Modulating cholesterol-rich lipid rafts to disrupt influenza A virus infection.

Influenza A virus (IAV) is widely disseminated across different species and can cause recurrent epidemics and severe pandemics in humans. During infection, IAV attaches to receptors that are predominantly located in cell membrane regions known as lipid rafts, which are highly enriched in cholesterol and sphingolipids. Following IAV entry into the host cell, uncoating, transcription, and replication of the viral genome occur, after which newly synthesized viral proteins and genomes are delivered to lipid rafts for assembly prior to viral budding from the cell. Moreover, during budding, IAV acquires an envelope with embedded cholesterol from the host cell membrane, and it is known that decreased cholesterol levels on IAV virions reduce infectivity. Statins are commonly used to inhibit cholesterol synthesis for preventing cardiovascular diseases, and several studies have investigated whether such inhibition can block IAV infection and propagation, as well as modulate the host immune response to IAV. Taken together, current research suggests that there may be a role for statins in countering IAV infections and modulating the host immune response to prevent or mitigate cytokine storms, and further investigation into this is warranted.

Treatment Outcome in Patients with Mycobacterium abscessus Complex Lung Disease: The Impact of Tigecycline and Amikacin.

BACKGROUND: The contemporary guidelines have recommended multiple antimicrobial therapies along with oral macrolides for the treatment of Mycobacterium abscessus complex lung disease (MABC-LD). However, there is little evidence supporting the parenteral tigecycline-containing regimens against MABC-LD. Therefore, we conducted this study to evaluate the effect of intravenous tigecycline-containing regimens on the treatment of MABC-LD. METHODS: A retrospective study was conducted in 6 medical centers. Patients with MABC-LD that were followed up at ≥12 months were enrolled. Mycobacterium abscessus subspecies were identified by hsp65, rpoB, secA1 gene PCR, and sequencing. Antimicrobial susceptibility was determined for 34 patients using broth microdilution methods following the Clinical and Laboratory Standards Institute (CLSI) guideline. The microbiology and treatment outcomes were defined as either success or failure. The impacts of tigecycline and amikacin were adjusted for age, comorbidities, surgical resection, and radiologic scores. RESULTS: During the study period, seventy-one patients were enrolled for final analysis. The microbiology failure rate was 61% (43/71) and the treatment failure rate was 62% (44/71). For M. abscessus complex, 97% (33/34) of tigecycline MIC were ≤1 mg/L. Amikacin also demonstrated great susceptibility (94.1%; 32/34). Treatment with regimens containing tigecycline plus amikacin provided better microbiology success (adjusted OR 17.724; 95% CI 1.227-267.206) and treatment success (adjusted OR 14.085; 95% CI 1.103-166.667). CONCLUSION: The outcome of MABC-LD is always unsatisfactory. Treatment regimens with oral macrolide in combination with tigecycline and amikacin were correlated with increased microbiology success and less treatment failure.

Structure-function analysis of neutralizing antibodies to H7N9 influenza from naturally infected humans.

Little is known about the specificities and neutralization breadth of the H7-reactive antibody repertoire induced by natural H7N9 infection in humans. We have isolated and characterized 73 H7-reactive monoclonal antibodies from peripheral B cells from four donors infected in 2013 and 2014. Of these, 45 antibodies were H7-specific, and 17 of these neutralized the virus, albeit with few somatic mutations in their variable domain sequences. An additional set of 28 antibodies, isolated from younger donors born after 1968, cross-reacted between H7 and H3 haemagglutinins in binding assays, and had accumulated significantly more somatic mutations, but were predominantly non-neutralizing in vitro. Crystal structures of three neutralizing and protective antibodies in complex with the H7 haemagglutinin revealed that they recognize overlapping residues surrounding the receptor-binding site of haemagglutinin. One of the antibodies, L4A-14, bound into the sialic acid binding site and made contacts with haemagglutinin residues that were conserved in the great majority of 2016-2017 H7N9 isolates. However, only 3 of the 17 neutralizing antibodies retained activity for the Yangtze River Delta lineage viruses isolated in 2016-2017 that have undergone antigenic change, which emphasizes the need for updated H7N9 vaccines.

Clinical characteristics and treatment outcomes of vancomycin-resistant Enterococcus faecium bacteremia.

BACKGROUND: Vancomycin-resistant Enterococcus faecium (VRE-fm) bacteremia causes significant mortality in hospitalized patients. We sought to investigate clinical characteristics, treatment outcomes, and microbiological eradication associated with VRE-fm bacteremia. METHODS: A retrospective cohort study was conducted and included 210 adult patients admitted between January 1, 2011 and December 31, 2015. RESULTS: The mean Pitt bacteremia score was 4.7. ICU stay (48.6%) and mechanical ventilation (46.2%) were common. Diabetes mellitus was the most common concomitant disease (43.3%), followed by malignancies, including hematologic malignancies (14.3%) and solid cancers (28.1%). The 14-day and 28-day mortality rates were 37.1% and 50.5%, respectively. Linezolid or daptomycin treatment for at least 10 days and higher Pitt bacteremia scores were independently associated with 14-day and 28-day mortality. Longer treatment duration of linezolid or daptomycin predicted microbiological eradication independently. Daptomycin-treated patients tended to have higher 14-day and 28-day mortality, and lower microbial eradication rates (20.8% versus 8.7%; 40.6% versus 26.1%; 14.1% versus 26.1%; respectively) than linezolid-treated patients, and cumulative survival rates at 14 and 28 days tended to be lower in patients who received low-dose daptomycin (<10 mg/kg/day) than that in those who received linezolid and high-dose daptomycin (≥10 mg/kg/day); however, the differences were not statistically significant. CONCLUSION: Higher disease severity and inappropriate treatment were associated with increased mortality and longer treatment duration of linezolid or daptomycin was associated with microbial eradication for the patient with VRE-fm bacteremia.

Predictive Symptoms and Signs of Laboratory-confirmed Influenza: A Prospective Surveillance Study of Two Metropolitan Areas in Taiwan.

Influenza infection poses annual threats and leads to significant morbidity and mortality. Early diagnosis is the key to successful treatment. Laboratory-based diagnosis has various limitations. Diagnosis based on symptoms or signs is still indispensable in clinical practice. We investigated the symptoms or signs associated with laboratory-confirmed influenza.A prospective study across 2 influenza seasons was performed from June 2010 to June 2012 at 2 branches (Taipei and Lin-Kou) of Chang Gung Memorial Hospital. Patients who visited outpatient clinics with suspected acute respiratory tract infection were sampled by throat swab or nasopharyngeal swab. RT-PCR and/or virus culture were used as a reference standard. We used logistic regression to identify the symptoms or signs associated with laboratory-confirmed influenza infection. We also evaluated the performance metrics of different influenza-like illness used in Taiwan, the USA, and WHO.A total of 158 patients were included in the study. The prevalence of influenza infection was 45% (71/158). Fever, cough, rhinorrhea, sneezing, and nasal congestion were significant predictors for influenza infection. Whereas fever + cough had a best sensitivity (86%; confidence interval [CI] 76%-93%), fever + cough and sneezing had a best specificity (77%; CI 62%-88%). Different case definitions of influenza-like illness had comparable accuracy in sensitivity and specificity.Clinical diagnosis based on symptoms and signs is useful for allocating resources, identifying those who may benefit from early antiviral therapy and providing valuable information for surveillance purpose.

Diagnostic capacity of rapid influenza antigen test: reappraisal with experience from the 2009 H1N1 pandemic.

BACKGROUND: The rapid influenza antigen test (RIAT) has been questioned because of its poor sensitivity. Clinicians are confused as to what diagnostic help it may provide. RIAT was reappraised by other laboratory confirmatory tests for its diagnostic capacity. METHODS: Records of RIAT, RT-PCR and virus culture, performed for upper respiratory tract samples during the period from July 2009 to January 2010, were reviewed. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of RIAT were evaluated with RT-PCR and virus culture as reference. RESULTS: With either positive RT-PCR or positive virus culture as confirmation of presence of the virus, the overall sensitivity was 44.2% (235/532) and the overall PPV was 91.1% (235/258). With both negative RT-PCR and negative virus culture as confirmation of absence of the virus, the overall specificity was 98.2% (239/264) and the overall NPV was 92.6% (239/258). The PPV reached 96.2% during peak prevalence of infection and the NPV increased to 91.7% with low prevalence. The sensitivity for seasonal H3N2 was 56% (56/100), significantly better than the 39.6%, (156/394) for 2009 pandemic H1N1. Although RIAT positivity correlated to the viral load in samples, a substantial amount of negative RIAT samples had high viral load, with 16.8% (260/1548) of Ct value less than 36 and 8.8% (136/1548) of Ct value less than 31. CONCLUSION: An algorithm is derived for the fast and inexpensive point-of-care laboratory test RIAT for appropriate application in clinical diagnosis of influenza virus infection. In peak seasons, positive RIAT confirms the diagnosis, with PPV over 96%. In low seasons, negative RIAT sufficiently excludes the diagnosis, with NPV over 91%. The sensitivity of RIAT may vary with different species of the influenza virus. Negative RIAT is not necessarily equal to low viral load in the upper respiratory tract or low infectivity of the patient.

Correcting the gas and particle partitioning of PCDD/F congeners in the flue gas of an iron ore sinter plant.

This study was aimed at developing an approach for correcting the gas and particle partitioning of PCDD/F congeners for samples collected from the flue gas of an iron ore sinter plant. An iron ore sinter plant equipped with an electrostatic precipitator (EP) and a selective catalytic reduction (SCR) was selected. Flue gas samples were collected at EP inlet, EP outlet and SCR outlet. Both particle- and gas-phase PCDD/Fs were analyzed for each collected sample. PCDD/F contents in EP ashes (EP(ash)) were also analyzed and used to correct the gas and particle partitioning of PCDD/F congeners of the collected flue gas samples. Results show that PCDD/Fs in the flue gas were dominated by the gas-phase. Before correction, the removal efficiencies for the gas- and particle-phase PCDD/Fs for EP were -58.1% and 64.3%, respectively, and SCR were 39.4% and 83.9%, respectively. The above results were conflict with the expected results for both EP and SCR indicating the need for correcting the gas and particle partitioning of PCDD/F congeners for all collected flue gas samples. After correction, the removal efficiencies become more reasonable for EP (=4.22% and 97.7%, respectively), and SCR (=54.7% and 62.0%, respectively). The above results confirm the effectiveness of the approach developed by this study.