Tocilizumab in COVID-19 interstitial pneumonia.

BACKGROUND: Published reports on tocilizumab in COVID-19 pneumonitis show conflicting results due to weak designs or heterogeneity in critical methodological issues. METHODS: This open-label trial, structured according to Simon's optimal design, aims to identify factors predicting which patients could benefit from anti-IL6 strategies and to enhance the design of unequivocal and reliable future randomized trials. A total of 46 patients with COVID-19 pneumonia needing of oxygen therapy to maintain SO2 > 93% and with recent worsening of lung function received a single infusion of tocilizumab. Clinical and biological markers were measured to test their predictive values. Primary end point was early and sustained clinical response. RESULTS: Twenty-one patients fulfilled pre-defined response criteria. Lower levels of IL-6 at 24 h after tocilizumab infusion (P = 0.049) and higher baseline values of PaO2/FiO2 (P = 0.008) predicted a favourable response. CONCLUSIONS: Objective clinical response rate overcame the pre-defined threshold of 30%. Efficacy of tocilizumab to improve respiratory function in patients selected according to our inclusion criteria warrants investigations in randomized trials.

Management of cytomegalovirus infection in pregnancy: is it time for valacyclovir?

BACKGROUND: Congenital cytomegalovirus (CMV) infection is the leading infectious cause of neurological impairment for which, currently, there are no approved antenatal treatment options. OBJECTIVES: The aim of this article was to summarize the available evidence on the use of valacyclovir during pregnancy to prevent and treat congenital CMV infection and disease. SOURCES: Two databases (PubMed and ClinicalTrial.gov) were reviewed. CONTENT: Six relevant documents were identified, namely one observational study, three clinical trials, two case reports. Most relevant findings were those from two clinical trials. A phase 2/3 placebo-controlled study showed a decrease of 71% (5 of 45 vs 14 of 47) in rate of CMV vertical transmission in women treated with 8 g/day valacyclovir following primary CMV infection in pregnancy. A phase 2, single-arm clinical trial, showed that 8 g/day valacyclovir administered to mothers of symptomatic infected foetuses increased the portion of asymptomatic neonates to 82% (34 of 41), compared with 43% (20 of 47) in untreated pregnancies from a historical cohort. IMPLICATIONS: Studies in favour of using valacyclovir during pregnancy for prevention and treatment of congenital CMV infection are emerging but are still few. Randomized clinical trials on large cohorts of patients investigating the efficacy on prevention and treatment of congenital CMV are required. Unfortunately, this will be probably not be feasible at least in the short period. In the meantime, data on the 'off label' use of valacyclovir for CMV in pregnancy could be collected within a multicentre observational study.

Comparison of different NAT assays for the detection of microorganisms belonging to the class Mollicutes.

BACKGROUND: Mollicutes detection can be cumbersome due to their slow growth in vitro. For this reason, the use of DNA based on generic molecular tests represents an alternative for rapid, sensitive and specific detection of these microorganism. For this reason, six previously described nucleic acid testing assays were compared to evaluate their ability to detect microorganisms belonging to the class Mollicutes. METHODS: A panel of 61 mollicutes, including representatives from the Mycoplasma, Acholeplasma, Mesoplasma, Spiroplasma and Ureaplasma genus, were selected to evaluate the sensitivity and specificity of these assays. A total of 21 non-mollicutes, including closely related non-mollicutes species, were used to evaluate specificity. Limits of detection were calculated to determine the analytical sensitivity of the assays. The two best performing assays were subsequently adapted into real-time PCR format, followed by melting curve analysis. RESULTS: Both assays performed satisfactorily, with a 100% specificity described for both assays. The detection limits were found to be between 10-4 and 10-5 dilutions, equivalent to 15 to 150 genome copies approximately. Based on our work, both van Kuppeveld and Botes real-time PCR assays were found to be the best performing tests in terms of sensitivity and specificity. Furthermore, Botes real-time PCR assay could detect phytoplasmas as well. CONCLUSIONS: These assays can be very useful for the rapid, specific and sensitive screening cell line contaminants, clinical samples as well as detecting non-culturable, unknown species of mollicutes or mollicutes whose growth is slow or difficult.

In vitro activity of tylvalosin against Spanish field strains of Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae is involved in the porcine enzootic pneumonia and respiratory disease complex; therefore, the search for new treatment options that contribute to the control of this organism is relevant. The minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations of tylvalosin and 19 other antimicrobial agents against 20 Spanish field isolates of M. hyopneumoniae were determined using the broth microdilution method, with the type strain (J) as a control strain. Tylvalosin had MIC50 and MIC90 values of 0.016 and 0.06 µg/ml, respectively, and was the second-most effective of the assayed antibiotics, after valnemulin. Tiamulin, tylosin and lincomycin were also among the antibiotics with the lowest MIC50 and MIC90 values against the 20 field isolates (0.06-0.25 µg/ml). However, resistance to tylosin and spiramycin, which like tylvalosin, are 16-membered macrolides, was observed. The MIC50 and MIC90 values for ciprofloxacin and enrofloxacin ranged from 0.125 to 1 µg/ml; the corresponding values ranged from 2 to 4 µg/ml for oxytetracyline, which was the most active tetracycline. Furthermore, tylvalosin and valnemulin exhibited the highest bactericidal activities. In conclusion, the macrolide tylvalosin and the pleuromutilin valnemulin exhibited the highest in vitro antimicrobial activities against M. hyopneumoniae field isolates in comparison with the other tested antibiotics.

Human case of autochthonous West Nile virus lineage 2 infection in Italy, September 2011.

On 10 September 2011, a patient in his 50s was admitted to hospital in Ancona, Italy, after six days of high fever and no response to antibiotics. West Nile virus (WNV) infection was suspected after tests to determine the aetiology of the fever were inconclusive. On 20 September, WNV-specific IgM and IgG antibodies were detected in the patient's serum. Genomic sequencing of the viral isolate showed that the virus belonged to WNV lineage 2.

Long-term outcomes of orthotopic liver transplantation in human immunodeficiency virus-infected patients and comparison with human immunodeficiency virus-negative cases.

Human immunodeficiency virus (HIV) positivity is no longer a contraindication for orthotopic liver transplantation (OLT) due to the efficacy of antiretroviral therapy. The aim of this study was to compare OLT among HIV-positive and HIV-negative cohorts; the results were also stratified for hepatitis C virus (HCV) coinfection. Between 2004 and 2009, all HIV-infected patients undergoing OLT from heart-beating deceased donors (n=27) were compared with an HIV-negative cohort (n = 27). The pure HCV infection rate was similar between HIV-positive and HIV-negative subjects (63% each). HIV-positive recipients were younger (P=.013). The CD4 count for HIV-positive subjects was 376 ± 156 at transplantation. The mean model for end-stage liver disease (MELD) score at transplantation was 15 ± 7 in both groups (P=.92). No differences were observed for donor age (P=.72) or time on the waiting list (P=.56). The median follow-up was 26 (range, 1-64) and 27 months (range, 1-48) for HIV and non-HIV recipients, respectively (P=.85). The estimated 1-, 3-, and 5-year patient and graft survival rates were 88%, 83%, and 83% versus 100%, 73%, and 73% (P=.95), and 92%, 87%, and 87% versus 95%, 88%, and 88% (P=.59) for HIV and non-HIV cases, respectively. HIV/HCV-coinfected patients were younger, namely 47 (range, 40-53) versus 52 years (range, 37-68; P=.003), and displayed lower MELD scores at transplantation compared with HCV-mono-infected patients 10 (range, 7-19) versus 17 (range, 8-30) (P=.008). For HIV/HCV-coinfected and HCV-mono-infected cases the estimated 1-, 3-, and 5-year patients and graft survival rates were respectively 93%, 76%, and 76% versus 100%, 70%, and 60% (P=.99) and 93%, 84%, and 84% versus 100%, 70%, and 60% (P=.64), respectively. No difference was observed in the histological severity of HCV recurrence. In conclusion, under specific, well-determined conditions, OLT can be a safe, efficacious procedure in HIV patients.

QnrB9 in association with TEM-116 extended-spectrum beta-lactamase in Citrobacter freundii isolated from sewage effluent: first report from Italy.

The aim of the present study was to investigate the mechanism of quinolone and beta-lactam resistance in an isolate of Citrobacter freundii PC2/08 collected from sewage effluent from l'Aquila, Italy. QnrB-9 and bla(TEM-116 )were co-expressed in a large plasmid identified in C. freundi PC2/08 strain. Compared to TEM-1, TEM-116 showed two single mutations: V84I and A184V. The plPC2/08 plasmid conferred resistance to several beta-lactams and fluoroquinolones. Tazobactam could be considered a good inhibitor whereas clavulanic acid was unable to restore susceptibility to amoxicillin. The QnrB-9 element seems to confer the same level of resistance to levofloxacin and ciprofloxacin with minimum inhibitory concentration (MIC) values of 4 mg/l for either. In this study, we confirm the common association of plasmid-mediated quinolone resistance with extended-spectrum beta-lactamase (ESbetaL) production. This is the first finding in Italy of qnrB9 and TEM-116 in a non-clinical or animal strain.

Constitutive SoxS expression in a fluoroquinolone-resistant strain with a truncated SoxR protein and identification of a new member of the marA-soxS-rob regulon, mdtG.

Elevated levels of fluoroquinolone resistance are frequently found among Escherichia coli clinical isolates. This study investigated the antibiotic resistance mechanisms of strain NorE5, derived in vitro by exposing an E. coli clinical isolate, PS5, to two selection steps with increasing concentrations of norfloxacin. In addition to the amino acid substitution in GyrA (S83L) present in PS5, NorE5 has an amino acid change in ParC (S80R). Furthermore, we now find by Western blotting that NorE5 has a multidrug resistance phenotype resulting from the overexpression of the antibiotic resistance efflux pump AcrAB-TolC. Microarray and gene fusion analyses revealed significantly increased expression in NorE5 of soxS, a transcriptional activator of acrAB and tolC. The high soxS activity is attributable to a frameshift mutation that truncates SoxR, rendering it a constitutive transcriptional activator of soxS. Furthermore, microarray and reverse transcription-PCR analyses showed that mdtG (yceE), encoding a putative efflux pump, is overexpressed in the resistant strain. SoxS, MarA, and Rob activated an mdtG::lacZ fusion, and SoxS was shown to bind to the mdtG promoter, showing that mdtG is a member of the marA-soxS-rob regulon. The mdtG marbox sequence is in the backward or class I orientation within the promoter, and its disruption resulted in a loss of inducibility by MarA, SoxS, and Rob. Thus, chromosomal mutations in parC and soxR are responsible for the increased antibiotic resistance of NorE5.

Perioperative considerations in HIV-infected liver transplanted patients.

UNLABELLED: The introduction of highly active antiretroviral therapy (HAART) has improved survival in HIV patients, allowing them to undergo liver transplantation (OLT) in cases of end-stage liver disease. HIV patients show a higher incidence of pulmonary hypertension. The aim of this study was to evaluate pulmonary and systemic hemodynamic changes in HIV-infected patients compared with a non-HIV-infected group of patients undergoing OLT. METHODS: We analyzed 20 HIV-infected patients and 20 non-HIV-infected patients who underwent OLT. We analyzed preoperative cardiovascular status, as well as intra- and postoperative hemodynamic data. Hemodynamic data were recorded at 4 predefined phases during OLT and at 24, 48, and 72 hours after intensive care unit (ICU) admission. We also evaluated the following perioperative aspects: transfusion requirements, postoperative mechanical ventilation time, ventilation time, and length of ICU and of hospital stay. RESULTS: HIV-positive patients were younger than controls with a greater incidence of coinfection with hepatotropic viruses. One HIV-infected patient died in the ICU. Hemodynamic data showed a higher cardiac index and higher pulmonary vascular resistance index among HIV-infected patients, but without any clinical impact. No significant difference in blood unit transfusions, postoperative time on mechanical ventilation, or length of ICU or hospital stay was observed between the groups. CONCLUSIONS: Although the number of patients studied is limited, we concluded that HIV-infected patients undergoing OLT showed similar perioperative courses as non-HIV-infected patients.

In vitro susceptibilities of field isolates of Mycoplasma agalactiae.

In order to determine how widespread antibiotic resistance has become to standard treatments, the in vitro susceptibilities of 28 Mycoplasma agalactiae Spanish field isolates to 16 antimicrobial agents were determined using a broth microdilution method. The most effective antimicrobials based on minimum inhibitory concentration (MIC)(90) values were fluoroquinolones, tetracyclines and macrolides. Two strains were tetracycline resistant. Streptomycin, erythromycin and nalidixic acid resistance was observed in all strains.

In vitro susceptibilities of Mycoplasma putrefaciens field isolates.

MICs were determined for 15 antimicrobial agents against 37 Mycoplasma putrefaciens isolates. The most effective antimicrobial drug classes were the fluoroquinolones, the tetracyclines, the lincosamide lincomycin, and the macrolides. The susceptibility profile of the isolates correlated with the geographic origin. This is the first report of decreased susceptibility to the macrolides, lincomycin, and the tetracyclines in M. putrefaciens strains.

In vitro selection and characterization of mutants in TEM-1-producing Escherichia coli by ceftazidime and ceftibuten.

The present work was undertaken to study the ability of ceftazidime and ceftibuten to selectin vitro Escherichia coli HB101 harboring bla(TEM-1) beta-lactamase gene. Minimum inhibitory concentrations (MICs) of ceftazidime and ceftibuten were increased by a factor of 32, overcoming in the case of ceftazidime the breakpoint for clinical resistance. Outer membrane protein analysis and PCR for bla(TEM )alleles revealed that ceftazidime and ceftibuten select for different resistance mechanisms. Ceftazidime created mutants that encode an extended-spectrum beta-lactamase (TEM-12) and exhibit decreased expression of OmpF. Ceftibuten was unable to select for extended-spectrum beta-lactamase expressing mutants but reduced the expression of two porins, OmpC and OmpF. The stability of ceftibuten to hydrolysis and the difference in the structure of these beta-lactam antibiotics could be responsible for the selection of different mechanisms of resistance.

In vitro susceptibilities of field isolates of Mycoplasma mycoides subsp. mycoides large colony type to 15 antimicrobials.

In vitro susceptibilities of 16 Mycoplasma mycoides subsp. mycoides large colony type field isolates to 15 antimicrobial agents were determined using a broth microdilution method. The most effective antimicrobials were fluoroquinolones, tetracyclines and macrolides, with MIC values under 2 microg/ml. Resistance to nalidixic acid, gentamicin, streptomycin and spectinomycin was observed.

Chronic hepatitis C in HIV infection: feasibility and sustained efficacy of therapy with interferon alfa-2b and tribavirin.

BACKGROUND: The role combination therapy with interferon alfa-2b and tribavirin (US: ribavirin) plays in producing sustained virological responses in patients with HIV and chronic hepatitis C (HCV) infection is still unknown. OBJECTIVES: To determine the feasibility and sustained response of interferon alfa-2b and tribavirin combination therapy. DESIGN: Phase II study. METHODS: Seventeen patients were enrolled at the National Cancer Institute, Aviano, Italy and received combination therapy with interferon alfa-2b 3 MIU subcutaneously three times a week plus tribavirin 1000-1200 mg/day for 24 weeks. Antiretroviral therapy was concomitantly given in all but one patient. RESULTS: At the end of treatment, five (31%) patients achieved clearance of HCV RNA and 11 (69%) showed normalized liver function enzyme levels. In three patients, serum HCV RNA concentration was still undetectable 24 weeks after treatment, with an overall sustained virological response rate of 19% The serum liver enzymes were still normal in 10 patients 24 weeks after treatment, the overall sustained biochemical response rate being 62% All patients with HCV RNA clearance at the end of treatment and 24 weeks after treatment had a concomitant biochemical response. Overall the combination treatment was well tolerated. CONCLUSIONS: Our data confirm that the combination of interferon alfa-2b and tribavirin is well tolerated and feasible in patients with HIV-HCV co-infection and it can be associated safely with highly active antiretroviral therapy. The sustained response achieved with the drug combination does not seem to be any better than that achieved with 12 months of monotherapy with interferon alfa-2b.

In vitro selected fluoroquinolone-resistant mutants of Citrobacter freundii: analysis of the quinolone resistance acquisition.

Ten quinolone-resistant mutants of Citrobacter freundii, which were selected in vitro with fluoroquinolones from two clinical isolates, were studied. The parent isolates were susceptible to quinolones in spite of showing a single substitution in the GyrB (His-417 --> Leu). No change was observed in the outer membrane proteins or in the lipopolysaccharide in any of the ten mutants studied with respect to their parent isolates. The development of quinolone resistance in selected mutants was associated with the appearance of a substitution in the GyrA (Thr-83 --> Ile) in nine of the ten mutants plus enhanced active efflux in all of them.

Decreased permeability and enhanced proton-dependent active efflux in the development of resistance to quinolones in Morganella morganii.

Five quinolone-resistant strains were developed from a clinical Morganella morganii isolate (M1 strain) which was susceptible to nalidixic acid and fluoroquinolones. All five in vitro selected mutants showed diminished norfloxacin accumulation and two of them also decreased the expression of the AgO in the outer membrane lipopolysaccharide with respect to their parent strain and to the M. morganii NCTC-235 type strain. Likewise, the M1 strain did not express a 37-38 kDa protein and accumulated less norfloxacin than the M. morganii NCTC-235 strain. The decreased norfloxacin uptake in the five mutants compared with the M. morganii NCTC-235 strain was due to an enhanced proton-dependent active efflux plus a pre-existent decreased expression of a 37-38 kDa protein in the parent strain.

Resolution of high-molecular-weight components in lipopolysaccharides of Escherichia coli, Morganella morganii, Citrobacter freundii and Citrobacter diversus strains with sodium dodecyl sulfate polyacrylamide gels.

The use of 0.5% sodium dodecyl sulfate in polyacrylamide separation gels allowed the resolution in several bands of high-molecular-mass components in smooth lipopolysaccharide of bacterial outer membrane from Escherichia coli, Morganella morganii, Citrobacter freundii and Citrobacter diversus. With or without 0.1% SDS, however, such a result was not possible.

Mechanisms involved in the development of resistance to fluoroquinolones in Escherichia coli isolates.

Eighteen quinolone-resistant isolates of Escherichia coli were selected by exposing ten clinical isolates to increasing concentrations of norfloxacin and lomefloxacin. The mutant isolates showed a multiple-antibiotic-resistance phenotype. All of them contained single mutations in gyrA consisting of the substitution of Ser-83-->Leu (n = 14), Val (n = 1) or Ala (n = 1) and the substitution of Asp-87-->Asn (n = 2). Only one concomitant mutation in parC (Ser-80-->Arg) was detected. Four parent isolates exhibited a single mutation in gyrA which required < or = 12 mg/L of norfloxacin to be inhibited. Fluoroquinolone resistance, in the 18 quinolone-resistant mutants, was a result of mutations affecting DNA gyrase plus decreased fluoroquinolone uptake. This latter mechanism of resistance was a combined effect of an absence of OmpF and an increase in active efflux in eight isolates, or an increased active efflux alone in the remaining ten selected mutants.

AIDS-associated Kaposi's sarcoma is more aggressive in women: a study of 54 patients.

OBJECTIVE: To describe the epidemiologic and clinical features of AIDS-associated Kaposi's sarcoma (KS) in women compared with men. METHODS: In a retrospective study, within the Italian Cooperative Group on AIDS and Tumors (GICAT), we compared selected characteristics of 54 women and 108 men with AIDS-associated KS, matched by date of KS diagnosis and referral hospital. The chi2 test was used to test differences among proportions; the Kaplan-Meier method to estimate the survival time, and the Cox proportional hazard model was used to assess the role of gender, age, and CD4 cell count on death's risk. RESULTS: KS occurred at an earlier age (p = .001), was associated with a more severe immunodeficiency (p = .03), more advanced stages of HIV disease (p = .05), and had more aggressive presentation and course in women than in men. At KS diagnosis, women had a significantly increased proportion of visceral disease (p = .009), in particular pulmonary involvement (p = .002) and atypical sites of involvement (p = .008). The number of deaths due to KS was significantly higher (p = .01) in female patients. Both the higher proportion of visceral disease and of KS-related deaths observed in women did not change after adjusting for CD4 cell count and age. Women showed a decreased overall survival compared with men (8.9 and 14.4 months, respectively; p = .07), and the CD4 cell count at diagnosis significantly influenced survival. CONCLUSIONS: This study suggests that KS is more aggressive and life threatening in female than in male patients. This peculiar clinical behavior may reflect an inherently more aggressive biology of KS in women, possibly mediated by the level of immunodeficiency.