RESUMEN
The newly identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, a pandemic respiratory disease. Moreover, thromboembolic events throughout the body, including in the CNS, have been described. Given the neurological symptoms observed in a large majority of individuals with COVID-19, SARS-CoV-2 penetrance of the CNS is likely. By various means, we demonstrate the presence of SARS-CoV-2 RNA and protein in anatomically distinct regions of the nasopharynx and brain. Furthermore, we describe the morphological changes associated with infection such as thromboembolic ischemic infarction of the CNS and present evidence of SARS-CoV-2 neurotropism. SARS-CoV-2 can enter the nervous system by crossing the neural-mucosal interface in olfactory mucosa, exploiting the close vicinity of olfactory mucosal, endothelial and nervous tissue, including delicate olfactory and sensory nerve endings. Subsequently, SARS-CoV-2 appears to follow neuroanatomical structures, penetrating defined neuroanatomical areas including the primary respiratory and cardiovascular control center in the medulla oblongata.
Asunto(s)
Encéfalo/virología , COVID-19/virología , Mucosa Olfatoria/virología , SARS-CoV-2/patogenicidad , Sistema Nervioso Central , Humanos , ARN Viral/genética , Olfato/fisiología , Internalización del VirusRESUMEN
Diagnostic electron microscopy (DEM) was an essential component of viral diagnosis until the development of highly sensitive nucleic acid amplification techniques (NAT). The simple negative staining technique of DEM was applied widely to smallpox diagnosis until the world-wide eradication of the human-specific pathogen in 1980. Since then, the threat of smallpox re-emerging through laboratory escape, molecular manipulation, synthetic biology or bioterrorism has not totally disappeared and would be a major problem in an unvaccinated population. Other animal poxviruses may also emerge as human pathogens. With its rapid results (only a few minutes after arrival of the specimen), no requirement for specific reagents and its "open view", DEM remains an important component of virus diagnosis, particularly because it can easily and reliably distinguish smallpox virus or any other member of the orthopoxvirus (OPV) genus from parapoxviruses (PPV) and the far more common and less serious herpesviruses (herpes simplex and varicella zoster). Preparation, enrichment, examination, internal standards and suitable organisations are discussed to make clear its continuing value as a diagnostic technique.
Asunto(s)
Microscopía Electrónica , Orthopoxvirus/ultraestructura , Infecciones por Poxviridae/diagnóstico , Infecciones por Poxviridae/virología , Animales , Exantema/diagnóstico , Exantema/virología , Herpesviridae/clasificación , Herpesviridae/ultraestructura , Infecciones por Herpesviridae/diagnóstico , Infecciones por Herpesviridae/virología , Humanos , Microscopía Electrónica/métodos , Orthopoxvirus/clasificación , Infecciones por Poxviridae/prevención & control , Viruela/diagnóstico , Viruela/virologíaRESUMEN
The 2014-2015 outbreak of the Ebola virus disease (EVD) in West Africa has been considered a major global health emergency by the WHO. Implications for health care providers including oral and maxillo-facial surgeons have been published by the WHO, the Centers for Disease Control and Prevention (USA), and other medical societies and public health organizations. While the risk of infection with the Ebola virus seems to be rather small in Europe, maxillo-facial and plastic surgeons often travel to Africa to treat patients with facial burns, cleft-lip and palate, and noma. The likelihood of an encounter with patients infected by Ebola virus in subsaharan and West Africa, therefore, has increased during the last 2 years. The purpose of this short overview was to summarize the virology of the Ebola virus, transmission, epidemiology, clinical features, oral manifestations, treatment, and possible implications for maxillo-facial surgeons of EDV.
Asunto(s)
Brotes de Enfermedades , Fiebre Hemorrágica Ebola/diagnóstico , Fiebre Hemorrágica Ebola/transmisión , Transmisión de Enfermedad Infecciosa de Paciente a Profesional , Cirugía Bucal , Cirugía Plástica , África del Sur del Sahara , África Occidental , Fiebre Hemorrágica Ebola/epidemiología , Fiebre Hemorrágica Ebola/virología , Humanos , Misiones Médicas , Factores de RiesgoRESUMEN
BACKGROUND: Even though smallpox was declared eradicated by WHO in 1980, it cannot be ruled out that the etiological variola virus could be used as a biological weapon. Undestroyed viruses from biowarfare programmes, virus strains left undetected in a freezer or dangerous recombinant poxvirus constructs could cause dangerous outbreaks in a relatively unprotected population. OBJECTIVES: Despite an abundance of studies performed during the eradication of smallpox, epidemiological data for preparedness planning and outbreak control in modern, industrialized countries are scarce. MATERIAL AND METHODS: Full-text hand search for the period from 1945 to 1975 in the main German public health journals. RESULTS: After World War II 12 smallpox outbreaks occurred in Germany. They were studied with the focus on the period of contagiousness, the protective effect of vaccination, booster-effect of revaccination and the place of infection. A total of 95 individuals contracted smallpox, including 10 fatalities. Despite having been previously vaccinated, 81 vaccinated persons came down with smallpox, yet 91% of them developed only mild symptoms. These patients presented a high risk for spreading the infection to contact persons due to misinterpretation of symptoms and the continuing social contacts. Basically, the risk of transmission in the first 2 to 3 days after onset of symptoms was low, thus facilitating antiepidemic measures. The importance of hospital preparedness is emphasized by the fact that most infections occurred in hospitals. CONCLUSION: The data analyzed provide valuable information for today's outbreak response planning and counter bioterrorism preparedness.
Asunto(s)
Bioterrorismo/prevención & control , Brotes de Enfermedades/prevención & control , Brotes de Enfermedades/estadística & datos numéricos , Vacuna contra Viruela/uso terapéutico , Viruela/mortalidad , Viruela/prevención & control , Bioterrorismo/estadística & datos numéricos , Alemania/epidemiología , Humanos , Incidencia , Factores de Riesgo , Viruela/diagnóstico , Tasa de SupervivenciaRESUMEN
Diagnostic electron microscopy (DEM) has conceptual predecessors â" the application of the sense of vision and of light microscopy in medicine. The evolvement of DEM and the role of its two branches - histopathology and rapid negative-contrast DEM - are described in this review, with a focus on the latter. By its resolving power of 2 nm in praxi, DEM is able to visualize all kinds of pathogens, bacteria, parasites, even the smallest viruses. In contrast to other laboratory diagnostic methods, DEM excels by speed and "open view". All structures on the support grid can be assigned directly by "pattern recognition" of their fine structure to a specific family of agents. The morphology-based "catch-all" diagnosis can be decisive as a differential diagnosis and will help as a preliminary diagnosis to select and apply proper diagnostic tools for typing of the observed agent. Based on two case reports, the advantages and possible pitfalls of DEM are exemplified and hints are given to make DEM reliable and effective. Finally the role of DEM in medicine and the wider fields of life sciences are described together with the organizational conditions to guarantee its future in laboratory diagnostics.
Asunto(s)
Microscopía Electrónica/métodos , Virosis/diagnóstico , Virus/ultraestructura , Animales , Historia del Siglo XVII , Historia del Siglo XVIII , Historia del Siglo XIX , Historia del Siglo XX , Humanos , Microscopía Electrónica/historia , Virosis/virología , Virus/aislamiento & purificaciónRESUMEN
A herpesvirus of smelt (Osmerus eperlanus) was identified by thin section electron microscopy. Degenerated cells of skin lesions located on the back fin of smelt showed either intranucleic- or cytoplasmic herpesvirus-specific structures. In the nuclei "naked" virus capsids with a diameter of about 100 nm were observed. The diameter of the complete virion including its unilaterally extended envelope ranged from 200 to 350 nm. Remarkably, in complete virions the electron-opaque tegument is completely filling the region between nucleocapsid and envelope and as another unique feature the virion shows a "comet-shape" due to a long unilateral extension of its envelope. This kind of shape had been not reported for any of herpesviruses known so far. Consequently this virus was termed herpesvirus of Osmerus eperlanus (HVOE1) or Comet herpesvirus of smelt. Due to the long time storage at the nonstandard temperature of smelt virus the biological and genomic analysis of the HVOE1 was hampered. All attempts to study host range of HVOE1 failed as no virus replication was observed, indicating that infectivity was lost or the suitable cell culture was missing. The genomic DNA of HVOE1 was analyzed by DNA restriction endonucleases.
Asunto(s)
Enfermedades de los Peces/virología , Infecciones por Herpesviridae/veterinaria , Herpesviridae/ultraestructura , Osmeriformes/virología , Animales , Genoma Viral , Herpesviridae/genética , Infecciones por Herpesviridae/virología , Microscopía Electrónica , Virión/ultraestructuraAsunto(s)
Enfermedades de los Trabajadores Agrícolas/virología , Exposición Profesional , Infecciones por Poxviridae/diagnóstico , Zoonosis/virología , Adulto , Enfermedades de los Trabajadores Agrícolas/diagnóstico , Animales , Animales Domésticos , Humanos , Masculino , Microscopía Electrónica/métodos , Parapoxvirus , Infecciones por Poxviridae/virologíaRESUMEN
The publisher regrets that this article is an accidental duplication of an article that has already been published in JMIC, 38/7, pages 699 doi:10.1016/j.micron.2007.05.007. The duplicate article has therefore been withdrawn.
RESUMEN
Oral amalgam tattoos (AT) are distinct pigmentations of the oral mucosa resulting from accidental incorporation of dental amalgam in the oral soft tissues. Dental amalgams and in particular mercury, one of the constituents of dental amalgams, have for long been considered toxic. Oral ATs are easily accessible to study soft tissue reaction to amalgam and its degradation products. In this study, 17 oral ATs were examined by transmission electron microscopy and energy dispersive X-ray microanalysis. Ultrastructurally, in the ATs, three kinds of electron-dense particles were observed. The largest particles ranged in size from 0.5 up to several 100 microm. Smaller electron-dense inclusions (0.5-0.1 microm) were seen extracellularly associated with meshworks of elastic fibers and collagen bundles. The third and smallest type of particles (5-30 nm in diameter) was found with basement membranes of small vessels and pericytes and particularly decorating collagen bundles. Element analysis regularly revealed the presence of silver, sulphur, copper and lead in the AT decay products. Mercury was found in only one instance. Tissue reactions due to ATs seem to be minimal. No acute inflammatory changes were seen. Larger inclusions occasionally were surrounded by macrophages and multinucleated cells. TEM and element analysis may in specific cases be helpful in the differential diagnosis of pigmented lesions of the oral mucosa.
Asunto(s)
Amalgama Dental/efectos adversos , Amalgama Dental/química , Amalgama Dental/metabolismo , Microanálisis por Sonda Electrónica , Cuerpos Extraños/metabolismo , Cuerpos Extraños/patología , Humanos , Metales Pesados/efectos adversos , Metales Pesados/análisis , Metales Pesados/metabolismo , Microscopía Electrónica , Mucosa Bucal/metabolismo , Mucosa Bucal/patología , Tamaño de la Partícula , TatuajeRESUMEN
The purpose of this study was to explore the turnaround times, section and image quality of a number of more "difficult" specimens destined for rapid diagnostic electron microscopy (EM) after microwave-assisted processing. The results were assessed and compared with those of conventionally processed samples. A variety of infectious agents, some with a potential for bioterrorism, and liver biopsies serving as an example for routine histopathology samples were studied. The samples represented virus-producing cell cultures (such as SARS-coronavirus, West Nile virus, Orthopox virus), bacteria suspensions (cultures of Escherichia coli and genetically knockout apathogenic Bacillus anthracis), suspensions of parasites (malaria Plasmodium falciparum, Leishmania major, Microsporidia cuniculi, Caenorhabditis elegans), and whole Drosophila melanogaster flies infected with microsporidia. Fresh liver samples and infected flies were fixed in Karnovsky-fixative by microwaving (20 min), all other samples were fixed in buffered glutaraldehyde or Karnovsky-fixative overnight or longer. Subsequently, all samples were divided to evaluate alternative processing protocols: one part of the sample was OsO4-postfixed, ethanol-dehydrated, Epon-infiltrated (overnight) in an automated tissue processor (LYNX, Leica), and polymerized at 60 degrees C for 48 h; in parallel the other part was microwave-assisted processed in the bench microwave device (REM, Milestone), including post-osmication and the resin block polymerization. The microwave-assisted processing protocol required at minimum 3 h 20 min: the respective epon resin blocks were uniformly polymerized allowing an easy sectioning of semi- and ultrathin sections. Sections collected on non-coated 200 mesh grids were stable in the electron beam and showed an excellent preservation of the ultrastructure and high contrast, thus allowing an easy, unequivocal and rapid assessment of specimens. Compared with conventional routine methods, microwave technology facilitates a significant reduction in sample processing time from days to hours without any loss in ultrastructural details. Microwave-assisted processing could, therefore, be a substantial benefit for the routine electron microscopic diagnostic workload. Due to its speed and robust performance it could be applied wherever a rapid electron microscopy diagnosis is required, e.g., if bioterrorism or emerging agents are suspected. Combining microwave technology with digital image acquisition, the 1-day diagnosis based on ultrathin section electron microscopy will become possible, with crucial or interesting findings being consulted or shared worldwide with experts using modern telemicroscopy tools via Internet.
Asunto(s)
Bioterrorismo , Técnicas Histológicas/métodos , Microscopía Electrónica/métodos , Animales , Biopsia , Humanos , Hígado/ultraestructura , Microondas , Enfermedades Parasitarias/diagnóstico , Factores de Tiempo , Virosis/diagnósticoRESUMEN
Starting in the 1960, electron microscopy (EM) became widely applied also in viral diagnosis. During the 1970th and 80th, many new agents were characterized from diagnostic cell cultures and clinical specimens. The wide introduction of ELISA- and PCR-techniques as well as cost-arguments recently reduced the role of EM in routine viral diagnosis. Compared to other diagnostic techniques, however, EM excells by speed and "open view", i.e. by the ability to detect also the "un-expected" without the need for specific reagents. As shown in 2003 by the elucidation of the SARS pandemia and the human monkeypox outbreak in US, EM is well suited as a safe, front-line diagnostic method in infectious diseases emergencies and/or in possible bioterrorist attacks.
Asunto(s)
Microscopía Electrónica , Virosis/diagnóstico , Virus/ultraestructura , Predicción , Humanos , Manejo de Especímenes , Coloración y Etiquetado , Virus/aislamiento & purificaciónRESUMEN
BACKGROUND: Serotyping of O-(lipopolysaccharide) and H-(flagellar) antigens is a wideley used method for identification of pathogenic strains and clones of Escherichia coli. At present, 176 O- and 53 H-antigens are described for E. coli which occur in different combinations in the strains. The flagellar antigen H4 is widely present in E. coli strains of different O-serotypes and pathotypes and we have investigated the genetic relationship between H4 encoding fliC genes by PCR, nucleotide sequencing and expression studies. RESULTS: The complete nucleotide sequence of fliC genes present in E. coli reference strains U9-41 (O2:K1:H4) and P12b (O15:H17) was determined and both were found 99.3% (1043 of 1050 nucleotides) identical in their coding sequence. A PCR/RFLP protocol was developed for typing of fliC-H4 strains and 88 E. coli strains reacting with H4 antiserum were investigated. Nucleotide sequencing of complete fliC genes of six E. coli strains which were selected based on serum agglutination titers, fliC-PCR genotyping and reference data revealed 96.6 to 100% identity on the amino acid level. The functional expression of flagellin encoded by fliC-H4 from strain U9-41 and from our strain P12b which is an H4 expressing variant type was investigated in the E. coli K-12 strain JM109 which encodes flagellar type H48. The fliC recombinant plasmid carrying JM109 strains reacted with both H4 and H48 specific antisera whereas JM109 reacted only with the H48 antiserum. By immunoelectron microscopy, we could show that the flagella made by the fliC-H4 recombinant plasmid carrying strain are constituted of H48 and H4 flagellins which are co-assembled into functional flagella. CONCLUSION: The flagellar serotype H4 is encoded by closely related fliC genes present in serologically different types of E. coli strains which were isolated at different time periods and geographical locations. Our expression studies show for the first time, that flagellins of different molecular weigh are functionally expressed and coassembled in the same flagellar filament in E. coli.
Asunto(s)
Antígenos Bacterianos/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Flagelina/genética , Flagelina/metabolismo , Genes Bacterianos/genética , Secuencia de Aminoácidos , Antígenos Bacterianos/química , Clonación Molecular , Escherichia coli/clasificación , Escherichia coli/citología , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Flagelina/química , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Filogenia , Alineación de Secuencia , Homología de Secuencia de Aminoácido , SerotipificaciónRESUMEN
Human monoclonal antibodies (MAbs) were selected from semisynthetic antibody phage display libraries by using whole irradiated severe acute respiratory syndrome (SARS) coronavirus (CoV) virions as target. We identified eight human MAbs binding to virus and infected cells, six of which could be mapped to two SARS-CoV structural proteins: the nucleocapsid (N) and spike (S) proteins. Two MAbs reacted with N protein. One of the N protein MAbs recognized a linear epitope conserved between all published human and animal SARS-CoV isolates, and the other bound to a nonlinear N epitope. These two N MAbs did not compete for binding to SARS-CoV. Four MAbs reacted with the S glycoprotein, and three of these MAbs neutralized SARS-CoV in vitro. All three neutralizing anti-S MAbs bound a recombinant S1 fragment comprising residues 318 to 510, a region previously identified as the SARS-CoV S receptor binding domain; the nonneutralizing MAb did not. Two strongly neutralizing anti-S1 MAbs blocked the binding of a recombinant S fragment (residues 1 to 565) to SARS-CoV-susceptible Vero cells completely, whereas a poorly neutralizing S1 MAb blocked binding only partially. The MAb ability to block S1-receptor binding and the level of neutralization of the two strongly neutralizing S1 MAbs correlated with the binding affinity to the S1 domain. Finally, epitope mapping, using recombinant S fragments (residues 318 to 510) containing naturally occurring mutations, revealed the importance of residue N479 for the binding of the most potent neutralizing MAb, CR3014. The complete set of SARS-CoV MAbs described here may be useful for diagnosis, chemoprophylaxis, and therapy of SARS-CoV infection and disease.
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Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Glicoproteínas de Membrana/inmunología , Proteínas de la Nucleocápside/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Proteínas del Envoltorio Viral/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Antivirales/química , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos , Sitios de Unión , Chlorocebus aethiops , Proteínas de la Nucleocápside de Coronavirus , Mapeo Epitopo , Humanos , Datos de Secuencia Molecular , Pruebas de Neutralización , Glicoproteína de la Espiga del Coronavirus , Células VeroRESUMEN
The sixth genomic segment of Thogoto virus (THOV) encodes two proteins, the viral matrix protein (M) and an accessory protein with an interferon (IFN)-antagonistic function named ML. M and ML are shown in this study to be structural components of the virion. Using an in vivo system based on the reconstitution of functional THOV ribonucleoprotein complexes from cloned cDNAs, it was demonstrated that M has an inhibitory effect on the viral RNA-dependent RNA polymerase (RdRP) and is essential for the formation of virus-like particles (VLPs). The functional domain responsible for the regulation of RdRP activity resides within the C-terminal half of M, while full-length M protein is required for VLP formation. The ML protein cannot complement M with respect to either RdRP downregulation or particle formation, although it is identical to M apart from a 38 aa extension at the C terminus. In contrast, ML, but not M, is able to prevent the induction of IFN-beta by double-stranded RNA. This function is contained within the C-terminal half of ML. These data suggest major structural differences between M and ML that could explain the different activities of the two proteins.
Asunto(s)
Interferón beta/antagonistas & inhibidores , Thogotovirus/fisiología , Proteínas de la Matriz Viral/fisiología , Proteínas Estructurales Virales/fisiología , Animales , Cricetinae , Humanos , Interferón beta/biosíntesis , Ratones , Replicón , Thogotovirus/genética , Virión/fisiología , Replicación ViralRESUMEN
SARS coronavirus continues to cause sporadic cases of severe acute respiratory syndrome (SARS) in China. No active or passive immunoprophylaxis for disease induced by SARS coronavirus is available. We investigated prophylaxis of SARS coronavirus infection with a neutralising human monoclonal antibody in ferrets, which can be readily infected with the virus. Prophylactic administration of the monoclonal antibody at 10 mg/kg reduced replication of SARS coronavirus in the lungs of infected ferrets by 3.3 logs (95% CI 2.6-4.0 logs; p<0.001), completely prevented the development of SARS coronavirus-induced macroscopic lung pathology (p=0.013), and abolished shedding of virus in pharyngeal secretions. The data generated in this animal model show that administration of a human monoclonal antibody might offer a feasible and effective prophylaxis for the control of human SARS coronavirus infection.
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Anticuerpos Monoclonales/uso terapéutico , Síndrome Respiratorio Agudo Grave/prevención & control , Animales , Femenino , Hurones , Humanos , Inmunización Pasiva , Inmunoglobulina G/inmunología , Pulmón/patología , Pulmón/virología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/aislamiento & purificación , Síndrome Respiratorio Agudo Grave/patología , Síndrome Respiratorio Agudo Grave/virología , Replicación ViralRESUMEN
Previously, we have demonstrated that hepatitis B virus (HBV) core particles tolerate the insertion of the amino-terminal 120 amino acids (aa) of the Puumala hantavirus nucleocapsid (N) protein. Here, we demonstrate that the insertion of 120 amino-terminal aa of N proteins from highly virulent Dobrava and Hantaan hantaviruses allows the formation of chimeric core particles. These particles expose the inserted foreign protein segments, at least in part, on their surface. Analysis by electron cryomicroscopy of chimeric particles harbouring the Puumala virus (PUUV) N segment revealed 90% T = 3 and 10% T = 4 shells. A map computed from T = 3 shells shows additional density splaying out from the tips of the spikes producing the effect of an extra shell of density at an outer radius compared with wild-type shells. The inserted Puumala virus N protein segment is flexibly linked to the core spikes and only partially icosahedrally ordered. Immunisation of mice of two different haplotypes (BALB/c and C57BL/6) with chimeric core particles induces a high-titered and highly cross-reactive N-specific antibody response in both mice strains.
Asunto(s)
Anticuerpos Antivirales/sangre , Antígenos del Núcleo de la Hepatitis B/inmunología , Proteínas de la Nucleocápside/inmunología , Orthohantavirus/inmunología , Proteínas Recombinantes de Fusión/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Antivirales/inmunología , Reacciones Cruzadas , Microscopía por Crioelectrón , Femenino , Orthohantavirus/clasificación , Infecciones por Hantavirus/prevención & control , Antígenos del Núcleo de la Hepatitis B/química , Antígenos del Núcleo de la Hepatitis B/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/genética , Proteínas Recombinantes de Fusión/genética , Vacunas Virales/administración & dosificaciónRESUMEN
We have conducted an international quality assurance study of filovirus, Lassa virus, and orthopox virus PCR with 24 participants. Of the participating laboratories, 45.8 and 66.7% detected virus in all plasma samples, which contained > or = 5,000 and > or = 100,000 copies per ml, respectively. Sensitivity levels were not significantly different between viruses. False-negative results were attributable to a lack of sensitivity.
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Bioterrorismo , Ebolavirus/aislamiento & purificación , Cooperación Internacional , Virus Lassa/aislamiento & purificación , Marburgvirus/aislamiento & purificación , Orthopoxvirus/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , ADN Viral/sangre , Ebolavirus/genética , Europa (Continente) , Humanos , Laboratorios , Virus Lassa/genética , Marburgvirus/genética , Orthopoxvirus/genética , Control de Calidad , Virosis/diagnóstico , Virosis/virologíaRESUMEN
BACKGROUND: We studied electron microscopy (EM) as an appropriate test system for the detection of polyomavirus in urine samples from bone marrow transplant patients. METHODS: We evaluated direct EM, ultracentrifugation (UC) before EM, and solid-phase immuno-EM (SPIEM). The diagnostic accuracy of EM was studied by comparison with a real-time PCR assay on 531 clinical samples. RESULTS: The detection rate of EM was increased by UC and SPIEM. On 531 clinical urine samples, the diagnostic sensitivity of EM was 47% (70 of 149) with a specificity of 100%. We observed a linear relationship between viral genome concentration and the proportion of urine samples positive by EM, with a 50% probability for a positive EM result for urine samples with a polyomavirus concentration of 10(6) genome-equivalents (GE)/mL; the probability of a positive EM result was 0% for urine samples with <10(3) GE/mL and 100% for urine samples containing 10(9) GE/mL. CONCLUSIONS: UC/EM is rapid and highly specific for polyomavirus in urine. Unlike real-time PCR, EM has low sensitivity and cannot quantify the viral load.