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A Negro Ponzi

Publications and source records attributed to A Negro Ponzi.

At least 19 recordsLinked to original sources
PubMed2006

Development and utilization of a quantitative polymerase chain reaction assay to evaluate human polyomavirus JC DNA load.

AIM: Quantitative polymerase chain reaction (PCR) analysis to evaluate virus load in comparison with the patient's base-line virus levels would be an optimal diagnostic approach to monitoring human polyomavirus infections and to investigate their possible involvement in the onset of nephropathy in this patient group. Studies on the correlation between viral burden and renal disease have pointed to the incidence of JC virus (JCV) related progressive multifocal leukoencephalopathy (PML) occurring in renal and haematopoietic stem cell transplant recipients. METHODS: We developed a reliable internally-controlled quantitative PCR assay to measure JCV-DNA in fluid samples of urine, serum and cerebrospinal fluid (CSF) by densitometric analysis of the amplification products. The assay was also used to evaluate the JCV load in CFS samples from patients with suspected demyelinating syndrome and in urine and serum samples from healthy subjects and renal transplant recipients. RESULTS: All CSF samples from the 51 patients with suspected demyelinating syndrome tested JCV-DNA negative: none of them had a diagnosed PML. Analysis of the prevalence of JCV-viruria and JCV-viraemia confirmed our previous data. JCV-viruria was detected in 17% of renal transplant recipients and 26.6% of healthy controls; JCV-viraemia was found in 3.4% of transplant patients and 0% in controls. Noteworthy was a lower prevalence of JCV-viraemia in the 116 (3.4%) renal transplant patients than the prevalence previously reported for the 51 (11.8%) patients with suspected demyelinating syndrome. The mean viral load of viruria was much higher in the healthy controls than in the transplant recipients [104020 DNA copies/mL (DS+/-62284) vs 4136 DNA copies/mL (DS+/-77371)]. CONCLUSIONS: The quantitative PCR assay developed in our lab offers in 2 h time a reliable true quantification of viral DNA by densitometric analysis of the amplification product. To check for the possible presence of potential Taq polymerase inhibitors an internal control (the homemade pJCV-C plasmid) is used. The relation between polyomavirus infections and their possible involvement in post-transplant pathologies need further investigation. It would be useful to monitor the JCV-DNA load in urine and serum from more renal transplant recipients, including patients with nephropathy or active graft rejection over a longer period of time.

Base Sequence↗
PubMed2006

Development of a quantitative-competitive PCR for quantification of human cytomegalovirus load and comparison with antigenaemia, viraemia and pp67 RNA detection by nucleic acid sequence-based amplification.

AIM: The human cytomegalovirus (HCMV) is an important pathogen in immunocompromised patients, such as transplant recipients. The use of sensitive and rapid diagnostic assays can have a great impact on antiviral prophylaxis and therapy monitoring and diagnosing active disease. Quantification of HCMV DNA may additionally have prognostic value and guide routine management. The aim of this study was to develop a reliable internally-controlled quantitative-competitive PCR (QC-PCR) for the detection and quantification of HCMV DNA viral load in peripheral blood and compare it with other methods: the HCMV pp65 antigenaemia assay in leukocyte fraction, the HCMV viraemia, both routinely employed in our laboratory, and the nucleic acid sequence-based amplification (NASBA) for detection of HCMV pp67-mRNA. METHODS: Quantitative-competitive PCR is a procedure for nucleic acid quantification based on co-amplification of competitive templates, the target DNA and a competitor functioning as internal standard. In particular, a standard curve is generated by amplifying 10(2) to 10(5) copies of target pCMV-435 plasmid with 10(4) copies of competitor pCMV-C plasmid. Clinical samples derived from 40 kidney transplant patients were tested by spiking 10(4) copies of pCMV-C into the PCR mix as internal control, and comparing results with the standard curve. RESULTS: Of the 40 patients studied, 39 (97.5%) were positive for HCMV DNA by QC-PCR. While the correlation between the number of pp65-positive cells and the number of HCMV DNA genome copies/mL and the former and the pp67mRNA-positivity were statistically significant, there was no significant correlation between HCMV DNA viral load assayed by QC-PCR and HCMV viraemia. CONCLUSIONS: The QC-PCR assay could detect from 10(2) to over 10(7) copies of HCMV DNA with a range of linearity between 10(2) and 10(5) genomes.

Adult↗
PubMed2004

Co-detection and discrimination of JCV and BKV DNA by duplex nested-PCR in renal transplant recipients.

AIM: Several studies have disclosed a correlation between human polyomavirus BK (BKV) and interstitial nephritis in renal transplant recipients. It has recently been hypothesized that some cases of nephropathy may be associated with human polyomavirus JC (JCV). METHODS: In this paper we describe the development of duplex nested-PCR assay which allows the simultaneous detection and discrimination of genomic sequences of JCV and BKV ''large T antigen'', resulting in amplicons of 150 bp and 278 bp, respectively. Thus, the presence of JCV and BKV DNA in urine and serum samples from 51 renal transplant recipients and 29 healthy controls was investigated and related to immunosuppressive regimens and renal function. RESULTS: The comparison between the incidence of the of BKV and/or JCV infections (detected by viruria and/or viraemia) in renal transplant recipients and the control group revealed a highly significant increase of the incidence of BKV infection in immunosuppressed patients vs healthy subjects (62.7% vs 27.6%; p=0.005). In particular, we found a significant increase of BKV-DNA viruria in renal transplant recipients vs healthy subjects (49% vs 17.2%; p=0.01), in agreement with the BKV urinary shedding in renal transplant recipients of the literature (5-45%). CONCLUSION: The nested-PCR technique is a valid diagnostic tool to detect viral presence in urine and its systemic diffusion. Our assay links the high sensitivity of nested amplification with the simultaneous detection and discrimination of genomic sequences of JC and BK polyomaviruses and thus provides a handy, rapid and sensitive means for DNA analysis of large numbers of samples.

Adult↗
PubMed2003

B19 virus infection in renal transplant recipients.

BACKGROUND: B19 virus infection with persistent anaemia has been reported in organ transplant recipients. Detection of B19 virus DNA in serum is the best direct marker of active infection. OBJECTIVE: The present study evaluated the incidence and clinical role of active B19 virus infection in renal transplant recipients presenting with anaemia. STUDY DESIGN: Forty-eight such recipients were investigated by nested PCR on serum samples. The controls were 21 recipients without anaemia. Active HCMV infection was also investigated as a marker of high immunosuppression. RESULTS AND CONCLUSIONS: In 11/48 (23%) patients B19 virus DNA was demonstrated in serum versus only 1/21 (5%) of the controls. Ten of these 11 patients had already been seropositive at transplantation and active infection occurred in eight of them during the first 3 months after transplantation. The remaining patient experienced a primary infection 9 months after transplantation. Eight (73%) of these 11 patients displayed a concomitant HCMV infection and four (36%) showed increasing serum creatinine levels but none developed glomerulopathy; 3/11 (27%) recovered spontaneously from anaemia whereas 8/11 (73%) needed therapy. In conclusion, the relatively high occurrence (23%) of B19 virus infection in patients presenting with anaemia, suggests that it should be considered in the differential diagnosis of persistent anaemia in renal transplant recipients. Presence of the viral DNA should be assessed early from transplantation and the viral load should be monitored to follow persistent infection and better understand the relation between active infection and occurrence of anaemia, and to assess the efficacy of IVIG therapy and/or immunosuppression reduction in clearing the virus.

Anemia↗
PubMed2003

Epstein Barr viral load monitoring by quantitative PCR in renal transplant patients.

Post-transplant lymphoproliferative disorders (PTLD), ranging from lymphoid hyperplasia to clonal malignancy, are a severe complication arising in solid organ transplant patients. Their reported incidence ranges from 1 to 20%, according to factors such as type of transplanted organ and the age of recipients. A strong correlation between Epstein Barr virus (EBV) infection, the grade and type of immunosuppression and the development of PTLD has been recognized. The detection and quantification of EBV-DNA load in peripheral blood have been utilized as prognostic markers for the development of PTLD, showing a correlation between high levels of EBV-DNA in the blood and the development of PTLD. In this study, we monitored EBV viral load monthly in 15 renal transplant recipients for six months. The number of EBV-DNA copies was measured in peripheral blood mononuclear cells (PBMC) and serum samples by a quantitative PCR protocol developed in our laboratory that employes a previous screening of samples containing a significant number of viral DNA copies (> or =1000 copies/10(5) PBMC or 100 microl serum) by semi-quantitative PCR followed by a precise quantification of the only significant samples by quantitative-competitive (QC)-PCR. Our 15 renal transplant patients neither developed PTLD nor had recurrent acute illnesses or acute graft rejections during the study. The results obtained in the monthly follow up of EB viral load in PBMC samples confirmed its fluctuation in asymptomatic patients reported in literature. In particular, 5/14 (35.7%) of EBV seropositive patients had an EBV-DNA load equal to 1000 EBV copies /10(5) PBMC (roughly corresponding to 10.000 copies/microg PBMC DNA), and 1/14 (7.1%) reached 5000 EBV copies /10(5) PBMC (roughly corresponding to 50.000 copies/microg PBMC DNA), at least once in our study. In the EBV seronegative patient, EBV-DNA in PBMC samples was always undetectable (less than 100 DNA copies/10(5) PBMC). EBV-DNA load in all serum samples was less than threshold value of our quantification protocol (<100 DNA copies/100 microl serum), supporting the literature data. With regard to immunosuppressive treatment, 66.7% of the six patients in whom EBV load reached values equal to or higher than 1000 DNA copies/10(5) PBMC, were on FK506 whereas only 33.3% of them were on CyA. In conclusion, further investigations are needed to better understand the role of EBV infection in the pathogenesis of PTLD in immunosuppressed patients. Given the high positive predictive value of EB viral load in peripheral blood for diagnosis of PTLD reported by several authors, and the described absence of correlation between the serological evidence of EBV reactivation and EB viral load, EBV viral load measurement in PBMC and serum samples using quantitative PCR techniques is a powerful diagnostic tool to monitor transplanted patients at risk to develop PTLD.

Cells, Cultured↗
PubMed2001

Quantitative PCR in EBV-infected renal transplant patients.

In this study we investigated the levels of Epstein Barr virus (EBV) DNA by quantitative polymerase chain reaction (Q-PCR) in serum, whole blood and peripheral blood mononuclear cells (PBMC) from anti-EA IgG seropositive or anti-EA IgG seronegative EBV infected renal transplant recipients. We compared serological data with the viral load to monitor the risk of developing post-transplant lymphoproliferative disorders (PTLD). All patients were asymptomatic and none of them developed PTLD at the time of the study. EBV DNA quantitation for each patient varied in whole blood and PBMC samples probably due to different numbers of mononuclear cells present in samples from which DNA was extracted (whole blood vs. purified PBMC). In 92% of the serum samples EBV DNA was undetectable probably due to absence of free genomes since the number of DNA copies detected in samples from whole blood and PBMC does not reach very high levels. The correlation between the presence of EA-antibody, considered serological evidence of EBV reactivation, and the viral load showed that 60% of EA-positive patients had quantifiable EBV DNA, whereas in 40% of EA-positive patients EBV DNA was undetectable, showing serological reactivity but no viral replication. Of the remaining EA-negative patients, EBV DNA could be detected in 71% of them, whereas 29% did not show EBV DNA, indicating no EBV replication. In conclusion, our results confirm that the presence of serum IgG anti-EA antibody is not a reliable marker of active EBV infection whereas the evaluation of the viral load in blood samples is a useful diagnostic tool to monitor and to better understand the course of EBV infection in immunocompromised renal transplant patients at risk of developing PTLD.

Adult↗
PubMed1996

[Epstein-Barr virus infection and lymphoproliferative disorders in patients after renal transplantation].

We have studied EBV infection in renal transplant patients during the first year after transplantation. At trasplantation all patients were EBV seropositive and reactivation of EBV infection was demonstrated in 54% cases after one year. CMV active infection was also demonstrated in 42% of patients with EBV reactivation. No correlation was observed between EBV reactivation and age, sex, immunosuppressive treatment, degree of immunosuppression or donor/recipient HLA matching. A correlation between immunosuppressive treatment, EBV infection and lymphoproliferative disorders (LD) is described in literature, however none of our patients developed LD so far, probably due to the different immunosuppressive protocol employed.

Adult↗
PubMed1989

The secretory immune system.

Several features distinguish mucosal from systemic immunity, depending on the anatomical and functional characteristics of the organs involved. Secretory IgA immunoglobulins are the main effectors of the mucosal immune system and their protective aspects are well documented. Serum IgA have received less attention but are equally important, since they control and remove in a non-phlogistic way antigens crossing the mucosal barriers.

Humans↗
PubMed1987

Specific serum IgA in rotavirus gastroenteritis.

Virus-specific serum IgM, IgA, and IgG were detected by ELISA in sera obtained from children with rotavirus gastroenteritis and fractionated by gel filtration. Specific IgM and IgG could be easily demonstrated, whereas IgA were very low. Moreover, polymeric IgA (p-IgA) were not present, whereas in the immune response against viruses causing systemic infections, they are synthetised in large amounts.

Antibodies, Viral↗
PubMed1986

[Effect of mercaptans on serum IgA].

Using reducing agents like dithiothreitol (DTT) 2.5 mM IgM lose their ability to bind antigens but they can be still detected by an anti-IgM antiserum. IgA are more resistant to reduction than IgM but using higher concentrations of DTT they lose both the ability to bind antigens and the possibility to be detected by an anti-IgA antiserum. Because of this fact IgA after mild reduction can result more or less inactivated according to the method employed to detect them. Using ELISA or similar methods that need an immunological detection of preformed immune complexes IgA-Ag, the loss of activity is more marked than using techniques such as HAI that detect them directly.

Antigen-Antibody Reactions↗
PubMed1985

Virus-specific polymeric immunoglobulin A antibodies in serum from patients with rubella, measles, varicella, and herpes zoster virus infections.

More than 85% of the immunoglobulin A (IgA) antibodies in normal adult serum are monomeric (m-IgA). By contrast, virus-specific IgA is mainly polymeric (p-IgA) in sera from patients with rubella, measles, and varicella. Specific m-IgA antibodies only reach quantitative significance in late convalescence. In patients with herpes zoster, on the other hand, a varying response was observed: in three of six sera, specific IgA was absent or at a very low titer, whereas in the remaining three cases, a high titer of both p-IgA and m-IgA was noted. These results suggest that in the initial response to rubella, measles, and varicella-zoster viruses, specific IgA first appears as p-IgA and only later becomes, or is replaced by, m-IgA.

Antibodies, Viral↗
PubMed1983

Streptococcal receptors for human immunoglobulins.

Six streptococcus strains with a high affinity for human serum Ig were examined under various experimental conditions to correlate their structures with specific IgG and IgA receptors. Treatment at 80 degrees C for 5 min appeared to have no effect on their ability to bind IgG and/or IgA, while long maintenance in culture determined a dissimilar partial loss of IgG and IgA binding ability. Proteolytic enzymes and hot HCl reduced the ability of these strains, especially those in group A, to absorb both IgG and IgA. It would seem that the protein most involved in this type of bond is the T protein.

Humans↗
PubMed1983

Pseudomonas aeruginosa beta-lactamases.

Forty recently isolated hospital strains of Pseudomonas aeruginosa were assayed to determine the part played by the production of beta-lactamase in their resistance to beta-lactamic antibiotics, together with the relative importance of constitutive and inducible enzymes in such resistance in the case of carbenicillin (CB). 62.5% were CB-resistant. In 56% of cases, this resistance was due to the production of a class Vd constitutive enzyme, whereas it was ascribable to intrinsic resistance in the other 44%.

Anti-Bacterial Agents↗
PubMed1983

Separation of IgM for diagnostic purposes by exclusion chromatography: a comparison between six gels.

Sera from rubella patients were fractionated with six different gels on a 0.9 X 30 cm column. Fractionation efficiency was evaluated by determining the IgM, IgA and IgG concentrations in each fraction and titring the rubella virus hemagglutination-inhibiting antibodies before and after treatment with 2-mercaptoethanol. All six gels were able to separate IgM from the other Ig, but the distance between their elution peaks varied considerably. Assessment of the usefulness of a gel for diagnostic purposes requires evaluation of its ability to separate IgM from the oligometric IgA often present in sera and a possible source of diagnostic error. Ultrogel AcA 22 and Bio-Gel A5m gave the best results. In addition to displaying good peak separation, they eluted IgM within their linear fractionation range.

Adult↗
PubMed1983

Comparison between five methods for the separation of IgM.

Five serum fractionation methods were examined with regard to their suitability for use in the demonstration of specific IgM antibodies. It was found that Protein A - Sepharose CL - 4B chromatography leads to higher IgM recovery than absorption with whole Staphylococcus aureus (58% as opposed to 40%). It is also cheaper, since this absorbent can be reused. Contamination with 2-5% IgG and about 70% IgA is encountered with both methods. Quaternary aminoethyl (QAE) - Sephadex chromatography gives excellent IgM recovery (97%) and its cost and execution time are the lowest. IgG and IgA residues are 2% and 53% respectively. Ultrafiltration results in poorer recovery of IgM (31%). However, the sample volume can be brought back to its initial value, or concentrated still further. IgG and IgA residues are 2% and 14%. The cost of the method and the time needed for its execution are relatively high. Gel chromatography is the only method to give a completely IgG- and IgA-free IgM fraction. The percentage of IgM recovered varies in accordance with the ratio between the volume of the sample and that of the column. Very high values may be obtained, but at the expense of heavy dilution. The cost is low, but the method requires a considerable amount of time. In short, none of the five methods is clearly superior to the others. Whether or not a given method is chosen will thus depend on the relative importance attached to the percentage of IgM recovered, the presence or otherwise of IgA, sample dilution, cost, and rapidity of execution.

Chromatography, Affinity↗
PubMed1977

[The immune response of the fetus in the diagnosis of intrauterine infections: IgM and IgA concentrations in the umbilical cord blood and titers of rubella and cytomegalic virus antibodies].

Two serological methods, hemagglutination and complement fixation, have been used respectively to determine HAI titers to rubella virus and C fixing antibodies to cytomegalovirus in cord blood of newborn infants delivered in Turin from August 1973 to April 1974. Sera have been divided according to antibody titer and aspecific IgM and IgA concentrations. By statistical analysis we observed a highly significant association between both IgM and IgA concentration and antibody titers to rubella virus.

Antibodies, Viral↗