Epidemiology and microbiology of prosthetic joint infections: a nine-year, single-center experience in Pavia, Northern Italy.

BACKGROUND: Prosthetic joint infections (PJIs) are a growing matter of concern due to their economic and social burden on health systems. In Italy, surgical data on PJIs are available in a national registry, but microbiological data are still scarce. MATERIALS AND METHODS: We performed a retrospective study at a single center with records of patients treated for primary PJIs of knee or hip from January 1, 2011, to May 30, 2018. Patients with infections of osteosynthesis means and external devices were excluded, as well as PJI recurrences and polytrauma patients. Infections were diagnosed according to IDSA and MSIS criteria. We collected data on demographics, risk factors and microbiology. All patients seen at our center undergo blood cultures and synovial fluid cultures, periarticular biopsy and prosthesis sonication by Bactosonic®. This was used only after 2014. Bacterial identification is achieved by MALDI-TOF, PHOENIX 100 and standard methods. Chi-square or Fisher tests were used to test statistical differences in proportions. RESULTS: Fifty-one patients matched our inclusion criteria. Of these, 16 (31.4%) were enrolled before 2014. The median age was 68.5 (range 22-88). The most common risk factors were obesity (34%), diabetes (21%) and chronic kidney disease (14%). Seventeen patients were diagnosed with a culture-negative PJIs (33.3%). Staphylococcus aureus was the most commonly isolated pathogen (14/51, 27.5%), followed by coagulase-negative staphylococci (7/51, 13.7%). Methicillin-resistant S. aureus rate was 28.6%. The rate of culture-negative PJIs dropped from 56 to 22% after 2014, with a significant difference between the two time periods (p = 0.016). CONCLUSIONS: The introduction of sonication dramatically increased our diagnostic accuracy. Our microbiological data are in line with those from other studies conducted in Italy.

Prosthetic Joint Infection from Carbapenemase-Resistant Klebsiella pneumoniae Successfully Treated with Ceftazidime-Avibactam.

Antimicrobial resistance in Gram-negative bacteria, particularly Enterobacteriaceae, has become a leading cause of morbidity and mortality and a serious public health concern. Gram-negative bacteria carrying extended-spectrum beta-lactamase (ESBL) enzymes now represent a significant proportion of all bacteria isolated from different countries worldwide. Furthermore, the increasing number of isolates carrying carbapenemases in recent years includes multidrug-resistant (MDR), extensively drug-resistant (XDR), and pandrug-resistant (PDR) bacteria. Here, we describe what, to our knowledge, is the first case of a patient with a prosthetic joint infection from carbapenemase-resistant Klebsiella pneumoniae (CRKP) successfully treated with ceftazidime-avibactam in Italy.

PEG-Ara-C conjugates for controlled release.

The antitumour agent 1-beta-D arabinofuranosilcytosyne (Ara-C) was covalently linked to poly(ethylene glycol) (PEG) in order to improve the in vivo stability and blood residence time. Eight PEG conjugates were synthesised, with linear or branched PEG of 5000, 10000 and 20000 Da molecular weight through an amino acid spacer. Starting from mPEG-OH or HO-PEG-OH, conjugation was carried out to the one or two available hydroxyl groups at the polymer's extreme. Furthermore, to increase the drug loading of the polymer, the hydroxyl functions of PEG were functionalised with a bicarboxylic amino acid yielding a tetrafunctional derivative and, by recursive conjugation with the same bicarboxylic amino acid, products with four or eight Ara-C molecules for each PEG chain were prepared. A computer graphic investigation demonstrated that aminoadipic acid was a suitable bicarboxylic amino acid to overcome the steric hindrance between the vicinal Ara-C molecules in the dendrimeric structure. In this paper we report the optimised conditions for synthesis and purification of PEG-Ara-C products with a low amount of remaining free drug, studies toward the hydrolysis of PEG-Ara-C and the Ara-C deamination by cytidine deaminase, pharmacokinetics in mice and cytotoxicity towards HeLa human cells were also investigated. Increased stability towards degradation of the conjugated Ara-C products, in particular for the highly loaded ones, improved blood residence time in mice and a reduced cytotoxicity with respect to the free Ara-C form was demonstrated.

New PEGs for peptide and protein modification, suitable for identification of the PEGylation site.

New PEG derivatives were studied for peptide and protein modification, based upon an amino acid arm, Met-Nle or Met-beta Ala, activated as succinimidyl ester. PEG-Met-Nle-OSu or PEG-Met-beta Ala-OSu react with amino groups in protein-yielding conjugates with stable amide bond. From these conjugates PEG may be removed by BrCN treatment, leaving Nle or beta Ala as reporter amino acid, at the site where PEG was bound. The conjugation of PEG and its removal by BrCN treatment was assessed on a partial sequence of glucagone and on lysozyme as model peptide or protein. Furthermore, insulin, a protein with three potential sites of PEGylation, was modified by PEG-Met-Nle, and the PEG isomers were separated by HPLC. After removal of PEG, as reported above, the sites of PEGylation were identified by characterization of the two insulin chains obtained after reduction and carboxymethylation. Mass spectrometry, amino acid analysis and Edman sequence, could reveal the position of the reporter norleucine that corresponds to the position of PEG binding.

In vitro and in vivo evaluation of a somatostatin analogue released from PLGA microspheres.

The purpose of this study was to design poly(lactide-co-glycolide) (PLGA) microspheres for the continuous delivery of the somatostatin analogue, vapreotide, over 2-4 weeks. The microspheres were produced by spray-drying and the desired characteristics, i.e. high encapsulation efficiency and controlled release over 2-4 weeks, achieved through optimizing the type of polymer, processing solvent, and co-encapsulated additive. The in vitro release was tested in fetal bovine serum preserved with 0.02% of thiomersal. Furthermore, formulations were injected intramuscularly into rats to obtain pharmacokinetic profiles. Encapsulation efficiency was between 34 and 91%, depending on the particular formulation. The initial peptide release (within 6 h) was lowest, i.e. <20%, when acetic acid was used as processing solvent and highest, i.e. 57%, with dichloromethane. The various co-encapsulated additives generally lowered the encapsulation efficiency by 15-30%. The best formulation in terms of low burst and effective drug serum levels (>1 ng/ml) over 21-28 days in rats was the one made with end-group uncapped PLGA 50:50, the solvent acetic acid and the additive polyethyleneglycol. In conclusion, the optimization of formulation parameters allowed us to produce vapreotide-loaded PLGA microspheres of suitable characteristics for therapeutic use.

Early immunisation with hepatitis B vaccine: a five-year study.

We evaluated the response to the recombinant Hepatitis B vaccine using an accelerated schedule versus the traditional schedule by studying the immunologic memory induced in 200 children with HBs-Ag negative mothers. At seroconversion, the traditional schedule presented a higher percentage of children with serum HBs-Ag concentrations over 100 mIU/ml than the accelerated schedule. After five years this difference was no longer statistically significant and children who presented anti-HBsAg concentrations below 10 mUI/ml received an additional booster dose which stimulated the antibody concentration to exceed 100 mIU/ml in all cases. Recombinant HBV vaccine induced better long term immunologic memory when it was administered earlier.

Importance of the test medium for the release kinetics of a somatostatin analogue from poly(D,L-lactide-co-glycolide) microspheres.

The determination of in vitro release kinetics of peptides from poly(d,l-lactide-co-glycolide) (PLGA) microspheres generally requires optimization of the test conditions for a given formulation. This is particularly important when in vitro/in vivo correlation should be determined. Here, the somatostatin analogue vapreotide pamoate, an octapeptide, was microencapsulated into PLGA 50:50 by spray-drying. The solubility of this peptide and its in vitro release kinetics from the microspheres were studied in various test media. The solubility of vapreotide pamoate was approximately 20-40 microg/ml in 67 mM phosphate buffer saline (PBS) at pH 7.4, but increased to approximately 500-1000 microg/ml at a pH of 3.5. At low pH, the solubility increased with the buffer concentration (1-66 mM). Very importantly, proteins (aqueous bovine serum albumin (BSA) solution or human serum) appeared to solubilize the peptide pamoate, resulting in solubilities ranging from 900 to 6100 microg/ml. The release rate was also greatly affected by the medium composition. Typically, in PBS of pH 7.4, only 33+/-1% of the peptide were released within 4 days, whereas 53+/-2 and 61+/-0.9% were released in 1% BSA solution and serum, respectively. The type of medium was found critical for the estimation of the in vivo release. The in vivo release kinetics of vapreotide pamoate from PLGA microspheres following administration to rats were qualitatively in good agreement with those obtained in vitro using serum as release medium. Finally, sterilization by gamma-irradiation had only a minor effect on the in vivo pharmacokinetics.

Polyphosphazene membranes and microspheres in periodontal diseases and implant surgery.

Membranes or microcapsules made from polyphosphazenes bearing amino acid side groups are proposed for the treatment of periodontal diseases. Polyphosphazene membranes, prepared with alanine ethyl ester and imidazole in the molar ratio of 80:20 as phosphorus substituents, gave a degradation rate that corresponded to the healing of the bone defect. These membranes were much more successful in promoting healing of rabbit tibia defects than polytetrafluoroethylene membranes. Antibacterial or anti-inflammatory drugs, useful in periodontal tissue regeneration, could be entrapped in the polyphosphazene membranes and released both in vitro and in vivo at a rate that ensured therapeutic concentrations in the surrounding tissue. Polyphosphazene microspheres, prepared with phenylalanine ethyl ester as a phosphorus substituent and loaded with succinylsulphathiazole or naproxen, were also obtained. The kinetics of release from these matrices were very convenient in yielding local concentrations of the two drugs that are useful per se or when mixed with hydroxyapatite for better bone formation.

Revaccination against hepatitis B virus of non-responding and low-responding infants immunised at birth. A parallel evaluation of rubella and tetanus vaccine.

The aim of the present study was to identify the true extent of the non-responsiveness in infants born from HBsAg-negative mothers, vaccinated against Hepatitis B Virus (HBV) at birth. Sixty-four non- and low-responding infants, selected from an initial cohort of 2009, were given two additional doses of recombinant HBV vaccine between the tenth and the twelfth month of age. A parallel evaluation was conducted on the response to anti-rubella and anti-tetanus vaccine. Only two infants remained non-responders, whereas 68% of the non-responders and 94% of the low responders after the primary vaccination schedule developed antibody titres over 100 mIU ml-1. No significant relationship between the specific antibody level against HBV and against rubella or tetanus 1 month after vaccination was observed.

Immunogenicity of hepatitis B vaccine in term and preterm infants.

Some studies have suggested that decreased seroconversion rates might be found in premature infants with low birthweight (< 2000g) following administration of hepatitis B vaccine at birth. The aim of the present investigation was to evaluate possible differences in seropositive rates between full-term and preterm infants after primary vaccination, in particular when gestational age or birthweight is very low. Two-thousand and nine neonates born to HBsAg-negative mothers were vaccinated with 10 microg of recombinant hepatitis B virus (HBV) vaccine, from May 1991 to October 1994. Children with infections, congenital malformations or serious illnesses were excluded. HBV vaccine was administered intramuscularly, on the fourth day of life and again at 1 and 6 months of age. A 1-ml blood sample was drawn from each infant 1 month after the third vaccine dose for determination of the level of anti-HBs antibody. The response to HBV vaccination was evaluated in 241 preterm (gestational age <38 weeks) infants and 1727 term neonates. No statistical difference was observed in the distribution of anti-HBs antibody level, either between preterm infants (<38 weeks) and newborns of normal gestational age, or between low birthweight (<2500 g) and normal weight infants. The results suggest that preterm and low birthweight infants (<2500g) respond to HBV vaccine in the same measure as normal-term infants.

Anti-HBV neonatal immunization with recombinant vaccine. Part I. Critical appraisal for a long-lived antibody course.

This study involved 912 infants born to HBsAg-negative mothers from 1 May 1991 to 30 June 1992. The subjects were randomly allocated to an accelerated (Group A) or traditional (Group B) immunization schedule and immunized with 10 micrograms of recombinant HBV vaccine. At the end of the vaccinal cycle 98.14% of both groups were protected against HBV with a high percentage of high responders (88.1% group B and 68% group A). Following a random plan, 345 of the initial 912 infants (144 group A and 201 group B) were serologically evaluated, 15-18 months after the booster dose, to identify the level of long-lasting specific antibody. The data obtained allowed us to identify the non-responder subjects after the seroconversion, to propose the evaluation of antibody titre after the booster dose of vaccine and, because one year after the booster dose 5.6% of the subjects responsive at seroconversion have shown undetectable anti-HBsAg titre, to propose the elevation of the antibody level considered as protective at the end of the vaccinal cycle.

Anti-HBV neonatal immunization with recombinant vaccine. Part II. Molecular basis of the impaired alloreactivity.

HLA study was performed in 9 absolute non-responder (serum titre of anti-HBsAg < 2 mIU ml-1) and 8 hyporesponder (serum antibody level between 2 and 9.9 mIU ml-1) babies who underwent, in neonatal period, HBV vaccination with Engerix B recombinant vaccine. The investigation pointed out that many of these subjects carry HLA haplotypes classically involved in autoimmune diseases: namely HLADR7; DQ2, DR4; DQ8 and DR3; DQ2. The genomic typing for DRB1, DRB3, DRB4, DRB5, DQA1, DQB1 and DPB1 genes revealed an increased frequency of the DRB1*0701; DQA1*0201; DQB1*0201 haplotype (23.5 vs 9.9% of the controls) and of DPB*0201 allele (42.3 vs 13.2% of controls). The polymorphism of Bf, C4A and C4B complement serum components, recognized as important "immune-function-related genes", pointed out an increased frequency of the null allele C4AQ0 (34.3 vs 6.8% of the controls) stressing the role of C4A serum complement component in response to foreign peptide. The immunogenetic investigation has been extended to 23 responder babies (titre of anti-HBsAg > 50 mIU ml-1), vaccinated with the same trial as the poor responders. The HLA frequencies observed in this group were comparable to those of control population and, with respect to the HLA markers cited above, absolutely different from the non/hyporesponder infants. From the HLA class II sequence analysis in the group of poor-responder babies some characteristics, peculiar to autoimmune diseases, have been observed: the majority of the infants showed at least an arginine at the 52 residue of the alpha chain of DQ molecule and a non-aspartic acid at the 57 position of the DQ beta chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Control of hepatitis B: evaluation of two different vaccinal schedules in newborns from HBsAg negative mothers.

504 healthy infants, born to HBsAg negative mothers from May 1st to December 31st 1991, were randomly allocated to an accelerated (group A) or traditional (group B) immunization schedule. The group A infants were immunized at 4 days, 1 month and 3 months of life with 10 micrograms of recombinant HBV vaccine (Engerix B, SKF) while the group B infants were immunized at 4 days, 1 month and 6 months of life with the same dose of vaccine. One month after the first dose of vaccine, 9.2% of the infants in both groups had an HBsAb serum level > 10 mIU/ml. One month after the booster dose, at 4 months of life for group A and at 7 months for group B, 97.40% and 98.53% of the infants presented a serum level > 10 mIU/ml respectively. None in group A and only 2 patients in group B could be considered non-responders (serum concentration below 2 mIU/ml) and 4 infants in group A and 4 in group B were considered hypo-responders (serum level between 2.1 and 9.9 mIU/ml). Immunogenetic study performed on the 2 non-responders and 6 of the hypo-responders, revealed the presence in all but two of the HLA haplotypes, classically involved in the lack of hyporesponsiveness to foreign peptides, namely: HLA-DR7; DQ2, DR4; DQ3, DR15; DQ6 and DR3; DQ2. Surprisingly, 2 hypo-responders carried the HLA haplotypes (DR11, DQ7 and DR13, DQ6), usually associated with hyperresponsiveness. Both vaccinal cycles provided evidence that infants respond well to vaccination, started at birth, against hepatitis B virus with a high degree of protection.(ABSTRACT TRUNCATED AT 250 WORDS)

Specificity and sensitivity of 3rd generation EIA for detection of HCV antibodies among intravenous drug-users.

Serum samples from 487 ambulatory I.V. drug users were screened for HIV and HCV antibodies to determine the prevalence of coinfection in this high risk group for AIDS. For anti-HCV antibody screening we first used a 3rd generation EIA using, as antigen synthetic peptides which were not subjected to false positive results due to antibodies against superoxide dismutase or against yeast proteins (which may copurify with the recombinant proteins employed in the first and second generation test). The specimens that were positive in the screening test were confirmed by a more specific EIA system that detect antibodies to proteins encoded by structural (HCV-st EIA) and non structural (HCV-nst-EIA) regions of the HCV genome. A second confirmation assay was also performed: sera were run in presence or absence of blocking reagents which inhibits antibodies to C200 and C22 HCV epitopes for binding to the solid phase. The sensitivity of the HCV EIA screening for human HCV antibody detection revealed a 100% positivity for HCV infection. The confirmatory strategy presented in this paper revealed an HCV EIA specificity of 98.6%. In this work we demonstrated a significantly higher prevalence (p < 0.001) of HCV exposure in HIV infected individuals compared to the general population. Our experimental data also confirmed that HBV infection in drug-users at high risk for HIV infection was significantly associated with HCV infection (p < 0.001). In contrast, the acquisition of HIV by sexual contact was not a statistically significant risk factor for HCV coinfection.

Mice infection with HIV-1: a new mouse model for HIV-1 in vivo research.

In mice experimentally infected with 1 x 10(5) UI/mouse of HTLV-IIIB IgM antibodies were detected 10-12 days after the infection, reaching peak values two weeks later; the IgM seratiter progressively decreased thereafter and was negative at ten-eleven weeks. HIV p24 antigen was detected ten-fifteen days after infection and reached peak values five-six weeks later. Antigenemia subsequently decreased and showed an oscillating course with a progressive decrease which persisted throughout the observation period. Two weeks after infection we detected IgG antibodies to the major core protein p24; reactivity to gp41 was observed as early as reactivity to p24 and persisted throughout observation period. The IgG antibodies to all HIV epitopes peaked two-three weeks after infection; the time course showed a decrease after ten weeks, progressively decreasing thereafter. After sixty-five weeks of infection the IgG seratiter value was lower but remained positive. Viruses indistinguishable from HIV were isolated from the peripheral blood mononuclear cells of infected mice 30, 60, 180 days after infection. These seroimmunological and virological data confirm that the immunocompetent mouse may serve as a low-cost reproducible model for HIV-1 in vivo research.

Duration of immune protection after hepatitis B vaccine in newborns from HBsAg-positive carrier mothers.

A group of 43 healthy neonates born from HBsAg-positive mothers were evaluated for the presence of protective HBsAb titre after administration of hepatitis B plasma-derived vaccine and hepatitis B hyperimmune-globulin. At 36 months of life we found that a significant number of children had a low HBsAb serum level. This suggests that a close serologic follow-up is necessary to evaluate the optimal timing for the administration of a booster dose of the vaccine.

Sensitivity and specificity of anti-HIV ELISA employing recombinant (p24, p66, gp120) and synthetic (gp41) viral antigenic peptides.

We have developed two immunoassay systems, one designated HIV (p24, p66, gp41) ELISA that uses as antigens the immunodominant epitopes mixed from each of three major groups of HIV-1 proteins: the core (p24), the pol (p66) and the env (gp41) gene. The other immunoassay system consists of four separate ELISAs for detection of single antibodies to HIV gag gene (p24), HIV pol gene (p66) and HIV env gene (gp41 and gp120). In the present study 200 specimens from patients with AIDS and 200 specimens from patients with ARC were repeatedly positive by HIV (p24, p66, gp41) ELISA. 1425 specimens from HIV drug addicts positive at W.B. were positive at HIV (p24, gp41, p66) ELISA. In addition, 60 samples that were indeterminate by W.B., were repeatedly positive at HIV (p24, p66, gp41) ELISA. The sensitivity and specificity of HIV (p24, p66, gp41) is estimated to be 100%. In this study 1507 specimens from HIV drug addicts, positive at W.B., were all positive (more than one test positive) at HIV p24 ELISA, HIV gp41 ELISA, HIV p66 ELISA and HIV gp120 ELISA used in combination. 135 samples from HIV positive drug addicts, positive at standard ELISA but indeterminate at W.B., were positive by HIV p24 ELISA, HIV gp41 ELISA, HIV p66 ELISA and HIV gp120 ELISA using the same criteria as in W.B. interpretation. The specificity (defined in terms of percentage of non-reacting persons in a low risk population) of HIV p24 ELISA, HIV gp41 ELISA, HIV p66 ELISA, HIV gp120 ELISA is 100%. In this work we demonstrated that: a) HIV (p24, p66, gp41) ELISA could be used as an adjunct or reliable alternative to standard ELISA for detection or confirmation of HIV antibodies in human sera; b) the specificity and sensitivity of antibodies to p24, p66, gp41, gp120 by ELISA used alone and/or in combination, is equal to or greater than W.B.

Endocytosis constitute the infectious route of HIV-1 entry in human and rabbit monocytes lacking the CD4 receptor.

To obtain "functionally" CD4 negative human monocytes (0-5 CD4 +/1 x 10(6)/cells), 50 ng/5.10(5) cells of OKT4A were added daily after a pre-incubation with OKT4A (100 ng/5.10(5) cells. In our experimental conditions the blocking the CD4 receptor of human monocytes with OKT4A monoclonal antibody did not prevent HIV-1 infection, although the level of virus replication appeared lower than that in cultures without OKT4A. "Naturally"CD4 negative rabbit monocytes infected with HIV-1 also released a detectable level of virus after 12-15 up 28-30 days. In "naturally" CD4 negative rabbit monocytes and "functionally" CD4 negative human monocytes, the virus particles entering via phagocytosis are not infectious because multiple well defined virions were observed in phagocytic vacuoles and the envelopes of these particles did not appear to interact with the vacuolar membrane. The infectious particles were represented by endocytic vesicles containing only the core of HIV after fusion between the viral envelope and endocytic membrane. Fusion between the viral envelope and plasma membrane on the cellular surface was never observed, in spite of examining greater than 1000 virions bound the surface of human and rabbit macrophage monocytes. The absence of cytopathic effect in the rabbit and human CD4 negative monocytes infected with HIV-1, and conversely the presence of specific sequences of HIV in the genomic DNA may indicate that the macrophages-monocytes serve as an important reservoir for the persistence of HIV in infected hosts, similar to the other related Lentiviruses. Our virological data have also demonstrated that virus infection can be transmitted from rabbit and human infected monocytes to uninfected H9 cells. This preliminary study may offer important evidence for the development and testing of vaccines and compounds that inhibit HIV penetration of susceptible cells.

Detection of antibodies to p24 and gp41 epitopes of HIV by ELISA using the recombinant core protein (p24) and envelope synthetic protein (gp41).

We have developed a system consisting of two separate ELISA, one designed to detect antibodies to HIV gag gene (p24) and the other to detect antibodies to HIV env gene (gp41). The antigen used in these ELISA was produced as recombinant DNA-derived proteins expressed in E. coli for HIV gag gene (p24) and synthetic peptide for the HIV env gene (gp41). These HIV (env-gag) ELISA, that provide independent determinations of the antibody response to the core and envelope proteins, are highly specific and sensitive. In this work we have demonstrated that determinations of antibodies such as those to p24 and gp41 by HIV (env-gag) ELISA are among the criteria for a confirmation procedure, and sensitivity one (gp41) and/or both these determination should be equal or greater than the sensitivity of W.B. In addition, the procedure should be objective and standardized and the antigen source used should be different from that adopted in the "classical" W.B. and screening test. In view of these considerations, this HIV (env-gag) ELISA could be used as a reliable alternative to W.B. for confirmation of antibody detection.