Viral Load In Urine Research Articles

BKV-DNA and JCV-DNA Co-Quantification Assay to Evaluate Viral Load in Urine and Serum

Infections from human polyomaviruses BK and JC (BKV and JCV) occur independently, but concomitant infections and the simultaneous persistence of both viruses have been observed in renal transplant recipients. Several studies have disclosed a correlation between BKV and interstitial nephritis in renal transplant recipients, and an association between JCV and some cases of nephropathy has recently been hypothesized. This article describes the development of a semiquantitative-nested polymerase chain reaction (PCR) assay to simultaneously detect BKV and JCV viral load in urine and serum. The first-round amplification step uses primers that amplify a 385-bp DNA fragment from the "large T antigen" region of both viruses. Samples testing positive in the first step are then run in the second step. In the second-round amplification, different inner primers are used to separately quantify BKV-DNA and/or JCV-DNA. The assay offers several advantages including: (1) rapid submission of clinical samples to screening; (2) verification of the absence of Taq polymerase inhibitors with the use of an internal control; (3) a sensitivity threshold of 10 copies/reaction; and (4) assay running is less labor intensive, cheap, and easy to perform. The assay may be easily used to monitor viral loads versus baseline levels in urine and serum samples from renal transplant recipients to detect those at risk of BKV- or JCV-related nephropathy, and to monitor their response to immunosuppression reduction therapy if it occurs.

Treatment of symptomatic congenital cytomegalovirus infection with valganciclovir

Cytomegalovirus (CMV) is the most common cause of congenital infection in humans. Some congenitally infected infants will develop sequelae later in life, especially sensorineural hearing loss (SNHL) and mental retardation. There is no generally accepted antiviral therapy for the treatment of symptomatic congenital CMV infections yet. We present a neonate with symptomatic congenital CMV infection, who was treated with intravenous (iv) ganciclovir (GCV) during 18 days and subsequently with oral valganciclovir (VGCV) for 5.5 months, in an attempt to prevent development of SNHL. GCV was given intravenously 10 mg/kg/day in two doses and VGCV doses ranged from 280-850 mg/m2 bidaily (bid). Our experience shows that it is not possible to give a fixed dosing regime for VGCV in neonates and that continuous adaptation of dose is necessary to achieve stable target levels of GCV and to keep the viral load in urine at undetectable level. At 18 months of age no hearing deterioration has occurred. While the current findings are encouraging, the limitations of a single case report with a relatively short follow-up emphasizes the need for further prospective randomized studies to evaluate pharmacokinetics, efficacy and safety of VGCV therapy in neonates with congenital CMV infection.

Cost efficiency in the prospective diagnosis and follow-up of polyomavirus allograft nephropathy

Evaluation of urine cytology (UC) for decoy cells and quantitative determinations of viruria (urine viral load [UPCR])and viremia (viral load in blood [VLB]) have been proposed as surrogate markers of polyomavirus allograft nephropathy (PVAN). In this study, we present the experience with the concurrent evaluation of UC, UPCR, and VLB in 349 patients (940 sets of samples). Results were correlated with each other and with a previous, concurrent, or subsequent biopsy diagnosis of PVAN. Patients were followed up for a mean of 27 months posttransplantation. We conclude that both UC and UPCR are useful for screening of renal transplant recipients. Simultaneous performance of both UC and UPCR does not add useful clinical information. In patients with positive UC, performance of UPCR, however, can allow for the distinction between BK and JC polyoma viruses. Quantitative measurement of viremia is not indicated in patients lacking viruria because no patients with PVAN present with this combination of findings. In patients with viruria, a positive viremia strongly correlates with PVAN. Rationale selection of screening protocols based on the current knowledge of the infection and tailored to the available laboratory capabilities in each transplantation center can optimize the use of resources.

BK Viruria in Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplantation: Increased Incidence in Patients Receiving Alemtuzumab (ALEM) as Conditioning Therapy and Lack of Efficacy of Cidofovir.

BK virus is a highly ubiquitous polyoma virus with a peak seroprevalence in childhood ranging from 60–100%. After primary infection, which is often asymptomatic or occasionally associated with fever and upper respiratory symptoms, the virus remains latent in the kidneys. During heavy immunosuppression, as occurs following agents such as Alem or anti-thymocyte globulin (ATG), the virus reactivates and can cause significant uroepithelial cell lysis with resultant hemorrhagic cystitis (HC). Between April 2003 and June 2004, 25 patients (15 males, 10 females) undergoing allogeneic (13 MSD, 12 VUD) hematopoietic stem cell transplantation for a variety of indications (18 MDS/AML, 3 NHL, 1 CLL, 2 MM, 1 CML) were found to have BK viruria. Patients ranged in age from 20–67 years. 16 patients underwent ablative conditioning (12 busulfex [Bu]-fludarabine [Flu]-Alem, 1 Bu-Flu-ATG, 2 total body irradiation (TBI)-cyclophosphamide (Cy), 1 TBI-etoposide) whereas 9 patients underwent reduced-intensity (RI) conditioning (5 Blu-Flu-Alem, 2 Bu-Flu-ATG, 1 Flu-Cy, 1 Cy-ATG). Patients treated on ablative regimens containing Bu received a total dose of 12.8 mg/kg whereas those treated with RI conditioning received 6.4 mg/kg. Those who received Flu got 30 mg/m2/d for 5 doses. Alem-based regimens contained 30 mg/d for 3 days or 20 mg/d for 5 days. Graft-versus-host disease (GVHD) prophylaxis consisted of: tacrolimus (FK) alone (n=16), FK + methotrexate (MTX) (n=7), FK + steroids (n=1), cyclosporine-MTX (n=1). 23 patients had BK viral loads in urine measured by PCR with peak levels ranging from 1500 copies/ml to > 200,000,000 copies/ml. BK virus was first detected in the urine anywhere from day −7 to day +85 post-transplant. The time to peak level of BK viruria ranged from day −2 to day + 85 (median 17 days). 40 % (10/25) of these BK viruric patients developed symptomatic HC and 16 % (4/25) developed uretostenosis and/or hydronephrosis (2 required ureteral stent placement). Only 1 of 14 patients treated with multiple doses of Cidofovir (range 3–26 doses) either cleared their viruria by qualitative assay or had greater than a 3 log reduction by quantitative PCR within 4 weeks of starting antiviral therapy; 2 of the 13 patients did have a 2 log reduction in viral load within 4 weeks of initiation of antiviral therapy. It is interesting that 27% (3/11) of patients not treated with Cidofovir cleared their BK viruria between 21–54 days post-transplant. Of note, an additional 6 patients were identified who received Alem; overall 21/23 patients who received Alem developed BK viruria. In conclusion, we found an extremely high incidence of BK viruria (91%) in patients receiving Alem as conditioning therapy for allogeneic SCT. This was associated with ureteral stenosis or hydronephrosis in 4 patients. Therapy with cidofovir given at two different doses had no impact on the excretion of BK virus.

Polyomavirus infection in pediatric renal transplant recipients: evaluation using a quantitative real-time PCR technique.

Polyomavirus infection and related nephropathy is being increasingly recognized as an important cause of allograft dysfunction in adult renal transplant recipients. We prospectively monitored pediatric renal transplant recipients for the presence of BK and JC polyomavirus in urine and blood using a quantitative PCR assay to evaluate the prevalence and clinical relevance of polyomavirus infection in the pediatric renal transplant population. Of 46 pediatric renal recipients who were evaluated, nine (20%) demonstrated isolated BKV viruria, while five (11%) had concomitant BKV viremia and viruria. JCV viruria was found in eight (17%) patients. BKV viremia was associated with a significantly higher urinary BKV viral load: median urinary viral load 1.9 x 10(9) copies/mL (range 6.7 x 10(2)-1.8 x 10(11)) for the group with concomitant viremia and viruria vs. 1.8 x 10(3) copies/mL (range 2.5 x 10(2)-4.5 x 10(6)) for the group with isolated viruria (p < 0.0001). In children that were followed prospectively since their transplantation, the BKV urinary viral load increased markedly before viremia became detectable a few weeks later. None of the patients with JCV viruria or isolated BKV viruria had renal dysfunction. Among the five patients with BKV in both urine and blood, two developed biopsy-proven BKV nephropathy associated with deterioration of the renal function. Management of the BKV nephropathy consisted of reduction of immunosuppression alone or in combination with antiviral treatment with cidofovir. This study shows that polyomavirus infection and related interstitial nephritis is a relevant clinical issue in the pediatric renal transplant population. Monitoring the polyomaviral load in the urine and the blood of the patients using a quantitative PCR technique is a useful tool in the diagnosis and subsequent management of this infection. Even before viremia is present, an important rise in the urinary viral load should draw the attention of the transplant clinician and raise the issue of adapting the immunosuppression.

Viral Loads in Clinical Specimens and SARS Manifestations

A retrospective viral load study was performed on clinical specimens from 154 patients with laboratory-confirmed severe acute respiratory syndrome (SARS); the specimens were prospectively collected during patients' illness. Viral load in nasopharyngeal aspirates (n = 142) from day 10 to day 15 after onset of symptoms was associated with oxygen desaturation, mechanical ventilation, diarrhea, hepatic dysfunction, and death. Serum viral load (n = 53) was associated with oxygen desaturation, mechanical ventilation, and death. Stool viral load (n = 94) was associated with diarrhea, and urine viral load (n = 111) was associated with abnormal urinalysis results. Viral replications at different sites are important in the pathogenesis of clinical and laboratory abnormalities of SARS.

Monitoring the progress of BK virus associated nephropathy in renal transplant recipients.

Nephropathy associated with BK virus (BKVAN) has recently emerged as an important cause of allograft failure following renal transplantation. The aim of this study was to evaluate the effectiveness of laboratory markers in the follow-up of patients with BKVAN. Serial samples from seven renal transplant recipients with biopsy proven BKVAN were studied. The median follow-up time from diagnosis was 76 weeks. Intervention after the diagnosis of BKVAN included immunosuppression dose reduction, alternative immunosuppressive agents and/or antiviral therapy with cidofovir. Serial urine samples (n = 127) were collected for electron microscopy (EM), decoy cell detection and quantitative urine BK viral load using real-time polymerase chain reaction. Serum BK viral load was also measured serially (n = 72). All patients showed a reduction in serum and urine viral load during the period of follow-up co-incident with the loss of decoy cells and negative urine EM. Urine samples that were negative for decoy cells or polyomavirus by EM had a urine viral load <10(6) copies/ml and a corresponding serum viral load <10(3) copies/ml. In paired serum/urine samples, there was a proportional relationship between serum and urine viral load with each urine viral load approximately 1000-fold higher than the corresponding serum level. Serum and urine viral loads that decreased to <200 and < 10(6) copies/ml, respectively, correlated with histological improvement. Negative EM and absence of decoy cells could be used as broad indicators of a response to intervention. However, measurement of BK virus DNA level provided a wider dynamic range and could be a better choice for determining the extent of viral control.

VIRAL LOAD MONITORING IN PATIENTS WITH POLYOMAVIRUS NEPHROPATHY WHO HAVE BEEN TREATED FOR REJECTION EPISODES

P823 Aims: Rejection occurring after reduction of immunosuppression for treatment of polyomavirus nephropathy (PVN) is a difficult clinical problem, as an increase in immunosuppression could result in a worsening of PVN. Recent anecdotal literature has suggested that treatment of rejection episodes followed by a rapid reduction of immunosuppression may prevent a relapse of PVN. The aim of this study was to determine the effects of a transient increase in immunosuppression for the treatment of acute rejection on viral load monitoring parameters in renal transplant recipients diagnosed with PVN. Methods: Four patients experienced six rejection episodes post PVN. BKV serum and urine viral loads were analyzed by Taqman PCR and anti-BKV antibody titers (ab) were analyzed by hemagglutination assay at the time of rejection, and at 8 and 24 weeks post rejection episode. Results: Four patients experienced six rejection episodes (multiple episodes in two patients) post PVN. Median time to acute rejection post PVN was 18 (2-60) weeks. Four of the rejections were mild, 1 was moderate and one patient developed humoral rejection after multiple acute cellular rejection episodes. Rejection treatment consisted of: none in 1, increased tacrolimus in 2, IVIG and increased tacrolimus in 1, leflunomide in 1 and IVIG alone in 1. Median BKV serum and urine viral loads did not deteriorate following the treatment of rejection (BKV serum viral load 5.04 x 10 4 at rejection vs 7.70 x 10 2 copies/mL post-rejection) and (BKV urine viral load 1.22 x 10 6 at rejection vs 1.52 x 10 4 copies/mL post-rejection). Median anti-BKV antibody titers were higher in patients post rejection (8192 at rejection vs 32968 HA units post rejection) and median serum creatinine values were not worse post rejection treatment followed by a reduction of immunosuppression (median serum creatinine at rejection 2.6 vs 2.1 mg/dL post rejection) Of patients who experienced post PVN rejection episodes, graft loss occurred in one patient (25%) who experienced multiple acute rejection episodes followed by humoral rejection and worsening PVN. Conclusions: 1) Transient increases in immunosuppression for the successful treatment of acute rejection episodes, in patients with PVN does not result in an increase in BKV viral loads. 2) Graft loss occurred only in 1 out of 4 patients experiencing rejection following diagnosis of PVN 3) Our results suggest that transient increases in immunosuppression for the treatment of rejection episodes in patients with PVN is indicated for the optimal management of these complex patients.

Polyomavirus infection and disease in renal transplant recipients

Asymptomatic urinary excretion of the human polyomaviruses, BK virus and JC virus, is common in recipients of renal allografts. However, as increasingly potent immunosuppressive strategies have been adopted, BK-associated interstitial nephritis involving the allografted kidney has emerged as a significant cause of chronic allograft dysfunction, occurring in 2 to 5% of patients. The relative roles of primary infection, reinfection with a second strain, or reactivation of BKV in disease development is not known. Plasma DNAemia and urine viral load assays are promising tools that might be used to develop cost-effective algorithms to diagnose disease and to identify patients at high risk for developing BK nephropathy. The histopathology of BK infection in the kidney shares features with acute rejection. It is important to distinguish the two entities, because reducing immunosuppression may be the most appropriate management. Antiviral therapy is not yet available, although new agents such as cidofovir show promise. The disorder has a poor prognosis, with approximately 50% of allografts failing over subsequent months.

Measurement of human cytomegalovirus-associated DNA polymerase activity in patient urine as a potential diagnostic tool.

Virus-associated DNA polymerase activity has recently been proposed for the detection of human cytomegalovirus (HCMV) in urine, a method that should allow rapid and quantitative determination of the viral load. In this report, the virus-associated DNA polymerase activity recovered from the urine of a group of patients shedding HCMV was measured using a poly(dA) oligo(dT) 12-18 synthetic template after polyethylene glycol precipitation of the virions. Detection of virus-associated DNA polymerase activity was compared to the classical methods most widely used to diagnose HCMV shedding in urines such as virus culture followed by indirect immunofluorescence and pp65 gene-specific polymerase chain reaction. Although less sensitive than the polymerase chain reaction and cross-reactive with other herpesvirus DNA polymerases, the activity measured in the urine samples was correlated with the number of positive nuclei found in shell vials (r = 0.89). The diagnostic threshold of the assay could be placed between 50 and 100 fluorescent nuclei per shell with a diagnostic sensitivity of 56%. Being simple and quantitative, the measurement of virus-associated DNA polymerase activity could be of value in some clinical conditions where it is necessary to assess viral load in urine. This method is proposed as an alternative to more laborious quantitative assays and to support qualitative polymerase chain reaction.