Automated Intelligent Microscopy for the Recognition of Decoy Cells in Urine Samples of Kidney Transplant Patients.

BACKGROUND: BK virus (BKV)-associated nephropathy is definitely involved in allograft failure after kidney transplant. Thus, the need for an early control of viral reactivation in immunocompromised patients is well established. Determination of urinary release of decoy cells (DC) and BK viral load in plasma and urine by polymerase chain reaction (PCR) usually precedes renal biopsy. The aim of the study is to assess viral reactivation by BKV-DNA PCR and DC detection in urinary sediment using automated intelligent microscopy. METHODS: Seventy-eight kidney transplant patients were analyzed for the presence of plasma BKV-DNA by quantitative TaqMan real-time PCR. Additionally, automated intelligent microscopy was used for urine sediment analysis, allowing to count cells with decoy feature, confirmed by phase contrast microscopic review. RESULTS: Plasma BKV-DNA PCR was detected in 14 (17.9%) patients. DC were identified in 19 (24.3%) urine sediments by automated analyzers and confirmed by microscopic observation. Two patients were BKV-DNA-positive/DC-negative; conversely, 7 subjects were DC-positive/BKV-DNA-negative. CONCLUSIONS: Plasma quantification of BK viral load is currently the best noninvasive method for the detection of viral reactivation. Nevertheless, automated methods to screen for the presence of DC in urine could facilitate early BK virus replication diagnosis and patient follow-up by quantitative and visual results.

Pre-Emptive Therapy for the Treatment of Cytomegalovirus After Kidney Transplantation.

BACKGROUND: Cytomegalovirus (CMV) represents the leading cause of viral infection in kidney transplantation patients. The aim of the present study was to evaluate the efficacy and safety of pre-emptive anti-CMV therapy. MATERIALS AND METHODS: We performed a retrospective analysis based on data from 227 consecutive patients transplanted from 2010 to 2015, of whom 38 (16.6%) were from a living donor, considering: incidence of rejection, CMV organ localization, and graft and patient survival. All patients underwent induction immunosuppressive therapy followed by maintenance therapy consisting of corticosteroids, antimetabolites, and tacrolimus (median basal dose = 5.3 ng/mL). The timing for the detection of plasma CMV-DNA in the post-transplantation period was: weekly (first month), quarterly (second through twelfth month), and then half-yearly. RESULTS: CMV viremia was positive in 98 of 227 (43.1%) patients, with an average of 248,482 copies/mL (range: 250 copies/mL to 9,745,000 copies/mL) and the first positivity after a median period of 2.5 months from kidney transplantation (range: 0.2 months to 43 months). A total of 49 of 227 (21.5%) patients were treated with antivirals: 27 of 49 (55.1%) because of CMV organ localization (gastrointestinal = 20, lungs = 3, kidney = 2, liver = 2). Fourteen of 227 (6.1%) patients had a rejection episode, 7 (3.1%) of which were CMV-related. Fifteen of 227 (6.6%) patients died (noninfectious CMV-related complications = 8, cardiovascular causes = 6, bleeding complications = 1). CONCLUSION: Our experience confirms the validity of the pre-emptive anti-CMV therapy in renal transplantation patients.

Multiplex polymerase chain reaction for the evaluation of cytomegalovirus DNA load in organ transplant recipients.

Because of the considerable impact of human cytomegalovirus (HCMV) infection, sensitive, specific, and standardized methods are required for rapid and accurate evaluation of viral load in monitoring transplant recipients. The aim of the present study was to evaluate the usefulness of a multiplex polymerase chain reaction (PCR) for the coamplification of HCMV-DNA and beta-globin genomic sequence in polymorphonuclear leukocytes (PMNL). Analysis and quantification of PCR products were carried out by a DNA enzyme immunoassay (DEIA), which is based on the hybridization of amplified DNA with a single-stranded DNA probe, which coats microtitre wells. Colorimetric detection of the DNA-antibody complex was carried out and optical density (O.D.) was recorded at 450/630 nm. To quantify HCMV/DNA load, a standard curve to which samples O.D. refer was obtained by amplifying serial dilutions of recombinant PGEM-3Z plasmid DNA containing a genomic fragment of glycoprotein B. 340 PMNL specimens from 102 solid organ recipients were tested for the detection of pp65 antigen and HCMV-DNA. The results showed a good correlation between viral load and clinical symptoms of HCMV infection; high specificity and predictive values for HCMV disease were found by PCR, using a cut-off limit of 10(3) genomic copies per 2 x 10(5) PMNL. These findings indicate that the system described is an efficient and reproducible diagnostic method easy to apply for routine diagnosis and therapeutic monitoring of transplanted patients.

Monitoring for cytomegalovirus infection in organ transplant recipients: analysis of pp65 antigen, DNA and late mRNA in peripheral blood leukocytes.

The use of sensitive and specific methods for rapid and reliable diagnosis is required due to the considerable impact of human cytomegalovirus (HCMV) in organ transplant recipients. For this purpose the demonstration of the presence of viral antigens in peripheral blood leukocytes (PMNLs) and of viral nucleic acids in the same cells or in sera would seem to be of valid support. The present study was designed to test pp65 antigen, HCMV DNA and HCMV late mRNA in order to provide clinical information for the management of the infection. Fifty solid organ recipients were monitored for six months after transplant. The data obtained from the various tests were analysed from the first evidence of HCMV infection revealed by positive antigenaemia and/or DNA-polymerase chain reaction (PCR). In 3 asymptomatic and in 7 symptomatic patients, PCR became positive 1-2 weeks before antigenaemia but PCR did not discriminate the clinical evolution of HCMV infection. The antigenaemia test well correlated to the development of viral infection being positive in all symptomatics and in 31, 2% of asymptomatics. The antigenic load > 100/2 x 10(5) positive cells was always associated with clinical signs of illness. The detection of late mRNA was more indicative of the virus replicative status in the follow-up of patients treated with ganciclovir. In some cases there was evidence, prior to the other two tests, the block of viral replication due to the antiviral therapy and in others the onset of HCMV infection relapse.

Differential effect of human and murine polyclonal and monoclonal antisera on TNF-alpha production by human monocytes.

The capacity of human and murine polyclonal and monoclonal antibodies to inhibit lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha (TNF-alpha) release from human monocytes was investigated. Human pooled immunoglobulin G (IVIG), human IgM monoclonal antibody (HA-1A) directed against the lipid A moiety of LPS, and murine IgG monoclonal antibody (MT-1F) raised in mice against antibiotic-treated Escherichia coli O6:K- were either added simultaneously with LPS to monocytes or preincubated for 1 h at 37 degrees C before being added to monocytes. TNF-alpha content in the monocyte supernatants was then tested. Simultaneous addition of increasing concentrations of IVIG (from 0.3 to 2.5 mg/ml) and 10 micrograms/ml of LPS to monocytes induced an enhanced release of TNF-alpha by monocytes in a dose dependent fashion. Preincubation of IVIG with LPS abolished the additive effect, but did not inhibit LPS-induced TNF-alpha release by monocytes. The simultaneous addition of LPS and HA-1A to monocytes had no additive effect nor did it inhibit TNF-alpha release. On the other hand, inhibition of TNF-alpha release was observed when HA-1A was preincubated with LPS before being added to monocytes. In all instances MT-1F inhibited TNF-alpha release when the monocytes were stimulated with smooth type LPS, but not with LPS isolated from rough mutants.

Effect of intravenous immunoglobulin on opsonic activity and TNF production in patients at high risk for sepsis syndrome.

In a randomized double blind study, we analyzed the efficacy of IVIG in the infectious complications in patients at high risk of developing sepsis syndrome. Two groups of twenty patients were enrolled, one receiving 250 mg/Kg of IVIG on the first and seventh day after admission and the other receiving sterile saline as placebo. Serum samples were drawn before IVIG administration and 24, 48 and 72 hours afterwards. The same schedule was used for patients treated with placebo. Sera pooled from healthy donors served as controls. On all the samples, opsonic and bactericidal activity as well as C3, total IgG and serum TNF content were tested. IVIG did not significantly affect total IgG and C3 content. Similarly, opsonic and bactericidal activity tested against E. coli 06 :K-, E. coli 0111 and SAC I was not modified ranging within HPS values. Furthermore, IVIG administration did not change the TNF level. A lower incidence of bacteremia in IVIG treated patients was observed.

Reactivity and protective capacity of a polyclonal antiserum derived from mice immunized with antibiotic exposed Escherichia coli.

The murine immune response to Escherichia coli O6:K-alone or pre-exposed to 0.1 x MIC of aztreonam was investigated. Relative to mice immunized with untreated bacteria, mice immunized with antibiotic-treated microorganisms presented a significantly enhanced protection towards a challenge of 100 x LD50 of viable E. coli O6:K-. Previous injection of 0.1 mL of serum drawn from mice immunized with treated and untreated bacteria protected non-immunized mice towards a challenge of 10 x LD50 of viable E. coli O6:K--. Serum from mice immunized with treated bacteria also protected non-immunized mice towards a lethal challenge of E. coli O111. The antiserum contained high titre of IgG antibodies that cross-reacted with lipopolysaccharide isolated from smooth and rough Gram-negative bacteria. Immunoblotting showed additional bands of reactivity to the untreated E. coli O6:K-. Immunization with antibiotic-treated bacteria led to the production of type specific and cross reactive antibodies that protected animals against viable homologous and heterologous lethal challenges.

Effect of cyclosporin A on B cell maturation and differentiation.

The activation and differentiation of resting B cells into Ig secreting cells are regulated by T cells, macrophages and their secreted factors. The present study evaluated the effect of cyclosporin A (CsA) on this process. Peripheral blood lymphomonocytes (PBMC) drawn from healthy donors were stimulated with protein A (PA) or with lipopolysaccharides plus pokeweed (LPS+PWM) in either the presence or the absence of CsA. Phenotypic B cell changes and immunoglobulin production was then analyzed. The data revealed that CsA decreased the expression of B cell surface receptors of the activation phase, and enhanced the resting phase receptors. Different effects of CsA were found on B cell differentiation, depending on its induction by PA or LPS+PWM. In the first system, CsA decreased the expression of differentiation phase receptors and the secretion of free Ig. In cultures stimulated with LPS+PWM, CsA increased the differentiated phase receptors and Ig secretion. Thus, CsA seemed to act as a blocking agent of the activation phase and as a modulator of the differentiation phase and of IgG secretion, depending upon the antigen used for stimulation.

Adherence of Pseudomonas aeruginosa elastase deficient mutant.

Pseudomonas aeruginosa produces several extracellular substances such as enzymes and toxins which seem to contribute to its pathogenicity. In particular, alkaline protease and elastase production seems to affect bacterial adherence. Aim of this study was to isolate an elastase deficient mutant of P. aeruginosa and to demonstrate a possible correlation between enzyme production and adherence to WEHI cells. Mutant strain showed a significant reduction of elastase and protease alkaline activity, as the decrease of absorbance values demonstrate. Furthermore the adherence to WEHI cells of mutant strain was strongly reduced with respect to the wild strain. Our results prove that proteolytic enzymes play an important role in adherence, probably modifying the cell surfaces and so enhancing adherence.

Role of alkaline protease and elastase in the adherence of Pseudomonas aeruginosa to WEHI cells.

Recent clinical isolates were tested for production of some extracellular factors such as alkaline protease and elastase. They were also assayed for adhesiveness to WEHI cells. It is well known that extracellular production of substances other than toxins is related to virulence and may increase adherence. The present investigation aimed to evaluate the role of extracellular proteins in adherence. Alkaline protease production was assayed using a test performed with casein as substrate while elastase activity was investigated with the elastin-congored method. Our results demonstrated that P. aeruginosa strains which are good alkaline protease and elastase producers adher better than those showing no or low protease and elastase activity.

[In vitro activity of norfloxacin against gram-positive and gram-negative organisms from recent clinical isolates]. / Attività in vitro della norfloxacina nei confronti di organismi gram positivi e gram negativi di recente isolamento clinico.

A total of 274 recently isolated Gram-positive e Gram-negative strains have been tested. The method used for the determination of the minimal inhibitory concentration of the antibiotics was by agar dilution and the minimal bactericidal concentrations were determined by "replica plating" system. All strains demonstrated a good sensitivity, above all, to Norfloxacin which inhibited Gram-positive bacteria at the concentration of 3.12 micrograms/ml and, concerning Gram-negative, at the concentration of 0.10 microgram/ml for K.E.S. group, 0.19 microgram/ml for Escherichia coli, 6.25 micrograms/ml for Proteus sp and 1.56 microgram/ml for Pseudomonas aeruginosa. Also with the other antibiotics we obtained good results especially with Nitrofurantoin concerning Enterococcus and Ceftazidime concerning all the strains. On the basis of these results we can conclude that Norfloxacin is one of the most active antibiotics among those used against strains implicated in urinary infections.

[Incidence of Pseudomonas sp., Staphylococcus sp. and Candida sp. in a high-risk population]. / Incidenza di Pseudomonas sp, Staphylococcus sp e Candida sp in un reparto ad alto rischio.

Since infections represent one of the greatest complications in immunocompromised hosts, our group, concerned with bacteriological monitoring, has applied its attention to microorganisms such as Pseudomonas sp, Staphylococcus sp and Candida sp. Pseudomonas sp strains have been identified using conventional techniques and the API system 20 NE. According to Varaldo's scheme, Staphylococci sp have been divided into six "lyogroup" and then, the typing of Candida sp has been checked by cultural and biochemical tests. The results obtained have demonstrated that the Pseudomonas aeruginosa species is prevalent not only in the urinary tract but also in the respiratory tract. As for the Staphylococci sp, the VI lyogroup showed the greatest percentage of strains and than, as regards Candida sp it has been possible to observe that, over a given period, the percentage of pluricontamination has been almost constant.