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.

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.

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.

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.

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.

[Epstein-Barr virus infection and lymphoproliferative disorders in patients after renal transplantation]. / Infezione da virus di Epstein-Barr e disordini linfoproliferativi in portatori di trapianto renale.

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.

[Early diagnosis of HCMV infections in patients undergoing transplantation of the kidney]. / Diagnosi precoce di infezione da HCMV in pazienti sottoposti a trapianto di rene.

HCMV infection is a major cause of morbidity and mortality following kidney transplantation. Clinical diagnosis is difficult, and rapid and sensitive diagnostic methods are needed since antiviral therapy is available. One hundred-forty-five consecutive kidney-transplanted patients were studied during a period of three months after transplantation. For laboratory diagnosis of HCMV infection, we looked for the presence of pp-65 antigen in polymorphonuclear leukocytes, HCMV-DNA and IgM. Demonstration of HCMV pp-65 antigen by immunofluorescence and HCMV DNA by PCR in leukocytes were efficient methods for early diagnosis of infection.

[Anti-CMV antibody affinity in renal transplant recipients]. / Affinità degli anticorpi anti-CMV in portatori di trapianto renale.

Variations in anti-CMV antibody affinity have been studied in 106 renal transplant patients. Maturation of immune response has been followed during two years after transplantation evaluating antibody affinity by ELISA before and after urea denaturation treatment. In primary infections while the antibody titer rises, the resistance to denaturing treatment rises as well, indicating an increased antibody affinity. In patients already seropositive at transplantation, the increase of antibody affinity has also been found: comparing the affinity index (AI) at transplantation and one year later, only 16.5% of patients showed an AI value between 80 and 100 at transplantation, whereas after one year 62.3% of patients reached such AI values (p = 0.0001).

[Epstein-Barr virus infection and immunoglobulin abnormalities in renal transplant recipients]. / Infezione da virus di Epstein-Barr ed anomalie immunoglobuliniche nei portatori di trapianto renale.

The relationship between immunoglobulin abnormalities and EBV infection has been investigated in 65 renal transplant patients. Immunoglobulins abnormalities were demonstrated in 44 (68%) patients, 8 of them (18%, 12% of all patients) had a monoclonal component. Up to now no lymphoproliferative disorder has been observed in these patients. All patients were EBV seropositive at the beginning of the study and in 22 of them (33%) a reactivation of EBV infection could be demonstrated. No relation has been observed between immunoglobulin abnormalities, EBV reactivation, age or sex. By contrast, a significant relation was found between EBV reactivation and immunosuppressive treatment: patients under triple therapy with Azathioprine, Cyclosporine A and Prednisone had less EBV reactivation compared to those under Cyclosporine A treatment.

[Infection by human herpesvirus type 6 (HHV-6) and renal transplantation]. / Infezione da virus erpetico umano di tipo 6 (HHV-6) e trapianto di rene.

Human herpes virus type 6 (HHV-6) infection was serologically investigated in renal transplant recipients. Before transplantation, 75.5% of patients was seropositive for HHV-6 and no correlation with age, sex and time on dialysis was found. During the first month after transplantation 66% of patients showed a variation in serological status against HHV-6 (seroconversion or fourfold increase of antibody titer). All patients who seroconverted had received the kidney from a HHV-6 seropositive donor, furthermore, in 11/13 (84.6%) pairs of patients receiving the kidney form the same seropositive donor, both members or had HHV-6 active infection or had no infection. The frequency of HHV-6 active infection in seropositive patients is almost the same in case of seronegative or seropositive donor. Comparing HHV-6 and CMV infections, they resulted independent as CMV infection in these patients occurs in a following period (II-III month). Notwithstanding a higher frequency of kidney rejection in patients with active HHV-6 infection, no significative correlation was found.

CMV-specific polymeric and monomeric IgA antibodies in serum of renal transplant patients.

CMV-specific IgA, IgM and IgG antibodies were detected by ELISA in sera from 81 renal transplant patients. Twenty-seven patients were followed from transplantation; 6 patients who underwent on transplantation before the beginning of the study were followed during admissions for graft failure or acute illness; 48 outpatients were periodically monitored. One of the patient followed from transplantation experienced a primary CMV infection, serologically demonstrated by the appearance of specific IgM and IgG. Specific IgA appeared at the same time as IgM and lasted for about six months. A specific IgA response was observed in all but five recurrent CMV infections too, even when specific IgM were not present. In all outpatients periodically monitored for CMV serology specific IgA were not found. About specific IgA polimerization, a transient marked polymeric IgA (p-IgA) response was observed in only the primary infection whereas in all the other IgA positive patients, specific IgA were represented by monomers (m-IgA).

Low prevalence of HTLV-I infection in drug addicts in Turin.

Anti-HTLV-I antibody presence in drug addicts dead in 1987-1988 in Turin, was evaluated. Comparing the prevalence of HTLV-I infection with that of HIV and HBV infections, a different way and/or rate of spread of HTLV-I infection is suggested.

Anti-HBc polymeric IgA antibodies in serum from acute hepatitis B patients.

Serum anti-HBc IgA antibodies can be demonstrated in acute hepatitis B patients. At first, they are mainly polymeric (p-IgA), whereas monomeric IgA (m-IgA) become detectable 3 months later. In most cases, anti-HBc IgA cannot be demonstrated after 6-12 months. From a diagnostic point of view, specific p-IgA can be regarded as a marker of active infection but, compared to IgM, their demonstration is more difficult.

Anti-HIV antibodies in postmortem vitreous humor.

Anti-HIV antibodies were detected in postmortem blood and vitreous humor samples from 60 drug addicts died in 1988 and medicolegally autopsied at the University Institute of Forensic Science of Turin. Fifteen subjects were positive both in blood and in vitreous samples confirming the possibility to detect anti-HIV antibodies in vitreous humor in the screening of high-risk population.

Anti-HIV and anti-HBc antibodies and HBsAg in postmortem blood of drug addicts: the picture for 1988.

Serological markers of HIV and HBV infections were studied in 90 drug addicts who died in 1988 and were medicolegally autopsied at the Institute of Forensic Sciences, University of Turin. Nineteen (21.1%) displayed evidence of HIV infection, demonstrated by the presence of anti-HIV antibodies; fifty-nine (65.5%) HBV infection, demonstrated by the presence of anti-HBc antibodies and/or HBsAg; nine (10%) had HBsAg, indicating potential infectiousness for HBV infection.

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.

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.

[Effect of mercaptans on serum IgA]. / Effetto dei mercaptani sulle IgA del siero.

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.

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.

[A fractionation method for human blood for the separation of subclasses of IgG]. / Un metodo di frazionamento del siero umano per la separazione delle sottoclassi delle IgG.

A serum fractionation method which permits a good separation among IgG subclasses is proposed. By loading serum on a Protein A-sepharose column, IgG3 are readily recovered as they do not bind to the Protein A. IgG of the other subclasses are eluted, equilibrated at pH 6 and loaded either on a chromatofocusing column, or on an anion exchanger gel. In these conditions IgG4 are retained, and then recovered by lowering the pH, whereas IgG1 and IgG2 pass through the column and can be recovered separately by absorption on a Protein A column and elution with a decreasing pH gradient.