Keith R. Jerome

Quantitation of BK Virus Load in Serum for the Diagnosis of BK Virus–Associated Nephropathy in Renal Transplant Recipients

BK virus-associated nephropathy is an increasingly recognized cause of graft dysfunction among kidney transplant recipients, and definitive diagnosis requires renal biopsy. By using a newly developed, quantitative, real-time polymerase chain reaction (PCR) assay for BK virus DNA, a retrospective analysis was done of sequential serum samples (n=28) from 4 transplant recipients with histopathologically documented BK virus nephropathy and from samples (n=76) from 16 transplant recipient control patients. BK virus DNA was detected in serum samples from all 4 case patients versus 0 of 16 control patients (P< .0001, Fishers exact test) at a median of 32 weeks (range, 17-61 weeks) before the diagnosis of BK virus nephropathy. BK virus load decreased in 3 of 3 patients after the reduction of immunosuppression and/or nephrectomy. It is concluded that quantitative PCR for BK virus DNA in serum is useful both for identifying transplant recipients at risk for BK virus nephropathy and for monitoring the response to therapy.

Quantitative Stability of DNA after Extended Storage of Clinical Specimens as Determined by Real-Time PCR

ABSTRACT Viral DNA stored for extended periods can be amplified by PCR. However, it is unknown whether stored specimens give accurate quantitative results by newer real-time PCR techniques. We therefore compared herpes simplex virus DNA levels in specimens before and after 16 months of storage. The levels of viral DNA remained stable whether the DNA was stored as purified DNA or unextracted DNA in a whole specimen.

Marked Variability of BK Virus Load Measurement Using Quantitative Real-Time PCR among Commonly Used Assays

ABSTRACT BK virus (BKV) is the infectious cause of polyomavirus-associated nephropathy. Screening guidelines for renal-transplant recipients define levels of viremia and viruria that are actionable for additional testing or intervention. However, standardized real-time PCR primers, probes, and standards are unavailable, and the extent of agreement among published assays is unknown. We compared seven TaqMan real-time PCR primer/probe sets (three designed at this institution, three described in the literature, and one purchased) in conjunction with two different standards to prospectively measure BKV titers in 251 urine specimens submitted to our clinical laboratory. We observed substantial disagreement among assays attributable both to features of primer and probe design and to choice of reference material. The most significant source of error among individual specimens was primer or probe mismatch due to subtype-associated polymorphisms, primarily among subtype III and IV isolates. In contrast, measurement of the most abundant subtypes (Ia, V, and VI) were typically uniform among all seven assays. Finally, we describe and validate a new clinical assay designed to reliably measure all subtypes encountered in our study population (Ia, Ic, III, IV, and VI). Consideration of available BKV sequence information in conjunction with details of subtype distribution allowed us to develop a redesigned assay with markedly improved performance. These results suggest that both accurate BKV measurement and the uniform application of BKV screening guidelines could be significantly improved by the use of standardized reference materials and PCR primers and probes.

Polyomavirus nephropathy in native kidneys of non-renal transplant recipients.

Chronic renal dysfunction is common in non-renal solid organ (SOT) and hematopoietic stem cell transplant (HSCT) recipients and is commonly attributed to calcineurin inhibitor toxicity, often without renal histopathologic evaluation. Polyomavirus nephropathy (PVN) is an important cause of allograft dysfunction in kidney transplant recipients but has rarely been reported in native kidneys of non-renal transplant recipients. We report the clinical, pathologic and virologic features of PVN in native kidneys of two allograft recipients. In both, severe renal dysfunction was accompanied by histopathologic evidence of PVN, including characteristic viral inclusions by routine stains, immunohistochemistry and electron microscopy. High levels of BK virus (BKV) DNA were detected in kidney tissue of patients using BKV-specific polymerase chain reaction (PCR). In 1 patient, high levels of BKV DNA were detected in plasma and urine, and administration of low-dose cidofovir was associated with clearance of BK viremia. These results extend the populations in which PVN has been documented in native kidneys to include heart and stem cell transplant recipients, and suggest that cidofovir has activity against BKV in vivo. Studies to define the incidence and potential contribution of PVN to chronic renal dysfunction commonly attributed to calcineurin inhibitor toxicity in non-renal transplant recipients are warranted.

Ontogeny and Specificities of Mucosal and Blood Human Immunodeficiency Virus Type 1-Specific CD8+ Cytotoxic T Lymphocytes

ABSTRACT Induction of adaptive immunity to human immunodeficiency virus type 1 (HIV-1) at the mucosal site of transmission is poorly understood but crucial in devising strategies to control and prevent infection. To gain further understanding of HIV-1-specific T-cell mucosal immunity, we established HIV-1-specific CD8+ cytotoxic T-lymphocyte (CTL) cell lines and clones from the blood, cervix, rectum, and semen of 12 HIV-1-infected individuals and compared their specificities, cytolytic function, and T-cell receptor (TCR) clonotypes. Blood and mucosal CD8+ CTL had common HIV-1 epitope specificities and major histocompatibility complex restriction patterns. Moreover, both systemic and mucosal CTL lysed targets with similar efficiency, primarily through the perforin-dependent pathway in in vitro studies. Sequence analysis of the TCRβ VDJ region revealed in some cases identical HIV-1-specific CTL clones in different compartments in the same HIV-1-infected individual. These results clearly establish that a subset of blood and mucosal HIV-1-specific CTL can have a common origin and can traffic between anatomically distinct compartments. Thus, these effectors can provide immune surveillance at the mucosa, where rapid responses are needed to contain HIV-1 infection.

Valganciclovir for the Prevention of Complications of Late Cytomegalovirus Infection After Allogeneic Hematopoietic Cell Transplantation: A Randomized Trial

Editors Notes Context Cytomegalovirus (CMV) disease is a late complication of allogeneic hematopoietic cell transplantation (HCT). Contribution In a multicenter, randomized, double-blind trial of patients who had HCT and were at high risk for late CMV disease, continuous valganciclovir prophylaxis was compared with a strategy of CMV polymerase chain reactionguided preemptive therapy with valganciclovir and ganciclovir. These 2 approaches did not differ in the occurrence of death, CMV disease, or other invasive fungal infections. Safety was similar. Caution More patients who received continuous valganciclovir required hematopoietic growth factors. Implication Both continuous valganciclovir prophylaxis and CMV polymerase chain reactionguided preemptive therapy are viable alternatives to prevent late CMV disease after HCT. Ganciclovir effectively prevents cytomegalovirus (CMV) disease during the first 3 months after hematopoietic cell transplantation (HCT) when given prophylactically at engraftment or preemptively for pp65 antigenemia or detection of CMV DNA by polymerase chain reaction (PCR). It improves survival in selected high-risk patients (1, 2). However, most cases of CMV disease now occur after withdrawal of ganciclovir (37), most frequently between 100 and 270 days after transplantation (3). In the absence of preventive strategies, both late CMV infection and disease are independent predictors for death after HCT (3). Although preemptive therapy based on virologic surveillance is the most commonly used strategy to prevent CMV disease during the first 3 months after HCT (8), maintaining surveillance is often difficult late after HCT because patients often return to remote locations and regular blood draws may be difficult to perform. The rationale for studying a prophylactic approach is supported by the observation that even asymptomatic CMV infection is associated with increased mortality rates, suggesting a role of indirect effects of CMV in the late period (3). However, the benefits of ganciclovir and valganciclovir prophylaxis are theoretically counterbalanced by their most common toxicity (neutropenia), which is also independently associated with death early after HCT (9, 10). Methods Design This was an investigator-initiated, multicenter, double-blind, placebo-controlled, randomized clinical trial. The Fred Hutchinson Cancer Research Center (FHCRC) held the Investigational New Drug application and served as the coordinating center. Patients were randomly assigned to receive valganciclovir (900 mg once per day) or matching placebo (Appendix Figure 1) between 1999 and 2008. Study patients, study personnel, and all clinical personnel were blinded. The study drug was withdrawn when CMV viral load was greater than 1000 copies/mL or greater than 5 times the baseline value, and preemptive therapy was started with intravenous ganciclovir (5 mg/kg twice daily) or valganciclovir (900 mg twice daily); foscarnet (90 mg/kg twice daily) was used instead if indicated due to neutropenia. All doses were adjusted to the creatinine clearance as per manufacturer recommendations. Weekly study samples were mailed to Seattle, Washington, and tested at the University of Washington clinical laboratories. Cytomegalovirus and chemistry testing results were made available in real time to study sites to allow initiation of open-label preemptive treatment of CMV disease, dose adjustment, drug withdrawal, or start of hematopoietic growth factors as prespecified in the protocol. The study drug was held and growth factors were started if the absolute neutrophil count decreased to less than 1.0109 cells/L. Appendix Figure 1. Study design. CMV = cytomegalovirus; HCT = hematopoietic cell transplantation. A CMV DNA level greater than 1000 copies/mL or consecutive positive results with increasing levels was used to withdraw the study drug and start preemptive treatment with open-label intravenous ganciclovir treatment. The protocol was amended halfway through the study to make valganciclovir open-label treatment available to patients for breakthrough preemptive therapy (instead of requiring intravenous treatment) and to formally include granulocyte colony-stimulating factor (G-CSF) treatment of neutropenia at an absolute neutrophil count of 1.0109 cells/L. Setting and Patients Allogeneic HCT recipients aged 16 years or older who were seropositive for CMV before transplant or had a seropositive donor were eligible. Seropositive recipients had to have either CMV infection with appropriate treatment course before random assignment; a history of graft-versus-host disease (GVHD) after transplantation requiring treatment with systemic corticosteroids at doses greater than 0.5 mg/kg at any time before enrollment; chronic, clinically extensive GVHD requiring treatment with corticosteroids; or receipt of ganciclovir, valganciclovir, foscarnet, or cidofovir prophylaxis between engraftment and random assignment. Seronegative recipients with seropositive donors had to have a CMV infection with appropriate treatment course before random assignment. A complete listing of inclusion and exclusion criteria is shown in Appendix 1. Randomization and Interventions Randomization occurred once patients were identified as eligible for the study at a median of 97 and 98 days after HCT for the valganciclovir and placebo groups, respectively (Table 1). We used an adaptive randomization scheme implemented using a statistical program written by an FHCRC statistician and run by staff of the FHCRC protocol office. Randomization was stratified by study site, previous neutropenia (presence or absence of absolute neutrophil count <1.0109 cells/L after initial engraftment), and presence or absence of refractory GVHD (requiring secondary therapy) at study enrollment (11, 12). Table 1. Demographic Characteristics of the Study Cohort, by Group* Outcomes and Follow-up The primary end point was a composite outcome consisting of CMV disease, invasive bacterial or fungal infections, or death (whichever occurred first), thereby assessing the net effect of the strategy, including consequences of neutropenia (9). Because most cases of late CMV disease occur between 4 and 9 months after HCT (35), the primary study period for the intervention was until 270 days after HCT. Follow-up was extended until 640 days after HCT for CMV disease, death, and CMV-specific immune reconstitution. Secondary end points were CMV disease (defined as per international guidelines [13]), death, invasive bacterial or fungal infections (defined as published [1, 14]) (Appendix 2), use of invasive tests (such as endoscopy procedures, including bronchoscopies), number of days alive without hospitalization during the active study period, treatment-emergent ganciclovir resistance, quality of life (QOL) as determined by the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire 30 (EORTC QLQ-30) (15), herpesvirus-specific T-cell function, and adverse events (clinical and laboratory). Laboratory Methods Cytomegalovirus viral load was determined at the University of Washington using a validated laboratory-developed quantitative PCR method; the assays conversion factor to IU/mL is 1.0 (16). Cytomegalovirus-specific T-cell function was determined by a multicolor flow cytometry assay designed to detect polyfunctional CD4+ and CD8+ T cells (17, 18). Ganciclovir resistance was examined by testing patients with persistent or increasing viral load (>1000 copies/mL) while receiving open-label treatment for mutations in the UL97 gene, using a rapid PCR- and sequencing-based assay as previously described (19). Safety laboratory testing (serum creatinine, alanine aminotransferase, blood differential, and platelet count) was done using established methods and limits for normal or abnormal at the University of Washington clinical laboratories. Statistical Analysis The frequency of the primary composite end point was 53% in our previous cohort (3). Our study was designed to test the superiority of the prophylactic strategy. To demonstrate a 45% reduction of the primary end point (which was deemed clinically meaningful), 184 randomly assigned patients (92 per treatment group) were needed to provide approximately 87% power (allowing for 1 interim analysis at the 0.005 level and a final analysis at the 0.048 level). All statistical tests were 2-sided. All patients who were randomly assigned and received at least 1 dose of study medication were included in the analysis. Descriptive statistics were used to summarize demographic and baseline characteristics of study patients. Comparisons between study groups of time to the primary end point from HCT to 270 days after HCT were evaluated using Cox regression models adjusted for the stratification factors of neutropenia presence before random assignment or absence of an absolute neutrophil count less than 1.0109 cells/L after initial engraftment and study site (FHCRC vs. other site). The number of patients with previous refractory GVHD was not sufficient to include this stratification factor. Secondary time-to-event outcomes were evaluated similarly; some outcomes were evaluated to 640 days, as noted in Table 2. Cumulative incidence curves were also used to evaluate time-to-event outcomes, with death as a competing risk for all outcomes except mortality (20). All time-to-event analyses were censored at the time of last contact in the absence of a competing risk or event of interest. Absolute differences and associated 95% CIs were evaluated using standard methods for risk differences for proportions (21), but SEs of cumulative incidence were used to construct CIs for cumulative incidence. Differences in medians with CIs were evaluated using HodgesLehmann estimation (22). Other secondary outcomes were compared between study groups with chi-square, Fisher exact, or Wilcoxon rank-sum tests, as appropriate. Subset analyses of efficacy and neutropenia en

Use of the MagNA Pure LC Automated Nucleic Acid Extraction System followed by Real-Time Reverse Transcription-PCR for Ultrasensitive Quantitation of Hepatitis C Virus RNA

ABSTRACT Hepatitis C virus (HCV) infection is an increasing health problem worldwide. Quantitative assays for HCV viral load are valuable in predicting response to therapy and for following treatment efficacy. Unfortunately, most quantitative tests for HCV RNA are limited by poor sensitivity. We have developed a convenient, highly sensitive real-time reverse transcription-PCR assay for HCV RNA. The assay amplifies a portion of the 5′ untranslated region of HCV, which is then quantitated using the TaqMan 7700 detection system. Extraction of viral RNA for our assay is fully automated with the MagNA Pure LC extraction system (Roche). Our assay has a 100% detection rate for samples containing 50 IU of HCV RNA/ml and is linear up to viral loads of at least 109 IU/ml. The assay detects genotypes 1a, 2a, and 3a with equal efficiency. Quantitative results by our assay correlate well with HCV viral load as determined by the Bayer VERSANT HCV RNA 3.0 bDNA assay. In clinical use, our assay is highly reproducible, with high and low control specimens showing a coefficient of variation for the logarithmic result of 2.8 and 7.0%, respectively. The combination of reproducibility, extreme sensitivity, and ease of performance makes this assay an attractive option for routine HCV viral load testing.

Inhibition of TCR Signaling by Herpes Simplex Virus

T lymphocytes are an essential component of the immune response against HSV infection. We previously reported that T cells became functionally impaired or inactivated after contacting HSV-infected fibroblasts. In our current study, we investigate the mechanisms of inactivation. We report that HSV-infected fibroblasts or HSV alone can inactivate T cells by profoundly inhibiting TCR signal transduction. Inactivation requires HSV penetration into T cells but not de novo transcription or translation. In HSV-inactivated T cells stimulated through the TCR, phosphorylation of Zap70 occurs normally. However, TCR signaling is inhibited at linker for activation of T cells (LAT) and at steps distal to LAT in the TCR signal cascade including inhibition of calcium flux and inhibition of multiple MAPK. Inactivation of T cells by HSV leads to the reduced phosphorylation of LAT at tyrosine residues critical for TCR signal propagation. Treatment of T cells with tyrosine phosphatase inhibitors attenuates inactivation by HSV, and stimulus with a mitogen that bypasses LAT phosphorylation overcomes inactivation. Our findings elucidate a potentially novel method of viral immune evasion that could be exploited to better manage HSV infection, aid in vaccine design, or allow targeted manipulation of T cell function.

CTL Are Inactivated by Herpes Simplex Virus-Infected Cells Expressing a Viral Protein Kinase

Numerous cell-to-cell signals tightly regulate CTL function. Human fibroblasts infected with HSV type 1 or 2 can generate such a signal and inactivate human CTL. Inactivated CTL lose their ability to release cytotoxic granules and synthesize cytokines when triggered through the TCR. Inactivation requires cell-to-cell contact between CTL and HSV-infected cells. However, inactivated CTL are not infected with HSV. The inactivation of CTL is sustainable, as CTL function remains impaired when the CTL are removed from the HSV-infected cells. IL-2 treatment does not alter inactivation, and the inactivated phenotype is not transferable between CTL, distinguishing this phenotype from traditional anergy and T regulatory cell models. CTL inactivated by HSV-infected cells are not apoptotic, and the inactivated state can be overcome by phorbol ester stimulation, suggesting that inactivated CTL are viable and that the signaling block is specific to the TCR. HSV-infected cells require the expression of US3, a viral protein kinase, to transmit the inactivating signal. Elucidation of the molecular nature of this signaling pathway may allow targeted manipulation of CTL function.

Measurement of CTL-induced cytotoxicity: The caspase 3 assay

Cytotoxic T lymphocytes (CTL) are critical effector cells of the immune system. Measurement of target cell damage has historically been an important measure of CTL function. CTL kill their target cells predominantly by inducing programmed cell death, or apoptosis. The gold standard for CTL-mediated cytotoxicity has been the 51Cr release assay. However, measurement of target cell lysis by 51Cr release does not provide mechanistic information on the fate of target cells, especially at the single cell level. Given the recent advances in our understanding of programmed cell death, newer assays are required which evaluate the status of the apoptotic pathways in target cells. We have developed a flow cytometry-based assay for CTL-mediated cytotoxicity based on specific binding of antibody to activated caspase 3 in target cells. Our assay is convenient and more sensitive than the 51Cr release assay. The use of this assay should allow mechanistic studies of the intracellular events resulting from CTL attack.