Multicenter comparison of molecular methods for detection of Legionella spp. in sputum samples.

Legionellosis can be diagnosed by PCR using sputum samples. In this report, the methods of nine laboratories for 12 sputum samples with Legionella pneumophila and Legionella longbeachae are compared. We conclude that (i) liquefaction prevents PCR inhibition, (ii) the employed mip gene PCRs detected L.

Functional polymorphisms in the mannan-binding lectin 2 gene: effect on MBL levels and otitis media.

Background: Mannan-binding lectin (MBL) can bind to microorganisms, initiating the lectin pathway of complement activation. Aberrant MBL serum levels, caused by MBL2 gene polymorphisms, are a possible risk factor for recurrent infections. Within the 7 common MBL haplotypes, still considerable variation in MBL serum levels exists.

Association of furunculosis and familial deficiency of mannose-binding lectin.

Background: Polymorphisms in the mannose-binding lectin gene reduce serum mannose-binding lectin levels and are associated with enhanced risk of infection. In a family with recurrent staphylococcal disease presenting as furunculosis or carbuncles, an association with mannose-binding lectin deficiency was investigated.

Materials And Methods: Levels of functional mannose-binding lectin were estimated and the genotypes of the mannose-binding lectin gene were analysed on blood samples, collected from the members of one particular family with a high prevalence of furunculosis.

Association between familial deficiency of mannose-binding lectin and mutations in the corresponding gene and promoter region.

In a recent report, our group presented clinical research data supporting the role of mannose-binding lectin (MBL) deficiency in susceptibility to meningococcal disease (W. A. Bax, O.

A case of familial meningococcal disease due to deficiency in mannose-binding lectin (MBL).

A case of familial meningococcal meningitis is described, which involved a 18-year old boy, his mother, and his grandfather, from who all three suffered from meningococcal disease at about similar age (17-19 y), albeit with one or two generations in between. By studying the genetic variants of MBL, we found out that all three family members described carried the B, D variant mbl genes instead of the homozygous mbl A, A trait. Serum MBL levels within the family varied from <0.

Glycosylation of the Enterobacter cloacae outer membrane protein OmpX in eukaryotic cells.

The topological model of the Enterobacter cloacae outer membrane protein OmpX showed three putative glycosylation sites. When OmpX was expressed in bacteria that were cultured under aerated conditions, no glycosylation was observed. The coupling of carbohydrate chains to the ompX gene product was also investigated in the eukaryotic baculovirus expression system.

Detection of parvovirus B19 infection in first and second trimester fetal loss.

Fetal and placental tissues and maternal sera from a series of 273 cases of first and second trimester fetal loss were collected to detect the frequency of parvovirus B19 infection. In addition, fetal tissues were studied for the presence of congenital anomalies. Serology of maternal sera, histology of fetal tissues and placenta, polymerase chain reaction (PCR), in situ hybridization (ISH), and immunohistochemistry (IHC) were used for the detection of parvovirus B19 infection.

Prevalence of parvovirus B19 infection in patients infected with human immunodeficiency virus.

Parvovirus B19 infection may cause chronic anemia in human immunodeficiency virus (HIV)-infected hosts. Small-scale studies and case reports have suggested that parvovirus B19 infection is a significant cause of anemia in HIV-infected patients. We studied single serum samples from 317 consecutive HIV-infected patients with use of parvovirus B19-specific serology and polymerase chain reaction for detection of viral DNA.

Recombinant parvovirus B19 capsids as a new substrate for detection of B19-specific IgG and IgM antibodies by an enzyme-linked immunosorbent assay.

A new enzyme-linked immunosorbent assay for the detection of B19-specific IgG and IgM antibodies was established using B19 capsids synthesized in a baculovirus expression system. These B19 capsids, consisting of either coat protein VP2 alone or of both VP1 and VP2, have been shown to be similar to native virus in size and appearance. The results obtained for the detection of B19-specific antibodies showed good correlations with a radioimmunoassay which uses native B19 virus and an immunofluorescence assay based on insect cells expressing coat protein VP1.

Detection of human parvovirus B19 DNA by dot-hybridization and the polymerase chain reaction: applications for diagnosis of infections.

The development of the polymerase chain reaction for the detection of human parvovirus B19 DNA in clinical specimens is described. The sensitivity of this detection is compared with detection of virus using radiolabeled DNA and RNA probes. The polymerase chain reaction was the most sensitive, detecting approximately 10 fg B19 DNA.

An immunofluorescence assay for the detection of parvovirus B19 IgG and IgM antibodies based on recombinant viral antigen.

An indirect immunofluorescence assay for serum IgG and IgM antibodies to human parvovirus B19 was established using recombinant B19 viral antigen, the capsid protein VP1, which had been produced in a baculovirus expression system. This protein gives a strong and characteristic signal in the immunofluorescence assay, making it a suitable candidate for this test system. The test results showed a good correlation with results obtained with a solid-phase capture radioimmunoassay (Cohen et al.

Antigenic parvovirus B19 coat proteins VP1 and VP2 produced in large quantities in a baculovirus expression system.

Two baculovirus expression vectors derived from Autographica californica nuclear polyhedrosis virus (AcNPV) were prepared containing the complete 2.5 kb coding region for parvovirus B19 coat protein VP1 (AcB19VP1L) and the 1.8 kb coding region for VP2 (AcB19VP2L), placed under the control of the polyhedrin promoter.

Rapid and simple method for purification of nucleic acids.

We have developed a simple, rapid, and reliable protocol for the small-scale purification of DNA and RNA from, e.g., human serum and urine.

High prevalence of latently present cytomegalovirus in arterial walls of patients suffering from grade III atherosclerosis.

The presence of cytomegalovirus (CMV) nucleic acids was demonstrated in arterial walls of patients with grade III and with maximally grade I atherosclerosis by dot blot and in situ DNA hybridization and by polymerase chain reaction (PCR) using probes and primers derived from immediate early (IE) and late (L) genomic regions. The presence of the complete viral genome could be demonstrated by both dot blot DNA hybridization and PCR. IE mRNA but not L mRNA could be demonstrated by in situ DNA hybridization, indicating the presence of latent CMV in the human arterial wall.

Fetal pathology in human parvovirus B19 infection.

In a current Netherlands study on the effects on mother and child of infection with the human parvovirus B19 during pregnancy, 10 pregnancies have been reported. Three of them ended before term: two in fetal death and one by elective abortion. In two of these fetuses B19 infection in cells other than those of the erythroid series was demonstrated, and in the one terminated, ocular malformation and extensive inflammatory reactions in all fetal and placental tissues were found.

Rapid detection of human cytomegalovirus DNA in peripheral blood leukocytes of viremic transplant recipients by the polymerase chain reaction.

Peripheral blood leukocyte samples (n = 458) of 24 bone marrow transplant and 52 kidney transplant patients were examined weekly for the presence of human cytomegalovirus (HCMV) using an improved culture technique (DEAFF; detection of early antigen fluorescent foci). In total 5 (21%) bone marrow transplant and 11 (21%) kidney transplant patients developed a viremia. Patients' samples were investigated for the presence of HCMV DNA using an in vitro DNA amplification technique, the polymerase chain reaction (PCR).

Detection of parvovirus B19 DNA in fetal tissues by in situ hybridisation and polymerase chain reaction.

Attempts were made to detect human parvovirus B19-DNA by in situ hybridisation and the polymerase chain reaction in placental and fetal tissues from a case of intrauterine fetal death. In the in situ hybridisation experiments radioactive and non-radioactive (labelled with 2-acetyl-aminofluorene, AAF) DNA probes were used. B19-DNA was detectable in paraffin wax embedded fetal tissue from the liver, heart, lung, brain and thymus.

Rapid detection of human parvovirus B19 DNA by dot-hybridization and the polymerase chain reaction.

The results of a comparison of three DNA-detection methods for human parvovirus B19 DNA are described. The sensitivity of detection of virus from hybridization assays using 32P-radiolabeled DNA and RNA probes was compared with a method for enzymatically amplifying specific target DNA sequences (polymerase chain reaction). B19 virus DNA was detected using a radiolabeled DNA probe at serum dilutions of 10(-3), equivalent to approximately 3 pg of viral DNA.

Affinity labeling by a photoreactive GTP analogue of the delta-subunit of eukaryotic initiation factor eIF-2 in different initiation complexes.

The interaction of GTP with initiation factor eIF-2 in different complexes was studied by affinity labeling using a derivative of [3H]GTP carrying a photoreactive group in the alpha-phosphate moiety. In the binary complex [eIF-2.GTP analogue], in the ternary complex [eIF-2.

Human parvovirus B19 DNA in synovial fluid.

We describe a 33-year-old woman with a serologically proven human parvovirus B19 infection, who developed synovitis. Using a dot-blot hybridization technique, we detected B19 DNA in her synovial fluid. To our knowledge, this is the first report of the isolation of B19 from synovial fluid.