Target imbalance: disparity of Borrelia burgdorferi genetic material in synovial fluid from Lyme arthritis patients.

Lyme arthritis is a late manifestation of Lyme disease that results in episodic synovial inflammation and swelling. Although this process is thought to be driven directly by the spirochetal etiologic agent, Borrelia burgdorferi, the organism itself has been recovered by culture only twice. In contrast, polymerase chain reaction (PCR) studies are usually positive.

Heritable susceptibility to severe Borrelia burgdorferi-induced arthritis is dominant and is associated with persistence of large numbers of spirochetes in tissues.

In human Lyme disease, symptoms with widely varying levels of severity have been observed. A mouse model of Lyme disease has been developed which allows analysis of mice with mild, moderate, and severe pathologies after inoculation with the spirochete Borrelia burgdorferi. To determine whether the differences in symptoms reflect differences in the number of spirochetes persisting in affected tissues, a sensitive PCR technique was developed to detect B.

Detection of Borrelia burgdorferi DNA by polymerase chain reaction in synovial fluid from patients with Lyme arthritis.

Background: Borrelia burgdorferi is difficult to detect in synovial fluid, which limits our understanding of the pathogenesis of Lyme arthritis, particularly when arthritis persists despite antibiotic therapy.

Methods: Using the polymerase chain reaction (PCR), we attempted to detect B. burgdorferi DNA in joint-fluid samples obtained over a 17-year period.

Nucleotide sequence analysis of the precore region in patients with fulminant hepatitis B in the United States.

Background: A precore defective hepatitis B virus (HBV) mutant unable to produce hepatitis B e antigen (HBeAg) has been associated with fulminant hepatitis B. We have studied the etiologic contribution of precore mutants among North American patients with this disorder.

Methods: We studied 39 patients with fulminant hepatitis B.

Dependence of polymerase chain reaction product inactivation protocols on amplicon length and sequence composition.

Specific diagnostic test results generated by polymerase chain reaction (PCR) depend upon control of amplicon contamination in the clinical laboratory. We compared photochemical (isopsoralen [IP]) and enzymatic (uracil N-glycosylase [UNG]) methods for their ability to prevent carryover of amplicons generated from genomic targets of five viruses. PCR products (amplicons) (herpes simplex virus, 342 bp; cytomegalovirus, 250 bp; Epstein-Barr virus, 240 bp) exposed to UV light in the presence of various concentrations of IP compound 10 (IP-10) resulted in apparent increased molecular sizes of the products, as indicated by migration patterns after gel electrophoresis, and were predictive of inactivation by the agent.

Preventing false positives: quantitative evaluation of three protocols for inactivation of polymerase chain reaction amplification products.

False-positive results because of carryover contamination by previously amplified nucleic acids are currently the greatest impediment to routine implementation of nucleic acid amplification protocols. We evaluated three methods for inactivation of a 156-bp Borrelia burgdorferi polymerase chain reaction (PCR) product: (i) post-PCR cross-linking with isopsoralen (IP), (ii) pre-PCR treatment of a dU-containing PCR product with uracil N-glycosylase (UNG), and (iii) post-PCR alkaline hydrolysis (primer hydrolysis) of PCR products synthesized by using primers containing 3' ribose residues. The sensitivities of the PCR performed under the conditions of each protocol were comparable.

Chronic Lyme borreliosis in the laboratory mouse.

C3H/HeJ mice were inoculated intraperitoneally with 10(7) uncloned Borrelia burgdorferi at 4 weeks of age and examined on days 30, 90, 180, and 360. Spirochetes were isolated from multiple tissues at all intervals. Joint and heart disease were present in all mice at 30 days and resolved after 90 days.

Babesiosis in Washington State: a new species of Babesia?

Objective: To characterize the etiologic agent (WA1) of the first reported case of babesiosis acquired in Washington State.

Design: Case report, and serologic, molecular, and epizootiologic studies.

Setting: South-central Washington State.

Elimination of contaminating DNA within polymerase chain reaction reagents: implications for a general approach to detection of uncultured pathogens.

Analysis based on comparisons of 16S rRNA sequences provides a rapid and reliable approach to identifying human pathogens. By directing oligonucleotide primers at sequences conserved throughout the eubacterial kingdom, bacterial 16S ribosomal DNA sequences of virtually any member of the eubacterial kingdom can be amplified by polymerase chain reaction and subsequently analyzed by sequence determination. Indeed, automated systems for broad-range amplification, sequencing, and data analysis are now feasible and may form the basis of the next generation of automated microbial identification systems.

Diagnostic molecular microbiology. Current challenges and future directions.

The advent of nucleic acid amplification techniques for the clinical laboratory provides not only new diagnostic opportunities but new responsibilities as well. Problems associated with the introduction of this technology include contamination with the products of amplification reactions, and difficulty in interpreting test results. Eventually, however, many of these problems will be overcome, and new applications of diagnostic molecular microbiology such as sequence-based microbial identification will become established.

A controlled trial of antimicrobial prophylaxis for Lyme disease after deer-tick bites.

Background: Borrelia burgdorferi, which causes Lyme disease, is transmitted by deer ticks (lxodes dammini) in the northeastern and midwestern United States. Although deer-tick bites are common in areas in which the disease is endemic, there is uncertainty about how to manage the care of persons who are bitten.

Methods: To assess the risk of infection with B.

Carditis in Lyme disease susceptible and resistant strains of laboratory mice infected with Borrelia burgdorferi.

The clinical and pathologic evolution of cardiac Lyme disease was evaluated in four-week-old susceptible C3H/He (C3H) and resistant C57Bl/6 (B6) mice on days 3, 6, 10, 15, 30, 60, and 90 after intradermal inoculation with Borrelia burgdorferi strain N40. Culture, DNA polymerase chain reaction, in situ nucleic acid hybridization, immunoperoxidase histochemical analysis, and silver stain were used to detect spirochetes. Spirochetes were first detected by culture on day 6 in two of four C3H mice.

Detection of Babesia microti by polymerase chain reaction.

Human babesiosis, which is caused by infection with the intraerythrocytic malarialike protozoan Babesia microti, has recently been diagnosed with increasing frequency in residents of New England. Diagnosis is difficult because of the small size of the parasite and the sparse parasitemia that is characteristic of most infections with this pathogen. We generated B.

Ribosomal DNA probe for differentiation of Babesia microti and B. gibsoni isolates.

The objective of this study was to determine whether different isolates of Babesia microti could be distinguished from morphologically similar isolates of B. gibsoni by using a ribosomal DNA (rDNA) probe. A Babesia-specific rDNA probe was obtained by polymerase chain reaction amplification of sequences from B.

Borrelia burgdorferi strain 25015: characterization of outer surface protein A and vaccination against infection.

Mice vaccinated with outer surface protein A (OspA) from Borrelia burgdorferi strain N40 are protected from challenge with an intradermal syringe inoculum of B. burgdorferi strains N40, B31, and CD16. Vaccination experiments were done to determine if protection extended to strains 297 and 25015.

Chemiluminescent universal probe for bacterial ribotyping.

We describe a novel procedure for direct covalent coupling of horseradish peroxidase to rRNA and ribotyping by using enhanced chemiluminescence. Compared with their 32P-end-labeled counterparts, chemiluminescent rRNA probes are stable and easy to synthesize and provide results as good as or superior to those obtained with isotopic labeling. Direct chemiluminescent labeling of Escherichia coli rRNA produces a sensitive, universal probe suitable for clinical laboratory use in the investigation of nosocomial outbreaks.

Kinetics of Borrelia burgdorferi dissemination and evolution of disease after intradermal inoculation of mice.

Borrelia burgdorferi dissemination to selected target organs was examined on days 1, 2, 3, 4, 7, 10, 15, 21, and 30 after intradermal inoculation of 4-week-old C3H mice. Infection was determined by culture (blood, spleen, kidney, ear punch); polymerase chain reaction (PCR) for outer surface protein A (OSP A) DNA (ear punch); histology and spirochete histochemistry (spleen, kidney, skin, heart, joints); and OSP A DNA in situ hybridization (joints, heart). Blood or spleen of most mice were culture positive by day 3 and ear punch by day 10.

Detection of Borrelia burgdorferi DNA in museum specimens of Ixodes dammini ticks.

In order to investigate the potential for Borrelia burgdorferi infection before the recognition of Lyme disease as a clinical entity, the polymerase chain reaction (PCR) was used to examine museum specimens of Ixodes dammini (deer ticks) for the presence of spirochete-specific DNA sequences. One hundred and thirty-six archival tick specimens were obtained representing various continental U.S.

Detection of Borrelia burgdorferi infection in Ixodes dammini ticks with the polymerase chain reaction.

The polymerase chain reaction (PCR) was used to amplify DNA sequences of the etiologic agent of Lyme disease, Borrelia burgdorferi, and was applied to the detection of the spirochete in its tick vector. The target for PCR amplification was the OSP-A gene of strain B31; analysis of isolates from different geographical areas indicated that this gene could be used to identify most North American isolates. These methods were extended to the analysis of colony-derived and field-collected Ixodes dammini.

In vitro amplification techniques for the detection of nucleic acids: new tools for the diagnostic laboratory.

The acceptance of nucleic acid probes as diagnostic tools for the clinical laboratory has been hampered by a number of factors, including laborious techniques and limited sensitivity. The focus of this review is on the recent development of amplification techniques to enhance the signal generated by nucleic acid-based detection systems. Three general areas are discussed: (1) amplification of target sequences using the polymerase chain reaction or the transcript amplification system, (2) amplification of the probe sequences using Q beta replicase, and (3) amplification of probe-generated signals with compound or "Christmas tree" probes.

The preS1 protein of hepatitis B virus is acylated at its amino terminus with myristic acid.

The preS/S coding region of hepatitis B virus encodes two polypeptides (preS1 and preS2) that are larger in size but less abundant than the major viral surface antigen (S) protein. Unlike the preS2 and S proteins, the preS1 protein is preferentially localized on circulating virus particles but is not efficiently secreted from mammalian cells in culture. To search for differences in protein processing that might relate to these properties, we determined whether any of the hepatitis B virus surface proteins are acylated with long-chain fatty acids.