Routine diagnosis of Borrelia burgdorferi (sensu lato) infections using a real-time PCR assay.

OBJECTIVE: To establish a one-tube fluorogenic real-time PCR assay for routine detection of Borrelia burgdorferi (sensu lato) DNA in various clinical specimens. METHODS: A fragment of the flagellin gene sequence was amplified with the TaqMan chemistry using primers and a probe common to Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii and Borrelia valaisiana. A recombinant plasmid containing the chromosomal gene coding for the flagellin protein was used as standard. RESULTS: The specificity of the assay was documented with 48 different clinically relevant Borrelia burgdorferi strains. No cross-reaction occurred with unrelated bacteria, viruses and fungi. At an analytic sensitivity of 10 copies, excellent precision within runs and between runs was observed. The potential presence of inhibitors of the Taq DNA polymerase was monitored by spiking aliquots of each sample with a plasmid containing the target sequence. Among 56 cerebrospinal fluid samples taken from 54 patients with clinical suspicion of neuroborreliosis, one (1.8%) tested positive for Borrelia burgdorferi sensu lato DNA. Borrelia burgdorferi DNA was also detected in five (17.9%) of 28 synovial fluid specimens and in one (20%) of five synovial membrane biopsies obtained from 31 patients with arthropathies. In order to test for the absence of false-positive results, 84 samples from 83 patients without evidence of Lyme disease were investigated. None of these samples showed measurable amounts of Borrelia burgdorferi DNA. CONCLUSION: By its established features, such as speed, reliability, sensitivity, specificity, the inclusion of carryover prevention and the monitoring of inhibitors in individual test tubes, this real-time PCR assay has proved to be a potent tool for the detection of Borrelia burgdorferi DNA under routine conditions in diagnostic laboratories.

Presence and significance of human parvovirus B19 DNA in synovial membranes and bone marrow from patients with arthritis of unknown origin.

Acute rheumatologic symptoms are frequently associated with human parvovirus B19 (B19) infections. A nested PCR (nPCR) assay was used to test for the presence of parvovirus B19 DNA in synovial fluid and/or synovial membrane specimens obtained from a total of 90 patients with arthritis of unknown origin. Whereas only one out of 73 synovial fluid samples were found positive, 15 (16.7%) out of 90 patients had parvovirus B19 DNA in the synovium. B19 virus DNA was detected in nine bone marrow aspirates subsequently obtained from these 15 patients (60%). Whereas each one of the 15 corresponding blood samples contained anti-B19 IgG antibody, none contained anti-B19 IgM antibody and only one was positive for B19 virus DNA. The blood and synovial fluid samples that contained B19 virus DNA were obtained from the same patient, who also had B19 DNA in synovium and bone marrow. For one patient, two distinct synovial membrane specimens collected 10 months apart tested positive for B19 virus DNA. Parvovirus B19 DNA was also detected in synovial tissue of one out of nine nonarthritic patients serving as control group, who also had anti-B19 IgG circulating antibody. These data illustrate that human parvovirus B19 may persist in bone marrow and synovial tissues of patients with arthritis of unknown origin. In contrast, persistence of B19 virus DNA in synovial fluid is rare. The significance of parvovirus B19 DNA in synovium of healthy patients has to be established.

A nested-PCR assay for the simultaneous amplification of HSV-1, HSV-2, and HCMV genomes in patients with presumed herpetic CNS infections.

To facilitate early diagnosis of herpes virus infection of the central nervous system (CNS), a nested polymerase chain reaction (nPCR) assay was developed to test simultaneously for the presence of HSV-1, HSV-2, and HCMV DNA in the cerebrospinal fluid (CSF) of patients with herpetic CNS disease suspected on clinical grounds. The virus type-specific PCR products were differentiated either by agarose gel electrophoresis or by DNA enzyme immunoassay. Using titrated viral stocks as standards, a sensitivity of at least 0.03 infectious units was obtained for HSV-1, HSV-2 and HCMV and no cross-reactions were recorded. Among 178 CSF specimens (171 patients), which were tested under routine conditions, three contained HSV-1 DNA, one contained HSV-2 DNA and one contained HCMV DNA. No double or triple infection was diagnosed. The presence of inhibitors of the Taq DNA polymerase was monitored by spiking aliquots of the above CSF samples with 3 infectious units each of HSV-1 and HSV-2 or HCMV. Whereas none of 93 samples spiked with HSV-1 and HSV-2 contained inhibitors, the PCR reaction was inhibited in three out of 175 samples (1.7%) spiked with HCMV. The complete procedure which requires only 150 microl of CSF is easily completed within 8 h. Through its speed, reliability and sensitivity, this nPCR assay has met the specific criteria of the diagnostic laboratory.

Evidence for persistence of human parvovirus B19 DNA in bone marrow.

A nested polymerase chain reaction assay (nPCR) was used to investigate the potential of human parvovirus B19 DNA to persist in blood or bone marrow samples obtained either from blood donors or cadaveric bone donors or from patients presenting with clinical signs of parvovirus B19 infection. The presence of parvovirus B19 specific antibody in blood was tested by enzyme immunoassay (EIA). B19 virus genome was not detected in any blood sample of 115 blood donors, of whom 92 (80%) had anti-B19 IgG antibody only as an indication of past infection. In contrast; B19 virus DNA was detected in the bone marrow of 4 out of 45 bone donors. Each one of the serum samples available for 3 of these 4 individuals contained anti-B19 IgG antibody. Among 84 patients with clinical manifestations of parvovirus B19 infection, 17 (20%) had B19 virus DNA in bone marrow. Eight of the latter patients had anti-B19 IgG antibody in their blood but neither anti-B19 IgG nor B19 virus DNA. These data document the ability of parvovirus B19 DNA to persist in the bone marrow of asymptomatic individuals and patients with parvovirus B19 infection suspected on clinical grounds.

Parvovirus [correction of Parovirus] B19-induced red cell aplasia in solid-organ transplant recipients. Two case reports and review of the literature.

Two solid-organ transplant recipients (one heart and one lung) developed severe anemia with reticulocytopenia. Both were heavily immunosuppressed. Bone marrow aspiration revealed almost complete absence of erythroid precursors. A few giant megaloblastic proerythroblasts with cytoplasmic vacuolisation and intranuclear inclusions were seen. Human parvovirus B19 (B19V)-DNA genome was found by nested-PCR assays in blood and bone marrow samples in both cases. Twelve similar cases are described in the literature. When looked for, B19V DNA was positive either in serum or bone marrow or both. Twelve of the fourteen patients were successfully treated by high dose i.v. immunoglobulin (IVIG). One patient recovered spontaneously and another after treatment with recombinant human erythropoietin (rHu-EPO) only. Transplant patients should be considered at risk for severe erythroblastopenic anemia due to B19V infection. Diagnosis is based on bone marrow examination and detection of B19V DNA by PCR in serum and/or marrow. IVIG is an effective and safe treatment. The role of erythropoietin in this indication needs further study.

Postpartum lupus erythematosus associated with parvovirus B19 infection.

We describe postpartum onset of systemic lupus erythematosus (SLE) associated with parvovirus B19 infection in a mother presenting with fever, polyarthritis, erythema, and multiorgan involvement. B19 infection was revealed by detection of B19 DNA and IgM antibodies. In addition, our patient showed low CD4+ (384 x 10(6)/l) and CD8+ (213 x 10(6)/l) T cells, high immunoglobulin values (23.77 g/l), hypocomplementemia, thrombocytopenia, leukopenia, and anemia. Her daughter had rash associated with increasingly high B19 IgG levels and transient antinuclear and anti-ds-DNA antibodies, suggesting that both development of SLE and active B19 infection occurred in pregnancy and B19 was transmitted prenatally. A 2 year followup showed persisting polyarthritis in the mother and atopy in the daughter.

Suitability and clinical application of a multiplex nested PCR assay for the diagnosis of herpes simplex virus infections.

A novel multiplex nested polymerase chain reaction (PCR) assay was designed and evaluated for routine diagnosis of herpes simplex virus (HSV) infections in patients with either putative HSV infection of the central nervous system or suspected HSV keratitis. Single-tube amplification of HSV type 1 (HSV-1) or type 2 (HSV-2) DNA extracted from cerebrospinal fluid (CSF) or from keratectomy specimens was followed by differentiation of the virus type-specific PCR products either by agarose gel analysis or by DNA enzyme immunoassay. Among 417 CSF specimens obtained from 395 consecutive patients with clinically suspected HSV infection, 11 (2.6%) were positive for HSV-1 DNA and four (1.0%) probes were positive for HSV-2 DNA. None of the specimens was positive for both HSV-1 and HSV-2 DNA. The genome of HSV-2 was detected in a CSF sample obtained from a woman with meningoencephalitis and genital herpes. The presence of PCR inhibitors was detected in six of 111 (5.4%) reconstructed CSF samples. Inhibition could be removed following extraction with a commercial kit. HSV-1 DNA, but no HSV-2 DNA, was detected in corneal buttons obtained from patients with suspected herpetic keratitis. No contamination has been recorded during the 2-year routine use of this test, which has met the specific requirements of a diagnostic laboratory.

Sequence variability among different parvovirus B19 isolates.

Parvovirus B19 is the causative agent of a variety of clinical manifestations, ranging from asymptomatic to severe infection. The basis for this complex pattern of B19-associated diseases is as yet poorly understood. In general there are two different possibilities: firstly, the infected individuals may have a genetic or acquired predisposition, which renders them susceptible for a certain course of infection; secondly, differences in the B19 genome may result in different outcomes of infection. In order to investigate this second possibility we have partially sequenced the genomes of 20 different B19 isolates derived from serum samples from patients with various B19-associated diseases. Four distinct regions, which cover nearly half of the genome and include parts of the coding regions of all three major B19 proteins-NS1, VP1 and VP2, were selected for sequencing. Comparisons between the different extracted virus isolates at the DNA and protein levels revealed that isolates from patients with persistent parvovirus B19 infection show a tendency towards higher genome variability with respect to isolates derived from persons with acute infection.

Identification of hepatitis A virus non-structural protein 2B and its release by the major virus protease 3C.

The RNA genome of hepatitis A virus (HAV) encodes a giant polyprotein that is putatively cleaved proteolytically into four structural and seven non-structural proteins. So far, most of the proposed non-structural proteins and their respective cleavage sites have not been identified. A vaccinia virus recombinant (vRGORF) containing the complete HAV ORF under the control of the bacteriophage T7 promoter was used to express HAV in recombinant animal cells (BT7-H) that constitutively expressed T7 DNA-dependent RNA polymerase. A HAV-specific 27.5 kDa expression product was identified as peptide 2B. The 27.5 kDa 2B antigen was also found in HAV-infected MRC-5 cells. The N-terminal amino acid residues of the new peptide 2B are Ala-Lys-Ile-Ser-Leu-Phe and polyprotein cleavage between 2A and 2B occurred at amino acids 836-837 (Gln-Ala). Furthermore, heterologous expression in the same system of regions P1-P2 and of the protease 3C (3Cpro) gene, showed that P1-P2 polyprotein is not cleaved autocatalytically but by 3Cpro. Hence, 3Cpro is effective in cleaving the polyprotein 2A-2B junction.

Acute bilateral carpal tunnel syndrome associated with human parvovirus B19 infection.

Human parvovirus B19 has been described as a causative agent of erythema infectiosum (a disease common in children), aplastic crisis in patients with hemolytic disorders, and arthralgias and arthritis. Joint involvement may be a prominent clinical feature of parvovirus B19 infection and may last for several weeks. We describe three cases of acute bilateral carpal tunnel syndrome associated with parvovirus B19 infection as evidenced by serological data and, in one case, by detection of parvovirus B19 DNA in blood with use of PCR.

[Diagnosis of Chlamydia trachomatis infections in routine ambulatory care of a gynecologic-obstetrical clinic: comparison of genome, antigen and cell culture detection methods for various indications]. / Diagnostik von Chlamydia-trachomatis-Infektionen im Routinebetrieb einer gynäkologisch-geburtshilflichen Klinik: Vergleich von Genom-, Antigen- und Zellkulturnachweisverfahren unter verschiedenen Indikationen.

OBJECTIVE: Comparison of four different assays for routine diagnosis of urogenital Chlamydia trachomatis infections. METHODS: Samples from 285 female patients were tested using each of the following tests: PCR (Amplicor Chlamydia trachomatis), ELFA (VIDAS Chlamydia), cell culture and direct immunofluorescence assay (DFA). RESULTS: C. trachomatis was detected by PCR in 13 endocervical swab specimens obtained from 189 female patients (6.9%). Among 13 PCR-positive samples, 10 tested positive by cell culture and DFA, and 8 were positive by ELFA. For 3 patients with pregnancy-related complications, a positive result was obtained by PCR only. Each of the 96 urethral swabs proved to be negative. CONCLUSIONS: PCR is more sensitive than cell culture, DFA and ELFA, especially in the context of C. trachomatis infection during pregnancy. In addition and in order to avoid false-negative PCR results, a careful collection of epithelial cells infected with C. trachomatis is imperative.

Detection of herpes simplex virus after penetrating keratoplasty by polymerase chain reaction: correlation of clinical and laboratory findings.

BACKGROUND: The study was carried out to investigate the possible correlation of clinical findings, histopathologic features and detection of herpes simplex virus DNA in corneal buttons obtained after penetrating keratoplasty. METHODS: We examined 47 consecutive corneal buttons sent for histopathologic examination by light microscopy and using the polymerase chain reaction for the detection of HSV1 and HSV2. Twenty-one corneal buttons from eyes with bullous keratopathy served as controls. RESULTS: The 47 cases were graded from the clinical information available as unproven, suspected and clinically proven cases of herpetic keratitis. This grading did not correlate to specific histopathologic features or to the results of HSV1 DNA testing. None of the cases were positive for HSV2 DNA. CONCLUSION: HSV DNA was detected in some of the cases of clinically unsuspected herpetic keratitis. This technique of demonstrating the presence or absence of HSV in the cornea after keratoplasty is more reliable than clinical data or histopathologic findings and may be important in cases of recurrent inflammatory episodes involving grafts after keratoplasty.

Association between human parvovirus B19 infection and arthritis.

OBJECTIVE: To gain information concerning the association between parvovirus B19 infection and arthritis. METHODS: Blood or synovial fluid, or both, from a total of 77 adult patients with various arthropathies (rheumatoid arthritis 13; mechanical arthropathies 11; crystal induced arthritis 13; idiopathic mono/oligoarthritis 25; suspicion of viral arthritis 15) were tested for the presence of the viral genome and anti-B19 antibodies. B19 DNA in blood and synovial fluid was investigated by nested polymerase chain reaction, and anti-B19 IgM and IgG antibodies were detected in blood by enzyme immunoassay. RESULTS: A recent parvovirus infection was documented by the presence of anti-B19 IgM antibodies in the blood of 13 patients. B19 DNA, together with anti-B19 IgM and IgG antibodies, were detected in the blood of seven patients who had an acute transient arthritis, putatively of viral origin. Viral DNA was detected in a synovial fluid sample and in the blood of one patient with monoarthritis who had an anti-B19 IgG response only. CONCLUSIONS: The prevalence of anti-B19 IgG antibody in these patients with various forms of arthritis (63%) was within the same range as that in the general population (blood donors). However, for the patients with clinical suspicion of viral arthritis, the increased seroprevalence of anti-B19 IgM and the presence of the B19 genome point to an association between human parvovirus infections and acute forms of arthritis.

[Parvovirus B19-induced arthritis/arthropathy--an important differential diagnosis of chronic polyarthritis]. / Parvovirus-B19-bedingte Arthritis/Arthropathie--eine wichtige Differentialdiagnose der chronischen Polyarthritis.

INTRODUCTION/AIMS: The differential diagnosis of rheumatoid arthritis (RA) and parvovirus-B19-induced arthritis/arthropathy (PBA) can be difficult, but is of importance because of the different therapeutic implications. The purpose is to describe characteristic features serving to differentiate between chronic PBA and RA, based on 6 personal cases and the literature. METHODS/PATIENTS: 6 patients presenting with acute (3 cases) or chronic PBA (3 cases) over the last 5 years are described. RESULTS/CONCLUSIONS: The demonstration of anti-parvovirus-B19-immunoglobulins (Ig)M in addition to anti-parvovirus-B19-IgG is the most important diagnostic finding. Measurement of IgM must be done within the first months after onset, as it disappears later on. Furthermore, history of disease (exposure, prodromi and acute onset of arthritis), clinical examination (rash) and further investigations (normal ESR and CRP, typical hematologic findings, examination of synovial tissue and fluid without inflammatory changes, demonstration of the genome of parvovirus B19 by polymerase chain reaction, no erosions on radiographs) support the diagnosis of PBA. 2 of the 3 patients with chronic PBA fulfilled the criteria for classification of RA. Therapeutic approaches in PBA are discussed. In contrast to the favourable effect in RA, immunosuppressive agents may prolong persistence of virus and disease in PBA.

Parvovirus B19 infection during pregnancy and development of hydrops fetalis despite the evidence for pre-existing anti-B19 antibody: how reliable are serological results?

BACKGROUND: Primary infection with human parvovirus B19 during pregnancy can lead to fetal hydrops, abortion, or stillbirth. However, reinfection in the presence of pre-existing anti-B19 antibody is generally assumed to have no significant effect on the developing fetus. OBJECTIVES: To describe a case of fetal loss at 28 weeks' gestation associated with parvovirus B19 infection which took place in a 26-year-old woman despite the evidence for pre-existing anti-B19 IgG antibodies. STUDY DESIGN: A nested-PCR assay for parvovirus B19 DNA was performed on maternal and fetal samples. Blood samples were tested by various enzyme immunoassays (EIA) for the presence of both anti-B19 IgM and IgG antibodies. RESULTS: B19 DNA together with anti-B19 IgG antibody were detected in maternal blood at the time of intrauterine fetal demise. Amniotic fluid, chorionic villi and various fetal tissues also tested positive for viral DNA. In retrospect, presence of anti-B19 IgG antibody, but no viral DNA, was repeatedly demonstrated in maternal blood before infection took place. However, the serological results differed with the test system used. CONCLUSIONS: Provided that the positive serological results are reliable, the presence of anti-B19 IgG in blood samples collected as early as four years before pregnancy neither protected the mother from reinfection not the fetus from transplacental infection with B19 virus. However, discrepant (negative) serological results were also obtained depending on the test system used. Therefore, and in the light of the possible severe consequences of B19-infection during pregnancy, the means for assessment of the significance of anti-B19 titers have to be urgently established by development of both qualitative and quantitative anti-B19 IgM and IgG standards.