HPyV6, HPyV7 and TSPyV DNA sequences detection in skin disease patients and healthy subjects.

BACKGROUND: The discovery, from 2007, of eight new human polyomaviruses (HPyVs) has revived interest in the Polyomaviridae family and their association with human diseases and cancer. In particular, HPyV6 and HPyV7 were discovered in skin swabs of healthy donors and TSPyV was discovered in a heart transplant recipient affected by virus-associated Trichodysplasia Spinulosa (TS), a rare skin disease, exclusively found in immunocompromised patients. OBJECTIVE: The presence of HPyV6, HPyV7 and TSPyV DNA in skin biopsies from patients affected by different skin diseases (cancers and inflammatory disorders) has been evaluated to confirm their skin tropism and the possible pathological association. METHODS: DNA extracted was amplified with HPyV6, HPyV7 and TSPyV specific PCR real time on Taqman platform with standard profile. RESULTS: HPyV7 and TSPyV sequences were not found in any skin specimen analysed. HPyV6, on the other hand, was detected in 30% of samples from healthy subjects vs. 14.3% of skin cancer patients and 2.9% of inflammatory disorders. HPyV6 sequences have been detected in primary cutaneous T-cell lymphoma (CTCL) patients (in 18.6% out of Mycosis Fungoides (MF) patients and in 16.7% out of CTCL not MF/SS(Sèzary syndrome) but have not been detected in primary cutaneous B-cell lymphoma (CBCL) patients. CONCLUSION: Our preliminary data suggest that these three novel human polyomaviruses seem not to play a significant role neither in the pathogenesis of cutaneous malignancies nor in that of inflammatory disorders but, according to literature, can inhabit the skin. On the basis of our data regarding the HPyV6 DNA presence with decreasing percentages in healthy subjects, skin cancer and inflammatory disorders patients, it could be an intriguing matter to study if the activated innate immune response in inflammatory disorders can suppress the virus. Further investigations are needed to better understand their relationship with the human host and its innate immune system.

White blood cells, TNF-α, and interleukin-6 in subjects with infantile colic treated with Lacticaseibacillus rhamnosus GG (ATCC 53103): a randomised prospective study.

Recent metanalysis reported that certain probiotic strains, such as Limosilactobacillus reuteri and Lacticaseibacillus rhamnosus (LGG), seem effective for treatment of infantile colic of exclusively breastfed infants; some reports have also linked probiotics to have an immunological effect, however further investigation are needed to fully understand the exact mechanism. The objective of this study was to assay white blood cells, tumour necrosis factor (TNF)-α and interleukin (IL)-6 values in peripheral blood in subjects treated in a randomised, double-blind, placebo-controlled trial for infantile colic with LGG. Fifty-eight infants were enrolled and followed for a study period of 28 days. Parent were asked to record daily crying time using a structured cry diary. Peripheral white blood cells was assessed and RNA (mRNA) expression of TNF-α and IL-6 was measured using TaqMan real-time PCR-maternal amplification. Infants with colic treated with LGG showed a reduction in daily crying duration after 28 days of treatment and a reduction in values of IL-6 ( P < 0.005) and TNF-α ( P < 0.05); we observe also a significantly decreasing of IL-6 in the placebo group while decrease of TNF-α was not significant in this group. A significant decreased values of monocytes ( P < 0.05) was observed in infants treated with LGG. Our data therefore showed, in addition to crying time reduction, a significant decrease of TNF-α and a significant reduction of monocytes cells in colicky infants treated with LGG, compared to placebo group. This observation supports the hypothesis that probiotics may have anti-inflammatory properties. Further studies are needed to better understand the influence of probiotic on immunity cells.

Th1, Th2, Th17 and regulatory T cell pattern in psoriatic patients: modulation of cytokines and gene targets induced by etanercept treatment and correlation with clinical response.

BACKGROUND: Psoriasis is sustained by pro-inflammatory CD4+ T helper cells mainly belonging to the Th1, Th17 and Th22 lineage. OBJECTIVE: To identify whether treatment with the anti-tumour-necrosis-factor antagonist etanercept is able to induce significant modulations in transcription factor and cytokine mRNA gene expressions related to the different T cell immune response polarization (Th1, Th2, Th17 and regulatory T cells, Treg and to correlate them with clinical response. METHODS: The study population included 19 psoriasis patients treated with etanercept and 19 healthy subjects. Blood samples were collected at baseline and every 4 weeks during treatment. Taqman quantitative real-time polymerase chain reaction was applied to analyse the expression of: Stat-4, T-bet, IL-12p35 and IFN-γ (Th1-related); GATA-3, IL-4 (Th2-related); Stat-3, RORγt, IL-23p19 (Th17-related); Foxp3, IL-2 (Treg-related). Flow cytometry was applied to analyse CD4+CD25+(bright)Foxp3+ cells in peripheral blood. RESULTS: Upregulation of Th1 and Th17 and downregulation of Treg subsets was found at baseline. The response to etanercept could be associated with a significant reversal of the Th1/Th17 activation, and a concomitant upregulation of Th2 and Treg subsets. CONCLUSION: Our data may contribute to a better understanding of the mechanisms underlying the achievement of clinical response in psoriasis and could be helpful for the identification of early predictive markers of response.

Detection of the novel variant of influenza virus A/H1N1v in bronchoalveolar lavage of adult hospitalized patients during the 2009/2010 winter season.

AIM: The aim of this study was to report most recent data regarding the occurrence of influenza A virus H1N1v in the lower respiratory tract from a cohort of hospitalized adult patients during the winter season 2009/2010 and investigated the main clinical features and outcomes. METHODS: A total of 130 consecutive BAL specimens (collected from October 2009-March 2010) of 101 patients were retrospectively analyzed for influenza A virus H1N1v positivity using a commercial kit. RESULTS: Overall, 19/130 (14.6%) BAL specimens from 17/101 (16.8%) patients were positive for the novel influenza A H1N1v virus. H1N1v resulted significantly more prevalent in immunocompetent subjects. As regards clinical features, H1N1v resulted more prevalent in respiratory insufficiency or acute respiratory illness. Thirteen patients died during the analytic period; three of them (23.1%) resulted positive to H1N1v but no direct association has been made. CONCLUSION: Our cohort study of influenza A H1N1v detection in BAL from hospitalized adult patients confirms the overall moderate clinical impact of this virus, as reported in most reports worldwide. It remains to be evaluated the role of reassortment with influenza virus strains circulating in the winter season 2010/2011 and its potential pathogenicity.

Prevalence of polyomaviruses BK, JC, SV40, KI, and WU in non-malignant tonsil specimens.

AIM: The recently described polyomaviruses KI and WU have been detected in respiratory samples, stools, tonsils, and blood, particularly in immunocompromised conditions, although little is known about tissue tropism. Herein we investigated the occurrence of KIV and WUV in non-malignant tonsillar specimens by Real-time quantitative PCR; the presence of polyomaviruses BK, JC and SV40-DNA was also evaluated. METHODS: Twenty-nine non-malignant tonsil specimens obtained from children and adults admitted for tonsillectomy were prospectively studied. Real-time quantitative TaqMan PCR for polyomaviruses KI, WU, BK, JC, and SV40 were performed. RESULTS: KI-DNA was positive in 2/29 tonsillar specimens (6.9%), while BK- DNA, JC-DNA, SV-40 DNA, and WU-DNA sequences were not identified. CONCLUSION: Few studies have investigated the prevalence of polyomaviruses in tonsil specimens, with varying results, and data are particularly scant as regards the newly discovered KIV and WUV. Two major questions remain to be definitely answered at this regard: the possibility that human tonsils represent the initial site of infection and/or a latency site and the biological and clinical meaning of KIV and WUV in different contexts and groups of patients, in that it is not clear whether they are simple bystanders or play a role in tonsil disease.

Circulating CD4+CD25 bright FOXP3+ T cells are up-regulated by biological therapies and correlate with the clinical response in psoriasis patients.

BACKGROUND: Regulatory T-cell (T(reg)) modulation is one of the potential mechanisms of anti-tumour-necrosis-factor biological agents. However, literature data on psoriasis patients are lacking. OBJECTIVE: To analyse the circulating CD4+CD25(bright)FOXP3+ subset in 30 patients with psoriasis vulgaris/arthropathic psoriasis treated with biologicals and to investigate its relationship with the clinical response. METHODS: The CD25(bright)FOXP3+ expression within the CD4+ subset was determined by multi-parameter flow cytometry at baseline and during treatment. FOXP3 mRNA expression was analysed by real-time reverse transcription PCR. RESULTS: A response was obtained in 16/17 patients (91.1%) with increased CD25(bright)FOXP3+ values and in only 3/11 patients (27.3%) who showed a CD25(bright)FOXP3+ decrease during biological treatment (p = 0.0001). Responders showed significantly higher values than did non-responders as from the first 2 months of treatment (p = 0.0032). A significantly higher posttreatment expression of mRNA FOXP3 was observed in responders compared to non-responders. CONCLUSION: Biological drugs induce a circulating T(reg) up-regulation in a significant percentage of patients; such an increase is an early predictive marker of response.

Lower respiratory tract viral infections in hospitalized adult patients.

AIM: The epidemiology of lower respiratory tract (LRT) viral infections in adults is probably underestimated and the high frequency of multiple viral infections complicates the evaluation of the possible role of the single viruses. The aim of this study was to investigate the clinical epidemiology and impact of respiratory viral pathogens, in particular of those singularly detected, in bronchoalveolar lavage (BAL) specimens from hospitalized adult patients. METHODS: A panel for the detection of 16 respiratory viruses was used to prospectively evaluate 324 consecutive specimens obtained from 219 patients over a full-year period. RESULTS: Two-hundred-twenty-one specimens (68.2%) were positive for at least one virus, 119/324 (36.7%) to a single viral agent. The most commonly detected viruses were herpesviruses HHV-7 (26.2%), human cytomegalo-virus (HCMV, 22.2%), HHV-6 (19.8%), EBV (12.7%), enteroviruses and rhinoviruses (both 11.7%), parainfluenza viruses (4.9 %), and metapneumovirus (4.0%). Human cytomegalo-virus was significantly more prevalent as single viral pathogen with a viral load >105 copies/ml associated to pneumonia in solid organ transplant recipients. Other viral pathogens might account for some cases of pneumonia or respiratory insufficiency, although multiple infections were common. CONCLUSIONS: The use of a comprehensive diagnostic panel for respiratory viral infections may be useful to clarify the epidemiology and clinical impact of viral pathogens in hospitalized adult patients. The occurrence of multiple infections is a common finding and results should be interpreted taking into account the clinical context as well as viral load and the biological characteristics of each virus.

Human herpesvirus 7 detection by quantitative real time polymerase chain reaction in primary cutaneous T-cell lymphomas and healthy subjects: lack of a pathogenic role.

BACKGROUND: Primary cutaneous T-cell lymphomas (CTCLs) are a heterogeneous group of lymphomas where the tumour population emerges within a multiple subclone pattern. Mycosis fungoides (MF) and Sézary syndrome (SS) are characterized by the expansion of clonal CD4+/CD45RO+ memory T cells. Lymphomatoid papulosis (LyP) is a chronic, lymphoproliferative disorder included in the CD30+ primary CTCL spectrum. Several studies have suggested a role of viral infection for super-antigenic activation of T lymphocytes; however, evidence of their association with CTCLs is still lacking. Human herpesvirus (HHV) 7 is a CD4+ T-lymphotropic herpesvirus; its restricted cellular tropism and the ability to induce cytokine production in infected cells could make it an important pathogenic cofactor in lymphoproliferative disorders. OBJECTIVES: To investigate the presence of HHV7 DNA on CTCL and healthy skin donors (HD). METHODS: We used quantitative real time polymerase chain reaction to evaluate the potential pathogenic role of HHV7. RESULTS: Twenty-seven of 84 (32.1%) HD were positive for HHV7 DNA. Twenty-one of 148 (14.2%) patients with CTCLs were positive for HHV7 DNA: nine of 39 (23.1%) SS, six of 14 (42.9%) CD30+ CTCLs and six of 24 (25.0%) LyP, and HHV7 DNA was negative in all 71 patients with MF. CONCLUSIONS: These results seem to exclude a pathogenic role of HHV7 in CTCLs, suggesting the possibility of skin as a latency site.

Severe crescentic BK virus nephropathy with favourable outcome in a transplanted patient treated with Leflunomide.

Herein reported is a severe case of BK virus nephropathy, probably caused by an intense/overimmunosuppression and identified 17 months after transplantation. The diagnostic biopsy showed extracapillary proliferation and typical cytopathic lesions, both in tubular epithelial cells and in those of the glomerular crescents. Severe inflammatory infiltrates, tubulitis, tubular atrophy and fibrosis were also observed. Immunohistochemistry and molecular biology disclosed the presence of an AS variant of the BK virus with a high viral load, both in renal tissue and urine. Immunosuppression was reduced and Leflunomide therapy administered for a month. Although this led to an improvement in the renal function, the therapy had to be suspended due to the onset of a skin rash. A second biopsy was performed 7 months later. The cellular crescents were no longer present and there was no evidence of either histologic or immunohistochemical findings consistent with an active BK virus infection. Tubular atrophy and interstitial fibrosis were still present. In addition, fibrotic crescents, which may be interpreted as late scarring changes of previous epithelial proliferation, were found. Although molecular investigation still showed the presence of the BK virus, the viral load in renal tissue, urine and serum was greatly reduced. Indeed, serum and urine viral load was still low at the last control, five months after the second biopsy. The morphologic and clinical evolution are reported and the possible role of the therapy is discussed.

BKV reactivation in renal transplant recipients: diagnostic and therapeutic strategy--case reports.

The Polyomaviridae family includes several viruses that are ubiquitous with specific host spectra. The human polyoma viruses BK and JC were discovered in 1971. Following primary infection, transmitted by the respiratory and probably the oral route, BK remains latent in uroepithelial cells, in B lymphocytes, or in other tissues (spleen, brain). Reactivation with asymptomatic viruria may occur in both immunocompetent subjects and immunocompromised patients. In renal transplant recipients, BKV replication may cause tubulointerstitial nephropathy (BKVAN) with increasing prevalence rates--1% in 1995, 8% in 2007--leading to the loss of the transplanted organ in 30% to 80% of cases. With the availability of diagnostic programs (decoy cells in urine, amplification of viral DNA by polymerase chain reaction (PCR) on serum and urine, real time (RT)-PCR test for mRNA VP1 urine (mRNA-VP1), and renal biopsy accompanied by reduction in immunosuppression, administration of leflunomide, cidofovir (after hydration), and N-acetylcysteine, as well as immunoglobulin by intravenous injection (IVIg), the incidence of renal loss caused by BKVAN infection has been reduced by 10% to 80%. In this study, we have described 12 patients: 6 treated with tacrolimus (FK), mycophenolate mofetil (MMF), and steroids, and 6 treated with cyclosporine or with mTOR inhibitors. Two patients from the first group showed BKVAN about 3 months posttransplantation. Early diagnosis and therapeutic intervention (cidofovir + IVIg) led to reduction in the viral load, with improvement and stabilization in renal function. Considering the high positive predictive value (98%) of mRNA VP1, it should be possible to avoid renal biopsy. The level of immunosuppression--rather than the immunosuppressive drug itself (FK and MMF)--seemed to be associated with BKV reactivation.

Monitoring of human Cytomegalovirus infection by antigenemia and viremia in the first 100 days following renal transplantation and relation to antiviral strategies.

AIM: Human Cytomegalovirus (HCMV) is a relevant pathogen in transplant recipients, particularly in the first three months post-transplantation. The use of antiviral prophylaxis and pre-emptive therapy is able to reduce incidence of HCMV infection and disease. The incidence of HCMV infection and disease in renal transplant recipients in the first 100 days post-transplantation was investigated, in relation with HCMV serological matching and therapeutic management. METHODS: Incidence of HCMV infection in the first 100 days post-transplantation was evaluated by pp65-antigenemia in 165 patients on a total number of 1241 clinical samples. Patients were divided in four groups according to donor/recipient serological matching: D(-)/R(-) (low risk of HCMV disease), D(-)/R+ and D+/R+ (intermediate risk) and D+/R(-) (high risk). Antiviral strategy (prophylaxis in high risk group; pre-emptive therapy in intermediate risk group, no therapy in low risk group) and immunosuppressive protocol were recorded. RESULTS: Incidence of antigenemia-positivity was as follows: 0/3 D(-)/R(-) patients; 59/130 (45.4%) D+/R+; 5/16 (31.3%) D(-)/R+; 4/16 D+/R(-). No significative difference was found between the four groups in terms of incidence of antigenemia-positivity in the first 100 days following transplantation. Antigenemia values >50 pp65-positive/2x10(5) peripheral blood leukocytes (used to start pre-emptive therapy) were present in 18/130 (13.8%) D+/R+; 1/16 (6.2%) D+/R(-); 0/16 D(-)/R+. Viral kinetics in patients with HCMV infection was described. CONCLUSION: No significative difference was found in terms of incidence of HCMV infection in the first 100 days post-transplantation in relation to immunosuppressive protocol and serological matching, suggesting the appropriateness of antiviral strategies and viral monitoring adopted in this setting.

Regulatory T cells and Toll-like receptor 2 and 4 mRNA expression in infants with colic treated with Lactobacillus reuteri DSM17938.

Regulatory T cells induce immune homeostasis and the expression of Toll like receptors (TLRs); subsequent inflammatory cytokine release may be involved. Recent studies have shown a microbial imbalance in the gut of colicky infants (with a prevalence of gram-negative bacteria, such as Escherichia coli), and accumulating evidence has shown the efficacy of a probiotic (Lactobacillus reuteri) in breastfed subjects, but the underlying mechanism remains undefined. The study enrolled 59 infants younger than 60 days, of whom 34 subjects had colic and 25 were healthy controls. With a double-blind, placebo-controlled randomised study performed in our unit from October 2016 to July 2017, infants with colic were randomly assigned to receive oral daily L. reuteri DSM17938 (1×108 cfu) or placebo for 28 days. Peripheral blood was collected to assess the expression of FoxP3, TLR2 and TLR4 mRNA using real-time TaqMan RT-PCR at baseline and after the study period. Our findings showed increased mRNA expression of the transcription factor forkhead box P3 (FoxP3) in infants treated with L. reuteri DSM 17938 for 28 days (P<0.009) and increased TLR2 and TLR4 mRNA expression in both treated and placebo subjects. After L. reuteri administration for 28 days in infants with colic, we observed a significant decrease in daily crying time (302.3±19.86 min/day on day 0 vs 76.75±22.15 min/day on day 28, P=0.001). This study provides evidence that the observed increase in FoxP3 expression and reduction in crying time might be responses to probiotic treatment, while the increase in TLR2 and TLR4 mRNA expression might be related to age. Exploiting these new findings may lead to an unprecedented level of therapeutic control over immune tolerance using probiotics.

Rapid shell vial culture for the detection of respiratory viruses from bronchoalveolar lavage in immunocompromised patients.

AIM: Viral lower respiratory tract infections (LRTI) are an important cause of morbidity in immunocompromised patients. The aim of this study was to evaluate the clinical impact of rapid shell vial cultures from bronchoalveolar lavage (BAL). METHODS: Sixty-seven BAL samples from 46 patients have been retrospectively examined: 51 from 31 transplant recipients and 16 from 15 immunocompromised patients. BAL were inoculated on human embryonic lung fibroblasts and VERO cells to isolate the following viruses: cytomegalovirus (CMV), herpesviruses, varicella-zoster virus, respiratory syncytial virus, adenovirus, Influenza viruses A and B and Parainfluenza viruses. Clinical, microbiological, laboratory, and radiological data were collected. RESULTS: A LRTI was present in 56.7% of cases: viral 40.3%, bacterial and/or fungal 23.9%, and mixed 7.5%. CMV accounted for 92.6% of viral LRTI. The prevalence of viral infections did not differ between symptomatic and asymptomatic patients; only bacterial and/or fungal infections were significantly more prevalent in symptomatic patients. No clinical, radiological or laboratory feature was significantly associated with the presence of a viral LRTI. In lung transplant recipients the rate of CMV infection was 50%. The result of BAL suggested commencement of antiviral chemotherapy in 25/67 episodes. CONCLUSION: Rapid shell vial culture and immunofluorescence techniques from BAL could play an important role in the clinical management of immunocompromised subjects.

The hazardous burden of Herpesviridae in inflammatory bowel disease: the case of refractory severe ulcerative colitis.

BACKGROUND: Herpesviridae infection or spread may be a hazard in immunodepressed patients. In the field of inflammatory bowel disease, refractory severe ulcerative colitis is a challenging condition, closely associated to immunosuppression both for inanition due to the disease activity and for immunosuppressive treatments. Cytomegalovirus (CMV) has been proposed as a major cause of refractoriness, while other Herpesviridae may be a risk factor in the long-term follow-up. AIM OF THE STUDY: To evaluate the positivity rates of CMV, Epstein-Barr (EBV) and Human herpes virus-8 (HHV8) in a consecutive group of ulcerative colitis patients who underwent colectomy for refractoriness to medical treatment compared to a control group, using state of the art methods. PATIENTS AND METHODS: Colonic specimens from 24 consecutive patients with ulcerative colitis submitted to colectomy for refractoriness and from 20 controls (submitted to colectomy for colorectal cancer) were studied. Standard histology and immunohistochemistry (IHC) for CMV and specific polymerase chain-reaction (PCR) for CMV, EBV and HHV8 were carried out. RESULTS: Regarding CMV, 1 case (4%) was positive at histology and IHC, whereas 3 cases (13%) were positive at PCR, compared to none in the control group (p=0.239). For EBV 2 cases (8%) and 2 controls (10%) were positive at PCR. None of the cases or of controls was positive for HHV8. The only clinical characteristic independently associated to CMV positivity was the white blood cell count at admission, higher among CMV positive patients (p<0.001). At the end of the post-surgery follow-up (median 7.3 years) none of the CMV positive cases experienced pouchitis, compared to 3/21 (14%) of the CMV negative cases (p=1.000). DISCUSSION: Our data suggest that CMV is uncommon (13%), even though PCR techniques, considered to be the most sensitive tools, were used for virus detection and the study population is made by highly selected patients with definite refractoriness. EBV and HHV8 may represent a theoretical risk of immunosuppressive therapy because of their potential role as cancer triggers; however in our study, results seem to be reassuring that UC patients undergoing immunosuppressive therapy are not exposed to an excessive risk of viral infection.

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.

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.

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.

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.