Predictive value of JC virus PCR in cerebrospinal fluid in the diagnosis of PML.

OBJECTIVE: To assess the predictive value of JC virus (JCV) PCR in cerebrospinal fluid (CSF) in the diagnosis of progressive multifocal leukoencephalopathy (PML). METHODS: We conducted a retrospective database query to identify patients with positive CSF JCV PCR. Clinical features, final diagnosis and quantitative PCR results were obtained. RESULTS: A positive CSF JCV PCR had a PPV of 10.4% for the diagnosis of PML. A weakly positive PCR had a PPV of 1.6%, whereas a moderately to highly positive PCR had a PPV of 92.3%. A PPV of 0.0% was observed in immunocompetent patients and in patients without compatible clinical or radiological features. CONCLUSIONS: A false-positive CSF JCV PCR is highly prevalent in our clinical practice. This test should be reserved for patients with a clinical suspicion of PML and the quantitative result of the PCR should be taken into account when making the diagnosis of PML.

Not a Disease of the Past: A Case Series of Progressive Multifocal Leukoencephalopathy in the Established Antiretroviral Era.

Progressive multifocal leukoencephalopathy (PML) and PML immune reconstitution inflammatory syndrome (PML-IRIS) can be devastating neurological processes associated with HIV, but limited knowledge of their characteristics in the established antiretroviral therapy (ART) era is available. We conducted a case series to evaluate the clinical course of PML and PML-IRIS at our urban safety-net hospital in Atlanta, GA. All HIV-positive individuals with a positive John Cunningham virus DNA polymerase chain reaction in the spinal fluid between May 1, 2013 to June 1, 2017 were identified from the electronic health records (EHRs) using the HIV Disease Registry. Demographics, symptom presentation, laboratory data, imaging results, treatment, and outcomes were abstracted from the EHR. PML and PML-IRIS were defined using the American Association of Neurology criteria. Of the 32 individuals identified, 6 (19%) were felt to have asymptomatic positive results. Of the remainder, 15 (58%) HIV-positive patients had PML and 11 (42%) PML-IRIS (2 with an unmasking presentation and 9 with a paradoxical presentation). The most common presenting symptoms were motor weakness (18, 69%), cognitive deficits (15, 58%), and dysarthria (11, 42%). Corticosteroids were used in 12 patients and maraviroc in 3 patients. Outcomes were dismal with 7 (47%) patients with PML and 9 (82%) with PML-IRIS dying or being referred to hospice, with median survival times of 266 days in the PML group and 109 days in the PML-IRIS group. Despite widespread access to ART, patients with PML continue to have poor outcomes, particularly among those who develop PML-IRIS. More research is needed to understand the risks for and prevention of PML-IRIS.

BK polyomavirus encephalitis in a patient with thrombotic microangiopathy after an allogeneic hematopoietic stem cell transplant.

To date, only one case of BK polyomavirus (BKPyV) encephalitis combined with transplant-associated thrombotic microangiopathy has been reported in an hematopoietic stem cell transplantation (HCT) recipient. We report the case of an HCT recipient who developed thrombotic microangiopathy and subsequent BKPyV encephalitis. She died despite treatment with cidofovir, ciprofloxacin, and intravenous immunoglobulin without improvement in mental status. Early suspicion of BKPyV encephalitis in an HCT recipient presenting with altered mental status and hemorrhagic cystitis is important.

Characteristics of progressive multifocal leukoencephalopathy clarified through internet-assisted laboratory surveillance in Japan.

BACKGROUND: Progressive multifocal leukoencephalopathy (PML), a rare but fatal demyelinating disease caused by JC virus (JCV), occurs mainly in immunocompromised patients. As PML develops in individuals with various underlying disorders sporadically and infrequently, a nationwide survey of PML is difficult. This study was conducted to elucidate the characteristics of PML in Japan through an internet-assisted laboratory surveillance program. METHODS: A diagnostic support system for PML was established using a real-time PCR assay of JCV DNA in cerebrospinal fluid (CSF), and requests for testing were received from clinicians via specialized websites. Medical histories of patients were collected through standardized questionnaires, and a database of CSF JCV loads and clinical information was created and analyzed. RESULTS: For 4 years from April 2007 to March 2011, CSF specimens from 419 patients were tested. Forty-eight individuals were found positive for JCV DNA in their CSF and were diagnosed with PML. PML primarily occurred not only in HIV-positive patients (33.3%) but also in patients with hematologic disorders after receiving stem cell transplantation, chemotherapy, and/or immunosuppressive treatment (39.6%). The frequencies of PML cases among the subjects in these two categories were 20.3% and 23.5%, respectively. Although no significant features were observed with respect to CSF JCV loads in PML patients with an HIV infection or hematologic disorder, males were predominant in both groups (100% and 89.5%, respectively). The proportion of PML cases with autoimmune disorders (6.3%) or solid-organ transplants (2.1%) was smaller than those with HIV infection or hematologic disorders, probably due to the limited availability of therapeutic monoclonal antibodies and transplantation from brain dead donors. CONCLUSIONS: The results suggest that the internet-assisted laboratory surveillance program might be a useful strategy for collecting precise real-time information on PML on a national level. The current database provides important background information for the diagnosis and treatment of patients with risk factors for PML.

BK polyomavirus with archetypal and rearranged non-coding control regions is present in cerebrospinal fluids from patients with neurological complications.

BK polyomavirus (BKPyV) has recently been postulated as an emerging opportunistic pathogen of the human central nervous system (CNS), but it is not known whether specific strains are associated with the neurotropic character of BKPyV. The presence of BKPyV large T-antigen DNA was examined in 2406 cerebrospinal fluid (CSF) samples from neurological patients with suspected JC polyomavirus infection. Twenty patients had a large T-antigen DNA-positive specimen. The non-coding control region (NCCR) of the BKPyV strains amplified from CSF from these 20 patients, strains circulating in renal and bone marrow transplant recipients and from healthy pregnant women was sequenced. The archetypal conformation was the most prevalent in all groups and 14 of the neurological patients harboured archetypal strains, while the remaining six patients possessed BKPyV with rearranged NCCR similar to previously reported variants from non-neurological patients. Transfection studies in Vero cells revealed that five of six early and four of six late rearranged promoters of these CSF isolates showed significantly higher activity than the corresponding archetypal promoter. From seven of the neurological patients with BKPyV DNA-positive CSF, paired serum samples were available. Five of them were negative for BKPyV DNA, while serum from the remaining two patients harboured BKPyV strains with archetypal NCCR that differed from those present in their CSF. Our results suggest that NCCR rearrangements are not a hallmark for BKPyV neurotropism and the dissemination of a rearranged NCCR from the blood may not be the origin of BKPyV CNS infection.

BK virus-associated infection in cerebrospinal fluid of neurological patients and mutation analysis of the complete VP1 gene in different patient groups.

While BK virus (BKV) is frequently associated with pathological conditions in bone marrow and renal transplant recipients, BKV infection in neurological individuals has been rarely reported. As a result of a BKV, JCV, and SV40 large T antigen-specific multiplex PCR on 2,062 cerebrospinal fluid (CSF) samples from neurological patients suspicious of JCV infection, we identified 20 subjects with at least 1 CSF specimen positive for BKV large T antigen DNA. Because VP1 protein has been suggested to influence the biological/pathological properties of BKV, we tried to sequence the entire VP1 gene in the BKV-positive neurological patients and succeeded in 14 of the 20 neurological patients. To compare the VP1 sequence of the BKV neurological strains with that of non-neurotropic strains in other clinical situations, full-length VP1 DNA was sequenced in 15 renal and 6 bone marrow transplant recipients positive to BKV-viremia, and in 8 pregnant women as non-pathological controls. An increased (respectively, decreased) tendency for mutations in the BC loop (respectively, EF loop) was observed, and no mutations were detected in the CD, GH, and HI loops. Subtype I was predominant (93%) and compared to archetypal BKV (WW), amino acid substitutions were detected in 4/14 neurological patients, 10/15 renal transplant recipients, 3/6 bone marrow transplant patients, and in all the pregnant women. Each patient group had distinctive VP1 mutations, but these unique substitutions were not present in all patients of this group. However, molecular modeling simulations of the VP1 mutants predicted changes in protein surface properties which might affect the VP1-receptor interaction.

Progressive multifocal leukoencephalopathy in a patient with B-cell lymphoma during rituximab-containing chemotherapy: case report and review of the literature.

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system caused by the JC polyomavirus. We describe a rare case of PML in a 48-year-old female patient with diffuse large B-cell lymphoma who received rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP) therapy. While she was undergoing five cycles of R-CHOP, she noticed gradually progressive neurological symptoms, such as slurred speech and gait disturbance, and she eventually developed high-grade fever. She also developed Pneumocystis jiroveci pneumonia. The neurological symptoms deteriorated thereafter, and she developed spastic quadriparesis and bulbar palsy. Magnetic resonance imaging showed hyperintensity within the right cerebellar hemisphere on T2-weighted images. Polymerase chain reaction-based tests of the cerebrospinal fluid revealed the presence of the JC virus. Despite intravenous and intrathecal cytarabine treatment, the patient died of PML 5 months after it was diagnosed. Retrospective analysis of her laboratory data showed that her CD4(+) T-cell count before R-CHOP therapy had decreased to 68 microL(-1). Thus, when administering rituximab-containing chemotherapy, even to patients with no prior history of opportunistic infections, attention should be paid to the potential occurrence of PML, particularly in patients with low CD4(+) T-cell counts.

JC virus in cerebrospinal fluid samples of multiple sclerosis patients at the first demyelinating event.

OBJECTIVE: To evaluate the possible involvement of JC virus (JCV) in the aetiology of multiple sclerosis (MS), through the comparison of DNA prevalences and viral loads of JCV in cerebrospinal fluid (CSF) of MS patients at the first demyelinating event and subjects suffering from other neurological diseases (OND). METHODS: Seventy-three CSF samples (43 from MS patients at the first demyelinating event, and 30 from patients with OND) were collected; all MS cases were followed up from 1 to 6.7 years after they were diagnosed with clinically definite MS. DNA was extracted and analysed by real-time PCR for the detection of JCV genomes. RESULTS: We found JCV DNA in the CSF of two MS patients (4.7%) with a mean viral load of 2.1 and 6.7 copies/mL of CSF. Among the patients of the OND group we did not find any positive sample. We did not find any difference in the course of the disease between MS patients with and without JCV genomes in their CSF along the follow up. CONCLUSION: JCV seems to be only a bystander in the pathology of MS, and the presence of cell-free viral particles could be related to the immunological activation of the disease, mainly during relapses.

New commercially available PCR and microplate hybridization assay for detection and differentiation of human polyomaviruses JC and BK in cerebrospinal fluid, serum, and urine samples.

JC and BK human polyomaviruses (family Polyomaviridae) may cause severe neurological or urinary tract pathologies in immunocompromised hosts. In the present study, we evaluated a new commercially available PCR and microplate colorimetric hybridization assay for the standardized differential detection of JC virus (JCV) and BK virus (BKV) genomes in clinical samples. This JC/BK Consensus test was first evaluated by testing serial dilutions of JCV or BKV plasmid DNA standards and was then compared with an in-house reference PCR assay for the detection of JC and BK virus genomes in 70 cerebrospinal fluid (CSF) samples of patients with neurological disorders and in 75 serum or plasma samples and 125 urine samples of renal graft recipients. This new test allowed a limit of detection of 10 copies and 1 copy of JC and BK virus genomes, respectively, and was able to differentiate various levels of JCV, BKV, and mixed JCV and BKV DNA genomes in a single reaction tube. Our results showed 100% specificity and sensitivity for the JC/BK Consensus test with CSF samples. With serum or plasma samples, this test had a sensitivity and a specificity of 100% for both JCV and mixed JCV and BKV DNA detection and a sensitivity and a specificity of 100 and 97.8% for BKV DNA detection, respectively. With urine samples, the sensitivity and specificity were 100 and 96.6%, respectively, for JCV DNA detection; 100 and 89.4%, respectively, for BKV DNA detection; and 44.4 and 100%, respectively, for mixed JCV and BKV DNA detection. In conclusion, our data indicate that this new test, the JC/BK Consensus test, is valuable for the sensitive and specific differential detection of single JCV and BKV infections in CSF, serum or plasma, and urine samples. The use of this reliable PCR assay would improve the routine virological diagnosis as well as the clinical care of immunocompromised patients with polyomavirus-related pathologies.

Comparison of PCR-southern hybridization and quantitative real-time PCR for the detection of JC and BK viral nucleotide sequences in urine and cerebrospinal fluid.

The presence of the human polyomaviruses JCV and BKV in immunocompromised patients can lead to lethal diseases and conditions including progressive multifocal leukoencephalopathy (PML), interstitial nephritis, hemorrhagic cystitis, and kidney allograft rejection. Typically, detection of JCV and BKV in clinical samples has employed standard PCR amplification for viral nucleotide sequences, with subsequent confirmation for viral genome specificity of PCR products by southern blot hybridization. Here, we directly tested a validated PCR-southern protocol with a TaqMan real-time PCR protocol (Applied Biosystems) to assay clinical samples of urine and cerebrospinal fluid. We found equal specificity and sensitivity with both methods. However, real-time allowed for absolute viral-genome quantitation without the use of radionucleotides and was performed more rapidly, in as little as 24 h. Such advantages are important to consider in the effort to establish international standardization of controls for the detection of JCV and BKV, which would aid in screening confidence and the reliable assessment of anti-viral therapies.

Detection of the archetypal regulatory region of JC virus from the tonsil tissue of patients with tonsillitis and tonsilar hypertrophy.

The regulatory regions of JC virus (JCV) DNAs in the brain of patients with progressive multifocal leukoencephalopathy (PML) (designated as PML-type regulatory regions) are hypervariable, whereas those in the urine and renal tissue of individuals without PML have the same basic structure, designated as the archetype. It is thought that JCV strains with the archetypal regulatory region circulate in the human population. Nevertheless, Monaco et al (J Virol 70: 7004-7012, 1996) reported that PML-type regulatory regions occur in human tonsil tissue. The purpose of this study is to confirm their findings. Using nested polymerase chain reaction (PCR), the authors detected the regulatory region of JCV DNA in the tonsil tissue from 14 (44%) of 32 donors with tonsillitis and tonsilar hypertrophy. Sequencing of the detected regulatory regions indicated that they were identical with the archetypal regulatory regions detected previously or, in a few cases, slightly deviated from the archetype. This finding suggests not only that tonsil tissue is the potential site of initial JCV infection but also that archetypal JCV strains circulate in the human population.

BK virus regulatory region rearrangements in brain and cerebrospinal fluid from a leukemia patient with tubulointerstitial nephritis and meningoencephalitis.

BK virus (BKV) was recovered by polymerase chain reaction (PCR) from brain, kidney, lung, urine, and cerebrospinal fluid (CSF) of a fatal case of BKV tubulointerstitial nephritis with dissemination to lung and brain. Viral regulatory regions in PCR-amplified urine and the lung samples were identical to the archetypal structure, WWT. In the brain and CSF, a rearranged sequence predominated, however. A 94-bp deletion preceded a 71-bp tandem duplication because the same 94-bp segment was deleted from both copies. PCR-amplified regulatory region products were cloned and sequenced to define further the extent of the rearranged structures. Two kidney clones were archetypal, whereas two others were rearranged differently from the brain and from each other. In contrast to the brain clones, the kidney rearrangements seemed to involve deletion after duplication. Three of four brain clones sequenced were identical to the rearrangement found to dominate in the PCR product. A fourth clone showed two short deletions without any duplication. The four CSF clones all showed rearrangements identical to that which was amplified by PCR from CSF and brain. This represents the first molecular analysis of a BKV strain obtained from a central nervous system infection, and it reveals regulatory region rearrangements reminiscent of those described in JC virus from brains with progressive multifocal leukoencephalopathy. We suggest that the presence in the CSF of BKV with a dominant rearranged regulatory region may be useful in the diagnosis of BKV meningoencephalitis secondary to BKV nephritis.