Screening for BKV-DNAEMIA after renal transplantation in a resource limited setting.

Costs may hinder the implementation of BK polyomavirus (BKV)-DNAemia screening in resource-limited kidney transplant (KT) centers. We analyzed data from two studies to assess the performance and potential cost saving of a dual-step screening strategy based on the use of a preliminary qualitative semi-nested PCR (snPCR) assay followed by BKV-DNAemia quantification after KT. In the preliminary study, in which 130 samples from 33 KT recipients were screened for BKV-DNAemia, the estimated positive and negative predictive values of snPCR, as compared to quantitative PCR (qPCR), were 88% and 99%, respectively. In the second study, which included 84 KT recipients, BKV-DNAemia was detected by snPCR in 28/472 (5.9%) samples and confirmed by qPCR in 26 samples of 21 (25%) subjects. No graft loss occurred among KT recipients who developed BKV-DNAemia. Cost analyses suggested that this strategy might be a cost saving alternative for BKV-DNAemia screening for some resource-limited settings.

Pre-Transplantation Assessment of BK Virus Serostatus: Significance, Current Methods, and Obstacles.

The immunosuppression required for graft tolerance in kidney transplant patients can trigger latent BK polyomavirus (BKPyV) reactivation, and the infection can progress to nephropathy and graft rejection. It has been suggested that pre-transplantation BKPyV serostatus in donors and recipients is a predictive marker for post-transplantation BKPyV replication. The fact that research laboratories have used many different assay techniques to determine BKPyV serostatus complicates these data analysis. Even studies based on the same technique differed in their standard controls choice, the antigenic structure type used for detection, and the cut-off for seropositivity. Here, we review the different BKPyV VP1 antigens types used for detection and consider the various BKPyV serostatus assay techniques' advantages and disadvantages. Lastly, we highlight the obstacles in the implementation of a consensual BKPyV serologic assay in clinics (e.g., the guidelines absence in this field).

The detection of BKPyV genotypes II and IV after renal transplantation as a simple tool for risk assessment for PyVAN and transplant outcome already at early stages of BKPyV reactivation.

BACKGROUND: After reactivation the BK-polyomavirus (BKPyV) associated nephropathy (PyVAN) is observed in 1-10% of renal transplant recipients, of which up to 80% undergo graft failure. BKPyV reactivation after renal transplantation was associated with donor-derived serotypes against which the recipient has no immunological protection. However, PyVAN risk assessment seroactivity testing is a time-consuming and cost intensive process. OBJECTIVES: Since BKPyV serotypes can be attributed to distinct genotypes I to IV, in the present study we retrospectively analyzed whether a simple PCR-based BKPyV genotyping assay might be a fast and inexpensive method to assess the risk for PyVAN and transplant outcome already at early stages of BKPyV reactivation. STUDY DESIGN: 56 patients who were renal transplanted and tested positive for BKPyV viremia were included into the study. The BKPyV-VP1-coding sequences were PCR-amplified, sequenced, and subjected to genotyping. For group specific analysis patients were grouped in genotype I (n = 46) and a second group including genotype II and IV (n = 10) and associated with their clinical outcomes. RESULTS: The most abundant genotype I was detected in 46 of 56 (82%) patients, however, in the genotype II and IV group PyVAN was twice as frequent as compared to the genotype I group 24 months after transplantation (8 of 10 (80%) vs. 17 of 46 (37%); p = 0.001). Accordingly, graft failure was significantly more frequent in the genotype II and IV group (3 of 10 (30%) vs. 2 of 46 (4%); p = 0.007). CONCLUSION: PCR-based BKPyV genotyping might represent a fast and inexpensive method to assess the risk for PyVAN and transplant outcome already at early stages of BKPyV reactivation even if matched samples of the donor are not available.

Comparative evaluation of laboratory developed real-time PCR assays and RealStar(®) BKV PCR Kit for quantitative detection of BK polyomavirus.

BACKGROUND: Quantitative, viral load monitoring for BK virus (BKV) by real-time PCR is an important tool in the management of polyomavirus associated nephropathy in renal transplant patients. However, variability in PCR results has been reported because of polymorphisms in viral genes among different subtypes of BKV, and lack of standardization of the PCR assays among different laboratories. In this study we have compared the performance of several laboratory developed PCR assays that target highly conserved regions of BKV genome with a commercially available, RealStar(®) BKV PCR Kit. METHOD: Three real-time PCR assays (i) VP1 assay: selected from the literature that targets the major capsid protein (VP1) gene (ii) VP1MOD assay: VP1 assay with a modified probe, and (iii) BKLTA assay: newly designed assay that targets the large T antigen gene were assessed in parallel, using controls and clinical specimens that were previously tested using RealStar(®) BKV PCR Kit (Altona Diagnostics GmbH, Hamburg, Germany). Nucleic acid from all samples were extracted using the QIA symphony virus/bacteria kit on an automated DNA extraction platform QIA symphony SP (Qiagen). Primer and probe concentration, and reaction conditions for laboratory developed assays were optimized and the limit of detection of different assays was determined. Positive control for laboratory developed BK assays was prepared through construction of a plasmid carrying respective amplicon sequences. RESULTS: The 95% detection limit of VP1, VP1MOD and BKLTA assays were 1.8×10(2), 3×10(3) and 3.5×10(2) genomic copies/ml, respectively, as determined by Probit regression analysis of data obtained by testing a dilution series of a titered patient specimen, using RealStar(®) BKV PCR Kit. The inter-assay and intra-assay, coefficient of variations of these assays using calibrated, plasmid standards were <1%. All assays, including the RealStar(®) BKV PCR assay, were highly specific when tested against a panel of external proficiency specimens containing both BK and JC viruses. All assays, except the VP1MOD assay determined BK viral load in proficiency specimens within the same log values. With reference to results obtained by RealStar(®) BKV PCR assay, the sensitivity and specificity of different assays tested in 116 serum specimens submitted for BK viral load assay were 91% and 97% for VP1 assay, 88% and 97% for VP1MOD assay, and 97% and 98% for BKLTA assay, respectively. BK Viral load in positive specimens determined by various assays was highly correlated (R(2)>0.97), based on linear regression analysis. CONCLUSIONS: The performance characteristics of the newly designed, BKLTA assay were highly comparable to RealStar(®) BKV PCR assay, and can be used for routine detection and viral load monitoring of BKV in a cost-effective manner.

A real time genotyping PCR assay for polyomavirus BK.

BACKGROUND: Polyomavirus BK (BKV) may cause nephropathy in renal transplant recipients and hemorrhagic cystitis in bone marrow recipients. We developed real-time PCRs (RT-PCR) to determine easily and rapidly the different BKV genotypes (BKGT) (I-IV). METHODS: On the VP1 gene a duplex of RT-PCRs was developed and validated to differentiate the four main BKGT. 212 BKV positive samples (21 plasma, 191 urine) were tested with these specific PCRs. Of these 212 samples, 55 PCR results were additionally confirmed by sequencing a VP1 gene fragment (nucleotide 1630-1956). RESULTS: For every genotype, a highly specific, precise and internally controlled assay was developed with a limit of detection of log 3 copies per ml. In 18 (8.5%) of these samples genotyping was not successful due to a low viral load. By sequence analysis, the genotype of 46 out of 55 and 2 out of 4 samples with double infection could be confirmed. CONCLUSIONS: This study describes RT-PCRs for detection of the main BKGT. It proved to be rapid, cheap and sensitive compared to sequencing. Double infections can also be detected. This method will be of value to investigate the role of BKV infection in relation to the genotype.

Clinical usefulness of BK virus plasma quantitative PCR to prevent BK virus associated nephropathy.

The present study investigated the clinical usefulness of plasma real-time polymerase chain reaction (PCR) (plasma-PCR) in the prevention of BK virus-associated nephropathy (BKVAN). First, we investigated the diagnostic value of plasma BK-PCR, urine BK-PCR, and urine cytology for the prediction of BKVAN retrospectively. Then we designed a prospective study of regular plasma-PCR monitoring and pre-emptive immunosuppression (IS) reduction based on the result. In the retrospective cohort, the prevalence of BKVAN was 3.7% (14/379) and the positive rate of decoy cells, urine-PCR (>1 × 10(10) copies/ml), and plasma-PCR (>1 × 10(4) copies/ml) was 18.6%, 11.1%, and 5.5%, respectively. Plasma-PCR was superior to urine-PCR or urine cytology in specificity and positive predictive value for detection of BKVAN. In prospective study, regular monitoring of plasma-PCR detected significant BKV viremia in 8.3% (12/145) and BKVAN in 1 patient (0.6%). After IS reduction, BKV viremia was eliminated in 91.6% (11/12) within 103 days (25-254). In patients with viremia, the frequency of acute rejection did not increase and allograft function did not differ significantly compared with those in patients without viremia during the first year post-transplant (P > 0.05, in both). Plasma-PCR is useful to predict an increased risk for BKVAN, and regular monitoring is effective to prevent the development of BKVAN.

Assessment of infection with polyomaviruses BKV, JCV and SV40 in different groups of Cuban individuals.

We investigated the frequency of BKV, JCV and SV40 reactivation in three groups of Cuban patients by multiplex nested PCR assay of 40 paraffin-embedded colorectal neoplasm tissues, 113 urine samples, and 125 plasma samples from 27 transplant recipients, and cerebrospinal fluid (CSF) from 67 HIV-1-infected individuals with central nervous system (CNS) disorders. None of these polyomaviruses were detected in colorectal neoplasms. JCV DNA was detected in 2 of 67 patients (2.9%) with CNS disorders, but neither BKV nor SV40 was identified. BKV was found in urine from 38.5% and 28.6% of adult and pediatric transplant recipients, respectively. In adult renal transplant recipients, excretion of BKV in urine was significantly associated with episodes of acute rejection (p=0.012) and with excretion of HCMV in urine (p= 0.008). In Cuba, the polyomaviruses studied here could not be related to colorectal neoplasms, and JCV was rarely detected in CSFs of HIV-1-infected individuals, whilst BKV reactivation was found to occur frequently in organ transplant recipients.

Detection of polyomavirus BK reactivation after renal transplantation using an intensive decoy cell surveillance program is cost-effective.

BACKGROUND: Reactivation of polyomavirus BK (BKV) after renal transplantation can lead to allograft dysfunction or loss with early detection improving outcomes. Current guidelines recommend quantitative polymerase chain reaction for surveillance; however, urinary decoy cell detection is a potentially cost-effective alternative. We present the outcomes from an early intensive BKV surveillance program using decoy cell detection for initial screening starting 2 weeks after transplantation. METHODS: Records for all recipients of kidney (n=211) or simultaneous kidney and pancreas (n=102) transplants performed over 2 years in a single center were reviewed. Follow-up was for a minimum of 1 year. Urine cytology screening was performed fortnightly from 0 to 3 months after transplantation, monthly from 3 to 6 months then every 2 months from 6 to 12 months. RESULTS: Decoy cell positivity occurred in 56 of 313 patients (17.9%) with sustained decoy cell positivity (≥2 positive urine samples >2 weeks apart) present in 32 patients (10.2%). Twenty-four patients (7.6%) became viremic and three patients (1%) developed polyoma virus nephropathy. The median time after transplantation until decoy cell positivity was 78 days, decreasing to 67 days for patients with sustained positivity and 57 days for patients who developed polyoma virus nephropathy. No grafts were lost due to BKV during the study period. Decoy cell screening resulted in savings of approximately £135,000 over 2 years, when compared with routine surveillance by quantitative polymerase chain reaction. CONCLUSIONS: Clinically significant BKV reactivation occurs early after transplantation and can be reliably detected by decoy cell screening. A surveillance strategy for detecting BKV reactivation based on urine cytology is cost-effective.

Inexpensive, universal serial bus-powered and fully portable lab-on-a-chip-based capillary electrophoresis instrument.

Capillary electrophoresis is a cornerstone of lab-on-a-chip (LOC) implementations for medical diagnostics. However, the infrastructure needed to operate electrophoretic LOC implementations tends to be large and expensive, hindering the development of portable or low-cost systems. A custom-designed and highly integrated microelectronic chip for high-voltage generation switching and interfacing is recently developed. Here, the authors integrate the microelectronic chip with a microfluidic chip, a solid-state laser, filter, lens and several dollars worth of electronic components to form an inexpensive and portable platform, which is the size of a mobile telephone. This compact system has such reduced power requirements that the complete platform can be operated using a universal serial bus link to a computer. It is believed that this system represents a significant advancement in practical LOC implementations for point-of-care medical diagnostics.

Case studies in cost effectiveness of molecular diagnostics for infectious diseases: pulmonary tuberculosis, enteroviral meningitis, and BK virus nephropathy.

Pathogen genome amplification is used to detect and identify microorganisms, assess response to therapy, and detect mutations associated with drug resistance. Nucleic acid amplification tests have been shown to be superior to conventional culture-based testing methods in many circumstances. However, the enthusiasm for the technology in clinical laboratories may be decreased by the practical considerations of cost, complexity of the technology, and lack of US Food and Drug Administration-approved tests. The impact of nucleic acid amplification tests on the diagnosis and management of patients with tuberculosis, enteroviral meningitis, and BK virus transplant nephropathy will be examined, with an emphasis on the potential for health care cost savings.

Identification of polyoma BK virus in kidney transplant recipients by shell vial cell culture assay and urine cytology.

Urine cytology often is used to identify BK virus in kidney transplant recipients in the cytology laboratory. To assess the usefulness of the shell vial cell culture assay to identify BK virus, urine samples from 42 kidney transplant recipients were tested by the urine cytology and shell vial cell culture assays. The shell vial cell culture assay is just as sensitive and specific as urine cytology for the identification of BK virus in kidney transplant recipients.

Comparative evaluation of three commercial systems for nucleic acid extraction from urine specimens.

A nucleic acid extraction system that can handle small numbers of specimens with a short test turnaround time and short hands-on time is desirable for emergent testing. We performed a comparative validation on three systems: the MagNA Pure compact system (Compact), the NucliSens miniMAG extraction instrument (miniMAG), and the BioRobot EZ1 system (EZ1). A total of 75 urine specimens submitted for polyomavirus BK virus detection were used. The human beta-actin gene was detected on 75 (100%), 75 (100%), and 72 (96%) nucleic acid extracts prepared by the miniMAG, EZ1, and Compact, respectively. The miniMAG produced the highest quantity of nucleic acids and the best precision among the three systems. The agreement rate was 100% for BKV detection on nucleic acid extracts prepared by the three extraction systems. When a full panel of specimens was run, the hands-on time and test turnaround time were 105.7 and 121.1 min for miniMAG, 6.1 and 22.6 min for EZ1, and 7.4 and 33.7 min for Compact, respectively. The EZ1 and Compact systems processed automatic nucleic acid extraction properly, providing a good solution to the need for sporadic but emergent specimen detection. The miniMAG yielded the highest quantity of nucleic acids, suggesting that this system would be the best for specimens containing a low number of microorganisms of interest.

Periodic assessment of urine and serum by cytology and molecular biology as a diagnostic tool for BK virus nephropathy in renal transplant patients.

OBJECTIVE: To investigate the significance of polyomavirus (PV) viruria and viremia by morphologic, immunohistochemical and molecular analysis (multiplex nested-polymerase chain reaction) in renal transplant patients. STUDY DESIGN: Urine (n=328), serum (n= 53) and renal biopsies (n=24) from renal transplant patients (n=106) were studied. RESULTS: Decoy cells were found in 53 samples (16%) from 19 patients (18%); viral DNA was amplified in all urinary samples and disclosed BK virus (BKV) (n=24), JC virus (JCV) (n=16), and JCV and BKV DNA (n=13). BKV was the prevailing genotype in patients with a high frequency of decoy cell excretion (p = 0.001). JCV excretion correlated with a low number (p = 0.01) and BKV with a high number of decoy cells (p=0.003). PV DNA was amplified from 30/53 serum samples (56.6%); BKV was the prevailing genotype (p = 0.04). On 24 renal biopsies (18 from the decoy cell-negative and 6 from the decoy cell-positive group) PV nephropathy (PVN) was identified and BKV DNA amplified in 4 biopsies, all from the group with a high frequency of decoy cell excretion. PVN was not identified in renal biopsies from the decoy cell-negative group. CONCLUSION: PV infection is frequent in renal transplant patients. The BKV genotype in urine and serum is significantly related to a high frequency and high number of decoy cells. PVN occurs only in patients with BKV viremia and a high number and frequency of decoy cell excretion in urine. In the absence of decoy cells, PVN can be excluded. Cytologic analysis of urine is an important diagnostic tool for screening renal transplant patients at risk of PVN.

Multiplex polymerase chain reaction for the simultaneous detection and typing of polyomavirus JC, BK and SV40 DNA in clinical samples.

A novel multiplex nested PCR (nPCR) method was developed for detecting and differentiating simultaneously the DNA of polyomaviruses JC, BK and SV40 in a single tube. In the first amplification step the same set of primers were used to amplify a conserved DNA region of the large T antigen gene of JCV, BKV and SV40. The second round of multiplex nPCR was carried out using a set of primers designed to render products of different size for each related virus. The thermocycling parameters and concentration of each reaction component were optimised systematically to achieve optimal specificity and sensitivity for the nPCR assay. The sensitivity of the method ranged between one and 10 copies of polyomavirus genome. Cerebrospinal fluid (CSF) was examined from AIDS patients with clinical and neuroradiological evidence of progressive multifocal leukoencephalopathy (PML) and CSF from AIDS patients with other neurological alterations. Urine specimens from bone marrow transplant recipients affected by haemorrhagic cystitis were also tested. The results obtained suggest that the assay is a good tool for supporting the diagnosis of polyomavirus infection and could be used for epidemiological purposes and in other studies in order to define better the role of polyomaviruses in human disease.