RESUMO
Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system caused by the JC polyoma virus. Electron microscopy and immunohistochemistry are the traditional methods of confirming the presence of the virus in brain biopsies from these patients. We studied the brain biopsies from 7 patients with PML and 6 patients without PML with chromogenic in situ hybridization (CISH) for the JC polyoma virus using a commercially available probe. The biopsies from the patients with the PML cases were proven to contain the JC polyoma virus by traditional and molecular methods. The CISH findings were compared with the known state of infection. All (7/7) of the biopsies from patients with PML were positive for the presence of polyoma virus by CISH, whereas the biopsies from patients without PML were uniformly negative. CISH seems to be a useful tool for the detection of the JC virus in brain biopsies from patients with PML, and is more accessible because a commercial probe is available.
Assuntos
Encéfalo/virologia , Vírus JC/genética , Vírus JC/isolamento & purificação , Leucoencefalopatia Multifocal Progressiva/virologia , Estudos de Casos e Controles , Compostos Cromogênicos , Humanos , Hibridização In Situ/métodos , Vírus JC/patogenicidade , Leucoencefalopatia Multifocal Progressiva/diagnóstico , Reação em Cadeia da PolimeraseRESUMO
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.
Assuntos
Vírus BK/isolamento & purificação , DNA Viral/isolamento & purificação , Infecções por Polyomavirus/virologia , Kit de Reagentes para Diagnóstico , Infecções Tumorais por Vírus/virologia , Urina/virologia , Actinas/genética , Vírus BK/genética , DNA Viral/genética , Humanos , Magnetismo , Reação em Cadeia da Polimerase , Infecções por Polyomavirus/diagnóstico , Kit de Reagentes para Diagnóstico/economia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Infecções Tumorais por Vírus/diagnósticoRESUMO
We evaluated 2 methods, a LightCycler PCR assay and pyrosequencing for the detection of the JC polyoma virus (JCV) in fixed brain tissue of 10 patients with and 3 control patients without progressive multifocal leukoencephalopathy (PML). Nucleic acid extraction was performed after deparaffinization and proteinase K digestion. The LightCycler assay differentiates the BK virus (BKV), JCV, and SV40 using melt curve analysis. Conventional PCR was used with the same primers to generate products for pyrosequencing. Two sequencing primers were used that differentiate the polyoma viruses. Seven of 11 biopsies (1 patient had 2 biopsies) with PML were positive for JCV by real-time PCR and/or PCR/pyrosequencing. Three of 4 remaining biopsies were positive by real-time PCR but had melting points between JCV and SV40. The 4 specimens that were negative or atypical by LightCycler PCR were positive by traditional PCR, but 1 had an amplicon of lower molecular weight by gel electrophoresis. These were shown to represent JCV by at least 1 of the 2 pyrosequencing primers. The biopsies from patients without PML were PCR negative. Both the LightCycler and pyrosequencing assays are useful for confirming JCV in brain biopsies from patients with PML, but variant JCVs may require supplementary methods to confirm JCV infection.
Assuntos
Encéfalo/virologia , Leucoencefalopatia Multifocal Progressiva/diagnóstico , Reação em Cadeia da Polimerase/métodos , Polyomavirus/isolamento & purificação , Análise de Sequência de DNA/métodos , Vírus BK/genética , Sequência de Bases , Encéfalo/patologia , Humanos , Vírus JC/genética , Leucoencefalopatia Multifocal Progressiva/patologia , Dados de Sequência Molecular , Polyomavirus/genética , RNA Viral/química , Estudos RetrospectivosRESUMO
The development of cytomegalovirus (CMV) disease and subsequent emergence of drug-resistant strains was examined in a large group of solid organ transplant recipients; drug-resistant CMV was detected in a total of 30 transplant recipients (20 lung, 5 kidney, 4 heart, and 1 liver). Drug resistance was confirmed both phenotypically and genotypically. The sequences of drug-resistant CMV strains from the same patient differed from drug-susceptible baseline sequences only at single sites previously confirmed to confer drug resistance. At least 1 isolate from each patient had a mutation in the UL97 phosphotransferase coding sequence. Mutations in the DNA polymerase gene were found in 6 of 38 sequenced strains. Lung transplant recipients had the highest incidence of drug-resistant virus: of the 30 patients, 28 were CMV-seronegative transplant recipients of CMV-seropositive organs, which strongly supports the premise that drug resistance is most prevalent in that transplant population.
