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. pneumophila only, and (iii) the 16S rRNA gene PCR detected both Legionella species and is preferred for the diagnosis of legionellosis.

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. RESULTS: Functional mannose-binding lectin levels in sera of 13 of the 28 members of one family showed deficiency. Furunculosis or carbuncles appeared to be present in nine of the 28 family members, seven of which showing the pBly allele and mannose-binding lectin deficiency. Four young family members of the second generation were pBly positive and mannose-binding lectin deficient, but had not shown furunculosis yet. CONCLUSION: Members of a particular family suffering from furunculosis differ from their 'healthy' relatives as to mannose-binding lectin genotypes, indicating the relevance of normal mannose-binding lectin levels in the defence against staphylococcal disease.

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. J. J. Cluysenaer, A. K. M. Bartelink, P. C. Aerts, R. A. B. Ezekowitz, and H. van Dijk, Lancet 354:1094-1095, 1999). This association was reported earlier by Hibberd et al. (M. L. Hibberd, M. Sumiya, J. A. Summerfield, R. Booy, M. Levin, and the Meningococcal Research Group, Lancet 353:1049-1053, 1999) but was not based on family data. Our study included three members of one family who had acquired meningococcal meningitis in early adulthood. The objective of the present study was to investigate whether the genotypes of the MBL gene in this family, analyzed by PCR, correlate with MBL concentrations. We found that genotype variants in the MBL gene and promoter region match the low functional MBL levels (<0.25 microg of equivalents/ml) in the sera of the three patients in this family and that a significant correlation between genotype MBL deficiency and meningococcal disease existed.

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. For this purpose, a recombinant ompX gene-containing baculovirus was made. Infection of insect cells with this recombinant virus resulted in the production of sufficient amounts of OmpX to study glycosylation. In this system, all potential N-glycosylation sites of OmpX were utilized. Furthermore, it became clear that glycosylated OmpX was retained in the insect cells and was not secreted in the medium. Given the fact that OmpX plays a role in the invasion of E. cloacae in rabbit enterocytes, glycosylation of this protein occurring only under specific conditions may be involved in this process.

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. Sera were tested for B19-specific immunoglobulin M (IgM) and/or IgG using an enzyme-linked immunosorbent assay technique. Based on serology, 149 cases not related to B19 infection were excluded from further analysis. Two of the remaining 124 cases (0.7% of all 273 cases) had parvovirus B19-specific IgM and IgG at the time of abortion, indicating a recent maternal parvovirus B19 infection. In our histological examination, 10 cases contained nuclear vacuolization in fetal erythroid progenitor cells, either in fetal tissues (n = 2) or in placental tissue (n = 8). However, this vacuolization was considered a fixation artifact and not identical to parvovirus B19-specific nuclear inclusions described in previous reports. Only 1 of these 10 cases had parvovirus B19 DNA detectable in placental tissue by PCR analysis. Neither in this case nor in any of the other cases tested was parvovirus B19 DNA or protein detectable by ISH or IHC, respectively. In none of 41 cases in which fetal tissues were available were congenital anomalies found. In conclusion, the frequency of maternal parvovirus B19 infection in this series of fetal losses is low (0.8%). This low frequency does not allow any conclusions with regard to the occurrence of congenital anomalies resulting from parvovirus B19 infection and the usage of nuclear histology for the detection of fetal parvovirus B19 infection is considered a nonspecific parameter that requires confirmation by PCR.

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. Anemia was noted in 176 patients (55.5%); 126 (39.9%) had < 0.10 x 10(9) CD4+ cells/L. In this study group, 191 (60.3%) of the patients were positive for parvovirus B19 IgG. Seroprevalence rates did not differ between patients with low and higher CD4+ cell counts or between anemic and nonanemic patients. Parvovirus B19 DNA was detected in none of the sera. In a control group of 226 healthy male blood donors, the seroprevalence of parvovirus B19 IgG was 68.1%; two IgG-positive sera also contained parvovirus B19 DNA. This study demonstrates that chronic parvovirus B19 infection should not be considered a frequent cause of anemia in HIV-infected individuals.

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. The course of the antibody response could be followed by determining the titers of sequential serum samples taken after a recent B19 infection. Both types of recombinant capsids form an excellent source of antigen for the detection of both B19 IgG and IgM antibodies and are a very promising substitute for native virus.

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. Several applications of the polymerase chain reaction for B19 DNA detection are discussed: i) Detection of B19 DNA sequences in paraffin-embedded fetal tissues in a case of intrauterine fetal death; ii) The presence of B19 DNA in a case of B19 infection after a bone marrow transplantation was demonstrated for a longer period of time as compared to detection with dot-hybridization; iii) In a considerable number of sera, proven to be B19 specific IgM positive, the presence of B19 DNA was demonstrated. The sensitivity of the polymerase chain reaction, combined with the simplicity, reduced time scale and range of applications, demonstrates the potential of this technique as an additional method for routine B19 diagnosis next to the detection of B19 specific antibodies.

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., 1983). 76% of sera from a random selection of blood donors were positive for B19 IgG which agrees with previous findings. The course of the IgM and IgG antibody response to B19 infection could be followed with the immunofluorescence assay by determining the titers of series of sera taken after a recent B19 infection. Investigation of 24 sera containing rubella-specific IgM showed no cross-reactivity with the recombinant B19 VP1 used in this test system. The test described here has the advantage of being based on a renewable source of antigen and will be further evaluated for routine diagnostic use in comparison with radioimmunoassay.

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. The recombinant viruses were used to infect Spodoptera frugiperda cells and the proteins expressed were analysed using appropriate antibodies. AcB19VP1L-infected cells produced B19 VP1 as shown by its reaction with 13 human sera containing B19-specific antibodies in Western blot analysis and indirect immunofluorescence. The signal seen with VP1 in immunofluorescence makes it suitable for the development of a diagnostic assay based on this technique. VP1 also reacted with two monoclonal antibodies (mAbs) specific for the B19 protein part of a 196 kDa beta-galactosidase B19 fusion protein expressed in E. coli. Cells infected with AcB19VP2L produced B19 VP2 which reacted with the same human sera in indirect immunofluorescence and with five of the 13 sera in Western blots. VP2 did not react with the fusion protein-specific mAbs. The large amounts of viral antigen produced in this system means the development of widely available diagnostic tests for B19 infection and the further characterization of the B19 structural proteins are within reach.

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. The method is based on the lysing and nuclease-inactivating properties of the chaotropic agent guanidinium thiocyanate together with the nucleic acid-binding properties of silica particles or diatoms in the presence of this agent. By using size-fractionated silica particles, nucleic acids (covalently closed circular, relaxed circular, and linear double-stranded DNA; single-stranded DNA; and rRNA) could be purified from 12 different specimens in less than 1 h and were recovered in the initial reaction vessel. Purified DNA (although significantly sheared) was a good substrate for restriction endonucleases and DNA ligase and was recovered with high yields (usually over 50%) from the picogram to the microgram level. Copurified rRNA was recovered almost undegraded. Substituting size-fractionated silica particles for diatoms (the fossilized cell walls of unicellular algae) allowed for the purification of microgram amounts of genomic DNA, plasmid DNA, and rRNA from cell-rich sources, as exemplified for pathogenic gram-negative bacteria. In this paper, we show representative experiments illustrating some characteristics of the procedure which may have wide application in clinical microbiology.

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. By PCR 90% of the samples obtained from atherosclerotic patients were shown to contain viral nucleic acids as compared to 53% of patients with maximally grade I atherosclerosis, thus substantiating a role for this virus in the pathogenesis of atherosclerosis.

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). From the statistically evaluable viremic patients (n = 13), 110 blood samples were analyzed. In 5 of these patients, the DEAFF and PCR led to identical results. In 8 patients however the PCR was more sensitive, i.e. HCMV DNA was detected for a longer period of time. Applying statistical analysis using the McNemar test, this result was significant (P less than 0.05). The PCR applied on leukocyte samples did not detect HCMV DNA in viruric patients without viremia. Moreover, the current PCR never led to positive results with peripheral blood leukocyte samples of healthy seropositive or seronegative controls. Since the PCR can be performed in 6 hr, this technique will contribute to rapid detection of HCMV DNA in peripheral blood leukocytes and therefore to optimal clinical management of HCMV-infected transplant recipients.

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. The presence of B19 DNA was demonstrated by dot hybridization in placental and fetal tissues. In the third no gross fetal abnormalities were found, although B19 DNA was detected in several fetal tissues by in-situ hybridization. Of the remaining seven pregnancies, six ended at term in the birth of apparently healthy babies. The other child was born near term with a low birthweight and multiple congenital malformations, but with no proof of intrauterine B19 infection. It is concluded that B19 infection in pregnancy can interfere with organ development and may lead to intrauterine fetal death.

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. The resolution with the AAF-labelled probes was higher than with the radiolabelled DNA. Parvovirus B19 DNA sequences were also detected in these tissues by an enzymatic in vitro amplification technique--the polymerase chain reaction. Amplification of a B19-DNA sequence before detection increases the rapidity and sensitivity of detection. The rapid, specific, and sensitive analysis of parvovirus B19 in normal and diseased tissues using these techniques may contribute considerably to determining the role of this virus as a risk factor in the outcome of pregnancy.

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. Using radiolabeled RNA probes even 0.3 pg of viral DNA was detectable. The polymerase chain reaction was more sensitive than the hybridization assays: 100 fg of viral DNA was easily detectable by electrophoresis on agarose and after subsequent hybridization with a radiolabeled probe approximately 10 fg of B19 DNA was detected. The sensitivity of the PCR, combined with the simplicity and reduced time scale, demonstrates the potential of this technique as an additional method for routine diagnosis of B19 infections.